Molecular mechanisms of the growth inhibitory effects of the bacterial metabolites, kinamycin A and C.

5546

The bacterial metabolites, kinamycin A and kinamycin C, are cytotoxic to cancer cells by some unknown mechanism. Both kinamycins were able to induce erythroid differentiation in K562 cells, a chronic myelogenous leukemia cell line, as measured by increased benzidine staining, an indicator of hemoglobin content. Because the cyclin dependent kinase inhibitor, p27^Kip1, is important for erythroid differentiation in K562 cells, protein levels of p27^Kip1 were examined after exposure to either kinamyicn A or C. To identify possible downstream targets of the kinamycins that are involved in cell cycle, protein levels of cyclins D1, D3, and E were studied in K562 cells exposed to either kinamycin A or C. Both kinamycin A and C contain an unusual and potentially reactive diazo group as well as a quinone group. The hypothesis that the kinamycins react with sulfhydryl groups was explored. When kinamycin C was pre-exposed to either dithiothreitol or glutathione (GSH), the IC₅₀ for K562 cells was increased 8- and 5-fold, respectively. Similarly, exposure of K562 cells to kinamycin A that had been pre-treated with GSH also increased the IC₅₀ 5-fold. The reaction of GSH with kinamycin A and C was examined spectrophotometrically and the compounds underwent a fast initial reaction with GSH. This was followed by a series of slower reactions. These data suggest that the reactivity of kinamycin A and C is modulated by GSH and also raises the possibility that the target may be an as of yet unknown sulfhydryl-containing protein. The GSH/kinamycin C system generated a free radical that could be detected by EPR spin trapping experiments. This idea, that the kinamycins may be reductively activated to their cytotoxic form, is actively being pursued. Support: MHRC fellowship (O’Hara, KA), IHR and a Canada Research Chair in Drug Development (Hasinoff, BB), NSERC (Dmitrienko, GI).