Study On The Mechansim Of Compounds-induced Apoptosis From Mirabilis Himalaica In Tumor Cells

In our previously study,(E)-3-(4-hydroxy-2-methoxyphenyl)-propenoic acid4-hydroxy-3-methoxyphenyl ester (1) and Mirabijalone E (2) were isolated from ethyl acetate extract of Mirabilis himalaica exhibited in vitro cytotoxicity. Compound1showed cytotoxicity against human lung cancer cells HepG2with IC50value at20.07μg/mL, and compound2against human hepatoma cells A549with IC50value at15.12μg/mL. In this paper, pharmacological effects on1and2were researched, and the studies were as follows:1. Brdu and soft agar assay detected if compound1affects HepG2cells proliferation. The results showed the numbers of BrdU-positive cells significantly decreased compared with that of DMSO-treated groups after treated with1for48h. The statistical analysis showed compound1induced approximately40%reduction in BrdU-positive cells. In addation, the clonogenic capacity of HepG2cells was affected of1in a soft agar culture system. Compared with DMSO-treated, HepG2cells gave rise to significantly fewer and smaller colonies after treated with1. In addition, colony numbers decreased about37%in1-treated cells. PI staining assay revealed that1induced HepG2S-phase cell cycle arrest. And the result showed that the percentage of S phase increased from18.8%to36.0%, and of G1phase decreased from70.8%to52.6%in1-treated cells compared with the control. No significant changes occurred in the percentage of G2/M phase. Several different cyclin-dependent kinases (CDKS) regulate the cell cycle progression by binding to different types of cyclins. Western blot analysis showed that the expression levels of CDK2, CDK4, CDK6, Cyclin D1and Cyclin A were upregulated. In our previously study, the results showed that1could induce apoptosis in HepG2cells. p53was found in human tumors, which as the most relevant genes by far, and more than50%of human cancers usually occurs p53genetic mutation. HepG2cells have endogenous wild-type p53as tumor suppressor gene. Thence, western blot analysis showed an increase in expression level of p53and p21in1-treated cells compared with DMSO-treated cells. It has been reported that p53can completed the regulation of apoptosis by Bax/Bcl-2, Fas/Apol, IGF-BP3proteins. Bcl-2family proteins are described as critical regulators of the mitochondrial apoptosis pathway. It is known that suppression of anti-apoptotic members or activation of pro-apoptotic members of the Bcl-2family, usually leads to an altered mitochondrial membrane permeability, which allows the release of cytochrome c into the cytosol and the subsequent activation of caspases. Therefore, we examined the effects of1on the expression level of Bax, Bcl-2and caspase-3in HepG2cells by western blot. Results showed that pro-apoptotic protein, Bax is upregulated by1compared with DMSO, while the expression of the anti-apoptotic protein, Bcl-2was downregulated. Futhermore, we found that overexpression of Bcl-2in HepG2cells employing retrovirus-mediated gene transfer dramatically eliminated the apoptotic effect induced by1. And endogenous p53gene was knoched down by the transfection with p53gene specific shRNA in HepG2cells, and followed by treatment of1for48h to evaluate the apoptotic effects employing flow cytometry assay. The results demonstrated that1slightly increased the cell apoptosis from10.1%to20.6%in p53si HepG2cells compared with the DMSO-treated groups. At the same time,1significantly caused the cell apoptosis from9.0%to68.8%in GFPsi HepG2cells. Above results illustrated that compound1induced apoptosis through activation of mitochondrial apoptosis pathway in p53-dependent manner. Moreover, In vivo xenograft study in nude mice showed that mirabijalone E inhibited tumor growth derived from A549cells, and we also kept track of the mice body weight and there was no significant change during the1treatment which implied no apparent side effect on the growth of mice.2. Brdu and soft agar assay detected if compound2affects A549cells proliferation. The results showed the numbers of BrdU-positive cells was significantly decreased by2compared with DMSO-treated group, and treatment with20μg/mL of2for48h resulted in a approximately60%reduction in the percentage of BrdU-positive cells compared to DMSO control. Addationly, A549cells gave rise to significantly fewer and smaller colonies after treated with2and the statistical analysis showed that colony numbers decreased about70%in2-treated cells. PI staining assay revealed the percentage of the cells in S phase obviously increased from24.09%to48.74%(control group:18.37%) after the cells were treated with2concentrations raising from20p.g/mL to40μg/mL for48h. Above observations demonstrated that2inhibited cell proliferation through cell cycle arrest. Annexin V-FITC and PI staining showed that the apoptotic cell gave rise to31.7%and to48.0%when treated with20μg/mL and40μg/mL of2for48h, respectively, while the control resulted in11.19%. Western blot analysis showed the Bax protein expression was increased significantly but Bcl-2protein expression was decreased, markedly. Caspase-3is considered to be one of the principal executing caspases in which all of the biochemical routes involved in the apoptosis response converge. To study if caspase-3could be activated, the protein expression was examined in A549cells using western blot analysis. The results showed that after treatment with2for48h, caspase-3was activated through proteolytic degradation of the inactive pro-enzyme (35KD) into the active form (17KD and12KD). Above datas illustrated that2may induce apoptosis through activation of mitochondrial apoptosis pathway. In addation, Furthermore, mirabij alone E suppressed the tumor growth tumor growth derived from A549cells in vivo.