Cell growth and topoisomerase IIα inhibitory activity of the bacterial metabolites kinamycin C and kinamycin A

Proc Amer Assoc Cancer Res, Volume 46, 2005 2392 The antibiotics kinamycin C (KC) and kinamycin A (KA) are naturally occurring bacterial metabolites isolated from Streptomyces murayamaensis that contain an unusual and potentially reactive diazo side chain. Although the antimicrobial activity of the kinamycins has been described, the mechanism of inhibition of cell growth by these compounds is unknown. Our results show that KC and KA strongly inhibit the growth of the human erythroleukemic K562 cells ( IC 50 of 0.2 and 0.3 μM, respectively). A short drug treatment of the cells also resulted in strong growth inhibition indicating a rapid onset of action. KC rapidly induced apoptosis in K562 cells as measured by flow cytometry using annexin V-FITC staining and PI staining of DNA content. When synchronized CHO cells were treated with KA, the cells were able to exit G0 and traverse the first G2/M phase, but were not able to enter the second G2/M phase. KC and KA also inhibited the catalytic activity of purified human DNA topoisomerase IIα (topo II), but not that of topoisomerase I. Preincubation of KC with topo II reduced topo II activity, indicating that KC may have inhibited topo II through a direct reaction. The inhibitory activities of KC and KA were modulated by the presence of dithiothreitol (DTT) in the assay mixture as an increase in the DTT concentration (from 0.1 to 250 μM) in the reaction mixture increased the IC 50 of KC from 9 to 108 μM, and of KA from 8 to 66 μM. Based on the ability of DTT to partially protect topo II from inhibition by these compounds, the possibility that these compounds inhibit by reacting with critical thiol groups on topo II is being explored. Neither KC nor KA were able to cross-link DNA, nor did they bind to DNA, which indicated that these compounds did not act directly on DNA. KA and KC were also examined for their ability to stabilize a DNA-topo II covalent complex and act as a topoisomerase IIα poison by measuring their ability to cleave pBR322 DNA to produce linear DNA. Neither KA nor KC induced DNA cleavage and, thus, it was concluded that they did not act as topo II poisons. Also the growth inhibitory effects of KC and KA on K562 and K/VP.5 cells (containing one-fifth the topo II content of the parental K562 cells) were not significantly different, a result that is also consistent with these compounds exerting their activity by mechanisms distinct from topo II poisoning. In summary these results indicate that even though KC and KA inhibited topo II catalytic activity, this may not be the mechanism by which they inhibited cell growth. Support: CIHR and a Canada Research Chair in Drug Development to BH, NSERC through a Discovery Grant to GID and ROI CA90787 to JCY.