Involvement of microtubules and mitochondria in the antagonism of arsenic trioxide on paclitaxel-induced apoptosis

Arsenic trioxide (As(2)O(3)) at low concentrations (1-10 microM) is effective in the treatment of acute promyelocytic leukemia (APL) and lymphoma and is in clinical trials for treatment of solid tumors. Paclitaxel, an antimicrotubule agent, is highly efficacious in the treatment of adult tumors and is in clinical evaluation in childhood tumors. This study is the first to investigate the combination of arsenic and paclitaxel in the range of clinically achievable concentrations. We found that the simultaneous combination was antagonistic on proliferation of the neuroblastoma SK-N-SH cell line by using the combination index (CI) method. Moreover, a 40+/-5% decrease in paclitaxel-induced apoptosis in cells co-treated with As(2)O(3) confirmed the antagonism. The mechanism of antagonism was studied at the cellular level with 200 nM paclitaxel, twice the IC(50) value, and with 1 microM As(2)O(3) which administered singly did not affect cell survival or the microtubule network. As(2)O(3) antagonized the effects of paclitaxel on tubulin and microtubules. Paclitaxel-induced mitotic block was decreased by 20+/-2% and bundles induced by 200 nM paclitaxel were less condensed in the presence of 1 microM As(2)O(3). As(2)O(3) (10-200 microM) induced a concentration-dependent inhibition of tubulin polymerization in vitro which was maintained in presence of paclitaxel. Spectrophotometric and spectrofluorometric measurements indicated an interaction of As(2)O(3) with tubulin SH groups, without modification of the stoichiometry of paclitaxel binding to tubulin. Moreover, 4 microM As(2)O(3) inhibited the release of cytochrome c from isolated mitochondria by 78+/-10%. Our results show that As(2)O(3) and paclitaxel act antagonistically on mitochondria and microtubules and illustrate the need for careful evaluation of drug combinations.