Treatment of hepatocellular carcinoma (HCC) cells with butyrate can induce apoptosis irrespective of hepatitis B virus integration.No information is available, however, regarding the effect of butyrate on HCC in the presence of hepatitis C virus (HCV) because some HCV proteins can regulate cell survival. By gene transfer, we found that HCV core enhances but HCV NS5A antagonizes sodium phenylbutyrate (NaPB)-induced apoptosis in HCC cells, which is independent of p53. We then chose the p53-negative Hep3B HCC cell to investigate the mechanism of anti-apoptosis mediated by NS5A. In the NaPB-treated Hep3B cells without NS5A expression, induction of apoptosis was associated with Bax redistribution from the cytosol to the nucleus interior and subsequently, to a nuclear membrane-bound form. In the NS5A expressing Hep3B cells, NaPB treatment also triggered relocalization of both Bax and NS5A from the cytosol to the nucleus interior but Bax retained inside the nucleus and did not finally move to the nuclear membrane. Using double immunofluorescence and coimmunoprecipitation, we demonstrated that NS5A co-localizes and interacts with Bax in the nucleus. The HCV NS5A protein was further found to contain Bcl-2 homology domains (BH3, BH1 and BH2). Additional studies using deleted NS5A constructs were carried out to determine whether the BH2 domain or nuclear localization signal (NLS) in NS5A is required for interaction with Bax in the nucleus or inhibition of apoptosis. NS5A with deletion of both BH2 domain and NLS localized in the cytoplasm, dissociated with Bax, and lost anti-apoptosis activity during NaPB treatment. In contrast, NS5A with intact BH domains except NLS still bound directly to Bax in the perinuclear region or the nucleus, but showed less association with Bax in the nucleus and lower effect in apoptosis inhibition than full-length NS5A. These results suggest that HCV NS5A as a Bcl-2 homologue interacts with Bax to protect p53-negative HCC cells from NaPB-induced apoptosis. © 2003 Wiley-Liss, Inc. Key words: hepatocellular carcinoma; hepatitis C virus; NS5A; Bax; phenylbutyrateThe majority of patients with hepatocellular carcinoma (HCC) are associated with chronic hepatitis B virus (HBV, a DNA virus) or hepatitis C virus (HCV, a RNA virus) infection. 1 They usually present with inoperable or unresectable diseases accompanied by various degrees of liver function impairment. 2 The most currently active anti-cancer drugs are not only ineffective in inhibiting HCC but also toxic to the liver. 3 Identification of effective drugs with less hepatic toxicity for advanced HCC remains the challenging areas of study. Experimental studies have shown that sodium butyrate, a low toxic histone deacetylase inhibitor, can remodel chromatins to epigenetically activate or repress multiple genes to suppress growth and induce apoptosis in HCC cells in vitro and in vivo, which is irrespective of the status of p53 mutation and HBV integration. 4 No information is available so far, however, regarding the effect of butyrate on HCC in th...

Purpose: To investigate the clinical benefit of additional radiotherapy to patients with unresectable hepatocellular carcinoma treated with transcatheter arterial chemoembolization (TACE) and the molecular effects of radiation on gene expression in hepatoma cells. Experimental Design: Between August 1996 and August 2003, 276 and 64 patients with American Joint Committee on Cancer stage T 3 N 0 M 0 hepatocellular carcinoma receiving TACE alone andTACE followed by three-dimensional conformal radiotherapy, respectively, at our institution were studied. Clinical outcome and pattern of failure were analyzed for the association of survival benefit with radiotherapy. The molecular effects of radiotherapy were studied in vitro and in vivo using human hepatoma cells with different p53 mutation and hepatitis B virus infection status. Results: Median follow-up and survival time in theTACE alone and TACE + radiotherapy groups were 39 and 19 months, and 51and 17 months, respectively. Additional radiotherapy toTACE did not improve overall survival (P = 0.65). However, different failure patterns were noted afterTACE and after radiotherapy. Although all irradiated tumors regressed substantially, radiotherapy rapidly enhanced both intrahepatic and extrahepatic tumor progression outside the radiotherapy treatment field in a significant portion of patients, which offset the benefit of radiotherapy on overall survival. In molecular analysis of the radiation effects on human hepatoma cells, radiotherapy rapidly induced p53-independent transcriptional up-regulation of vascular endothelial growth factor (VEGF), increased VEGF secretion in a dose-, time-, and cell type^dependent manner, and promoted hepatoma cell growth in vivo with enhanced intratumor angiogenesis, which correlated well with elevated levels of serumVEGF. Conclusions: Radiotherapy to eradicate a primary hepatocellular carcinoma might result in the outgrowth of previously dormant microtumors not included in the radiotherapy treatment field. Radiotherapy-inducedVEGF could be a paracrine proliferative stimulus.Therapeutic implications of the study justify the combination of three-dimensional conformal radiotherapy with anti-VEGF angiogenic modalities for the treatment of unresectable hepatocellular carcinoma to reduce relapses.

In addition to genetic changes, epigenetic aberrations also play important roles in radiation- and chemical-induced disorders and carcinogenesis. The present study investigated whether epigenetic therapy with a histone deacetylase (HDAC) inhibitor has dual benefits for radiation-induced oral mucositis and chemical-induced oral carcinogenesis, which should be treated at the same time. The HDAC inhibitor phenylbutyrate was first tested to determine if it influences DNA damage repair and survival in irradiated normal cells in vitro by investigating the patterns and dynamics of phospho-gammaH2AX foci, Rad51 foci and phospho-gammaH2AX/Rad51 colocalization and using the comet and clonogenic assays. Oral mucositis or carcinogenesis was induced in hamsters using radiation or 7,12-dimethylbenz[a]anthracene (DMBA) irritation to the cheek pouch. The ability of phenylbutyrate formed in proper carriers to prevent radiation-induced oral mucositis and inhibit chemical-induced oral carcinogenesis was assessed. The treated or untreated irradiated or DMBA-irritated oral tissues or mucosal epithelia were subjected to the studies of histology, immunohistochemistry, gene expression, comet assay, HDAC activity or oxidative stress. We found that phenylbutyrate promoted DNA repair and survival in normal cells after radiation. Compared with blank or vehicle-treated hamsters, the irradiated mucosa treated with phenylbutyrate had significantly lower oxidative stress and tumor necrosis factor-alpha expression and less severe oral mucositis of a shorter duration. A reduction of the oral tumor incidence, burden and progression by phenylbutyrate correlated with the suppression of oncomiRs and Rad51 overexpression, the upregulation of differentiation markers and the decrease of intracellular HDAC activity and oxidative stress during DMBA-induced oral carcinogenesis. Thus, epigenetic therapy using the HDAC inhibitor as an adjuvant to radiotherapy for chemical-induced oral cancer may provide a promising strategy combining the prevention of radiation-induced oral mucositis and the inhibition of oral carcinogenesis.

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