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Areca (Areca catechu L.) Seeds Ethanolic Extract and Its ChloroformFraction Increase Apoptotic Effect of Doxorubicin on Human Colon CancerCells

Edy Meiyanto*)

, Sri Handayani, R.A. Susidarti, and Riris Istighfari JenieCancer Chemoprevention Research Center, Faculty of Pharmacy, Gadjah Mada University

*)Correspondence: Dr. Edy Meiyanto, MSi., Apt., Faculty of Pharmacy, Gadjah Mada UniversityYogyakarta;

e-mail: [email protected]

AbstractPrevious study on cytotoxic activity of Areca catechuL. on human colon cancer

WiDr cells showed that the IC50 values of areca ethanolic extract (AE) and areca

chloroform fraction (ACF) were 124 g/mL and 119 g/mL respectively. The aim of thisresearch is to examine the effect of combination between AE and ACF with doxorubicin,

a widely used chemotherapeutic agent and whether these combinations induce apoptosison human colon cancer WiDr cells.

Ethanol 96% was carried out in extraction of A. catechu seed powders.

Furthermore, the extract was fractioned with partition by hexane and chloroform to

collect chloroform fraction. Cytotoxic activity of AE, ACF, and Dox on single andcombination was examined using MTT assay. Apoptosis was observed using acrydine

orange-ethidiumbromide double staining. Immunocytochemistry assay was carried out to

detect COX-2 and Bax expression.

Combination of AE and ACF with Dox showed synergistic effect on WiDr withcombination index (CI)


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may affect the growth inhibition of cancer cells. This rationale is supported by researchreports on proanthocyanidin activity from other plants.

Flavonoid fraction (flavonol, anthocyanin, flavan-3-ol, and proanthocyanidin)

from cranberry extract inhibit cells growth with G1 and G2/M arrest, and induce

apoptosis on human breast cancer MDA-MB-435 cells (Ferguson et al., 2004).

Proanthocyanidin from grape extract induce apoptosis with downregulation of Bcl-Xlexpression (Leigh, 2003). Sharma et al.(2004) reported that combination of grape seeds

extract and doxorubicin shows a strong synergistic effect (CI


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isopropanol-acid reagent (HCl 4N (Merck)-isopropanol (Merck), 1:100) and was shook

for 10 minute. Obtical densities were measured at 595 nm using ELISA reader (Bio-Rad),

the IC50 was determined using probit analysis (SPSS 11.5). The combined growth-

inhibitory effect was analyzed with CI method (Reynolds and Maurer, 2005).

Table 1.Concentration ratio of the combination treatmentDoxorubicin

(IC50)

AE or ACF (IC50)1/10

1/3 1/10

1/10 :

1/10

1/10 :

1/10 :

1/3

1/10 :

: 1/10 : :1/3 :

1/31/10 :

1/31/3 :

1/3 :1/3

1/3 :

1/10 : : :1/3 :

Apoptosis Detection.

Apoptosis was detected using acrydine orange-ethidium bromide staining (AO/EB

double staining). WiDr cells (5 x 104 cells/well) were seeded on coverslips in 24-well

plates until 50-60% confluent. Cells were then incubated with AE (60 g/mL), ACF (60g/mL), Dox (500 nM) and combination of Dox-AE and Dox-ACF for 48 h. Culture

medium was removed and cells were washed with PBS. Coverslips were placed into

object-glass and added with 10 L 1X working solution acrydine orange (Sigma)-ethidium bromide (Sigma), observed using fluoroscence microscopy (Zeiss MC 80).

Apoptosis were observed and at least 100 cells/field were evaluated. The results wereobtained from average of 3 fields.

Immunocytochemistry for COX-2 and Bax.WiDr cells (5 x 104 cells/well) were seeded on coverslips in 24-well plates until

80% confluent. Then, cells were incubated with ACF (60 g/mL), Dox (500 nM) and

combination of Dox-ACF for indicated time. Culture medium was removed and cellswere washed in PBS. Cells were fixed with cold methanol for 10 minutes and washed by

PBS. Then, cells were added with H2O2 as blocking solution for 10 minutes and removed.Cells were added with normal mouse serum for 10 minutes, removed, and incubated with

monoclonal antibody anti-COX-2 (Dako) or Bax (Sigma) at 4o C over night. Then, cells

were washed with PBS and incubated with biotinylated universal secondary antibody for10 minutes, removed, and washed with PBS. Cells were incubated with streptavidin-

peroxidase complex reagent for 10 minutes, removed, and washed with PBS. Cells were

stained with substrate solution DAB for 10 minutes, removed, and washed with aquadest.

Cells were stained with Mayer Haematoxylin for 3 min, removed, and washed withaquadest. Coverslips were moved into object-glass and fixed with ethanol and xylol.

After that, coverslips were added with mounting media and covered by other coverslips.

Protein expression were observed using light microscope (Nikon YS 100). Cells withpositive expression appears in brown/dark, while cells with negative expression appears

in blue/violet.

Determination of CI.

The CI was calculated by the Chou-Talalay equation (Reynolds and Maurer,

2005), which takes into account the potency (IC50). The Combination Index (CI) wasobtained from this following equation:


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CI= (D)1/(Dx)1+ (D)2/(Dx)2

where (Dx)1 and (Dx)2 in the denominators are the doses (or concentrations) ofD1 (drug

#1, for example, AE or ACF) and D2 (drug #2, for example, Dox) alone that gives x%

inhibition, whereas (D)1 and (D)2 in the numerators are the doses of D1 and D2 in

combination that also inhibitsx% (i.e., isoeffective). The various degrees of synergism orantagonism have been interpreted by CI values as Table 2 (Reynolds and Maurer, 2005).

Table 2. Interpretation of CI values (Reynolds and Maurer, 2005)

Statistical Analysis.One-way Anova was used to assess differences among treatment groups (P


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Figure 1. Combination effect of Dox-AE on WiDr cells growth inhibition. (A) Combination of

AE (12-60 g/mL) and Dox (100-500 nM) on WiDr cells viability. (B) Synergistic effect of Dox-AE with

CI


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Figure 2. Combination effect of Dox-ACF on WiDr cells growth inhibition. (A) Combination of ACF

(12-60 g/mL) and Dox (100-500 nM) on WiDr cells viability. (B) Synergistic effect of Dox-

ACF with CI


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Figure 3. Effect of Dox-AE and Dox-ACF combination induced apoptosis on WiDr cells. WiDr cells(5x10

4) incubated with AE (60 g/mL), ACF (60 g/mL) and Dox (500 nM) in single and

combination for 48 h. (A) untreated cells, (B) cells with Dox 500 nM, (C) cells with AE 60 g/mL,cells with ACF 60 g/mL, (D) cells with Dox-AE (500 nM-60 g/mL), and (D) cells with Dox-ACF(500 nM-60 g/mL). Cells were stained with acrydine orange-ethidium bromide and saw influoroscence microscope. 100x magnification. apoptosis, viable cells.

Tabel 5. Apoptotic effects of Dox-AE and Dox-ACF against WiDr cells.

No Treatment Apoptosis (%)*)

1 Untreated cells 0

2 Dox 500 nM 20

3 AE 60 g/mL 22

4 Dox-AE 25

5 ACF 60 g/mL 16

6 Dox-ACF 26*)

means of 3 field from sum of apoptotic cells from at least 100 cells/field.

Effect of Dox-ACF combination at COX-2 and Bax expression mediated apoptosis

on WiDr cells.

Expression of COX-2 and Bax was examined using immunocytochemistry assay.

The results showed that ACF treatment on WiDr cells caused different level of COX-2(Figure 4C), but not Bax (Figure 5C) expression. On the other hand, Dox caused different

level of Bax (Figure 5D), but not COX-2 (Figure 4D), expression. This is interesting that

combination of Dox-ACF decreased COX-2 expression (Figure 4E) and increased Baxexpression (Figure 5E). These findings indicated that within this combination, ACF

contributed on the decreasing of COX-2 expression, while Dox on the increasing of Bax

expression.

A B C

D E F


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Figure 4. COX-2 expression on WiDr cells after Dox-ACF treatment. WiDr cells (5 X 104) incubated withACF (60 g/mL) and Dox (500 nM) in single and combination for 6 h. (A) cells without COX- 2antibody, (B) untreated cells, (C) cells with Dox 500 nM, (D) cells with ACF 60 g/mL, (E) cells withDox-ACF (500 nM-60 g/mL). 400x magnification. positive, negative.

Figure 5. Bax expression on WiDr cells after Dox-ACF treatment. WiDr cells (5 X 104) incubated with

ACF (60 g/mL) and Dox (500 nM) in single and combination for 6 h. (A) cells without Baxantibody, (B) untreated cells, (C) cells with Dox 500 nM, (D) cells with ACF 60 g/mL, (E) cells withDox-ACF (500 nM-60 g/mL). 400x magnification. positive, negative.

Discussions

Cytotoxic effect of AE and ACF on WiDr cells is promising on combinationtherapy (Meiyanto et al., 2008) since WiDr is a chemotherapeutic agent resistance cell

A B C

ED

A B

D

C

E


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(Peters et al., 1999). Interestingly, both of Dox-AE and Dox-ACF combination showed

strong synergistic effect on WiDr cells (CI=0.1). Double staining AO/EB assay resultedthat combination of Dox-AE and Dox-ACF induced apoptosis higher than that caused byeither of the agent alone. Furthermore, we investigated the possible mechanism mediated

apoptosis induction of the combination.

Figure 6: Possibly synergistic mechanism induce apoptosis by Dox-ACF on WiDr cells. ACF may

decrease COX-2 expression via inhibition of NF B activation, followed with inhibition of cells

proliferation and induction of ceramide formation. Ceramide induce apoptosis in p53-dependentor p53-independent. On the other hand, Dox induce DNA break and followed by Fas-L

expression. Fas-L binding on Fas receptor activate caspase 8 causing Bid activation (tBid) and

followed by Bax localization on mitochondria which then increase cytochrome C release and

induce apoptosis. Decreasing of COX-2 expression by ACF sensitized WiDr cells against

doxorubicin via Bax expression mediated apoptosis induction.

Based on immunocytochemistry observation, ACF decreased COX-2 expression,while doxorubicin increased Bax expression on WiDr cells. Over expression of COX-2

and PGE2 synthesis mediate neoplastic transformation via the increasing of proliferation

and metastasis (Davies et al., 2002). The decreased of COX-2 expression causingaccumulation of arachidonic acid which then induces sphyngomyelinase, a catalist of

ceramide formation. Ceramide induces apoptosis (Simstein et al., 2003) by increasing

p53 expression (Kim et al.,2002).ACF decreased COX-2 expression possibly due to inhibition of NF B activation.

Our previous study exhibited thatA. catechuextract contains flavonoid (Meiyanto et al.,

2008). Flavonoid containing in AE/ACF presumably mediated the inhibition of NF Bactivation. A number of flavonoids have been reported to inhibit protein kinase, such as

IKK, which is important for NF B activation (Sukla and Gupta, 2004). On the otherhand, Dox acted by inducing DNA break and followed by Fas-L expression where itsbinding on Fas receptor activated caspase 8. Furthermore, caspase 8 induced Bid

activation (tBid), causing Bax localization on mitochondria outer membrane, increased

cytochrome C release and eventually induced apoptosis. Decreasing of COX-2expression by ACF may increase sensitivity of WiDr cells against doxorubicin via Bax

expression-mediated apoptosis induction (Figure 6).

ArachidonicAcid

COX-2

ACF

PGE

P53

Mitochondria Cyt Crelease

Bax

Cellproliferation

NF B

Ceramide

Apoptosis

Dox

Fas

Caspase 8

FADD

Bid

t-Bid

FasL

?


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These results showed that either AE or ACF sensitize human colon cancer WiDr

cells against doxorubicin, they perform synergistic effect and induce apoptosis whencombined with doxorubicin. This finding exhibits the relationship of synergistic effect of

Dox-ACF, the decreasing of COX-2 and increasing of Bax expression on WiDr cells.

Nevertheless, the clear mechanism is unknown and need further investigation.

Acknowledgement

This work was supported by Hibah Bersaing XV from Direktorat Jenderal

Pendidikan Tinggi (DIKTI).

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