cytochrome-c-t and beta-hederin

α-Hederin, a monodesmosidic triterpenoid saponin, exhibited promising antitumor potential against a variety of human cancer cell lines. However, few related studies about effects of α-hederin on gastric cancer are available. Herein, our results showed that α-hederin significantly inhibited the proliferation of gastric cancer cells and arrested the cell cycle in G1 phase in vitro (p < .05). Further research of the potential mechanism reflected that α-hederin could induce intracellular glutathione decrement, adenosine triphosphate level, and mitochondrial membrane potential variation via inducing reactive oxygen species accumulation during the apoptosis of gastric cancer cells. Moreover, the detection of mitochondrial and cytosol proteins with apoptosis-inducing factor, apoptosis protease activating factor-1, and cytochrome C showed an increase in the cytosol, followed by a decrease of Bcl-2 levels and increases of caspase-3, caspase-8, caspase-9, and Bax, which revealed that α-hederin induced apoptosis via triggering activation of the mitochondrial pathway. Furthermore, the above changes were amplified when pretreated with buthionine sulfoximine, whereas attenuated in the group pretreated with NAC than α-hederin alone (p < .05). In addition, α-hederin significantly inhibited the growth of xenografted gastric tumors with favorable safety. In conclusion, α-hederin could inhibit the proliferation and induce apoptosis of gastric cancer accompanied by glutathione decrement and reactive oxygen species generation via activating mitochondrial dependent pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Glutathione; Humans; Membrane Potential, Mitochondrial; Mitochondria; Oleanolic Acid; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Saponins; Stomach Neoplasms

α-Hederin has been shown promising anti-tumor potential against various cancer cell lines. However, reports about effects of α-hederin on esophageal squamous cell carcinoma (ESCC) are still unavailable.. To investigate the inhibitory effects of α-hederin on ESCC and explore the underlying mechanism.. Human esophageal carcinoma cell line (Eca-109) was used for the experiment. Cell Counting Kit-8, flow cytometry, Hoechst 33258 staining, enhanced ATP assay kit, 2',7'-dichlorofluorescin diacetate, JC-1 kit, and Western bolt were used to assess the cell viability, cycle, apoptosis, cellular ATP content, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. Xenografted tumor model was constructed to evaluate the in vivo anti-tumor effects of α-hederin.. Compared with control group, α-hederin significantly inhibited the proliferation, induced apoptosis of ESCC, and arrested the cell cycle in G1 phase (P < 0.05). α-Hederin induced the accumulation of ROS, decrement of ATP levels, and disruption of MMP (P < 0.05). The detection of mitochondrial and cytosol proteins showed that AIF, Apaf-1, and Cyt C were released and increased in cytoplasm, and then, caspase-3, caspase-9, and Bax were involved and increased, while Bcl-2 level was decreased (P < 0.05). Furthermore, the above changes were amplified in the group pretreated with L-buthionine sulfoximine, while N-acetyl-L-cysteine plays an opposite role (P < 0.05). Meanwhile, α-hederin significantly inhibited the growth of xenografted tumors with favorable safety.. α-Hederin could inhibit the proliferation and induce apoptosis of ESCC via dissipation of the MMP with simultaneous ROS generation and activation of the mitochondrial pathway.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Apoptosis Inducing Factor; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Flow Cytometry; Humans; In Situ Nick-End Labeling; In Vitro Techniques; Male; Membrane Potential, Mitochondrial; Mice, Nude; Mitochondria; Neoplasm Transplantation; Oleanolic Acid; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Saponins; Xenograft Model Antitumor Assays

alpha-Hederin, a pentacyclic triterpene saponin isolated from the seeds of Nigella sativa, was recently reported to have potent in vivo antitumor activity against LL/2 (Lewis Lung carcinoma) in BDF1 mice. In this study we observed that alpha-hederin caused a dose- and time-dependent increase in apoptosis of murine leukemia P388 cells. In order to evaluate the possible mechanisms for apoptosis, the effects of alpha-hederin on intracellular thiol concentration, including reduced glutathione (GSH), and protein thiols, and the effects of pretreatment with N-acetlycysteine (NAC), a precursor of intracellular GSH synthesis, or buthionine sulfoxime (BSO), a specific inhibitor of intracellular GSH synthesis, on alpha-hederin-induced apoptosis were investigated. It was found that alpha-hederin rapidly depleted intracellular GSH and protein thiols prior to the occurrence of apoptosis. NAC significantly alleviated alpha-hederin-induced apoptosis, while BSO augmented alpha-hederin-induced apoptosis significantly. The depletion of cellular thiols observed after alpha-hederin treatment caused disruption of mitochondrial membrane potential (deltapsi(m)) and subsequently increased the production of reactive oxygen species (ROS) in P388 cells at an early time point. Bongkrekic acid (BA), a ligand of the mitochondrial adenine nucleotide translocator, and cyclosporin (CsA) attenuated the alpha-hederin-induced loss of deltapsi(m), and ROS production. Thus, oxidative stress after alpha-hederin treatment is an important event in alpha-hederin-induced apoptosis. As observed in this study, permeability transition of mitochondrial membrane occurs after depletion of GSH and precedes a state of reactive oxygen species (ROS) generation. Further, we observed that alpha-hederin caused the release of cytochrome c from the mitochondria to cytosol, leading to caspase-3 activation. Our findings thus demonstrate that changes in intracellular thiols and redox status leading to perturbance of mitochondrial functions are important components in the mechanism of alpha-hederin-induced cell death.

Topics: Animals; Apoptosis; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; Glutathione; Humans; Intracellular Membranes; Leukemia P388; Mice; Mitochondria; Nigella sativa; Oleanolic Acid; Oxidative Stress; Plant Extracts; Reactive Oxygen Species; Saponins; Tumor Cells, Cultured