Glaucocalyxin B Induces Apoptosis And Cell Cycle Arrest Through JNK And P38 MAPK Pathways In Human Cervical Cancer Cells

BackgroundCervical cancer is the fourth leading cause of cancer-related death among women worldwide.At present,chemotherapy is still the main treatment option for patients with advanced and recurrent cervical cancer,but large doses of chemotherapy drugs cause adverse reaction and drug resistance phenomenon caused by the long-term use of chemotherapy drugs,make its in the clinical application is limited by certain,so looking for a new,low toxic and efficient treatment is an urgent problem to be solved.JNK and p38 MAPK signaling pathways are involved in cell proliferation,differentiation,apoptosis,cell cycle arrest and other physiological processes.The relationship between the anti-tumor effect of glaucocalyxin B and the JNK and p38 MAPK signaling pathways,as well as the specific molecular mechanism of action,are still unclear.ObjectivesIn this study,the relationship between the cytotoxic effects of glaucocalyxin B on human cervical cancer and the JNK and p38 MAPK signaling pathways was studied,to further clarify the relevant molecular mechanism of glaucocalyxin B in cervical cancer cells,and to lay a theoretical foundation for the treatment of cervical cancer with calycetin.Methods1.The proliferation activity of HeLa and SiHa cells in different treatment groups were determined by MTT assay.2.The changes of apoptosis and cell cycle distribution of HeLa and SiHa cells in different treatment groups were detected by flow cytometry.3.Western blot was used to detect the changes of relevant protein expression levels.4.The mRNA expression levels of relevant molecules were detected by real-time fluorescence quantitative PCR(qRT-PCR).Results1.MTT results showed that the proliferation of HeLa and SiHa cells was significantly reduced in a dose-dependent manner by glaucocalyxin B,while the activity of cells was significantly increased after adding specific inhibitors of p38 MAPK(SB203580)and JNK(SP600125).The results were statistically significant(P<0.05).2.Flow cytometry results indicated that the apoptosis of HeLa and SiHa cells was significantly enhanced after treatment with Glaucocalyxin B for 24 h,while the apoptosis of HeLa and SiHa cells was significantly reduced after pretreatment with SB203580 or SP600125.Glaucocalyxin B induced cell cycle arrest at the G2/M stage in a dose-dependent manner,while after adding SB203580 or SP600125,the cells in the G2/M stage were significantly reduced.The results were statistically significant(P<0.05).3.Western-blot results showed that treatment HeLa and SiHa cells with Glaucocalyxin B for 24 h,P-JNK,P-p38,Bax,p21 protein expression levels increased,while Bcl-2,cyclin D1,MMP-9,VEGF protein expression levels decreased,T-JNK,T-p38 protein expression levels did not change,compared with the control group.After pretreatment with SB203580 or SP600125,the expression of P-JNK,P-p38,Bax,p21 decreased,while the expression of Bcl-2,cyclin D1 increased.The results were statistically significant(P<0.05).4.qRT-PCR results revealed that treatment HeLa and SiHa cells with Glaucocalyxin B for 24 h,Fas,FasL,Bax,p21 mRNA expression increased,while Bcl-2,cyclin D1,MMP-9,VEGF mRNA expression is reduced,compared with the control group.After pretreatment with SB203580 or SP600125,Fas,FasL,Bax,p21 mRNA expression decreasd,while Bcl-2,cyclin D1 mRNA expression increases.The results were statistically significant(P<0.05).Conclusion1.Glaucocalyxin B inhibits the proliferation and promotes apoptosis in HeLa and SiHa cells by activating the JNK and p38 MAPK signaling pathways.2.Glaucocalyxin B induces G2/M cell cycle arrest which is associated with up-regulation of p21 and down-regulation of cyclin D1 protein expression and,may be regulated by the JNK and p38 MAPK signaling pathways.