cytochrome-c-t and magnolol

Magnolol, a small-molecule hydroxylated biphenol, isolated from the root and stem bark of Magnolia officinalis, has been shown to possess antiproliferative effect on various cancer cell lines. In the current study, we found that magnolol potently inhibited proliferation and induced apoptosis in MCF-7 human breast cancer cells. Further mechanistic studies revealed that induction of apoptosis is associated with cell cycle arrest at G2/M phase, increased generation of reactive oxygen species (ROS), reduced mitochondrial membrane potential (MMP), release of cytochrome c (Cyto c) and apoptosis inducing factor (AIF) from mitochondria to cytosol, upregulation of Bax, p21 and p53, and down-regulation of Bcl-2, cyclin B1 and cyclin-dependent kinase 1 (CDK1). Our findings indicated that magnolol induced apoptosis in MCF-7 cells via the intrinsic pathway with release of AIF from mitochondrial and G2/M phase arrest pathway. Therefore, magnolol might be a potential lead compound in the therapy of breast cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; Biphenyl Compounds; Bisbenzimidazole; Breast Neoplasms; Caspases; Cell Division; Cell Line, Tumor; Cell Proliferation; Coloring Agents; Cytochromes c; Female; Flow Cytometry; G2 Phase; Genes, bcl-2; Humans; Indicators and Reagents; Lignans; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles

Colon cancer is the third most common malignancy around the world. Surgery, chemotherapy, and radiotherapy are generally used to treat colon cancer, but no effective therapy for advanced colon carcinoma is available. Therefore, there is a need to identify other therapeutic agents against this disease. Magnolol, a hydroxylated biphenyl compound present in Magnolia officinalis, exerts anticancer potential and low toxicity. Emerging evidence has suggested that activation of AMP-activated protein kinase (AMPK), a potential cancer therapeutic target is involved in apoptosis in colon cancer cells. However, the effects of magnolol on human colon cancer through activation of AMPK remain unexplored. In this study, we explored whether magnolol exerts an antiproliferative effect, and induces apoptosis in HCT-116 human colon cancer cells. Magnolol displayed several apoptotic features, including propidium iodide labeling, DNA fragmentation, and caspase-3 and poly(ADP-ribose) polymerase cleavages. We showed that magnolol induced the phosphorylation of AMPK in dose- and time-dependent manners. The selective AMPK inhibitor compound C abrogated the effect of magnolol on AMPK activation, suppression of proliferation, and caspase-3 cleavage. Magnolol downregulated expression of the antiapoptotic protein Bcl2, upregulated expression of pro-apoptotic protein p53 and Bax, and caused the release of mitochondrial cytochrome c. Magnolol-induced p53 and Bcl2 expression was abolished in the presence of compound C. Magnolol inhibited migration and invasion of HCT-116 cells through AMPK activation. These findings demonstrate that AMPK mediates the anticancer effects of magnolol through apoptosis in HCT-116 cells.

Topics: AMP-Activated Protein Kinases; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Biphenyl Compounds; Caspase 3; Cell Movement; Colonic Neoplasms; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; HCT116 Cells; Humans; Lignans; Magnolia; Mitochondria; Phosphorylation; Phytotherapy; Plant Extracts; Poly(ADP-ribose) Polymerases; Propidium; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Suppressor Protein p53

Although magnolol is cytoprotective against warm ischemia/reperfusion injury, its effect on cold preservation has not been fully investigated. This study aimed at examining whether magnolol maintains the liver graft integrity after cold preservation and elucidating the underlying mechanisms in terms of apoptotic signaling under both normothermic and hypothermic conditions. After being preserved in Ringer's lactate (RL) at 4 degrees C for 6h ex vivo, the magnolol-treated grafts demonstrated significantly higher AST, ALT, and LDH levels in perfusates than those from negative controls. TUNEL staining showed no difference in the number of apoptotic nuclei in both groups, whereas a more intense apoptotic signal in magnolol-treated grafts was shown as compared with the controls. In vitro data showed no significant difference in viability of RL-preserved clone-9 hepatocytes between the magnolol-treated and control groups, while magnolol pretreatment at 30min before cold preservation prominently induced hepatocyte cell death. RT-PCR and Western blotting analyses revealed a suppression in Bcl-2, but an up-regulation in Bax expression in clone-9 cells after magnolol treatment. Magnolol suppressed the ratios of NF-kappaB to I-kappaBalpha protein contents and I-kappaBalpha phosphorylation induced by TNF-alpha, and potentiated mitochondrial cytochrome c release and subsequent caspase-3 cleavage. Conversely, caspase-3 inhibitor attenuated magnolol-induced hepatotoxicity. We concluded that magnolol could not protect liver grafts from cold ischemia/reperfusion injury. High concentration of magnolol under serum-reduced conditions attenuates NF-kappaB-mediated signaling and induces intrinsic apoptotic pathway, thereby inducing in vitro hepatotoxicity.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Biphenyl Compounds; Blotting, Western; Caspase 3; Chemical and Drug Induced Liver Injury; Cold Temperature; Cryopreservation; Cytochromes c; I-kappa B Proteins; In Situ Nick-End Labeling; Lignans; Liver; Liver Transplantation; Magnolia; Male; Mitochondria; NF-kappa B; Plant Bark; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Serum; Signal Transduction; Tumor Necrosis Factor-alpha

We observed that treatment of prostate cancer cells for 24 h with magnolol, a phenolic component extracted from the root and stem bark of the oriental herb Magnolia officinalis, induced apoptotic cell death in a dose- and time-dependent manner. A sustained inhibition of the major survival signal, Akt, occurred in magnolol-treated cells. Treatment of PC-3 cells with an apoptosis-inducing concentration of magnolol (60 microM) resulted in a rapid decrease in the level of phosphorylated Akt leading to inhibition of its kinase activity. Magnolol treatment (60 microM) also caused a decrease in Ser((136)) phosphorylation of Bad (a proapoptotic protein), which is a downstream target of Akt. Protein interaction assay revealed that Bcl-xL, an anti-apoptotic protein, was associated with Bad during treatment with magnolol. We also observed that during treatment with magnolol, translocation of Bax to the mitochondrial membrane occurred and the translocation was accompanied by cytochrome c release, and cleavage of procaspase-8, -9, -3, and poly(ADP-ribose) polymerase (PARP). Similar results were observed in human colon cancer HCT116Bax(+/-) cell line, but not HCT116Bax(-/-) cell line. Interestingly, at similar concentrations (60 microM), magnolol treatment did not affect the viability of normal human prostate epithelial cell (PrEC) line. We also observed that apoptotic cell death by magnolol was associated with significant inhibition of pEGFR, pPI3K, and pAkt. These results suggest that one of the mechanisms of the apoptotic activity of magnolol involves its effect on epidermal growth factor receptor (EGFR)-mediated signaling transduction pathways.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; bcl-X Protein; Biphenyl Compounds; Cell Line, Tumor; Cytochromes c; Epidermal Growth Factor; ErbB Receptors; Humans; Lignans; Male; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction

Oxidative stress plays a significant role in the progression of cataract. We aimed to investigate the protective effect of magnolol, a compound extracted from the Chinese herb Magnolia officinalis, against oxidative stress in human lens epithelial (HLE) cells as well as the possible molecular mechanism involved. In this study, magnolol was observed to protect against H2O2-induced cytotoxicity in HLE B-3 cells. Magnolol inhibited the generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential (Delta psi m) and release of cytochrome c from mitochondria caused by H2O2 into cytosol in HLE B-3 cells. Magnolol also inhibited H2O2-induced expressions of caspase-9 and caspase-3 and reduction of Bcl-2/Bax ratio. Moreover, magnolol attenuated the deactivation of ERK/MAPK (extracellular signal-regulated kinase/mitogen activated protein kinase) and the enhanced activation of p38, JNK (c-Jun N-terminal kinase) induced by H2O2. Magnolol could be useful in protecting against oxidative stress in HLE cells, suggesting a potential protective effect against cataractogenesis effect against cataractogenesis.

Topics: bcl-2-Associated X Protein; Biphenyl Compounds; Blotting, Western; Caspase 3; Caspase 9; Cell Line; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Epithelial Cells; Flow Cytometry; Humans; Hydrogen Peroxide; Lens, Crystalline; Lignans; Magnolia; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Oxidants; Oxidative Stress; Protective Agents; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Magnolol, a compound extracted from the Chinese medicinal herb Magnolia officinalis, has several biological effects. However, its protective effects against endothelial injury remain unclear. In this study, we examined whether magnolol prevents oxidized low density lipoprotein (oxLDL)-induced vascular endothelial apoptosis. Incubation of oxLDL with magnolol (2.5-20 microM) inhibited copper-induced oxidative modification via diene formation, thiobarbituric acid reactive substances (TBARS) assay and electrophoretic mobility assay. Apoptotic cell death as characterized by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain. We measured the production of reactive oxygen species (ROS) by using the fluorescent probe 2',7'-dichlorofluorescein acetoxymethyl ester (DCF-AM), and observed the activity of antioxidant enzymes. Furthermore, several apoptotic signaling pathways which showed NF-kappaB activation, increased cytosolic calcium, alteration of mitochondrial membrane potential, cytochrome c release and activation of caspase 3 were also investigated. We demonstrated that magnolol prevented the copper-induced oxidative modification of LDL. Magnolol attenuated the oxLDL-induced ROS generation and subsequent NF-kappaB activation. Furthermore, intracellular calcium accumulation and subsequent mitochondrial membrane potential collapse, cytochome c release and activation of caspase 3 caused by oxLDL were also inhibited by magnolol. Our results suggest that magnolol may have clinical implications in the prevention of atherosclerotic vascular disease through decreasing the oxLDL-induced ROS production.

Topics: Antioxidants; Apoptosis; Biphenyl Compounds; Calcium; Caspase 3; Cell Survival; Cells, Cultured; Copper Sulfate; Cytochromes c; Dose-Response Relationship, Drug; Endothelial Cells; Humans; Lignans; Lipid Peroxidation; Lipoproteins, LDL; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; NF-kappa B; Oxidation-Reduction; Reactive Oxygen Species; Signal Transduction; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Time Factors

Magnolol, a substance purified from the bark of Magnolia officialis, inhibits cell proliferation and induces apoptosis in a variety of cancer cells. The aim of this study was to study the effects of magnolol on CGTH W-2 thyroid carcinoma cells. After 24 h treatment with 80 microM magnolol in serum-containing medium, about 50% of the cells exhibited apoptotic features and 20% necrotic features. Cytochrome-c staining was diffused in the cytoplasm of the apoptotic cells, but restricted to the mitochondria in control cells. Western blot analyses showed an increase in levels of activated caspases (caspase-3 and -7) and of cleaved poly (ADP-ribose) polymerase (PARP) by magnolol. Concomitantly, immunostaining for apoptosis inducing factor (AIF) showed a time-dependent translocation from the mitochondria to the nucleus. Inhibition of either PARP or caspase activity blocked magnolol-induced apoptosis, supporting the involvement of the caspases and PARP. In addition, magnolol activated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and inactivated Akt by decreasing levels of phosphorylated PTEN and phosphorylated Akt. These data suggest that magnolol promoted apoptosis probably by alleviating the inhibitory effect of Akt on caspase 9. Furthermore, inhibition of PARP activity, but not of caspase activity, completely prevented magnolol-induced necrosis, suggesting the notion that it might be caused by depletion of intracellular ATP levels due to PARP activation. These results show that magnolol initiates apoptosis via the cytochrome-c/caspase 3/PARP/AIF and PTEN/Akt/caspase 9/PARP pathways and necrosis via PARP activation.

Topics: Apoptosis; Apoptosis Inducing Factor; Biphenyl Compounds; Blotting, Western; Caspase 3; Caspase 7; Cell Line, Tumor; Cytochromes c; Flow Cytometry; Humans; Immunohistochemistry; Lignans; Necrosis; Poly(ADP-ribose) Polymerases; Signal Transduction; Thyroid Neoplasms

Magnolol, isolated from the stem bark of Magnolia officnalis, was found to inhibit proliferation of human HL-60 cells and Jurkat T leukemia cells via inducing apoptosis in a dose- and time-dependent manner. By contrast, magnolol did not cause apoptosis in neutrophils and peripheral blood mononuclear cells of healthy donors. Apoptosis was determined by detection of DNA fragmentation in gel electrophoresis, morphological alternations by flow cytometry, quantification of phosphatidylserine externalization by Annexin V labeling and oligonucleosomal DNA content by TUNEL labeling. Activation of caspase-9, -3 and -2, and the proteolytic cleavage of poly(ADP-ribose) polymerase were found during apoptosis induced by magnolol. In addition, both pan-caspase and selective caspase-9 inhibitor blocked magnolol-induced apoptosis. The apoptosis could also be partially attenuated by caspase-3 and -2 inhibitors. Magnolol induced the reduction of mitochondrial transmembrane potential and the release of cytochrome c into cytoplasm. In conclusion, our findings indicate that magnolol-induced apoptotic signaling is carried out through mitochondria alternations to caspase-9 and that then the downstream effector caspases are activated sequentially. Magnolol could be a potentially effective drug for leukemia with low toxicity to normal blood cells and it merits further investigation.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Caspases; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Electrophoresis, Agar Gel; Enzyme Activation; Flow Cytometry; HL-60 Cells; Humans; In Situ Nick-End Labeling; Inhibitory Concentration 50; Jurkat Cells; Lignans; Magnolia; Membrane Potentials; Mitochondria; Poly(ADP-ribose) Polymerases; Time Factors