Experimental Researches Of PPARγ/RXRα Targeting Treatment In Multiple Myeloma

1.Influence of PPARγligand rosiglitazone and ATRA on myeloma cell apoptosis and its potential machanism studyObjectives To investigate the effects of rosiglitazone (RGZ) as well as combined with all trans-retinoic acid (ATRA) on human myeloma cell lines and try to address its potential mechanism.Methods U266 and RPMI-8226 cells were treated with different concentration of RGZ in the presence or absence of ATRA and various biological responses were studied by the methods of [3H] thymidine incorporation, Annexin V-PI staining, RT-PCR, caspase-3 activity assay and tumor formation test in BALB/c nude mice.Results Exposure to RGZ induced proliferation inhibition in a dose-dependent manner in both U266 (r2= 0.991, p<0.01) and RPMI-8226 cells (r2= 0.961, p<0.01). A combination of RGZ with ATRA can enhance the proliferation inhibition effect of RGZ(p<0.001 in U266, p<0.01 in RPMI8226. TUNEL examination showed that apoptosis index (AI) increased when U266 cells were treated with 5μmol/L RGZ (10.4±2.6)% and the apoptosis was more apparent when RGZ concentration increased to 10μmol/L (28.6±7.7)% or ATRA was combined. Annexin-V/PI staining confirmed that 12h incubation with 5μmol/L RGZ induced apoptosis in (9.8±1.7)% U266 cells and (10.7±3.3) % RPMI8226 cells, a time-course and dose dependent increase of apoptosis was also observed and ATRA can also intensified the apoptosis induction effects of RGZ(p <0.01 in both cell lines). The mRNA expressions of FLIP, XIAP and survivin were detected in both cell lines and the levels decreased significantly after cultured with RGZ, the addition of ATRA in culture medium made these changes more apparently. Caspase-3 activity increased substantially along with the increase of RGZ concentration and the addition of ATRA in culture medium shows similar synergism effect on caspase-3 activation.Conclusion The down-regulation of FLIP, XIAP and Survivin induced by RGZ can furthermore activate caspas-3, which finally induced apoptosis and proliferation inhibition in myeloma cells. ATRA can enhance these effects of RGZ on multiple myeloma cells and combined use of these two drugs shows synergistic effect on myeloma cells.2.The influence of PPARγligand rosiglitazone and ATRA on myeloma cells differrentiation and its potential mechanism studyObjectives To investigate the effects of PPARγligand (rosiglitazone, RGZ) as well as combined with all trans-retinoic acid (ATRA) on human myeloma cells and try to address its potential mechanism.Methods U266 and RPMI-8226 cells were treated with RGZ in the presence or absence of ATRA, cell proliferation was evaluated by [3H] thymidine incorporation, cell cycle distribution and CD49e expression were analyzed by flow cytometry, morphology change was evaluated by Wright-Giemsa staining, p27Kip1,p21Waf1 expression was detected by western-blotting.Results Our study demonstrated that exposure to RGZ induced dose-dependantly cell cycle arrest in myeloma cells. A combination of RGZ with all-trans retinoic acid (ATRA) can enhance the cell cycle arrest effects of RGZ. Further study shows that RGZ treated myeloma cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when RGZ was combined with ATRA. These changes were confirmed by the detection of CD49e expression and light chain protein secretion. The expression of p27Kip1 and p21Waf1 in myeloma cells was up-regulated by RGZ and this change was more apparent when RGZ was combined with ATRA.Conclusion RGZ can induce cell cycle arrest and cell differentiation in myeloma cells which may caused by up-regulation of p27Kip1,p21Waf1 expression. ATRA can enhance these effects of RGZ on multiple myeloma cells and combined use of these two drugs shows synergistic effect on myeloma cells.3.Influence of RGZ and ATRA on primary CD138+ myeloma cellsObjectives To investigate the influence of rosiglitazone (RGZ) as well as combined with all trans-retinoic acid (ATRA) on primary CD138+ myeloma cells.Methods Mononuclear cells were freshly isolated from the bone marrow of five patients with multiple myeloma by Ficoll–Hypaque density gradient centrifugation. Myeloma cells were purified with CD138 positive selection method using CD138 immunomagenetic beads and auto MACS magnetic cell sorter machine. After incubation with RGZ and ATRA, the primary CD138+ myeloma cells were collected for MTT assay, morphological evaluation, analysis of apoptosis and CD49e expression. The culture supernatants were collected for measurement of light chain protein.Results MTT assay showed that there was a decrease of cell viability in RGZ-treated group as compared to vehicle control. ATRA 1μM alone showed no significant inhibition on cell viability of primary myeloma cells. But when ATRA was combined with RGZ, the viability was inhibited more profoundly than that of RGZ groups. Morphological evaluation showed CD138+ cells from myeloma patients also underwent morphological maturation and apoptosis. More obvious morphological changes can be observed when RGZ was combined with ATRA. Compare to control group, an increase of apoptosis was observed in RGZ treated primary myeloma cells(p<0.05,31.6±5.7% vs 10.2±2.7%). There was no significant increase of apoptosis cells in ATRA group, while ATRA can intensified the apoptosis induction effects of RGZ(p <0.05). Analysis of CD49e expression shows that ATRA 1μM caused no significant increase of CD49e on primary cells, while the CD49e expression was obviously up-regulated in RGZ 10μM treated primary cells and the up-regulation was more significant when RGZ was combined with ATRA. Further measurement of the light chain protein in culture supernatant showed that the secretions of light chain protein in ATRA 1μM treated primary cells were not significantly higher than that of the control. But after exposure to RGZ 10μM, the secretions of all patients'myeloma cells were increased remarkably. When RGZ was combined with ATRA, much higher levels of light chain in culture supernatant were observed in 4/5 patientsConclusion In this study, we demonstrated that the activation of PPARγby RGZ may cause apoptosis and partial re-differentiation in primary CD138+ cells from myeloma patients, and that ATRA could sensitize primary myeloma cells to the differentiation and apoptosis induction effects of RGZ.4. Influence of RGZ and ATRA on angiogenesis and growth of myeloma cell xenograft in nude miceObjectives To explore the influence of RGZ and ATRA on VEGF expression in myeloma cells, and to observe the effects of RGZ and ATRA on the growth of myeloma cells and angiogenesis of myeloma cell xenograft in nude mice.Methods VEGF gene expression in U266 and RPMI-8226 cells were analyzed by semi-quantitive RT-PCR after incubation with RGZ, ATRA, RGZ+ATRA for 24h. Myeloma cell xenograft was established by subcutaneous injection of 108 U266 cell in nude mice. 7days after U266 cells injection, nude mice were administrated with RGZ and ATRA by intraperitoneal injection once every day for 21 days. Control mice were given equal volumes of normal saline instead of the drug. The tumors were monitored every 3 days. At the end of 21d treatment, tumors were peeled off and measurement of the volume and weight was made. Tumor microvessel density (MVD) and VEGF expression in the tumors were analyzed by immunohistochemical staining.Results VEGF mRNA was expressed in both u266 and RPMI-8226 cells and the expression levels were decreased in dose dependant manner after incubation with RGZ. The levels were decreased more significantly when RGZ was combined with ATRA. The xenograft of U266 cells were developed in all nude mice. The volume and weight of U266 cell xenograft in RGZ BALB/c nude mice decreased significantly (785±262mm3 and 1748±365mg respectively) and the inhibition was more potential by the combination use of RGZ and ATRA (154±89mm3 and 626±102mg respectively)(p<0.01 ATRA+RGZ group versus RGZ group). RGZ caused angiogenesis inhibition in U266 xenograft and immunohistochemical staining showed tumor MVD and VEGF expression were significantly decreased by treatment of RGZ. More evident signs of angiogenesis inhibition were observed when RGZ was combined with ATRA.Conclusion In vivo of nude mice, the growth and proliferation of myeloma cells can also be inhibited by RGZ and ATRA. Besides by the way of differentiation and apoptosis induction, RGZ can also inhibite the formation of myeloma cell xenograft through down regulation of VEGF expression and subsequent angiogenesis. In vivo, ATRA can also ameliorate the effect RGZ on myeloma cells.