Study On The Effect Of RCE-4,the Active Ingredient Of Reineckia Carnea,on Cell Cycle Of Cervical Cancer Ca Ski Cells

Research background RCE-4 is a steroidal saponins monomer compound extracted from Reineckia carnea（Andr.）Kunth of Lilaceae,which has been recorded in Standards for local Chinese medicinal materials in Hubei Province as a symbolic component of Reineckia carnea.Current studies have shown that RCE-4 has anti-cervical cancer effects,which can inhibit the proliferation of cervical cancer cells and promote cell apoptosis,but the mechanism of action is not fully clear.Objective To study the inhibitory effect of RCE-4 on the proliferation of human cervical cancer Ca Ski cells,and to explore the effects of RCE-4 on the endoplasmic reticulum stress and cell cycle arrest of Ca Ski cells and its molecular mechanism,so as to provide theoretical basis for the future clinical application of RCE-4.Methods Etramethylazolyl blue method（MTT）was adopted to observe in the inhibitory effects of different concentrations of RCE-4 on the growth of Ca Ski cells at 24h、48h and 72h,and then calculated its half maximal inhibitory concentration(IC₅₀).Clone formation assay was used to detect the proliferation inhibitory effect of RCE-4 on Ca Ski cells.Flow cytometry was used to investigate the distribution of cell cycle of Ca Ski cells after treated with RCE-4.Western blot was used to detect the effect of RCE-4 on proteins levels which are related to ER stress and cell cycle signaling pathways.Results（1）RCE-4 has an significant inhibitory effect on the proliferation of human cervical cancer Ca Ski cells,with the IC₅₀of 8.61、4.69、4.34μmol·L^-1at 24h、48h and 72h respectively.（2）Clone formation assay results show that the number of Ca Ski cell colonies formed was significantly reduced after treated with 8,12,16μmol·L^-1RCE-4 respectively compared with the control group.（3）Flow cytometry results show that RCE-4 can significant induce G₂/M phase cell cycle arrest in Ca Ski cells at 24h.（4）Western blot results show that the protein expression levels of endoplasmic reticulum-related classic molecules PERK,ATF-4,CHOP,p-e IF2αand the protein levels of cell cycle-related regulatory molecules p21,p27,ATM,Cyclin E1 were significantly increased in Ca Ski cells;CDK1,cdc25C,p-cdc25C,Cyclin B1,Cyclin D1,Chk2,p-Chk2,GADD45α,MDM2 protein levels were significantly reduced in Ca Ski cells,which shows that RCE-4 triggered endoplasmic reticulum stress by activating PERK/e IF2α/ATF4/CHOP signaling pathway,also induced G₂/M phase cell cycle arrest by inhibiting the expression of Cyclin B1 and CDK1 via ATM/Chk2/cdc25C signaling pathway,some molecules like p21,p27,GADD45αand MDM2 are involved in this process.Conclusion RCE-4 may induce G₂/M phase arrest in Ca Ski cells through endoplasmic reticulum stress,thereby inhibiting the proliferation of human cervical cancer Ca Ski cells.