Stimulation of α2-adrenergic receptor inhibits cholangiocarcinoma growth through modulation of Raf-1 and B-Raf activities

Growth factor signaling, mediated by the mitogen-activated protein kinase (MAPK) cascade, induces cell mitosis. Adenosine 3',5'-monophosphate (cAMP) may inhibit or stimulate mitosis (depending on the cell type) through the activation of MAPK and Raf proteins. Among Raf proteins, Raf-1 and B-Raf differentially regulate mitosis. Our aims were to evaluate the role and mechanisms of action of the alpha(2)-adrenergic agonist UK14,304 in the regulation of growth of the human cholangiocarcinoma cell line Mz-ChA-1. Immunocytochemistry and immunoblotting for alpha(2A)-, alpha(2B)-, or alpha(2C)-adrenergic receptor subtypes showed positive reaction in Mz-ChA-1 cells. We found that physiological concentrations of UK14,304 increased cAMP levels and inhibited proliferation and MAPK activity in Mz-ChA-1 cells. Mz-ChA-1 cells expressed Raf-1 and B-Raf. Epidermal growth factor (EGF) immediately and transiently stimulated Raf-1 activity, whereas B-Raf activity was increased with prolonged EGF stimulation. EGF-stimulated Raf-1 and B-Raf activities were both inhibited by UK14,304. UK14,304 did not affect Ras activity. In Mz-ChA-1 cells, alpha(2)-adrenoreceptor stimulation causes up-regulation of cAMP, which inhibits EGF-induced MAPK activity through an acute increase of Raf-1 and sustained activation of B-Raf. In conclusion, because alpha(2)-AR inhibition of growth occurred downstream of Ras, adrenergic stimulation or other stimulants of cAMP may overcome the Ras mutations and offer a new therapeutic approach for patients with cholangiocarcinoma.