Vascular damage and anti-angiogenic effects of tumor vessel-targeted adenovirus-mediated herpes simplex virus thymidine kinase gene

AIM： To explore the therapeutic efficacy and mechanism of herpes simplex virus-thymidine kinase （HSV-tk） targeting angiogenesis against hepatocellular carcinoma in vivio and in vitro. METHODS： Recombinant adenovirus containing kinase domain insert with receptor （KDR） or cytomegalovirus （CMV） promoter-controlled HSV-tk gene （AdKDR-tk and AdCMV-tk） was constructed using pAdeasy system. The expression of KDR antigen in human umbilical venous endothelial cells （HUVEC） and HepG2 was detected with histological analysis of cells. The virus was used to infect HUVEC and HepG2. Following administration of ganciclovir （GCV）, the survival rate of gene-transfected HUVEC and HepG2 was evaluated by MTT method. To develop hepatocarcinomas in 32 Balb/C mice with HepG2 cells, the mice were divided into four groups： ganciclovir group （Ⅰ）, Ad group （Ⅱ）, AdCMV-tk group （Ⅲ） and AdKDR-tk group （Ⅳ）. Then selective administration of recombinant adenovirus or Ad via the intratumorial was given to all rats. Ganciclovir （GCV） was given at a dose of 100 mg·kg^-1·d^-1 （ip） started on the following day and lasted 10 d. Microvessel density （MVD） of tumor in all the treated animals were examined by the immunohistochemical methods and tumor burden was evaluated 10 d before and alter the last GCV dose.RESULTS： Immunocytochemical staining indicated the expression of KDR antigen in HUVEC. Under adenovirus infection index of 100, with increasing GCV concentration from 0 up to 50 mg/L, the survival rate of AdKDR-tk- transfected HUVEC and HepG2 decreased from 100% to （28.94 ± 5.67）% and （75.45 ± 2.91）% at proper order, respectively （P 〈 0.01）, while the survival rate of AdCMV- tk-transfected HUVEC and HepG2 declined from 100% to （17.56 ± 2.48）% and （23.15± 5.72）%, respectively （P 〉 0.05）. Compared with group I, there was a decrease of tumor weight by 14.7% in group Ⅲ and by 23.6% in group Ⅳ. And there was a distinct difference between group M and Ⅳ （P 〈 0.05）. The median MVD for all groups was 37.4 ± 8.6, 30.6 ± 7.8, 27.6 ± 7.1, and 10.7 ± 4.1 （microvessels/mm^2） in group Ⅰ, Ⅱ, M and IV, respectively. And there was a marked difference between group M and Ⅱ （P 〈 0.05）, Ⅳ and Ⅱ （P 〈 0.01）, and Ⅳ and M （P 〈 0.01）. CONCLUSION： KDR promoter-HSV-tk gene may effectually restrain the growth of tumor via targeting angiogenesis for hepatocellular carcinoma with treatment of GCV.

AIM: To explore the therapeutic efficacy and mechanism of herpes simplex virus-thymidine kinase (HSV-tk) targeting angiogenesis against hepatocellular carcinoma in vivio and in vitro. METHODS: Recombinant adenovirus containing kinase domain insert with receptor (KDR) or cytomegalovirus (CMV) promoter-controlled HSV-tk gene (AdKDR-tk and AdCMV-tk) was constructed using pAdeasy system. The expression of KDR antigen in human umbilical venous endothelial cells (HUVEC) and HepG2 was detected with histological analysis of cells. The virus was used to infect HUVEC and HepG2. Following administration of ganciclovir (GCV), the survival rate of gene-transfected HUVEC and HepG2 was evaluated by MTT method. To develop hepatocarcinomas in 32 Balb/C mice with HepG2 cells, the mice were divided into four groups: ganciclovir group (Ⅰ), Ad group (Ⅱ), AdCMV-tk group (Ⅲ) and AdKDR-tk group (Ⅳ). Then selective administration of recombinant adenovirus or Ad via the intratumorial was given to all rats. Ganciclovir (GCV) was given at a dose of 100 mg·kg-1·d-1 (ip) started on the following day and lasted 10 d. Microvessel density (MVD) of tumor in all the treated animals were examined by the immunohistochemical methods and tumor burden was evaluated 10 d before and after the last GCV dose.RESULTS: Immunocytochemical staining indicated the expression of KDR antigen in HUVEC. Under adenovirus infection index of 100, with increasing GCV concentration from 0 up to 50 mg/L, the survival rate of AdKDR-tk- transfected HUVEC and HepG2 decreased from 100% to (28.94 ± 5.67)% and (75.45 ± 2.91)% at proper order, respectively (P < 0.01), while the survival rate of AdCMV- tk-transfected HUVEC and HepG2 declined from 100% to (17.56 ± 2.48)% and (23.15 ± 5.72)%, respectively (P > 0.05). Compared with groupⅠ, there was a decrease of tumor weight by 14.7% in group Ⅲ and by 23.6% in group Ⅳ. And there was a distinct difference between group Ⅲ and Ⅳ (P < 0.05). The median MVD for all groups was 37.4 ± 8.6, 30.6 ± 7.8, 27.6 ± 7.1, and 10.7 ± 4.1 (microvessels/mm2) in groupⅠ, Ⅱ, Ⅲ and Ⅳ, respectively. And there was a marked difference between group Ⅲ and Ⅱ (P < 0.05), Ⅳ and Ⅱ (P < 0.01), and Ⅳ and Ⅲ (P < 0.01). CONCLUSION: KDR promoter-HSV-tk gene may effectually restrain the growth of tumor via targeting angiogenesis for hepatocellular carcinoma with treatment of GCV.