Apoptotic signaling pathways are the most promising therapeutic targets for cancer treatment [17C26]. Cisplatin induces a significant apoptotic response in GSK 366 cancer cells, and impaired apoptosis is one of the molecular mechanisms of chemoresistance to cisplatin in cancer cells. Bak). The extrinsic pathway is usually brought on by ligand binding to specific death receptors around the cell surface. Both pathways lead to the activation of caspase cascades. Apoptotic signaling pathways are the most promising therapeutic targets for cancer treatment [17C26]. Cisplatin induces a significant apoptotic response in cancer cells, and impaired apoptosis is one of the molecular mechanisms of chemoresistance to cisplatin in cancer cells. Considering that -elemene blocks the cell cycle at G2/M phase and that cells accumulated in G2/M phase often enter the apoptotic process, we hypothesized that -elemene sensitizes resistant human ovarian cancer cells to cisplatin through the induction of apoptosis. To test this hypothesis, we designed a series of experiments to detect apoptotic responses in cancer cells treated with either -elemene or cisplatin alone, or the combination of both drugs. We found that -elemene dramatically increased cisplatin anticancer activity in resistant human ovarian cancer cells by the induction of a remarkable apoptotic response mediated by a mitochondria- and caspase-dependent cell death pathway. These findings may have profound implications in ovarian cancer chemotherapy. Materials GSK 366 and methods Chemicals and immunoreagents The (?)–elemene (98 % purity) was obtained from Yuanda Pharmaceuticals, Ltd, Inc. (Dalian, China). Cisplatin, dimethyl sulfoxide (DMSO), and propidium iodide (PI) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Antibodies against caspase-3, caspase-8, caspase-9, Bax, Bcl-2, Bcl-XL, cytochrome release into the cytosol The mitochondrial and cytosolic fractions were isolated using Mitochondrial Isolation Kit (Sigma-Aldrich). Briefly, 3 107 cells were harvested and washed with PBS. The GSK 366 cells were suspended in 10 volumes of mitochondrial extraction buffer A containing 2 mg/ml albumin and homogenized on ice by a Wheaton Dounce homogenizer. Unbroken cells and nuclei were removed by centrifugation at 600for 5 min at 4 C. The supernatant was further centrifuged at 11,000for 10 min. The supernatant was saved as a cytosolic fraction while the precipitate was dissolved in storage buffer A and saved as the mitochondrial fraction. The cytosolic fraction was analyzed by Western blotting with an anti-cytochrome monoclonal antibody. Measurement of mitochondrial membrane potential by flow cytometry using BD MitoSensor? reagent Changes in mitochondrial membrane potential (for 5 min. The cell pellet was suspended in incubation buffer and analyzed by Rabbit polyclonal to COT.This gene was identified by its oncogenic transforming activity in cells.The encoded protein is a member of the serine/threonine protein kinase family.This kinase can activate both the MAP kinase and JNK kinase pathways. flow cytometry. The green fluorescence represented the geometric mean fluorescence of the cells. Higher geometric mean fluorescence indicated lower test was used to analyze the differences between the means of treatment groups and the control group. Differences with a value of less than 0.05 were considered statistically significant. Results -Elemene enhanced cisplatin-induced apoptotic membrane changes in ovarian cancer cells, as detected by annexin V binding Translocation of phosphatidylserine to the outer surface of the cytoplasmic membrane is an early feature of apoptosis. Annexin V and propidium iodide (PI) binding was used to evaluate the surface expression of phosphatidylserine. Cells staining with annexin V alone have early apoptotic changes and intact cell membranes, whereas cells staining with annexin V and PI have membrane disintegration consistent with necrosis or a late stage of apoptosis. A2780/CP cells treated with GSK 366 both -elemene and cisplatin for 48 h exhibited a significant increase in apoptosis and necrosis, as compared with untreated control cells, cells treated with -elemene alone, or cells treated with cisplatin alone (Fig. 1a). The percentages of apoptosis plus necrosis in A2780/CP cells after each treatment were 1.35 % (untreated control cells), 20.17 % (-elemene alone), 7.09 % (10 M cisplatin alone), 10.41 % (20 M cisplatin alone), 54.74 % (-elemene plus 10 M cisplatin), and 59.98 % (-elemene plus 20 M cisplatin). Similar data were obtained in MCAS cells (Fig. 1b). The percentages of early plus late apoptosis in MCAS cells after each treatment were 0.1 % (untreated control cells), 24.81 % (-elemene alone), 8.54 % (cisplatin alone), and 58.15 % (-elemene plus cisplatin). The increases in the surface expression of.