cytochrome-c-t and tetrandrine

Although neoadjuvant chemotherapy has improved the survival rate of osteosarcoma patients, drug resistance remains a predominant obstacle to improving efficacy and necessitates the development of novel chemotherapeutical agents. The aim of this study was to investigate whether tetrandrine (TET) induces apoptosis in the U-2OS and MG-63 osteosarcoma cell lines and to further determine the underlying mechanism. This study investigated the effects of TET on osteosarcoma in vitro. To examine the antitumor effects of TET on osteosarcoma, the two osteosarcoma cell lines were treated with TET and subjected to apoptosis assays. The results revealed that TET induced the apoptosis of osteosarcoma cells in a time- and dose-dependent manner. Furthermore, the apoptosis of osteosarcoma cells was accompanied by increased cytochrome c (Cyto-C), apoptotic protease-activating factor (Apaf)-1, Bid and Bax activation and reduced Bcl-2 and Bcl-xl activation, demonstrating that the apoptosis may have occurred through the mitochondrial pathway. In conclusion, the results suggest that TET is a promising agent for osteosarcoma therapy.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Benzylisoquinolines; BH3 Interacting Domain Death Agonist Protein; Caspases; Cell Line, Tumor; Cytochromes c; Humans; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Currently, multi-drug resistance (MDR) to anticancer drugs is a major obstacle to successful treatment of cancer. Looking for novel compounds with anti-MDR activity is an effectively way to overcome cancer drug resistance. Here, we found that H1, a novel derivate of Tetrandrine, displayed anti-MDR activity in vitro and in vivo. Average resistant factor of H1 is only 1.6. In KB and KBv200 cancer cells xenograft mice, H1 also displayed favorable anti-MDR activity. It could induce typical apoptosis as indicated by morphologic changes, DNA fragmentation in sensitive and resistant cancer cells. Further studies showed that H1 treatment resulted in the increase of ROS generation, elevation of the Bax/Bcl-2 ratio, loss of mitochondrial transmembrane potential (ΔΨ(m)), release of cytochrome c and AIF from mitochondria into cytosol, and activation of caspase-9 and caspase-3, but had no effect on activation of caspase-8 and the expression of Fas/FasL. On the other hand, H1 also inhibited survival pathways such as the activation of Erk1/2 and Akt1/2. In conclusion, H1 exerts good anti-MDR activity in vitro and in vivo, its mechanisms may be associated with initiating intrinsic apoptosis pathway and inhibiting the activation of Erk1/2 and Akt1/2. These findings further support the potential of H1 to be used in clinical trial of MDR cancer treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzylisoquinolines; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cytochromes c; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Enzyme Activation; Female; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Transplantation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Tetrandrine, a bisbenylisoquinoline alkaloid isolated from the dried root of Stephenia tetrandra (S Moore), possesses a remarkable pharmacological profile. However, the mechanisms of tetrandrine hepatotoxicity remain to be elucidated. In this study, we first proved apoptosis and mitochondrial dysfunction induced by tetrandrine in Sprague-Dawley rat liver in vivo. By further assuming apoptosis as an important mechanism in tetrandrine-induced hepatotoxicity, we focused on mitochondria-initiated apoptosis in primary hepatocytes isolated from Sprague-Dawley male rats. Tetrandrine treatment led to significant release of cytochrome c and downregulation of Bcl-X(L) accompanied by caspase 3 activation, and ultimately, DNA fragmentation. Caspase 3 activation was markedly inhibited by cyclosporin A (CsA) and Ac-DEVD-CHO. Furthermore, Endo G, a caspase-independent apoptotic protein, was detected for its expression and DNase activity. CsA blocked the release both of Endo G and cytochrome c significantly. Additionally, the generation of reactive oxygen species (ROS) increased in a time-dependent manner corresponding with a fall in intracellular GSH content after 10 microM tetrandrine treatment in 4h. Tetrandrine also induced mitochondrial dysfunction indicated by transition of mitochondrial transmembrane potential and decrease of intracellular ATP level. The findings indicated that the caspase-dependent mitochondrial apoptosis pathway was primarily involved in tetrandrine-induced apoptosis in rat primary hepatocytes. In addition, a caspase-independent pathway indicated by Endo G also contributed to apoptosis caused by tetrandrine. Meanwhile, ROS was proved an important inducer in this apoptosis process.

Topics: Alkaloids; Animals; Apoptosis; bcl-X Protein; Benzylisoquinolines; Blotting, Western; Caspase 3; Caspase Inhibitors; Caspases; Cell Survival; Cytochromes c; Drugs, Chinese Herbal; Electrophoresis, Polyacrylamide Gel; Endodeoxyribonucleases; Enzyme Inhibitors; Hepatocytes; In Situ Nick-End Labeling; Intracellular Membranes; Male; Membrane Potentials; Mitochondria, Liver; Mitochondrial Swelling; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction

Studies have shown that silica induces apoptosis through mechanisms that also regulate the inflammatory responses of lung cells to silica exposure. Although implicated in cell culture studies, the major in vivo pathway through which silica induces apoptosis has not been characterized. The present study is to study the role of mitochondria in silica-induced oxidative stress and apoptosis in vivo. Rats were intratracheally instilled with saline or silica (20 mg/kg) and sacrificed at 3 days post-exposure unless otherwise specified. Alveolar macrophages (AM) were harvested by bronchoalveolar lavage and measured for apoptosis and secretion of inflammatory mediators in the presence or absence of appropriate inhibitors. Concurrent studies were carried out to determine the presence of intracellular reactive oxygen species (ROS) via confocal microscopy, mitochondrial trans-membrane potential by flow cytometry, mitochondrial release of cytochrome c, and the activation of caspase activities in AM by Western blot analysis. Silica was shown to induce elevated levels of intracellular ROS, resulting in a marked decrease in intracellular glutathione (GSH) and cysteine and a sustained presence of apoptotic AM in silica-exposed rats up to two weeks post-exposure. The apoptotic AM were characterized by decreased mitochondrial trans-membrane potential, increased mitochondrial release of cytochrome c, activated caspase 9 (but not caspase 8) and caspase 3 activities, and PARP degradation, comparing to cells from the saline control. Silica induced AM production of IL-1 and TNF-alpha, which may be inhibited by ex vivo treatment of cells with N-acetylcysteine (NAC) or microtubule modifiers such as tetrandrine and taxol. NAC was shown to prevent intracellular GSH depletion and silica-induced production of IL-1beta and TNF-alpha but not apoptosis in AM from silica-exposed rats. These results show that silica-induced apoptosis is mediated through the mitochondrial pathway but not through cellular production of inflammatory cytokines, ROS generation, however, induces both apoptosis and cellular secretion of inflammatory mediators.

Topics: Acetylcysteine; Alkaloids; Animals; Anti-Inflammatory Agents; Apoptosis; Apoptosis Regulatory Proteins; Benzylisoquinolines; Caspase 3; Caspases; Cysteine; Cytochromes c; Disease Models, Animal; Glutathione; Inflammation Mediators; Macrophages, Alveolar; Male; Membrane Potentials; Microscopy, Confocal; Mitochondria; Oxidative Stress; Paclitaxel; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Silicon Dioxide; Sulfhydryl Compounds; Tumor Necrosis Factor-alpha