sirolimus and cobaltous-chloride

Topics: Adenine; Animals; Autophagosomes; Autophagy; Cell Line; Chemical and Drug Induced Liver Injury; Chromones; Cobalt; Disease Models, Animal; Erythropoietin; Humans; Liver; Liver Function Tests; Mice; Mice, Inbred C57BL; Morpholines; Peptide Fragments; Proto-Oncogene Proteins c-akt; Random Allocation; Reperfusion Injury; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Malignant glioma is the most aggressive brain tumor. Hypoxic condition has been explored for killing cancer stem cells or drug-resistant tumor cells. This study investigated the effects of hypoxia on autophagic death and the possible mechanisms. Exposure of human malignant glioma U87-MG cells to cobalt chloride (CoCl2) increased cellular hypoxia-inducible factor-1α levels and concurrently decreased cell viability concentration- and time-dependently. In parallel, treatment with CoCl2 suppressed proliferation of human U87-MG cells. Autophagic cells and levels of LC3-II were concentration- and time-dependently induced in human U87-MG cells after exposure to CoCl2. However, pretreatment with 3-mehyladenine (3-MA) and chloroquine, inhibitors of cell autophagy, caused significant alleviations in CoCl2-induced cell autophagy. In contrast, exposure to rapamycin, an inducer of cell autophagy, synergistically induced hypoxia-induced autophagy of U87-MG cells. Administration of human U87-MG cells with CoCl2 triggered caspase-3 activation and cell apoptosis. Interestingly, pretreatment with 3-MA and chloroquine remarkably suppressed CoCl2-induced caspase-3 activation and cell apoptosis. Application of p53 small interference (si)RNA into human U87-MG cells downregulated levels of this protein and simultaneously lowered hypoxia- and 3-MA-induced alterations in cell autophagy, apoptosis, and death. The hypoxia-induced autophagy and apoptosis of DBTRG-05MG cells were significantly lowered by 3-MA pretreatment and p53 knockdown. Therefore, the present study shows that CoCl2 treatment can induce autophagy of human glioma cells and subsequent autophagic apoptosis via a p53-dependent pathway. Hypoxia-induced autophagic apoptosis may be applied as a therapeutic strategy for treatment of glioma patients.

Topics: Adenine; Antimutagenic Agents; Apoptosis; Autophagy; Brain Neoplasms; Caspase 3; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chloroquine; Cobalt; Glioma; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Microtubule-Associated Proteins; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Sirolimus; Tumor Suppressor Protein p53

It has been reported that cerebral ischemia/reperfusion (I/R) injury can activate autophagy. However, the role of autophagy in cerebral I/R injury remains controversy. Two major proteins, mTOR and Beclin-1, govern the formation of autophagosomes to regulate autophagy activity. However, the cross-talking between Beclin-1 and mTOR in cerebral I/R injury remains elusive. In this study, global cerebral I/R injury animal model and focal cerebral I/R injury animal model were induced to test the variation of Beclin-1 level in vivo. To further confirm the variation of Beclin-1 level and investigate the cross-talking between Beclin-1 and mammalian target of rapamycin (mTOR) in I/R injury, we used cobalt chloride (CoCl2) to develop an I/R injury cell model in HT22 cell line. Our data showed that the levels of Beclin-1 and phosphorylated mammalian target of rapamycin (p-mTOR) were clearly induced by I/R injury in vitro. And the time course studies suggested that the Beclin-1 and mTOR may have coordinated regulation in ischemia stages but not in reperfusion stages. Moreover, inhibitor of mTOR could prevent Beclin-1 decreasing, but this prevention may play opposite roles in different stages of I/R injury. We conclude that this study represents a major advance in our understanding of the cross-talking of two key proteins, Beclin-1 and mTOR, in autophagy and the role of autophagy in cerebral I/R injury.

Topics: Analysis of Variance; Animals; Antimutagenic Agents; Apoptosis Regulatory Proteins; Beclin-1; Brain Ischemia; Cell Line; Cell Survival; Cobalt; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression Regulation; Male; Mice; Neurons; Phosphorylation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus

Hypoxia-induced stress plays a central role in retinal vascular disease and cancer. Increased hypoxia-inducible factor-1 alpha (Hif-1 alpha) expression leads to HIF-1 formation and the production of vascular endothelial growth factor (VEGF). Cytokines, including insulin-like growth factor-1 (IGF-1), also stimulate VEGF secretion. In this study, we examined the relationship between IGF-1 signaling, HIF-1 alpha protein turnover and VEGF secretion in the ARPE-19 retinal pigment epithelial cell line. Northern analysis revealed that IGF-1 stimulated Hif-1 alpha message expression, whereas the hypoxia-mimetic CoCl2 did not. CoCl2 treatment increased Hif-1 alpha protein accumulation to a greater extent than IGF-1 treatment. However, IGF-1 stimulated a more significant increase in VEGF secretion. IGF-1-stimulated VEGF promoter activity was phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR (mammalian target of rapamycin)-dependent, whereas VEGF secretion was only partially reduced by inhibition of PI3K/Akt/mTOR and HIF-1 activities. Analysis of VEGF promoter truncation mutants indicated that sensitivity to CoCl2 was hypoxia response element (HRE)-dependent with the region upstream of the HRE conferring IGF-1 sensitivity. In conclusion, IGF-1 regulates VEGF expression and secretion via HIF-1-dependent and -independent pathways.

Topics: Anti-Bacterial Agents; Cell Line; Cobalt; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Immunoblotting; Immunohistochemistry; Insulin-Like Growth Factor I; Ligands; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; RNA, Messenger; Signal Transduction; Sirolimus; Stimulation, Chemical; Transfection; Vascular Endothelial Growth Factor A

Cancer cells frequently evade apoptosis during tumorigenesis by acquiring mutations in apoptotic regulators. Chronic activation of the PI 3-kinase-Akt pathway through loss of the tumor suppressor PTEN is one mechanism by which these cells can gain increased protection against apoptosis. We report here that REDD1 (RTP801) can act as a transcriptional downstream target of PI 3-kinase signaling in human prostate cancer cells (PC-3). REDD1 expression is markedly reduced in PC-3 cells treated with LY294002 (LY) or Rapamycin and strongly induced under hypoxic conditions in a hypoxia-inducible factor-1 (HIF-1)-dependent manner. Loss of function studies employing antisense molecules or RNA interference indicate that REDD1 is essential for invasive growth of prostate cancer cells in vitro and in vivo. Reduced REDD1 levels can sensitize cells towards apoptosis, whereas elevated levels of REDD1 induced by hypoxia or overexpression desensitize cells to apoptotic stimuli. Taken together our data designate REDD1 as a novel target for therapeutic intervention in prostate cancer.

Topics: Apoptosis; Cell Hypoxia; Cell Line, Tumor; Chromones; Cobalt; Dimethyl Sulfoxide; Gene Expression; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Morpholines; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Prostatic Neoplasms; RNA, Antisense; Signal Transduction; Sirolimus; Transcription Factors; Up-Regulation

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor containing an inducibly expressed HIF-1alpha subunit and a constititutively expressed HIF-1beta subunit. Under hypoxic conditions, the HIF-1alpha subunit accumulates due to a decrease in the rate of proteolytic degradation, and the resulting HIF-1alpha-HIF-1beta heterodimers undergo posttranslational modifications that promote transactivation. Recent studies suggest that amplified signaling through phosphoinositide 3-kinase, and its downstream target, mTOR, enhances HIF-1-dependent gene expression in certain cell types. In the present study, we have explored further the linkage between mTOR and HIF-1 in PC-3 prostate cancer cells treated with hypoxia or the hypoxia mimetic agent, CoCl(2). Pretreatment of PC-3 cells with the mTOR inhibitor, rapamycin, inhibited both the accumulation of HIF-1alpha and HIF-1-dependent transcription induced by hypoxia or CoCl(2). Transfection of these cells with wild-type mTOR enhanced HIF-1 activation by hypoxia or CoCl(2), while expression of a rapamycin-resistant mTOR mutant rendered both HIF-1alpha stabilization and HIF-1 transactivating function refractory to inhibition by rapamycin. Studies with GAL4-HIF-1alpha fusion proteins pinpointed the oxygen-dependent degradation domain as a critical target for the rapamycin-sensitive, mTOR-dependent signaling pathway leading to HIF-1alpha stabilization by CoCl(2). These studies position mTOR as an upstream activator of HIF-1 function in cancer cells and suggest that the antitumor activity of rapamycin is mediated, in part, through the inhibition of cellular responses to hypoxic stress.

Topics: Cell Hypoxia; Chromones; Cobalt; DNA-Binding Proteins; Enzyme Inhibitors; Glucose Transporter Type 1; Helix-Loop-Helix Motifs; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Monosaccharide Transport Proteins; Morpholines; Nuclear Proteins; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Protein Kinases; Recombinant Fusion Proteins; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor involved in normal mammalian development and in the pathogenesis of several disease states. It consists of two subunits, HIF-1alpha, which is degraded during normoxia, and HIF-1beta, which is constitutively expressed. Activated HIF-1 induces the expression of genes involved in angiogenesis, erythropoiesis, and glucose metabolism. We have previously reported that insulin stimulates vascular endothelial growth factor (VEGF) expression (). In this study, we show that insulin activates HIF-1, leading to VEGF expression in retinal epithelial cells. Insulin activates HIF-1alpha protein expression in a dose-dependent manner with a maximum reached within 6 h. The expression of HIF-1alpha is correlated with the activation of HIF-1 DNA binding activity and the transactivation of a HIF-1-dependent reporter gene. Insulin does not appear to affect HIF-1alpha mRNA transcription but regulates HIF-1alpha protein expression through a translation-dependent pathway. The expression of an active form of protein kinase B and treatment of cells with specific inhibitors of phosphatidylinositol 3-kinase (PI3K), MAPK, and target of rapamycin (TOR) show that mainly PI3K and to a lesser extent TOR are required for insulin-induced HIF-1alpha expression. HIF-1 activity and VEGF expression are also dependent on PI3K- and TOR-dependent signaling. In conclusion, we show here that insulin regulates HIF-1 action through a PI3K/TOR-dependent pathway, resulting in increased VEGF expression.

Topics: Animals; Cell Line; Cell Nucleus; Cobalt; DNA-Binding Proteins; Endothelial Growth Factors; Gene Expression Regulation; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin; Kinetics; Lymphokines; Mitogen-Activated Protein Kinases; Nuclear Proteins; Phosphatidylinositol 3-Kinases; Pigment Epithelium of Eye; Plasmids; Protein Biosynthesis; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Transcription Factors; Transcription, Genetic; Transfection; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors