Growth inhibition of gastric cancer cell lines by the tyrphostin RG13022 and its effects on intracellular signalling

Abstract

Overexpression of EGFr and c-erbB-2 is related to poor prognosis in a variety of cancers including gastric cancer. Thus: the ability to modulate the functional activity of these receptors is an attractive target for diagnostic intervention. In this study we examined the effect of a well characterised tyrosine kinase inhibitor (RG13022) on the cellular proliferation and EGF activated tyrosine kinase signalling pathway of two primary gastric cell lines: MKN45 and N87. RG13022 has a dose dependent, antiproliferative effect on both gastric cell lines when grown either in serum-free conditions or in the presence of FCS. Western blotting revealed RG13022 caused an inhibition of EGF stimulated tyrosine phosphorylation of EGFr in A431 cells and both EGFr and c-erbB-2 in MKN45 cells. No clear modulation of EGFr or c-erbB-2 phosphorylation was observed in N87 cells. In both A431 cells and N87 cells (which overexpress EGFr and c-erbB-2 respectively) exposed to EGF, MAP2 kinase immunoblot analysis resulted in the detection of a second protein band with reduced migration in SDS-PAGE. In N87 cells, this protein appeared to co-mi,orate with a strongly tyrosine phosphorylated protein, which suggests that it is a hyper-phosphorylated form of MAP2 kinase. However, treatment with RG13022, whilst inhibiting phosphorylation of this protein, did not prevent a shift in gel mobility (suggestive of activation) of MAP2 kinase in response to EGF. These findings demonstrate that the tyrphostin RG13022 inhibits cell proliferation of two primary gastric cancer cell lines. Investigation of intracellular signalling pathways suggests that alterations in intracellular signalling are responsible for the actions of RG 13022 in these cells. The biochemical analysis revealed that in N87 and A431, cells which overexpress c-erbB-2 and EGFr respectively, the tyrphostin affects the MAP2 kinase immunoreactivity and migration on SDS gels but fails to affect this protein in the MKN45 cell line. This data questions the usefulness of MAP2 kinase gel shift assays as markers of activation but supports the further development of tyrosine kinase inhibitors as potential inhibitors of gastric tumour proliferation.