Hepatocellular carcinoma (HCC) is one of the

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1 Original Article / Liver Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B Xin-Bo Xue, Kun Chen, Cong-Jun Wang, Jian-Wei Zheng, Yuan Yu, Zhi-Hai Peng and Zai-De Wu Wuhan, China BACKGROUND: Melanoma differentiation associated gene-7 (mda-7) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways. In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma (HCC) in vitro. METHODS: Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modified Eagle's medium without serum (control). The others were transfected with adenovirus expressing the mda-7 gene (Ad.mda-7) or adenovirus vector serving as negative control (Ad.vec). The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was confirmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and flow cytometry were used to assess tumor cell proliferation and the cell cycle. Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells. The expression of MDA-7, Bcl-2 and Bax proteins were detected by Western blotting. Author Affiliations: Department of Biliary and Pancreatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan , China (Xue XB, Chen K, Zheng JW, Yu Y and Wu ZD); Department of General Surgery, First People's Hospital Affiliated Shanghai Jiaotong University, Shanghai , China (Wang CJ and Peng ZH) Corresponding Author: Xin-Bo Xue, MD, Department of Biliary and Pancreatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan , China (Tel: ; xiangfanck@163.com) 2008, Hepatobiliary Pancreat Dis Int. All rights reserved. RESULTS: The mda-7 gene was expressed in Hep3B and L-02 cells. The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml, respectively. mda-7 induced Hep3B growth suppression and apoptosis, compared with Ad.mda-7 and control (P<0.01). In addition, cell block in G2/M was identified by exposure of HCC cells to secreted MDA-7 protein, but this was not found in L-02. The gene expression of Bcl-2 was markedly decreased in Hep3B but not in L-02. CONCLUSIONS: mda-7 selectively induces growth inhibition and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the antiapoptosis protein Bcl-2. It could be an ideal gene for gene therapy in HCC. (Hepatobiliary Pancreat Dis Int 2008; 7: ) KEY WORDS: replication-incompetent adenovirus vector; melanoma differentiation associated-7 gene; carcinoma, hepatocellular; Bcl-2 Introduction Hepatocellular carcinoma (HCC) is one of the most common lethal malignant diseases in the world, resulting in thousands of deaths every year. Unfortunately, more than 50% of new cases are found in China. [1] The clinical effects of most gene therapies are limited because the genes are not specifically responsive to tumor cells. Melanoma differentiation associated gene-7 (mda-7) was identified by a combination of recombinant fibroblast interferon (IFN-β) and the protein kinase C activator mezerein (MEZ) subtraction hybridization by Fisher in [2] Recent studies indicate that overexpression of mda-7 by a replication-defective adenovirus vector results in growth suppression and apoptosis in a broad spectrum of human carcinomas Hepatobiliary Pancreat Dis Int,Vol 7,No 5 October 15,

2 Hepatobiliary & Pancreatic Diseases International both in vitro and in vivo. [3-10] Although mda-7 has potential for killing many different kinds of cancer cells, it has no harmful effects on normal cells. [11] In the present study, we investigated the impact of adenovirus expressing the mda-7 gene (Ad.mda-7) on growth, the cell cycle and survival of a HCC cell line, and a human liver cell line. In these contexts, the study provides important support for the use of Ad.mda-7 in selective gene therapy for HCC. Methods Cell lines and culture conditions The HCC cell line Hep3B was obtained from the Institute of HCC at Fudan University, and the human liver cell line L-02 was a gift from Dr. Jian Guan. The cell lines were cultured in high glucose DMEM (HyClone, Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, USA) in a 5% CO 2 humidified atmosphere at 37. Virus construction, identification and purification The recombinant replication-defective Ad.mda-7 virus was created in our laboratory. [12] Briefly, human mda-7 cdna was directionally cloned into psgcv to produce psgcmv-mda-7. Using a plasmid transfection method, we co-transfected the psgcmvmda-7 and adenovirus skeletal plasmid into HEK293 cells to construct the recombinant adenovirus vector Ad.mda-7, carrying the mda-7 gene, by intracellular homologous recombination. The genomes were analyzed to confirm the recombinant structure and then the virus was plaque purified and amplified in HEK293 cells. RNA isolation and RT-PCR Hep3B and L-02 cells infected with 250 virus particles/cell (vp/cell) of Ad.vec and Ad.mda-7 were harvested at 24, 48 and 72 hours. Total cell RNA was extracted with the Qiagen RNeasy mini kit (USA) according to the manufacturer's protocol, and RT-PCR was performed to quantify the expression of mda-7 mrna. The sequences of primers of mda-7 mrna were: upstream, 5'-GGG CTG TGA AAG ACA CTA T-3', downstream, 5'-GCA TCC AGG TCA GAA GAA-3'. The sequences of primers of glyceraldehyde phosphate dehydrogenase (GAPDH) were: upstream, 5'-CCT TCC TGG GCA ATG GAG TCC T-3', downstream, 5'-GGA ACA ATG ATC TTG ATC TT-3'. Reaction mixtures with corresponding primers were carried out under the following conditions: denaturation at 95 (3 minutes); 30 cycles at 94 (30 seconds), 55 (30 seconds), and 72 (30 seconds); then extension at 72 (5 minutes). The PCR products underwent 1% agarose gel electrophoresis. Concentration of MDA-7 in supernatant The cell culture supernatant was collected at 24, 48 and 72 hours and stored at -20. The concentrations of MDA-7 in the supernatant at different times were evaluated by ELISA. Samples from each group were placed in triple wells and analyzed following the instructions with the ELISA kit. MTT assay to determine cell proliferation Briefly, Hep3B and L-02 cells were seeded at /well (containing cells/ml) in 96-well plates and incubated in 100 μl of culture medium overnight. According to the optimal dose for infection, the cells were then transfected with Ad.mda-7 or Ad.vec and cultured in DMEM without FBS. After cultivation for 24 hours, 20 μl stock MTT solution (0.5 mg/ml; Roche Diagnostics GmbH Co., Germany) was added to each well, and further incubated at 37 for 4 hours. Then the culture was incubated for a further 15 minutes at 37 with gentle shaking. The absorbance of samples was detected at 540 nm on a microplate reader. Fluorescence microscopy evaluation of cell apoptosis Fourty-eight hours after infection, cells were washed once with PBS and fixed in 4% paraformaldehyde for 30 minutes at room temperature. After two washes with PBS, the cells were stained with 0.05 mg/ml Hoechst (Sigma, USA) in PBS for 15 minutes in the dark at room temperature. Nuclear fragmentation was visualized using a fluorescence microscope equipped with a UV-2A filter and Olympus BX60 photographic camera. Apoptotic cells were identified by condensation and fragmentation of nuclear chromatin. Fluorescence-activated cell sorter (FACS) analysis and Annexin V/PI assay Treated with PBS, Ad.vec and Ad.mda-7 cells were trypsinized and washed once with complete media 24, 48 or 72 hours later. Aliquots of cells ( ) were resuspended in complete media (0.5 ml) and stained with FITC-labeled Annexin-V (kit from Jinmei Co, China) according to the manufacturer's instructions. Propidium iodide (PI) was added to the samples after staining with Annexin-V to distinguish late apoptotic and necrotic cells. The cells were then processed for FACS analysis (Becton Dickinson, San Jose, CA, USA). 510 Hepatobiliary Pancreat Dis Int,Vol 7,No 5 October 15,2008

3 Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B DNA staining with PI for cell-cycle analyses For cell cycle analysis, when the cells grew to about 30%, they were synchronized by serum starvation for 24 hours and induced to re-enter the cell cycle by an exchange of 5% fetal bovine serum. In DMEM without FCS, Ad.vec- and Ad.mda-7-treated cells were prepared as a single cell suspension of cells/ml in PBS. After the cells were fixed in 70% cold ethanol overnight at -20, they were washed with PBS, and then stained with PI at final concentration of 50 μg/ml with RNAse at 1 mg/ml in PBS. After 30 minutes of incubation, the cells were analyzed by flow cytometry using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). Treated cells were then evaluated by FACS analysis for identifying the cells at different stages of the cell cycle. Determination of expression of Bcl-2 family by Western blotting Hep3B and L-02 cell lines were cultured on 10-cm plates, and cells receiving different treatments were collected at the indicated times. Protein extracts were prepared with radio immunoprecipitation assay buffer containing a cocktail of protease inhibitors. A total of 50 mg of protein was applied to 15% SDS-PAGE and transferred to nitrocellulose/polyvinylidene fluoride membranes. The membranes were probed with polyclonal antibodies to MDA-7, Bcl-2, Bax and total β-actin, and blots were visualized with DAB. Statistical analysis All tests were performed at least three times. Results were expressed as mean±sd. Statistical comparisons were made using one-way ANOVA and Student's t test. Significance was set at P<0.05. Results Ad.mda-7 transduced HCC cells to express high levels of mda-7 mrna and MDA-7 protein The expression of mda-7 mrna was markedly increased after adenovirus V-mediated transduction of mda-7 compared with the control and Ad.vec groups (Fig. 1). Treatment of HCC (Hep3B) cells and human liver (L-02) cells with DMEM without FBS and Ad.vec or Ad.mda-7 (250 vp/ml) indicated that the secreted and intracellular MDA-7 proteins were expressed after infection of these cells with Ad.mda-7. The concentrations of MDA-7 protein in supernatants by ELISA assay were 130, 260, and 790 ng/l after transduction for 24, 48, and 72 hours in L-02. Meanwhile, the concentrations of MDA-7 protein in supernatants were 110, 250, and 810 ng/l in Hep3B. In Ad.mda-7-treated L-02 and Hep3B cells, MDA-7 protein concentrations increased in a time-dependent manner. Endogenous MDA-7 expression was not detected in cells treated with Ad.vec or DMEM without FBS (Fig. 2). Ad.mda-7 reduced the proliferation of HCC cells To investigate whether Ad.mda-7 can inhibit cell proliferation, Hep3B and L-02 cells were treated with DMEM without FBS and Ad.vec or Ad.mda-7 (250 vp/ml). Analysis of cells after treatment showed that Ad.mda-7 inhibited tumor cell proliferation compared with cells treated with DMEM without FBS or Ad.vec. The inhibition rate was 72%. In contrast, no growth inhibitory effects were found in L-02 cells treated with Ad.mda-7 compared with the other treatment groups (Fig. 3). These results indicated that Ad.mda-7 reduced the proliferation of HCC cells but not that of normal cells. Ad.mda-7 induced apoptosis in HCC cells as demonstrated by Hoechst staining Hoechst staining showed that the rates of apoptosis- M M bp L-02 mda-7 Hep3B mda-7 Fig. 1. mrna expression of the adenovirus-mediated mda-7 gene in hepatoma cells and human liver cells (M: Marker; 1: control; 2: Ad.vec group; 3: Ad.mda-7 group). MDA-7 Bcl-2 Bax β-actin L-02 Hep3B Fig. 2. Determination of MDA-7, Bcl-2, and Bax protein expression by Western blotting. Lane 1: control; 2: Ad.vec; 3: 24 hours after Ad.mda-7; 4: 48 hours after Ad.mda-7; 5: 72 hours after Ad.mda-7. bp Hepatobiliary Pancreat Dis Int,Vol 7,No 5 October 15,

4 Hepatobiliary & Pancreatic Diseases International The number of cells The number of cells Time (day) Control Hep3B Ad.vec Ad.mda Control Ad.vec Ad.mda-7 L Time (day) Fig. 3. Ad.mda-7 suppresses HCC cell growth. Control Ad.vec Ad.mda-7 L-02 Hep3B Fig. 4. mda-7 selectively induces apoptosis of HCC cells (Hoechst staining, original magnification 400). positive cells treated with DMEM without FBS or Ad.vec were 5.8%, and 7.1%, respectively (t=-1.76; P>0.05) (Fig. 4). Ad.mda-7-mediated apoptosis in Hep3B cells was higher than in the other groups (1000 cells counted consecutively) (F=149.12, P<0.05). In contrast, there were no apparent changes in L-02 cells; the apoptosis rates were 2.1%, 2.1% and 2.2% (F=1.86, P>0.05). These data indicated that mda-7 induced apoptosis in HCC cells. Expression of mda-7 induced apoptosis in HCC cells Annexin-V/PI staining assays with flow cytometry showed that the rate of apoptotic HCC cells increased in Ad.mda-7-infected cells compared to control and Apoptotic cells Table. mda-7 selectively kills HCC cells (FACScan) Ad.vec infected cells (F= ; P<0.05), whereas there was no change in L-02 (F=2.98; P>0.05) (Table). These results showed that Ad.mda-7 infection selectively killed HCC cells but not normal cells. Ad.mda-7 induced cell cycle block in HCC cells Ad.mda-7 induced a G2/M accumulation in Hep3B cells, and the rate in the G2/M phase was 48.29%. The Ad.mda-7 group differed from the control (2.44%) or Ad.vec groups (3.49%) (F= ; P<0.01). However, the rate of G2/M phase cells was 6.95%, 6.65% and 5.54%, respectively (F=2.31; P>0.05), only minimal accumulation was seen in L-02. These results indicated that Ad.mda-7 infection induced an increase in the G2/M population of the cell cycle in HCC cells but not in normal cells. Modulation of Bcl-2 family proteins in HCC cells following Ad.mda-7 infection To investigate further the mechanisms of cell death induced by Ad.mda-7 in HCC cells, we performed Western blotting assay in L-02 and Hep3B. Previous studies showed that Bcl-2 family proteins change in many Ad.mda-7-treated cancer cell lines and play a role in apoptosis. [13] Based on these reports, we analyzed the effect of Ad.mda-7 treatment on the expression levels of Bcl-2 and Bax. Ad.mda-7 upregulated the expression of Bax proteins and downregulated the expression of Bcl-2 proteins compared with the control or Ad.vec groups. Transduction of HCC cells with Ad.mda-7 resulted in time-dependent cell death. The results showed significant changes in the expression of Bcl-2 and Bax at 48 and 72 hours. However, we did not find increased activation of these molecular markers in Ad-mda7-treated normal cells (L-02), compared with PBS-treated or Ad.vec- treated normal cells (Fig. 2). Discussion L-02 Hep3B Control Ad.vec Ad.mda-7 Control Ad.vec Ad.mda-7 Early Late Total HCC presents as one of the most aggressive malignancies with an extremely poor prognosis. The clinical therapies for HCC include surgical resection, liver transplantation, and non-surgical 512 Hepatobiliary Pancreat Dis Int,Vol 7,No 5 October 15,2008

5 Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B treatment. [14] The recurrence rate of the former two treatment strategy is still very high. However, cancer gene therapies that transduce tumor suppressor/ apoptosis-inducing genetic elements into tumor cells by viruses or other approaches have been remarkably successful. The novel therapeutic cytokine mda-7 inhibits the growth of a large spectrum of cancer cells, without exerting side-effects on normal human epithelia or fibroblasts. [15] Transfection of this cancerspecific apoptosis-inducing gene, mda-7, represents a rational and potentially successful approach for HCC treatment. The studies in this context were designed to determine the impact of the novel gene mda-7 on growth and apoptosis induction in a HCC cell line. Preliminary experiments showed that the expression of secreted and intracellular MDA-7 proteins increased in a time-dependent manner in Ad.mda- 7-treated cells, suggesting that the enhanced killing was due to increased MDA-7 protein expression. The apoptosis-inducing effect of mda-7 is positively correlated with the duration of protein action and the protein concentration. [16] The results were significantly different between HCC cells and normal cells after expression of MDA-7 proteins and suggested that MDA-7 reduced cell proliferation and accelerated cell death in HCC cells but not in normal cells. Previous studies from several laboratories have demonstrated that mda-7 possesses growth suppressive properties in a wide variety of human cancer cell lines, without inducing harmful effects on normal cells. [17] Our studies show that mda-7 is highly selective to tumor cells, and extend the anti-tumor spectrum of mda-7, especially in HCC. In the present study, expression of MDA-7 protein by Ad.mda-7 transfection of a HCC cell line significantly suppressed cell proliferation by a pathway which was mediated via G2/M cell cycle arrest and resulted in apoptosis. But it is still unknown whether mda-7 induces G2/M cell cycle arrest directly or indirectly by alternative mechanisms. The G2/M checkpoint prevents the cell from entering mitosis. As cells from G2 approach M phase, the activity of cyclins, cyclin-dependent kinases (CDK), and CDK inhibitors are necessary in the progression. These include s, which binds to phosphorylated Cdc2- cyclin B kinase and exports it from the nucleus; [18] GADD45, which apparently binds to and dissociates the Cdc2-cyclin B kinase; [19] and p21cip1, an inhibitor of a subset of the cyclin-dependent kinases including Cdc2 (CDK1). [20] In previous studies, G2/M arrest after Ad.mda-7 infection was associated with an increased expression of one of the CDK inhibitors, p21, which is a key component for G2/M transition. [21] The mechanism of G2/M arrest induced by mda-7 in HCC cells is unknown, so further studies are required. Overexpression of the anti-apoptotic proteins Bcl-2 and Bcl-xL is a frequent occurrence in HCC development and progression. [22] In many cancer subtypes, Ad.mda-7 infection reduces the levels of anti-apoptosis proteins, including Bcl-2 and/or Bcl-xL, and enhances expression of pro-apoptosis proteins, including Bax and/or Bak, thus shifting the balance towards an apoptotic phenotype and tumor cell death. [13] To explore the potential molecular mechanism, we assessed the expression of apoptosisrelated molecules, the Bcl-2 family proteins, in vitro. The present study showed that the expression of Bcl-2 in HCC transfected with Ad.mda-7 gradually decreased with time. However, the expression of Bcl-2 did not change in L-02 cells. In addition, the increased expression of Bax that correlated with MDA-7 expression was observed in Hep3B cells treated with Ad.mda-7. Therefore, Ad.mda-7 altered the antiapoptotic/pro-apoptotic protein ratio, thus promoting hepatoma apoptosis. In conclusion, the present study provides support for future clinical applications of mda-7 in gene therapy for HCC. Overexpression of mda-7 clearly induces apoptosis and growth suppression in the HCC cell line Hep3B via downregulating the anti-apoptosis protein Bcl-2 and upregulating Bax protein, without any harmful effect on the normal liver cell line L-02. We are optimistic that with this new knowledge of mda-7, the intriguing cytokine may provide a new method of gene therapy for HCC. Funding: This work was supported by a grant from the Key Project of the China Hubei Provincial Science and Technology Department (2006AA304B52-4). Ethical approval: Not needed. Contributors: XXB and CK wrote the first draft, and CK analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. XXB is the guarantor. Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. References 1 Yao DF, Dong ZZ, Yao M. Specific molecular markers in hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int 2007;6: Jiang H, Lin JJ, Su ZZ, Goldstein NI, Fisher PB. Subtraction hybridization identifies a novel melanoma Hepatobiliary Pancreat Dis Int,Vol 7,No 5 October 15,

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