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-alpha-glycyrrhetinic acid (AGA) respectively. Then the killing rate on PC-3m cells was examined by MTT.
Results: After 72 h treatment of 100 micro mol/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 of AGA. Apigenin of the concentration of 10 micro mol/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.