单纯疱疹胸苷激酶/更昔洛韦系统杀伤前列腺癌细胞的“旁观者效应”及其调控

Bystander effect mediated by herpes simplex virus-thymidine kanase/ganciclovir approach on prostatic cancer cells and its regulation

目的 评价单纯疱疹胸苷激酶 更昔洛韦 (HSV TK GCV)系统杀伤前列腺癌细胞PC 3m的“旁观者效应” ,探讨连接蛋白 (Cx)介导的细胞间隙连接交流 (GJIC)在该系统“旁观者效应”中的作用 ,观察apigenin对PC 3m细胞连接蛋白 43(Cx43)表达及GJIC状况的调控。方法 MTT法评价TK基因系统杀伤PC 3m细胞的“旁观者效应” ,划痕标记染料示踪技术 (SLDT)比较几种代表细胞系GJIC功能状况 ,检测HSV TK GCV对其“旁观者效应”。逆转录 -聚合酶链式反应 (RT PCR)法和SLDT检测PC 3m细胞Cx43表达和GJIC状况 ,并观察apigenin对Cx43表达、GJIC状况及“旁观者效应”的调控作用。结果 HSV TK GCV系统杀伤PC 3m细胞时“旁观者效应”程度较弱 ,TK基因阳性细胞比例约为1 0 %的混合细胞经 1 0 0 μmol LGCV处理 72h后 ,仅 2 3 .5 %± 3 .2 1 %细胞被杀灭。该自杀基因系统对GJIC功能强大的NIH 3T3、Cos 7和L 0 2细胞“旁观者效应”程度远较GJIC功能低下的ACHN和HeLa细胞者强 (P <0 .0 0 1 )。PC 3m细胞Cx43mRNA表达降低 ,GJIC功能低下 ,apigenin不仅可上调PC 3m细胞Cx43表达、促进GJIC功能 ,还可使其“旁观者效应”明显改观 (P <0 .0 0 1 )。结论 细胞内在的GJIC功能与HSV TK GCV对其“旁观者效应”程度有正向关系 ,PC

Objective To estimate the bystander effect mediated by herpes simplex virus thymidine kanase/ganciclovir (HSV TK/GCV) suicide gene therapy approach on PC 3m, a prostate cancer cell line, to explore the role of connexin (Cx) mediated gap junctional intercellular communication (GJIC) in the procedure of bystander effect of HSV TK/GCV system and to investigate the modulation of apigenin, a Cx expression up regulator on the connexin43 (Cx43) expression and GJIC of PC 3m cells. Methods PC 3m cells were cultured and PC 3m cells transfected with EBV based expression vector containing HSV TK gene (TK + PC 3m cells ) and TK - PC 3m cells were mixed at the ratio of 1∶9. GCV was added into the mixture. The bystander effect was evaluated by MTT assay. GJIC and HSV TK/GCV induced bystander effect in several typical cell lines, such as NIH 3T3, Cos 7, and L 02 cells, were determined by crape loading dye tracing (SLDT) and MTT assay respectively. Cx43 mRNA expression and inherent GJIC capacity of PC 3m cells were examined by RT PCR and SLDT. TK + PC 3m cells and TK - cells were mixed and divided into 4 groups and added with GCV, apigenin, apigenin+GCV, and apigenin+GCV+18 α glycyrrhetinic acid (AGA) respectively. Then the killing rate on PC 3m cells was examined by MTT. Results After 72 h treatment of 100 μmol/L GCV on the mixture of wild type PC 3m cells and HSV TK gene modified PC 3m cells, only 23.5%±3.2% cells were killed. The magnitude of HSV TK/GCV bystander effect were more powerful in NIH 3T3, Cos 7, and L 02 cells which manifested excellent GJIC than in ACHN and HeLa cells ( P <0.001). Expression of Cx43 mRNA was shown by RT PCR, however, it is weaker than that in ACHN cells and normal prostate tissue. With the administration of apigenin, the expression of Cx43 mRNA and the GJIC function of PC 3m cells were increased by 2.2 times ( P <0.01) The enhancing effect of apigenin on GJIC function of PC 3m cells lasted 48 hours and could be inhibited by addition ofAGA. Apigenin of the concentration of 10 μmol/L could obviously improve the bystander effect of TK system on PC 3m cells ( P <0.001). The killing rate of GCV on the mixed PC 3m cells was 59.86%±2.44%, and was only 25.34%±2.89% with the addition of AGA. Conclusion There is a positive correlation between the magnitude of bystander effect mediated by HSV TK/GCV approach and the potency of internal GJIC in the target cells. Down regulated Cx43 expression and disrupted inherent GJIC potential of PC 3m cells result in the poor magnitude of HSV TK/GCV bystander effect. Chemical agent like apigenin up modulates Cx43 expression and invokes GJIC capacity of PC 3m cells, thus enhancing the bystander effect and augmenting the efficacy of TK suicide therapy.