Study On The Mechanism Of Adipocyte’s Regulation Of Growth And Drug Resistance In Multilple Myeloma Cells

Part1Assessment of serum adipokines levels in multiple myeloma patients and their relation with clinical significanceObjective:To investigate the serum adipokines levels in multiple myeloma patients and their relation with clinical characteristics.Methods:Serum concentrations of four adipokines (leptin, adiponectin, resistin, and visfatin) were studied in28newly diagnosed MM patients. Serum levels of adipokines were assessed by ELISA. The control group was comprised of28age-and sex-matched healthy volunteers. We investigated the role of serum adipokines levels in the etiopathogenesis of MM and we explored their association with disease stage as well as MM prognostic biological parameters such as bone marrow plasma cells%, CRP, LDH, β2-microglobulin and ER.Results:leptin was significantly higher in MM patients compared with the control group, also a significant difference in leptin level has been observed between lower stages (stage Ⅰ Ⅱ) and higher stage (stage Ⅲ). Adiponectin was significantly lower in MM patients compared with the control group, and lower in the higher stage (stage Ⅲ) than lower stages (Ⅰ Ⅱ). It indicates that lower serum adiponectin levels and higher serum leptin levels were associated with higher risk of MM. Adiponectin may have a protective role in MM whereas leptin promote the development of the disease. Furthermore, a significant positive relation was found between leptin and IgG level, ER, bone marrow plasma cells%as well as β2MG, but not the LDH, hemoglobin or Ca2+. Adiponetin level was inversely correlated with IgG level, β2M and ER. Whereas, there were no significant differences in resistin level and visfatin level between MM patients and healthy individuals. Also we haven’t been able to find either a significant association between resistin level and MM prognostic biological parameters or a significant association between visfatin level and MM prognostic biological parameters. Part2adipocytes support myeloma cells proliferation and drug resistanceObjective:We have found adipokines levels in multiple myeloma patients were associated with clinical characteristics. Furthermore, bone marrow cavity is filled with adipocytes and the adipocyte numbers in the bone marrow strongly correlate with age. In this part, we investigate the effect of adipocyte on the biological of multiple myeloma and the related mechanisms in vitro.Methods:3T3-L1cells were used to differentiate into adipocytes. We also isolated human MSCs from iliac crest bone marrow aspirates of6healthy volunteers. The MSCs were later differentiated into adipocytes. Both of the two kinds of adipocytes were submitted to oil stain. The concentrations of leptin in the culture supernatant of both adipocytes were tested by ELISA. We then coculture myeloma cells and3T3-L1adipocytes with or without anti-leptin receptor antibody. Proliferation assays using CFSE staining were performed after3d of coculture. Flow cytometry was used to measure the effects of adipocytes on the cell cycle of MM cells. Western blotting was used to observed the effect of adipocytes on the expression levels of cell cycle related proteins as well as signal proteins. Last, we add Bortezomib or Dexamethasone into the coculture system. Flow cytometry was used to measure the effects of adipocytes on chemotherapy drugs induced apoptosis of MM cells. Western blotting was used to observed the effect of adipocytes on the expression levels of apoptosis related proteins.Results:During the adipocyte-differentiation culture period, more and more3T3-L1cells or MSCs showed an accumulation of multiple lipid droplets.80%-90%of the3T3-L1cells were differentiated into adipocytes while20%-30%of the MSCs were differentiated into adipocytes. Both of the differentiated adipocytes secreted leptin. Levels secreted by undifferentiated MSCs (0.4+0.08ng/ml) increased to1.52±0.12ng/ml. levels secreted by3T3-L1adipocytes were0.88±0.17ng/ml. Coculture myeloma cells with adipocytes improves the proliferation of MM cells especially U266cell and NCI-H929. While add anti-leptin receptor antibody, the proliferation effect was inhibited. Coculture promotes cell cycle and increase cyclinD1expression. Cocultured with adipocytes increased the phosphorylation of AKT and STAT3, while adding anti-leptin receptor antibody reduced it.3T3-L1adipocytes significantly impaired the anti-myeloma efficacy of Bortezomib. Adipocytes prevented chemotherapy-induced apoptosis, and this was associated with increased expression of Bcl-2and decrease expression of caspase-3.Conclution:Both of the differentiated adipocytes secreted leptin. We chose a highly differentiated cell line3T3-L1in later experiments. Aidpocytes promoted proliferation of MM cells through activating AKT and STAT3signal. Also adipocytes protect MM cells from Bortezomib induced apoptosis through regulate expression of apoptosis related protein. Part3Leptin stimulated MM cell proliferation trough activating JAK/STAT-PI3K/AKT pathwayObjective:We found in part2that adipocytes can promote MM cell proliferation through leptin/leptin receptor. In this part we investigate the detail mechanisms of leptin promoting MM cell growth.Methods:We examined the effect of leptin on MM cells proliferation using cck8. U266and929were treated with various concentrations (25ng-200ng/ml) of recombinant human leptin for different time intervals (12,24, and48h). We next examined the changes in signal transduction pathways involved in mediating leptin action. Total cellular proteins were extracted from cells treated with50ng/ml leptin for various time periods, then we used western blot to assess the expression level of AKT, pAKT, STAT3and PSTAT3. Next, we studied the effect of pharmacologic inhibitors of JAK/STAT (AG490) and PI3K (LY294002) on leptin-induced stimulation of proliferation and the involved cell signal. We treated MM in different conditions:non treatment, add leptin, add AG490; add AG490and leptin; add LY2904002; add LY2904002and leptin. We assess the expression level of AKT, pAKT, STAT3and PSTAT3by western blot, assess the cell proliferation using cck8.Results:Leptin treatment stimulated the growth of U266and929cells in a time-dependent manner, but not a dose-dependent manner. Leptin stimulated the growth of both cells most at the concentration of50ng/ml. AKT phosphorylation was increased to peak at1h after treatment, while the AKT protein expression was not changed. STAT3phosphorylation was increased to peak at6h of treatment and the increase was not due to the increased STAT3protein expression. LY294002specifically inhibited the phosphorylation of AKT, without affecting the expression of AKT or level of phosphorylated STAT3. Treatment with AG490blocked leptin-induced hyperphosphorylation of both ERK and AKT. Simultaneous treatment with leptin and AG490could not restore the level of phosphorylation of STAT3and AKT. These data suggest that activation of JAK/STAT is upstream of the activation of AKT pathway. Both AG490and LY2904002inhibited leptin-induced proliferation of MM cells. Conclution:leptin stimulated MM cell growth through activating JAK/STAT-PI3K/AKT pathway...