Potent inhibition of rhabdoid tumor cells by combination of flavopiridol and 4OH-tamoxifen

Rhabdoid Tumors (RTs) are highly aggressive pediatric malignancies with poor prognosis. There are currently no standard or effective treatments for RTs in part because treatments are not designed to specifically target these tumors. Our previous studies indicated that targeting the cyclin/cdk pathway is a novel therapeutic strategy for RTs and that a pan-cdk inhibitor, flavopiridol, inhibits RT growth. Since the toxicities and narrow window of activity associated with flavopiridol may limit its clinical use, we tested the effect of combining flavopiridol with 4-hydroxy-Tamoxifen (4OH-Tam) in order to reduce the concentration of flavopiridol needed for inhibition of RTs.

Combining flavopiridol with 4OH-Tam potently inhibited the growth of RT cells by increasing the ability of either drug alone to induce caspases 2 and 3 thereby causing apoptosis. The potency of flavopiridol was enhanced by abrogation of p53. Our results warrant further studies investigating the combinatorial effects of flavopiridol and 4OH-Tam as a novel therapeutic strategy for RTs and other tumors that have been shown to respond to flavopiridol.

The effects of flavopiridol, 4OH-Tam, and their combination on RT cell cycle regulation and apoptosis were assessed by: i) cell survival assays, ii) FACS analysis, iii) caspase activity assays, and iv) immunoblot analysis. Furthermore, the role of p53 in flavopiridol- and 4OH-Tam-mediated induction of cell cycle arrest and apoptosis was characterized using RNA interference (siRNA) analysis. The effect of p53 on flavopiridol-mediated induction of caspases 2, 3, 8 and 9 was also determined.

We found that the combination of flavopiridol and 4OH-Tam potently inhibited the growth of RT cells. Low nanomolar concentrations of flavopiridol induced G&sub2; arrest, which was correlated to down-modulation of cyclin B1 and up-regulation of p53. Addition of 4OH-Tam did not affect flavopiridol-mediated G&sub2; arrest, but enhanced caspase 3,7-mediated apoptosis induced by the drug. Abrogation of p53 by siRNA abolished flavopiridol-induced G&sub2; arrest, but enhanced flavopiridol- (but not 4OH-Tam-) mediated apoptosis, by enhancing caspase 2 and 3 activities. Conclusions: Combining flavopiridol with 4OH-Tam potently inhibited the growth of RT cells by increasing the ability of either drug alone to induce caspases 2 and 3 thereby causing apoptosis. The potency of flavopiridol was enhanced by abrogation of p53. Our results warrant further studies investigating the combinatorial effects of flavopiridol and 4OH-Tam as a novel therapeutic strategy for RTs and other tumors that have been shown to respond to flavopiridol.