Effects And Mechanisms Of Xanthatin On Proliferation And Apoptosis Of Tumor Cells

Cancer is a leading systemic disease in both incidence and mortality worldwide. The most commom tumors in developing countries are lung, liver, gastric and cervical cancer. Surgical ablation, radiotherapy and conventional chemotherapy are three primary strategies for cancer treatment, but none of them has been proven completely effective due to their side effects and general toxicity. However, searching antitumor therapeutics from naturally occurring products has been demonstrated to be a promising approach for cancer patients.Among the natural product-derived candidates in preclinical or clinical development, a large group of compounds belonging to sesquiterpene lactones (SLs) have attracted much attention recently. For example, thapsigargin from Thapsia and parthenolide from Tanacetum parthenum that have already been in clinical trials, can selectively inhibit tumors. Therefore, identification of potential anticancer drugs from SLs arguably becomes a prosperous anti-cancer practice today. It is noteworthy that more than 3000 SLs with interesting biological activities have been isolated from plants of asteraceae family. Xanthatin is a bicyclic sesquiterpene lactone isolated mainly from xanthium plants, which has a wide range of pharmacological activity. Observational studies demonstrated that xanthatin had significant antitumor activity in a variety of cell culture systems implicated in colon, breast, lung, gastric and skin cancers. More recently, xanthatin was found to be a selective inducer of GADD45gamma and a highly effective inhibitor of MDA-MB-231 cell growth by inducing caspase-independent cell death. Xanthatin exerted fundamental therapeutic values for other crucial and specific molecular mechanisms, which needs full exploration.Objectives1 To investigate the effect of xanthatin on survival in a variety of tumor cells2 To investigate the effect of xanthatin on proliferation, apoptosis and glutathione S-transferase P1 of these selected tumor cells; to evaluate the inducing apoptosis of xanthatin in tumor cells and to explore its possible molecular mechanisms.Methods1 The effect of xanthatin on proliferation of tumor cells was determined by the MTS assay. After being cultured overnight, tumor cells were exposed to 0.1,1,2,4,8,10,20, 40,80 μmol/L xanthatin for 24 h. DMSO was used as a negative control. Inhibitory effect of of xanthatin was used the value of OD in the human hepatocellular carcinoma cell line HepG2, human lung adenocarcinoma epithelial cell line A549, human cervical carcinoma squamous cell carcinoma cell line SiHa and human cervical adenocarcinoma cell line Hela.2 HepG2 and SiHa cells were exposed to 0.1,1,5,10,20,40 μmol/L xanthatin for 12, 24,48 h. MTS method was detected the growth inhibition in tumor cells. Morphological changes on tumor cells were observed with fluorescence microscope. The apoptosis rate of HepG2 and SiHa cells exposed to different concentration of xanthatin was detected by flow cytometry. These changes of GSTP1 expression and activity in HepG2 and SiHa cells exposed to different concentration of xanthatin were determined by immuohistochemistry and colorimetric method. Changes of p-JNK expression in SiHa cells exposed to different concentration of xanthatin were determined by western blot. These expressions of GSTP1、JNK、p-JNK、Bax、Bcl-2、Caspase-3、Caspase-9、 Cytochrome c (Cyt c) in HepG2 cells were assayed by western blot.Results1 Screening effects of xanthatin on proliferation and apoptosis of tumor cells0.1,1,2,4,8,10,20,40,80μmol/L xanthatin exerted a potent antiproliferative effect on HepG2, A549, SiHa, Hela cells in a dose-dependent manner. IC50 values of HepG2, A549, SiHa, Hela cells exposed to xanthatin for 24 h were 21.5,45,18,35.9 umol/L. Flow cytometry of HepG2 and SiHa cells at 24 h after expossed to xanthatin showed that a dose-dependent increase of early apoptotic cells. The double positive staining of particular cells (which were in a late apoptotic stage or necrotic)were not changed. However, xanthatin induced apoptosis at a late apoptotic stage in A549 cells and no changes in Hela cells.2 Effects and mechanisms of xanthatin on proliferation and apoptosis of HepG2 and SiHa cells2.1 Xanthatin inhibited HepG2 and SiHa cells growthXanthatin(0-40 μmol/L) significantly inhibited the proliferation on HepG2, SiHa cells in a time and dose-dependent manner. The morphological changes observed in xanthatin-treated cells included cell shrinkage, roundup and even some apoptotic bodies existed.2.2 Xanthatin induced apoptosis in HepG2 and SiHa cellsAfter xanthatin treatment for 24 h in HepG2, SiHa cells, the apoptotic ratios were 5.72%,10.26%,20.5%,33.9%,40.1% and 5.02%,9.62%,18.5%,26.4%,37.5%. With concentration of xanthatin at 0,5,10,20,40 μmol/L. Compared with the control, apoptosis rate increased obviously.2.3 Molecular virtual docking about xanthatin and GSTP1Xanthatin and unbound protein in docking operation were found with the H site and substrate binding site G site binding. Agreement with positive control group with EA docking.2.4 Effect of xanthatin on the expression and activity of GSTP1 in HepG2 and SiHa cellsHepG2, SiHa cells exposed to increasing concentration of xanthatin(1～40 μmol/L) led to expression and activity of GSTP1 down-regulated.2.5 Effect of xanthatin on the expression of p-JNK in SiHa cells and GSTP1,JNK, p-JNK,Bax,Bcl-2,Caspase-3,Caspase-9,Cyt c in HepG2 cellsXanthatin increased the expression of p-JNK, inhibited the expression of GSTP1, improved the ratio of Bax/Bcl-2 in HepG2 cells. Furthermore, xanthatin induced the release of Cyt c, caspase-3 and caspase-9 in HepG2 cells. SiHa cells exposed to increasing concentration of xanthatin led to the expression of p-JNK up-regulated.ConclusionsThese results demonstrate that xanthatin exerts anti-tumor effects on HepG2, SiHa cells through its anti-proliferative and pro-apoptotic roles. The molecular mechanism for pro-apoptotic effect may be related to its down-regulated effects on GSTP1,up-regulated effects in c-Jun N-terminal kinase (JNK) pathway.