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Expression of kinase-defective mutants of c-Src in human metastatic colon cancer cells decreases Bcl-x(L) and increases oxaliplatin- and Fas-induced apoptosis

Durham University, Durham, England, United Kingdom
Journal of Biological Chemistry (Impact Factor: 4.6). 11/2004; 279(44):46113-21. DOI: 10.1074/jbc.M408550200
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ABSTRACT Tumor resistance to current drugs prevents curative treatment of human colon cancer. A pressing need for effective, tumor-specific chemotherapies exists. The non-receptor-tyrosine kinase c-Src is overexpressed in >70% of human colon cancers and represents a tractable drug target. KM12L4A human metastatic colon cancer cells were stably transfected with two distinct kinase-defective mutants of c-src. Their response to oxaliplatin, to SN38, the active metabolite of irinotecan (drugs active in colon cancer), and to activation of the death receptor Fas was compared with vector control cells in terms of cell cycle arrest and apoptosis. Both kinase-defective forms of c-Src co-sensitized cells to apoptosis induced by oxaliplatin and Fas activation but not by SN38. Cells harboring kinase-defective forms of c-Src carrying function blocking point mutations in SH3 or SH2 domains were similarly sensitive to oxaliplatin, suggesting that reduction in kinase activity and not a Src SH2-SH3 scaffold function was responsible for the observed altered sensitivity. Oxaliplatin-induced apoptosis, potentiated by kinase-defective c-Src mutants, was dependent on activation of caspase 8 and associated with Bid cleavage. Each of the stable cell lines in which kinase-defective mutants of c-Src were expressed had reduced levels of Bcl-x(L.) However, inhibition of c-Src kinase activity by PP2 in vector control cells did not alter the oxaliplatin response over 72 h nor did it reduce Bcl-x(L) levels. The data suggest that longer term suppression of Src kinase activity may be required to lower Bcl-x(L) levels and sensitize colon cancer cells to oxaliplatin-induced apoptosis.

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    • "Irinotecan is a prodrug that, in presence of hepatic or gastrointestinal carboxylesterase, is metabolized to its active form 7-ethyl-10-hydroxy camptothecin (SN-38). Irinotecan and SN-38 bind to the topoisomerase DNA complex preventing religation of the singlestrand breaks in the DNA molecule and causing DNA double strand breaks, which can lead to apoptosis (Catley et al., 2004; Griffiths et al., 2004). The dose-limiting toxic effects of irinotecan are myelosuppression and diarrhea. "
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    ABSTRACT: Thesis (Master of Fine Arts)--University of Cincinnati, 1990.
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