cytochrome-c-t and triptolide

The hepatotoxicity of Tripterygium wilfordii Hook. f. (TW), due to the presence of triptolide (TP), limits its therapeutic potential. Based on the traditional Chinese medicine theory, the theory of "Yi lei xiang zhi" was proposed that Chinese herbs with different efficacy can restrict each other to achieve the least adverse reactions.. To observe the effects of Catapol (CAT) and Panax notoginseng saponins (PNS), active ingredients in Rehmannia glutinosa (RG) and Panax notoginseng (PN) respectively, on reducing TP-induced hepatotoxicity, and further to explore the mechanisms.. The human hepatic cell line L-02 was cultured and treated with CAT, PNS or Combinations, and then treated with TP. The cytotoxic assay, the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), apoptosis, mitochondrial membrane potential and the expressions of NF-E2-related factor 1 (Nrf1) and its downstream targets were detected. Rats were treated with TP, TP + CAT, TP + PNS, or the combinations for 4 weeks. The levels of ALT, AST and LDH in serum, apoptosis of liver cells, mitochondria injury and the protein expressions of Caspase 3 and Nrf1 were investigated.. CAT, PNS or CAT+PNS pre-treatment inhibited TP-induced toxicity in L-02 cells, distinctly decreased the apoptosis, alleviated the reduction of mitochondrial membrane potential, and modulated the expressions of Nrf1 and its downstream target, the mitochondrial transcription factor A (TFAM) and cytochrome C (Cyt-C). CAT, PNS or CAT+PNS inhibited the TP-induced hepatotoxicity in SD rats by reducing the mitochondria injury, decreasing the cells apoptosis and increasing the Nrf1 protein expression. Noticeably, TP + PNS + CAT combinations exhibited more effective than any single ingredient alone.. PNS and CAT were able to effectively attenuate TP-induced hepatotoxicity. The efficiency benefits from their modulating Nrf1 and its downstream genes TFAM and Cyt-C, and further influencing mitochondrial functions and cells apoptosis. The combination is more effective than single ingredient alone.

Topics: Animals; Apoptosis; Biomarkers; Caspase 3; Cell Line; Chemical and Drug Induced Liver Injury; Cytochromes c; Cytoprotection; Disease Models, Animal; Diterpenes; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Therapy, Combination; Drugs, Chinese Herbal; Epoxy Compounds; Female; Humans; Liver; Membrane Potential, Mitochondrial; Mitochondria, Liver; Mitochondrial Proteins; NF-E2-Related Factor 1; Panax; Phenanthrenes; Phytotherapy; Plants, Medicinal; Quaternary Ammonium Compounds; Rats, Sprague-Dawley; Saponins; Transcription Factors

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Autophagy; B-Lymphocytes; Caspase 3; Caspase 8; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Diterpenes; DNA Damage; Endoplasmic Reticulum Chaperone BiP; Epoxy Compounds; Leukemia; Lymphocyte Activation; Macrophages; Male; Medicine, Chinese Traditional; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Phenanthrenes; Reactive Oxygen Species; Spleen; T-Lymphocytes; Transplantation, Homologous

Triptolide, a diterpenoid trioxide purified from the Chinese herb Tripterygium wilfordii Hook F, has been used as a natural medicine in China for hundreds of years. Several reports have demonstrated that triptolide inhibits the proliferation of cancer cells in vitro and reduces the growth of several types of tumors in vivo. To address the potential of triptolide as a novel therapeutic agent for patients with multiple myeloma, we investigated the effects of triptolide on the induction of apoptosis in human multiple myeloma cells in vitro. Triptolide rapidly induces apoptotic cell death in various myeloma cell lines. Triptolide-induced apoptosis in myeloma cells is associated with the loss of mitochondrial transmembrane potential (∆ψm), the release of cytochrome c and Smac/DIABLO from mitochondria into the cytosol, and the activation of caspase-3 and caspase-9. Furthermore, triptolide induces a rapid decline in the levels of Mcl-1 protein that correlates with caspase activation and induction of apoptosis. Inhibition of Mcl-1 synthesis by triptolide occurs at the level of mRNA transcription and is associated with an inhibition of phosphorylation of RNA polymerase II CTD. These results indicate that Mcl-1 is an important target for triptolide-induced apoptosis in myeloma cells that occurs via inhibition of Mcl-1 mRNA transcription coupled with rapid protein degradation through the ubiquitin-proteasome pathway.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Diterpenes; Epoxy Compounds; G1 Phase Cell Cycle Checkpoints; Humans; Intracellular Signaling Peptides and Proteins; Medicine, Chinese Traditional; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Proteins; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Phenanthrenes; Transcription, Genetic

Triptolide (TP), a major active ingredient extracted from the widely used Chinese herb Tripterygium wilfordii Hook f. (TWHF), has been demonstrated to possess various biological activities. However, the clinical applications of TP are limited by its narrow therapeutic window and severe toxicity. The current study aimed to investigate the roles of reactive oxygen species (ROS) and mitochondria in TP-induced cardiac injury. Male BALB/C mice were intravenously (i.v.) treated with a single dose of TP (1.2 mg/kg). After 24h, TP induced the oxidative stress, mitochondrial dysfunction, apoptotic damage, and pathological changes of heart tissue. In vitro studies also indicated that the cytotoxic effects of TP involved the ROS-mediated mitochondria-dependent pathway in H9c2 cells. TP treatment increased the accumulation of ROS and subsequently triggered cell apoptosis by depolarizing the mitochondrial membrane potential (ΔΨm), reduced the ratio of Bax/Bcl-2, released cytochrome c and, ultimately, activated caspase-3. Nrf2, as well as its downstream antioxidants, were also suppressed by TP. Taken together, these results suggest that TP induces cardiotoxicity in vivo and in vitro via oxidative stress, which was associated with down regulated Nrf2 activation, and the mitochondria-mediated apoptotic signaling pathway.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line; Cytochromes c; Diterpenes; Epoxy Compounds; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mitochondria; Myocytes, Cardiac; NF-E2-Related Factor 2; Oxidative Stress; Phenanthrenes; Rats; Reactive Oxygen Species; Signal Transduction; Up-Regulation

Malignant peripheral nerve sheath tumours (MPNSTs) are invasive, hard-to-treat, soft tissue sarcomas. In this study, in vitro and in vivo effects of triptolide were investigated using human MPNST cell lines.. Cultured STS-26T and ST88-14 cells were treated with 0-100 ng/ml triptolide (for determination of cell proliferation by sulphorhodamine B assay), with 12.5 ng/ml or 25 ng/ml triptolide (for analysis of caspase activity, effects on apoptotic pathway intermediates [by Western blots and flow cytometry], and for measurement of vascular endothelial growth factor [VEGF] and epidermal growth factor receptor [EGFR] levels by enzyme-linked immunosorbent assay). A xenograft model was established by injection of STS-26T cells into nude mice, and the effects of 250 μg/kg triptolide on tumour growth and apoptosis were compared with controls.. Triptolide significantly inhibited cell proliferation and induced apoptosis in vitro, through activation of caspases, in a dose- and time-dependent manner; VEGF and EGFR levels were suppressed. In vivo, triptolide inhibited the growth of STS-26T xenografts and reduced apoptosis.. Triptolide may have a therapeutic benefit in MPNST treatment.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Diterpenes; Enzyme Activation; Epoxy Compounds; ErbB Receptors; Female; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Neurilemmoma; Phenanthrenes; Proto-Oncogene Proteins c-bcl-2; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Renal cell carcinoma (RCC) is the most frequent type of renal-originated malignancy. Although nephrectomy is successfully used to save the lives of patients with localized RCC, treatment of advanced and other refractory RCCs is poor and still inadequate. Here, we show that triptolide, a small molecule and a well-known anti-inflammatory and anti-immunity agent used in the clinic, is capable of inducing cell apoptosis via the mitochondrial pathway in the 786-0 RCC cell line. This induction occurred in concert with reduced expression of genes related to the stabilization of mitochondria such as Bcl-2 and Bcl-XL. Cell cycle analysis showed that exposure to triptolide decreased the proportion of cells in the G0/G1 and G2/M phases, and increased the proportion of cells in the S phase. Cell accumulation in the S phase can be attributed to reduced expression of cell cycle checkpoint regulators such as cyclin A, cyclin B, CDK1, CDK2 and retinoblastoma proteins (Rb). These results raise the possibility that triptolide-induced apoptosis is mediated by cell cycle arrest. Similarly, in another human RCC cell line, OS-RC-2, triptolide-induced apoptosis and cell accumulation in S phase were also observed. Therefore, triptolide emerges as a stimulator of apoptosis by influencing coordinate regulation of proliferation and apoptosis, and may be applicable to the treatment of human renal cell carcinoma.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Carcinoma, Renal Cell; Caspases; Cell Cycle; Cytochromes c; Diterpenes; Epoxy Compounds; Humans; Kidney Neoplasms; Mitochondria; Phenanthrenes; Tumor Cells, Cultured

Triptolide, a purified diterpenoid triepoxide compound derived from a traditional Chinese medicine, Tripterygium wilfordii HOOK. f (TWHf), has been used in the treatment of autoimmune and inflammatory diseases. However, the toxicity of triptolide limits its application to a great extent. In the present study, we treated human normal liver L-02 cells (L-02 cells) with triptolide in vitro and investigated its toxic effects. The cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cellular viability and by flow cytometry and Hoechst 33258 staining for apoptosis. The mitochondrial membrane potential (delta psi m) was evaluated by flow cytometry with JC-1 as probe. After treatment with triptolide, a decrease in the viability of L-02 cells and increase in apoptosis were observed. Triptolide-induced apoptosis was accompanied by loss of mitochondrial membrane potential and release of cytochrome c (cyt-c) from the mitochondria to the cytosol and down-regulation of anti-apoptotic protein Bcl-2 levels with concurrent up-regulation in pro-apoptotic protein Bax levels and tumor suppressor protein p53 levels. Triptolide-increased activity of caspase 9 and caspase 3 was also observed. These results indicate that triptolide induced cytotoxicity in L-02 cells by apoptosis, which is mediated through mitochondrial pathway.

Topics: Annexin A5; Antineoplastic Agents, Alkylating; Apoptosis; bcl-2-Associated X Protein; Bisbenzimidazole; Blotting, Western; Caspase 3; Caspase 8; Caspase 9; Cell Line; Coloring Agents; Cytochromes c; Diterpenes; Epoxy Compounds; Humans; Liver; Membrane Potentials; Mitochondria; Mitochondrial Membranes; Phenanthrenes; Proto-Oncogene Proteins c-bcl-2

Triptolide, a major active component extracted from the root of Tripterygium wilfordii Hook f, has been shown to possess potent immunosuppressive and anti-inflammatory properties. In the present report, we reported that triptolide increased the generation of reactive oxygen species (ROS) and nitric oxide (NO) and induced apoptosis of RAW 264.7 cells in a dose-dependent manner (5-25 ng/ml). The antioxidant, reduced glutathione (GSH), significantly inhibited triptolide-induced apoptosis and inhibited the degradation of Bcl-2 protein, disruption of mitochondrial membrane potential, release of cytochrome c from mitochondria into the cytosol, activation of caspase-3, and cleavage of poly-(ADP-ribose)-polymerase. The inducible nitric oxide synthase-specific inhibitor 1400w blocked triptolide-induced apoptosis, but did not alter mitochondria disruption and caspase-3 activation. These results, for the first time, implicated that the increased endogenous ROS and NO co-mediated triptolide-induced apoptosis in macrophages. ROS initiated triptolide-induced apoptosis by the mitochondria signal pathway, while the apoptotic cell death mediated by NO was not via mitochondria collapse and caspase-3 activation. In addition, combining mathematical calculation and computer simulation based on our conventional experimental results, we set and validated the apoptotic model and provided more dynamic processes of triptolide-induced apoptotic cascade in macrophages.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents, Alkylating; Apoptosis; Caspases; Cytochromes c; Diterpenes; Enzyme Activation; Epoxy Compounds; Humans; Macrophages; Membrane Potential, Mitochondrial; Mice; Nitric Oxide; Nitric Oxide Synthase Type II; Nitrites; Phenanthrenes; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; U937 Cells

Pancreatic cancer is highly resistant to current chemotherapy agents. We therefore examined the effects of triptolide (a diterpenoid triepoxide) on pancreatic cancer growth and local-regional tumor spread using an orthotopic model of pancreatic cancer. We have recently shown that an increased level of HSP70 in pancreatic cancer cells confers resistance to apoptosis and that inhibiting HSP70 induces apoptosis in these cells. In addition, triptolide was recently identified as part of a small molecule screen, as a regulator of the human heat shock response. Therefore, our aims were to examine the effects of triptolide on (a) pancreatic cancer cells by assessing viability and apoptosis, (b) pancreatic cancer growth and local invasion in vivo, and (c) HSP70 levels in pancreatic cancer cells. Incubation of PANC-1 and MiaPaCa-2 cells with triptolide (50-200 nmol/L) significantly reduced cell viability, but had no effect on the viability of normal pancreatic ductal cells. Triptolide induced apoptosis (assessed by Annexin V, caspase-3, and terminal nucleotidyl transferase-mediated nick end labeling) and decreased HSP70 mRNA and protein levels in both cell lines. Triptolide (0.2 mg/kg/d for 60 days) administered in vivo decreased pancreatic cancer growth and significantly decreased local-regional tumor spread. The control group of mice had extensive local invasion into adjacent organs, including the spleen, liver, kidney, and small intestine. Triptolide causes pancreatic cancer cell death in vitro and in vivo by induction of apoptosis and its mechanism of action is mediated via the inhibition of HSP70. Triptolide is a potential therapeutic agent that can be used to prevent the progression and metastases of pancreatic cancer.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Caspase 3; Cell Growth Processes; Cell Line, Tumor; Cytochromes c; Diterpenes; Epoxy Compounds; HSP70 Heat-Shock Proteins; Humans; Mice; Mice, Nude; Pancreatic Neoplasms; Phenanthrenes; Xenograft Model Antitumor Assays

Triptolide, a bioactive component of the Chinese medicinal herb Tripterygium wilfordii Hook F., induces p53-mediated apoptosis in cancer cells. This study demonstrated that triptolide activated an alternative p53-independent apoptotic pathway in HL-60 cells. In the absence of an intact p53 and without changing Bax level, at nM range triptolide induced apoptosis with concomitant DNA fragmentation, S phase cell cycle arrest, mitochondrial cytochrome c release and the activation of caspases. Besides, both caspases 8 and 9 were activated and the simultaneous inhibition of both was required to completely block triptolide's apoptotic effect. Importantly, triptolide induced the appearance of a truncated 23kD Bcl-2 which was inhibited by the general caspase inhibitor Z-VAD-FMK. In the MCF-7 cells that possessed the wild type p53 but lacked caspases 3, triptolide induced cell death with an increase in p53 but Bcl-2 remained unaltered. On the other hand, transfected cells overexpressing the 28kD Bcl-2 became more resistant to triptolide and upon triptolide treatment accumulated in the G(1) instead of S phase. After 36h treatment, triptolide activated JNK pathways, at the same time inactivated the ERK and p38 pathways. However, SP600125, a specific JNK inhibitor, could not inhibit the triptolide-mediated cleavage of caspase 3, indicated that activation of JNK might not be related to the apoptotic effects of triptolide. Our data suggest that in the absence of an intact p53 and without altering Bax level triptolide induces apoptosis activates a positive amplification loop involving caspase-mediated Bcl-2 cleavage/activation, mitochondrial cytochrome c release and further activation of caspases.

Topics: Caspases; Cell Cycle; Cytochromes c; Diterpenes; Enzyme Activation; Epoxy Compounds; Extracellular Signal-Regulated MAP Kinases; HL-60 Cells; Humans; Hydrolysis; Mitochondria; p38 Mitogen-Activated Protein Kinases; Phenanthrenes; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53