Leinamycin is a structurally novel Streptomyces-derived
natural product that displays very potent activity against various human
cancer cell lines (IC(50) values in the low nanomolar range). Previous in vitro biochemical studies have revealed that
leinamycin alkylates
DNA, generates apurinic (AP) sites and
reactive oxygen species (ROS), and causes
DNA strand breaks. However, it is not clear whether these events occur inside cells. In the present study, we have determined the endogenous amount of AP sites and
DNA strand breaks in genomic
DNA and the amount of oxidative stress in a human
pancreatic carcinoma cell line, MiaPaCa, treated with
leinamycin by utilizing the
aldehyde-reactive probe assay, the comet assay, and
fluorescent probes, respectively. We demonstrated that AP sites are formed rapidly following exposure to
leinamycin, and the number of AP sites was increased up to seven-fold in a dose-dependent manner. However, only 25-50% of these sites remain 2 h after media containing
drug molecules were aspirated and replaced with fresh media. We also observed
leinamycin-induced ROS generation and a concomitant increase in apoptosis of MiaPaCa cells. Because both AP sites and ROS have the potential to generate strand breaks in cellular
DNA, the comet assay was utilized to detect damage to nuclear
DNA in
leinamycin-treated MiaPaCa cell cultures. Both alkaline and neutral electrophoretic analysis revealed that
leinamycin produces both single- and
double-stranded DNA damage in
drug-treated cells in a dose-dependent manner. Taken together, the results suggest that rapid conversion of
leinamycin-
guanine (N7) adducts into AP sites to produce
DNA strand breaks, in synergy with
leinamycin-derived ROS, accounts for the exceedingly potent
biological activity of this
natural product.