GROWTH-INHIBITION OF GASTRIC-CANCER CELL-LINES BY THE TYRPHOSTIN RG13022 AND ITS EFFECTS ON INTRACELLULAR SIGNALING

Overexpression of EGFr and c-erbB-2 is related to poor prognosis in a variety of cancers including gastric cancer. Thus: the ability to modu late the functional activity of these receptors is an attractive targe t for diagnostic intervention. In this study we examined the effect of a well characterised tyrosine kinase inhibitor (RG13022) on the cellu lar 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 gr own either in serum-free conditions or in the presence of FCS. Western blotting revealed RG13022 caused an inhibition of EGF stimulated tyro sine 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 phosphorylatio n was observed in N87 cells. In both A431 cells and N87 cells (which o verexpress EGFr and c-erbB-2 respectively) exposed to EGF, MAP2 kinase immunoblot analysis resulted in the detection of a second protein ban d with reduced migration in SDS-PAGE. In N87 cells, this protein appea red to co-mi,orate with a strongly tyrosine phosphorylated protein, wh ich suggests that it is a hyper-phosphorylated form of MAP2 kinase. Ho wever, treatment with RG13022, whilst inhibiting phosphorylation of th is protein, did not prevent a shift in gel mobility (suggestive of act ivation) of MAP2 kinase in response to EGF. These findings demonstrate that the tyrphostin RG13022 inhibits cell proliferation of two primar y gastric cancer cell lines. Investigation of intracellular signalling pathways suggests that alterations in intracellular signalling are re sponsible 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 immun oreactivity and migration on SDS gels but fails to affect this protein in the MKN45 cell line. This data questions the usefulness of MAP2 ki nase gel shift assays as markers of activation but supports the furthe r development of tyrosine kinase inhibitors as potential inhibitors of gastric tumour proliferation.