Abstract：Objective To investigate the effects of cinobufacini on the radiosensitivity of Bel-7402 hepatocellular carcinoma cells and its molecular mechanisms.Methods Bel-7402 hepatocellular carcinoma cells were treated with 0.125 μg/mL, 0.250 μg/mL, 0.500 μg/mL, 1.000 μg/mL cinobufacin for 24 h, 48 h and 72 h, following the detection of cell proliferation by MTT assay. And the concentration and time of action for cinobufacini were selected according to the half inhibitory concentration (IC50). Furthermore, Bel-7402 cells were treated with 0 μg/mL (control group) or 0.4 μg/mL cinobufagin and irradiated with 6 MV X-rays at doses of 0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy at the same time. Clonogenic assays were performed to determine the cellular radiosensitivity. IC50 of 0.4 μg/mL cinobufacini and 6 Gy were used alone or in combination to treat Bel-7402 hepatocellular carcinoma cells. After 48 h, cell cycle and apoptotic rate were measured by flow cytometry, while the expression of p50, p65, Bcl-2 and cyclin D1 protein were detected by Western blot.Results Cinobufacini could significantly inhibit the proliferation of Bel-7402 cells. With the increase of the concentration and the prolongation of the time of action, the inhibition rate was increased gradually, showing a dose-time dependent effect. Meanwhile, cinobufacini could inhibit the colonization ability of Bel-7402 cells after irradiation, and its survival rate was significantly lower than that of the control group (P<0.05). In addition, when compared with the control group, the percentages of Bel-7402 cells in G0/G1 phase in the cinobufacini group, radiotherapy group and the combination group were all significantly increased (all P values<0.01), while the proportions of S phase cells were significantly decreased (all P values<0.01), and the apoptotic rate was significantly increased (all P values<0.01), but the expression levels of p50, p65, Bcl-2 and cyclin D1 protein were significantly decreased (all P values<0.01), and the differences in cell cycle, apoptotic rate and protein expression in the combination group were more significant than those of the cinobufotalin group and the radiotherapy group (all P values<0.01).Conclusions Cinobufacini can enhance the radiosensitivity of Bel-7402 cells, and the mechanism may be related to the inhibitory effect of cinobufagin on nuclear factor-κB signaling pathway and down-regulation of Bcl-2 and cyclin D1 protein expression, thereby inhibiting cell proliferation and inducing cell cycle arrest and apoptosis.
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2018, Vol. 23 
