Cardamonin Possesses Various Activitis Against Multiple Myeloma In Vitro

Objective: To investigate the effect of cardamonin on the viability, proliferation, apoptosis, cell cycle, and angiogenesis in multiple myeloma (MM) cells and the mechanisms involving in the process.Methods: MTT assay was used to screen natural products with anti-myeloma activity. CCK-8 assay was performed to evaluate the effect of cardamonin on the viability of MM cells and PBMNCs. was used to observe the influence of cardamonin on the proliferation of MM cells. Flow cytometry was used to measure the effect of cardamonin on the cell cycle and apoptosis of MM cells. The apoptosis changes were further examined by double staining with ethidium bromide (EB) and acridine orange (AO). Western blot was performed to observe the effect of cardamonin on the activation of caspase-3 and PARP in MM cells. Finally, tube formation assay was used to investigate the effect of cardamonin on angiogenesis.Results: Among the four natural products being screened, cardamonin exhibited inhibitory activity against myeloma cells, which was also confirmed by CCK-8 assay in a time- and dose-dependent manner. The IC50 of 48h on RPMI 8226, U266, and ARH-77 cells were 14.69μM, 13.06μM, and 10.82μM respectively, whereas little influence was seen in PBMNCs, except when the concentration of cardamonin reached 200μM. EdU assay showed that cardamonin largely inhibited the proliferation of myeloma cells. Flow cytometry revealed that cardamonin arrested myeloma cells at G2/M phase and induced apoptosis of myeloma cells. AO/EB staining confirmed the pro-apoptotic effect of cardamonin. Western blot found that cardamonin induced the activation of caspase-3 and PARP of myeloma cells. Tube formation assay showed the anti-angiogenesis effect of cardamonin against myeloma cells. Conclusion: The multiple effect of cardamonin on myeloma included the inhibition of viability and proliferation, arrest of cell phase, induction of apoptosis, inhibition of angiogenesis, etc. The pro-apoptotic effect was associated with activate-on. Thus, further research should be carried out on the anti-myeloma effect of cardamonin. ple myeloma cellsObjective: To explore the regulating effect of cardamonin on NF-κB pathway in multiple myeloma cells and the involved mechanisms.Methods: Western blot analysis was used to measure the effect of cardamonin on NF-κB activation. Immunofluoence test was performed to observe the subcellular location of NF-κB. Western blot assay was applied to measure the protein levels of IκBα, IKKα, IKKβ, and AKT. Meanwhile, the levels of Bcl-2, Bcl-xL, surviving, XIAP, cIAP-1, cIAP-2 (related to anti-apoptotic effect) as well as ICAM-1 (related to immigration), COX-2 (related to proliferation), and VEGF (related to angiogenesis) were measured. CCK-8 assay was used to observe the effect of cardamonin in combination with vincristine or dexamethasone on myeloma cells.Results: Cardamonin time- and dose-dependently inhibited p65 phosphorylation. Nuclear translocation of NF-κB was also suppressed by cardamonin. Western blot assay showed decreased level of IκBαphosphorylation, time- and dose-dependent inhibition of IKKαand IKKβ, and down-regulated level of AKT. Moreover, cardamonin time-dependently suppressed the expression of Bcl-2, Bcl-xL, surviving, XIAP, cIAP-1, cIAP-2, ICAM-1, COX-2, and VEGF. CCK-8 assay demonstrated synergic inhibitory effect of cardamonin combined with vincristine or dexamethasone on myeloma cells. Conclusion: Through inhibiting AKT, IKKα, and IKKβ, cardamonin suppressed the expression of NF-κB and its downstream proteins, which might be one of the mechanisms of cardamonin against myeloma. tiple myeloma cellsObjective: To investigate the regulating effect of cardamonin on STAT3 pathway in multiple myeloma cells and the related mechanisms. Methods: ELISA assay was used to detect IL-6 level, western blot analysis for constitutive activation of STAT3, and immunofluorescence assay for subcellular location of STAT3. Western blot was also used to measure the inducible activation of STAT3 by IL-6, effect of cardamonin on the IL-6-inducible actiovation of STAT3, and the expression of downstream gene products of STAT3, such as cyclin D1, Bcl-2, and VEGF.Results: Cardamonin time-dependently inhibited IL-6 secretion by U266 cells. Western blot showed that cardamonin suppressed STAT3 activation in U266 cells in a time- and dose-dependent manner. Nuclear translocation of STAT3 in U266 cells was also inhibited. Western blot revealed that IL-6 could induce activation of STAT3 in RPMI8226 cells, while cardamonin suppressed STAT3 activaton induced by IL-6. In the meantime, cardamonin suppressed the expression of cyclin D1, Bcl-2, and VEGF.Conclusion: Cardamonin downregulated IL-6 expression, inhibited constitutive and IL-6-inducible activation of STAT3, and suppressed downstream genes of STAT3, which might be another important mechanism for anti-myeloma effect of cardamonin.