Multimodal therapies play important roles in the treatment of osteosarcoma (OS) and Ewing's family of tumors (EFTs), two most frequent malignant bone tumors. Although the clinical outcome of primary OS and EFTs is greatly improved, the relapsed cases often are associated with multidrug resistance of the tumors and the prognosis of these patients is still poor. Flavopiridol, a pan cyclindependent kinase (CDK) inhibitor is a novel antitumor agent that can induce cell cycle arrest and apoptosis in many cancer cells. However, there have been no studies about the effects of flavopiridol on drug-resistant OS and EFTs. Here, we demonstrated that flavopiridol induced the cleavage of poly-ADP-ribose polymerase (PARP) in a time and dose dependent manner in adriamycin-resistant OS and EFTs cells expressing P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP 1 ) as effectively as in their parental cells. Our data also showed that flavopiridol caused the release of mitochondrial cytochrome c and the activation of caspase-9, caspase-8 and caspase-3, with an increase ratio of the proapoptotic protein level (Bax) to the antiapoptotic protein level (Bcl-2 and Bcl-X L ), while apoptosis was inhibited by pan caspase inhibitor (Z-VAD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK), not by caspase-8 inhibitor (Z-IETD-FMK). The treatment with flavopiridol further inhibited the tumor growth in mouse models of the drug-resistant OS and EFTs. These results suggest that flavopiridol might be promising in clinical therapy for the relapsed OS and EFTs. ' 2007 Wiley-Liss, Inc.Key words: CDK inhibitor; flavopiridol; multidrug resistance; apoptosis Flavopiridol, a small-molecule cyclin-dependent kinase (CDK) inhibitor is a novel and promising antitumor reagent. Previous studies have demonstrated that flavopiridol can inhibit the activity of CDKs mainly via binding to the ATP-binding pocket of the molecule, 1,2 and inhibit RNA transcription via inhibition of the transcription elongation factor, P-TEFb to regulate the cell cycle progression, 3,4 and induce apoptosis 5,6 in malignant tumors. It has been reported that flavopiridol exhibits strong cytotoxic synergy with other chemotherapeutic reagents in Phase I and/or II clinical trials for non-small cell lung cancer (NSCLC), gastric cancer, leukemia, colon and renal cell cancers. 7 Recent studies showed that the inhibition by flavopiridol of cdk9, as well as cdk7, had profound effects on cellular transcription, and preferentially affected the expression of mRNAs for antiapoptotic proteins, cell cycle regulators and signal transduction molecules in p53 and nuclear factor-kappa B (NF-jB) pathways. 8,9 Osteosarcoma (OS) and Ewing's family of tumors (EFTs) are the two most frequent primary malignant bone tumors in childhood and adolescence. Although the marked improvements in the multimodal therapies and outcome for primary OS and EFTs have been reported, systemic and/or local relapses are still observed in approximately 30 AE 40% of the cases leading to poor prognosis. [10][11][12][1...