Study On Antitumor Activity And Mechanism Of Brevilin A

Aim: To explore the anti-tumor activity and mechanism of Brevilin A and to provide some experimental basis for its clinical application.Methods: The effect of Brevilin A on the cell proliferation of human lung cancer A549 cells,human hepatocellular carcinoma HepG2 cells,human cervical cancer Hela cells,human melanoma A875 cells and mice colon cancer CT26 cells were detected by MTT assay,cells were treated with various concentrations of Brevilin A for 48 h.Observe the mouse status,tumor growth and changes of organs on the CT26 solid tumor model which treated by Brevilin A.Immunohistochemistry was carried out to observe the expression of cleaved-caspase-3 and LC3-Ⅱin tumor tissues.Annexin V/PI dobule staining method was applied to investigate the apoptosis of CT26 cells which treated with Brevilin A for 72 hours.the apoptosis and autophagy related proteins were detected by Western Blot,and confocal laser scanning microscopy was applied to observe the expression and distribution of autophagy marker,LC3.Result: Compared to the control group,Brevilin A exerted a significant inhibitory effect on human lung cancer A549,human hepatocellular carcinoma HepG2,cervical carcinoma Hela,human melanoma A875 and murine colon carcinoma CT26 proliferation in a dose-dependent manner.And the IC50 valus was 5.98 μg/mL,5.72 μg/mL,4.41 μg/mL,8.17 μg/mL,3.28 μg/mL.The inhibitory effect of Brevilin A on colon cancer CT26 was the best of them,therefore,we taken CT26 cell as the subject to study anti-tumor mechanism.In vivo experiment indicated that there was significant difference in body weight between the Brevilin A group and Adriamycin group with the model group(P<0.01).The tumor weight in Brevilin A group or Adriamycin treated was both lighter than that in the model one(P<0.01、P<0.05),and statistically significant of tumor weight between the Brevilin A group with the Adriamycin group(P<0.01).Compared with the model group,the liver index and kindney index of Brevilin A group were significantly different(P<0.01,P<0.05).However,there was no significant difference in spleen index between the Brevilin Agroup and the model group.The liver index,spleen index and kidney index of Adriamycin group was significantly lower than that of the modle group(P<0.01).The liver index and spleen index of Adriamycin group was significantly lower than that of Brevilin A group(P < 0.01,P <0.01).Compared with the model group,the expression of cleaved-caspase-3 and LC3-Ⅱin tumor tissues of Brevilin A group and Adriamycin group were enhanced,and the expression of Brevilin A were higher than the Adriamycin.Flow cytometry results showed that with the increase of the concentration of Brevilin A,the apoptotic rate was increased and higher than that of Adriamycin group.The apoptotic rate of Brevilin A group at 4 μg/mL(42.92%)was similar to that at 2 μg/mL(45.55%).The late-phase apoptotic rate of Brevilin A at 4 μg/mL(33.29%)was higher to that at 2 μg/mL(9.63%).The expression of cleaved-caspase-8,cleaved-caspase-9,cleaved-caspase-3 and Bax protein in the treated CT26 cells were promoted in a dose-dependent manner in Brevilin A-treated,the expression of Bcl-2 protein was reduced.Furthermore,the expression of p-PI3 K,p-AKT,p-mTOR protein were down-regulated while the expression of LC3-Ⅱ,Beclin 1,Atg5 were up-regulated.Confocal laser scanning microscopy results showed that autophagy marker protein LC3 of CT26 in Brevilin A treatment group was induced to accumulate and form autophagosomes.The accumulation of LC3 protein was enhanced when it co-treatment of Brevilin A and rapamycin,and compared with Brevilin A group,the accumulation of LC3 protein was significantly less to that co-treatment by Brevilin A and 3-MA.Conclusion: Brevilin A can suppress the growth of CT26 in vitro and vivo.The probable anti-tomor mechanism is related to the cell apoptosis via regulating the mitochondrial pathway and cell autophagy via inhibiting PI3 K / Akt / mTOR pathway.