ORIGINAL PAPER

Anti-cancer effect of a quinoxaline derivative GK13as a transglutaminase 2 inhibitor

Seon-Hyeong Lee • Nayeon Kim • Se-Jin Kim •

Jaewhan Song • Young-Dae Gong • Soo-Youl Kim

Received: 28 January 2013 / Accepted: 27 March 2013 / Published online: 21 April 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract

Purpose Transglutaminase 2 (TGase 2), a cross-linking

enzyme, plays an important role in both pro-survival and

anti-apoptosis during oncogenesis. For instance, TGase 2

induces NF-jB activation through I-jBa polymerization,

which leads to the increase of pro-survival factors such as

BCl-2. TGase 2 also suppresses apoptosis via depletion of

caspase 3 and cathepsin D. Therefore, a specific TGase 2

inhibitor may become a very useful treatment for cancer

showing high levels of TGase 2 expression.

Methods By small-molecule library screening, we man-

aged to locate a competitive TGase 2 inhibiting quinoxa-

line compound (GK13) from 50 other quinoxaline

derivatives. The 50 compounds that were screened repre-

sent a thousand structurally diverse, potentially pharma-

ceutical heterocyclic compound libraries, including

benzopyrans, oxadiazoles, thiadiazoles, and quinoxalines.

By measuring GI50, TGI, and LC50 using SRB assay,

GK13 was selected.

Results In vitro enzyme kinetics using guinea pig liver

TGase 2 showed that IC50 value was about 16.4 E-6 M.

GK13 inhibits TGase 2-mediated I-jBa polymerization in

a dose-dependent manner. LC50 of GK13 showed greater

efficacy as 4.3E-4 M than LC50 of doxorubicin that

showed efficacy as 3.87E-3 M in NCC72 composing 11

tissue origins and 72 cancer cell lines.

Conclusion GK13 showed a possibility that quinoxaline

derivatives may be effective for anti-cancer activity via

TGase 2 inhibition.

Keywords Quinoxaline derivative � Transglutaminase 2 �Apoptosis � Anti-cancer drug

Introduction

Transglutaminase 2 (TGase 2, E.C. 2.3.2.13, protein–glu-

tamine c-glutamyltransferase) is a calcium-dependent

cross-linking enzyme making isopeptide bonds between

protein-bound glutamine and protein-bound lysine and

ubiquitous expression (Folk 1980). These covalent e-(c-

glutamyl) lysine cross-links are stable and resistant to

enzymatic, chemical, and mechanical disruption that is

widely used in many biological systems for generic tissue

stabilization purposes or immediate defenses for infection

(Iismaa et al. 2009; Kim 2011). TGase 2 activates NF-jB

via an IKK-independent pathway involving I-jBa poly-

merization (Lee et al. 2004). Since the treatment of cancer

cells with TGase 2 inhibitors reduced NF-jB activity in a

dose-dependent manner, blocking of TGase 2 activity can

be suggested as a novel strategy to ameliorate NF-jB-

mediated cancer progression (Kim et al. 2006, 2009a;

Verma and Mehta 2007; Gupta et al. 2010; Lin et al. 2011;

Shao et al. 2009). Previously, we have reported that TGase

2 plays an important role of NF-jB activation (Kim 2011).

TGase 2 inhibition, indeed, has a huge benefit on many

cases of inflammatory diseases (Kim 2006). However,

S.-H. Lee � J. Song

Department of Biochemistry, Yonsei University,

Seoul, Republic of Korea

S.-H. Lee � S.-J. Kim � S.-Y. Kim (&)

Cancer Cell and Molecular Biology Branch, Division of Basic

Science, Research Institute, National Cancer Center,

Goyang, Kyonggi-do, Republic of Korea

e-mail: kimsooyoul@gmail.com

N. Kim � Y.-D. Gong (&)

Innovative Drug Library Research Center, Dongguk University,

Pil-dong 3-ga, Jung-gu, Seoul, Republic of Korea

e-mail: ydgong@dongguk.edu

123

J Cancer Res Clin Oncol (2013) 139:1279–1294

DOI 10.1007/s00432-013-1433-1

TGase 2 has another very important protective role in

tissue damage by blocking caspase 3 (Delhase et al.

2012) or cathepsin D (Kim et al. 2011) to prevent

apoptotic process. Due to the loss of this protective role,

TGase 2 knockout mice are more vulnerable to septic

shock such as TNF-a treatment in the liver tissue than in

the liver of wild-type mice (Yoo et al. 2013). TGase

2-mediated protective role was adopted by cancer cells

for survival. We also found that knockout of TGase 2

expression using siRNA of TGase 2 induces apoptosis

efficiently on renal cell carcinoma (unpublished).

Therefore, TGase 2 inhibitors may be useful for certain

type of cancer therapeutics.

Many groups developed TGase 2 inhibitors that are

competitive amine inhibitors, reversible inhibitors, and

irreversible inhibitors. A review about TGase 2 inhibitors

was reported by Siegel (Siegel and Khosla 2007). Poly-

amines are good TGase 2 substrates modifying proteins

via cross-linking (Folk et al. 1980). Therefore, poly-

amines can be used as competitive TGase 2 inhibitors,

such as cystamine, at very high concentrations. Com-

petitive peptidic TGase 2 inhibitor mimicking natural

protein substrate demonstrated anti-inflammatory effect

(Sohn et al. 2003). Cinnamoyl triazole derivatives as

reversible inhibitors were competing with acyl donor

TGase 2 substrates (Pardin et al. 2008). Dipeptide-based

sulfonium peptidylmethylketones derived from 6-diazo-

5-oxo-L-norleucine (DON) were introduced as water-

soluble inhibitors of extracellular TGase 2 (Griffin et al.

2008). PQP-(DON)-LPF-aldehydes were developed via

structure-based study as TGase 2 inhibitor (Siegel et al.

2007). There are also various irreversible active-site

TGase 2 inhibitors (Lorand and Graham 2003). Recent

results showed ZM 39923, ZM 449829, tyrphostin 47,

and vitamin K were found as TGase 2 inhibitors in part a

thiol-dependent mechanism (Lai et al. 2008). However,

none of them have been tested as a cancer sensitizer or

for anti-cancer effects. There was a report about dihyd-

roisoxazole derivative KCC009 as a TGase 2 inhibitor

against glioblastoma tumors (Yuan et al. 2007; Choi

et al. 2005). KCC009 treatment with anti-cancer drug

decreased tumor size based on weight by 50 % compared

to the control (Yuan et al. 2007). However, specificity

of KCC009 against TGase 2 was not warranted for ani-

mal experiments because IC50 of KCC009 was over

100 lM.

We found out a hit of competitive TGase 2 inhibitor

GK13 among 1,000 structurally diverse and druggable,

heterocyclic compound libraries, including quinoxaline

(Gong et al. 2011), benzopyrans (Lee and Gong 2012),

oxadiazoles and thiadiazoles (Gong and Lee 2010), and

quinoxalines produced by Gong’s laboratory. We have

tested GK13 for anti-cancer activity using various

cancer cell lines. After cell line screening, the hollow

fiber assay was performed using selected cell lines. We

have established 18 cell lines for hollow fiber assay in

NCC. We adopted the screening panel including 12 cell

lines from stomach and liver in addition to NCI 60,

called NCC72 (Kim 2010). We found that TGase 2

inhibitor may have anti-cancer effect in various cancer

cells.

Methods

Cell culture and reagents

NCI 60 cell lines were obtained from NCI (MTA Number:

2702-09). The National Cancer Center (NCC) 72 cell lines

were composed of 60 NCI cells (Shoemaker 2006),

including 10 lung cancer cells, 6 ovarian cancer cells, 5

CNS cancer cells, 6 hematopoietic cancer cells, 7 colon

cancer cells, 8 renal cancer cells, 8 melanomas, 5 breast

cancer cells, 2 prostate cancer cells, and 12 NCC cells

including 7 stomach cancer cells (SNU-16, Kato-III, SNU-

216, MKN-28, MKN-45, SNU-484, and SNU-668), and 5

liver cancer cells (Hep3B, Huh7, SNU-354, SNU-423, and

SNU-449) (Ku and Park 2005). Those were cultured in 5 %

CO2 and 100 % humidity at 37 �C in complete RPMI 1640

containing 10 % fetal bovine serum. Cell cultures were

passaged using trypsin–EDTA to detach cell. Doxorubicin

was purchased from Sigma and GK13 was obtained from

the drug synthesis. Compounds were dissolved in DMSO

and diluted into complete medium before addition to cell

culture. Cystamine (Sigma-Aldrich) and Z006 (Zedira)

were purchased.

SRB test

For SRB test, cells are incubated into 96-well microtiter

plates in 100 ll from 5,000 to 40,000 cells/well depending

on the doubling time of individual cell lines. After 24 h,

drugs were prepared for the appropriate concentration

(100 lM) and added 100 ll to each well, and cultures were

incubated for 48 h at 37 �C. Fixation was done by adding

50 ll of 50 and 80 % cold trichloroacetic acid (TCA) for

adherent cell lines and for suspension cell lines, respec-

tively. The plate is incubated for a minimum of 1 h and a

maximum of 3 h at 4 �C. After 1 h, it removed the liquid

from the plate and rinsed the plate 5 times with water.

Then, the plate is dried at room temperature (R.T.) for

approximately 12–24 h. The fixed cells are stained with

100 ll sulforhodamine B (SRB) for 5 min at R.T. After

staining, the plate is washed 3 times with 1 % glacial acetic

1280 J Cancer Res Clin Oncol (2013) 139:1279–1294

123

acid and is dried at R.T. for approximately 12–24 h. The

SRB stain is solubilized with 10 mM Trizma base and the

absorbance is read at a wavelength of 515 nm. The effect

of drug was expressed as GI50 (50 % growth inhibition),

TGI (total growth inhibition), and LC50 (lethal

concentration).

Immunoblotting

Nuclear protein extraction is prepared using a CelLytic

NuCLEAR Extraction Kit (Sigma). Proteins were isolated

to 4–12 % SDS-polyacrylamide gel (Invitrogen) and

transferred to PVDF membranes (Bio-Rad). Membranes

were blocked in TBS-T (TBS containing 0.1 % Tween 20)

containing 5 % BSA for 1 h at R.T. and then incubated

with primary antibody overnight at 4 �C. Membranes were

washed in TBS-T at R.T. for 1 h and incubated with

horseradish peroxidase-conjugated secondary antibody in

TBS-T containing 1 % BSA for 1 h at R.T. Finally,

membranes were washed in TBS-T for 1 h at R.T. Proteins

were detected using enhanced chemiluminescence (Pierce).

General for synthesis

All chemicals were reagent grade and used as purchased.

Reactions were monitored by thin layer chromatography

(TLC) analysis using Merck silica gel 60 F-254 thin layer

plates or attenuated total reflection Fourier transform

infrared (ATR-FTIR) analysis using TravelIRTM (SensIR

Technology). Flash column chromatography was carried

out on Merck silica gel 60 (230–400 mesh). The crude

products were purified by parallel chromatography using

Quad3TM. 1H NMR and 13C NMR spectra were recorded in

d units relative to deuterated solvent as an internal refer-

ence using a Bruker 500 MHz NMR instrument. Liquid

chromatography–mass spectrometry (LC–MS) analysis

was performed on an electrospray ionization (ESI) mass

spectrometer with photodiode-array detector (PDA)

detection. LC–MS area percentage purities of all products

were determined by LC peak area analysis (XTerraMS C18

column, 4.6 mm 9 100 mm; PDA detector at 200–400

nm; gradient, 5–95 % CH3CN/H2O). High-resolution mass

spectrometry fast-atom bombardment (HRMS-FAB) spec-

tra were obtained using API 4000Q TRAP LC/MS/MS

system (Applied Biosystems).

Synthetic procedures for the preparation of 2-(phenyl-

ethynyl)-3-(2-(pyrrolidin-1-yl) ethoxy) quinoxaline (lead

compound GK13).

Synthesis of quinoxaline-2,3-diol (2) A solution of

benzene-2,3-diamine (1) (5.0 g, 45.8 mmol) and oxalic

acid (4.8 g, 53.3 mmol) in 3 N aq. HCl (100 ml) was

stirred at reflux condition for 24 h. The resulting mixture

was filtered and then washed with cold water and dried in a

vacuum oven at 50 �C. The desired product 2, quinoxaline-

2,3-diol, was obtained in good yield (89 %, 6.7 g). 1H

NMR (500 MHz, DMSO) d 7.13 (m, 1H), 7.46 (dd,

J = 1.4, 6.3 Hz, 1H), 8.07 (m, 1H), 11.98 (s, 1H), 12.33 (s,

1H); MS (ESI) m/z 163 ([M ? H]?).

Synthesis of 2,3-dichloroquinoxaline (3): To a stirred

solution of quinoxaline-2,3-diol (2) (4.2 g, 26.0 mmol) in

chloroform (CHCl3, 100 ml) were added thionyl chloride

(9.3 g, 78.0 mmol) and N,N-dimethylformamide (DMF,

0.5 ml) at reflux condition for 24 h. The resulting mixture

was concentrated in vacuo to remove the solvent and then

water was added. The desired product was filtered and

washed with water and dried in a vacuum oven at 50 �C.

The desired product 3, 2,3-dichloroquinoxaline, was

obtained in good yield (78 %, 4.8 g). 1H NMR (500 MHz,

DMSO) d 7.81 (m, 1H), 8.43 (dd, J = 6.6, 1.7 Hz, 1H),

9.19 (dd, J = 3.1, 1.5 Hz, 1H); MS (ESI) m/z 200

([M ? H]?).

Synthesis of 2-chloro-3-(phenylethynyl)quinoxaline (4):

To a stirred solution of 2,3-dichloroquinoxaline (3) (4.76 g,

18.5 mmol) in dimethylsulfoxide (DMSO, 2 ml) solution

were added phenylacetylene (2.3 ml, 21.3 mmol), trieth-

ylamine (18.0 ml, 129.6 mmol), palladium(II) acetate

(290 mg, 1.3 mmol), copper(I) iodide (437 mg, 1.7 mmol)

and triphenylphosphine (388 mg, 2.0 mmol) at 80 �C for

2 h. The resulting mixture was concentrated in vacuum to

remove the solvent and then water was added. The mixture

was extracted with ethyl acetate and the organic layer was

washed with water and dried over MgSO4. After removal

of solvent in vacuum, the residue was purified by SiO2

column chromatography (CH2Cl2:n-hexane = 3:2) to yield

the desired compound 4, 2-chloro-3-(phenylethynyl)quin-

oxaline (83 %, 4.6 g). 1H NMR (500 MHz, CDCl3) d 7.46

(m, 6H), 7.73 (m, 6H), 8.34 (d, J = 8.3 Hz, 2H), 9.19 (dd,

J = 2.31, 1.8 Hz, 2H); MS (ESI) m/z 265 ([M ? H]?).

Synthesis of 2-(phenylethynyl)-3-(2-(pyrrolidin-1-

yl)ethoxy) quinoxaline (lead compound, GK13): To a

stirred solution of 2-(pyrrolidin-1-yl)ethanol (1.34 g,

11.6 mmol) in tetrahydrofuran (THF; 10 ml) solution was

added sodium hydride dispersion (60 %) in mineral oil

(743 mg, 18.6 mmol) at R.T. for 20 min, after which THF

(10 ml) solution of the prepared compound 4,2-chloro-3-

(phenylethynyl)quinoxaline (2.47 g, 9.3 mmol), was

dropped for 1 h. Stirring was continued at R.T. for 8 h. The

resulting mixture was concentrated in vacuo to remove the

solvent and then water was added. The mixture was

extracted with ethyl acetate and the organic layer was

washed with water and dried over MgSO4. After removal

of solvent in vacuo, the residue was purified by SiO2 col-

umn chromatography (CH2Cl2:ethanol = 9:1) to yield

(83.1 %, 3.35 g) the desired compound GK13, 2-(phe-

nylethynyl)-3-(2-(pyrrolidin-1-yl)ethoxy)quinoxaline: 1H

NMR (500 MHz, CDCl3) d 1.81 (s, 4H), 2.77 (s, 4H), 3.05

J Cancer Res Clin Oncol (2013) 139:1279–1294 1281

123

(t, J = 5.7 Hz, 2H), 4.72 (t, J = 5.7 Hz, 2H), 7.40-7.42 (m,

3H), 7.56 (t, J = 4.2 Hz, 1H), 7.65 (dd, J = 1.8, 6.3 Hz,

2H), 8.95 (dd, J = 1.8, 2.4 Hz, 1H); MS (ESI) m/z 344

([M ? H]?).

TGase activity assay

The inhibitory effect of each compound on TGase 2

activity was determined by measuring the incorporation of

[1,4-14C] putrescine into succinylated casein. Following a

10-min pre-incubation of 1.0 milliunits (mU) of TGase 2

from guinea pig liver (Sigma) with various concentrations

of GK13 in 0.1 ml of reaction buffer solution with or

without 10 mM CaCl2, 0.4 ml of substrate solution con-

taining 2 % of succinylated casein and 100 nCi of [1,4-14C]

putrescine was added. After incubation at 37 �C for 1 h,

the reaction was terminated by the addition of 4 ml of cold

(4 �C) 7.5 % (w/v) TCA. TCA-insoluble precipitates were

collected in GF/A glass fiber filters (Millipore), washed

with cold 5 % (w/v) TCA, dried, and assessed for the

incorporation of radiolabel using a scintillation counter

(Beckman Coulter). TGase 2 pre-incubated with buffer

alone was used as the positive control. The scintillation

counts were compared with that of the positive control, and

the IC50 value was determined using a logistic linear

regression method. The data were presented as the means

of three independent experiments.

In vitro inhibitory effect of GK13 on I-jBacross-linking by TGase 2

For polymerization of I-jBa by TGase 2, purified recom-

binant I-jBa (0.5 lg) was incubated with TGase 2 in 20 ll

reaction buffer (0.1 M Tris–Cl, pH 8.0, 0.15 M NaCl, and

10 mM CaCl2) at 37 �C for 1 h. To examine the inhibitory

effect of GK13 on polymerization of I-jBa by TGase 2,

0 20 40 60 80 100 1200

20

40

60

80

100

120

IC50: 16.4 E-6M

I-κBα

TGase 2

+ + + + +

- + + + +

GK13 0 0 0.5 0.1 0.05 μM

I-κBαmonomer

a

b

c

0 1 2 4 8 16 (µM)

I- B

-actin

d

TG

a se

2ac

tivity

(com

par a

tive,

%)

Fig. 1 Inhibitory effect of

GK13 on TGase 2 activity. The

process of GK13 synthesis was

introduced in method section

(a). IC50 of GK13 is 16.4E-

6 M (b). For the analysis,

guinea pig liver TGase 2,

succinylated casein, and C14-

putrescine were employed for

competition with GK13.

Competition assay using GK13

reversed free I-jBa level dose-

dependently in TGase

2-mediated I-jBa depletion (c).

GK13 treatment for 12 h in

Hep3B cells rescued depletion

of I-jBa in a dose-dependent

manner (d)

1282 J Cancer Res Clin Oncol (2013) 139:1279–1294

123

Fig. 2 Cytotoxic effect made comparison between GK13 and

doxorubicin. NCC 72-cell line screen data (GI50, TGI, and LC50

values) for GK13 against a panel of human cancer cell lines. The

midline of each portion of the graph represents the mean for that

endpoint, calculated across all 72 cell lines. This mean value is then

subtracted from the value for each individual cell line and plotted.

Cell lines more sensitive to GK13 are visualized as bars deflecting to

the right, while more resistant cell lines have bars extending to the

left of the mean. Average GI50 over all cell lines are 5.92E-5 M.

Average TGI over all cell lines are 5.22E-5 M. Average LC50 over

all cell lines are 4.3E-4 M (a). Dose–response curves for GK13.

Three endpoints (negative log10 of the concentration inhibiting the

growth of 50 % of the cells (GI50), total growth inhibition (TGI) and

negative log10 concentration need to kill 50 % of the cells (LC50)h)

are calculated from 7-log dose–response curves for compounds tested

using 72 human tumor cell line screen. A leukemia, B lung, C colon,

D CNS, E melanoma, F ovarian, G renal, H prostate, I breast, J liver,

K stomach (b). NCC 72-cell line screen data (GI50, TGI, and LC50

values) for doxorubicin against a panel of human cancer cell lines (c).

Average GI50 over all cell lines are 6.68E-6 M. Average TGI over

all cell lines are 5.58-5M. Average LC50 over all cell lines are

3.87E-3 M. Dose–response curves for doxorubicin. A leukemia,

B lung, C colon, D CNS, E melanoma, F ovarian, G renal, H prostate,

I breast, J liver, K stomach (d)

J Cancer Res Clin Oncol (2013) 139:1279–1294 1283

123

GK13 (0.05, 0.1, 0.5 lM) was pre-incubated with 0.5 mU

TGase 2 in 20 ll reaction mixture (0.1 M Tris–Cl, pH 8.0,

and 0.15 M NaCl) at 37 �C for 10 min. After pre-incuba-

tion, purified I-jBa (0.5 lg) and CaCl2 (10 mM final

concentration) were added, and the mixture was incubated

at 37 �C for 1 h.

Hollow fiber assay

Efficacy was evaluated in vivo using a hollow fiber animal

model in which polyvinylidene fluoride (PVDF) hollow

fibers containing cancer cell lines (OVCAR-5, SW620,

U251, UACC62) in triplicate were implanted subcutane-

ously and intraperitoneally into mice as described previ-

ously (Hollingshead et al. 1995). Briefly, PVDF hollow

fibers with 1.0-mm inner diameter and a molecular weight

cutoff point of 500 kD (S9320101: Spectrum Laboratories,

Rancho Dominquez, CA) were individually flushed and

incubated in 70 % ethanol at R.T. for [72 h. After being

washed with deionized water, the fibers are autoclaved and

flushed with RPMI 1640 (WelGENE, Daegu, Korea) con-

taining 20 % FBS (WelGENE, Daegu, Korea). The cancer

cell lines (OVCAR-5, SW620, U251, UACC62), which

were grown in RPMI 1640 containing 10 % FBS, were

Fig. 2 continued

1284 J Cancer Res Clin Oncol (2013) 139:1279–1294

123

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration(Molar)

A

CCRF-CEM

HL-60(TB)

K-562

MOLT-4

RPMI-8226

SR-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration(Molar)

BA549/ATCC

EKVX

HOP-62

HOP-92

NCI-H226

NCI-H23

NCI-H322M

NCI-H460

NCI-H522

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration(Molar)

C

COLO 205

HCC-2998

HCT-116

HCT-15

HT29

KM12

SW-620 -100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration(Molar)

D

SF-268

SF-295

SF-539

SNB-19

SNB-75

U251

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration(Molar)

E LOX IMVI

MALME-3M

M14

MDA-MB-435

SK-MEL-2

SK-MEL_28

SK-MEL-5

UACC-257

UACC-62-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration(Molar)

F

IGR-OV1

OVCAR-3

OVCAR-4

OVCAR-5

OVCAR-8

NCI/ADR-RES

SK-OV-3

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration(Molar)

G

786-O

A498

ACHN

CAKI-1

RXF 393

SN12C

TK-10

UO-31-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration(Molar)

H

PC-3

DU-145

b

Fig. 2 continued

J Cancer Res Clin Oncol (2013) 139:1279–1294 1285

123

harvested with trypsin/EDTA, pelleted by centrifugation,

suspended in conditioned medium, and diluted with RPMI

1640 containing 20 % FBS (inoculation density

2–10 9 106/ml). The fibers were filled with the cell sus-

pension via a 20 gauge needle. Each fiber was then heat-

sealed by clamping preheated smooth-jawed needle-hold-

ers across the fiber every 2 cm along its length. The sam-

ples were incubated for 1 or 2 nights at 37 �C in a 5 % CO2

incubators prior to implantation into mice. Three subcuta-

neous fibers were implanted by caudally inserting a trocar

containing the fibers through a skin incision made at the

nape of the neck of each 7–8-week Balb/C (nu/nu) female

mouse (Orient Bio, Sungnam, Korea), after inhalational

isoflurane (Choongwae, Seoul, Korea) anesthesia. Three

intraperitoneal fibers were inserted into the peritoneal

cavity of the same mouse in a craniocaudal direction using

an incision through the abdominal wall. Two layers of

sutures were used to close the abdominal incision.

GK13 treatment (50 mg/kg) was started 3 or 4 days

after implantation of fibers into mice. As a positive control,

30 mg/kg of paclitaxel (Bristol-Meyers Squibb Korea,

Seoul, Korea), diluted as 3 mg/ml, was given to mice

intraperitoneally for 4 consecutive days. Mice were killed

the next day after the last drug treatment. MTT (3-[4,5-

dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)

dye conversion assay was performed to define the viable

cell mass within the fiber.

Analysis of apoptosis induced by GK13

by fluorescence-activated cell sorting (FACS)

Analysis of annexin V binding was carried out using an

Annexin V-FITC Apoptosis Detection Kit (BD Biosci-

ences), according to the manufacturer’s instructions.

Briefly, cells were collected, washed twice with cold PBS,

and then subjected to centrifugation at 1,500 rpm for

5 min. The cell pellet was resuspended in 19 binding

buffer at a concentration of 1 9 106 cells per ml, and then

100 ll of the cell suspension was transferred to a 5 ml

culture tube, to which 5 ll of annexin V-FITC and 5 ll of

PI were added. The cells were gently vortexed and then

incubated for 15 min at R.T. in the dark. Finally, 400 ll of

19 binding buffer was added to each tube and the samples

were analyzed by flow cytometry. For each sample, 10,000

ungated events were acquired; PI(-)/annexin(?) cells

were taken as the early apoptotic population.

Annexin V-FITC/PI double-staining assay

Cells were washed three times with cold PBS and trans-

ferred to 100 ll of 19 binding buffer (Annexin V-FITC

Apoptosis Detection Kit, BD Biosciences) with 1 lg/ll of

DAPI, 5 ll of annexin V-FITC, and 5 ll of PI for 15 min

at R.T. in the dark. After incubation, the cells were washed

three times in binding buffer and then mounted on glass

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

K

SNU-16

KATO

SNU-216

MKN-28

MKN-45

SNU-484

SNU-668

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

J

Hep3B

Huh7

SNU-354

SNU-423

SNU-449

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

I

MCF7

MDA231

MDA468

HS578T

BT-549

T-47D

Mean Values

GI50 5.92E-5M

TGI 5.22E-5M

LC50 4.3E-4M

Fig. 2 continued

1286 J Cancer Res Clin Oncol (2013) 139:1279–1294

123

slides. The slides were examined with Zeiss Axiovert 200 M

microscope (Carl Zeiss Microimaging, Thornwood, NY).

Results

GK13 showed TGase 2 inhibition activity

We have screened TGase 2 inhibitor in DGG drug library

developed by Gong et al. After third-round screening, we

have narrowed down to quinoxaline derivative of

2-(phenylethynyl)-3-(2-(pyrrolidin-1-yl) ethoxy)quinoxa-

line (GK13) as a hit compound of TGase 2 inhibitor

(Fig. 1a). The synthesis of GK13 compound was explained

in the method section. In in vitro enzyme kinetics using

guinea pig liver TGase 2, IC50 value was obtained as

16.4 lM against putrescine (Fig. 1b). GK13 inhibits TGase

2-mediated I-jBa polymerization in a dose-dependent

manner (Fig. 1c). GK13 treatment in Hep3B rescued also

I-jBa level in a dose-dependent manner (Fig. 1d).

Fig. 2 continued

J Cancer Res Clin Oncol (2013) 139:1279–1294 1287

123

GK13 showed anti-cancer effect on 72 cancer cell lines

GK13 has been tested for anti-cancer effect using 72 cancer

cell lines (NCC 72): NCI 60 cancer cell lines; and 7

stomach cancer cell lines including SNU16, Kato-III, SNU-

216, MKN-28, MKN-45, SNU-484, and SNU-668; and 5

liver cancer cell lines including Hep3B, Huh7, SNU-354,

SNU-423, and SNU-449. Interestingly, GK13 showed

moderate anti-cancer effect throughout the cancer cell

lines. In the aspects of GI50, TGI, and LC50, GK13

showed good anti-cancer effects especially in colon and

renal cancer cell lines (Fig. 2a), while doxorubicin as a

positive control showed good anti-cancer effects in

melanoma, CNS, and renal cancer cells (Fig. 2c). GK13

showed about 10 times less GI50 than doxorubicin showed

(Fig. 2b, d). However, GK13 showed almost the same

effect of TGI and 10 times better effect of LC50 when

compared to the doxorubicin.

GK13 showed anti-cancer effect on hollow fiber assay

on U251 and UACC62

After in vitro screening process, further evaluation in

in vivo models of the compounds identified as anti-cancer

was needed as the next step prior to further development.

However, the cost, time, and expense of running

Fig. 2 continued

1288 J Cancer Res Clin Oncol (2013) 139:1279–1294

123

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

A

CCRF-CEM

HL-60(TB)

K-562

MOLT-4

RPMI-8226

SR-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

BA549/ATCC

EKVX

HOP-62

HOP-92

NCI-H226

NCI-H23

NCI-H322M

NCI-H460

NCI-H522

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

C

COLO 205

HCC-2998

HCT-116

HCT-15

HT29

KM12

SW-620 -100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

D

SF-268

SF-295

SF-539

SNB-19

SNB-75

U251

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

ELOX IMVI

MALME-3M

M14

MDA-MB-435

SK-MEL-2

SK-MEL_28

SK-MEL-5

UACC-257

UACC-62

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

F

IGR-OV1

OVCAR-3

OVCAR-4

OVCAR-5

OVCAR-8

NCI/ADR-RES

SK-OV-3

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

H

PC-3

DU-145

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

G

786-O

A498

ACHN

CAKI-1

RXF 393

SN12C

TK-10

UO-31

d

Fig. 2 continued

J Cancer Res Clin Oncol (2013) 139:1279–1294 1289

123

conventional xenograft models with empirical dosing

strategies for all such lead compounds, or developing

pharmacokinetic assays for each compound to be evaluated

in vivo, would be critical rate limiting step. To address this

problem, a short-term in vivo assay was developed by NCI

people (Hollingshead et al. 1995) in which cells growing in

polyvinylidene fluoride (PVDF) ‘‘hollow fibers’’ are placed

in various body compartments of mice. The anti-cancer

effect of GK13 has been tested using hollow fiber assay, as

a semi-in vivo assay, using four different cancer cell lines,

including OVCAR5 (ovary cancer), SW620 (colon cancer),

U251 (CNS), and UACC62 (melanoma). The cancer cell

lines are cultivated and harvested by standard trypsiniza-

tion technique and resuspended at the desired cell density.

The cell suspension is flushed into 1 mm (internal diame-

ter) polyvinylidene fluoride hollow fibers with a molecular

weight exclusion of 500 kDa. The hollow fibers are

implanted into mouse with 3 intraperitoneal implants (1 of

each tumor line) and 3 subcutaneous implants (1 of each

tumor line). Mice are treated with experimental agents

starting on day 3 or 4 following fiber implantation and

continuing daily for 4 days. The fibers are collected from

the mice on the day following the fourth compound treat-

ment and subjected to the stable endpoint MTT assay

(details in the method). Interestingly, GK13 showed good

anti-cancer effect on U251 and UACC62 (Fig. 3). This

result concords to the result from SRB test in Fig. 2.

GK13 treatment induced apoptosis

U251 and UACC62 cells were treated with or without

GK13 for 6 h. Apoptosis was measured by flow cytometric

analysis using annexin V–PI staining (Fig. 4a). The total

apoptotic cells (early and late-stage apoptosis) were over

twofold increased in U251 and over sevenfold increased in

UACC62 by FACS analysis (Fig. 4a). The cells treated

with GK13 were stained with annexin V-FITC and PI and

examined by fluorescence microscopy. Early apoptotic

cells stained with annexin V appeared green colored and

PI-stained cells (red) were observed (Fig. 4b).

Anti-cancer effect of TGase 2 inhibitors or

Wnt/b-catenin inhibitor compared to GK13

To compare anti-cancer effect of TGase 2 inhibitor GK13

against various TGase 2 inhibitors such as KCC009 (Yuan

et al. 2007), cystamine (Caccamo et al. 2010), and Z006

(Verhaar et al. 2011), UACC62 cell was treated with the

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

K

SNU-16

KATO

SNU-216

MKN-28

MKN-45

SNU-484

SNU-668

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

J

Hep3B

Huh7

SNU-354

SNU-423

SNU-449-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4

Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

I

MCF7

MDA231

MDA468

HS578T

BT-549

T-47D

Mean Values

GI50 6.68E-6M

TGI 5.58-5M

LC50 3.87E-3M

Fig. 2 continued

1290 J Cancer Res Clin Oncol (2013) 139:1279–1294

123

indicated concentration of GK13 or TGase 2 inhibitors.

Cell viability using SRB assay showed that GK13 has 10

times greater anti-cancer effect that others have (Fig. 5a).

GK13 may trigger growth inhibition through Wnt/b-

catenin inhibition due to the structural similarity (Gong

et al. 2011). To clarify this possibility in cancer cell lines,

we employed Wnt/b-catenin signaling inhibitor such as

cardamonin to test whether Wnt inhibition mimics GK13

effect in cancer cells. However, Wnt/b-catenin signaling

inhibition did not fully mimic GK13 inhibition in

UACC62 that presents sensitive growth inhibition on

GK13 treatment (Fig. 5b). Figure 5 shows us that GK13

presents cell growth inhibitory effect that appears to affect

TGase 2 activity rather than affecting Wnt/b-catenin

activity.

Discussion

Previously, Dr. Rich’s group reported a small molecule

derived from dihydroisoxazole KCC009 containing a

TGase 2 inhibitory effect, which showed increase of anti-

cancer drug sensitivity against glioblastoma tumors (Yuan

et al. 2007; Choi et al. 2005) and lung cancer cells (Frese-

Schaper et al. 2010). Although IC50 of KCC009 was over

100 lM, KCC009 demonstrated that TGase 2 inhibition

has a benefit to increase chemosensitivity (Yuan et al.

2007; Frese-Schaper et al. 2010). Following the discovery

that TGase 2 can activate NF-jB activity (Lee et al. 2004)

as well as extend NF-jB activation (Park et al. 2011)

through depletion of I-jBa via cross-linking (Lee et al.

2004; Park et al. 2006), several groups, including us, have

0.0

50.0

100.0

150.0

Con

trol

GK

13

Tax

ol

Con

trol

GK

13

Tax

ol

Con

trol

GK

13

Tax

ol

Con

trol

GK

13

Tax

ol

OVCAR5 SW620 U251 UACC62

Hollow Fiber (S.C.)

0.0

50.0

100.0

150.0

Con

trol

GK

13

Tax

ol

Con

trol

GK

13

Tax

ol

Con

trol

GK

13

Tax

ol

Con

trol

GK

13

Tax

ol

OVCAR5 SW620 U251 UACC62

Hollow Fiber (I.P.)

Fig. 3 Hollow fiber assay using

GK13. The hollow fibers were

implanted in subcutaneous

(S.C.), and intraperitonial (I.P.).

GK13 treatment (50 mg/kg) was

started 3 or 4 days after

implantation of fibers into mice

intraperitoneally for 4

consecutive days. When mice

were killed the next day after

the last drug treatment, hollow

fibers were recovered for MTT

assay (detailed in ‘‘Methods’’)

J Cancer Res Clin Oncol (2013) 139:1279–1294 1291

123

GGK13, 0 µM GK13, 16 µM

U25

1U

AC

C62

9.65% 55.53%

7.8% 61.57%

16 µM0 µM 16 µM0 µM

U251 UACC62

Ann

exin

VP

IM

erge

DA

PI

a

b

Fig. 4 Inhibition of TGase2 by

GK13-induced apoptosis.

Annexin V–PI staining of

GK13-treated U251 and

UACC62 cells. The cells treated

with or without GK13 for 6 h.

Apoptosis was further measured

by flow cytometric analysis (a).

The total apoptotic cells (early-

and late-stage apoptosis) are

represented by the right side of

the panel. Fluorescence

micrographs of the cells treated

with GK13. Cells, treated with

GK13, were stained with

annexin V-FITC and PI and

examined by fluorescence

microscopy (b). Original

magnifications 9200. For

16 lM GK13 treatment, early

apoptotic cells stained with

annexin V appeared green

colored. PI-stained cells (red)

were seen

1292 J Cancer Res Clin Oncol (2013) 139:1279–1294

123

reported therapeutic possibilities of TGase 2 inhibitors as

an anti-cancer drug sensitizer (Kim et al. 2006; Mann et al.

2006). Inhibition of TGase 2 turned out to increase anti-

cancer drug sensitivity because TGase 2 can activate NF-

jB, which has been previously demonstrated using cysta-

mine with doxorubicin (Kim et al. 2006). We have also

found a safe natural product containing the TGase 2

inhibitory effect, which is glucosamine. Glucosamine also

showed an anti-cancer sensitization effect in use with

doxorubicin (Kim et al. 2009a). The effective concentra-

tion of glucosamine cannot be reached in the serum level

by oral administration. However, via infusion administra-

tion, glucosamine was shown to have an anti-cancer effect

on Walker 256 carcinoma (Molnar and Bekesi 1972).

To improve specificity and efficacy of TGase 2 inhibi-

tion in cancer, we have tried to obtain proper lead com-

pounds by screening the small-molecule library from Dr.

Gong. We found that the quinoxaline derivative GK13

contains its TGase 2 inhibitory effect in vitro as well as in

cell. In this study, we found a lead compound of TGase 2

inhibitor, GK13, which showed IC50 about 16 lM using

purified guinea pig liver TGase 2. In theory, based upon

our findings of TGase 2-mediated NF-jB activation, TGase

2 inhibition may trigger cell death in cancer cells due to

decrease of NF-jB down stream including BCl-2 (Kim

et al. 2009b). To test whether TGase 2 inhibition alone may

influence cell growth and survival, we have tested GK13 on

NCC72 cell lines composing 11 tissue origins and 72

cancer cell lines. Interestingly, LC50 of GK13 showed

greater efficacy as 4.3E-4 M than LC50 of doxorubicin

that showed efficacy as 3.87E-3 M (Fig. 2). In hollow

fiber assay, GK13 showed distinguishable growth inhibi-

tion of CNS and melanoma cell lines (Fig. 3). This result

implicated that GK13 potentially has a good PK character

in an animal’s physiological condition.

In conclusion, previously, a small molecule containing a

TGase 2 inhibitory effect demonstrated anti-cancer drug

sensitivity under high concentration of KCC009

(*500 lM for 48 h) (Frese-Schaper et al. 2010) due to

less specificity. In our study, we introduced a quinoxaline

derivative, containing a TGase 2 inhibitory effect, which

presented anti-cancer effects approximately in the 10 lM

range. Further development may increase specificity as

well as anti-cancer effects in the near future.

Acknowledgments This work was supported by a research grant

(NCC1110011-2) from the National Cancer Center in Korea to S.Y.K.

and National R&D Program for Cancer Control (No. 1020050) in

Korea to Y.D.G. We declare that none of the authors have a financial

interest related to this work, and none of the authors have any

financial support beyond the research grant mentioned above.

References

Caccamo D, Curro M, Ientile R (2010) Potential of transglutaminase

2 as a therapeutic target. Expert Opin Ther Targets 14(9):989–

1003. doi:10.1517/14728222.2010.510134

Choi K, Siegel M, Piper JL, Yuan L, Cho E, Strnad P, Omary B, Rich

KM, Khosla C (2005) Chemistry and biology of dihydroisox-

azole derivatives: selective inhibitors of human transglutaminase

2. Chem Biol 12(4):469–475. doi:10.1016/j.chembiol.2005.

02.007

Delhase M, Kim SY, Lee H, Naiki-Ito A, Chen Y, Ahn ER, Murata K,

Kim SJ, Lautsch N, Kobayashi KS, Shirai T, Karin M, Nakanishi

M (2012) TANK-binding kinase 1 (TBK1) controls cell survival

through PAI-2/serpinB2 and transglutaminase 2. Proc Natl Acad

Sci USA 109(4):E177–E186. doi:10.1073/pnas.1119296109

Folk JE (1980) Transglutaminases. Annu Rev Biochem 49:517–531.

doi:10.1146/annurev.bi.49.070180.002505

Folk JE, Park MH, Chung SI, Schrode J, Lester EP, Cooper HL

(1980) Polyamines as physiological substrates for transgluta-

minases. J Biol Chem 255(8):3695–3700

Frese-Schaper M, Schardt JA, Sakai T, Carboni GL, Schmid RA,

Frese S (2010) Inhibition of tissue transglutaminase sensitizes

TRAIL-resistant lung cancer cells through upregulation of death

receptor 5. FEBS Lett 584(13):2867–2871. doi:10.1016/j.febslet.

2010.04.072

-80

-60

-40

-20

0

20

40

60

80

100

120

0 -10 -9 -8 -7 -6 -5 -4Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

GK13

Cardamonin

-100

-50

0

50

100

150

0 -10 -9 -8 -7 -6 -5 -4Per

cent

Gro

wth

Log10 of Sample Concentration (Molar)

GK13

KCC009

Cystamine

Z006

a

b

Fig. 5 Anti-cancer activity of TGase 2 inhibitors or Wnt/b-catenin

inhibitor, cardamonin compared to GK13. a UACC62 cell was treated

with the indicated concentration of GK13 or TGase 2 inhibitors, such

as KCC009 (Yuan et al. 2007), cystamine (Caccamo et al. 2010), and

Z006 (Verhaar et al. 2011). Cell viability was determined using SRB

assay. b UACC62 cell was treated with the indicated concentration of

GK13 or Wnt/b-catenin inhibitor, such as cardamonin. Cell viability

was determined using SRB assay

J Cancer Res Clin Oncol (2013) 139:1279–1294 1293

123

Gong YD, Lee T (2010) Combinatorial syntheses of five-membered

ring heterocycles using carbon disulfide and a solid support.

J Comb Chem 12(4):393–409. doi:10.1021/cc100049u

Gong YD, Dong MS, Lee SB, Kim N, Bae MS, Kang NS (2011) A

novel 3-arylethynyl-substituted pyrido[2,3,-b]pyrazine deriva-

tives and pharmacophore model as Wnt2/beta-catenin pathway

inhibitors in non-small-cell lung cancer cell lines. Bioorg Med

Chem 19(18):5639–5647. doi:10.1016/j.bmc.2011.07.028

Griffin M, Mongeot A, Collighan R, Saint RE, Jones RA, Coutts IG,

Rathbone DL (2008) Synthesis of potent water-soluble tissue

transglutaminase inhibitors. Bioorg Med Chem Lett 18(20):

5559–5562. doi:10.1016/j.bmcl.2008.09.006

Gupta R, Srinivasan R, Nijhawan R, Suri V (2010) Tissue transglu-

taminase 2 as a biomarker of cervical intraepithelial neoplasia

(CIN) and its relationship to p16INK4A and nuclear factor

kappaB expression. Virchows Arch 456(1):45–51. doi:10.1007/

s00428-009-0860-5

Hollingshead MG, Alley MC, Camalier RF, Abbott BJ, Mayo JG,

Malspeis L, Grever MR (1995) In vivo cultivation of tumor cells

in hollow fibers. Life Sci 57(2):131–141

Iismaa SE, Mearns BM, Lorand L, Graham RM (2009) Transgluta-

minases and disease: lessons from genetically engineered mouse

models and inherited disorders. Physiol Rev 89(3):991–1023.

doi:10.1152/physrev.00044.2008

Kim SY (2006) Transglutaminase 2 in inflammation. Front Biosci

11:3026–3035. doi:10.1007/s00432-009-0681-6

Kim SY (2010) A new paradigm for cancer therapeutics development.

BMB Rep 43(6):383–388

Kim SY (2011) Transglutaminase 2: a new paradigm for NF-kappaB

involvement in disease. Adv Enzymol Relat Areas Mol Biol

78:161–195

Kim DS, Park SS, Nam BH, Kim IH, Kim SY (2006) Reversal of drug

resistance in breast cancer cells by transglutaminase 2 inhibition

and nuclear factor-kappaB inactivation. Cancer Res 66(22):

10936–10943. doi:10.1158/0008-5472.CAN-06-1521

Kim DS, Park KS, Jeong KC, Lee BI, Lee CH, Kim SY (2009a)

Glucosamine is an effective chemo-sensitizer via transglutamin-

ase 2 inhibition. Cancer Lett 273(2):243–249. doi:10.1016/j.

canlet.2008.08.015

Kim DS, Park KS, Kim SY (2009b) Silencing of TGase 2 sensitizes

breast cancer cells to apoptosis by regulation of survival factors.

Front Biosci 14:2514–2521. doi:10.2741/3394

Kim SJ, Kim KH, Ahn ER, Yoo BC, Kim SY (2011) Depletion of

cathepsin D by transglutaminase 2 through protein cross-linking

promotes cell survival. Amino Acids. doi:10.1007/s00726-011-

1089-6

Ku JL, Park JG (2005) Biology of SNU cell lines. Cancer Res Treat

37(1):1–19. doi:10.4143/crt.2005.37.1.1

Lai TS, Liu Y, Tucker T, Daniel KR, Sane DC, Toone E, Burke JR,

Strittmatter WJ, Greenberg CS (2008) Identification of chemical

inhibitors to human tissue transglutaminase by screening existing

drug libraries. Chem Biol 15(9):969–978. doi:10.1016/j.chembiol.

2008.07.015

Lee T, Gong YD (2012) Solid-phase parallel synthesis of drug-like

artificial 2H-benzopyran libraries. Molecules 17(5):5467–5496.

doi:10.3390/molecules17055467

Lee J, Kim YS, Choi DH, Bang MS, Han TR, Joh TH, Kim SY (2004)

Transglutaminase 2 induces nuclear factor-kappaB activation via

a novel pathway in BV-2 microglia. J Biol Chem 279(51):

53725–53735. doi:10.1074/jbc.M407627200

Lin CY, Tsai PH, Kandaswami CC, Chang GD, Cheng CH, Huang

CJ, Lee PP, Hwang JJ, Lee MT (2011) Role of tissue

transglutaminase 2 in the acquisition of a mesenchymal-like

phenotype in highly invasive A431 tumor cells. Mol Cancer

10:87. doi:10.1186/1476-4598-10-87

Lorand L, Graham RM (2003) Transglutaminases: crosslinking

enzymes with pleiotropic functions. Nat Rev Mol Cell Biol

4(2):140–156. doi:10.1038/nrm1014

Mann AP, Verma A, Sethi G, Manavathi B, Wang H, Fok JY,

Kunnumakkara AB, Kumar R, Aggarwal BB, Mehta K (2006)

Overexpression of tissue transglutaminase leads to constitutive

activation of nuclear factor-kappaB in cancer cells: delineation

of a novel pathway. Cancer Res 66(17):8788–8795. doi:

10.1158/0008-5472.CAN-06-1457

Molnar Z, Bekesi JG (1972) Cytotoxic effects of D-glucosamine on

the ultrastructures of normal and neoplastic tissues in vivo.

Cancer Res 32(4):756–765

Pardin C, Pelletier JN, Lubell WD, Keillor JW (2008) Cinnamoyl

inhibitors of tissue transglutaminase. J Org Chem 73(15):

5766–5775. doi:10.1021/jo8004843

Park SS, Kim JM, Kim DS, Kim IH, Kim SY (2006) Transgluta-

minase 2 mediates polymer formation of I-kappaBalpha through

C-terminal glutamine cluster. J Biol Chem 281(46):34965–

34972. doi:10.1074/jbc.M604150200

Park KS, Kim DS, Ko C, Lee SJ, Oh SH, Kim SY (2011) TNF-alpha

mediated NF-kappaB activation is constantly extended by

transglutaminase 2. Front Biosci (Elite Ed) 3:341–354

Shao M, Cao L, Shen C, Satpathy M, Chelladurai B, Bigsby RM,

Nakshatri H, Matei D (2009) Epithelial-to-mesenchymal transi-

tion and ovarian tumor progression induced by tissue transglu-

taminase. Cancer Res 69(24):9192–9201. doi:10.1158/0008-

5472.CAN-09-1257

Shoemaker RH (2006) The NCI60 human tumour cell line anticancer

drug screen. Nat Rev Cancer 6(10):813–823. doi:10.1038/nrc

1951

Siegel M, Khosla C (2007) Transglutaminase 2 inhibitors and their

therapeutic role in disease states. Pharmacol Ther 115(2):232–

245. doi:10.1016/j.pharmthera.2007.05.003

Siegel M, Xia J, Khosla C (2007) Structure-based design of alpha-

amido aldehyde containing gluten peptide analogues as modu-

lators of HLA-DQ2 and transglutaminase 2. Bioorg Med Chem

15(18):6253–6261. doi:10.1016/j.bmc.2007.06.020

Sohn J, Kim TI, Yoon YH, Kim JY, Kim SY (2003) Novel

transglutaminase inhibitors reverse the inflammation of allergic

conjunctivitis. J Clin Invest 111(1):121–128. doi:10.1172/JCI

15937

Verhaar R, Jongenelen CA, Gerard M, Baekelandt V, Van Dam AM,

Wilhelmus MM, Drukarch B (2011) Blockade of enzyme

activity inhibits tissue transglutaminase-mediated transamidation

of alpha-synuclein in a cellular model of Parkinson’s disease.

Neurochem Int 58(7):785–793. doi:10.1016/j.neuint.2011.03.004

Verma A, Mehta K (2007) Transglutaminase-mediated activation of

nuclear transcription factor-kappaB in cancer cells: a new

therapeutic opportunity. Curr Cancer Drug Targets 7(6):559–565

Yoo H, Ahn ER, Kim SJ, Lee SH, Oh SH, Kim SY (2013) Divergent

results induced by different types of septic shock in transglu-

taminase 2 knockout mice. Amino Acids 44(1):189–197. doi:

10.1007/s00726-012-1412-x

Yuan L, Siegel M, Choi K, Khosla C, Miller CR, Jackson EN,

Piwnica-Worms D, Rich KM (2007) Transglutaminase 2 inhib-

itor, KCC009, disrupts fibronectin assembly in the extracellular

matrix and sensitizes orthotopic glioblastomas to chemotherapy.

Oncogene 26(18):2563–2573. doi:10.1038/sj.onc.1210048

1294 J Cancer Res Clin Oncol (2013) 139:1279–1294

123