Flavopiridol Decreases the Level of p21 and Induces Apoptosis in Human Myeloma Cell Lines. (2005)

Abstract Flavopiridol is a cyclin dependent kinase (CDK) inhibitor with a broad spectrum of activity both in vitro and in vivo and has been demonstrated to inhibit cell growth and induce apoptosis in cancer cells. We have evaluated the effect of Flavopiridol on a panel of human myeloma cell lines (HMCL)( NCI H929, LP-1, RPMI 8226, OPM-2 and U266) and in an in vivo murine model of multiple myeloma (5T33). Dose response was determined by MTS assays with a range of Flavopiridol doses (0–5mM) for 72 hours. The profiles of the cells after treatment with Flavopiridol were evaluated via FACS and cell cycle analysis. MTS assays showed a dose and time dependent Flavopiridol-induced cell killing in HMCL with effective concentrations of Flavopiridol ranging from 10nM - 5mM. This observation was associated with increasing number of cells in the late apoptotic stage and accumulation of cells in the SubG0 and G0/G1 phases. Western analysis using antibodies against p21, cyclin A, p27, Mcl-1, Bcl-XL, Bcl-2a, pRB, and PARP were performed to help characterize the mechanism(s) of cell killing. Cleavage of PARP in NCI H929 was noted within 24 hours of Flavopiridol treatment indicating early Flavopiridol-induced HMCL apoptosis. Expression of p21 protein, a member of the Cip/Kip family of CDK that can bind and obstruct a broad range of cyclin/Cdk complexes, was found to decrease following 24 hours of exposure to Flavopiridol. A reduction of another CDK member, Cyclin A, was apparent following Flavopiridol treatment whilst p27 expression was unchanged even after 72 hours. Three anti apoptotic proteins, Mcl-1, Bcl-2a and Bcl-XL protein expression were evaluated. Mcl-1 protein expression increased with time in all three cell lines tested while Bcl-2a was unaltered and Bcl-XL decreased following treatment. Retinoblastoma protein (RB), a tumour suppressor which acts as a cell cycle checkpoint between G1 and S phases was evaluated. A decrease of hyperphosphorylated RB (ppRB) was demonstrated in all 3 cell lines indicating a decrease in proliferating cells. Furthermore, silencing of p21 by siRNA enhanced Flavopiridol induced apoptosis indicating cell death occurred via a p21 dependent mechanism. The impact of combination and scheduling with bortezomib was evaluated in NCI-H929 and primary myeloma cells and it was found that addition of bortezomib subsequent to Flavopiridol improved cell killing compared to single-agent treatment. Reversal of drug treatment was found to be not as efficient. 2.5 mg/ml Flavopiridol and 1% DMSO/saline (vehicle) were administered to a 5T33 murine model of multiple myeloma for 5 days intraperitoneally. Hind limp paralysis was observed later in the Flavopiridol treated mice compared to vehicle treated mice with the median survival for vehicle treated mice of 23 days compared to 24.5 days in the AZA treatment (p= 0.018). These data provide a rationale for further evaluation of Flavopiridol as a therapeutic agent in multiple myeloma. Furthermore, we propose that Flavopiridol induced killing of HMCL may be mediated by a novel mode of action involving suppression of p21.