H3 histamine receptor-mediated activation of protein kinase Cα inhibits the growth of cholangiocarcinoma in vitro and in vivo

Abstract

Histamine regulates functions via four receptors (HRH1, HRH2, HRH3, and HRH4). The D-myo-inositol 1,4,5-trisphosphate (IP3)/Ca2+/protein kinase C (PKC)/mitogen-activated protein kinase pathway regulates cholangiocarcinoma growth. We evaluated the role of HRH3 in the regulation of cholangiocarcinoma growth. Expression of HRH3 in intrahepatic and extrahepatic cell lines, normal cholangiocytes, and human tissue arrays was measured. In Mz-ChA-1 cells stimulated with (R)-(á)-(-)-methylhistamine dihydrobromide (RAMH), we measured (a) cell growth, (b) IP3 and cyclic AMP levels, and (c) phosphorylation of PKC and mitogen-activated protein kinase isoforms. Localization of PKCá was visualized by immunofluorescence in cell smears and immunoblotting for PKCá in cytosol and membrane fractions. Following knockdown of PKCá, Mz-ChA-1 cells were stimulated with RAMH before evaluating cell growth and extracellular signal-regulated kinase (ERK)-1/2phosphorylation. In vivo experiments were done in BALB/c nude mice. Mice were treated with saline or RAMH for 44 days and tumor volume was measured. Tumors were excised and evaluated for proliferation, apoptosis, and expression of PKCá, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF receptor 2, and VEGF receptor 3. HRH3 expression was found in all cells. RAMH inhibited the growth of cholangiocarcinoma cells. RAMH increased IP3 levels and PKCá phosphorylation and decreased ERK1/2phosphorylation. RAMH induced a shift in the localization of PKCá expression from the cytosolic domain into the membrane region of Mz-ChA-1 cells. Silencing of PKCá prevented RAMH inhibition of Mz-ChA-1 cell growth and ablated RAMH effects on ERK1/2 phosphorylation. In vivo, RAMH decreased tumor growth and expression of VEGF and its receptors; PKCá expression was increased. RAMH inhibits cholangiocarcinoma growth by PKCá-dependent ERK1/2dephosphorylation. Modulation of PKCá by histamine receptors may be important in regulating cholangiocarcinoma growth. Copyright © 2009 American Association for Cancer Research.