Abstract
BackgroundRhabdoid 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.MethodsThe 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.ResultsWe found that the combination of flavopiridol and 4OH-Tam potently inhibited the growth of RT cells. Low nanomolar concentrations of flavopiridol induced G2 arrest, which was correlated to down-modulation of cyclin B1 and up-regulation of p53. Addition of 4OH-Tam did not affect flavopiridol-mediated G2 arrest, but enhanced caspase 3,7-mediated apoptosis induced by the drug. Abrogation of p53 by siRNA abolished flavopiridol-induced G2 arrest, but enhanced flavopiridol- (but not 4OH-Tam-) mediated apoptosis, by enhancing caspase 2 and 3 activities.ConclusionsCombining 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.
Highlights
Rhabdoid Tumors (RTs) are highly aggressive pediatric malignancies with poor prognosis
We previously reported that down-modulating cyclin D1 and inhibiting cyclin dependent kinases using either flavopiridol or a combination of N-(4-hydroxyphenyl)retinamide (4-HPR) with 4OH-Tam is effective in inhibiting RTs in vitro and in xenograft tumor models in vivo [11,13]
Since the efficacy of flavopiridol in xenograft RTs was correlated with down-modulation of cyclin D1 and up-regulation of p21Waf1[13], we considered combining 4OH-Tam with flavopiridol to enhance its therapeutic efficacy in RT cells
Summary
Rhabdoid Tumors (RTs) are highly aggressive pediatric malignancies with poor prognosis. We have found that cyclin D1 is de-repressed in human and mouse RTs and is required for rhabdoid tumorigenesis in mouse models [9,11,12] Such studies indicated that therapeutic targeting of cyclin D1 and its pathway could be an effective and novel therapeutic strategy for RTs. We previously reported that down-modulating cyclin D1 and inhibiting cyclin dependent kinases (cdks) using either flavopiridol or a combination of N-(4-hydroxyphenyl)retinamide (4-HPR) with 4OH-Tam is effective in inhibiting RTs in vitro and in xenograft tumor models in vivo [11,13]. The effectiveness of 4-HPR and flavopiridol was correlated with down-modulation of cyclin D1 in xenograft tumors [13]
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