title: Potent inhibition of rhabdoid tumor cells by combination of flavopiridol and 4OH-tamoxifen.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  We 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.
  
  
  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.
authors.affiliation: Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Velasco
authors.initials: V
authors.lastname: Cimica
authors.forename: Melissa E
authors.initials: ME
authors.lastname: Smith
authors.forename: Zhikai
authors.initials: Z
authors.lastname: Zhang
authors.forename: Deepti
authors.initials: D
authors.lastname: Mathur
authors.forename: Sridhar
authors.initials: S
authors.lastname: Mani
authors.forename: Ganjam V
authors.initials: GV
authors.lastname: Kalpana
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis; D020169:Caspases / Q000096:biosynthesis; D002453:Cell Cycle; D002470:Cell Survival; D004354:Drug Screening Assays, Antitumor; D005419:Flavonoids / Q000008:administration & dosage*; D005434:Flow Cytometry; D016195:G2 Phase; D006801:Humans; D010880:Piperidines / Q000008:administration & dosage*; D034622:RNA Interference; D018335:Rhabdoid Tumor / Q000188:drug therapy*; D013629:Tamoxifen / Q000008:administration & dosage / Q000031:analogs & derivatives*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D005419:Flavonoids; D010880:Piperidines; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D013629:Tamoxifen; C016601:afimoxifene; C077990:alvocidib; D020169:Caspases