Arsenic trioxide-mediated growth inhibition in MC/CAR myeloma cells via cell cycle arrest in association with induction of cyclin-dependent kinase inhibitor, p21, and apoptosis

We investigated the in vitro effect of As2O3 on proliferation, cell cycle r egulation, and apoptosis in human myeloma cell lines. As2O3 significantly i nhibited the proliferation of all of eight myeloma cell lines examined in a dose-dependent manner with IC50 of similar to 1-2 mu M. DNA flow cytometri c analysis indicated that As2O3 (2 mu M) induced a G(1) and/or a G(2)-M pha se arrest in these cell lines. To address the mechanism of the antiprolifer ative effect of As2O3, we examined the effect of As2O3 on cell cycle-relate d proteins in MC/CAR cells in which both G(1) and G(2)-M phases were arrest ed. Western blot analysis demonstrated that treatment with As2O3 (2 mu M) f or 72 h did not change the steady-state levels of CDK2, CDK4, cyclin D1, cy clin E, and cyclin B1 but decreased the levels of CDK6, cdc2, and cyclin A. The mRNA and protein levels of CDKI, p21 were increased by treatment with As2O3, but those of p27 were not. In addition, As2O3 markedly enhanced the binding of p21 with CDK6, cdc2, cyclin E, and cyclin A compared with untrea ted control cells. Furthermore, the activity of CDK6-associated kinase was reduced in association with hypophosphorylation of Rb protein. The activity of cdc2-associated kinase was decreased, which was accompanied by the up-r egulation of cdc2 phosphorylation (cdc2-Tyr(15) phosphorylation) resulting from reduction of cdc25B and cdc25C phosphatases. As2O3 also induced apopto sis in MC/CAR cells as evidenced by flow cytometric detection of sub-G(1) D NA content and annexin V binding assay. This apoptotic process was associat ed with down-regulation of Bcl-2, loss of mitochondrial transmembrane poten tial (Delta psi(m)), and an increase of caspase-3 activity. These results s uggest that As2O3 inhibits the proliferation of myeloma cells, especially M C/CAR cells, via cell cycle arrest in association with induction of p21 and apoptosis.