cytochrome-c-t and tanshinone

Previous studies have shown that Tanshinone (Tan) IIA exerts obvious antitumor efficacy; however, its molecular mechanism remains unclear. This study was conducted to identify the influence of Tan IIA on Eca-109 cell apoptosis, explore its molecular mechanism, and provide a theoretical basis for clinical application.. Eca-109 cells were cultured in vitro and treated with different concentrations of Tan IIA. Morphologic changes were viewed under inverted fluorescence microscope with dual acridine orange/ethidium bromide staining assay. Methyl-thiazolyl-tetrazolium and Annexin V propidium iodide assays were respectively used to measure cell viability and apoptosis rate. The protein and messenger (m)RNA expression of binding immunoglobulin protein (BIP), mitochondrial cytochrome c (CytC), and caspase-9 were detected by Western blot and quantitative real-time PCR, respectively.. Cell viability decreased and the apoptosis rate significantly increased with increasing concentrations of Tan IIA (0, 20, 40, 60 μg/mL), which indicated that Tan IIA inhibited the proliferation and induced the apoptosis of Eca-109 cells in a dose-dependent manner. Eca-109 cells treated with 60 μg/mLTan IIA showed typical morphological changes of apoptosis under the inverted microscope. Moreover, compared with the negative control group, protein and mRNA expression of BIP decreased significantly (P < 0.05), whereas protein and mRNA expression of CytC and caspase-9 increased significantly (P < 0.05).. Tan IIA can induce apoptosis in human esophageal carcinoma Eca-109 cells by regulating BIP, CytC, and caspase-9 expression. Endoplasmic reticulum stress and mitochondrial-dependent may be involved in Tan IIA-induced Eca-109 cell apoptosis.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Signal Transduction

Dihydrotanshinone, a functional food in China, is an effective anti-cardiovascular disease substance isolated from Salvia miltiorrhiza (S. miltiorrhiza). Glioma is considered to be fatal due to its poor prognosis and few effective therapeutic options. In this study, we investigated the anticancer effects of S. miltiorrhiza extract dihydrotanshinone on human glioma SHG-44 cells, by using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide assay, Hoechst 33258 nuclear staining, Annexin V/propidium iodide double staining, as well as western blot analysis. The results showed that dihydrotanshinone effectively suppressed SHG-44 cells proliferation and induced apoptosis in both dose- and time-dependent manner. Moreover, we demonstrated that dihydrotanshinone increased the activation of caspases (caspase-3 and caspase-9) and the release of cytochrome c in SHG-44 cells. Overall, dihydrotanshinone could induce apoptosis and inhibit proliferation of glioma cells by regulating caspases and cytochrome c. This study suggests that dihydrotanshinone may serve as a potential treatment option for patients with glioma.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Brain Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Glioma; Humans; Plant Extracts; Salvia miltiorrhiza

Tanshinone IIA (TSIIA), a natural diterpene quinone in the traditional Chinese medicinal herb Dan-Shen (Salvia miltiorrhiza), has extensively exerted antitumor activity in cellular and animal models. However, the molecular mechanisms underlying the antitumor effects of TSIIA remain largely unknown. The in vitro effects of TSIIA on apoptosis were investigated in A549 non-small cell lung cancer (NSCLC) cells. The data showed that TSIIA significantly suppressed the proliferation of A549 cells in a dose-dependent manner, with IC50 values of 16.0±3.7 and 14.5±3.3 µM at 48 h as determined by Cell Counting Kit-8 (CCK-8) assay and clone formation assay, respectively. The change of mitochondrial morphology and the loss of mitochondrial membrane potential (MMP) were observed during the induction. Furthermore, TSIIA induced A549 cell apoptosis as confirmed by typical morphological changes, with cytochrome c release from the mitochondria and Bax translocation to the mitochondria. Caspase activity data indicated that TSIIA activated caspase-9 and caspase-3 of mitochondria-mediated apoptosis, but not caspase-8 of receptor-mediated apoptosis, which could be largely rescued by SP600125 (JNK inhibitor). Taken together, these findings provide the first evidence that TSIIA inhibits growth of NSCLC A549 cells, induces activation of JNK signaling and triggers caspase cascade apoptosis mediated by the release of cytochrome c, which provides a better understanding of the molecular mechanisms of TSIIA on lung cancer.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Fluorescent Antibody Technique; Humans; Lung Neoplasms; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Microscopy, Fluorescence

Tanshinone IIA (Tan IIA), a phytochemical derived from the roots of Salvia miltiorrhiza BUNGE, has been documented with anti-tumor, pro-apoptotic, and anti-inflammatory activities. Salvia miltiorrhiza has long been used to treat rheumatoid arthritis (RA). Apoptosis induction of RA-fibroblast-like synoviocytes (FLS) was suggested to be a potential therapeutic approach for RA. The aim of this study was to investigate whether Tan IIA promotes apoptosis in RA-affected FLS. In this study, the viability of an immortalized FLS cell line derived from RA patients was assessed by 3-(4,5-dimethylthiazol-2-yl)-5,3-carboxymethoxyphenyl-2,4-sulfophenyl-2H-tetrazolium (MTS) assay after Tan IIA treatment. Apoptosis was measured by terminal deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay and flow cytometry. Cell cycle was evaluated by flow cytometry. The expressions of mitochondrial apoptosis-related molecules, including Bcl-2, Bax, mitochondrial cytochrome c (Cyt-c), cytosolic Cyt-c, apoptotic protease activating factor 1 (Apaf-1), procaspase-9, procaspase-3, caspase-9, and caspase-3 were determined by Western blotting. Our data demonstrate that Tan IIA induced apoptosis of RA-FLS, blocked the cell cycle in the G2/M phase, and regulated the protein expression of Bcl-2, Bax, and Apaf-1, the release of mitochondrial Cyt-c, and the activation of caspase-9 and caspase-3. The results support the conclusion Tan IIA treatment likely induces apoptosis of RA-FLS through blockade of the cell cycle in the G2/M phase and a mitochondrial pathway. These data suggest that Tan IIA may have therapeutic potential for RA.

Topics: Abietanes; Apoptosis; Apoptotic Protease-Activating Factor 1; Arthritis, Rheumatoid; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Cycle; Cell Line; Cytochromes c; Fibroblasts; Humans; Mitochondria; Proto-Oncogene Proteins c-bcl-2

Mitochondrial apoptosis pathway is an important target of cardioprotective signalling. Tanshinones, a group of major bioactive compounds isolated from Salvia miltiorrhiza, have been reported with actions against inflammation, oxidative stress, and myocardial ischemia reperfusion injury. However, the actions of these compounds on the chronic hypoxia-related mitochondrial apoptosis pathway have not been investigated. In this study, we examined the effects and molecular mechanisms of two major tanshonones, tanshinone IIA (TIIA) and cryptotanshinone (CT) on hypoxia induced apoptosis in H9c2 cells. Cultured H9c2 cells were treated with TIIA and CT (0.3 and 3 μΜ) 2 hr before and during an 8 hr hypoxic period. Chronic hypoxia caused a significant increase in hypoxia inducible factor 1α expression and the cell late apoptosis rate, which was accompanied with an increase in caspase 3 activity, cytochrome c release, mitochondria membrane potential and expression of pro-apoptosis proteins (Bax and Bak). TIIA and CT (0.3 and 3 μΜ), in concentrations without affecting the cell viability, significantly inhibited the late apoptosis and the changes of caspase 3 activity, cytochrome c release, and mitochondria membrane potential induced by chronic hypoxia. These compounds also suppressed the overexpression of Bax and reduced the ratio of Bax/Bcl-2. The results indicate that TIIA and CT protect against chronic hypoxia induced cell apoptosis by regulating the mitochondrial apoptosis signaling pathway, involving inhibitions of mitochondria hyperpolarization, cytochrome c release and caspase 3 activity, and balancing anti- and pro-apoptotic proteins in Bcl-2 family proteins.

Topics: Abietanes; Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Cell Hypoxia; Cell Survival; Cyclosporine; Cytochromes c; Cytoprotection; Enzyme Activation; Hypoxia-Inducible Factor 1, alpha Subunit; Membrane Potential, Mitochondrial; Mitochondria; Myocytes, Cardiac; Phenanthrenes; Protein Stability; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Rats

Tanshinone IIA (Tan-IIA) is extracted from Danshen and known to inhibit proliferation and induce apoptosis in many cancer cells. We aimed to elucidate its anticancer activity and molecular mechanism in human lung cancer A549 cells. The cytotoxicity of Tan-IIA in A549 cells were measured by the MTT assay. The effects of Tan-IIA on the cell cycle, mitochondrial membrane potential (MMP), calcium and reactive oxygen species (ROS) released in A549 cells were detected by flow cytometry. The protein expressions of p53, Bax, Bcl-2 and beta-actin in A549 cells were tested by Western blotting. The proliferative rates of A549 cells were obviously inhibited by Tan-IIA in a dose- and time-dependent manner. The results of FACS showed that the sub-G1 phase was increased when A549 cells were cultured with various concentrations of Tan-IIA (control, 2.5, 5 and 10 microg/ml) for 48 h. Tan-IIA induced the production of ROS, Ca+2 and decreased MMP. The outcome of Western blotting showed that protein expressions of p53 and bax were increased, but proto-oncogene bcl-2 was notably decreased, after culturing with Tan-IIA (5 microg/ml) for 6, 12 and 24 h. Tan-IIA inhibited the proliferation of non-small cell lung cancer A549 cells, possibly by decreasing the MMP and inducing apoptosis due to the induction of a higher ratio of Bax/Bcl-2.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Calcium; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Fragmentation; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Phenanthrenes; Proto-Oncogene Mas; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Tumor Suppressor Protein p53

A novel compound, acetyltanshinone IIA (ATA) was obtained from chemical modifications of tanshinone TIIA (TIIA) isolated from a medicinal plant, Salvia miltiorrhiza. ATA exhibited increased water solubility and stronger apoptotic activity on multiple cancer cell lines than TIIA. ATA displayed a higher growth inhibition ability on breast cancer especially HER2 positive cells than normal cells and it inhibited xenografted tumor growth in mice. Mechanistic studies showed that ATA could induce significant reactive oxygen species (ROS) generation, Bax translocation to mitochondria, resulting in mitochondria damage, cytochrome c release, caspase-3 activation and apoptotic cell death. ATA-mediated ROS production and its downstream apoptotic events could be blocked by an antioxidant agent, propyl gallate, indicating the prominent role of ROS in ATA-induced apoptosis. Overexpression of Bcl-2 protein reduced ATA-induced cell death. In conclusion, ATA is a novel anticancer agent with potent in vitro and in vivo anticancer ability. ROS-mediated Bax activation should be the mechanism by which ATA induces apoptosis and inhibits tumor growth.

Topics: Abietanes; Animals; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Doxorubicin; Female; HeLa Cells; Hep G2 Cells; HL-60 Cells; Humans; Inhibitory Concentration 50; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Neoplasms; Paclitaxel; Phenanthrenes; Propyl Gallate; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptor, ErbB-2; Solubility; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

Oxidative stress caused by amyloid β-peptide (Aβ) may play an important role in the pathogenesis of Alzheimer disease (AD). Aβ is known to be directly responsible for the production of reactive oxygen species (ROS) and induction of apoptosis. Tanshinone IIA (Tan IIA) is extracted from a traditional herbal medicine Salvia miltiorrhiza BUNGE, which has been shown to protect against oxidative stress and cell death. In this study, we investigated the neuroprotective effect of Tan IIA against Aβ₂₅₋₃₅-induced cell death in cultured cortical neurons. Exposure of cortical neurons to 30μM Aβ₂₅₋₃₅ caused a significant viability loss, cell apoptosis and decreased activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) as well as increased levels of malondialdehyde (MDA) production. In parallel, Aβ₂₅₋₃₅ significant increased the intracellular ROS elevation and decreased mitochondrial membrane potential (MMP). However, pretreatment of the cells with Tan IIA prior to Aβ₂₅₋₃₅ exposure suppressed these Aβ₂₅₋₃₅-induced cellular events noticeably. In addition, Tan IIA reduced the Aβ₂₅₋₃₅-induced increase of caspase-3 activity, and reduced cytochrome c translocation into the cytosol from mitochondria. Furthermore, Tan IIA also ameliorated the Aβ₂₅₋₃₅-induced Bcl-2/Bax ratio reduction in cortical neurons. Taken together, these data indicate that Tan IIA protected cultured cortical neurons against Aβ₂₅₋₃₅-induced neurotoxicity through its antioxidative potential. Our results strongly suggest that Tan IIA may be effective in treating AD associated with oxidative stress.

Topics: Abietanes; Amyloid beta-Peptides; Animals; Apoptosis; Cell Survival; Cells, Cultured; Cerebral Cortex; Cytochromes c; Lipid Peroxidation; Membrane Potential, Mitochondrial; Mitochondria; Neurons; Neuroprotective Agents; Peptide Fragments; Phenanthrenes; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

to examine the effects of tanshinone IIA, the main effective component of Salvia miltiorrhiza (known as 'Danshen' in traditional Chinese medicine) on angiotensin II (Ang II)-mediated cardiomyocyte apoptosis.. rat neonatal cardiomyocytes were primarily cultured with Ang II or Ang II plus tanshinone IIA. Myocyte apoptosis was evaluated by caspase-3 activity and DNA strand break level with TdT-mediated dUTP nick-end labeling (TUNEL) staining. Western blot analysis was employed to determine the related protein expression and flow cytometry assay was used to determine the TUNEL positive cells and the intracellular reactive oxygen species (ROS) production. SiRNA targeted to Akt was used.. ang II (0.1 micromol/L) remarkably increased caspase-3 activity, TUNEL positive cells, and cleaved caspase-3 and cytochrome c expression, but reduced Bcl-X(L) expression. These effects were effectively antagonized by pretreatment with tanshione IIA (1-3 micromol/L). Tanshinone IIA had no effect on basal ROS level, while attenuated the ROS production by Ang II. Interestingly, tanshione IIA significantly increased the phosphorylated Akt level, which was countered by the PI3K antagonist wortmannin or LY294002. Knockdown of Akt with Akt siRNA significantly reduced Akt protein levels and tanshinone IIA protective effect.. tanshinone IIA prevents Ang II-induced apoptosis, thereby suggesting that tanshinone IIA may be used for the prevention of the cardiac remodeling process.

Topics: Abietanes; Angiotensin II; Animals; Animals, Newborn; Antioxidants; Apoptosis; Caspase 3; Cell Survival; Cells, Cultured; Cytochromes c; Myocytes, Cardiac; Phenanthrenes; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species; Salvia miltiorrhiza

The role of reactive oxygen species (ROS) and p38 mitogen-activated protein kinases (MAPK) in tanshinones-induced apoptosis was investigated in HepG2 cells in this study. The major tanshinones (cryptotanshinone, dihydrotanshinone, tanshinone I, tanshinone IIA), isolated from Salvia miltiorrhiza, inhibit cell growth and induce caspase-dependent apoptosis concentration-dependently, with dihydrotanshinone being the most potent. All four tanshinones were found to induce ROS generation, but only dihydrotanshinone can induce activation of p38 MAPK. The p38 MAPK activation by dihydrotanshinone was inhibited by N-acetyl cysteine pretreatment. It is thus concluded that ROS-mediated p38 MAPK activation plays a vital role in dihydrotanshinone-induced apoptosis in HepG2 cells.

Topics: Abietanes; Acetylcysteine; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Furans; Humans; Imidazoles; Inhibitory Concentration 50; JNK Mitogen-Activated Protein Kinases; L-Lactate Dehydrogenase; Liver Neoplasms; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phenanthrenes; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Protein Transport; Pyridines; Quinones; Reactive Oxygen Species; Time Factors

Tanshinone IIA, a major component extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, is known to exhibit potent cytotoxicity against various human carcinoma cells in vitro. However, the mechanism by which tanshinone IIA produces this anti-tumor effect remains unknown. Since anti-neovascularization has generally been regarded as an effective strategy for anti-cancer therapy, we decided to investigate the mechanism underlying tanshinone IIA-mediated death of human endothelial cells. In this study, we demonstrate that tanshinone IIA elicits human endothelial cell death independent of oxidative stress. These events are partially calcium-dependent and actually dependent upon NAD(P)H: quinone oxidoreductase (NQO1) activity. Tanshinone IIA induces an increase in intracellular calcium, which triggers the release of cytochrome c, thus causing loss of the mitochondrial membrane potential (MMP), resulting in the subsequent activation of caspases. Blocking the induction of Ca2+ perturbation with BAPTA-AM partially rescued cells from tanshinone IIA-induced cytotoxicity. Additionally, blocking NQO1 activity with dicoumoral or inhibiting caspase activities with the general caspase inhibitor, z-VAD-fmk, prevented cell death induced by tanshinone IIA. Therefore, our results imply that tanshinone IIA-mediated cytotoxicity against human endothelial cells may occur through activation of NQO1, which induces a calcium imbalance and mitochondrial dysfunction, thus stimulating caspase activity.

Topics: Abietanes; Acridine Orange; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Calcium; Caspase Inhibitors; Caspases; Cell Cycle; Cell Death; Cytochromes c; Dicumarol; Drugs, Chinese Herbal; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; Humans; Membrane Potentials; Mitochondria; Models, Biological; NAD(P)H Dehydrogenase (Quinone); Oxidative Stress; Phenanthrenes; Plant Extracts; Reactive Oxygen Species; Salvia miltiorrhiza; Time Factors