Abstract. Momilactone B, a terpenoid phytoalexin present in rice bran, has been shown to exhibit several biological activities. The present study was conducted using cultured human leukemia U937 cells to elucidate the possible mechanisms by which momilactone B exerts its anticancer activity, which to date has remained poorly understood. Momilactone B treatment of U937 cells resulted in a dose-dependent inhibition of cell growth and induced apoptotic cell death as detected by chromatin condensation, DNA fragmentation, the cleavage of poly(ADP-ribose) polymerase and Annexin V-FITC staining. Flow cytometric analysis revealed that momilactone B resulted in G1 arrest in cell cycle progression, which was associated with the dephosphorylation of retinoblastoma protein (pRB) and enhanced binding of pRB with the E2F transcription factor family proteins. Treatment with momilactone B also increased the expression of cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1 in a p53-independent manner, without any noticeable changes in G1 cyclins and cyclin-dependent kinases (Cdks), except a slight decrease in cyclin E. Moreover, in vitro kinase assay indicated that momilactone B significantly decreased Cdk4-and Cdk6-associated kinase activities through a notably increased binding of p21 to Cdk4 and Cdk6. Our results demonstrated that momilactone B caused G1 cell cycle arrest and apoptosis in U937 cells through the induction of p21 expression, inhibition of Cdk/cyclin-associated kinase activities, and reduced phosphorylation of pRB, which may be related to anticancer activity.
IntroductionImpaired deregulated cell cycle progression and induction of apoptosis are the primary characteristics of cancer cells due to an imbalance between proliferation and cell death. In terms of cell cycle regulation, the cyclin-dependent kinases (Cdks) are the key regulators of eukaryotic cell cycle progression in cooperation with various endogenous cyclins and Cdks (1,2). An alteration in the cooperation may lead to increased or decreased cell growth and proliferation followed by differentiation and/or cell death by apoptosis (3,4). Therefore, the key regulators of cell cycle progression and apoptotic induction may be important molecular targets for therapeutic intervention, and inhibition of cell cycle regulation may be particularly useful in the treatment of diseases caused by uncontrolled cell proliferation, such as cancer.In general, passage through G1 into the S phase is regulated by the activities of D-type cyclin-and cyclin E-associated Cdks. D-type cyclins bind to existing Cdk4 and Cdk6, forming active complexes. The complexes in turn phosphorylate the retinoblastoma susceptibility protein (pRB). The hyperphosphorylated pRB dissociates from the E2F/DP1/pRB Momilactone B induces apoptosis and G1 arrest of the cell cycle in human monocytic leukemia U937 cells through downregulation of pRB phosphorylation and induction of the cyclin-dependent kinase inhibitor p21Waf1/Cip1