Neuroendocrine tumor cell growth inhibition by ZM336372 through alterations in multiple signaling pathways.

Abstract

We have shown previously that activation of the Raf-1/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK)1/2 signaling pathway by ZM336372 inhibits carcinoid cells growth. In the present study, we further characterize the molecular details of the growth inhibition by the signaling-based compound ZM336372 in neuroendocrine neoplasms (NENs). NEN cells were treated with ZM336372 (20 to 100 mumol/L) or carrier (DMSO). Western Blot was used to determine the activation of the Raf-1/MEK/ERK, other pathways activation, and cellular bioactive hormone production. ZM336372 in NEN cells resulted in increasing raf-1 activation and inactivation of glycogen synthase kinase-3 beta (GSK-3beta) as measured by phosphorylation of ERK1/2 and GSK-3beta, respectively. There was no alteration in the levels of phosphorylated Akt, an important mediator of the phosphatidyl inositol 3 kinase pathway. Importantly, blocking of raf-1 pathway by U0126, a potent inhibitor, in the presence of ZM336372 did not reduce the levels of p-GSK-3beta, indicating that GSK-3beta inactivation is independent of raf-1 pathway activation. Moreover, the levels of chromogranin A and achaete-scute complex like-1 reductions were persistent even after blocking the raf-1 pathway. Treatment with ZM336372 in the presence of small interfering RNA against raf-1 resulted in an increase in Raf-1 production, suggesting that ZM336372 upregulates raf-1 at the transcriptional level. This is the first description of a novel compound ZM336372 that regulates multiple pathways in NEN cells.