Regulation Of Apoptosis By SPK-S1P Signal In Multiple Myeloma And Studies On Its Correlative Mechanism

Multiple myeloma is a malignant B cell disorder characterized by the uncontrolled proliferation of monoclonal plasma cells in the bone marrow and the presence of monoclonal immunoglobulin in serum and/or urine. It is currently an incurable plasma cell malignancy. Elucidating the nature of proliferation and apoptosis of MM cells will be very helpful for the diagnosis and therapy of this disease.The proliferation and apoptosis in human multiple myeloma cells are regulated by cytokines and bone marrow microenvironment which composed of various stromal cells plays a crucial role in the pathogenesis of MM cell growth and survival. The stromal cells of marrow microenviroment regulate the proliferation, apoptosis and drug-resistence of MM cells through cell-cell interaction or secretion of cytokines. It is established that IL-6, produced in either an autocrine or paracrine manner, has an essential role in the malignant progression of MM by regulating the growth, survival and drug-resistence of tumor cells. Multiple pathways including JAK-Stat, PI-3K, MAPK and Rho pathway are the central players in this process. And it is belived that, besides theses signals, other pathways might be involved in. Recently, the sphingolipid metabolites, including ceramide (Cer), sphingosine (Sp), and sphingosine 1-phospate (S1P), have been proved to play important roles in the regulation of cell proliferation, survival, and apoptosis. The cell levels of these sphingolipid metabolites are regulated by sphingosine kinase (SPK). However, the roles of SPK-S1P signal in the regulation of the proliferation and survival of MM cells remain unclear. In this study, we detected the expression of SPK-S1P related molecules by MM cells. And we found that S1P upregultes the expression of mcl-1, leading to reduce Dex-induced apoptosis of MM cells. We further demostrate that SPK-S1P signal mediates the suppressive effect of IL-6 on apoptosis of MM cells.To detect the expression of SPK-SIP related molecules by MM cells, we have isolated the CD 138+ primary MM cells from patient bone marrow by using MACS sorting techniques. We detected the expression of SPK and SIP receptors by RT-PCR and Northern Blot in both primary MM cells and MM cell lines. . We found that MM cells express the SPK and SIP receptor such as EDG|, EDG3 and EDG5. These results suggest that SPK-SIP signal might play a physiological role in regulation of proliferation and survival of MM cells. And we investigated the effects of SIP on the apoptosis of MM cells. Treatment of MM cells with Dex induces apoptosis of XG-7 cells. However, addition of SIP reduces Dex-induced apoptosis and SIP also upregultes the expression of Mel-1 protein by XG-7 cells. The SIP receptors, EGDs, could be blocked by G Protein coupled receptor inhibitor, PTX. Treatment of XG-7 cells with PTX results in blocking SIP-induced upregulation of mcl-1. We demostrate that SIP reduce the Dex-induced apoptosis of MM cells via upregulation of Mcl-1 protein expression.Several lines of evidences show that sphingosine kinase (SPK), a key enzyme catalyzing the formation of SIP, could be activated by cytokines and growth factors. To determine whether SPK-SIP signal involve in the IL-6 signal in MM cells, we first tested the SPK activity of XG-7 cells treated with IL-6. We found that IL-6 could activate cellular SPK in a concentration-dependent manner. IL-6 is known to activate several signals such as PI3K, ERK/MAPK, Jak/STAT and Rho signal, which play important role in the proliferation and apoptosis of MM cells. To examine the roles of these signal cascades in the activation of SPK by IL-6 in multiple myeloma cells, we pretreated XG-7 cells with PD98059, Y27632, AG490 and LY294002, specific inhibitors of MAPK, Rho, Jak/STAT and PI3K respectively, and then assayed the IL-6-induced SPK activity. LY294002, PD98059 and AG490 could abolish the activation of SPK induced by IL-6. This indicates that IL-6 activates SPK via PI3K, MAPK and Jak/STAT pathways.To elucidate the role of activation SPK-SIP signal in the regulation of apoptosis...