Research ArticleAntiproliferative Effects of Methanolic Extracts ofCryptocarya concinna Hance Roots on Oral Cancer Ca9-22 andCAL 27 Cell Lines Involving Apoptosis ROS Induction andMitochondrial Depolarization

Hurng-Wern Huang1 Yi-An Chung1 Hsun-Shuo Chang23 Jen-Yang Tang456

Ih-Sheng Chen23 and Hsueh-Wei Chang678

1 Institute of Biomedical Science National Sun Yat-Sen University Kaohsiung 80424 Taiwan2Graduate Institute of Natural Products College of Pharmacy Kaohsiung Medical University Kaohsiung 80708 Taiwan3 School of Pharmacy College of Pharmacy Kaohsiung Medical University Kaohsiung 80708 Taiwan4Department of Radiation Oncology Faculty of Medicine College of Medicine Kaohsiung Medical UniversityKaohsiung 80708 Taiwan

5Department of Radiation Oncology Kaohsiung Medical University Hospital Kaohsiung 80708 Taiwan6Cancer Center Translational Research Center Kaohsiung Medical University Hospital Kaohsiung Medical UniversityKaohsiung 80708 Taiwan

7 Institute of Medical Science and Technology National Sun Yat-sen University Kaohsiung 80424 Taiwan8Department of Biomedical Science and Environmental Biology Kaohsiung Medical University Kaohsiung 80708 Taiwan

Correspondence should be addressed to Ih-Sheng Chen m635013kmuedutw and Hsueh-Wei Chang changhwkmuedutw

Received 16 July 2014 Accepted 25 August 2014 Published 15 October 2014

Academic Editor Shao-Yu Chen

Copyright copy 2014 Hurng-Wern Huang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Cryptocarya-derived natural products were reported to have several biological effects such as the antiproliferation of some cancersThe possible antioral cancer effect of Cryptocarya-derived substances was little addressed as yet In this study we firstly used themethanolic extracts of C concinna Hance roots (MECCrt) to evaluate its potential function in antioral cancer bioactivity Wefound that MECCrt significantly reduced cell viability of two oral cancer Ca9-22 and CAL 27 cell lines in dose-responsive manners(119875 lt 001) The percentages of sub-G1 phase and annexin V-positive of MECCrt-treated Ca9-22 and CAL 27 cell lines significantlyaccumulated (119875 lt 001) in a dose-responsive manner as evidenced by flow cytometry These apoptotic effects were associated withthe findings that intracellular ROS generation was induced in MECCrt-treated Ca9-22 and CAL 27 cell lines in dose-responsiveand time-dependent manners (119875 lt 001) In a dose-responsive manner MECCrt also significantly reduced the mitochondrialmembrane potential in these two cell lines (119875 lt 001ndash005) In conclusion we demonstrated thatMECCrtmay have antiproliferativepotential against oral cancer cells involving apoptosis ROS generation and mitochondria membrane depolarization

1 Introduction

Oral squamous cell carcinoma (OSCC) is a type of cancer thatfrequently occurs in oral cavity Although it is comparativelyeasy to clinically inspect by a dentist or to detect by someOSCC tumormarkers [1 2] this carcinoma is usually ignored

by patients especially for the early stage Subsequently OSCCis frequently diagnosed at advanced stages which then leadto high mortality [3] Therefore the drug development ofantioral cancer is still necessary and remains to be a challenge

Natural products have improved the drug discoveryfor anticancer therapy [4] For example some anticancer

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 180462 10 pageshttpdxdoiorg1011552014180462

2 The Scientific World Journal

drugs derived from natural products were approved by theUnited States Food and Drug Administration [5] In basicresearches natural products with antioral cancer effects haveincreasingly being reported This holds for the ethanolic andmethanolic extracts of red algaGracilaria tenuistipitata [6 7]crude extracts of Selaginella tamariscina (oriental medicinalherb) [8] green tea [9] goniothalamin from Goniothalamusspecies [10] and 4120573-hydroxywithanolide E fromgolden berry[11]

Cryptocarya plants (family Lauraceae) comprising about350 species worldwide are widely distributed in the tropicsand subtropics [12] This plant group is well known forits common secondary metabolites containing alkaloidsflavonoids and 120572-pyrones [12ndash15] Several biological effectsof Cryptocarya-derived natural products have been reportedthat include anti-dengue virus [16] anti-HIV [17] anti-tuberculosis [18] antiplasmodial [19] antitrypanosomal [20]and anti-inflammatory [21] function

Anticancer effects of crude extracts of Cryptocarya plantare known as well For example the ethanolic extracts of fruitand trunk bark of C obovata showed 56 and 23 growthinhibition of human KB cells at 10 120583gmL respectively [22]Methanolic extracts of the leaves of C griffithiana providecytotoxicity forhuman HL60 promyelocytic leukemia cells[23]

Recently accumulating findings for anticancer effectsof pure compounds isolated from Cryptocarya plants werereported especially from methanolic extracts For examplecompounds isolated frommethanol extracts of the trunk barkof C infectoria [24] the trunk bark of C costata [25] andthe wood of C konishii [26] were reported to be cytotoxicto leukemia cells Compounds from methanolic extracts ofleaves of C chinensis were shown to be cytotoxic to humanlung cancer and glioblastoma cells [27] These drugs wereisolated from the trunk bark wood and leaves ofCryptocaryasp However the bioactivity of the roots of Cryptocaryaplants remained little investigated particularly with respectto antioral cancer

Because C concinna Hance is an evergreen plant com-monly distributed in low-altitude forests in Taiwan [28] it iseasy to preparemethanolic extracts of the roots ofC concinnaHance (namely forMECCrt)We therefore chose twoOSCCcell lines that is Ca9-22 and CAL 27 to evaluate the possibleanticancer function of MECCrt and investigate their drugmechanisms in terms of cell viability cell cycle distributionapoptosis reactive oxygen species (ROS) generation andmitochondrial depolarization

2 Materials and Methods

21 Cell Cultures and Methanolic Extracts of C concinnaTwo human OSCC cell lines Ca9-22 and CAL 27 purchasedfrom the Cell Bank RIKEN BioResource Center (TsukubaJapan) and the American Type Culture Collection (ATCCVirginia USA) respectively were incubated in DMEMF12(3 2) medium (Gibco Grand Island NY USA) supple-mented with 10 fetal bovine serum (Gibco) 100UmLpenicillin 100 120583gmL streptomycin and 003 glutamine

These two cell lines were humidly incubated at 37∘C with 5CO2in the humid atmosphere

C concinna was identified by one of the authors (Ih-Sheng Chen) and its roots were collected at Mudan PingtungCounty Taiwan in May 2004 A voucher specimen (Chen6153) has been deposited in the Herbarium of the Schoolof Pharmacy College of Pharmacy Kaohsiung MedicalUniversity The dried roots of C concinna were processedby slicing and cold methanol-extraction for three times atroom temperature Finally the solutionwas evaporated underreduced pressure to yield the methanolic extract (MECCrt)MECCrt was stored at minus20∘C and dissolved in dimethylsulfoxide (DMSO) before treatment

22 Cell Viability Cell viability was measured by theCellTiter 96 AQueous one solution cell proliferation assay(MTS) (Promega Corporation Madison WI USA) as pre-viously described [11] Ca9-22 and CAL 27 cell lines wereseeded at a density of 1 times 105 and 2 times 105 cells per well ina 6-well plate respectively After plating for 24 h these cellswere incubated with different concentrations of MECCrt for24 h and finally subjected to a MTS assay applying an ELISAreader at 490 nm

23 Cell Cycle Progression and Sub-G1 Population Propidiumiodide (PI Sigma St Louis MO USA) was added to stainthe cellular DNA content [29] In brief 3 times 105 cells perwell in 6 well plates were plated for 24 h and then treatedwith vehicle (DMSO 1120583L2mL culture medium) as a controlor 5 10 15 20 and 25 120583gmL of MECCrt for 24 h Afterexposure termination cells were centrifuged washed twicewith PBS fixed overnight with 70 ethanol and centrifugedSubsequently the cell pellets were resuspended in 50120583gmLPI reagent and stand for 30min at 37∘C in darkness Cellcycle distribution was evaluated by a flow cytometer (BDAccuri C6 Becton-Dickinson Mansfield MA USA) and aBD Accuri C6 Software (version 10264)

24 Apoptosis To validate apoptosis in MECCrt-treated oralcancer cells annexin V (Strong Biotect Corporation TaipeiTaiwan) [30]PI (Sigma St Louis MO USA) method wasused [31] Briefly 3 times 105 cells per well in 6 well plates wereplated for 24 h and then treated with vehicle or indicated con-centrations of MECCrt for 24 h Subsequently apoptotic cellswere stained for 30minwith 100120583L binding buffer containing2 120583L of annexin-V-fluorescein isothiocyanate (FITC) stock(025 120583g120583L) and 2120583L of PI stock (1mgmL) Finally it wassuspended with 400 120583L PBS for analysis of a flow cytometer(BD Accuri C6 Becton-Dickinson) and its software

25 Intracellular ROS The dye 2101584071015840-dichlorodihydroflu-orescein diacetate (DCFH-DA) was used to detect ROS byits fluorescence change [7] Cells at the density of 3 times 105 in2mL medium per well in 6 well plates were plated for 24 hDifferent concentrations of MECCrt were added to Ca9-22cells for 6 h and 12 h After washing with PBS 100 nMDCFH-DA in PBS was added to cells in 6 well plates at cell cultureincubator for 30min After trypsinization PBS washing and

The Scientific World Journal 3

120

100

80

60

40

20

0

0 5 10 2015

Cel

l via

bilit

y (

)Ca9-22

MECCrt (120583gmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

0 5 10 2015

Cel

l via

bilit

y (

)

MECCrt (120583gmL)

CAL 27140

120

100

80

60

40

20

0

lowastlowast

lowastlowast

lowastlowastlowastlowast

(b)

Figure 1 Cell viability of two oral cancer cells was inhibited by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with variousconcentrations ofMECCrt (0 5 10 15 and 20 120583gmL) for 24 hThe cell viability was measured by theMTS assay Data means plusmn SDs (119899 = 18)lowastlowast

119875 lt 001 against vehicle

centrifugation cell pellets were resuspended in 1mL PBSbefore analyzing by a flow cytometer (BDAccuri C6 Becton-Dickinson) and its software

26 Mitochondrial Membrane Potential MitoProbeDiOC

2(3) assay kit (Invitrogen Eugene OR USA) was

applied to analyze mitochondrial membrane potential(MMP) as described previously [10] Briefly 3 times 105 cellsin 2mL medium per well in 6 well plates were plated for24 h After MECCrt treatment 10 120583L of 10 120583M DiOC

2(3)

was added per well and incubated in a cell culture incubatorfor 20min After being harvested cells were washedand resuspended in 1mL PBS for analysis using a flowcytometer (BD Accuri C6 Becton-Dickinson) and itssoftware

27 Statistical Analysis The significance of differences wasdetermined by Studentrsquos 119905-test compared with the test datawith the vehicle controls Data are expressed as means plusmn SDs

3 Results

31 Antiproliferation in MECCrt-Treated Two Oral CancerCell Lines Based on MTS assay (Figure 1) the relative cellviability () of oral cancer Ca9-22 cells at indicated concen-trations of MECCrt (0 5 10 15 and 20120583gmL) was 1000 plusmn07 933 plusmn 23 714 plusmn 30 576 plusmn 16 and 484 plusmn 12 after24 h respectively The relative cell viability () of CAL 27cells at indicated concentrations of MECCrt (0 5 10 15 and20120583gmL) was 1000 plusmn 081193 plusmn 49 869 plusmn 100 298 plusmn 62and 284 plusmn 55 respectively The MTS-based cell viabilities ofMECCrt-treated twooral cancerCa9-22 andCAL27 cell linessignificantly reduced in a dose-responsive manner (119875 lt 001compared to the vehicle)

32 Sub-G1 Population in MECCrt-Treated Two Oral Can-cer Cell Lines The MECCrt-treated effects of cell cycledistribution profiles are demonstrated in Figure 2(a) AfterMECCrt treatment (Figure 2(b)) the sub-G1 populations ()ofMECCrt- (0 5 10 15 20 and 25 120583gmL) treated oral cancerCa9-22 cells were 50 plusmn 03 67 plusmn 01 181 plusmn 06 179 plusmn 07165plusmn03 and 224plusmn13 and those ofMECCrt-treatedCAL 27cells were 67plusmn17 51plusmn11 79plusmn01 283plusmn10 526plusmn02 and691 plusmn 01 respectively These sub-G1 changes significantlyaccumulated in a dose-responsive manner (119875 lt 001)

33 Apoptosis of MECCrt-Treated Two Oral Cancer Cell LinesTo validate the possible outcome of apoptosis in MECCrt-induced sub-G1 accumulation of these two oral cancer cellsannexin VPI profiles of flow cytometry were generated(Figure 3(a)) In Figure 3(b) the percentages of annexin V-positive intensities forMECCrt (0 5 10 15 20 and 25120583gmL)treatment of Ca9-22 cells were 93 plusmn 02 86 plusmn 03 117 plusmn 09223 plusmn 08 400 plusmn 02 and 544 plusmn 17 and those of MECCrt-treated CAL 27 cells were 248 plusmn 01 175 plusmn 03 207 plusmn 04598plusmn17 794plusmn02 and 853plusmn05 respectively AccordinglyMECCrt treatments significantly increased in annexin V-positive intensities of two oral cancer Ca9-22 and CAL 27 celllines in a dose-responsive manner (119875 lt 001)

34 ROSGeneration inMECCrt-Treated TwoOral Cancer CellLines To validate the role of ROS in the MECCrt-inducedapoptosis of two oral cancer cell lines a DCFH-DA assay offlow cytometry was chosen Figures 4(a) and 4(b) show therelative ROS-positive staining () of two oral cancer Ca9-22 and CAL 27 cell lines for the different concentrations ofMECCrt treatment for 6 and 12 h incubation After MECCrttreatment for 6 h the relative ROS-positive staining () of 05 10 15 20 and 25 120583gmL MECCrt-treated Ca9-22 cells was

4 The Scientific World Journal

Cou

ntCa

9-22

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmL

400

300

200

100

0

DNA content DNA content DNA content

400

300

200

100

0

Sub-G147G1

492S

177G2M288

Sub-G168G1

529S

182G2M232

Sub-G1171

G1370

S127G2M285

Sub-G1158

G1307

S179G2M265

Sub-G1141

G1266

S212G2M235

Sub-G1200

G1293

S201G2M208

(a)

Cou

ntC

ount

0120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmL

400

300

200

100

0

DNA content DNA content DNA content

400

300

200

100

0

Sub-G148G1

539S

201G2M216

Sub-G151G1

548S

214G2M192

Sub-G1G1

539S

201G2M216

Sub-G1279

G1322

S230G2M184

Sub-G1535

G1248

S122G2M109

Sub-G1699

G1181

SG2M

48

78

54

CAL 27

CAL 27

(b)

Sub-

G1

()

MECCrt (120583gmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast20

20

10

10

5

5

0

0

25

25

15

15

(c)

Sub-

G1

()

lowastlowast

lowastlowast

lowastlowast

0

10

20

30

40

50

60

70

80

MECCrt (120583gmL)201050 2515

(d)

Figure 2 The sub-G1 accumulation of two oral cancer cells was induced by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treatedwith 0 5 10 15 20 and 25 120583gmL of MECCrt for 24 h ((a) and (b)) Representative cell cycle distribution profiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 and CAL 27 cells and vehicles at 24 h respectively ((c) and (d)) Statistics analyses for the percentages ofsub-G1 population in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

1000 plusmn 18 1067 plusmn 04 1312 plusmn 08 1406 plusmn 16 1506 plusmn 03and 1831 plusmn 78 and that of MECCrt-treated CAL 27 cells was1000 plusmn 08 1141 plusmn 14 1423 plusmn 15 1611 plusmn 07 1798 plusmn 11and 1851 plusmn 14 respectively After MECCrt treatment for12 h the relative ROS-positive staining () of 0 5 10 1520 and 25120583gmL MECCrt-treated Ca9-22 cells was 1000 plusmn23 1241 plusmn 04 1664 plusmn 12 1848 plusmn 06 1933 plusmn 03 and2005 plusmn 00 and that of MECCrt-treated CAL 27 cells was1000plusmn09 1407plusmn14 1771plusmn03 1938plusmn01 1986plusmn01 and1994 plusmn 01 respectively Accordingly MECCrt treatmentssignificantly increased in both dose-responsive and time-dependent manners in these two oral cancer cell lines (119875 lt005) (Figures 4(c) and 4(d))

35 MMP Depolarization in MECCrt-Treated Two Oral Can-cer Cell Lines Figures 5(a) and 5(b) show the MMP profilesof DiOC

2(3)-positive intensities for the vehicle andMECCrt-

treated oral cancer cell lines in 24-hour treatments Treated

with MECCrt (0 5 10 15 20 and 25120583gmL) for 24 hthe DiOC

2(3)-positive () intensities of Ca9-22 cells were

1000 plusmn 29 961 plusmn 24 940 plusmn 16 768 plusmn 14 456 plusmn 14and 250 plusmn 11 respectively Similarly the percentages ofDiOC

2(3)-positive () intensities ofMECCrt-treatedCAL27

cells were 1000 plusmn 15 1142 plusmn 07 1089 plusmn 13 513 plusmn 05273 plusmn 05 and 75 plusmn 04 respectively Accordingly MECCrtsignificantly reduced DiOC

2(3)-positive intensities of two

oral cancer Ca9-22 and CAL 27 cell lines in a dose-responsivemanner (119875 lt 001ndash005)

4 Discussion

We discovered for the first time that methanolic extracts ofthe roots ofC concinnaHance have an antiproliferative effecton two oral cancer cell lines The proliferation inhibitingfunction of MECCrt against oral cancer Ca9-22 and CAL 27cell lines was dose-responsive (Figure 1)

The Scientific World Journal 5

104

105

106

1027

104

105

106

1027

1031

104

105

10610

6410

3110

410

510

610

6410

3110

410

510

610

64

Ca9

-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity

PI in

tens

ityCa

9-22

PI in

tens

ity

(a)

15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity

104

105

106

1027

104

105

106

1027

1031

104

105

10610

6410

3110

410

510

610

6410

3110

410

510

610

64

0 120583gmL 5120583gmL 10120583gmL

PI in

tens

ityPI

inte

nsity

CAL 27

CAL 27

(b)

Apop

totic

cells

()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

60

50

40

30

20

10

0

0 5 10 15 20 25

MECCrt (120583gmL)

(c)

Apop

totic

cells

()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

10

20

30

40

50

60

70

80

90

0 5 10 15 20 25

MECCrt (120583gmL)

(d)

Figure 3 Apoptosis of two oral cancer cells was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with 0ndash25120583gmLof MECCrt for 24 h ((a) and (b)) Representative results of annexin VPI double staining of flow cytometry for MECCrt-treated oral cancerCa9-22 and CAL 27 cell lines and vehicle controls at 24 h respectively ((c) and (d)) Quantification analysis of apoptosis for MECCrt-treatedoral cancer Ca9-22 and CAL 27 cell lines in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

6 The Scientific World Journal

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL

400

300

200

100

0

Cou

ntCa

9-22

400

300

200

100

0

400

300

200

100

0

400

300

200

100

0

10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL 15120583gmL

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL

6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h

V2-L500

V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R V2-L V2-R

V3-L V3-R V3-L V3-R

V3-L V3-R

V3-L V3-R

V3-L V3-R V3-L V3-R

500 500 500

916 962 996

172 828618382471 529 347 653

298 702 255 745 119 881 84 38 04

(a)

Cou

nt

0120583gmL 5120583gmL 10120583gmL

15120583gmL

400

300

200

100

0

Cou

nt

400

300

200

100

0

400

300

200

100

0

400

300

200

100

0

10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL 15120583gmL

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL

6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h

V2-L500

V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R

V2-L V2-R

V2-L V2-R

V2-L V2-R V2-L V2-R

500 500 500

970 993 998

112 888700300497 503 386 614

298 702 173 827 921 3079 07 02

CAL 27

CAL 27

(b)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Relat

ive R

OS-

posit

ive (

)

220

200

180

160

140

120

100

80

0 5 10 15 20 25

6h12h

MECCrt (120583gmL)

(c)

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

Relat

ive R

OS-

posit

ive (

)

220

200

180

160

140

120

100

80

0 5 10 15 20 25 25

6h12h

MECCrt (120583gmL)

(d)

Figure 4 Reactive oxygen species (ROS) generation of two oral cancer cell lines was induced by MECCrt Oral cancer Ca9-22 and CAL 27cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 6 and 12 h ((a) (b)) Representative ROS profiles of flowcytometry for MECCrt-treated oral cancer Ca9-22 and CAL 27 cell lines ((c) and (d)) Statistics analysis of relative ROS intensity in (a) and(b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

The Scientific World Journal 7

500V1-L V1-R

500 517V1-L V1-R

483 539V1-L V1-R

461

620V1-L V1-R

380 770V1-L V1-R

230 872V1-L V1-R

128

Cou

ntCa

9-22

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

(a)

500V2-L V2-R

500 440V2-L V2-R

560 455V2-L V2-R

545

744V2-L V2-R

256 868V2-L V2-R

132 964V2-L V2-R

Cou

ntC

ount

0120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

36

CAL 27

CAL 27

(b)

lowast

lowastlowast

lowastlowast

lowastlowast

120

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(c)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

120

140

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(d)

Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle

8 The Scientific World Journal

The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC

50values of 2101584041015840-dihydroxy-5101584061015840-

dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC

50

values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50

values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC

50values ranging from 08 to 11 120583M This is

close to our preliminary result that the IC50

of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC

50values of the MECCrt in oral

cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC

50values of the

MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots

Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27

In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described

in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future

5 Conclusions

We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing

References

[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009

[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013

[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003

[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011

[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012

[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012

[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways

The Scientific World Journal 9

in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013

[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012

[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012

[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013

[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989

[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006

[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000

[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000

[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011

[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012

[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011

[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013

[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010

[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012

[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004

[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009

[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001

[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006

[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010

[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010

[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996

[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011

[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013

[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011

[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002

[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009

[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013

[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013

[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004

[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006

[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999

[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006

10 The Scientific World Journal

[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010

[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013

[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007

[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

2 The Scientific World Journal

drugs derived from natural products were approved by theUnited States Food and Drug Administration [5] In basicresearches natural products with antioral cancer effects haveincreasingly being reported This holds for the ethanolic andmethanolic extracts of red algaGracilaria tenuistipitata [6 7]crude extracts of Selaginella tamariscina (oriental medicinalherb) [8] green tea [9] goniothalamin from Goniothalamusspecies [10] and 4120573-hydroxywithanolide E fromgolden berry[11]

Cryptocarya plants (family Lauraceae) comprising about350 species worldwide are widely distributed in the tropicsand subtropics [12] This plant group is well known forits common secondary metabolites containing alkaloidsflavonoids and 120572-pyrones [12ndash15] Several biological effectsof Cryptocarya-derived natural products have been reportedthat include anti-dengue virus [16] anti-HIV [17] anti-tuberculosis [18] antiplasmodial [19] antitrypanosomal [20]and anti-inflammatory [21] function

Anticancer effects of crude extracts of Cryptocarya plantare known as well For example the ethanolic extracts of fruitand trunk bark of C obovata showed 56 and 23 growthinhibition of human KB cells at 10 120583gmL respectively [22]Methanolic extracts of the leaves of C griffithiana providecytotoxicity forhuman HL60 promyelocytic leukemia cells[23]

Recently accumulating findings for anticancer effectsof pure compounds isolated from Cryptocarya plants werereported especially from methanolic extracts For examplecompounds isolated frommethanol extracts of the trunk barkof C infectoria [24] the trunk bark of C costata [25] andthe wood of C konishii [26] were reported to be cytotoxicto leukemia cells Compounds from methanolic extracts ofleaves of C chinensis were shown to be cytotoxic to humanlung cancer and glioblastoma cells [27] These drugs wereisolated from the trunk bark wood and leaves ofCryptocaryasp However the bioactivity of the roots of Cryptocaryaplants remained little investigated particularly with respectto antioral cancer

Because C concinna Hance is an evergreen plant com-monly distributed in low-altitude forests in Taiwan [28] it iseasy to preparemethanolic extracts of the roots ofC concinnaHance (namely forMECCrt)We therefore chose twoOSCCcell lines that is Ca9-22 and CAL 27 to evaluate the possibleanticancer function of MECCrt and investigate their drugmechanisms in terms of cell viability cell cycle distributionapoptosis reactive oxygen species (ROS) generation andmitochondrial depolarization

2 Materials and Methods

21 Cell Cultures and Methanolic Extracts of C concinnaTwo human OSCC cell lines Ca9-22 and CAL 27 purchasedfrom the Cell Bank RIKEN BioResource Center (TsukubaJapan) and the American Type Culture Collection (ATCCVirginia USA) respectively were incubated in DMEMF12(3 2) medium (Gibco Grand Island NY USA) supple-mented with 10 fetal bovine serum (Gibco) 100UmLpenicillin 100 120583gmL streptomycin and 003 glutamine

These two cell lines were humidly incubated at 37∘C with 5CO2in the humid atmosphere

C concinna was identified by one of the authors (Ih-Sheng Chen) and its roots were collected at Mudan PingtungCounty Taiwan in May 2004 A voucher specimen (Chen6153) has been deposited in the Herbarium of the Schoolof Pharmacy College of Pharmacy Kaohsiung MedicalUniversity The dried roots of C concinna were processedby slicing and cold methanol-extraction for three times atroom temperature Finally the solutionwas evaporated underreduced pressure to yield the methanolic extract (MECCrt)MECCrt was stored at minus20∘C and dissolved in dimethylsulfoxide (DMSO) before treatment

22 Cell Viability Cell viability was measured by theCellTiter 96 AQueous one solution cell proliferation assay(MTS) (Promega Corporation Madison WI USA) as pre-viously described [11] Ca9-22 and CAL 27 cell lines wereseeded at a density of 1 times 105 and 2 times 105 cells per well ina 6-well plate respectively After plating for 24 h these cellswere incubated with different concentrations of MECCrt for24 h and finally subjected to a MTS assay applying an ELISAreader at 490 nm

23 Cell Cycle Progression and Sub-G1 Population Propidiumiodide (PI Sigma St Louis MO USA) was added to stainthe cellular DNA content [29] In brief 3 times 105 cells perwell in 6 well plates were plated for 24 h and then treatedwith vehicle (DMSO 1120583L2mL culture medium) as a controlor 5 10 15 20 and 25 120583gmL of MECCrt for 24 h Afterexposure termination cells were centrifuged washed twicewith PBS fixed overnight with 70 ethanol and centrifugedSubsequently the cell pellets were resuspended in 50120583gmLPI reagent and stand for 30min at 37∘C in darkness Cellcycle distribution was evaluated by a flow cytometer (BDAccuri C6 Becton-Dickinson Mansfield MA USA) and aBD Accuri C6 Software (version 10264)

24 Apoptosis To validate apoptosis in MECCrt-treated oralcancer cells annexin V (Strong Biotect Corporation TaipeiTaiwan) [30]PI (Sigma St Louis MO USA) method wasused [31] Briefly 3 times 105 cells per well in 6 well plates wereplated for 24 h and then treated with vehicle or indicated con-centrations of MECCrt for 24 h Subsequently apoptotic cellswere stained for 30minwith 100120583L binding buffer containing2 120583L of annexin-V-fluorescein isothiocyanate (FITC) stock(025 120583g120583L) and 2120583L of PI stock (1mgmL) Finally it wassuspended with 400 120583L PBS for analysis of a flow cytometer(BD Accuri C6 Becton-Dickinson) and its software

25 Intracellular ROS The dye 2101584071015840-dichlorodihydroflu-orescein diacetate (DCFH-DA) was used to detect ROS byits fluorescence change [7] Cells at the density of 3 times 105 in2mL medium per well in 6 well plates were plated for 24 hDifferent concentrations of MECCrt were added to Ca9-22cells for 6 h and 12 h After washing with PBS 100 nMDCFH-DA in PBS was added to cells in 6 well plates at cell cultureincubator for 30min After trypsinization PBS washing and

The Scientific World Journal 3

120

100

80

60

40

20

0

0 5 10 2015

Cel

l via

bilit

y (

)Ca9-22

MECCrt (120583gmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

0 5 10 2015

Cel

l via

bilit

y (

)

MECCrt (120583gmL)

CAL 27140

120

100

80

60

40

20

0

lowastlowast

lowastlowast

lowastlowastlowastlowast

(b)

Figure 1 Cell viability of two oral cancer cells was inhibited by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with variousconcentrations ofMECCrt (0 5 10 15 and 20 120583gmL) for 24 hThe cell viability was measured by theMTS assay Data means plusmn SDs (119899 = 18)lowastlowast

119875 lt 001 against vehicle

centrifugation cell pellets were resuspended in 1mL PBSbefore analyzing by a flow cytometer (BDAccuri C6 Becton-Dickinson) and its software

26 Mitochondrial Membrane Potential MitoProbeDiOC

2(3) assay kit (Invitrogen Eugene OR USA) was

applied to analyze mitochondrial membrane potential(MMP) as described previously [10] Briefly 3 times 105 cellsin 2mL medium per well in 6 well plates were plated for24 h After MECCrt treatment 10 120583L of 10 120583M DiOC

2(3)

was added per well and incubated in a cell culture incubatorfor 20min After being harvested cells were washedand resuspended in 1mL PBS for analysis using a flowcytometer (BD Accuri C6 Becton-Dickinson) and itssoftware

27 Statistical Analysis The significance of differences wasdetermined by Studentrsquos 119905-test compared with the test datawith the vehicle controls Data are expressed as means plusmn SDs

3 Results

31 Antiproliferation in MECCrt-Treated Two Oral CancerCell Lines Based on MTS assay (Figure 1) the relative cellviability () of oral cancer Ca9-22 cells at indicated concen-trations of MECCrt (0 5 10 15 and 20120583gmL) was 1000 plusmn07 933 plusmn 23 714 plusmn 30 576 plusmn 16 and 484 plusmn 12 after24 h respectively The relative cell viability () of CAL 27cells at indicated concentrations of MECCrt (0 5 10 15 and20120583gmL) was 1000 plusmn 081193 plusmn 49 869 plusmn 100 298 plusmn 62and 284 plusmn 55 respectively The MTS-based cell viabilities ofMECCrt-treated twooral cancerCa9-22 andCAL27 cell linessignificantly reduced in a dose-responsive manner (119875 lt 001compared to the vehicle)

32 Sub-G1 Population in MECCrt-Treated Two Oral Can-cer Cell Lines The MECCrt-treated effects of cell cycledistribution profiles are demonstrated in Figure 2(a) AfterMECCrt treatment (Figure 2(b)) the sub-G1 populations ()ofMECCrt- (0 5 10 15 20 and 25 120583gmL) treated oral cancerCa9-22 cells were 50 plusmn 03 67 plusmn 01 181 plusmn 06 179 plusmn 07165plusmn03 and 224plusmn13 and those ofMECCrt-treatedCAL 27cells were 67plusmn17 51plusmn11 79plusmn01 283plusmn10 526plusmn02 and691 plusmn 01 respectively These sub-G1 changes significantlyaccumulated in a dose-responsive manner (119875 lt 001)

33 Apoptosis of MECCrt-Treated Two Oral Cancer Cell LinesTo validate the possible outcome of apoptosis in MECCrt-induced sub-G1 accumulation of these two oral cancer cellsannexin VPI profiles of flow cytometry were generated(Figure 3(a)) In Figure 3(b) the percentages of annexin V-positive intensities forMECCrt (0 5 10 15 20 and 25120583gmL)treatment of Ca9-22 cells were 93 plusmn 02 86 plusmn 03 117 plusmn 09223 plusmn 08 400 plusmn 02 and 544 plusmn 17 and those of MECCrt-treated CAL 27 cells were 248 plusmn 01 175 plusmn 03 207 plusmn 04598plusmn17 794plusmn02 and 853plusmn05 respectively AccordinglyMECCrt treatments significantly increased in annexin V-positive intensities of two oral cancer Ca9-22 and CAL 27 celllines in a dose-responsive manner (119875 lt 001)

34 ROSGeneration inMECCrt-Treated TwoOral Cancer CellLines To validate the role of ROS in the MECCrt-inducedapoptosis of two oral cancer cell lines a DCFH-DA assay offlow cytometry was chosen Figures 4(a) and 4(b) show therelative ROS-positive staining () of two oral cancer Ca9-22 and CAL 27 cell lines for the different concentrations ofMECCrt treatment for 6 and 12 h incubation After MECCrttreatment for 6 h the relative ROS-positive staining () of 05 10 15 20 and 25 120583gmL MECCrt-treated Ca9-22 cells was

4 The Scientific World Journal

Cou

ntCa

9-22

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmL

400

300

200

100

0

DNA content DNA content DNA content

400

300

200

100

0

Sub-G147G1

492S

177G2M288

Sub-G168G1

529S

182G2M232

Sub-G1171

G1370

S127G2M285

Sub-G1158

G1307

S179G2M265

Sub-G1141

G1266

S212G2M235

Sub-G1200

G1293

S201G2M208

(a)

Cou

ntC

ount

0120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmL

400

300

200

100

0

DNA content DNA content DNA content

400

300

200

100

0

Sub-G148G1

539S

201G2M216

Sub-G151G1

548S

214G2M192

Sub-G1G1

539S

201G2M216

Sub-G1279

G1322

S230G2M184

Sub-G1535

G1248

S122G2M109

Sub-G1699

G1181

SG2M

48

78

54

CAL 27

CAL 27

(b)

Sub-

G1

()

MECCrt (120583gmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast20

20

10

10

5

5

0

0

25

25

15

15

(c)

Sub-

G1

()

lowastlowast

lowastlowast

lowastlowast

0

10

20

30

40

50

60

70

80

MECCrt (120583gmL)201050 2515

(d)

Figure 2 The sub-G1 accumulation of two oral cancer cells was induced by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treatedwith 0 5 10 15 20 and 25 120583gmL of MECCrt for 24 h ((a) and (b)) Representative cell cycle distribution profiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 and CAL 27 cells and vehicles at 24 h respectively ((c) and (d)) Statistics analyses for the percentages ofsub-G1 population in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

1000 plusmn 18 1067 plusmn 04 1312 plusmn 08 1406 plusmn 16 1506 plusmn 03and 1831 plusmn 78 and that of MECCrt-treated CAL 27 cells was1000 plusmn 08 1141 plusmn 14 1423 plusmn 15 1611 plusmn 07 1798 plusmn 11and 1851 plusmn 14 respectively After MECCrt treatment for12 h the relative ROS-positive staining () of 0 5 10 1520 and 25120583gmL MECCrt-treated Ca9-22 cells was 1000 plusmn23 1241 plusmn 04 1664 plusmn 12 1848 plusmn 06 1933 plusmn 03 and2005 plusmn 00 and that of MECCrt-treated CAL 27 cells was1000plusmn09 1407plusmn14 1771plusmn03 1938plusmn01 1986plusmn01 and1994 plusmn 01 respectively Accordingly MECCrt treatmentssignificantly increased in both dose-responsive and time-dependent manners in these two oral cancer cell lines (119875 lt005) (Figures 4(c) and 4(d))

35 MMP Depolarization in MECCrt-Treated Two Oral Can-cer Cell Lines Figures 5(a) and 5(b) show the MMP profilesof DiOC

2(3)-positive intensities for the vehicle andMECCrt-

treated oral cancer cell lines in 24-hour treatments Treated

with MECCrt (0 5 10 15 20 and 25120583gmL) for 24 hthe DiOC

2(3)-positive () intensities of Ca9-22 cells were

1000 plusmn 29 961 plusmn 24 940 plusmn 16 768 plusmn 14 456 plusmn 14and 250 plusmn 11 respectively Similarly the percentages ofDiOC

2(3)-positive () intensities ofMECCrt-treatedCAL27

cells were 1000 plusmn 15 1142 plusmn 07 1089 plusmn 13 513 plusmn 05273 plusmn 05 and 75 plusmn 04 respectively Accordingly MECCrtsignificantly reduced DiOC

2(3)-positive intensities of two

oral cancer Ca9-22 and CAL 27 cell lines in a dose-responsivemanner (119875 lt 001ndash005)

4 Discussion

We discovered for the first time that methanolic extracts ofthe roots ofC concinnaHance have an antiproliferative effecton two oral cancer cell lines The proliferation inhibitingfunction of MECCrt against oral cancer Ca9-22 and CAL 27cell lines was dose-responsive (Figure 1)

The Scientific World Journal 5

104

105

106

1027

104

105

106

1027

1031

104

105

10610

6410

3110

410

510

610

6410

3110

410

510

610

64

Ca9

-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity

PI in

tens

ityCa

9-22

PI in

tens

ity

(a)

15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity

104

105

106

1027

104

105

106

1027

1031

104

105

10610

6410

3110

410

510

610

6410

3110

410

510

610

64

0 120583gmL 5120583gmL 10120583gmL

PI in

tens

ityPI

inte

nsity

CAL 27

CAL 27

(b)

Apop

totic

cells

()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

60

50

40

30

20

10

0

0 5 10 15 20 25

MECCrt (120583gmL)

(c)

Apop

totic

cells

()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

10

20

30

40

50

60

70

80

90

0 5 10 15 20 25

MECCrt (120583gmL)

(d)

Figure 3 Apoptosis of two oral cancer cells was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with 0ndash25120583gmLof MECCrt for 24 h ((a) and (b)) Representative results of annexin VPI double staining of flow cytometry for MECCrt-treated oral cancerCa9-22 and CAL 27 cell lines and vehicle controls at 24 h respectively ((c) and (d)) Quantification analysis of apoptosis for MECCrt-treatedoral cancer Ca9-22 and CAL 27 cell lines in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

6 The Scientific World Journal

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL

400

300

200

100

0

Cou

ntCa

9-22

400

300

200

100

0

400

300

200

100

0

400

300

200

100

0

10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL 15120583gmL

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL

6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h

V2-L500

V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R V2-L V2-R

V3-L V3-R V3-L V3-R

V3-L V3-R

V3-L V3-R

V3-L V3-R V3-L V3-R

500 500 500

916 962 996

172 828618382471 529 347 653

298 702 255 745 119 881 84 38 04

(a)

Cou

nt

0120583gmL 5120583gmL 10120583gmL

15120583gmL

400

300

200

100

0

Cou

nt

400

300

200

100

0

400

300

200

100

0

400

300

200

100

0

10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL 15120583gmL

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL

6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h

V2-L500

V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R

V2-L V2-R

V2-L V2-R

V2-L V2-R V2-L V2-R

500 500 500

970 993 998

112 888700300497 503 386 614

298 702 173 827 921 3079 07 02

CAL 27

CAL 27

(b)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Relat

ive R

OS-

posit

ive (

)

220

200

180

160

140

120

100

80

0 5 10 15 20 25

6h12h

MECCrt (120583gmL)

(c)

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

Relat

ive R

OS-

posit

ive (

)

220

200

180

160

140

120

100

80

0 5 10 15 20 25 25

6h12h

MECCrt (120583gmL)

(d)

Figure 4 Reactive oxygen species (ROS) generation of two oral cancer cell lines was induced by MECCrt Oral cancer Ca9-22 and CAL 27cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 6 and 12 h ((a) (b)) Representative ROS profiles of flowcytometry for MECCrt-treated oral cancer Ca9-22 and CAL 27 cell lines ((c) and (d)) Statistics analysis of relative ROS intensity in (a) and(b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

The Scientific World Journal 7

500V1-L V1-R

500 517V1-L V1-R

483 539V1-L V1-R

461

620V1-L V1-R

380 770V1-L V1-R

230 872V1-L V1-R

128

Cou

ntCa

9-22

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

(a)

500V2-L V2-R

500 440V2-L V2-R

560 455V2-L V2-R

545

744V2-L V2-R

256 868V2-L V2-R

132 964V2-L V2-R

Cou

ntC

ount

0120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

36

CAL 27

CAL 27

(b)

lowast

lowastlowast

lowastlowast

lowastlowast

120

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(c)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

120

140

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(d)

Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle

8 The Scientific World Journal

The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC

50values of 2101584041015840-dihydroxy-5101584061015840-

dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC

50

values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50

values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC

50values ranging from 08 to 11 120583M This is

close to our preliminary result that the IC50

of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC

50values of the MECCrt in oral

cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC

50values of the

MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots

Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27

In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described

in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future

5 Conclusions

We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing

References

[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009

[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013

[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003

[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011

[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012

[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012

[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways

The Scientific World Journal 9

in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013

[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012

[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012

[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013

[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989

[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006

[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000

[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000

[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011

[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012

[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011

[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013

[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010

[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012

[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004

[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009

[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001

[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006

[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010

[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010

[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996

[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011

[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013

[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011

[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002

[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009

[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013

[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013

[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004

[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006

[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999

[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006

10 The Scientific World Journal

[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010

[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013

[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007

[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

The Scientific World Journal 3

120

100

80

60

40

20

0

0 5 10 2015

Cel

l via

bilit

y (

)Ca9-22

MECCrt (120583gmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

0 5 10 2015

Cel

l via

bilit

y (

)

MECCrt (120583gmL)

CAL 27140

120

100

80

60

40

20

0

lowastlowast

lowastlowast

lowastlowastlowastlowast

(b)

Figure 1 Cell viability of two oral cancer cells was inhibited by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with variousconcentrations ofMECCrt (0 5 10 15 and 20 120583gmL) for 24 hThe cell viability was measured by theMTS assay Data means plusmn SDs (119899 = 18)lowastlowast

119875 lt 001 against vehicle

centrifugation cell pellets were resuspended in 1mL PBSbefore analyzing by a flow cytometer (BDAccuri C6 Becton-Dickinson) and its software

26 Mitochondrial Membrane Potential MitoProbeDiOC

2(3) assay kit (Invitrogen Eugene OR USA) was

applied to analyze mitochondrial membrane potential(MMP) as described previously [10] Briefly 3 times 105 cellsin 2mL medium per well in 6 well plates were plated for24 h After MECCrt treatment 10 120583L of 10 120583M DiOC

2(3)

was added per well and incubated in a cell culture incubatorfor 20min After being harvested cells were washedand resuspended in 1mL PBS for analysis using a flowcytometer (BD Accuri C6 Becton-Dickinson) and itssoftware

27 Statistical Analysis The significance of differences wasdetermined by Studentrsquos 119905-test compared with the test datawith the vehicle controls Data are expressed as means plusmn SDs

3 Results

31 Antiproliferation in MECCrt-Treated Two Oral CancerCell Lines Based on MTS assay (Figure 1) the relative cellviability () of oral cancer Ca9-22 cells at indicated concen-trations of MECCrt (0 5 10 15 and 20120583gmL) was 1000 plusmn07 933 plusmn 23 714 plusmn 30 576 plusmn 16 and 484 plusmn 12 after24 h respectively The relative cell viability () of CAL 27cells at indicated concentrations of MECCrt (0 5 10 15 and20120583gmL) was 1000 plusmn 081193 plusmn 49 869 plusmn 100 298 plusmn 62and 284 plusmn 55 respectively The MTS-based cell viabilities ofMECCrt-treated twooral cancerCa9-22 andCAL27 cell linessignificantly reduced in a dose-responsive manner (119875 lt 001compared to the vehicle)

32 Sub-G1 Population in MECCrt-Treated Two Oral Can-cer Cell Lines The MECCrt-treated effects of cell cycledistribution profiles are demonstrated in Figure 2(a) AfterMECCrt treatment (Figure 2(b)) the sub-G1 populations ()ofMECCrt- (0 5 10 15 20 and 25 120583gmL) treated oral cancerCa9-22 cells were 50 plusmn 03 67 plusmn 01 181 plusmn 06 179 plusmn 07165plusmn03 and 224plusmn13 and those ofMECCrt-treatedCAL 27cells were 67plusmn17 51plusmn11 79plusmn01 283plusmn10 526plusmn02 and691 plusmn 01 respectively These sub-G1 changes significantlyaccumulated in a dose-responsive manner (119875 lt 001)

33 Apoptosis of MECCrt-Treated Two Oral Cancer Cell LinesTo validate the possible outcome of apoptosis in MECCrt-induced sub-G1 accumulation of these two oral cancer cellsannexin VPI profiles of flow cytometry were generated(Figure 3(a)) In Figure 3(b) the percentages of annexin V-positive intensities forMECCrt (0 5 10 15 20 and 25120583gmL)treatment of Ca9-22 cells were 93 plusmn 02 86 plusmn 03 117 plusmn 09223 plusmn 08 400 plusmn 02 and 544 plusmn 17 and those of MECCrt-treated CAL 27 cells were 248 plusmn 01 175 plusmn 03 207 plusmn 04598plusmn17 794plusmn02 and 853plusmn05 respectively AccordinglyMECCrt treatments significantly increased in annexin V-positive intensities of two oral cancer Ca9-22 and CAL 27 celllines in a dose-responsive manner (119875 lt 001)

34 ROSGeneration inMECCrt-Treated TwoOral Cancer CellLines To validate the role of ROS in the MECCrt-inducedapoptosis of two oral cancer cell lines a DCFH-DA assay offlow cytometry was chosen Figures 4(a) and 4(b) show therelative ROS-positive staining () of two oral cancer Ca9-22 and CAL 27 cell lines for the different concentrations ofMECCrt treatment for 6 and 12 h incubation After MECCrttreatment for 6 h the relative ROS-positive staining () of 05 10 15 20 and 25 120583gmL MECCrt-treated Ca9-22 cells was

4 The Scientific World Journal

Cou

ntCa

9-22

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmL

400

300

200

100

0

DNA content DNA content DNA content

400

300

200

100

0

Sub-G147G1

492S

177G2M288

Sub-G168G1

529S

182G2M232

Sub-G1171

G1370

S127G2M285

Sub-G1158

G1307

S179G2M265

Sub-G1141

G1266

S212G2M235

Sub-G1200

G1293

S201G2M208

(a)

Cou

ntC

ount

0120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmL

400

300

200

100

0

DNA content DNA content DNA content

400

300

200

100

0

Sub-G148G1

539S

201G2M216

Sub-G151G1

548S

214G2M192

Sub-G1G1

539S

201G2M216

Sub-G1279

G1322

S230G2M184

Sub-G1535

G1248

S122G2M109

Sub-G1699

G1181

SG2M

48

78

54

CAL 27

CAL 27

(b)

Sub-

G1

()

MECCrt (120583gmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast20

20

10

10

5

5

0

0

25

25

15

15

(c)

Sub-

G1

()

lowastlowast

lowastlowast

lowastlowast

0

10

20

30

40

50

60

70

80

MECCrt (120583gmL)201050 2515

(d)

Figure 2 The sub-G1 accumulation of two oral cancer cells was induced by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treatedwith 0 5 10 15 20 and 25 120583gmL of MECCrt for 24 h ((a) and (b)) Representative cell cycle distribution profiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 and CAL 27 cells and vehicles at 24 h respectively ((c) and (d)) Statistics analyses for the percentages ofsub-G1 population in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

1000 plusmn 18 1067 plusmn 04 1312 plusmn 08 1406 plusmn 16 1506 plusmn 03and 1831 plusmn 78 and that of MECCrt-treated CAL 27 cells was1000 plusmn 08 1141 plusmn 14 1423 plusmn 15 1611 plusmn 07 1798 plusmn 11and 1851 plusmn 14 respectively After MECCrt treatment for12 h the relative ROS-positive staining () of 0 5 10 1520 and 25120583gmL MECCrt-treated Ca9-22 cells was 1000 plusmn23 1241 plusmn 04 1664 plusmn 12 1848 plusmn 06 1933 plusmn 03 and2005 plusmn 00 and that of MECCrt-treated CAL 27 cells was1000plusmn09 1407plusmn14 1771plusmn03 1938plusmn01 1986plusmn01 and1994 plusmn 01 respectively Accordingly MECCrt treatmentssignificantly increased in both dose-responsive and time-dependent manners in these two oral cancer cell lines (119875 lt005) (Figures 4(c) and 4(d))

35 MMP Depolarization in MECCrt-Treated Two Oral Can-cer Cell Lines Figures 5(a) and 5(b) show the MMP profilesof DiOC

2(3)-positive intensities for the vehicle andMECCrt-

treated oral cancer cell lines in 24-hour treatments Treated

with MECCrt (0 5 10 15 20 and 25120583gmL) for 24 hthe DiOC

2(3)-positive () intensities of Ca9-22 cells were

1000 plusmn 29 961 plusmn 24 940 plusmn 16 768 plusmn 14 456 plusmn 14and 250 plusmn 11 respectively Similarly the percentages ofDiOC

2(3)-positive () intensities ofMECCrt-treatedCAL27

cells were 1000 plusmn 15 1142 plusmn 07 1089 plusmn 13 513 plusmn 05273 plusmn 05 and 75 plusmn 04 respectively Accordingly MECCrtsignificantly reduced DiOC

2(3)-positive intensities of two

oral cancer Ca9-22 and CAL 27 cell lines in a dose-responsivemanner (119875 lt 001ndash005)

4 Discussion

We discovered for the first time that methanolic extracts ofthe roots ofC concinnaHance have an antiproliferative effecton two oral cancer cell lines The proliferation inhibitingfunction of MECCrt against oral cancer Ca9-22 and CAL 27cell lines was dose-responsive (Figure 1)

The Scientific World Journal 5

104

105

106

1027

104

105

106

1027

1031

104

105

10610

6410

3110

410

510

610

6410

3110

410

510

610

64

Ca9

-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity

PI in

tens

ityCa

9-22

PI in

tens

ity

(a)

15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity

104

105

106

1027

104

105

106

1027

1031

104

105

10610

6410

3110

410

510

610

6410

3110

410

510

610

64

0 120583gmL 5120583gmL 10120583gmL

PI in

tens

ityPI

inte

nsity

CAL 27

CAL 27

(b)

Apop

totic

cells

()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

60

50

40

30

20

10

0

0 5 10 15 20 25

MECCrt (120583gmL)

(c)

Apop

totic

cells

()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

10

20

30

40

50

60

70

80

90

0 5 10 15 20 25

MECCrt (120583gmL)

(d)

Figure 3 Apoptosis of two oral cancer cells was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with 0ndash25120583gmLof MECCrt for 24 h ((a) and (b)) Representative results of annexin VPI double staining of flow cytometry for MECCrt-treated oral cancerCa9-22 and CAL 27 cell lines and vehicle controls at 24 h respectively ((c) and (d)) Quantification analysis of apoptosis for MECCrt-treatedoral cancer Ca9-22 and CAL 27 cell lines in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

6 The Scientific World Journal

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL

400

300

200

100

0

Cou

ntCa

9-22

400

300

200

100

0

400

300

200

100

0

400

300

200

100

0

10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL 15120583gmL

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL

6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h

V2-L500

V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R V2-L V2-R

V3-L V3-R V3-L V3-R

V3-L V3-R

V3-L V3-R

V3-L V3-R V3-L V3-R

500 500 500

916 962 996

172 828618382471 529 347 653

298 702 255 745 119 881 84 38 04

(a)

Cou

nt

0120583gmL 5120583gmL 10120583gmL

15120583gmL

400

300

200

100

0

Cou

nt

400

300

200

100

0

400

300

200

100

0

400

300

200

100

0

10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL 15120583gmL

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL

6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h

V2-L500

V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R

V2-L V2-R

V2-L V2-R

V2-L V2-R V2-L V2-R

500 500 500

970 993 998

112 888700300497 503 386 614

298 702 173 827 921 3079 07 02

CAL 27

CAL 27

(b)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Relat

ive R

OS-

posit

ive (

)

220

200

180

160

140

120

100

80

0 5 10 15 20 25

6h12h

MECCrt (120583gmL)

(c)

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

Relat

ive R

OS-

posit

ive (

)

220

200

180

160

140

120

100

80

0 5 10 15 20 25 25

6h12h

MECCrt (120583gmL)

(d)

Figure 4 Reactive oxygen species (ROS) generation of two oral cancer cell lines was induced by MECCrt Oral cancer Ca9-22 and CAL 27cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 6 and 12 h ((a) (b)) Representative ROS profiles of flowcytometry for MECCrt-treated oral cancer Ca9-22 and CAL 27 cell lines ((c) and (d)) Statistics analysis of relative ROS intensity in (a) and(b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

The Scientific World Journal 7

500V1-L V1-R

500 517V1-L V1-R

483 539V1-L V1-R

461

620V1-L V1-R

380 770V1-L V1-R

230 872V1-L V1-R

128

Cou

ntCa

9-22

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

(a)

500V2-L V2-R

500 440V2-L V2-R

560 455V2-L V2-R

545

744V2-L V2-R

256 868V2-L V2-R

132 964V2-L V2-R

Cou

ntC

ount

0120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

36

CAL 27

CAL 27

(b)

lowast

lowastlowast

lowastlowast

lowastlowast

120

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(c)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

120

140

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(d)

Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle

8 The Scientific World Journal

The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC

50values of 2101584041015840-dihydroxy-5101584061015840-

dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC

50

values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50

values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC

50values ranging from 08 to 11 120583M This is

close to our preliminary result that the IC50

of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC

50values of the MECCrt in oral

cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC

50values of the

MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots

Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27

In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described

in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future

5 Conclusions

We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing

References

[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009

[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013

[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003

[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011

[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012

[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012

[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways

The Scientific World Journal 9

in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013

[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012

[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012

[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013

[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989

[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006

[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000

[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000

[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011

[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012

[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011

[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013

[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010

[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012

[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004

[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009

[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001

[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006

[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010

[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010

[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996

[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011

[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013

[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011

[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002

[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009

[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013

[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013

[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004

[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006

[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999

[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006

10 The Scientific World Journal

[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010

[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013

[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007

[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

4 The Scientific World Journal

Cou

ntCa

9-22

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmL

400

300

200

100

0

DNA content DNA content DNA content

400

300

200

100

0

Sub-G147G1

492S

177G2M288

Sub-G168G1

529S

182G2M232

Sub-G1171

G1370

S127G2M285

Sub-G1158

G1307

S179G2M265

Sub-G1141

G1266

S212G2M235

Sub-G1200

G1293

S201G2M208

(a)

Cou

ntC

ount

0120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmL

400

300

200

100

0

DNA content DNA content DNA content

400

300

200

100

0

Sub-G148G1

539S

201G2M216

Sub-G151G1

548S

214G2M192

Sub-G1G1

539S

201G2M216

Sub-G1279

G1322

S230G2M184

Sub-G1535

G1248

S122G2M109

Sub-G1699

G1181

SG2M

48

78

54

CAL 27

CAL 27

(b)

Sub-

G1

()

MECCrt (120583gmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast20

20

10

10

5

5

0

0

25

25

15

15

(c)

Sub-

G1

()

lowastlowast

lowastlowast

lowastlowast

0

10

20

30

40

50

60

70

80

MECCrt (120583gmL)201050 2515

(d)

Figure 2 The sub-G1 accumulation of two oral cancer cells was induced by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treatedwith 0 5 10 15 20 and 25 120583gmL of MECCrt for 24 h ((a) and (b)) Representative cell cycle distribution profiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 and CAL 27 cells and vehicles at 24 h respectively ((c) and (d)) Statistics analyses for the percentages ofsub-G1 population in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

1000 plusmn 18 1067 plusmn 04 1312 plusmn 08 1406 plusmn 16 1506 plusmn 03and 1831 plusmn 78 and that of MECCrt-treated CAL 27 cells was1000 plusmn 08 1141 plusmn 14 1423 plusmn 15 1611 plusmn 07 1798 plusmn 11and 1851 plusmn 14 respectively After MECCrt treatment for12 h the relative ROS-positive staining () of 0 5 10 1520 and 25120583gmL MECCrt-treated Ca9-22 cells was 1000 plusmn23 1241 plusmn 04 1664 plusmn 12 1848 plusmn 06 1933 plusmn 03 and2005 plusmn 00 and that of MECCrt-treated CAL 27 cells was1000plusmn09 1407plusmn14 1771plusmn03 1938plusmn01 1986plusmn01 and1994 plusmn 01 respectively Accordingly MECCrt treatmentssignificantly increased in both dose-responsive and time-dependent manners in these two oral cancer cell lines (119875 lt005) (Figures 4(c) and 4(d))

35 MMP Depolarization in MECCrt-Treated Two Oral Can-cer Cell Lines Figures 5(a) and 5(b) show the MMP profilesof DiOC

2(3)-positive intensities for the vehicle andMECCrt-

treated oral cancer cell lines in 24-hour treatments Treated

with MECCrt (0 5 10 15 20 and 25120583gmL) for 24 hthe DiOC

2(3)-positive () intensities of Ca9-22 cells were

1000 plusmn 29 961 plusmn 24 940 plusmn 16 768 plusmn 14 456 plusmn 14and 250 plusmn 11 respectively Similarly the percentages ofDiOC

2(3)-positive () intensities ofMECCrt-treatedCAL27

cells were 1000 plusmn 15 1142 plusmn 07 1089 plusmn 13 513 plusmn 05273 plusmn 05 and 75 plusmn 04 respectively Accordingly MECCrtsignificantly reduced DiOC

2(3)-positive intensities of two

oral cancer Ca9-22 and CAL 27 cell lines in a dose-responsivemanner (119875 lt 001ndash005)

4 Discussion

We discovered for the first time that methanolic extracts ofthe roots ofC concinnaHance have an antiproliferative effecton two oral cancer cell lines The proliferation inhibitingfunction of MECCrt against oral cancer Ca9-22 and CAL 27cell lines was dose-responsive (Figure 1)

The Scientific World Journal 5

104

105

106

1027

104

105

106

1027

1031

104

105

10610

6410

3110

410

510

610

6410

3110

410

510

610

64

Ca9

-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity

PI in

tens

ityCa

9-22

PI in

tens

ity

(a)

15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity

104

105

106

1027

104

105

106

1027

1031

104

105

10610

6410

3110

410

510

610

6410

3110

410

510

610

64

0 120583gmL 5120583gmL 10120583gmL

PI in

tens

ityPI

inte

nsity

CAL 27

CAL 27

(b)

Apop

totic

cells

()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

60

50

40

30

20

10

0

0 5 10 15 20 25

MECCrt (120583gmL)

(c)

Apop

totic

cells

()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

10

20

30

40

50

60

70

80

90

0 5 10 15 20 25

MECCrt (120583gmL)

(d)

Figure 3 Apoptosis of two oral cancer cells was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with 0ndash25120583gmLof MECCrt for 24 h ((a) and (b)) Representative results of annexin VPI double staining of flow cytometry for MECCrt-treated oral cancerCa9-22 and CAL 27 cell lines and vehicle controls at 24 h respectively ((c) and (d)) Quantification analysis of apoptosis for MECCrt-treatedoral cancer Ca9-22 and CAL 27 cell lines in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

6 The Scientific World Journal

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL

400

300

200

100

0

Cou

ntCa

9-22

400

300

200

100

0

400

300

200

100

0

400

300

200

100

0

10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL 15120583gmL

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL

6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h

V2-L500

V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R V2-L V2-R

V3-L V3-R V3-L V3-R

V3-L V3-R

V3-L V3-R

V3-L V3-R V3-L V3-R

500 500 500

916 962 996

172 828618382471 529 347 653

298 702 255 745 119 881 84 38 04

(a)

Cou

nt

0120583gmL 5120583gmL 10120583gmL

15120583gmL

400

300

200

100

0

Cou

nt

400

300

200

100

0

400

300

200

100

0

400

300

200

100

0

10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL 15120583gmL

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL

6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h

V2-L500

V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R

V2-L V2-R

V2-L V2-R

V2-L V2-R V2-L V2-R

500 500 500

970 993 998

112 888700300497 503 386 614

298 702 173 827 921 3079 07 02

CAL 27

CAL 27

(b)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Relat

ive R

OS-

posit

ive (

)

220

200

180

160

140

120

100

80

0 5 10 15 20 25

6h12h

MECCrt (120583gmL)

(c)

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

Relat

ive R

OS-

posit

ive (

)

220

200

180

160

140

120

100

80

0 5 10 15 20 25 25

6h12h

MECCrt (120583gmL)

(d)

Figure 4 Reactive oxygen species (ROS) generation of two oral cancer cell lines was induced by MECCrt Oral cancer Ca9-22 and CAL 27cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 6 and 12 h ((a) (b)) Representative ROS profiles of flowcytometry for MECCrt-treated oral cancer Ca9-22 and CAL 27 cell lines ((c) and (d)) Statistics analysis of relative ROS intensity in (a) and(b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

The Scientific World Journal 7

500V1-L V1-R

500 517V1-L V1-R

483 539V1-L V1-R

461

620V1-L V1-R

380 770V1-L V1-R

230 872V1-L V1-R

128

Cou

ntCa

9-22

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

(a)

500V2-L V2-R

500 440V2-L V2-R

560 455V2-L V2-R

545

744V2-L V2-R

256 868V2-L V2-R

132 964V2-L V2-R

Cou

ntC

ount

0120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

36

CAL 27

CAL 27

(b)

lowast

lowastlowast

lowastlowast

lowastlowast

120

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(c)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

120

140

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(d)

Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle

8 The Scientific World Journal

The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC

50values of 2101584041015840-dihydroxy-5101584061015840-

dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC

50

values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50

values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC

50values ranging from 08 to 11 120583M This is

close to our preliminary result that the IC50

of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC

50values of the MECCrt in oral

cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC

50values of the

MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots

Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27

In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described

in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future

5 Conclusions

We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing

References

[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009

[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013

[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003

[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011

[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012

[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012

[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways

The Scientific World Journal 9

in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013

[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012

[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012

[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013

[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989

[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006

[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000

[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000

[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011

[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012

[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011

[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013

[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010

[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012

[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004

[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009

[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001

[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006

[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010

[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010

[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996

[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011

[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013

[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011

[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002

[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009

[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013

[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013

[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004

[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006

[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999

[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006

10 The Scientific World Journal

[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010

[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013

[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007

[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

The Scientific World Journal 5

104

105

106

1027

104

105

106

1027

1031

104

105

10610

6410

3110

410

510

610

6410

3110

410

510

610

64

Ca9

-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity

PI in

tens

ityCa

9-22

PI in

tens

ity

(a)

15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity

104

105

106

1027

104

105

106

1027

1031

104

105

10610

6410

3110

410

510

610

6410

3110

410

510

610

64

0 120583gmL 5120583gmL 10120583gmL

PI in

tens

ityPI

inte

nsity

CAL 27

CAL 27

(b)

Apop

totic

cells

()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

60

50

40

30

20

10

0

0 5 10 15 20 25

MECCrt (120583gmL)

(c)

Apop

totic

cells

()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

10

20

30

40

50

60

70

80

90

0 5 10 15 20 25

MECCrt (120583gmL)

(d)

Figure 3 Apoptosis of two oral cancer cells was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with 0ndash25120583gmLof MECCrt for 24 h ((a) and (b)) Representative results of annexin VPI double staining of flow cytometry for MECCrt-treated oral cancerCa9-22 and CAL 27 cell lines and vehicle controls at 24 h respectively ((c) and (d)) Quantification analysis of apoptosis for MECCrt-treatedoral cancer Ca9-22 and CAL 27 cell lines in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

6 The Scientific World Journal

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL

400

300

200

100

0

Cou

ntCa

9-22

400

300

200

100

0

400

300

200

100

0

400

300

200

100

0

10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL 15120583gmL

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL

6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h

V2-L500

V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R V2-L V2-R

V3-L V3-R V3-L V3-R

V3-L V3-R

V3-L V3-R

V3-L V3-R V3-L V3-R

500 500 500

916 962 996

172 828618382471 529 347 653

298 702 255 745 119 881 84 38 04

(a)

Cou

nt

0120583gmL 5120583gmL 10120583gmL

15120583gmL

400

300

200

100

0

Cou

nt

400

300

200

100

0

400

300

200

100

0

400

300

200

100

0

10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL 15120583gmL

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL

6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h

V2-L500

V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R

V2-L V2-R

V2-L V2-R

V2-L V2-R V2-L V2-R

500 500 500

970 993 998

112 888700300497 503 386 614

298 702 173 827 921 3079 07 02

CAL 27

CAL 27

(b)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Relat

ive R

OS-

posit

ive (

)

220

200

180

160

140

120

100

80

0 5 10 15 20 25

6h12h

MECCrt (120583gmL)

(c)

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

Relat

ive R

OS-

posit

ive (

)

220

200

180

160

140

120

100

80

0 5 10 15 20 25 25

6h12h

MECCrt (120583gmL)

(d)

Figure 4 Reactive oxygen species (ROS) generation of two oral cancer cell lines was induced by MECCrt Oral cancer Ca9-22 and CAL 27cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 6 and 12 h ((a) (b)) Representative ROS profiles of flowcytometry for MECCrt-treated oral cancer Ca9-22 and CAL 27 cell lines ((c) and (d)) Statistics analysis of relative ROS intensity in (a) and(b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

The Scientific World Journal 7

500V1-L V1-R

500 517V1-L V1-R

483 539V1-L V1-R

461

620V1-L V1-R

380 770V1-L V1-R

230 872V1-L V1-R

128

Cou

ntCa

9-22

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

(a)

500V2-L V2-R

500 440V2-L V2-R

560 455V2-L V2-R

545

744V2-L V2-R

256 868V2-L V2-R

132 964V2-L V2-R

Cou

ntC

ount

0120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

36

CAL 27

CAL 27

(b)

lowast

lowastlowast

lowastlowast

lowastlowast

120

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(c)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

120

140

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(d)

Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle

8 The Scientific World Journal

The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC

50values of 2101584041015840-dihydroxy-5101584061015840-

dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC

50

values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50

values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC

50values ranging from 08 to 11 120583M This is

close to our preliminary result that the IC50

of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC

50values of the MECCrt in oral

cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC

50values of the

MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots

Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27

In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described

in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future

5 Conclusions

We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing

References

[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009

[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013

[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003

[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011

[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012

[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012

[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways

The Scientific World Journal 9

in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013

[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012

[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012

[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013

[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989

[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006

[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000

[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000

[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011

[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012

[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011

[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013

[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010

[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012

[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004

[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009

[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001

[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006

[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010

[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010

[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996

[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011

[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013

[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011

[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002

[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009

[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013

[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013

[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004

[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006

[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999

[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006

10 The Scientific World Journal

[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010

[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013

[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007

[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

6 The Scientific World Journal

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL

400

300

200

100

0

Cou

ntCa

9-22

400

300

200

100

0

400

300

200

100

0

400

300

200

100

0

10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL 15120583gmL

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL

6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h

V2-L500

V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R V2-L V2-R

V3-L V3-R V3-L V3-R

V3-L V3-R

V3-L V3-R

V3-L V3-R V3-L V3-R

500 500 500

916 962 996

172 828618382471 529 347 653

298 702 255 745 119 881 84 38 04

(a)

Cou

nt

0120583gmL 5120583gmL 10120583gmL

15120583gmL

400

300

200

100

0

Cou

nt

400

300

200

100

0

400

300

200

100

0

400

300

200

100

0

10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL 15120583gmL

ROS intensity20120583gmL

ROS intensity ROS intensity25120583gmL

6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h

V2-L500

V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R V2-L V2-R

V2-L V2-R V2-L V2-R

V2-L V2-R

V2-L V2-R

V2-L V2-R V2-L V2-R

500 500 500

970 993 998

112 888700300497 503 386 614

298 702 173 827 921 3079 07 02

CAL 27

CAL 27

(b)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Relat

ive R

OS-

posit

ive (

)

220

200

180

160

140

120

100

80

0 5 10 15 20 25

6h12h

MECCrt (120583gmL)

(c)

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

Relat

ive R

OS-

posit

ive (

)

220

200

180

160

140

120

100

80

0 5 10 15 20 25 25

6h12h

MECCrt (120583gmL)

(d)

Figure 4 Reactive oxygen species (ROS) generation of two oral cancer cell lines was induced by MECCrt Oral cancer Ca9-22 and CAL 27cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 6 and 12 h ((a) (b)) Representative ROS profiles of flowcytometry for MECCrt-treated oral cancer Ca9-22 and CAL 27 cell lines ((c) and (d)) Statistics analysis of relative ROS intensity in (a) and(b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle

The Scientific World Journal 7

500V1-L V1-R

500 517V1-L V1-R

483 539V1-L V1-R

461

620V1-L V1-R

380 770V1-L V1-R

230 872V1-L V1-R

128

Cou

ntCa

9-22

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

(a)

500V2-L V2-R

500 440V2-L V2-R

560 455V2-L V2-R

545

744V2-L V2-R

256 868V2-L V2-R

132 964V2-L V2-R

Cou

ntC

ount

0120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

36

CAL 27

CAL 27

(b)

lowast

lowastlowast

lowastlowast

lowastlowast

120

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(c)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

120

140

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(d)

Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle

8 The Scientific World Journal

The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC

50values of 2101584041015840-dihydroxy-5101584061015840-

dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC

50

values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50

values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC

50values ranging from 08 to 11 120583M This is

close to our preliminary result that the IC50

of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC

50values of the MECCrt in oral

cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC

50values of the

MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots

Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27

In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described

in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future

5 Conclusions

We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing

References

[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009

[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013

[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003

[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011

[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012

[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012

[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways

The Scientific World Journal 9

in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013

[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012

[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012

[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013

[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989

[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006

[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000

[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000

[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011

[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012

[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011

[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013

[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010

[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012

[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004

[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009

[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001

[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006

[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010

[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010

[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996

[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011

[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013

[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011

[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002

[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009

[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013

[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013

[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004

[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006

[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999

[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006

10 The Scientific World Journal

[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010

[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013

[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007

[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

The Scientific World Journal 7

500V1-L V1-R

500 517V1-L V1-R

483 539V1-L V1-R

461

620V1-L V1-R

380 770V1-L V1-R

230 872V1-L V1-R

128

Cou

ntCa

9-22

Cou

ntCa

9-22

0 120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

(a)

500V2-L V2-R

500 440V2-L V2-R

560 455V2-L V2-R

545

744V2-L V2-R

256 868V2-L V2-R

132 964V2-L V2-R

Cou

ntC

ount

0120583gmL 5120583gmL 10120583gmL

15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity

1200

1000

500

0

1200

1000

500

0

10110

210

310

410

510

610

7210

110

210

310

410

510

610

7210

110

210

310

410

510

610

72

36

CAL 27

CAL 27

(b)

lowast

lowastlowast

lowastlowast

lowastlowast

120

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(c)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

120

140

100

80

60

40

20

0

Relat

ive M

MP-

posit

ive (

)

0 5 10 15 20 25

MECCrt (120583gmL)

(d)

Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle

8 The Scientific World Journal

The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC

50values of 2101584041015840-dihydroxy-5101584061015840-

dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC

50

values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50

values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC

50values ranging from 08 to 11 120583M This is

close to our preliminary result that the IC50

of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC

50values of the MECCrt in oral

cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC

50values of the

MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots

Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27

In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described

in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future

5 Conclusions

We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing

References

[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009

[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013

[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003

[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011

[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012

[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012

[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways

The Scientific World Journal 9

in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013

[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012

[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012

[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013

[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989

[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006

[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000

[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000

[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011

[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012

[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011

[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013

[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010

[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012

[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004

[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009

[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001

[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006

[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010

[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010

[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996

[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011

[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013

[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011

[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002

[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009

[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013

[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013

[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004

[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006

[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999

[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006

10 The Scientific World Journal

[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010

[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013

[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007

[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

8 The Scientific World Journal

The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC

50values of 2101584041015840-dihydroxy-5101584061015840-

dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC

50

values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50

values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC

50values ranging from 08 to 11 120583M This is

close to our preliminary result that the IC50

of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC

50values of the MECCrt in oral

cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC

50values of the

MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots

Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27

In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described

in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future

5 Conclusions

We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing

References

[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009

[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013

[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003

[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011

[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012

[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012

[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways

The Scientific World Journal 9

in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013

[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012

[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012

[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013

[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989

[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006

[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000

[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000

[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011

[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012

[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011

[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013

[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010

[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012

[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004

[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009

[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001

[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006

[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010

[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010

[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996

[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011

[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013

[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011

[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002

[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009

[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013

[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013

[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004

[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006

[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999

[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006

10 The Scientific World Journal

[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010

[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013

[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007

[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

The Scientific World Journal 9

in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013

[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012

[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012

[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013

[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989

[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006

[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000

[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000

[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011

[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012

[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011

[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013

[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010

[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012

[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004

[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009

[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001

[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006

[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010

[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010

[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996

[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011

[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013

[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011

[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002

[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009

[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013

[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013

[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004

[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006

[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999

[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006

10 The Scientific World Journal

[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010

[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013

[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007

[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

10 The Scientific World Journal

[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010

[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013

[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007

[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of