endothelin-1 and rottlerin

Rottlerin, a natural product purified from Mallotus philippinensis, has a number of target molecules and biological effects. We recently found that Rottlerin caused growth arrest in MCF-7 breast cancer cells and human immortalized keratinocytes, through inhibition of NFκB and downregulation of cyclin D-1. To evaluate whether this effect could be generalized to primary cells, human microvascular endothelial cells were treated with Rottlerin. In this study, we demonstrated that Rottlerin prevents basal and TNFα-stimulated NFκB nuclear migration and DNA binding also in human microvascular endothelial cell, where NFκB inhibition was accompanied by the downregulation of NFκB target gene products, such as cyclin D-1 and endothelin-1, which are essential molecules for endothelial cell proliferation and survival. Rottlerin, indeed, inhibited human microvascular endothelial cells proliferation and tube formation on Matrigel. Rottlerin also increases cytoplasmic free calcium and nitric oxide levels and downregulates endothelin converting enzyme-1 expression, thus contributing to the drop in endothelin-1 and growth arrest. These results suggest that Rottlerin may prove useful in the development of therapeutic agents against angiogenesis.

Topics: Acetophenones; Active Transport, Cell Nucleus; Angiogenesis Inhibitors; Aspartic Acid Endopeptidases; Benzopyrans; Calcium; Cell Proliferation; Cells, Cultured; Cyclin D1; DNA; Endothelial Cells; Endothelin-1; Endothelin-Converting Enzymes; Endothelium, Vascular; Gene Expression Regulation; Humans; Mallotus Plant; Metalloendopeptidases; Neovascularization, Physiologic; NF-kappa B; Nitrogen Oxides; RNA, Messenger; Tumor Necrosis Factor-alpha

1. Cardiomyocyte apoptosis plays an important role in the transition from cardiac hypertrophy to heart failure. Hyper-trophic cardiomyocytes show an increased susceptibility to apoptotic stimuli, but the mechanisms remain unclear. 2. We hypothesized that activated protein kinase Cδ (PKCδ) associated with cardiomyocyte hypertrophy could move from the cytoplasm to mitochondria, and subsequently trigger the apoptotic signalling pathway. 3. Hypertrophy was induced in cultured neonatal rat cardiomyocytes using endothelin-1 (ET-1), insulin-like growth factor-1 (IGF-1), thyroid hormone (T(3) ) or angiotensin-II (AngII). AngII at high concentrations (1 and 10 nmol/L) also induced apoptosis. Hypertrophic cells were then treated with AngII with or without specific inhibitors of the angiotensin receptors AT(1) and AT(2) (losartan and PD123319, respectively), endothelin receptor A (BQ-123) and PKCδ (rottlerin). ET-1 plus AngII had a threefold and significant increase in apoptosis in the hypertrophic cultures compared with AngII alone. In association with the increase in apoptosis, this treatment also promoted mitochondrial translocation of PKCδ, and increased expression of cleaved caspase 9 and activity of caspase 3. All of these increases were modulated by concurrent use of the PKCδ inhibitor, rottlerin. 4. The results suggest that apoptotic signalling in hypertrophic cardiomyocytes is determined by mitochondrial pathways involving PKCδ.

Topics: Acetophenones; Angiotensin II; Animals; Apoptosis; Benzopyrans; Cardiomegaly; Caspase 3; Caspase 9; Cells, Cultured; Endothelin-1; Imidazoles; Insulin-Like Growth Factor I; Mitochondria; Myocytes, Cardiac; Peptides, Cyclic; Protein Kinase C-delta; Protein Transport; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Endothelin; Signal Transduction; Triiodothyronine

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