Flow Cytometric Determination of Apoptosis Apoptosis rate of DU145 cells was examined by flow cytometry by using annexin V-FITC/PI staining [11]. regulation of pro-apoptotic Bax protein. However, pre-treatment with the antioxidant N-acetylcysteine (NAC) significantly inhibited the activation of Bax and prevented down-regulation of STAT3 target genes. Collectively, our findings suggest that altholactone induces DU145 cells death through inhibition of NF-B and STAT3 activity. spp., which belongs to the styryl-lactone family, has been reported to display anticancer activities in human colorectal cancer (HCRC) cells through caspases-dependent and independent apoptotic pathways [12], in the cervical carcinoma HeLa cell line by decreasing Bcl-2 and increasing p53 expression [13] and in leukemia HL-60 cells by induction of apoptosis via oxidative stress [14]. We previously reported that altholactone inhibited cell growth and induced apoptosis in human bladder cancer T24 cells by causing mitochondrial membrane potential imbalance followed by MAPK-p38 activation and suppression of the Akt pathway [15]. However, the details of the mechanism of action of altholactone remain unclear. Open in a separate window Figure 1 Chemical structure of altholactone and its effects on cell viability: (A) Chemical structure; (B) Altholactone inhibited the cell Pantoprazole (Protonix) growth and induced cell death in prostate cancer cells. LNCaP, PC-3 and DU-145 cells were treated with the indicated doses of altholactone for 48 h and cell viability was measured by MTT assays. Data are expressed as mean SD (= 3). To date there are no reports of chemo-therapeutic effects of altholactone on human prostate cancer. Therefore, investigations have now been done for the first time to demonstrate the anti-proliferative potential of altholactone against human prostate cancer cells and then to delineate its underlying mechanisms of action. In this study, we revealed, by using DU145 cells as model, that altholactone inhibits transcriptional activity and phosphorylation levels of STAT3 in a dose-dependent manner. Further we present evidence that Pantoprazole (Protonix) altholactone results in induction of reactive oxygen species (ROS) generation in prostate cancer DU145 cells, followed by activation of Bax and suppression of STAT3 target gene products, including Bcl2, and survivin. 2. Results and Discussion 2.1. Altholactone-Induced Cell Growth Inhibition in Prostate Cancer Cells Natural plant products are an excellent potential source of novel anticancer agents. Over 70% of anticancer drugs developed in the last 30 years either are natural product-derived compounds from animals, plants and microorganisms [16]. The current study has been performed after random screening of Nature-derived drugs formerly selected from our own repositories. We choose compounds those were representative of specific classes of natural products we had previously reported [10,11,17,18]. The aim of this screening was to identify compounds that target ROS metabolism in cancer. Recently, we reported that altholactone induced ROS-mediated apoptosis in bladder cancer cells [15]. Here, we extend those previous Pantoprazole (Protonix) studies to examine the cytotoxic potential of altholactone on prostate cancer cells. MTT assays were performed on two androgen-independent human prostate cancer cell lines (PC-3 and DU145) and an androgen-dependent cell line (LNCaP) to assess the dose-dependent cytotoxicity of the compound. Drug concentration, altholactone, and cell viability work inversely, as cell viability decreases in DU145 cells expressing constitutively active STAT3 as the drug concentration increases, with an IC50 (concentration to achieve 50% of cell growth) value of 38.5 M. However drug exerted the lesser effect Pantoprazole (Protonix) SPP1 on PC-3 and LNCaP cells as comparedto the DU145 cells (Figure 1B). To support our previous results [15] that altholactone induces cytotoxic effects by targeting the ROS metabolism, pretreatment of DU145 with NAC (5 mM, a specific ROS inhibitor) was performed. The results showed that 5 mM NAC diminished the effect of alhtolactone on DU145 cells and support the notion that alhtolactone induces cytotoxic effects by targeting the ROS metabolism (data not shown). These findings are the parallel with the previous studies in which alhtolactone has been reported as a growth inhibitor of different cancer cell lines such as bladder cancer T24 cells [15], cervical carcinoma HeLa cells [13] and human colorectal cancer cells [12]. 2.2. Effect of Altholactone on Induction of DU145 Cell Cycle Recent reports related to cell cycle regulation have disclosed that cell cycle progression is tightly controlled by various checkpoints in normal cells and alterations in these checkpoints of cell cycle progression may lead to aberrant cell proliferation and development of cancer [19]. Cancer cells frequently acquire defects in the checkpoints and these results in cell cycle deregulation, which leads to unrestrained cell proliferation. Pharmacological corrections of these checkpoints followed Pantoprazole (Protonix) by proper progression of cell cycle can be an effective strategy to control the growth and proliferation of cancer cells [19,20]. In order to evaluate the whether the.