GELDANAMYCIN-INDUCED CYTOTOXICITY IN HUMAN COLON-CANCER CELL-LINES - EVIDENCE AGAINST THE INVOLVEMENT OF C-SRC OR DT-DIAPHORASE

We investigated two of the major proposed modes of action of the benzo quinoid ansamycin geldanamycin using a pair of human colon-carcinoma c ell lines, BE and HT29. One potential mechanism of action in colorecta l cancer is the inhibition of c-Src kinase activity, since this proto- oncogene is hyperexpressed in human large-bowel rumours. Our results s how that despite the 9-fold higher level of c-Src kinase activity foun d in HT29 cells, there was only a 1.4-fold difference in cytotoxicity as compared with BE cells, the latter being the most sensitive. Moreov er, even at concentrations of geldanamycin that resulted in cell kill of 80% or more after a 24-h period of exposure, there was no effect on c-Src kinase activity in HT29 cells, although c-Src protein was deple ted at supralethal levels of exposure. We also investigated the metabo lism of the quinone moiety of geldanamycin by DT-diaphorase, an enzyme that activates certain quinone antibiotics such as mitomycin C and is hyperexpressed in colorectal cancer cells. Geldanamycin was shown to be a substrate for DT-diaphorase present in HT29 cells. However, the l ack of a major differential in cytotoxicity between HT29 and BE cells indicates that this is unlikely to be pharmacologically significant, s ince the former contains high levels of enzyme activity, whereas BE ce lls have no significant activity due to a point mutation in the DT-dia phorase (NQO1) gene. Although reduction of geldanamycin was also catal ysed by non-DT-diaphorase reductases in HT29 and BE cells, providing t he potential for free radical induction, this is unlikely to be signif icant since studies previously reported by us elsewhere showed that ce lls exposed to geldanamycin exhibited no evidence of DNA damage. Thus, as far as the mode of action of geldanamycin in human colon-carcinoma cells is concerned, the present results rule out two major possibilit ies; namely, the involvement of c-Src tyrosine kinase inhibition and D T-diaphorase metabolism.