Genistein Suppressed NF-κB Expression Of Human Multiple Myeloma Cells And Marrow Stromal Cells

ObjectivesTo investigate effect of Genistein on proliferation of XG-1,RPMI8226 multiple myeloma cells and myeloma bone marrow stromal cells and to investigate influence of the apoptosis and myeloma cells.To investigate effect of Genistein on proliferation of human myeloma bone marrow stromal cells.To study effect of Genistein on NF-κB expression of XG-1,RPMI8226 multiple myeloma cells and bone marrow stromal cells. To investigate effect of Genistein on bcl-2,bcl-xl,CyclinDl,ICMA-1 and IL-6 gene expression of myeloma cells and Bone Marrow Stromal Cells.To examine effect of Genistein on expression of the Akt and Phospho- Akt in XG-1 cells;To observe the growth of XG-1 cells in Genistein-treated bone marrow stromal cells.MethodXG-1,RPMI8226 multiple myeloma cells and myeloma bone marrow stromal cells were treated with a series of concentrations of Genistein.The proliferation of XG-1,RPMI8226 multiple myeloma cells and myeloma bone marrow stromal cells was measured with MTT assay.RPMI8226 multiple myeloma cells were treated with a series of concentrations of Genistein.The apoptosis of was measured with flow cytometry assay.XG-1,RPMI8226 multiple myeloma cells and myeloma bone marrow stromal cells were treated with a series of concentrations of Genistein.The gene expression of the IL-6,bcl-xl,cyclinD1,ICAM-1,bcl-2 of cells was detected by RTPCR. XG-1 and RPMI8226 multiple myeloma cells were treated with different concentrations of Genistein.The nuclear retention of p65 was shown by immunocytochemistry.XG-1,RPMI8226 multiple myeloma cells,myeloma bone marrow stromal cells and normal bone marrow stromal cells were treated with different concentrations of Genistein.The NF-κB in cells was indicated by electrophoretic mobility gel shift assay.XG-1 multiple myeloma cells were treated with different concentrations of Genistein.The nuclear retention of p65 was shown by immunocytochemistry.The expression of Akt and Phospho- Akt in XG-1 cells was detected by western blot assay.The growth model in vitro was a co-culture of XG-1 cells out of rIL-6 and myeloma Bone Marrow Stromal Cells.XG-1 cells out of rIL-6 was cultured in four different supernatant:RPMI1640 of the rIL-6(1 ng/ml);normal myeloma bone marrow stromal cells;Genistein-treated myeloma bone marrow stromal cells;common RPMI1640,respectively.The proliferation of XG-1 cells was measured with MTT assay.ResultGenistein led to the suppression of proliferation of XG-1,RPMI8226 multiple myeloma cells and myeloma bone marrow stromal cells and induction of apoptosis of RPMI8226 multiple myeloma cells.Genistein affected XG-1 cells that IC50 of 72 hours is 8μg/ml.Genistein affected RPMI8226 cells that IC50 of 72 hours is 15μg/ml.Genistein affected bone marrow stromal cells that IC50 of 72 hours is 10μg/ml.The treatment of RPMI8226 and XG1 cells for genistein and doxorubicin resulted in inhibition of cell proliferation.The inhibition of cell growth was dose-and time-dependent.Genistein was weaker than doxorubicin about inhibition of cell proliferation,but Genistein contributed toward enhancing the inhibition of cell proliferation of doxorubicin,leading to greater antitumor activity in vitro.Genistein down-regulated the expression of NF-κB-regulated gene products,including bcl-2, bcl-xl,cyclin D1,IL-6 and ICAM-1 in XG-1,RPMI8226 multiple myeloma cells and myeloma bone marrow stromal cells.NF-κB was constitutively active in two human myeloma cell lines examined and that Genistein down-regulated NF-κB in two cell lines and myeloma bone marrow stromal ceils as indicated by electrophoretic mobility gel shift assay but not normal bone marrow stromal cells and prevented the nuclear retention of p65 as shown by immunocytochemistry.XG-1 myeloma cells showed consitutively active Akt and Phospho-Akt.Genistein suppressed Akt phosphorylation but not Akt.Result is clear that the XG-1 cells can recover quickly in the RPMI1640 of the rIL-6(1ng/ml);Can also recover in the normal bone marrow stromal cells quality cell ascend pure liquid,but the speed and degrees is slightly bad; XG-1 cells can't recover in the Genistein-treated bone marrow stromal cells.XG-1 cells also can't recover in the common RPMI1640. ConclusionNF-κB was constitutively active in two human myeloma cell lines examined and myeloma bone marrow stromal cells and that Genistein down-regulated NF-κB and suppressed the constitutive Akt phosphorylation.Genistein also down-regulated the expression of NF-κB-regulated gene products,including bcl-2,bcl-xl,cyclin DI,IL-6 and ICAM-1.Genistein-treated myeloma bone marrow stromal cells could suppress proliferation of XG-1 cells in the growth model in vitro.