Pigment Epithelium-Derived Factor (PEDF) Inhibits Multiple Myeloma through Suppressing NADPH Oxidase ROS Generation.

Abstract Interaction of multiple myeloma (MM) cells and bone marrow stromal cells (BMSC) plays a crucial role in the pathogenesis of multiple myeloma (MM). IL-6 secreted by BMSCs promotes proliferation and survival of MM cells. IL-6 also enhaces the vascular endothelial growth factor (VEGF) production of MM cells, which in turn promotes secretion of IL-6 from BMSCs. Thus VEGF is both an autocrine growth factor and IL-6 mediated paracrine growth factor of MM cells. VEGF is also angiogenic factor as increasing microvessel density in the BM of MM patients. VEGF stimulates Rac1 dependent NADPH oxidase to produce reactive oxygen species (ROS), which is an important factor for angiogenic response. Pigment epithelium-derived factor (PEDF) is a glycoprotein with M.W. 50,000, belongs to serine protease inhibitor superfamily, and is produced and secred by broad variety of cells. PEDF functions as the most potent inhibtor of angiogenesis in the mammalian eyes. Recently, we reported that PEDF effectively inhibits growth factor-induced ROS generation by suppressing NADPH oxidase. Accordingly, we hypothesized that PEDF would exhibit anti-MM effect by reducing ROS generation. We first examined the role of VEGF-induced ROS generation in MM cell proliferation by using the fluorescent probe CM-H2DCFDA and measuring 3H-thymidine incorporation. We found that VEGF promoted ROS generation more effectively than IL-6 stimulation in both MM cell lines (RPMI 8226 and U-266). VEGF- and IL-6-induced MM cell proliferation and ROS generation were completely abrogated by treatment with antioxidants, gluthatione peroxidase mimetic, ebselen(50uM). Then, we examined whether ROS generation would be associated with Myeloid cell leukemia-1(Mcl-1) which was shown to be regulated by VEGF as well as IL-6, and necessary for MM cell proliferation. VEGF- and IL-6-induced Mcl-1 up-regulation was also inhibited by antioxidant, ebselen, in consistent with ROS generation. Next, we examined whether PEDF (30nM) would affect on MM cell proliferation and ROS generation on MM cells. We found that PEDF prevented MM cell proliferation induced by VEGF (50ng/ml) and IL-6 (50ng/ml), and decreased ROS generation. Moreover, PEDF suppressed the expression of Mcl-1, cyclin E and also p22phox up-regulated by VEGF. These PEDF inhibitory effects occurred more prominent on VEGF stimulation, whereas lesser on IL-6 stimulation. These data suggest that PEDF may exhibit anti-MM effects through suppressing NADPH oxidase, because NADPH oxidase is activated by VEGF, not but IL-6. The addition of NADPH oxidase inhibitor (Diphenyleneiodonium) decreased the 3H-thymidine incorporation as well as Mcl-1 expression on U-266. Furthermore, to investigate the therapeutic role of PEDF in patients with MM, we studied the apoptotic effects of PEDF on MM cells from three patients. The addition of PEDF resulted in increased apoptotic cells on VEGF-and IL-6-treated primary myeloma cells (CD138 positive cells). Our data suggest that PEDF is the putative endogenous inhibitory factor against human MM, and administration of PEDF could be a novel therapeutic way for MM.