Experimental Studies For The Inhibitory Effect Of Trichosanthin On The Growth Of CaSki Cervical Cancer Cells And Its Underlying Mechanism

Objective The purpose of this study is to analyze the effects of TCS on the proliferation,autophagy and apoptosis of Ca Ski cervix cancer cells,and to further elucidate the molecular basis of TCS in the cervical cancer therapy.Methods(1)MTT assay was used to analyze the inhibitory effect of TCS on the proliferation of Ca Ski cells and the effective drug concentration;(2)The effects of TCS on cell morphology was observed by light microscope;(3)Western blot assay was used to detect the expression levels of autophagy-related and apoptosis-related proteins in Ca Ski cells;(4)The subcellular distribution of GFP-LC3 fusion protein in Ca Ski was observed by laser confocal microscopy;(5)The effect of TCS treatment on cell apoptosis and cell cycle of Ca Ski cells was detected by flow cytometry;(6)The effect of TCS on the formation of apoptotic bodies and autophagosomes in Ca Ski cells was observed by transmission electron microscope.Results(1)TCS could effectively inhibit the proliferation of Ca Ski cells.The IC50 values of TCS in the 48 h treated cells was 178.5 μg/m L;(2)Compared with control Ca Ski cells,the morphology of TCS-treated cells changed from the regular long spindle shape to the swollen,poorly adherent,irregular polygonal or round shape,at the same time,the exfoliated cells increased;(3)TCS can reduce the expression of autophagic protein Beclin1 and LC3A/B,but increase the accumulation of autophagic substrate protein P62 in Ca Ski cells;(4)Compared with control Ca Ski cells,the formation of the spotted green fluorescent sinal(autophagosomes which binded with GFP-LC3 fusion protein)decreased in Ca Ski cells treated with TCS;(5)TCS could induce apoptosis of Ca Ski cells,downregulate the expression levels of anti-apoptosis protein Bcl2 and apoptotic substrate protein PARP,but increase the expression level of pro-apoptotic protein Bax;(6)TCS could interfere with the Ca Ski cell cycle,leading to an increased proportion of the cells in S and G2/M phase;(7)Compared with the control group,the apoptosis of the cells treated with TCS was significantly increased than that of the control group.And the formation of apoptotic bodies in the cells was increased,while the formation of autophagosomes was significantly inhibited.Conclusion TCS can effectively inhibit the proliferation of cervical cancer Ca Ski cells,and its mechanism may be closely related to its abilities in interfering with cell cycle,inducing cell apoptosis and inhibiting cell autophagy.