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Enhanced antitumor activity by combining an adenovirus harboring ING4 with cisplatin for hepatocarcinoma cells

The inhibitor of growth (ING) family proteins have been defined as candidate tumor suppressors. ING4 as a novel member of the ING family has potential tumor-suppressive effects. In this study, we explored the combined effect of adenovirus-mediated ING4 (Ad-ING4) gene transfer plus chemotherapy drug cisplatin (CDDP) on SMMC-7721 human hepatocarcinoma cells in vitro and in vivo, and its underlying mechanism. We demonstrated that Ad-ING4 plus CDDP induced synergistic growth inhibition, enhanced apoptosis, and had an additive effect on upregulation of Fas, Bax, Bak, cleaved Bid, cleaved caspase-8, caspase-9, caspase-3 and cleaved PARP, and on downregulation of Bcl-2 and Bcl-XL in SMMC-7721 hepatocarcinoma cells. Moreover, Ad-ING4 plus CDDP synergistically suppressed in vivo SMMC-7721 hepatocarcinoma subcutaneous (s.c.) xenografted tumor growth and reduced tumor vessel CD34 expression and microvessel density (MVD) in athymic nude mice. Most importantly, Ad-ING4 plus CDDP did not have overlapping toxicities in HL-7702 normal human liver cells and normal liver tissues of mice. The in vitro and in vivo enhanced antitumor effect elicited by Ad-ING4 plus CDDP was closely associated with the cooperative regulation of extrinsic and intrinsic apoptotic pathways and synergistic inhibition of tumor angiogenesis. Thus, our results indicate that Ad-ING4 plus CDDP is a potential combined treatment strategy for hepatocarcinoma.


Introduction
Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world, and is the third highest cause of cancer-related mortality. 1 The incidence is increasing worldwide because of the dissemination of hepatitis B and C virus infection. 2,3 Surgical resection is the mainstay of curative treatment for early HCC, but prognosis remains unsatisfactory because of frequent recurrence. 4 Liver transplantation is another potentially curative treatment for HCC, but its application is limited by the shortage of grafts. 5 For unresectable HCC, various locoregional therapies may be used to palliate symptoms and prolong survival. 6 Furthermore, conventional chemotherapy has not been shown to be effective in HCC, and there is no proven effective systemic therapy for HCC patients with metastatic disease. 7 Hence, the search for novel therapeutic modalities for HCC is of paramount importance.
Cancer gene therapy represents a new and promising therapeutic modality for cancers, which is based on the introduction of genetic material into cells to generate a curative biological effect. A variety of gene therapy-based anticancer strategies have been effective in animal tumor models, including replacement of tumor suppressor genes, selective activation of prodrugs, genetic immunotherapy and antiangiogenic actions. Inhibitor of growth 4 (ING4), a novel member of the ING tumor suppressor family, was first isolated and characterized by Shiseki et al. 8 Recently, ING4 has attracted much attention as a strong candidate tumor suppressor that has an important role in oncogenesis, DNA repair, tumor growth, angiogenesis, migration and gene transcription regulation. ING4 has a functionally conserved plant homeodomain-finger motif in the COOH-terminal region involved in chromatin remodeling and subsequent gene transcriptional regulation by its interaction with histone acetyltransferase and histone deacetylase complexes. [9][10][11] It also has a potential bipartite nuclear localization signal domain in the middle region, which is essential for nuclear localization of ING4 and its binding and functional interaction with p53. 12 Previous studies showed that ING4 was dramatically downregulated in glioblastoma, 13 head and neck carcinoma, 14 HCC, 15 melanoma 16,17 and gastric carcinoma, 18 which was closely associated with tumor grade, metastasis and prognosis. ING4 can significantly inhibit tumor cell growth and induce cell cycle alteration and apoptosis in different tumor types 8,13,16,17,[19][20][21][22] such as colorectal, glioblastoma, melanoma, hepatocellular, myeloma, lung and pancreatic carcinomas, and enhance chemosensitivity to doxorubicin and etoposide in HepG2 hepatocarcinoma cells. 19 ING4 can also suppress the activity of NF-kB and HIF-1a, leading to inhibition of tumor angiogenesis. 13,20 In addition, ING4 can suppress the loss-of-contact inhibition elicited by MYCN or MYC. 23 More recently, it has been reported that ING4 can exhibit a marked inhibitory effect on tumor cell spreading, migration and invasion. 16,21,24 Therefore, ING4 is a potent tumor suppressor that exerts its tumor-suppressive effect via multiple pathways in a variety of tumors.
Chemotherapy is one of the most conventional therapeutic strategies for human cancers. Conventional chemotherapy drugs include cisplatin, also named cisdiamminedichloroplatinum (CDDP), adriamycin and 5-fluorouracil. CDDP is deemed to be the 'penicillin of cancer drugs' because of its universal, early and effective treatment for many cancers. 25 Unlike many anticancer drugs, CDDP is an inorganic molecule with a simple structure that is often used as an attractive chemotherapy drug and broadly used for the treatment of various forms of malignant tumors. Although the mechanism has not yet been fully understood, CDDP is generally believed to kill cancer cells by binding to DNA and interfering with the cell's repair mechanism, which eventually leads to cell death. 26 Despite these merits, severe toxic side effects and drug resistance that limit its efficacy are major clinical obstacles associated with CDDP-based chemotherapy. 27,28 Thus, it is urgent to explore novel approaches to reduce drug dosage, minimize side effects and enhance therapeutic efficacy to promote the application of CDDP in cancer chemotherapy.
Combination therapy with multiple drugs or modalities such as the combined treatment of gene therapy and conventional chemotherapy (chemo-gene therapy) or radiotherapy is a common practice in the treatment of cancers, which can achieve greater therapeutic benefit and reduce the side effects and resistance to drugs. 29,30 Interestingly, the tumor suppressor ING4 can sensitize HepG2 hepatocarcinoma cells to DNA-damaging agents such as doxorubicin and etoposide, 19 suggesting that it may be used as a chemosensitive modulator in the combination therapy with chemotherapy drugs for cancers. Therefore, we hypothesized that a combination of adenovirus-mediated ING4 (Ad-ING4) gene transfer and chemotherapy drug CDDP would exhibit an enhanced antitumor activity for human cancers. To date, the therapeutic effect of the conjugation of Ad-ING4 with CDDP on human cancers has not been reported. To enhance the therapeutic efficacy and develop a novel combination therapeutic modality for HCC, in this study, we investigated the potential combined effect of Ad-ING4 plus CDDP against SMMC-7721 human hepatocarcinoma cells in vitro and in vivo in an athymic nude mouse model, and also elucidated its underlying molecular mechanism.

Adenoviruses, cell lines, reagents and mice
The Ad-ING4 and Ad-green fluorescent protein (GFP) replication-incompetent Ad5E1-and E3-deleted adenoviruses were constructed in our laboratory, 21 Cell and Molecular Biology Institute, College of Medicine, Soochow University (Suzhou, China). The QBI-293A human embryonic kidney cell line was kindly provided by Prof. Jiang Zhong, Fudan University (Shanghai, China). The SMMC-7721 and HepG2 human hepatocarcinoma cell lines and HL-7702 normal human liver cells were purchased from the American Type Culture Collection (ATCC) (Shanghai, China). The QBI-293A, SMMC-7721, HepG2 and HL-7702 cells were cultured in RPMI1640 (GIBCO, Shanghai, China) supplemented with 10% fetal bovine serum (Hyclone, Shanghai, China), respectively. Trizol was purchased from Invitrogen (Shanghai, China). The reverse transcriptase polymerase MuMLV was purchased from MBI (Shanghai, China). The polyclonal goat anti-ING4 antibody was purchased from Abcam (Shanghai, China). The CDDP was kindly provided by Dr Yehan Zhu, the first hospital affiliated to Soochow University (Suzhou, China). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit was purchased from Sigma (Shanghai, China). The Annexin V-PE/7-AAD apoptosis detection kit was purchased from BD Biosciences (Shanghai, China). The antibodies specific for Fas, Bcl-2, Bcl-X L , Bax, Bak, b-actin and CD34 were purchased from Santa Cruz (Shanghai, China). The antibodies specific for Bid, caspase-3, caspase-8, caspase-9 and PARP were purchased from Cell Signaling (Shanghai, China). The SuperEnhanced chemiluminescence detection kit was purchased from Applygen Technologies Inc. (Beijing, China). The UltraSensitiveTM SP kit was purchased from Maixin (Fuzhou, China). The mouse alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Uscn Life Science (Wuhan, China). The female athymic nude mice were purchased from Shanghai Experimental Animal Center (Shanghai, China) and maintained in the animal facility at Soochow University according to the animal research committee's guidelines of Soochow University.

Analysis of adenoviral infection efficiency and ING4 transgene expression
The recombinant replication-incompetent Ad5E1-and E3-deleted adenovirus with two independent cytomegalovirus promoters driving ING4 and GFP expression, Ad-ING4 and its control adenovirus Ad-GFP expressing GFP under the control of cytomegalovirus promoter were prepared as described previously. 21 The titer of Ad-ING4 and Ad-GFP adenoviruses was determined using the gene transfer unit (GTU) method by calculating the number of the reporter gene GFP-expressing QBI-293A cells under fluorescence microscopy within 18 h after adenoviral infection. To assess the optimal ratio of infectious adenovirus (GTU) to target cells, called multiplicity of infection (MOI), for a maximal infection and transgene expression in SMMC-7721, HepG2 and HL-7702 cells, the SMMC-7721 and HepG2 human hepatocarcinoma cells and HL-7702 normal human liver cells were infected with Ad-GFP and Ad-ING4 at various MOIs (0, 1, 10, 25, 50, 100 and 200), respectively. The adenoviral infection efficiency was examined according to GFP expression by fluorescence microscopy. Furthermore, the ING4 transgene expression mediated by adenoviral infection in SMMC-7721 and HepG2 human hepatocarcinoma cells and HL-7702 normal human liver cells was determined by reverse transcriptase-PCR and western blot analysis. Total cellular RNAs were extracted from Ad-ING4-or Ad-GFP-infected and uninfected SMMC-7721, HepG2 and HL-7702 cells using Trizol, and firststrand complementry DNA was reversely transcribed with RNA as template and oligo d(T) 18 as primer. The PCR reaction was carried out using complementry DNA as template and ING4-F (5 0 -GCGTCGACATGGATGA TGGGATGTATTTGGAAC-3 0 ) and ING4-R (5 0 -GCAA GCTTCTATTTCTTCTTCCGTTCTTGGGAG-3 0 ) as primers under the conditions 1 cycle at 94 1C (2 min) and then 72 1C (10 min) followed by 35 cycles at 94 1C (50 s), 58 1C (50 s) and 72 1C (55 s). All PCR reaction products were analyzed on 1% agarose gel electrophoresis with ethidium bromide staining. Total cellular lysates derived from Ad-ING4 or Ad-GFP-infected and uninfected SMMC-7721, HepG2 and HL-7702 cells were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a polyvinylidene difluoride membrane. The membrane was blocked by incubation with 5% (w/v) nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 1 h at 37 1C. For western blot analysis, the membrane was incubated with primary antibody polyclonal goat anti-ING4 (1:1000) in blocking solution for 1 h at 37 1C. The membrane was washed with TBST and incubated with peroxidase horseradish peroxidase-conjugated secondary antibody rabbit anti-goat IgG (1:3000) in blocking solution for another 1 h at 37 1C. The membrane was then washed and developed by use of a SuperEnhanced chemiluminescence detection kit. The protein bands were visualized after exposure of the membranes to Kodak X-ray film.

Cell viability assay
To assess the cytotoxic effect of Ad-ING4 or CDDP alone on SMMC-7721, HepG2 and HL-7702 cells, the SMMC-7721 and HepG2 human hepatocarcinoma cells and HL-7702 normal human liver cells were dispensed in a 96-well culture plate at a density of 1 Â 10 4 cells per well, respectively, and incubated at 37 1C. After 24-h incubation, the SMMC-7721, HepG2 and HL-7702 cells were treated with Ad-ING4 at the optimal MOIs, 50 (SMMC-7721), 25 (HepG2) and 25 (HL-7702), or various doses of CDDP (0.75, 1.5, 3, 6 or 12 mg ml À1 ) for 3 days, respectively. The medium containing Ad-GFP was used as a blank adenovirus control, whereas the medium containing phosphate-buffered saline (PBS) without Ad-ING4, Ad-GFP or CDDP was used as a cell control (PBS control). At 3 days after treatment, the SMMC-7721, HepG2 and HL-7702 cells were incubated with 10 ml MTT (5 mg ml À1 ) for 4 h at 37 1C. The formazan crystals in the cells were solubilized with stop solution and the plate was then read at 570 nm using a Microplate Reader Model 550 (BIO-RAD, Shanghai, China). To further determine the combined effect of Ad-ING4 and CDDP on hepatocarcinoma cells, the SMMC-7721 hepatocarcinoma cells, a relatively non-sensitive hepatocarcinoma cell line to chemotherapy drug CDDP, and an optimal dose of 1.5 mg ml À1 of CDDP were employed in the in vitro combination therapy studies. Briefly, the SMMC-7721 human hepatocarcinoma cells were dispensed in a 96-well culture plate at a density of 1 Â Flow-cytometric analysis of apoptosis Ad-ING4 plus CDDP-induced apoptosis in SMMC-7721 human hepatocarcinoma cells was assessed by flow cytometric analysis using Annexin V-PE/7-AAD double staining following the manufacturer's instructions. Briefly, the SMMC-7721 tumor cells (1 Â 10 6 ) were treated with Ad-ING4 (50 MOI), Ad-GFP (50 MOI) or CDDP (1.5 mg ml À1 ) alone, Ad-ING4 (50 MOI) plus CDDP (1.5 mg ml À1 ), or Ad-GFP (50 MOI) plus CDDP (1.5 mg ml À1 ). The medium containing PBS without Ad-ING4, Ad-GFP, CDDP, Ad-ING4 plus CDDP, or Ad-GFP plus CDDP was used as a cell control (PBS control). After 24 h, the treated and untreated SMMC-7721 tumor cells were collected and washed with cold PBS, respectively. Then the cells (1 Â 10 5 ) were suspended in 100 ml of 1Â binding buffer and incubated with 5 ml Annexin V-PE and 5 ml 7-AAD at room temperature. After 15-min incubation, 400 ml of 1 Â binding buffer was added and the apoptotic cells were analyzed by flow cytometry.

Western blot analysis
The SMMC-7721 human hepatocarcinoma cells were treated with Ad-ING4 (50 MOI), Ad-GFP (50 MOI) or CDDP (1.5 mg ml À1 ) alone, Ad-ING4 (50 MOI) plus CDDP (1.5 mg ml À1 ) or Ad-GFP (50 MOI) plus CDDP . Total cellular proteins were extracted and the protein concentration was determined by BCA protein assay using a spectrophotometer. To elucidate the molecular mechanism involved in Ad-ING4 plus CDDP-induced enhancement of growth inhibition and apoptosis in SMMC-7721 hepatocarcinoma cells, the cellular lysates containing 100 mg of total protein were subjected to western blot analysis as described above using primary antibodies specific for Fas, caspase-8, Bid, Bcl-2, Bcl-X L , Bax, Bak, caspase-9, caspase-3 and PARP, respectively.

Animal studies
Female athymic nude mice were subcutaneously (s.c.) inoculated on the armpits of their right anterior limbs with 2 Â 10 6 SMMC-7721 human hepatocarcinoma cells, and then monitored daily for tumor growth. Tumor volume was measured with a caliper and calculated by the formula, tumor size ¼ ab 2 /2, where a is the larger and b is the smaller of the two dimensions. When the tumors grew up to a mean tumor volume of around 0.2-0.3 cm 3 , SMMC-7721 human hepatocarcinoma s.c. xenografted tumor-bearing mice were subjected to in vivo CDDP-dosefinding study and Ad-ING4 plus CDDP combination treatment, respectively. In the CDDP-dose study, tumorbearing mice (three mice each group) were intraperitoneally (i.p.) injected with various doses of CDDP (1, 3, 5 or 10 mg kg À1 body weight) or PBS every other day for a total of five times. Tumor volume and body weight were monitored over time. According to the preliminary CDDP-dose pilot study, an optimal dose of 1 mg kg À1 of CDDP was selected for the subsequent Ad-ING4 plus CDDP in vivo combination treatment. In the in vivo combination treatment, tumor-bearing mice were randomly divided into six groups (six mice each group) and intratumorally injected with Ad-ING4 (1 Â 10 8 GTU), Ad-GFP (1 Â 10 8 GTU) or PBS alone, or i.p. injected with CDDP (1 mg kg À1 ) alone or plus Ad-ING4 (1 Â 10 8 GTU) or Ad-GFP (1 Â 10 8 GTU) by intratumoral injection every other day for a total of five times, respectively. Tumor progression and body weight were monitored daily. In addition, the tumor-bearing mice were killed 3 weeks after single or combination treatment, and the SMMC-7721 hepatocarcinoma s.c. xenografted tumors and normal liver tissues of mice were removed, weighted, fixed by 10% neutral formalin and embedded in paraffin for hematoxylin and eosin staining and immunohistochemistry analysis, respectively. On the day of euthanization, blood samples were also collected by tail veil breeding for measurement of liver serum enzymes such as ALT and AST by ELISA using mouse ALT and AST ELISA kits according to the manufacturer's instructions.

CD34 immunostaining and microvessel density (MVD) counting
The expression of CD34 in SMMC-7721 human hepatocarcinoma xenografted tumors was tested by immunohistochemistry analysis using an UltraSensitiveTM SP kit. The presence of buffy or brown diaminobenzidine precipitates is indicative of positive reactivity for CD34. MVD, detected by immunostaining for CD34, was determined as previously described by Weidner et al. 31 Any endothelial cell cluster immunoreactive for CD34 clearly separated from adjacent microvessels was considered as a single countable vessel. Each value represents the number of vessels counted at a high-power view ( Â 400) by microscopy. The mean value represents the average number derived from five high-power fields of each case.

Calculation of synergistic indexes
The interactive effects of Ad-ING4 and CDDP were evaluated by combination index (CI) calculated by the formula as described previously, 32

Statistical analysis
All data are presented as the mean ± s.d. The significance of the difference between groups was evaluated by oneway and two-way repeated-measures analysis of variance (ANOVA), multiple comparisons and Student's t test with SPSS 10.0 software. A value of Po0.05 was considered statistically significant.

Ad-ING4 transgene expression
To determine the optimal MOI for a maximal infection  (Figure 2b). Furthermore, SMMC-7721 and HepG2 hepatocarcinoma cells and HL-7702 normal liver cells are different in their sensitivity to chemotherapy drug CDDP. The moderate reduction in SMMC-7721 hepatocarcinoma cell viability was observed at 1.5 mg ml À1 of CDDP (19% reduction), and significant reduction was observed starting at 3 mg ml À1 (37% reduction), with maximum reduction observed at 12 mg ml À1 (69% reduction). Unexpectedly, the significant reduction in HepG2 hepatocarcinoma cell viability was observed starting at the lowest concentration of CDDP (0.75 mg ml À1 ) (49% reduction), with maximum reduction observed at 12 mg ml À1 (89% reduction). However, the significant reduction in HL-7702 normal liver cell viability was observed at 6 (29% reduction) and 12 mg ml À1 (47% reduction), with slight reduction observed at 3 mg ml À1 (14% reduction). These results indicated that HepG2 hepatocarcinoma cells are much more sensitive to chemotherapy drug CDDP than SMMC-7721 hepatocarcinoma cells, and only a higher concentration of CDDP ranging from 6 to 12 mg ml À1 exhibits significant cytotoxic effect on HL-7702 normal liver cells. Therefore, we selected the relatively non-sensitive hepatocarcinoma cell line to CDDP, SMMC-7721 and the optimal dose of 1.5 mg ml À1 of CDDP in the in vitro combination treatment to minimize toxicity to normal cells and elicit only modest antitumor activity when used as single therapy, while leaving a window for observation of the additive or synergistic effects of Ad-ING4 plus CDDP combination therapy.  To determine the optimal dose of CDDP employed in vivo in an athymic nude mouse animal model combination treatment, a dose-finding study was performed in which SMMC-7721 human hepatocarcinoma s.c. xenografted tumor-bearing mice (three mice each group) were treated with a range of CDDPs (1, 3, 5 or 10 mg kg À1 ) by i.p. injection. Tumor volume and body weight were monitored over time. Mice treated with higher doses of CDDP (5 or 10 mg kg À1 ) resulted in a significant loss of body weight and had to be euthanized within a week, whereas those treated with 1 or 3 mg kg À1 of CDDP showed no significant body weight losses with only minimal toxicity (data not shown). Compared with the PBS-treated control group, tumor progression was significantly retarded in the mice treated with 3 mg kg À1 of CDDP, but moderately retarded in those treated with 1 mg kg À1 of CDDP (date not shown). Therefore, an optimal dose of 1 mg kg À1 of CDDP was selected for the Ad-ING4 plus CDDP in vivo combination treatment. To further explore whether the combination of

Discussion
Recent studies have shown that ING4 as a novel tumor suppressor is involved in a variety of processes including oncogenesis, cell cycle control, apoptosis, DNA repair, angiogenesis, migration and gene transcription regulation. ING4 can significantly inhibit tumor cell growth and induce cell cycle alteration and apoptosis in a variety of tumors. ING4 can also suppress brain-tumor angiogenesis by inhibiting the activity of NF-kB leading to transcriptional repression of NF-kB-responsive genes such as IL-6, IL-8 and Cox-2 by physically interacting with the p65 subunit of NF-kB. 13 Nozell et al. 33 further confirmed that ING4 can specifically regulate the activity Chemotherapy is one of the commonly used strategies in HCC treatment, especially for unresectable patients. Conventional chemotherapy drugs often have severe side effects that limit their efficacy. Moreover, tumor cells are able to acquire resistance to chemotherapy. The increased dose of chemotherapy drug that is necessary to overcome resistance of tumor cells, even a small increase, may result in severe cytotoxicity to normal cells. Therefore, it is urgent to explore novel approaches to broaden the application of the chemotherapy drug in cancer therapy. To enhance therapeutic effect, eliminate drug resistance and reduce side effects, several conjugated strategies such as the combined treatment of gene therapy and conventional chemotherapy (chemo-gene therapy) have been applied successfully in cancer chemotherapy. Our previous studies demonstrated that Ad-ING4 gene transfer can induce growth inhibition and apoptosis in human lung and pancreatic carcinoma cells. 21,22 A recent study has shown that ING4 can enhance chemosensitivity to doxorubicin and etoposide in HepG2 hepatocarcinoma cells, 19 suggesting that it may be used as a chemosensitive modulator in the combination therapy with chemotherapy Ad-ING4 plus cisplatin exerts enhanced antitumor effect Y Xie et al drugs. However, the combined effect of Ad-ING4 gene therapy and CDDP, an attractive chemotherapy drug, on human cancers, is still unknown. Therefore, in this study, we were interested in addressing the therapeutic effect of chemo-gene therapy by combining Ad-ING4 with CDDP on human hepatocarcinoma cells in vitro and in vivo in an athymic nude mouse model and its potential mechanism.
Before initiating in vitro combination therapy, we first assessed the cytotoxic effect of Ad-ING4 or CDDP alone on SMMC-7721 and HepG2 hepatocarcinoma cells and HL-7702 normal liver cells. We found that Ad-ING4 significantly inhibited SMMC-7721 human hepatocarcinoma cell growth, whereas it moderately inhibited HepG2 hepatocarcinoma cell growth. However, Ad-ING4 had only a minimal effect on HL-7702 normal human liver cells, indicating that transgene ING4 overexpression exerts selective tumor-killing activity in hepatocarcinoma cells. In addition, chemotherapy drug CDDP induced a dose-dependent growth inhibition in SMMC-7721 and HepG2 hepatocarcinoma cells or HL-7702 normal liver cells. Furthermore, the HepG2 hepatocarcinoma cells were more sensitive to CDDP than SMMC-7721 hepatocarcinoma cells. Different tumor cell lines are different in their susceptibility to drugs. In this study, the HepG2 and SMMC-7721 hepatocarcinoma cell lines are both adherent and epithelial-like cells, but from different origins. The former was established from the tumor tissue of a 15-year-old Argentine boy with HCC in 1975, which was reported to produce a variety of proteins such as alphafetoprotein, albumin, alpha2-macroglobulin, alpha1-antitrypsin and transferrin. The latter was established from the tumor tissue of a 56-year-old Chinese man with HCC in 1980. The different origins and different biological activities may be one of the reasons for the different susceptibility to Ad-ING4 and CDDP. In addition, higher concentration of CDDP (6 or 12 mg ml À1 ) exhibited significant growth-suppressive effect on HL-7702 normal liver cells, while 3 mg ml À1 of CDDP only induced slight cytotoxicity. To minimize toxicity to normal cells and produce only modest antitumor efficacy when used as monotherapy, while leaving a window for observation of additive or synergistic effects of Ad-ING4 plus CDDP combination therapy, we selected the relatively nonsensitive hepatocarcinoma cell line to CDDP, SMMC-7721, and the optimal dose of 1.5 mg ml À1 of CDDP in the in vitro combination treatment. We demonstrated that Ad-ING4 plus CDDP combination treatment selectively exerted in vitro synergistic therapeutic activity in SMMC-7721 hepatocarcinoma cells, but not in HL-7702 normal liver cells. Moreover, treatment of SMMC-7721 hepatocarcinoma cells with Ad-ING4 plus CDDP resulted in enhancement of apoptosis. To further address the underlying mechanism involved in Ad-ING4 plus CDDPinduced enhanced antitumor activity, the expression of apoptosis-related proteins such as Fas, caspase-8, Bid, Bcl-2, Bcl-X L , Bax, Bak, caspase-9, caspase-3 and PARP in different treatments of SMMC-7721 hepatocarcinoma cells was assessed by western blot analysis. Fas as an important apoptotic marker has been shown to regulate FasL-Fas extrinsic apoptotic pathway. 35 Associated with the increase in Fas expression is the cleavage of Caspase-8 and Bid, downstream targets of Fas, that have a important role in the extrinsic apoptotic pathway. 35 Moreover, caspase-8-mediated cleavage of Bid, which translocates to the mitochondria, also results in the activation of intrinsic apoptotic pathway. 36,37 Bcl-2 protein family is known to be a key regulator of apoptosis and an important determinant of cell fate. 38 The ratio of anti-to pro-apoptotic molecules (Bcl-2, Bcl-X L ; Bax, Bak) constitutes a rheostat that sets the threshold of susceptibility to apoptosis for the intrinsic apoptotic pathway, which promotes pore formation in the mitochondrial outer membrane leading to loss of mitochondrial integrity and the release into the cytosol of cytochrome c followed by the activation of caspase-9, caspase-3 and cleavage of downstream substrate PARP. 38,39 Western blot results showed that Ad-ING4 plus CDDP combination treatment elicited an additive effect on the altered expression of apoptosis-related proteins, leading to the cooperative activation of extrinsic and intrinsic apoptotic pathways, which may closely account for the Ad-ING4 plus CDDP-mediated synergistic growth inhibition and apoptosis in SMMC-7721 hepatocarcinoma cells. In the in vivo combination treatment, a CDDP-dose-finding study was also primarily performed in SMMC-7721 human hepatocarcinoma s.c. xenografted tumor-bearing athymic nude mice by i.p. injection with various doses of CDDP. Mice treated with higher CDDP doses (5 or 10 mg kg À1 ) caused significant toxicity, while those treated with 1 or 3 mg kg À1 of CDDP showed only minimal toxicity. Moreover, tumor progression was significantly retarded in the mice treated with 3 mg kg À1 of CDDP, but moderately retarded in those treated with 1 mg kg À1 of CDDP. Therefore, an optimal dose of 1 mg kg À1 of CDDP was selected for the in vivo combination treatment. We demonstrated that Ad-ING4 plus CDDP also synergistically suppressed in vivo SMMC-7721 hepatocarcinoma s.c. xenografted tumor growth in an athymic nude mouse model without additive toxicity in vivo. The progressive growth and metastasis of solid tumors is dependent on the process of angiogenesis. 40,41 Tumor angiogenesis has a critical role in the development and progression of HCC. It has been shown that ING4 can suppress tumor angiogenesis by inhibiting the activity of NF-kB and HIF-1a. 13,20 It has also been demonstrated that CDDP can exert a potent antiangiogenic activity during CDDP-based cancer chemotherapy. 42,43 To explore the combined effect of Ad-ING4 plus CDDP on tumor angiogenesis in vivo, the MVD in SMMC-7721 hepatocarcinoma xenografted tumor tissues was determined by CD34 immunohistochemistry analysis. We further found that Ad-ING4 plus CDDP synergistically downregulated CD34 expression and suppressed angiogenesis in SMMC-7721 hepatocarcinoma s.c. xengrafted tumors, which may be another important mechanism involved in Ad-ING4 plus CDDP-mediated in vivo synergistic growth inhibition of SMMC-7721 hepatocarcinoma xenografted tumor in an athymic nude mouse model. Taken together, Ad-ING4 plus CDDP combined treatment resulted in in vitro and in vivo synergistic growth inhibition, enhanced apoptosis, had an additive effect on upregulation of Fas, Bax, Bak, cleaved Bid, cleaved caspase-8, caspase-9, caspase-3 and cleaved PARP, and on downregulation of Bcl-2 and Bcl-X L in SMMC-7721 hepatocarcinoma cells, and synergistically reduced CD34 expression and MVD in SMMC-7721 hepatocarcinoma s.c. xenografted tumors. Most importantly, the combination of Ad-ING4 with CDDP did not have overlapping toxicities in in vitro HL-7702 normal liver cells and in vivo normal liver tissues of mice. The enhanced antitumor effect in vitro and in vivo elicited by Ad-ING4 plus CDDP was closely associated with the increased Fas expression and cleavage of Caspase-8 and Bid, and decrease in the ratio of anti to pro-apoptotic molecules (Bcl-2, Bcl-X L ; Bax, Bak) followed by activation of Caspase-9 leading to Caspase-3 activation and apoptosis by cooperatively regulating extrinsic and intrinsic apoptotic pathways, and the in vivo synergistic inhibition of tumor angiogenesis. Thus, our results indicate that Ad-ING4 plus CDDP is a potential combined treatment strategy for hepatocarcinoma.

Conflict of interest
The authors declare no conflict of interest.