rottlerin and Glioma

Oxidative phosphorylation and glycolysis are important features, by which cells could bypass oxidative stress. The level of oxidative stress, and the ability of cells to promote oxidative phosphorylation or glycolysis, significantly determined proliferation or cell demise. In the present work, we have employed selective mitochondrial probe MitoTracker™ Orange CMTM/Ros (MTO) to estimate the level of oxidative stress in cancer cells at different stressed conditions. MTO is partially sensitive to decrease of mitochondrial membrane potential and to reactive oxygen species (ROS) generated in mitochondria. We have demonstrated, that fluorescence lifetime of MTO is much more sensitive to oxidative stress than intensity-based approaches. This method was validated in different cancer cell lines. Our approach revealed, at relatively low ROS levels, that Gö 6976, a protein kinase C (PKC) α inhibitor, and rottlerin, an indirect PKCδ inhibitor, increased mitochondrial ROS level in glioma cell. Their involvement in oxidative phosphorylation and apoptosis was investigated with oxygen consumption rate estimation, western blot and flow-cytometric analysis. Our study brings new insight to identify feeble differences in ROS production in living cells.

Topics: Acetophenones; Antimycin A; Apoptosis; Benzopyrans; Carbazoles; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Line, Tumor; Flow Cytometry; Glioma; Glutathione; Humans; Kinetics; Microscopy, Fluorescence; Mitochondria; Molecular Imaging; Oligomycins; Oxidative Stress; Oxygen Consumption; Rotenone; Superoxides; Time Factors

Gliomas belong to the most infiltrative types of tumors. Photodynamic therapy (PDT) can be applied to regulate glioma cell proliferation. The inhibitors of PKCs (Protein Kinase C) are very promising drugs that can mediate glioma cells apoptosis in PDT. Hypericin is one of PKCs regulators, and thanks to its physicochemical properties it can be used in PDT. Rottlerin is also considered to be the PKCδ inhibitor. Its implementation in PDT may significantly influence glioma cells response to PDT.. The viability of U87 MG glioma cells in the presence of rottlerin and hypericin was assessed by MTT assay and flow cytometry in the absence and presence of light. The flow cytometric data were analyzed through Shannon entropy. The oxidative stress and immunocytochemistry of PKCδ and phosphorylated Bcl-2 (the regulators of apoptosis) were observed using fluorescence microscopy.. A pretreatment of glioma cells with rottlerin before hypericin induced PDT led to significant increase in apoptosis accompanied by the decrease of intracellular oxidative stress and increase of phosphorylated Bcl-2 in the cytoplasm of U87 MG cells.. In conclusion, we assume that the synergism between rottlerin and hypericin leads firstly to activation of rescue mechanisms in the glioma cells, but finally this cooperation triggers apoptosis rather than necrosis.

Topics: Acetophenones; Angiogenesis Inhibitors; Anthracenes; Antineoplastic Agents; Apoptosis; Benzopyrans; Cell Line, Tumor; Cell Survival; Gene Expression Regulation; Glioma; Humans; Light; Perylene; Photochemotherapy; Protein Kinase C-delta; Radiation-Sensitizing Agents

Rottlerin, isolated from a medicinal plant Mallotus phillippinensis, has been demonstrated to inhibit cellular growth and induce cytoxicity in glioblastoma cell lines through inhibition of calmodulin-dependent protein kinase III. Emerging evidence suggests that rottlerin exerts its antitumor activity as a protein kinase C inhibitor. Although further studies revealed that rottlerin regulated multiple signaling pathways to suppress tumor cell growth, the exact molecular insight on rottlerin-mediated tumor inhibition is not fully elucidated. In the current study, we determine the function of rottlerin on glioma cell growth, apoptosis, cell cycle, migration and invasion. We found that rottlerin inhibited cell growth, migration, invasion, but induced apoptosis and cell cycle arrest. Mechanistically, the expression of Cdc20 oncoprotein was measured by the RT-PCR and Western blot analysis in glioma cells treated with rottlerin. We observed that rottlerin significantly inhibited the expression of Cdc20 in glioma cells, implying that Cdc20 could be a novel target of rottlerin. In line with this, over-expression of Cdc20 decreased rottlerin-induced cell growth inhibition and apoptosis, whereas down-regulation of Cdc20 by its shRNA promotes rottlerin-induced anti-tumor activity. Our findings indicted that rottlerin could exert its tumor suppressive function by inhibiting Cdc20 pathway which is constitutively active in glioma cells. Therefore, down-regulation of Cdc20 by rottlerin could be a promising therapeutic strategy for the treatment of glioma.

Topics: Acetophenones; Apoptosis; Benzopyrans; Brain Neoplasms; Cdc20 Proteins; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Glioma; Humans; Neoplasm Invasiveness; RNA, Small Interfering

Despite advances in radiotherapeutic and chemotherapeutic techniques and aggressive surgical resection, the prognosis of glioblastoma patients is dismal. Accumulation of evidence indicates that some cancer cells survive even the most aggressive treatments, and these surviving cells, which are resistant to therapy and are perhaps essential for the malignancy, may be cancer stem cells. The CD133 surface marker is commonly used to isolate these extremely resistant glioma-initiating cells (GICs). In the present study, GICs which tested positive for the CD133 marker (CD133+) were isolated from both the established U251 cell line and the 5310 xenograft glioma cell line to study the events related to the molecular pathogenesis of these cells. Simultaneous down-regulation of uPAR and cathepsin B by shRNA (pUC) treatment caused the disruption of radiation-induced complex formation of pPKC θ/δ, integrin β1 and PKC ζ, integrin β1 in glioma cells. Further, pUC treatment inhibited PKC/integrin signaling via FAK by causing disassociation of FAK and the cytoskeletal molecules vinculin and α-actinin. Also, we observed the inhibition of ERK phosphorylation. This inhibition was mediated by pUC and directed a negative feedback mechanism over the FAK signaling molecules, which led to an extensive reduction in the signal for cytoskeletal organization generating migratory arrest. Altogether, it can be hypothesized that knockdown of uPAR and cathepsin B using shRNA is an effective strategy for controlling highly invasive glioma cells and extremely resistant glioma-initiating cells.

Topics: Acetophenones; Animals; Antigens, Differentiation; Benzopyrans; Cathepsin B; Cell Adhesion Molecules, Neuronal; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Cytoskeleton; Extracellular Matrix; Gene Expression; Gene Knockdown Techniques; Glioma; Humans; Integrin beta1; Integrins; Mice; Mice, Nude; Neoplastic Stem Cells; Protein Binding; Protein Kinase C; Radiation Tolerance; Receptors, Urokinase Plasminogen Activator; RNA Interference; RNA, Small Interfering; Signal Transduction; Spheroids, Cellular; Xenograft Model Antitumor Assays

Paraquat (1,1'-dimethyl-4,4'-bipyridinium) is structurally similar to the neurotoxin 1-methyl-4-phenyl-4-phenylpyridium ion (MPP+), the active metabolite of the parkinsonism-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which can induce the parkinsonism property in rodents, nonhuman primates, and human. In contrast to the neurotoxic effects of paraquat, little is known about its effects on glial cells. Here, we examined the mechanisms of paraquat toxicity in glial cells in culture. Paraquat treatment also reduced the viability of C6 glial cells in primary astrocyte cultures, and cell death was mostly apoptotic in nature. PKCdelta played a central role in the paraquat-induced glial cell death: (1) the PKCdelta-specific inhibitor rottlerin blocked paraquat-induced glial cell death; (2) paraquat induced tyrosine and threonine phosphorylation of PKCdelta; and (3) transfection of the dominant-negative mutant of PKCdelta attenuated paraquat toxicity. PKCdelta was also involved in the generation of reactive oxygen species (ROS), which mediated the paraquat toxicity. The nicotinamide adenine dinucleotide phosphate (reduced form) oxidase (NADPH oxidase) inhibitor diphenyleneiodonium blocked the paraquat-induced ROS production and subsequent cell death, indicating the involvement of NADPH oxidase in the cytotoxic action of paraquat in glia. PKCdelta was also important in glial cell death induced by MPP+ but not in that induced by rotenone. Last, Rac1 appeared to antagonize paraquat toxicity in glia. These results indicate a gliotoxic effect of paraquat and an opposing role of PKCdelta and Rac1 in paraquat-induced glial cell death.

Topics: Acetophenones; Animals; Apoptosis; Astrocytes; Benzopyrans; Cell Death; Cell Line, Tumor; Cell Survival; Cells, Cultured; Enzyme Inhibitors; Glioma; Neuroglia; Paraquat; Protein Kinase C-delta; rac1 GTP-Binding Protein; Rats; Recombinant Proteins; Transfection

Mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) are activated in the majority of gliomas and contribute to tumor cell growth and survival. Sorafenib (Bay43-9006; Nexavar) is a dual-action Raf and vascular endothelial growth factor receptor inhibitor that blocks receptor phosphorylation and MAPK-mediated signaling and inhibits growth in a number of tumor types. Because our initial studies of this agent in a series of glioma cell lines showed only partial growth inhibition at clinically achievable concentrations, we questioned whether inhibition of PKC signaling using the PKC-delta inhibitor rottlerin might potentiate therapeutic efficacy. Proliferation assays, apoptosis induction studies, and Western immunoblot analysis were conducted in cells treated with sorafenib and rottlerin as single agents or in combination. Sorafenib and rottlerin reduced proliferation in all cell lines when used as single agents, and the combination produced marked potentiation of growth inhibition. Flow-cytometric measurements of cells stained with Annexin V-propidium iodide and immunocytochemical assessment of cytochrome c and apoptosis-inducing factor release demonstrated that addition of rottlerin resulted in significantly higher levels of apoptosis than sorafenib alone. In addition, the combination of sorafenib and rottlerin reduced or completely inhibited the phosphorylation of extracellular signal-regulated kinase and Akt and down-regulated cell cycle regulatory proteins such as cyclin-D1, cyclin-D3, cyclin-dependent kinase (cdk)4, and cdk6 in a dose- and time-dependent manner. Our results clearly indicate that inhibition of PKC-delta signaling enhances the antiproliferative effect of sorafenib in malignant human glioma cell lines and support the examination of combinations of signaling inhibitors in these tumors.

Topics: Acetophenones; Annexin A5; Antineoplastic Agents; Apoptosis; Benzenesulfonates; Benzopyrans; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Cell Survival; Clone Cells; Drug Synergism; Enzyme Inhibitors; Glioma; Humans; Immunohistochemistry; Microscopy, Fluorescence; Niacinamide; Phenylurea Compounds; Platelet-Derived Growth Factor; Protein Folding; Protein Kinase C; Pyridines; Sorafenib; Vascular Endothelial Growth Factor A

In the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant glioma cells, treatment with TRAIL in combination with subtoxic doses of rottlerin induced rapid apoptosis. While the proteolytic processing of procaspase-3 by TRAIL was partially blocked in these cells, treatment with rottlerin efficiently recovered TRAIL-induced activation of caspases. Treatment with rottlerin significantly decreased Cdc2 activity through the downregulation of cyclin A, cyclin B, and Cdc2 proteins, whereas the sensitizing effect of rottlerin on TRAIL-induced apoptosis was independent of PKCdelta activity. Furthermore, treatment with rottlerin downregulated the protein levels of survivin and X-chromosome-linked IAP (XIAP), two major caspase inhibitors. Forced expression of Cdc2 together with cyclin B attenuated rottlerin-potentiated TRAIL-induced apoptosis by over-riding the rottlerin-mediated downregulation of survivin and XIAP protein levels. Taken together, inhibition of Cdc2 activity and the subsequent downregulation of survivin and XIAP by subtoxic doses of rottlerin contribute to amplification of caspase cascades, thereby overcoming resistance of glioma cells to TRAIL-mediated apoptosis. Since rottlerin can sensitize Bcl-2- or Bcl-xL-overexpressing glioma cells but not human astrocytes to TRAIL-induced apoptosis, this combined treatment may offer an attractive strategy for safely treating resistant gliomas.

Topics: Acetophenones; Antigens, Neoplasm; Apoptosis; Apoptosis Regulatory Proteins; Benzopyrans; Caspase Inhibitors; CDC2 Protein Kinase; Cell Line, Tumor; Enzyme Inhibitors; Glioma; Humans; Inhibitor of Apoptosis Proteins; Membrane Glycoproteins; Microtubule-Associated Proteins; Neoplasm Proteins; Proteins; Survivin; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; X-Linked Inhibitor of Apoptosis Protein

Activation of the A(2A) adenosine receptor (A(2A)AR) contributes to the neuromodulatory and neuroprotective effects of adenosine in the central nervous system. Here we demonstrate that, in rat C6 glioma cells stably expressing an epitope-tagged canine A(2A)AR, receptor phosphorylation on serine and threonine residues can be increased by pretreatment with either the synthetic protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or endothelin 1, which increases PKC activity via binding to endogenous endothelin(A) receptors. Under conditions in which PMA was maximally effective, activation of other second messenger-regulated kinases was without effect. While basal and PMA-stimulated phosphorylation were unaffected by the A(2A)AR-selective antagonist ZM241385, they were both blocked by GF109203X (a selective inhibitor of conventional and novel PKC isoforms) and rottlerin (a PKCdelta-selective inhibitor) but not Go6976 (selective for conventional PKC isoforms). However, coexpression of the A(2A)AR with each of the alpha, betaI, and betaII isoforms of PKC increased basal and PMA-stimulated phosphorylation. Mutation of the three consensus PKC phosphorylation sites within the receptor (Thr298, Ser320, and Ser335) to Ala failed to inhibit either basal or PMA-stimulated phosphorylation. In addition, phosphorylation of the receptor was not associated with detectable changes in either its signaling capacity or cell surface expression. These observations suggest that multiple PKC isoforms can stimulate A(2A)AR phosphorylation via activation of one or more downstream kinases which then phosphorylate the receptor directly. In addition, it is likely that phosphorylation controls interactions with regulatory proteins distinct from those involved in the classical cAMP signaling pathway utilized by this receptor.

Topics: Acetophenones; Animals; Benzopyrans; Carbazoles; CHO Cells; Consensus Sequence; Cricetinae; Cyclic AMP; Dogs; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Glioma; Indoles; Isoenzymes; Maleimides; Mutagenesis, Site-Directed; Phosphorylation; Protein Kinase C; Rats; Receptor, Adenosine A2A; Receptors, Purinergic P1; Signal Transduction; Tetradecanoylphorbol Acetate; Triazines; Triazoles; Tumor Cells, Cultured