Antiproliferative Action of Cudraflavone B, Isolated From Cudrania tricuspidata, Through the Downregulation of pRb Phosphorylation in Aortic Smooth Muscle Cell Proliferation Signaling

Cudrania tricuspidata has been proposed to possess anti-inflammatory, antioxidant, hepatoprotective, and antitumor activities. Although cudraflavone B, isolated from the root bark of C. tricuspidata, has a variety of pharmacological effects, its effects on rat aortic smooth muscle cells (RASMCs) are unclear. In the present study, cudraflavone B was found to inhibit cell proliferation and DNA synthesis in cultured RASMCs. Pretreatment with cudraflavone B (0.1-4 μM) suppressed platelet-derived growth factor-BB (PDGF-BB)-stimulated cell number in a concentration-dependent manner. The inhibition percentages were 19.7%, 36.4%, 52.3%, and 99.1% at concentrations of 0.1, 1, 2, and 4 μM, respectively. Moreover, cudraflavone B inhibited [H]-thymidine incorporation into DNA in RASMCs in response to 25 ng/mL PDGF-BB. PDGF-BB-stimulated DNA synthesis was significantly reduced by 15.9%, 31.7%, 43.1%, and 78.2% at concentrations of 0.1, 1, 2, and 4 μM, respectively. Thus, cudraflavone B blocked the PDGF-BB-inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Furthermore, PDGF-BB-induced phosphorylation of retinoblastoma protein (pRb), the hyperphosphorylation of which is a hallmark of the G1-S transition in the cell cycle, was significantly inhibited by cudraflavone B. Because pRb phosphorylation is regulated by cyclin-dependent kinases (CDKs), we investigated the expression of CDK2, CDK4, cyclin E, and cyclin D1 and the CDK inhibitors p21 and p27. Treatment with cudraflavone B downregulated the cyclins and CDKs and upregulated the expression of p21 and p27, a CDK inhibitor. These findings suggest that cudraflavone B inhibits RASMC proliferation via the induction of p21 and p27 expression and subsequent cell cycle arrest with reduction of pRb phosphorylation at the G1-S phase.