Lysate array analyses of signal transduction inhibitors in tumor cell lines

Abstract

We probed the ability of reverse lysate array technology to help explain potential differences in the responses of cancer cells to various small-molecule kinase inhibitors. To understand the antitumor potential of signal transduction inhibitors and their effects on signaling pathways downstream of Src, we used reverse lysate array technology to study SIG11293, a selective inhibitor of Src and LcK kinases, and AEE788, a selective inhibitor of Kdr (VEGFR1) and epidermal growth factor receptor/ErbB-2 that also has affinity for Src, c-abl, c-fms, and Flt-1. We observed the effects of drug dose on cell killing and expression and phosphorylation of various signal transduction proteins in MDA435 and MDA231 human breast cancer cells and U251HF glioblastoma cells. After 24 h, SIG11293 induced the least amount of cell killing in MDA435 cells; decreased Stat3(pY705) and Src(pY529) in all cell lines; decreased Src(pY418) and total Src in MDA231 and MDA435 cells, but not U251 cells; and in U251 cells, uniquely increased activated caspase 3, Src(pY418), panSrc, and p70S6K. AEE788 decreased Src(pY529) and Stat3(pY705) in U251HF and MDA435 cells. In regard to Src phosphorylation, both drugs decreased the negative regulatory site, Src (pY529), more than the positive regulatory site, Src(pY418), relative to total Src. These observations suggest that the two drugs have complex and different effects on Src signaling pathways. Although this general conclusion could be predicted, we believe that these studies exemplify the ability and robustness of reverse lysate arrays to measure signaling pathway modulation in tumor cells. Our hope is that these techniques will help to develop more robust preclinical and, eventually, clinical treatment paradigms.