The Molecular Mechanism Of Sophoridine Inhibiting Breast Cancer Cells Growth

Objective: As a quinorizidine alkaloid from Sophora alopecuroides L,sophoridine shows well anti-tumor effect.In this study,to explore the effect and molecular mechanism of SRI on the growth of MCF-7 and MDA-MB-231 cells through experiments in vitro,network pharmacology and molecular docking.Meathods: 1.MTT assays were used to detect the effect of SRI on the viability of MCF-7 and MDA-MB-231 cells at different concentrations and times,and colony formation assays were used to detect the effect of SRI on MCF-7 and MDA-MB-231 cell growth.2.Flow cytometry was used to detect the effect of SRI on MCF-7 and MDA-MB-231 cell apoptosis rate,and q PCR and WB experiments were used to detect the expressions of anti-apoptosis factor Bcl-2 and pro-apoptosis factor Bax at transcription and translation level.3.Based on network pharmacology,the SRI targets were predicted on Pharm Mapper database,and the luminal A and triple negative breast cancer targets were obtained on Gene Cards and Dis Ge Net database.The Venn diagrams were drawn to obtain the commom targets of SRI-luminal A breast cancer and SRI-triple negative breast cancer.4.The most promising target for SRI inhibiting breast cancer was gradually selected from SRI-luminal A breast cancer and SRI-triple negative breast cancer common targets through q PCR and molecular docking,and then verified by WB experiments.5.We explored the mechanism of SRI inhibiting breast cancer cell growth based on this target,and then verified the mechanism by detecting expression of pathway related proteins through WB experiment.Results: 1.The results of MTT assay indicated that SRI inhibited MCF-7 and MDAMB-231 cell viability and the inhibition in MCF-7 cells was time-and concentrationdependent,as well as the results of colony fromation assay indicated that SRI significantly inhibited the cell colony froming ability.These above results showed that SRI inhibited breast cancer cell growth.2.After being treated 24 h by SRI,the apoptosis rate of MCF-7 and MDAMB-231 cells were significantly increased.The gene and protein expressions of Bcl-2were inhibited,while the gene and protein expressions of Bax were promoted by SRI.These results indicated that SRI promoted breast cancer cell apoptosis.3.Among 41 common targets between SRI and luminal A breast cancer,targets with Norm Fit above 0.5 were CASP7,NQO2,PIM1,EPHB4,GSK3 B,CDK2,DPP4 and GSTP1.The q PCR experiment results showed that GSK3 B gene expression was not regulated by SRI,molecular docking results showed the affinity between SRI with CASP7,NQO2,EPHB4,CDK2,GSTP1 were beyond-8.0Kcal/mol,SRI with PIM1,DPP4 were no more than-8.0Kcal/mol.These results indicated that PIM1 and DPP4 were the most promising targets for SRI inhibiting luminal A breast cancer.4.Among 35 common targets between SRI and triple negative breast cancer,targets with Norm Fit above 0.5 were CASP7,NQO2,PIM1,GSK3 B,CDK2 and GSTP1.The q PCR experiment results showed that CASP7 gene expression was not regulated by SRI,molecular docking results showed the affinity between SRI with NQO2,GSK3 B,CDK2,GSTP1 were beyond-8.0Kcal/mol,SRI with PIM1 were below-8.0Kcal/mol.These results indicated that PIM1 was the most promising target for SRI inhibiting triple negative breast cancer.5.PIM1 was a promising target for SRI in treatment of both luminal A and triple negative breast cancer,thus it was assumed that PIM1 was the target for SRI in treatment of breast cancer.The WB experiment results showed that PIM1 protein expression in MCF-7 and MDA-MB-231 cells was significantly inhibited by SRI,and the inhibitory effect was dose dependent,indicating that PIM1 was indeed the target for SRI inhibiting triple negative breast cancer.6.After being treated 48 h by SRI,the expression of PIM1,p-ASK1(Ser83)and caspase3 in MCF-7 and MDA-MB-231 cells were significantly decreased,p-JNK and p-p38 MAPK were increased,while ASK1,JNK and p38 MAPK were not changed.These results were statistically significant,indicating that SRI might promote breast cancer cell apoptosis through PIM1/MAPK signaling pathway,and then inhibit cell growth.Conclusion: SRI could inhibit the growth of human breast cancer cells MCF-7 and MDA-MB-231,and its mechanism might be that it promotes the activation of caspase3 through PIM1/MAPK signaling pathway,and then induces breast cancer cell apoptosis.