Hepatocellular carcinoma (HCC) is one of the most common cancerous diseases worldwide. The annual occurrences exceed one million peoples affected. Currently, the treatment modalities for HCC include surgical resection, trans-arterial embolization (TAE) and chemotherapy. However, these modalities are not completely effective, underscoring the need for development of novel therapeutic approaches. PTEN, a tumor suppressor that antagonizes the PI3K pathway, is frequently mutated or deleted in various human cancers. Loss of PTEN occurs in 40-50% of surgical resected HCC samples and predicts poor prognosis for HCC patients, suggesting PTEN restoration may constitute a treatment alternative for HCC. Since PTEN increased ethanol-induced cytotoxicity in hepatoma cells, PTEN gene delivery may serve as an adjuvant therapy in conjunction with ethanol TAE for HCC. In the present study, we evaluated the efficacy of PTEN gene therapy and its combination with ethanol in a syngenic Novikoff hepatoma model by implantation of N1-S1 cells into livers of Sprague Dawley rats. Adenovirus encoding PTEN (Ad-PTEN) or green fluorescent protein (Ad-GFP) was generated for gene delivery studies. The optimal condition for adenovirus vectors to infect N1-S1 cells was determined at multiplicity of infection (MOI) of 100-200. Infection of N1-S1 cells with Ad-PTEN, but not Ad-GFP, increased PTEN levels and led to 40-50% inhibition of cell proliferation via cell cycle arrest. Besides, the half maximal -inhibitory concentration (IC50) for ethanol in N1-S1 cells was determined at 6%. Combination with PTEN gene delivery further augmented the cytotoxicity of ethanol in N1-S1 cells from 40% to 70% inhibition. To evaluate the prevention efficacy of PTEN gene delivery, N1-S1 cells were infected with adenovirus vectors then implanted into livers of Sprague-Dawley rats to induce Novikoff hepatoma. Injection of PBS- or Ad-GFP-treated N1-S1 cells led to large hepatoma (with an average size of 3-4 cm) with tumor incidence of 80-90%. In contrast, injection of Ad-PTEN-infected N1-S1 cells only induced one hepatoma (with size of 0.1 cm) in six rats, suggesting that pretreatment with PTEN gene delivery effectively abolished the tumorigenic potential of N1-S1 hepatoma cells in vivo. In summary, these results validate the feasibility of PTEN gene delivery as a new promising therapeutic strategy for the treatment of orthotopic hepatoma in immune-competent rats.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. Current therapeutic approaches for HCC including surgical resection and trans-arterial embolization (TAE) remain largely ineffective, underscoring the need for development of novel therapeutic strategies. Because HCC is high vascularized, continuous administration angiogenesis inhibitor using gene therapy approach may facilitate long-term blockade tumor vasculature, thereby perturbing the growth of HCC. Vasostatin 112 (VS112) encodes an alternatively spliced fragment of angiogenesis inhibitor vasostatin, which encompasses residues 1-64 and 133-180 of calreticulin. In this study, recombinant adenovirus encoding VS112 (Ad-VS112) was generated to evaluate its potential for suppression of orthotopic Novikoff hepatoma in syngenic Sprague-Dawley (SD) rats. Adenovirus-mediated VS112 overexpression significantly inhibited the migration and tube formation of endothelial cells, indicating the anti-angiogenic potency of VS112 gene delivery. However, VS112 overexpression had no influence on the viability of N1-S1 Novikoff hepatoma cells. To investigate the prophylactic effect of VS112 expression on hepatoma growth, N1-S1 cells were infected with Ad-VS112 or adenovirus encoding green fluorescent protein (Ad-GFP) then implanted into the liver of SD rats. After 14 days, rats implanted with VS112-expressing showed significantly reduced incidence and size of hepatoma compared with those implanted with Ad-GFP-infected cells. To investigate the therapeutic efficacy of VS112 gene delivery, the SD rats were implanted with N1-S1 cells on day 0, treated with adenovirus vectors (2 x 1010 plaques forming units) via intravenous route on day 1, then sacrificed on day 14 to monitor hepatoma growth. By measuring tumor weight, it was found that Ad-VS112-treated rats exhibited significantly decreased tumor burden compared with control groups, which was in accordance with their lower serum GOT level. Histological analysis revealed a significant reduction of vWF-positive blood vessels in Ad-VS112-treated tumors, which was accompanied with a decrease in Ki-67-positive proliferating cells and an increase in TUNEL-positive apoptotic cells. Moreover, the expression of pro-inflammatory nuclear factor kappa B (NF£eB) and cyclooxgenase II (COXII) was also effectively attenuated in Ad-VS112-treated hepatoma. In conclusion, prior or post VS112 gene delivery potently suppresses the growth of orthotopic hepatoma,thereby holding promises for future treatment of HCC.