Prostaglandin E receptors (EP2 and EP4) mediated NNK promoted gastric carcinogenesis

Abstract

BACKGROUND: Cigarette smoking is associated with various cancer deaths including gastric cancer. Ample evidence demonstrated that increased cumulative dose of cigarette smoke associated with increased risk of gastric cancer. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most important components in cigarette smoke contributes to gastric cancer development. However, the molecular mechanism by which NNK promotes gastric carcinogenesis is unclear. We have previously reported that nicotine promoted gastric cancer growth through cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway. AIM: This study aimed to examine the roles of PGE2 receptors (EP) in NNK promoted gastric cancer growth. Methods: Gastric cancer cell line MKN45 was cultured in presence or absence of NNK (10-100nM) treated with or without antagonists of EPs. Nude mice implanted with MNK-45 were used as human gastric cancer xenograft model. Cells were incubated with or without NNK for 24 hrs prior to subcutaneous injection into flank of mice. Cell growth was measured by [3H]-thymidine incorporation assay, cell proliferation by proliferating cell nuclear antigen, angiogenesis by von Willebrand factor and apoptosis by TUNEL. mRNA and protein levels were determined by real time RT-PCR and western blot respectively. RESULTS: Both In Vitro and In Vivo studies showed that NNK promoted cancer cell growth concomitant with COX-2/PGE2 upregulation. In cell line, mRNA levels of EP1-4 were constitutively expressed in basal condition, and NNK upregulated EP2 and EP4 expression but not EP1 and EP3. Such induction was significantly blunted by antagonists of EP2 (AH6809, 3uM) and EP4 (AH23848, 30uM). On the other hand, NNK (10-100nM) promoted tumor growth in a dose dependent manner In Vivo. Pretreatment with EP2 and EP4 antagonists repressed the tumor growth, concomitant with reduced cell proliferation, angiogenesis and increased apoptosis. Moreover, EP2 and EP4 antagonists reversed NNK induced tumorigenic properties through repression of PGE2, VEGF and VEGFR-2 levels. VEGFR inhibitor (CBOP11, 10uM) abrogated NNK-induced COX-2 and PGE2 levels, leading to retardation of cell proliferation. In addition, NNK caused upregulation of Bcl2, cyclin D1 and the associated cyclin-dependent kinase (CDK) 4/6, and downregulation of caspase-3, p21 and p27, which was abrogated by EP2 and EP4 antagonists. Conclusion: These data strongly indicate that EP2 and EP4 are important for NNK promoted gastric carcinogenesis. EP2 and EP4 antagonists therefore represent putative novel molecular targets for chemopreventive therapy or to inhibit gastric cancer growth for smokers.