Effects Of Sophoridine Combined With Cisplatin On Proliferation Inhibition Of Cervical Adenocarcinoma Hela Cells

Objective:To discuss the influence of Sophoridine combined with Cisplatin on proliferation inhibition of Cervical adenocarcinoma Hela cells, and provide a new thoughts and theoretical basis of laboratory to prevent and cure the cervical adenocarcinoma in clinic.Methods:Cervical adenocarcinoma Hela cells were cultured in vitro:The proliferation inhibition of Hela cells respectively treated with different concentrations of Sophoridine, Cisplatin and different concentrations of Sophoridine combined with Cisplatin for24h,48h and72h were detected by MTT; and then the50%Inhibitory Concentration (IC50) was calculated, and the combined effects of two medicines was determined by Jin Zhengjun Q value method; Morphological alterations of Hela cells was observed by Inverted phase contrast microscope; Hela cells treated with different concentrations of Sophoridine combined Cisplatin for24h were stained by Hoechst33342to observe the morphological changes through fluorescence microscopy; The changes of cell cycle distribution and apoptotic rate of Hela cells treated with different concentrations of Sophoridine combined Cisplatin for24h were analyzed by Flow cytometry technology; The expression changes of apoptosis related proteins (Bax, Bcl-2, P53and Caspase-3) in Hela cells treated with different concentrations of Sophoridine combined Cisplatin for24h were detected by Western blot.Results:(1) The results of MTT assay indicated Sophoridine could markedly inhibit the proliferation of Cervical adenocarcinoma Hela cells in a dose and time-dependent manner (P<0.05); The IC50values were5.5mg/ml,4.2mg/ml and2.1mg/ml) in24h,48h and72h,respectively. The results of the combined effects of two medicines showed that:Hela cells was treated with different concentrations of Sophoridine combined Cisplatin for24h, the combined effects of two medicine were antagonism (Q<0.85), when the concentrations of Sophoridine was less than1.0mg/ml; when the concentrations of Sophoridine was equal or greater than1.0mg/ml, the combined effects of two medicines were synergistic (Q>0.85). while Hela cells was treated with different concentrations of Sophoridine combined Cisplatin for48h and72h, the combined effects of two medicine were synergistic(Q>0.85); The morphological feature of Hela cells changed significantly with the increase of the concentration of Sophoridine after treated for24h.(2) Hoechst33342staining determined the cell apoptosis in Sophoridine combined Cisplatin treated Hela cells and also typical apoptosis characteristics in Hela cells.(3) The results of Cell cycle:Flow cytometry analyzed that Cisplatin group and Sophoridine combined Cisplatin groups could affect cell cycle distribution of Hela cells. The percentages of G0/G1phase cells of Cisplatin group and Sophoridine combined Cisplatin groups were lower than the negative control group, the percentage of S phase cells were higher than the negative control group (P<0.05).In addition, as compared with Cisplatin group, the percentages of S phase cells were lower than the cisplatin group, the percentages of G0/G1phase cells were higher than the Cisplatin group(P<0.05), while the concentrations of Sophoridine was0.5mg/ml. The percentages of S phase cells were higher than the cisplatin group, the percentages of GO/Glphase cells were lower than the Cisplatin group (P<0.05), while the concentrations of Sophoridine was equal or greater than1.0mg/ml.(4) The results of Cell apoptosis rate:Flow cytometry analyzed that Cisplatin group and Sophoridine combined Cisplatin groups could induce Hela cells apoptosis, the apoptosis rate of Cisplatin group and Sophoridine combined Cisplatin groups were higher than the negative control group (P<0.05). In addition, as compared with Cisplatin group, the apoptosis rate is lower than the cisplatin group (P<0.05), while the concentrations of Sophoridine was0.5mg/ml. The apoptosis rate were higher than the cisplatin group (P<0.05), while the concentrations of Sophoridine was equal or greater than1.0mg/ml.(5) The results of Western blot:Western blot confirmed that Cisplatin group and Sophoridine combined Cisplatin groups could down-regulate the expression of Bcl-2protein, also significantly up-regulate the expression of the P53protein and Bax protein and Caspase-3protein (P<0.05), when compared with negative control. In addition, as compared with Cisplatin group, the expression of Bcl-2protein was superior than Cisplatin group, the expression of the P53protein and Bax protein and Caspase-3protein were inferior to Cisplatin group, while the concentrations of Sophoridine was0.5mg/ml; The expression of Bcl-2protein was inferior to Cisplatin group, the expression of the P53protein and Bax protein and Caspase-3protein were superior than Cisplatin group, while the concentrations of Sophoridine was equal or greater than1.0mg/ml (P<0.05).Conclusion:(1) Sophoridine could inhibit the proliferation of Cervical adenocarcinoma Hela cells in a dose and time-dependent manner;The combined effects of Sophoridine and Cisplatin were synergistic.(2)Sophoridine combined Cisplatin could inhibit the proliferation of Cervical adenocarcinoma Hela cells, Its mechanism may be related to the induction of Hela cells apoptosis and S phase retardation, down-regulated expression of Bcl-2, and up-regulated expression of P53and Bax and Caspase-3, down-regulated the ratio of Bcl-2/Bax.Its suggested that Sophoridine combined Cisplatin induced Cervical adenocarcinoma Hela cells apoptosis may be done by P53dependent apoptosis pathway.