Investigation Of The Effect Of Human Umbilical Cord Blood-derived Stromal Cells On Proliferation And Apoptosis Of Myeloma Cells

Multiple myeloma (MM) derived from terminal differentiating B lymphocyte cells belongs to the family of plasma cell cancer and usually occurs in the elderly.It accounts 10% in hematopoietic malignancies and 1% in all malignant tumors. MM is characterized by the clonal expansion of plasma cells accumulating in the bone marrow (BM), the appearance of monoclonal protein in the serum and Bence Jones in the urine, the inhibition of normal immunoglobulins, bone destruction and osteoporosis.In recent years, the pathogenesis of MM has been focused on abnormal cytogenetics, interactions between hematopoietic inductive microenvironment (HIM)and myeloma cells, resistance, NF-κB and Notch signaling pathways.Hematopoietic inductive microenvironment(HIM),the site for establishment, proliferation, differentiation and maturation of hematopoietic stem/ progenitor cells in bone marrow,is the internal environment for sustaining and regulating the growth and development of hematopoietic cell.On the other hand,HIM acts as an important role in the generation and progress of hematologic diseases,as well as,in the proliferation, differentiation and apoptosis of tumor cells.The foregoing role of HIM is especially significant for multiple myeloma. Bone marrow stromal cells(BMSCs) as the main component and the functional unit of HIM, affect the growth,survival,resistance and immigration of myeloma cells by adhering MM cells and secreting cytokines.Over the last 150 years, neither the introduction of melphalan nor the large dose chemotherapy combined hematopoietic stem cell transplantation couldn't decrease the high relapse rate.Although the therapeutic effect rate and the complete remission rate increase,MM is still an incurable disease. In recent years,because of the development of biotechnology such as gene chips and proteomics,the targeted therapies of HIM and cytokines network are especially advised. Accumulating studies have confirmed that reforming the abnormal HIM is in favor of clearance of myeloma cells.Can we enhance the clearance of myeloma cells by using non-bone marrow derived stromal cells to substitute the abnormal HIM?Now, the stromal cells originated from the liver,lung,thymus and skin don't have the ideal functions of HIM in vitro. Our group confirmed the existence of precursor cells of the stromal cells in human umbilical cord blood and successfully acquired human umbilical cord blood-derived stromal cells(hUCBDSCs) by expansion in vitro. Interestingly, it was found that many stromal cell specific features were possessed by hUCBDSCs. Moreover, we found that compared with BMSCs, hUCBDSCs suppressed proliferation and induced apoptosis of Jurkat cells more obviously. HIM plays a more important role in the occurrence and the development of MM compared with leukemia, then can hUCBDSCs suppress proliferation and induce apoptosis of myeloma cells?In order to investigate the effect of hUCBDSCs on the proliferation and apoptosis of myeloma cells, based on the KM3 cells/hUCBDSCs co-culture model, the suppression of proliferation and induction of apoptosis of KM3 cells were observed.In this study,the mechanisms were focused on the following two aspects:IL-6/IL-6R pathway,NF-κB and NF-κB regulating genes pathway.Some experimental data might be obtained to provide new adjuvant treatment for enhancing clearance of myeloma cells.Methods:1. To observe the influence of hUCBDSCs on the proliferation and apoptosis of myeloma cell line KM3.Groups:KM3 suspended culture group;KM3/hUCBDSCs co-culture group; KM3/MM-BMSCs co-culture group.The spatial relationship between the KM3 cells and stromal cells was observed under the inverted microscope and scanning electron microscope,then CCK8 assay was applied to determine the proliferation of KM3 cells.After this, the cell cycle distribution and the rate of apoptosis/necrosis of KM3 cells were assessed by flow cytometry.At last,the ultrastructure of KM3 cells was revealed under the transmission electron microscope.2. Possible role performed by the IL-6/IL-6R pathway.The concentration of IL-6 and sIL-6R in the culture supernatant of KM3 cells, hUCBDSCs and MM-BMSCs were detected by ELISA assay before and after co-culture.The expression of IL-6R at mRNA and protein levels in KM3 cells under different culture conditions was determined by real time PCR,confocal microscopy and flow cytometry.3. Possible role performed by the NF-κB pathways.3.1 NF-κB activity of KM3 cells was inhibited by hUCBDSCsNF-κB activity in KM3 cells was detected by EMSA and confocal microscopy under the different culture conditions.The expression of IκBαin KM3 cells was detected by Western blot under different culture conditions.3.2 NF-κB regulating genes were regulated by hUCBDSCsThe expression of ICAM-1 at mRNA and protein levels in KM3 cells was determined by real time PCR,confocal microscopy and flow cytometry under different culture conditions. The expressions of Bcl-2,Bcl-XL at mRNA and protein levels in KM3 cells were determined by real time PCR,confocal microscopy and Western blot under different culture conditions.Results:1. It was revealed by the inverted microscopy that many KM3 cells attached on the hUCBDSCs.Under the scanning electron microscope,some of them adhered to the hUCBDSCs surface through pseudopodia,the others inserted into the meshes formed by several fused hUCBDSCs.The quantity of attached-KM3 cells increased gradually as the culturing time prolonged.Compared with the other groups,KM3 cells in the KM3/hUCBDSCs co-culture group grew slowest.In the KM3/hUCBDSCs co-culture group ,the percentages of KM3 cells in S phase was highest ,and inversely the percentages of KM3 cells in G2/M phase was lowest.The rate of apoptosis was highest in KM3/hUCBDSCs co-culture group,but the rate of necrosis in different groups was not statistically significant.2. It was revealed by ELISA assay that the concentration of IL-6 secreted by hUCBDSCs was lower than that of MM-BMSCs did before and after co-cultured with KM3 cells and KM3 cells could significantly stimulate hUCBDSCs and MM-BMSCs to secrete IL-6. It was revealed by ELISA assay that the concentration of sIL-6R secreted by KM3 cells obviously decreased after co-cultured with hUCBDSCs.The expression of IL-6R at mRNA and protein levels markedly decreased as determined by real time PCR, and flow cytometry after KM3 cells co-cultured with hUCBDSCs.3. After co-cultured with hUCBDSCs, NF-κB activity of KM3 cells was lowest and NF-κB was high expressed in cytoplasm but low or no expressed in nucleus as determined respectively by EMSA and confocal microscopy.Inversely,it was revealed by Western blot that the expression of IκBαof KM3 cells was markedly enhanced by hUCBDSCs. Otherwise,it was showed that the mean fluorescence of ICAM-1 on KM3 cells was obviously weakened in KM3/hUCBDSCs co-culture group as determined by flow cytometry and confocal microscopy.The expression of Bcl-2,Bcl-XL at mRNA and protein levels markedly decreased as determined by real time PCR,confocal microscopy and flow cytometry after KM3 cells co-cultured with hUCBDSCsConclusion:1. The proliferation of KM3 cells can be inhibited and the apoptosis can be induced by hUCBDSCs in vitro.2. KM3 cells can stimulate both hUCBDSCs and MM-BMSCs to secrete IL-6,but lower level of IL-6 is secreted by hUCBDSCs.3. hUCBDSCs suppress the proliferation of KM3 cells by down-regulating the expression of IL-6R.4. hUCBDSCs inhibit the proliferation of KM3 cells and induce the apoptosis by inhibiting NF-кB activity and down-regulating the NF-кB regulating genes such as ICAM-1,Bcl-2 andBcl-XL.