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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Hepatocellular carcinoma (HCC) is an aggressive cancer with a poor prognosis. The specific cellular gene alterations responsible for hepatocarcinogenesis are not well known. Previous works showed that loss of TIP30, also called CC3, a putative tumor suppressor, increased the incidence of hepatocellular carcinoma in mice, and some clinical samples of human HCC tissues had aberrant expression of TIP30. Here, we report that the introduction of TIP30 by an adenovirus vector into HCC cell lines that had decreased expressions of TIP30 inhibited cell proliferation, decreased anchorage-dependent growth, suppressed invasion through the extracellular matrix, and inhibited tumorigenesis in nude mice. Moreover, exogenous expression of Tip30 sensitized HCC cells to cytotoxic drugs and to apoptosis induced by tumor necrosis factor–related ligands in vitro. Ectopic expression of TIP30 in HCC cells enhanced p53 expression and decreased Bcl-2/Bcl-xL expression. Treatment of nude mice bearing subcutaneously established HCC tumors with a combination of an adenovirus expressing TIP30 and the cytotoxic drug 5-fluorouracil completely suppressed tumor growth and prolonged survival. In conclusion, TIP30 may play an important role in the suppression of hepatocarcinogenesis by acting as a tumor suppressor. Overexpression of TIP30 might be a promising candidate as a treatment for HCC that would increase sensitivity to chemotherapeutic drugs. (HEPATOLOGY 2006;44:205–215.)

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, especially in Asia and Africa. Infection by hepatitis B and C viruses, exposure to aflatoxin B1, and cirrhosis of any etiology are considered the major risk factors for the development of HCC.1 The ability to diagnose and treat HCC is limited. To date, surgery is offered as the main treatment, but improved approaches to long-term survival are urgently needed. The poor prognosis of HCC is largely a result of high recurrence after surgery and of resistance to chemotherapy. Therefore, it is necessary to find new clues to understand hepatocarcinogenesis and to explore new targets for the diagnosis of HCC and the development of effective therapeutic strategies.

TIP30, also called CC3 or HTIP2, is a putative tumor suppressor. It was initially identified by a differential display analysis of mRNA from the highly metastatic human variant small cell lung carcinoma (v-SCLC) versus less metastatic classic small cell lung carcinoma (c-SCLC) cell lines.2 Its expression has been detected in many human normal tissues; however, in some tumor types such as melanoma, breast cancer, neuroblastoma, glioblastoma, colon cancer, and hepatocellular carcinoma, its expression was found to be decreased.2–6 Studies of Tip30-deficient mice revealed a high incidence of hepatocellular carcinoma and other tumors with a relatively long latency.6 Expression of TIP30 was reduced in about 33% of surgical specimens from HCC patients. Some of these carcinomas harbored missense mutations in exon 3 of the Tip30 gene, which caused instability or abnormal cellular distributions of the TIP30 protein.6 These data suggest that TIP30 may function as a tumor suppressor and play an important role in the suppression of hepatocarcinogenesis.

The antitumor effect of TIP30 might be ascribed partly to its proapoptotic property.2, 7–9 The introduction of TIP30 into v-SCLC and other tumor cell lines up-regulated expression of a subset of proapoptotic genes and angiogenic inhibitors and down-regulated expression of angiogenic stimulators.3, 8 Thus, TIP30 may suppress tumor growth and metastasis through regulating expression of genes involved in apoptosis and metastasis. Recently, TIP30 was found to interact with coactivator independent of AF-2 function (CIA) and to negatively regulate estrogen receptor α–mediated c-myc expression.10The mechanism by which TIP30 regulates gene expression is not clear. Crystal structure analysis showed that TIP30 had a short-chain dehydrogenase reductase fold and binding specificity for NADPH, which was important for the biological activity of TIP30, including interaction with CIA.11 This suggested that TIP30 might be a metabolism-related transcription factor that serves as a sensor of metabolism to regulate transcription.12

To further explore the role of TIP30 in hepatocarcinogenesis and its potential in the treatment of HCC, we delivered the Tip30 gene into HCC cells by an adenovirus vector. We showed that ectopic expression of TIP30 by the adenovirus inhibited HCC cell proliferation, invasion through the extracellular matrix (ECM) in vitro, and tumorigenesis in nude mice. Overexpression of TIP30 sensitized HCC cells to cytotoxic drugs and to apoptosis induced by tumor necrosis factor–related ligands (TRAIL). A combination of Ad-TIP30 with 5-fluorouracil (5-FU) completely eliminated established HCC xenografts in vivo. In brief, we present evidence that TIP30 might play an important role in the suppression of hepatocarcinogenesis by acting as a tumor suppressor. Overexpression of TIP30 might be a promising candidate for being a treatment for HCC by sensitizing it to chemotherapeutic drugs.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Chemicals and Antibodies.

5-FU, etoposide (VP16), TRAIL, and cycloheximide were purchased from Calbiochem (San Diego, CA). Anti-TIP30 antibody was kindly provided by Dr. Hua Xiao (University of Nebraska Medical Center, Omaha, NE). Anticaspase-8, anticaspase-9, and anti-poly(ADP-ribose) polymerase (PARP) antibodies were purchased from R&D Systems, Inc. (Minneapolis, MN). Anti-p53, anti-Bcl-2, anti-Bcl-xL, and anti-MDM2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Culture and Transfection.

HCC cell lines HepG2, Hep3B, and PLC/PRF/5 and transformed liver cell line Chang liver were propagated at 37°C in an atmosphere containing 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. To generate an antisense p53 construct, full-length p53 cDNA, isolated from a pCMV-p53 plasmid (kindly provided by Dr. Bert Vogelstein, Johns Hopkins Oncology Center, Baltimore, MD) by BamHI digestion was subcloned into a pcDNA3 vector (Invitrogen, Carlsbad, CA) in the reverse direction. The full-length Tip30 gene (kindly provided by Dr. Hua Xiao, University of Nebraska Medical Center, Omaha, NE) was subcloned into a pcDNA3 vector in the reverse direction to generate an antisense Tip30 plasmid. HepG2 or Chang liver cells were stably transfected with pc-anti-p53 or pc-anti-Tip30 plus the pcDNA3 vector by jetPET™ cationic polymer transfection reagent (PolyPlus-transfection, France). Positive clones were selected with 800 μg/mL G418 and isolated.

Adenovirus Infection and Immunohistochemistry.

Preparation and construction of Ad-TIP30 and Ad-GFP vector, and adenovirus titration were carried out as previously described.13 HCC cells were infected with adenoviruses at multiplicities of infection (MOI) of 20. One day after infection, cells were subjected to immunohistochemistry analysis. After a reaction with the anti-TIP30 antibody, the antibody staining signals were amplified by goat anti-rabbit IgG conjugated to peroxidase labeled-dextran polymer (EnVision™ system, DAKO, Glostrup, Denmark) and visualized by 3,3′-diaminobenzidine.

Cell Proliferation and Viability Assay.

HCC cells were subjected to MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Sigma, St. Louis, MO) in order to detect cell proliferation. Alternatively, cell viability was detected using the Trypan blue staining assay.

Anchorage-Independent Growth and Cell Invasion Assay.

Cells were seeded into 0.3% Bacto-agar over a 0.6% agar bottom layer in triplicate and incubated for 2 weeks. Colonies greater than 100 μm in diameter were counted. Invasion of cells through the ECM was determined by a Cell Invasion Assay Kit (Chemicon, Temecula, CA) according to the manufacturer's instructions.

Flow Cytometry and DNA Fragmentation Analysis.

Both floating and adherent cells were harvested and resuspended with propidium iodide. The apoptotic cells (sub-G1 population) were quantified on a Becton Dickinson FACscan. The same cells were collected and lysed in DNA extract buffer [10 mmol/L Tris, 110 mmol/L EDTA (pH 8.0), 0.5% SDS], treated with 20 μ μg/mLL RNase A and proteinase K (100 μ μg/mL). DNA samples were electrophoresed in 2% agarose gel.

Real-Time PCR.

mRNA expression of p53 was determined by real-time PCR using SYBR Premix Ex Taq (TaKaRa Biotechnology Co., Ltd., Dalian, People's Republic of China). ACTIN was used as an endogenous control to normalize for differences in the amount of total RNA in each sample. Relative expression of genes was calculated and expressed as 2−ΔΔCT, as previously described.14

Western Blotting Analysis.

Total cell lysate was prepared in 1× SDS buffer. Proteins were separated by SDS-PAGE and transferred onto PDVF membranes. Membranes were then blotted with individual antibodies. Antigen-antibody complexes were visualized with the enhanced chemiluminescence reagent Supersigal (Pierce Biotechnology, Rockford, IL).

Protein Stability Assay.

To inhibit protein synthesis, cycloheximide (50 μ μg/mLL) was added to the medium 24 h after infection with Ad-TIP30 or Ad-GFP. Cell lysates were extracted at the indicated times and subjected to Western blotting analysis.

Animals.

Female Balb/c nude mice 4-6 weeks old were purchased from the Shanghai Experimental Animal Center of Chinese Academic of Sciences (Shanghai, China). Animals were placed in a pathogen-free environment and allowed to acclimate for a week before being used in the study. All procedures were performed according to institutional guidelines and conformed to the National Institutes of Health guidelines on the ethical use of animals.

Tumorigenicity Assay.

Twenty-four hours after adenovirus infection, HepG2 cells were trypsinized and resuspended in PBS. About 1 × 107 cells in a 200-μL volume were injected subcutaneously per mouse (n = 6 mice/group). Tumor size was determined weekly as [length (mm) × width (mm)2]/2. The life span of each animal was recorded.

Treatment of Mice With HCC Xenografts.

Mice were randomized into six groups (n = 6 mice/group) and injected subcutaneously with 1 × 107 HepG2 cells into the right flank of each animal. Mice were treated intratumorally with 1 × 109 plaque-forming units of Ad-TIP30 or Ad-GFP daily for 5 days. Injection of PBS was used as a blank control. One day after adenovirus injection, the mice were dosed intraperitoneally with or without 5-FU at a concentration of 10 mg/kg body weight daily for 5 days. Tumor size was monitored weekly, and the life span of each mouse was recorded.

In Vivo Apoptosis Assay.

Tumor tissue samples from mice subjected to different treatments were taken on day 30 and prepared with a routine pathological procedure. Tumor sections were deparaffinized and subjected to immunohistochemistry analysis as described in the Adenovirus Infection and Immunohistochemistry section. In situ apoptosis assay was performed with the DeadEnd colorimetric TUNEL system (Promega, Madison, WI). Apoptotic nuclei were stained dark brown.

Statistic Analysis.

Statistical analysis was performed using the Analysis ToolPack of Microsoft Excel. A two-sample Student t test assuming unequal variances was used to determine the equality of means of two samples. Results were considered statistically significant at P < .05. Life span was compared for statistic significance using the log-rank test (SAS 8.2 software). Differences in survival were considered statistically significant at P < .05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Overexpression of TIP30 Inhibited Cell Proliferation and Colony Formation in Human HCC cells.

Compared to the level of TIP30 protein in a transformed liver cell line, Chang liver cells, TIP30 expression was completely abolished in HepG2 and Hep3B cells and significantly decreased in PLC/PRF/5 cells (Fig. 1A). To further investigate the biological function of TIP30 on HCC, we infected HCC cell lines with a replication deficient adenovirus expressing the human Tip30 gene, which caused similar expression of TIP30 in the three HCC cells as detected by immunohistochemistry analysis (Fig. 1B). Ectopic expression of Tip30 strongly inhibited cell proliferations in HCC cells compared to cells infected with Ad-GFP, which had proliferation rates similar to those of the parental cells (Fig. 1C). However, HepG2 cells that contained the wild-type p53 gene were more sensitive to Ad-TIP30-induced inhibition of cell growth than Hep3B cells lacking the wild-type p53 gene and PLC/PRF/5 cells containing a mutant p53 gene. Loss of anchorage-dependent growth is a characteristic of malignantly transformed cells. In the present study, we observed that overexpression of TIP30 significantly reduced colony formation in HepG2 and Hep3B cells, but not in PLC/PRF/5 cells, compared to colony formation in Ad-GFP-infected or PBS-treated cells (Fig. 1D). Together, these results indicated that TIP30 had an inhibitory effect on the growth of human HCC cells.

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Figure 1. TIP30-inhibited cell proliferation and colony formation in human HCC cells. (A) Western blotting analysis of TIP30 expression in HCC cells. Transformed liver cell line, Chang liver, was used as the control. (B) Immunohistochemistry of HCC cells infected with Ad-TIP30 or Ad-GFP was determined by reaction with anti-TIP30 antibody. Cells positive for TIP30 expression were stained brown (original magnification ×200). (C) HCC cells were infected with Ad-TIP30 or Ad-GFP or were untreated as controls. Cell proliferation was analyzed by the MTT assay various days after infection. Data are shown as the mean ± SD of three independent experiments. (D) Growth of HCC cells in a semisolid soft agar medium. The numbers represent the mean number of colonies of three independent experiments ± SD (*P < .05).

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TIP30 Suppressed HCC Cell Invasion Through the ECM.

TIP30 was first identified as a metastasis suppressor that could suppress metastasis of v-SCLC in SCID-hu-L mice and mouse melanoma B16 cells in vivo.2, 4 In the present study, we detected the invasion of HCC cells through the ECM in vitro. Ectopic expression of Tip30 significantly suppressed invasion of HepG2 and Hep3B cells through the ECM, compared to cells treated with PBS (P < .05) 36 h after incubation with ECM (Supplementary Fig. 1; supplementary material for this article can be found on the HEPATOLOGY website [http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html]). The suppression became more effective (P < .01) after 72 hours of incubation (Supplementary Fig. 1). However, TIP30 had no obvious effect on the PLC/PRF/5 cells, which have less potential for invasion through the ECM, and on human fibroblasts, which served as a negative control and could hardly invade through ECM. Thus, functional TIP30 suppressed HCC cell invasion in vitro.

TIP30 Inhibited Tumorigenesis of HCC Cells in Nude Mice.

To determine the tumor suppression effect of TIP30 in vivo, we examined the tumorigenicity of HepG2 cells in nude mice. It took a longer time for mice injected with Ad-TIP30-infected HepG2 cells to develop tumors than for mice injected with Ad-GFP-infected or PBS-treated cells (Fig. 2A). Infection with Ad-TIP30 significantly inhibited HepG2 cell growth in nude mice compared to that with Ad-GFP infection or PBS treatment (Fig. 2B). Infection with Ad-TIP30 also significantly increased survival of tumor-bearing mice compared to that with Ad-GFP infection or PBS treatment (Fig. 2C). Our data suggested that TIP30 exerted a negative effect on regulation of tumorigenesis of HCC in vivo, confirming the role of TIP30 in tumor suppression.

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Figure 2. TIP30-inhibited tumorigenesis of HepG2 cells in nude mice. (A) Time to formation of tumors of HepG2 cells infected with Ad-TIP30 or Ad-GFP or treated with PBS as the control. (B) Survival of mice inoculated with HepG2 cells infected with either Ad-TIP30 or Ad-GFP or treated with PBS as the control. Shown are the fractions of animals alive in the different treatment groups plotted against number of days after inoculation(*P < .05, log-rank). (C) Tumor volume in the same mice as in B was monitored weekly. Data represent the mean ± SD of tumor volume derived from each group (*P < .05).

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TIP30 Enhanced p53 and Suppressed Bcl-2/Bcl-xL Expression.

Antiapoptotic Bcl-2 family members such as Bcl-2 and Bcl-xL are known to be abnormally expressed in HCC, which is related to hepatocarcinogenesis and resistance to chemotherapeutic drugs.15–18 We therefore investigated Bcl-2 and Bcl-xL expression upon TIP30 induction. Bcl-xL was reduced in both HepG2 and Hep3B cells on infection with Ad-TIP30. Bcl-2 was observed to have reduced expression in HepG2 cells and was not detectable in Hep3B cells (Fig. 3). These data indicated that TIP30 might promote apoptosis by suppressing Bcl-2/Bcl-xL expression independently of p53.

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Figure 3. TIP30-suppressed Bcl-2/Bcl-xL expression in HCC cells. Total protein was isolated from HCC cells at various times after Ad-TIP30 infection and subjected to Western blotting analysis with anti-Bcl-2 and anti-Bcl-xL antibodies.

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We further explored the role of p53 in TIP30-mediated apoptosis. The expression of p53 protein was greatly enhanced in Ad-TIP30-infected HepG2 cells (Fig. 4A). In contrast, no p53 protein could be detected in Hep3B cells, and no obvious change was detected in PLC/PRF/5 cells. We further analyzed p53 mRNA level by real-time PCR. The expression of p53 mRNA was significantly enhanced after Ad-TIP30 infection in HepG2 cells (Fig. 4B). Interestingly, the half-life of p53 protein was reduced after infection with Ad-TIP30, as measured by the cycloheximide inhibition assay (Fig. 4C). The reduction of the half-life of the p53 protein might be a result of the enhanced expression of MDM2 (Fig. 4D), which is a transcription target of p53 and functions as a ubiquitin ligase to target p53 for degradation by proteasomes.19, 20 These results suggested that TIP30 might promote apoptosis by enhancement of p53 mRNA and protein expression in HepG2 cells.

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Figure 4. TIP30-enhanced p53 expression in HCC cells. (A) Total proteins were isolated from HCC cells infected with Ad-TIP30 or Ad-GFP and mock control cells. Western blotting analysis was performed with anti-p53 and anti-TIP30 antibodies. (B) RNA was extracted from HepG2 cells infected with Ad-TIP30 or Ad-GFP and untreated cells. mRNA expression of p53 was normalized to endogenously expressed ACTIN in the same samples. Relative expression of p53 was calculated and expressed as 2−ΔΔCT (*P < .05). (C) HepG2 cells were infected with Ad-TIP30 or Ad-GFP for 24 hours. The cells were treated with cycloheximide (50 μ μg/mLL), and then total proteins were harvested at various times. Proteins were subjected to Western blotting with anti-p53 and anti-TIP30 antibodies. (D) Total proteins were harvested from HepG2 cells infected with Ad-TIP30 or Ad-GFP and untreated cells and then subjected to Western blotting with anti-MDM2 antibody.

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To explore whether Ad-TIP30-induced cell death in HepG2 was p53 dependent, we generated a HepG2 cell line stably expressing antisense p53. The reduced p53 expression was confirmed by Western blotting and real-time PCR analysis (Supplementary Fig. 2A-B). Ad-TIP30-induced cell death was significantly more attenuated in pc-anti-p53-expressing cells than in pcDNA3-vector-transfected or irrelevant pc-anti-GFP-expressing cells (Supplementary Fig. 2C). However, Ad-TIP30-induced cell death was not completely abolished. This confirmed our previous hypothesis that Ad-TIP30 might induce apoptosis through both p53-dependent and -independent pathways.

TIP30 Sensitized HCC Cells to Cytotoxic Drug-Induced Cell Death.

HCC is resistant to chemotherapeutic drugs, but ectopic expression of Tip30 has been reported to predispose v-SCLC cells to apoptosis induced by etoposide and cisplatin.2, 7 We therefore examined whether overexpression of TIP30 would sensitize HCC cells to apoptosis induced by cytotoxic drugs. Infection of Ad-TIP30 significantly enhanced inhibition of etoposide- and 5-FU-induced cell proliferation (Fig. 5A). The percentage of apoptosis of HepG2 cells treated with Ad-TIP30 combined with etoposide increased to 63%, compared to 10% and 37% in cells infected with Ad-TIP30 alone or Ad-GFP combined with etoposide, respectively (Fig. 5B). DNA fragmentation analysis showed that the combination of Ad-TIP30 with etoposide produced a stronger DNA ladder than did Ad-TIP30 or etoposide treatment alone (Fig. 5C). We further measured the activation of caspase-9 and the cleavage of PARP on treatment with various concentrations of etoposide. The activated caspase-9 and cleaved PARP could be detected even without etoposide treatment and became obvious in Ad-TIP30-infected cells at 5 μg/mL of etoposide. In contrast, the cleaved bands became detectable when the concentration of etoposide reached 10 μg/mL in Ad-GFP-infected cells (Fig. 5D).

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Figure 5. TIP30-enhanced cytotoxic drug-induced apoptosis. (A) HCC cells were infected with Ad-TIP30 or Ad-GFP, or treated with PBS as a control. Twenty-four hours later, the cells were treated with 8 μg/mL etoposide or 15 μg/mL 5-FU and then subjected to the MTT assay 48 and 72 hours after cytotoxic drug exposure (*P < .05, * *P < .01). (B) HepG2 cells were infected with Ad-TIP30 and Ad-GFP individually and then treated with 8 μg/mL etoposide 24 hours after infection. The treated cells were subjected to flow cytometric analysis for apoptotic cell by PI staining. (C) Total DNA samples from the same cells as in B were analyzed electrophoretically on 2% agarose gel and visualized by ethidium bromide. (D) HepG2 cells were infected with Ad-TIP30 or Ad-GFP; 24 hours later cells were treated with various concentrations of etoposide. Total cellular protein was isolated 24 hours after etoposide treatment and subjected to Western blotting with anti-caspase-9, anti-PARP, and anti-TIP30 antibodies.

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To exam the effect of Ad-TIP30 on normal cells expressing endogenous TIP30, Chang liver cells were infected with Ad-TIP30 and treated with etoposide. The percentage of cells undergoing apoptosis was slightly higher in Ad-TIP30-infected cells than in Ad-GFP-infected cells after etoposide stimulation; however, this difference was not statistically significant (P > .05; Supplementary Fig. 3A). To further validate Ad-TIP30-induced sensitization of HCC cells to cytotoxic drugs occurring as a result of the abnormal expression of TIP30 in HCC cells, we generated a Chang liver cell stably expressing antisense Tip30. The expression of TIP30 was confirmed by Western blotting analysis (Supplementary Fig. 3B). Disruption of TIP30 expression by antisense Tip30 made Chang liver cells less sensitive to etoposide-induced cell death, compared to pcDNA3-transfected cells; however, the difference had no statistical significance (P > .05; Supplementary Fig. 3C). Infection of anti-Tip30-expressing Chang liver cells with Ad-TIP30 increased expression of TIP30 to a level similar to that of the endogenous protein (Supplementary Fig. 3D). Hence, anti-Tip30-expressing Chang liver cells significantly enhanced etoposide-induced cell death (Supplementary Fig. 3E). Thus, Ad-TIP30 only sensitized HCC cells with abnormal TIP30 expression to cytotoxic-drug-induced cell death, but no such biological effect was observed in normal cells.

TIP30 Sensitized HCC Cells to TRAIL-Induced Cell Death.

Previous work has demonstrated that HCC cells display high resistance to TRAIL-mediated cell death.21–23 The present study showed that infection of Ad-TIP30 significantly increased TRAIL-induced cell death in HCC cells compared to in Ad-GFP-infected cells (Fig. 6A). Infection of Ad-TIP30 also enhanced activation of caspase-8 and promoted cleavage of PARP after exposure to TRAIL (Fig. 6B). Thus, TIP30 was able to sensitize HCC cells to TRAIL-induced cell death.

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Figure 6. HCC cells sensitized by TIP30 to TRAIL-induced cell death. (A) HCC cells were treated with different concentrations of TRAIL 24 hours after infection with Ad-TIP30 or Ad-GFP. Trypan-blue-staining positive cells were counted under a microscope 48 hours after TRAIL treatment. Data shown as mean ± SD of three independent experiments (*P < .05). (B) HepG2 cells infected with Ad-TIP30 or Ad-GFP were treated with various concentrations of TRAIL. Twenty-four hours after treatment, total cellular protein was isolated and subjected to Western blotting with anti-caspase-8, anti-PARP, and anti-TIP30 antibodies.

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Combination of Ad-TIP30 With 5-FU Eliminated Established HCC Xenografts in Nude Mice.

To investigate the antitumor effect of Ad-TIP30 combined with chemotherapeutic drugs in vivo, HepG2 cells were implanted subcutaneously to form tumors in immunodeficient Balb/C nude mice. Intratumoral injection of Ad-TIP30 alone significantly increased mice survival compared to injection of Ad-GFP or PBS alone (Fig. 7A). Furthermore, a combination of intratumoral injection of Ad-TIP30 with intraperitoneal injection of 5-FU resulted in a significant increase in survival compared to treatment with 5-FU alone or with Ad-GFP combined with 5-FU (Fig. 7A).

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Figure 7. Antitumor effect of Ad-TIP30 combined with 5-FU in HepG2 xenografts in mice. (A) Survival of animals in HepG2 xenografts with different treatments. Shown are the fractions of animals alive in the different treatment groups plotted against number of days after treatments (*P < .05, log-rank). (B) Tumor growth rate in animals that received the same treatment as in A. Tumor volume was measured weekly. Data are the mean ± SD of the tumor volume derived from each group (*P < .05).

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Intratumoral administration of Ad-TIP30 or intraperitoneal injection of 5-FU alone significantly inhibited tumor growth compared to treatment with Ad-GFP or PBS alone (Fig. 7B). However, this inhibition effect was limited. Notably, intratumoral administration of Ad-TIP30 in combination with systemic administration of 5-FU resulted in complete inhibition of tumor growth (Fig. 7B). The results clearly indicated that combined therapy of Ad-TIP30 and 5-FU completely eliminated HCC xenografts in vivo.

Ad-TIP30 Enhanced 5-FU-Induced Apoptosis in vivo.

Strong TIP30 expression was observed in the tumor specimens from mice that had received Ad-TIP30 and Ad-TIP30 plus 5-FU treatments as detected by immunohistochemistry analysis (Fig. 8A), a finding confirmed by Western blotting analysis (Fig. 8B). The same specimens were subjected to the in situ apoptosis TUNEL assay. Apoptotic nuclei, stained brown, were detected in tumor specimens derived from mice that had received Ad-TIP30, 5-FU, Ad-GFP plus 5-FU, and Ad-TIP30 plus 5-FU treatment (Fig. 8C). However, apoptotic cells significantly increased in tumors injected with Ad-TIP30 along with 5-FU compared to that in tumors injected with Ad-GFP or PBS plus 5-FU (Fig. 8D). These results indicate that Ad-TIP30 induced tumor cell apoptosis in vivo and that this biological effect could be greatly enhanced when combined with chemotherapeutic drugs.

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Figure 8. Detection of TIP30 expression and apoptosis in tumor sections. (A) Immunohistochemistry of the tumor sections was determined by reaction with anti-TIP30 antibody. Cells positive for TIP30 expression were stained brown (original magnification ×200). (B) Total protein was extracted from tumor tissue and subjected to Western blotting with anti-TIP30 antibody. (C) In situ TUNEL apoptosis analysis of tumor sections derived from mice receiving different treatments. The apoptotic nuclei were stained dark brown (original magnification ×400). (D) Percentage of apoptotic cells calculated by counting >100 cells from three randomly chosen fields in each section. Data are shown as mean ± SD of three independent experiments (*P < .05).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The aggressive cancer cell phenotype is the result of various genetic and epigenetic alterations leading to deregulation of the intracellular signaling pathway.24 The major molecular features of HCC include aneuploidy and chromosomal aberrations, activation of oncogenes, and inactivation of tumor-suppressor genes.25, 26 A number of tumor-suppressor genes or tumor-suppressor-like genes, including p53, Rb1, p73, mdm2, APC, β-catenin, E-cadherin, PTEN, and BRCA, have been implicated in the molecular pathogenesis of HCC.1

TIP30 is a novel tumor-suppressor gene that executes its antitumoral effect by promoting apoptosis.2, 7–9 Studies on TIP30-deficient mice suggested that abnormal expression of TIP30 was strongly related to hepatocarcinogenesis.6 To further explore the role of TIP30 in hepatocarcinogenesis, we investigated the biological effects of TIP30 in HCC cells both in vitro and in vivo. Ectopic expression of Tip30 in HCC cell lines with low expression of TIP30 greatly inhibited tumor cell proliferation, colony formation in soft agar, and evasion through ECM in vitro; suppressed tumorigenicity; inhibited growth of HCC xenografts; and prolonged survival in nude mice models. These in vitro and in vivo data strongly indicate that TIP30 acts as a tumor suppressor in suppressing hepatocarcinogenesis.

The mechanism by which TIP30 inhibits tumorigenesis is still under investigation. Here we present evidence that TIP30 may inhibit tumorigenesis by enhancing p53 expression and suppressing Bcl-2/Bcl-xL expression. A key regulator of cell homeostasis, p53 is frequently mutated in various types of human cancer.27 However, only 30% of hepatocellular carcinomas contain the p53 gene mutation (data from http://p53.curie.fr). It remains unclear why wild-type p53 fails to suppress tumor growth in 70% of hepatocellular carcinomas. Previous studies have demonstrated that the interaction with HBV X-antigen,28, 29 overexpression of MDM2 and inactivation of p14ARF might abrogate wild-type p53 activity, which contributed to hepatocarcinogenesis.30–33 Here we showed that overexpression of TIP30 elevated p53 mRNA and protein expression in HepG2 cells. The expression of MDM2, one of the transcription targets of p53, also increased. Although the half-life of the p53 protein decreased, which might have been a result of the enhanced expression of MDM2, the accumulation of p53 protein remained. This might be because the amount of p53 protein produced in the Ad-TIP30-infected cells was much more than the amount degraded. Thus, TIP30 might be an upstream regulator of p53. Deficiency of TIP30 might allow tumor cells to bypass the tumor-suppression effect of p53, thereby contributing to hepatocarcinogenesis.

The importance of Bcl-2 family members, especially Bcl-xL, in the development of HCC has been previously demonstrated.15, 16 Recent studies have suggested that TIP30 might inhibit metastasis through down-regulation of Bcl-2.2 In the present work, we found that overexpression of TIP30 suppressed Bcl-2/Bcl-xL expression in both HCC cells containing the wild-type p53 gene and those containing the null p53 gene. Thus, decreased expression of TIP30 in HCC might contribute to hepatocarcinogenesis by eliminating suppression of Bcl-2/Bcl-xL.

Overexpression of TIP30 promoted cytotoxic-drug- and TRAIL-induced cell death in HCC cells with abnormal TIP30 expression but not in Chang liver cells, which have a normal TIP30 expression. Most important, the combination of intratumoral injection of Ad-TIP30 with systemic administration of 5-FU completely eradicated established human hepatoma in nude mice. This antitumor therapeutic efficacy was achieved by enhancing tumor cell apoptosis in vivo. Given that HCC is refractory to chemotherapeutic drugs,34 our finding provides a new clue to a treatment of HCC that would work by sensitizing HCC to both cytotoxic drugs and TRAIL-mediated cell death.

In conclusion, our data demonstrated an important role for TIP30 in the suppression of hepatocarcinogenesis. The heightened sensitivity of tumor cells to cytotoxic drugs and TRAIL-mediated apoptosis by restoring TIP30 expression would provide a new avenue for further developing a therapeutic strategy for the treatment of HCC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Dr. Xiao Hua for providing the Tip30 gene and anti-TIP30 antibody, Dr. Karl Erik Hellstrom for critical review of the manuscript, and Xiaodong Li, Yan Huang, and Qirui Liu for technical support.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supplementary material for this article can be found on the H EPATOLOGY website ( http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html )

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jws-hep.21213.fig1.tif4729KSuppl Fig 1. TIP30 suppressed HCC cell invasion through ECM. HCC cells and human fibroblasts were incubated with ECM. Thirty-six (A) and 72 hours (B) latter, invasive cells were stained and counted. Data were presented as means?SD from three independent experiments. * P .05, **P .01.
jws-hep.21213.fig2.eps1242KSuppl Fig 2. TIP30-induced apoptosis was partly through p53. (A) HepG2 cells were stably transfected with pc-anti- p53or pcDNA3 vector. Total proteins were isolated and subjected to Western Blotting with anti-p53 antibody. (B) RNA was extracted from the same cells as above and subjected to real-time PCR analysis as described previously. (C) HepG2 cells stably expressing pc-anti- p53,pc-anti- GFPor pcDNA3 were infected with Ad-TIP30. The dead cells indicated by Trypan blue staining were counted under microscope at various times following infection. Data were presented as means?SD from three independent experiments. *P .01.
jws-hep.21213.fig3.tif746KSuppl Fig 3. Ad-TIP30 did not sensitize Chang liver cells to cytotoxic drug. (A) Chang liver cells were infected with Ad-TIP30 or Ad-GFP for 24 hours and then treated with etoposide at various concentrations for another 24 hours. Positive cells stained with Trypan blue were counted under microscope. Data were present as means?SD from three independent experiments. (B) Chang liver cells were stably transfected with anti-senseTip30or pcDNA3. Total proteins were isolated and subjected to Western Blotting with anti-TIP30 antibody. (C) Chang liver cells stably transfected with anti-senseTip30or pcDNA3 were treated with etoposide at various concentrations. The positive cells stained with Trypan blue were counted under microscope. Data were present as means?SD from three independent experiments. (D) Chang liver cells stably transfected with anti-senseTip30or pcDNA3 were infected with Ad-TIP30 or Ad-GFP. Total proteins were extracted and subjected to Western Blotting with anti-TIP30 antibody. (E) Chang liver cells stably transfected with anti-senseTip30were infected with Ad-TIP30 or Ad-GFP for 24 hours and then treated with etoposide at various concentrations. Trypan blue staining positive cells were counted under microscope. Data were present as means?SD from three independent experiments.*P .01.

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