Anti-hepatoma Activity And Mechanisms Of Extracts From Endophytic Fungus Of Bupleurum Scorzonerifolium Willd.

Objective:Extract and separate the anti-hepatoma active components from endophytic fungus CHS5 from Bupleurum scorzonerifolium Willd.and test the anti-hepatoma activity and possible anti-hepatoma mechanisms of each fractionMethods:a)The anti-hepatoma active components of endophvtic fungus CHS5 from Bupleurum scorzonerifolium Willd were extracted and separated and we got petroleum ether fraction,ethyl acetate fraction,n-butanol fraction and water-soluble fraction,respectively The MTT method was used to test the proliferation inhibitory effect on HepG2 cell of each fraction.The concentration of each fraction used for follow-on experiments was determined according to the results.b)After treatment with petroleum ether fraction and ethvl acetate fraction of endophytic fungus CHS5 from Bupleurum scorzonerifolium Willd,the cell morphology of hepatoma cell HepG2 was observed under optical microscope,and the cell nuclear morphological and staining conditions of hepatoma cell HepG2 by Hoechst 33258 staining was observed under fluorescence microscope,apoptosis rate of hepatoma cell HepG2 was detected by flow cytometry with Annexin V-FITC/PI double staining,and the expression condition of apoptosis related proteins and NF-κB signaling pathway related proteins were analyzed by Western Blot.c)The invasion and migration conditions of hepatoma cell HepG2 with the treatment of petroleum ether fraction and ethyl acetate fraction of endophytic fungus CHS5 from Bupleurum scorzonerifolium Willd were evaluated by Transwell method.and the expression condition of extracellular angiogenesis related proteins was tested by ELISA.Results:The effects of petroleum ether fraction and ethyl acetate f-raction of endophytic fungus CHS5 from Bupleuum scorzonerifolium Willd on inhibiting proliferation of hepatoma cell HepG2 are positive correlation with time and concentration.The IC50 of ethyl acetate fraction are 32.85 μg/mL at 24h and 28.49 μg/mL at 48h,and the IC50 of petroleum ether fraction are 56.70 μg/mL at 24h and 55.07 μg/mL at 48h.Through the morphological observation and the apoptotic analysis,it is identified that the promoting apoptotic activities of the both fractions are positive correlation with concentration.The results from Western Blot shows that the expression are increasing in Cleaved PARP and Bax and descreasing in Caspase 3,Caspase 9 and Bcl-2.However,the down-regulation of NF-κB.IκB-α and IKK-βexpression indicates that inducing apoptosis of hepatoma cell HepG2 is through mitochondrial pathway but not NF-κB signaling pathway.The inhabition of migration and invasion of the cancer cells is also positive correlation with concentration.The results from ELISA turns out that the expression of angiogenic related proteins are inhibited by the both fractions.Conclusion:The results indicate that petroleum ether fraction and ethyl acetate fraction of endophytic fungus CHS5 from Bupleurum scorzonerifolium Willd are capable of inhibiting proliferation of hepatoma cell HepG2 via promoting apoptosis and suppressing migration and invasion of cancer cells.