The Effect And Mechanism Of Telomerase Inhibitor MST-312 On Radiosensitivity Of Human Hepatoma HepG2 Cells

Purpose:To investigate the effect and mechanism of telomerase inhibitor MST-312 on radiosensitivity of human hepatoma HepG2 cells.Materials and Methods:The cytotoxicity of MST-312 was measured with the MTT method for screening the suitable concentration of MST-312. HepG2 cells were pretreated with MST-312 for 2 h, followed by 2 Gy X-ray treatment, and then were performed to evaluate the cell surviving fractions and determine the radiation-sensitizing effect of MST-312 by clonogenic assay. The cell cycle distribution and apoptosis after radiation were detected with flow cytometry analysis and Hoechst 33258 fluorescent staining. To estimate the levels of protein expression that was involved with cells damage, repair and apoptosis by Western blot. The telomerase activity was detected by real-time fluorescent quantitative PCR. Rad51, XRCC4 and y-H2AX were detected by immunofluorescence, which could measure DNA damages and repair. Mitochondrial membrane potential (△Ψm) that indicated initiation of cell apoptosis was quantified by staining with the targeting probe JC-1.Results:1. MTT assay showed that MST-312 decreased the proliferation of HepG2 cells in a dose-dependent manner.4 μM of MST-312 inhibited proliferation of HepG2 cells was less during the test period, and had no significant effect on the cell morphology. The radio-sensitivity of HepG2 cells was examined by clonogenic assay, which revealed that colony-forming efficiency of the MST-312 pretreatment followed by X-ray was obviously lower with the comparison of irradiation group. Flow cytometry analysis showed that X-ray could lead to cell cycle arrest at 12h post-irradiation. However, at 24 h post-irradiation, the proportion of Sub-Gl was increased and G2/M phase fraction was decreased in the combined treatment group, when compared with the irradiation alone group. This study further confirmed the apoptosis fraction of HepG2 cell after X-ray radiation with Hoechst33258 staining, and the results showed higher level of cell apoptosis in the combined treatment group. The Western Blot results indicated a prolonged higher expression level of y-H2AX in combined treatment group, when compared with the irradiation group, implying an enhanced DNA damaging effect.2. Real-time fluorescent quantitative PCR validated that MST-312 inhibited inherent and irradiation-induced hTERT mRNA expression were closely associated with telomerase activity of HepG2 cells. The immunofluorescence staining results showed that a number of γ-H2AX foci were presented in the combined treatment group. However, the formation of Rad51 foci in the combined treatment group was blocked outside the nuclear of HepG2 cells, when compared with the irradiation alone group. In the combined treatment group, JC-1 staining showed that the green/red fluorescence intensity ratio (△Ψm) was increased compared with X-ray irradiation alone. MST-312 pretreatment followed by X-ray irradiation elevated expression of p53 protein, decreased expression of caspase-3 and fraction of Bcl-2/Bax.Conclusion:The telomerase inhibitor MST-312 pretreatment enhanced the radiosensitivity of HepG2 cells to X-ray irradiation. This increased radiosensitivity was due to inhibition of telomerase activity that could result in telomerase dysfunction and impair HR repair processes. MST- 312 not only increased irradiation-induced DNA damages, but impaired DNA repair, which led to HepG2 cells apoptosis through p53-dependent mitochondrial pathway.