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Inhibitory effect and mechanism of betulin acid on human cervical cancer HeLa cell lines

CHEN Xiu-wei,WANG Dan-yun,LIU Jia,YIN Ming-zhu,LOU Ge,GAO Ying,KANG Xi-xiong,ZHANG Guang-mei(Department of Gynecology,The Third Clinical College of Harbin Medical University,Harbin Heilongjiang 150081,China)  
Background and purpose:Betulinic acid,a pentacyclic triterpenoid isolated from birch bark,has been reported that it could inhibit multiple cancer cells proliferation,but there is no report concerning cervical cancer.Therefore,we investigated the effect of betulinic acid on the proliferation of human cervical cancer HeLa cells in vitro and its mechanisms.Methods:MTT assay was used to determine the inhibitory rate of betulinic acid on the proliferation of HeLa cells.Apoptosis induced by betulinic acid was detected by Hoechst33258 staining.Apoptotic rate was quantified by flow cytometry(FCM) using Annexin-Ⅴ/PI dual staining method.Western blot was used to evaluate the changes of caspase-3 and Cyto C proteins in HeLa cells.Results:Betulinic acid induced HeLa cells apoptosis in a time-and dose-dependent manner.The 50% inhibiting concentration(IC50) at 24,48 and 72 h of HeLa cells were 20.36,15.45 and 12.02 μg/mL,respectively.Typical morphological changes of apoptosis were observed in HeLa cells with Hoechst33258 staining after induced by betulinic acid.FCM showed the early apoptotic cells by Annexin-Ⅴ/PI staining.The protein expressions of caspase-3 and Cyto C in HeLa cells were both up-regulated after treatment with betulinic acid.Conclusion:Betulinic acid could significantly inhibit the proliferation of HeLa cells and induce apoptosis through caspase-3 pathway by up-regulating caspase-3 and Cyto C proteins.
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