Inducing Apoptosis Of Acanthopanax Senticosus On Renal Carcinoma Cell Line 786-O

Acanthopanax senticosus(Rupr.et Maxim.)Harms(AS)is classified into the family of Araliaceae,which has been recorded in the Pharmacopoeia of China as a traditional Chinese herb.This plant has multiple pharmacological activities,such as anti-cancer,antioxidation,Hypoglycemic effect,improving microcirculation, protecting myocardial ischemie,stimulating rat-blood macrophages to perform phagocytosis,treatment of arthritis and treatment of neurasthenia,et al.The incidence of renal cell carcinoma in urinary and reproductive system is just second to bladder carcinoma,and not sensitive to chemotherapy,radiotherapy and endocrine therapy. It's a research focus in searching for anti-tumor drugs from natural products.The AS leaves hasn't been recorded in Chinese Pharmacopeia.Whether the AS leaves have the similar medical value as the traditional medical parts or not needs to be studied further.The renal cancer cell line(786-O)is used to as the object of this study,for investigating the anti-proliferative effect of Acanthopanax senticosus leaves on 786-O cells and a preliminary study on the mechanism of inducing apoptosis.This study is firstly on the detecting and analyzing the main components of AS leaves,in which the content of flavonoids is 13.47%±0.5,the content of saponins is 4.17%±0.3,the content of polysaccharides is 1.94%±0.002,the content of lipid is 2.09%±0.0006.These results have proved AS leaves has the similar pharmaceutical components as the traditional medical parts,which has Ginseng-like medicinal components such as saponins,polysaccharides and flavonoids.In order to find the best anti-tumor component in AS leaves,five different components water extract of Acanthopanax Senticosus(WEAS),ethanol extract of Acanthopanax Senticosus(EEAS),Acanthopanax Senticosus flavonoids(ASF), Acanthopanax Senticosus saponins(ASS)and petroleum ether extract of Acanthopanax Senticosus(PEEAS)were extracted and separated.The anti-tumor effect of these five components were to be evaluated by MTT and SRB assay.The results showed that the ASS had the strongest anti-tumor effect,then the PEEAS, EEAS,WEAS,and the ASF had almost no inhibiting effect on 786-O within the dose range of 0-120μg/mL.Then the cytotoxic effect of ASS on 786-O were compared with that on human normal kidney cell line(HEK-293).The results showed that ASS a significant inhibitory effect on 786-O within the dose range of 20-100μg/mL which was in a dose dependent manner,and nearly no inhibitory effect on the proliferation of HEK-293 within the dose range of0-150μg/mL. In order to further investigate the effect of ASS induced-apoptosis on 786-O, several methods of morphological observation were used,such as observed by inverted light microscope,Wright staining,acridine orange staining,DAPI staining and electron microscope,the results showed that the morphological changes were induced by ASS with apoptosis features on 786-O,but the DNA Ladder wasn't tested probably because the rate of apoptosis was very low.In addition,the effects of ASS on the cell cycle and the expression of Bcl-2 protein were determined by flow cytometry(FCM).NF-κB were detected by immunofluorescent staining.The results were follows:①typical morphological changes:the cell shrinked,showed deformation and loosed contact to its neighbouring cells.Its chromatin condensed and marginated at the nuclear membrane,the plasma membrane was blebbing or budding, and finally the cell was fragmented into compact membrane-enclosed structures, called 'apoptotic bodies' which contain cytosol,the condensed chromatin,and organelles.But DNA ladder assay and FCM showed the apoptosis rate was low.②the cell cycle had changed,the cell ratio of G0/G1 phase increased significantly,and that of S phase reduced,which showed that ASS could inhibit the 786-O cells in G0/G1 phase.③The expressions of Bcl-2 protein was down-regulated,which might be the molecular mechanism of ASS inducing apoptosis on 786-O.④NF-κB might not be the signal pathway for ASS inducing apoptosis on 786-O.This study preliminary confirmes the effect of ASS inducing apoptosis on 786-O, and investigates that may relate to the change of the cell cycle and the down-regulation expression of Bcl-2 protein.This provides a basis for the further study of ASS inducing apoptosis at molecular level.