Introduction: Hepatocellular carcinoma (HCC) has previously shown characteristics similar to that of neuroendocrine tumors (NETs). This includes a similar response to Notch1 activation, which in both cases results in growth inhibition and cell cycle arrest. Chromogranin A secretion can occur in both HCC and NETs. In addition, NETs and HCC uncommonly possess activating mutations of the Ras/Raf-1/MEK/ERK pathway, and have actually shown anti-neoplastic responses to ERK activation. This has previously been shown in NETs through an estrogen-inducible Raf-1 construct. In the HepG2 hepatocellular carcinoma cell line growth inhibition has been shown with ERK activation secondary to both induced constitutive expression of Ha-Ras and treatment with hepatocyte growth factor. ZM336372, a Raf-1 activating agent, has previously been shown to cause growth inhibition and suppression of hormone secretion in a neuroendocrine cell line. Here we examine treatment of the HepG2 cell line with ZM336732 to determine if a similar anti-proliferative response will be obtained.
Methods: The HepG2 cells were treated with (1-25 μM) ZM336372 or solvent (DMSO). The result on the proliferation of this cell line was measured using the MTT assay. Western blot analysis was performed to examine the activation of the Raf-1/MEK/ERK pathway and chromogranin A production. UO126, a MEK inhibitor was used to aid in mechanistic studies.
Results: Growth inhibition was observed using concentrations of ZM336372 greater than 1 μM. Suppression of growth increased as the doses of ZM336372 escalated until complete growth suppression was observed at a concentration of 15 μM. No baseline phosphorylation of ERK 1/2 was observed, however activation was observed after treatment with ZM336372. In addition, ERK1/2 phosphorylation due to ZM336372 treatment was inhibited with UO126 treatment. The presence of chromogranin A secretion from the HepG2 cell line was confirmed. Chromogranin A secretion was suppressed due to treatment with ZM336372.
Conclusion: ZM336372 causes growth inhibition and suppression of hormone secretion in a human hepatocellular carcinoma cell line, similar to that previously seen in NETs.