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Rapid growth of residual tumors can occur as a result of their recurrence and progression. The present study aimed to investigate the expression of hypoxia inducible factor-2 subunit a (HIF-2a), vascular endothelial growth factor A (VEGFA), erythropoietin-producing hepatocellular A2 (EphA2) and angiogenesis in residual hepatocellular carcinoma (HCC), following treatment with high-intensity focused ultrasound (HIFU) ablation, in order to investigate the association between protein expression and tumor recurrence and growth. Athymic BALB/c (nu/nu) mice were subcutaneously inoculated with the HCC cell line HepG2, in order to create xenograft tumors. Approximately 30 days post-inoculation, eight mice were treated with HIFU, whereas eight mice received no treatment and acted as the control group. Residual tumor tissues were obtained from the experimental groups after one month. Levels of HIF-2a, VEGFA, EphA2 and cluster of differentiation 31 (CD31) expression was measured by immunohistochemical staining. CD31-positive vascular endothelial cells were counted to calculate microvascular density (MVD), and western blot analysis was performed to determine levels of HIF-2a, VEGFA, and EphA2 protein. It was found that the expression levels of HIF-2a, VEGFA, EphA2, and MVD proteins in residual HCC tissues were significantly higher than in the control group tissues (P<0.05). Tumor MVD was strongly correlated with VEGFA (R=0.957, P<0.01) and EphA2 (R=0.993, P<0.01) protein expression levels. Furthermore, there was a significant positive correlation between HIF-2a and EphA2 expression (R=0.991, P<0.01). The correlation between VEGFA and EphA2 expression was also positive (R=0.985, P<0.01). These data suggest that overexpression of HIF-2a, VEGFA and EphA2 is related to angiogenesis in residual HCC following HIFU ablation, potentially via their association with key mediators of recurrence. [ABSTRACT FROM AUTHOR]

Normal hepatocytes express connexin32 (Cx32), which forms gap junctions at cell-cell contact areas. The aim of the present study was to investigate whether Cx32 mediates the cell death-inducing effects of ultrasound microbubbles carrying the herpes simplex virus thymidine kinase (HSV-TK) suicide gene against hepatocellular carcinoma cells in vitro and in vivo. HepG2 cells were exposed to different concentrations of trans-retinoic acid (ATRA) in culture, to evaluate the intrinsic antitumor effect of ATRA. Detailed in-vitro and in-vivo investigations on the antitumor effects of ATRA via Cx32 mediation were performed, and the possible underlying mechanisms of action of the compound were then examined. The gene expression of HSV-TK transfected by ultrasound wave irradiation in the HepG2 cells was quantified using reverse transcription-quantitative polymerase chain reaction analysis. The effects on cell death were assessed using an MTT assay. The protein expression levels of Cx32 in ATRA-untreated or ATRA-treated tissues were quantified by immunohistochemical analysis and Western blot assays. The HSV-TK gene was successfully transfected into the HepG2 cell using ultrasound wave irradiation, and was stably expressed. Compared with the other groups, the HSV-TK gene group treated with ATRA exhibited an increased number of apoptotic cells (P<0.05) and improved tumor suppression (P<0.05). ATRA significantly increased the expression of Cx32 in the hepatoma tissues (P<0.01). The present study demonstrated that ATRA elevated the protein expression of Cx32 and enhanced the bystander effect of the HSV-TK/GCV suicide gene therapy system, which may provide a potential strategy for hepatocellular carcinoma treatment. [ABSTRACT FROM AUTHOR]