Research On HepG-2 Cell Apoptosis And Its Mechanisms Of Quaternary Ammonium Hydroxide From Capparis Spionosa L.

This study explored the effect of HepG-2 cells cultured in vitro by quaternary ammonium hydroxide of Capparis spinosa L(C.S) and investigated the apoptotic induction effects of it on tumor cells and the relationship between mitochondrial apoptosis and mitochondrial transmembrane potential (△Ψm), relative apoptotic genes and proteins.In in vitro studies, it had been found that quaternary ammonium hydroxide of CS could inhibit the growth of HepG-2 tumor cells.Measurements using mononuclear cell direct cytotoxicity assay (the MTT method) showed that its in vitro IC50, or inhibition concentration for 50% reduction, was about 178.87μg/ml.Resulted from SRB assay showed that the anticancer effect of quaternary ammonium hydroxide of Capparis spinosa was cytostatic at low concentration, with the GI50 being 35.93μg/ml. The quaternary ammonium hydroxide of Capparis spinosa inhibited the formation of HepG-2 colonies at TGI50 of 127.12μg/ml, all with marked results.FCM and CLSM were used to detect the criticality events of mitochondrial pathway.Result of CLSM indicated that action of different concentration of quaternary ammonium hydroxide of Capparis spinosa could make the PT pore opened,and the effect of quaternary ammonium hydroxide of Capparis spinosa was dosage-dependent.The mitochondrial membrane potential was degrade.The concentration of cyto in endochylema Caspase-9 and Caspase-3 were all increased.All the results above indicated that quaternary ammonium hydroxide of CS induced apoptosis by starting the mitochondrial pathway.Colorimetric method was used to measure Caspase-9 and Caspase-3 activities in HepG-2 cells which were treated with 100μg/ml,200μg/ml,400μg/ml quaternary ammonium hydroxide of CS for 48h.The results showed that all drug groups of quaternary ammonium hydroxide of CS can rise Caspase-9 and Caspase-3 activity with dosage dependence. Then flow cytometry was used to analyze the expression of bcl-2,bax and Cty-c.The results showed that expression of Bcl-2 protein in HepG-2 cells which were treated with 100μg/ml,200μg/ml,400μ/ml quaternary ammonium hydroxide of CS for 48h decreased but expression of Bax protein increase.And expression of Cty-c protein which were treated with 100μg/ml,200μg/ml,400μg/ml quaternary ammonium hydroxide of CS for 48h was increased. All results showed that pathway of quaternary ammonium hydroxide of CS inducing tumour cell apoptosis may be mitochondrion way:first through increasing expression of Bax protein make bax transfer onto mitochondrion,and distroyed the stability of membrane of mitochondrion.Then mitochondrion released a great deal of cytochrome-c to cytoplasm,and cytochrome-c combine with Apaf-l,then activated Caspase-9,further activated Caspase-3.In the end induced Caspase chain reaction.Besides,through dreasing expression of Bcl-2 weakened its restrain apoptosis effect.From the above, it could be concluded that quaternary ammonium hydroxide of CS could inhibit in vitro the growth of HepG-2 cells, and that it had this effect probably because it could accommodate the Bcl-2 family proteins. In other words, it induced cell apoptosis by the up-regulated expression of Bax protein and the down-regulated expression of Bcl-2 protein. Then the mitochondria permeability transition pore was open. The loss of mitochondrial transmembrane potential caused cascade reaction of Caspases by releasing cytochrome c and thus activating Caspase-9 and Caspase-3.