targeting constitutive & interleukin-6 inducible signal transducer and activator of transcription-3...

7/29/2019 Targeting Constitutive & Interleukin-6 Inducible Signal Transducer and Activator of Transcription-3 (STAT-3) Signaling

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Targeting constitutive & interleukin-6 inducible

signal transducer and activator of transcription-

3 (STAT-3) signaling cascade in hepatocellular

carcinoma by a novel histone acetyltransferaseinhibitor.

Esteehara Bte Rosli

U061851J

Undergraduate Research Opportunities in Science

PROJECT REPORT

Submitted to the

Department of Pharmacology

National University of Singapore

LSM 3288: Advanced UROPS in Life Sciences I (8MC)

October 2009

4942 words


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Abstract

Constitutive activation of STAT3 has been shown in several human cancers

and transformed cell lines including hepatocellular carcinoma (HCC). Thus, agents

that suppress STAT3 phosphorylation have a potential for the treatment of HCC. In

the present report, we investigated whether garcinol (camboginol), a

polyisoprenylated benzophenone derivative can modulate the STAT3 signaling

pathway. We found that garcinol inhibited constitutive and inducible STAT3

phosphorylation in HCC cells and this correlated with the inhibition of JAK1, and

JAK2 activation. Vanadate, however, reversed the garcinolinduced downregulation

of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase.

Indeed, we found that garcinol can induce the expression of Src homology

phosphatase 1 (SHP1) that correlated with STAT3 inhibition. Consistent with these

results, garcinol also inhibited proliferation of different HCC cell lines and also

significantly potentiated the apoptotic effects of doxorubicin and paclitaxel in HCC

cells. Overall, these results suggest that garcinol is a novel blocker of the STAT3

activation pathway, with a potential role in the prevention and treatment of HCC and

other cancers.

(170 words)


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INTRODUCTION

Hepatocellular carcinoma (HCC) is one of the most common occurring tumors

worldwide and causes approximately one million deaths each year (Seow et. al2001).

It is the fifth common cancer worldwide (Bosch, 1997) and about three quarters of the

cases of liver cancer are found in Southeast Asia namely China, Hong Kong, Taiwan,

Korea, and Japan. In Singapore, according to a Singapore Cancer Registry report, the

incidence of liver cancer is the fourth most common cancer in men and the second

most fatal cancer. In more than 80% of cases, HCC development has been linked to

chronic infection with hepatitis B and C viruses (Wu et. al2002). Other risk factors

include alcohol-related cirrhosis and dietary exposure to aflatoxin B1 (Schafer and

Sorell, 1999). Systemic chemotherapy represents a palliative treatment and currently,

there are first line chemotherapeutic drugs used which include doxorubicin,

paclitaxel, gemcitabine, mitomyocin C and fluorouracil (Pastorelli et. al2006; Zangos

et. al, 2007). However, the use of cytotoxic agents in advanced HCC has been

disappointing with few agents showing response rates (RRs) above 20% (Nowaket.

al2004). This is evident in the case of Nexavar, which is the only approved systemic

drug therapy by U.S Food and Drug Administration for HCC treatment where patients

suffer from a myriad of side effects and develop chemoresistance (Aggarwal et. al,

2006). Hence, it is imperative to develop more efficient therapies for the treatment of

HCC.

Signal transducer and activator of transcription-3 (STAT3) protein is part of a

family of six different transcription factors, which play major roles in cytokine

signaling (Shuai et. al, 1993; Costantino and Barlocco, 2008; Gao and Bromberg,

2006). A typical STAT protein consists of a coiled-coil domain, a DNA-binding


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domain, a linker, an SH2 domain, and a transactivation domain (TAD) (Aggarwal et.

al2006). STAT3 can be activated by a myriad of agents; such as cytokines and

growth factors including the epidermal growth factor (EGF) where 30% of all tumor

cells are triggered, platelet-derived growth factor (PDGF), oncostatin M,

thrombopoietin, transforming growth factor- (TGF-) and IL-5, IL-6, IL-9, IL-10,

IL-12, IL-22 as well as oncogenic proteins Src and Ras. STAT3 can also be activated

by oxidative stress, tobacco chewing, hepatitis C virus, ultraviolet B,

lipopolysaccharide, osmotic shock and progestins (Aggarwal et. al, 2009; Aggarwal

et. al, 2006). The binding of these factors to receptors on the cell-surface leads to

receptor autophosphorylation. STAT3 proteins in the cytosol will be recruited upon

recognizing the phosphotyrosine due to its SH2 domain and will associate with the

activated receptor. The activation of STAT3 involves the phosphorylation of tyrosine

residue at position 705 within the carboxy-terminal region either directly by the

receptor or by a receptor-associated Janus-activated kinases (JAK) of which JAK2 is

one of the major mediators of STAT3 phosphorylation (Bhutani et. al, 2007 ;

Aggarwal et. al, 2006).

Several other kinases have also been implicated in STAT3 phosphorylation which

includes members of the Src family (hck, src), Erb B1, Erb B2, anaplastic lymphoma

kinase, protein kinase C (PKC)-, c-fes, gp130, and epithelial growth factor (EGF)

receptor (Aggarwal et. al, 2006). Upon activation, STAT3 undergoes

homodimerization, leading to nuclear translocation, DNA binding, and subsequent

gene transcription (Kwang et. al, 2008). STAT3 is also acetylated on a single lysine

residue 685 by histone acetyltransferase (HAT) p300 and acetylation of STAT3 is

considered essential for it to form stable dimers, which are required for cytokine-

stimulated DNA binding and transcriptional regulation. However, unphosphorylated


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or tyrosine-mutated STAT3 can still form dimers and induce transcription (Braunstein

et. al., 2003). Others have found that STAT3 dimerization is regulated by reversible

acetylation of lysine at residue 685 in the SH2 domain of STAT3 (Yuan et. al., 2005).

IL-6-induced acetylation of the STAT3 N terminus is necessary for acute phase

induction of angiotensinogen (Ray et. al., 2002). These observations indicate that

site-specific acetylation of STAT3 is an important regulatory modification that

influences protein-protein interaction and transcriptional regulation.

STAT3 activation is negatively regulated through numerous mechanisms, which

involve suppressors of cytokine signaling (SOCS), protein inhibitor of activated

STAT (PIAS), protein phosphatases (SHP1, SHP2, CD45, PTEN), and ubiquitination-

dependent proteosomal degradation. In normal cells, activation of STAT3 is strictly

controlled to prevent unscheduled gene regulation however it was found that STAT3

is constitutively active in large number of tumors (Aggawal et. al, 2006) through up-

regulation of genes encoding apoptosis inhibitors such as Bcl-2, Bcl-xL survivin, and

Mcl-1, cell cycle regulators, e.g cyclins and c-Myc, and also inducers of angiogenesis,

such as vascular endothelial growth factor (Germain and Frank, 2007; Turkson and

Jove, 2000).

STAT3 represents a promising target for HCC therapy since inhibition of STAT3

induces growth arrestand apoptosis of human HCC cells (Choudhari et al., 2007;

Kusaba et al., 2007; Li et al., 2006; Lin et al., 2009; Sun et al., 2008; Tatebe et al.,

2008). One potential source for the inhibition of STAT3 is drugs derived from natural

products. For many centuries, natural products have been used as means of therapy

and thus envisioned as safe (Prasad et. al, 2009). It was found that as many as 70% of

all drugs approved for cancer treatment between 1981 and 2002 were either natural

products or based on natural products (Newman et. al, 2003). We describe here the


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identification of a novel compound Garcinol (camboginol), which is a

polyisoprenylated benzophenone derivative, derived from the dried rind of the fruit

Garcinia indica which is used as a spice and as a traditional medicine for the

treatment of diabetes, obesity, and ulcer.

Garcinol is structurally similar to a well-known anti-oxidant, curcumin, which

contains both phenolic hydroxyl groups and a -ketone moiety (Liao et. al, 2004). It

has been shown to exhibit health-promoting properties such as anti-oxidant, anti-

biotic and anti-inflammatory activities, inhibit protein glycation and induction of

apoptosis in a wide variety of tumor cells including leukemia, colon cancer and

gastrointestinal cancer cells (Liao et. al, 2004). The mechanisms of how this

benzophenone exhibits all its effects are not fully understood but it has been shown to

suppress the expression of inducible nitric oxide synthase (iNOS) and

cyclooxygenase-2 (COX-2) by inhibiting NF-B activation (Liao et. al, 2004), block

phosphorylation of cPLA2, and decrease iNOS protein by inhibiting STAT1

activation (Hong et. al, 2006); repress chromatin transcription and global gene

expression through inhibition of histone acetyltransferases p300 and PCAF but has no

effect on the deacetylation of histones (Balasubramanyam et. al, 2004); and induce

apoptosis through the activation of caspase-2, caspase-3 and caspase-9 leading to

cleavage of PARP, D4-GDI and DFF-45 (Pan et. al, 2001). Therefore, these results

and evidences mentioned above may contribute to its chemopreventive functions and

plausibly make garcinol a suitable candidate for a potential cancer chemopreventive

agent.

Because of the critical role of STAT3 activation in tumor cell survival,

proliferation, and angiogenesis, we hypothesized that garcinol must mediate its effects

through the suppression of the STAT3 pathway. In our experiments on human


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hepatocellular carcinoma cells, we found that garcinol does indeed suppress both

constitutive and inducible STAT3 activation, down-modulated activation of upstream

kinases, induced the activation of a phosphatase, inhibited proliferation and

potentiated the apoptotic effects of doxorubicin and paclitaxel in HCC cells.


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MATERIALS & METHODS

Reagents

Garcinol, and LTK14 were a kind gift from Prof. Tapas K. Kundu, Molecular Biology

and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research

(JNCASR), Bangalore, India. A 50 mM solution of garcinol and LTK14 was

prepared in dimethyl sulfoxide (DMSO), stored as small aliquots at -20C, and then

diluted as needed in cell culture medium. MTT, Tris, , glycine, NaCl, SDS, b-actin

and bovine serum albumin were purchased from Sigma-Aldrich (St. Louis, MO).

Minimum Essential Medium (MEM), Dulbeccos Modified Eagle Medium (DMEM),

fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA). Antibodies

to p-STAT3 (Tyr705), STAT3, p-JAK1 (Tyr1022/1023) and p-JAK2, JAK1, and

JAK2 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to

SHP1 as well as goat anti- mouse and anti-rabbit horseradish peroxidase were

obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Bacteria-derived

recombinant human IL-6 was purchased from ProSpec-Tany TechnoGene Ltd.

(Rehovot, Israel). FuGENE 6 Transfection Reagent was obtained from Roche. Goat

anti-rabbit Alexa 594 purchased from Invitrogen.

Cell Lines

Human hepatocellular carcinoma cell lines C3A (ATCC CRL -10741), HepG2

(ATCC HB 8065) and PLC/PRF5 (ATCC CRL 8024) were obtained from the

American Type Culture Collection (Manassas, VA). Human hepatoma HUH7 cells

were a kind gift from Prof. Hui Kam Man at National Cancer Center, Singapore. The

HepG2 cell line was isolated from a liver biopsy of a 15-year-old male Caucasian

with a well-differentiated hepatocellular carcinoma. These cells secrete a variety of


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major plasma proteins e.g. albumin, 2-macroglobulin, 1-antitrypsin, transferrin, and

plasminogen. HepG2 and C3A cells were maintained in monolayer culture at 37C

and 5% CO2 in MEM containing 1x penicillin-streptomycin solution (Invitrogen)

with 10% FBS. PLC/PRF5 and HUH7 cells were cultured in DMEM containing 1x

penicillin- streptomycin solution, non-essential amino acids, sodium pyruvate, and L-

glutamine with 10% FBS.

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay

The effect of garcinol and LTK14 treatment on the viability of four HCC cell

lines was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

bromide (MTT) dye uptake method (Sigma-Aldrich) based on the ability of live and

active cells to cleave the MTT (tetrazolium salt) to purple formazan that is absorbed

at 570 nm. 2.5 x 104 cells/well (200ml) were seeded in 96-well plates for 24 h and

the cells were further incubated with different concentrations of garcinol and LTK14

in medium containing 1x penicillin-streptomycin with 10% FBS. MTT reagent was

dissolved at a concentration of 5mg/ml in sterile PBS at room temperature, filtered

and stored at 4C in the dark. After 12, 24, and 48 h, 20ml MTT reagent was added to

each well, and the cells were further incubated at 37C for 2 h. MTT lysis buffer was

prepared with 20% W/V SDS dissolved at 37C in a solution of 50% of each DMF

(N,N-dimethyl Foramide) and dH2O; pH was adjusted to 4.7 by adding concentrated

hydrochloric acid. 100ml of MTT lysis buffer was added to each well for 4 h,

followed by reading on a scanning multi-well spectrophotometer (TECAN) at 570nm.

Untreated cells were used as controls. Experiments were performed with 6 wells per

concentration.


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Western blotting

For detection of different proteins, garcinol-treated whole-cell extracts (1 x

106 cells/well, 2ml) were lysed in lysis buffer (20 mM Tris (pH 7.4), 250 mM NaCl, 2

mM EDTA (pH 8.0), 0.1% TritonX-100, 0.01 mg/ml aprotinin, 0.005 mg/ml

leupeptin, 0.4 mM PMSF,and 4 mM NaVO4). Lysates were then spun at 13,300 rpm

for 5 min at 4C to remove insoluble material and resolved on an 8% SDS gel. After

electrophoresis, the proteins were electrotransferredto a nitrocellulose membrane,

blocked with 5% non-fat milk, andprobed with anti-STAT3 and anti-phospho-STAT3

antibodies (1:1000) overnight at 4C. Theblot was washed, exposed to HRP-

conjugated secondary antibodies for1 h, and finally examined by chemiluminescence

(ECL; AmershamPharmacia Biotech). For detection of non-phosphorylated proteins,

the respective nitrocellulose membranes were stripped with stripping buffer (Thermo

Scientific), blocked with blocking buffer, and probed with antibodies against total

STAT3 overnight at 4C, and then detected by enhanced chemiluminescence (GE

Health care). -actin was detected as a housekeeping protein to ensure equal amounts

of protein was loaded per sample in each blot; anti-mouse secondary antibodies were

used.

Immunocytochemistry for STAT3 localization

C3A cells were plated in chamber slides in DMEM containing 10% FBS and

allowed to adhere for 24 h. On the next day, the cells are treated with 50 M of

garcinol and then incubated for 4 h in 37CO. Then the cells were fixed with cold

acetone for 10 min, washed with PBS and blocked with 5% normal goat serum for 1

h. The cells were then incubated with rabbit polyclonal anti-humanSTAT3 Antibody

(dilution, 1/100). After overnight incubation, the cells were washed and then


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incubated with goat anti-rabbitIgG-Alexa 594 (1/100) for 1 h and counterstained for

nucleiwith Hoechst (50 ng/ml) for 5 min. Stained cells were mountedwith mounting

medium (Sigma-Aldrich) and analyzed under an fluorescence microscope (Olympus,

Japan).

Live/Dead Assay

Viability of cells was also determined by Live/Dead assay (Molecular Probes,

Eugene, OR, USA) that measures intracellular esterase activity and plasma membrane

integrity. The polyanionic dye calcein is well retained within live cells, producing an

intense uniform green fluorescence in live cells while EthD-1 enters cells with

damaged membranes and produces a bright red fluorescence in dead cells. Briefly,

1 X106 cells were incubated with different concentrations of garcinol/doxorubicin/

paclitaxel alone or in combination for 24 h at 37C and then washed with D-PBS.

Cells were stained with the Live/Dead reagent (5 M ethidium homodimer, 5 M

calcein-AM) and then incubated at 37C in the dark for 30 min. The staining solution

was discarded; cells were washed with PBS and were analyzed under a fluorescence

microscope(Olympus, Japan).


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RESULTS

The present study was undertaken to determine the effect of garcinol on

constitutive and IL6-inducible STAT3 activation in human hepatocellular carcinoma

cells. We also evaluated the effect of garcinol on various mediators of STAT3

signaling pathway. The structure of garcinol is shown in Fig. 1A.

Garcinol inhibits constitutive STAT3 phosphorylation in C3A cells:

Whether garcinol can modulate the constitutive STAT3activation in HCC

cells, was investigated. C3A cells were incubated withdifferent concentrations of

garcinol for 6 h, prepared the whole cell extracts and examined for phosphorylated

STAT3 by Western blot analysis using antibodies which recognize STAT3

phosphorylated at tyrosine 705. As shown in Fig.1B, garcinol inhibited the

constitutive activation of STAT3 in C3A cells in a dose-dependent manner, with

maximum inhibition occurring at around 50 M. Garcinol had no effect on the

expression of STAT3 protein (Fig. 1B; lower panel). As shown in Fig.1C, the

inhibition was time-dependent, with maximum inhibition occurring at around 4 h,

again with no effect on the expression of STAT3 protein (Fig. 1C; lower panel).

Garcinol depletes nuclear pool of STAT3 in HCC cells:

Because nuclear translocation is central to the function of transcription factors

and because it is not certain whether phosphorylation is mandatory for nuclear

transport of STAT3 and its oncogenic functions (Bowman et al., 2000), we

determined whether garcinol suppresses nuclear translocation of STAT3. Fig. 1D

clearlydemonstrates that garcinol inhibited the translocation of STAT3 to the nucleus

in C3A cells.


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Garcinol inhibits inducible STAT3 phosphorylation in HCC cells:

Because IL-6 induces STAT3 phosphorylation (Moran et al., 2008;

Zauberman et al., 1999), we determined whether garcinol could inhibit IL-6-induced


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STAT3 phosphorylation. In HUH7 cells incubated with garcinol for different times,

IL-6-inducedSTAT3 phosphorylation was suppressed by garcinol in a time-

dependentmanner. Exposure of cells to garcinol for 6 h was sufficient to completely

suppress IL-6-induced STAT3 phosphorylation (Fig 2A).

Garcinol suppresses constitutive activation of J AK1 and J AK2:

STAT3 has been reported to be activated by soluble tyrosine kinases of the

Janus family (JAKs), so we determined whether garcinol affects constitutive

activation of JAK1 in C3A cells. We found that garcinol suppressed the constitutive

phosphorylation of JAK1 (Fig 2B). The levels of non-phosphorylated JAK1 remained

unchanged under the same conditions (Fig. 2B, bottom panel). To determine the effect

of garcinol on JAK2 activation, C3A cells were treated with different concentrations

and time intervals with garcinol and phosphorylation of JAK2 was analyzed by

Western blot. As shown in Fig.2C, JAK2 was constitutively active in C3A cells and

pretreatment with garcinol suppressed this phosphorylation in a time-dependent

manner.

Tyrosine phosphatases are involved in garcinol-induced inhibition of STAT3

activation:

Because protein tyrosine phosphatases have also been implicated in STAT3

activation, we determine whether garcinol-induced inhibition of STAT3 tyrosine

phosphorylation could be due to activation of a protein tyrosine phosphatase

(PTPase). Treatment of C3A cells with the broad-acting tyrosine phosphatase

inhibitor sodium pervanadate prevented the garcinol-induced inhibition of STAT3


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activation (Fig 2D). This suggests that tyrosine phosphatases are involved in garcinol-

induced inhibition of STAT3 activation.

Garcinol induces the expression of SHP1 in HCC cells

SHP1 is a non-transmembrane protein tyrosine phosphatase expressed most

abundantly in hematopoietic cells (Wu et al., 2003). We therefore examined whether

garcinol can modulate the expression of SHP1 in C3A cells. Cells were incubated

with different concentrations of garcinol for 4 h, and whole-cell extracts were

prepared and examined for SHP1 protein by Western blot analysis. As shown in

Fig.2E, garcinol induced the expression of SHP1 protein in C3A cells.


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Garcinol is more potent than its analogue LTK14 in suppressing the proliferation

of HCC cells

Next we compared the anti-proliferative effects of garcinol with its analogue

LTK14 in four different HCC cells by using the MTT analysis method. As shown in


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Figs 3A and 3B, garcinol was more potent than LTK14 in inhibiting HCC cell

proliferation.

Garcinol potentiates the apoptotic effect of doxorubicin and paclitaxel in HCC cells

Among chemotherapeutic agents, doxorubicin, an anthracycline antibiotic, and

paclitaxel, a mitotic inhibitor, have been widely used for HCC treatment (Burden and


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Osheroff,1998). We examined whether garcinol can potentiate the effect of these

drugs.

C3A cells were treated with garcinol together with either doxorubicin or

paclitaxel, and then apoptosis was measured by the live and dead assay. As shown in

Fig.4, garcinol significantly enhanced the apoptotic effects of doxorubicin from 10 to

42% and of paclitaxel from 12 to 60%.


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DISCUSSION

The goal of this study was to determine whether garcinol exerts its anti-

proliferative effects through the abrogation of the STAT3 signaling pathway in HCC

cells. We found that this benzophenone suppressed both constitutive and IL-6-

inducible STAT3 activation (Fig. 1B, 1C and 2A) in parallel with the inhibition of

JAK1 and JAK2 (Fig. 2B and 2C). Garcinol specifically depleted the nuclear pool of

STAT3; stimulated the expression of non-transmembrane protein tyrosine

phosphatase SHP1, suppressed the proliferation more potently than LTK14, and

significantly potentiated the apoptotic effects of doxorubicin and paclitaxel on HCC

cells (Fig. 4).

We report for the first time that garcinol could suppress both constitutive and

inducible STAT3 activation in HCC cells and that these effects correlated with the

suppression of upstream protein tyrosine kinases JAK1 and JAK2. The activation of

STAT3 can be induced by a wide variety of growth factors including IL-6, EGF,

interferon-g, and lipopolysaccharide (Aggarwal et. al., 2006). We found that

activation of STAT3 induced by IL-6 was completely suppressed by garcinol. How

garcinol inhibits IL-6 is not clear, howeverthe roles of JAK1, JAK2, mitogen-

activated protein kinase, and Akt have been implicated in IL-6-induced STAT3

activation (Pandey et.al, 2008). Constitutive activation of STAT3 has been reported in

a large number of tumors, including breast cancer, prostate cancer, head and neck

squamous cell carcinoma, lymphomas and leukemias, brain tumor, colon cancer,

Ewing sarcoma, gastric cancer, esophageal cancer, ovarian cancer, nasopharyngeal

cancer, and pancreatic cancer (Aggarwal et. al., 2006). Hence the suppression of

constitutively active STAT3 in HCC cells raises the possibility that this novel STAT3

inhibitor might also exhibit cytotoxicity against other types of cancer cells that


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display constitutively active STAT3.

It has been previously reported that garcinol is an inhibitor of NF-B activation

(Liao et. al, 2004). Whether suppression of STAT3 activation by this benzophenone is

linked to inhibition of NF-B is not clear. The p65 subunit of NF-B has been shown

to interact with STAT3 (Yu et. al, 2002), but activation of STAT3 and NF-B are

dependent on different cytokines. Although IL-6 is a major activator of STAT3,

tumor necrosis factor is a potent activator of NF-B. Interestingly; JAK2 kinase

needed for STAT3 activation has been shown to be required for erythropoietin-

induced NF-B activation (Digicaylioglu et. al, 2001). Thus, it is possible that the

suppression of JAK2 activation is the potential link for inhibition of both NF-B and

STAT3 activation by garcinol.

We also found evidence that the garcinol-induced inhibition of STAT3

activation involves a protein tyrosine phosphatase (PTP); as its STAT3 inhibitory

effects were reversed by broad-spectrum phosphatase inhibitor, pervanandate.

Numerous protein tyrosine phosphastases (PTPs) have been implicated in STAT3

signaling, including SHP1, SHP2, T-cell PTP, PTEN, PTP-1D, CD45, PTPe, and low

molecular weight PTP (Pathaket. al, 2007). Indeed we found for the first time that

garcinol stimulates the expression of SHP1 protein in HCC cells, which correlated,

with down-regulation of constitutive STAT3 phosphorylation. However, several

groups have isolated putative inhibitors for the JAK/STAT pathways by functional or

molecular screening of cDNA libraries (Chung et al., 1997; Endo et al., 1997; Starret

al., 1997). Aberrant methylation of SH2 domain-containing protein known as

suppressor of cytokine signaling (SOCS1) which is a negative regulator of STAT3

pathway has been found in 65% of human primary HCC tumor samples (Yoshikawa


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et al., 2001). Another group identified a family of proteins with a putative zinc-

binding motif that was named PIAS, for protein inhibitor of activated STAT (Chung

et al., 1997). It was observed that PIAS3 specifically interacts with activated STAT,

thereby inhibiting its DNA-binding activity and induction of gene expression.

Although further investigation is required for elucidation of the molecular mechanism

for functions of these inhibitors, it is conceivable that JAK/STAT pathways might be

regulated at multiple levels.

When compared with its analogue LTK14, garcinol was more effective in

inhibiting the proliferation of various HCC cell lines. Doxorubicin and paclitaxel are

commonly used chemotherapeutic drugs for the treatment of HCC (Burden and

Osheroff, 1998). We found that garcinol potentiates the apoptotic effect of these drugs

in HCC cells (Fig. 4).There is emerging evidence which suggests that garcinol could

be useful as an anti-cancer agent, and it is increasingly being realized that garcinol is

a pleiotropic agent capable of modulating key regulatory cell signaling pathways

(Padhye et. al, 2009). Hence further studies are warranted, as the available data is

promising in order to fully appreciate its potential against various cancers. In

conclusion, our results clearly demonstrated that the anti-proliferative and anti-

carcinogenic activities of garcinol are mediated through the inhibition of STAT3

signaling cascade.


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