Effect Of PTL On Angiogenesis Induced By Multiple Myeloma Cell Line In Vitro

Part I The effect of PTL on angiogenesis induced by multiple myeloma cell line in vitro[Objective]The growth and metastasis of malignant tumor depeneds on angiogenesis, solid tumor can secrete tumor vessel growth factors to stumulate vascular proliferation and it is verified that angiogenesis is very important to the development of multiple myeloma(MM). Someone had reported that parthenolide(PTL) had inhibitory effect on MM, but nobody had reported the effect on angiogeneis induced by MM. So we investigate if there is some effects of PTL on the angiogenesis induced by MM cell line in vitro. In order to find a noval drug to treat MM.[Method]The multiple myeloma cell line RPMI8226 and human umbilical vein endothelial cell(HUVEC) were cultured in vitro. RPMI8226 cells were treated with PTL at various concentrations for 48 hours, and endothelial cell migration assay and tubule formation assay were applied to assay the angiogenic activity.[Results]The RPMI8226 cell supernatant significantly induced endothelial cell migration and tubule formation in vitro.PTL suppressed the migration of endothelial cell induced by RPMI8226 cell supernatant, and the numbers of migrated cells were 310±56 at 3.5μmol/L, 207±28 at 5μmol/L, 127±21 at 7.5μmol/L, 49±10 at 10μmol/L.The areas of the capillary-like structures decreased while the concentration of PTL incresed, 0.092±0.003mm2 at 3.5μmol/L, 0.063±0.002 mm2 at 5μmol/L, which were less than the control(p<0.01).[Conclusion]PTL can depress the activity of NF-κB, inhibit the expressions of VEGF and IL-6, but have no effect on the expressions of MMP2 and MMP9. It indicates that the effect of PTL on angiogenesis induced by MM is in part due to the inhibitory effect on the activity of NF-κB and downregulation of mRNA expression and secretion of VEGF and IL-6. Therefore, PTL could serve as a novel drug to treat multiple myeloma. Part II The exploration of the mechanism of the inhibitory effect of PTL on angiogenensis induced by multiple myeloma cells[Objective]It is verified that PTL can strongly inhibit angiogenesis induced by MM in vitro,but the mechanism is still unclear. NF-κB is a very important transcription factor of VEGF and IL-6 ,which are related to the angiogenic effect of MM. So we are order to explore the mechanisms of the inhibitory effect of PTL on angiogenensis induced by multiple myeloma cells.[Methods]The multiple myeloma cells were cultured in vitro. RPMI8226 cells were treated with PTL at various concentrations for different periods, and MTT assays were used to measure the proliferative activity. After cultured with PTL,the NF-κB activity of cells were examined by western bloting assay. The expression levels of vascular endothelial growth factor(VEGF) and interleukin-6(IL-6) in RPMI8226 cell supernatant were detected by ELISA. The proteins of matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9(MMP9) were detected by immunohistochemistry staining.The mRNAs of VEGF ,IL-6 , MMP2 and MMP9 were detected by RT-PCR.[Results]PTL could obviously inhibit the growth and proliferation of the MM cells,which showed the dependment on dose and time with the concentration of 1 to 10μmol/L. After being treated with PTL for 48h, the activity of p65 went upward while the content of IκB-αwent downward as the increase of the concentration of PTL,which demonstrated inhibition to the activity of NF-κB. 48h after treatment of PTL at concentrations of 1,2,3.5,5,7.5,10μmol/L, the levels of VEGF in the supernatant were 2667.2±360.8,2664.2±398.0,2373.4±392.2,1982.3±293.3, 1247.0±338.4 and 936. 5±168.5pg/ml, respectively. Except groups of 1μmol/L and 2μmol/L, the others showed significant changes while compared with the control(p<0.05). The levels of IL-6 in the supernatant of MM treated with 7.5μmol/L and 10μmol/L PTL were 59.6±2.8pg/ml,41.4±9.8pg/ml,respectively,which were less than the control(p<0.05). Furthermore, PTL depressed the mRNA expression of VEGF and IL-6 in MM cells in a dose-dependent manner. PTL had nearly none effect on the exprssion and secretion of MMP 2 and MMP9.[Conclusion]PTL can significantly inhibit angiogenic ability of MM cells, which is in part due to the inhibitory effect on the activity of NF-κB and downregulation of mRNA expression and secretion of VEGF and IL-6. Therefore, PTL could serve as a novel drug to treat multiple myeloma.