| Section 1 BBD9 inhibited cell growth and induced apoptosisin myeloma cellsObjective:To study the effects and the mechanisms of BBD9 on myeloma cell growth.Methods:Cell growth was measured by MTT assay in myeloma cell lines and primary cellsfrom patients with MM.Cell morphology was observed by Wright-Giemsa stain.AV-PIstaining was used for detecting apoptosis,PI staining was used for cell cycle analysis,and western blot was used for detecting caspase,BCL-2 family proteins and cell cycleregulating proteins.Results:The cell viabilities of 4 myeloma cell lines,including RPMI8226,U266,MM.1S,MM.1R,were significantly inhibited by BBD9 at the concentration of 0-4μg/ml.Thecell survival rate was decreased with the increasing concentration of BBD9. Furthermore,BBD9 inhibited cell growth in all 4 cell lines in a time dependent manner.If treated at the same concentration of BBD9,fewer cells would survive when the cellswere treated for an increasing time.The 24h IC50 of BBD9 on RPMI8226,U266,MM.1S,MM.1R were 2.30μg/ml,1.87μg/ml,2.39μg/ml,1.54μg/ml,respectively.Primary cells from three patients with MM were treated with BBD9 for 24h,andshowed growth inhibition in a dose dependent manner.The IC50 for primary cells was1.08μg/ml.The cell growth inhibition induced by BBD9 was also measured inmononuclear cells form normal bone marrow by MTT assay.The results showed thatthe cell growth of normal mononuclear cells was inhibited,in a dose dependant manner,by BBD9 at the concentration of 0-4μg/ml.However,normal mononuclear cells wereless sensitive to BBD9 treatment than primary MM cells were,and the 24h IC50 was3.24μg/ml.After treated by BBD9 for 24h,U266 and RPMI8226 cells,observed byWright-Giemsa stain,showed a typical apoptotic morphology,nuclear fragmentationand apoptotic body.Typical DNA ladders were also seen in RPMI8226 cells treated byBBD9 for 24h.With the concentration of BBD9 increasing,the DNA ladder becamemore significant.BBD9 could induce apoptosis in MM cells,as measured by AV-PIassay.After treated by 0,1,2,3μg/ml BBD9 for 24h,the RPMI8226 cells in earlyapoptosis were 1.57±0.37%,4.79±2.47%,16.18±5.45%,42.94±13.99%,respectively.Meanwhile,the U266 cells in early apoptosis were 2.69±1.28%,7.36±2.22%,19.67±2.83%,50.26±16.29%,respectively.Furthermore,caspase-3,-8,-9 and PARP werecleaved and activated,as measured by western blot,in U266 cells and RPMI8226 cellstreated by BBD9.In addition,pro-apoptotic protein Bak,Bax and Bim wereup-regulated in U266 cells and RPMI8226 cells treated by BBD9 and anti-apoptoticprotein Mcl-1 were down-regulated.However,another two important anti-apoptoticprotein,BCL-2 and BCL-XL,had not changed in protein expression level,as wasmeasured by western blot assay.In addition,the results of cell cycle analysis showedthat BBD9 induced cell cycle arrest in G2/M phase.After treated by 0,1,2,3μg/ml BBD9 for 24h,the RPMI8226 cells arrested in G2/M phase were 4.49±3.79%,13.61±2.09%,41.03±2.09%,35.99±9.05%,respectively.After being treated by 0,1,2,3μg/ml BBD9 for24h,the U266 cells arrested in G2/M phase were14.14±0.6%,19.48±2.84%,26.54±3.44%,33.62±3.0%,respectively.Cell cycle regulators cyclin A,cyclin B1 were bothdown-regulated significantly,as measured by western blot assay,in U266 cells andRPMI8226 cells treated by BBD9 for 24h,whereas,CDK1 and P27 showed no anydistinct down-or up-regulation.Conclusions:(1)The cell growth of 4 MM cell lines and primary cells from 3 patients with MMcould be inhibited by BBD9.(2)BBD9 induced apoptosis in MM cells.(3)Pro-apoptotic protein Bak,Bax and Bim were up-regulated and anti-apoptoticprotein Mcl-1 was down-regulated by BBD9 in MM cells.(4)BBD9 induced cell cycle arrested in G2/M phase through down-regulation ofcyclin A and cuclin B1.Section 2 BBD9 induced apoptosis in MM cells accompaniedby the activation of FOXO3a/BimObjective:To study the mechanisms of the up-regulation of Bim protein in apoptotic MMcells induced by BBD9.Methords:Real-time PCR was used for quantifying Bim mRNA and mir-17-5p,western blotused for FOXO3a and pFOXO3a analysis,immunofluorescent staining was used forobserving the distribution of FOXO3a in cells. Results:The copies of Bim mRNA significantly increased in U266 cells treated by BBD9for 8h.In addition,mir-17-5p,the negative regulator of Bim mRNA,was down-regulated significantly.Mir-17-5p in U266 cells treated with 3μg/ml BBD9 wasdown-regulated to 0.35±0.23 folds of that in control group.Western blot assay showedthat the expression of FOXO3 factor,one of pivotal transcription factors of Bim gene,was significantly higher in nuclear of U266 cells treated with BBD9 than control.Onthe contrary,phosphor-FOXO3a(pFOXO3a)was decreased with the increasing ofBBD9 concentration.Observations using immunofluorescent microscope showed thatwhen U266 cells were not treated with BBD9,FOXO3a in nuclear was lower than thatin cytoplasm.After U266 cells were treated with 2μg/ml BBD9 for 24h,theimmunofluorescence of FOXO3a in nuclear was higher than that in cytoplasm.Likewise,before RPMI8226 cells were treated with BBD9,FOXO3a in nuclear waslower than that in cytoplasm.After RPMI8226 cells were treated with 2μg/ml BBD9 for24h,the immunofluorescence of FOXO3a in nuclear was higher than that in cytoplasm.Conclusions:(1)BBD9 up-regulated Bim protein in MM cells through up-regulation ofFOXO3a factor in nuclear and subquently promotion of the Bim gene transcription,BimmRNA up-regulation.(2)BBD9 down-regulated mir-17-5p which resulted in decreased degradation ofBim mRNA or in decreased inhibition of Bim mRNA translation.Section 3 BBD9 inhibited IL-6 signaling in MMObjective:To better understand the relationship between the apoptosis induced by BBD9 inMM cells and IL-6 signaling pathway. Methods:MTT was used for assaying the effect of extraneous IL-6 on cell growth inhibitioninduced by BBD9 in U266 cells.ELISA was used for measuring IL-6 concentration inU266 culture supematant.Membrane IL-6R was measured by FACS,IL-6mRNA andIL-6R mRNA were measured by Real-time PCR,and protein levels were measured bywestern blot.Results:Extraneous IL-6 abrogated the cell growth inhibition induced by BBD9 in U266cells.The cell survival rate,which was 88.83% in U266 cells treated with 1μg/ml BBD9for 24h,significantly increased to 104.83% in U266 cells treated with 150ng/ml IL-6and 1μg/ml BBD9.The U266 cell survival rate,which was only 43.03% when cellswere treated with 2μg/ml BBD9 for 24h,significantly increased to 55.93% in U266cells treated with 150ng/ml IL-6 and 2μg/ml BBD9.IL-6 concentration in U266 cellculture was determined using ELISA assay and a decreased auto-secreted IL-6 wasfound.IL-6 concentrations in culture supernatant of U266 cells treated with 0,1,2,3μg/ml BBD9 for 24h were 33.49±7.92pg/ml,10.34±S.83pg/ml,13.59±7.22pg/ml,10.78±6.67pg/ml,respectively.In addition,membrane IL-6R was also slightlydown-regulated.Compared with the untreated control group,the expressions of IL-6mRNA were 0.9,3,2.7 folds,whereas the expressions of IL-6 mRNA were not changed.Furthermore,the phosphorylation level of two important kinases,STAT3 and AKT,were significantly down regulated in U266 cells treated with BBD9,while theexpression of total STAT3 and total AKT kept intact.Conclusions:(1)BBD9 inhibited MM cells auto-secretions of IL-6 and the production ofmembrane IL-6R.But they were not regulated at mRNA level.(2)Two of IL-6 signaling pathways,STAT3 signaling pathway and AKT signaling pathway were inhibited by BBD9.(3)The blockage of IL-6 signaling pathway was an important mechanism in BBD9induced MM apoptosis.Section 4 BBD9 increases sensitivity to adriamycin in multi-drugresistant K562/A02 cellsObjective:To investigate the role of BBD9 in anti-multi-drug resistant acute leukemia and itsmechanisms.Methods:MTT was used for detecting multi-drug resistance of leukemia cells,apoptosis,theconcentration of ADM in the cells and membrane p-gp were measured by FACS,mdr-1mRNA was measured by Real-time PCR.Results:24h IC50 of ADM on K562/A02 was 7.57μg/ml,the IC50 of ADM on K562 was0.651μg/ml,and the resistance index was 11.65.K562/A02 cells were treated with2μg/ml ADM and/or 1μg/ml BBD9 for 24h,and the results showed that the apoptosisrate in 2μg/ml ADM group was 6.04±2.28%,the apoptosis rate in 1μg/ml BBD9 groupwas 8.21±5.74%,the apoptosis rate in control group was 6.59±2.56%.However,theapoptosis rate in 2μg/ml ADM combined with 1μg/ml BBD9 group was 35.93±1.87%,and comparing with control,P＜0.05.In addition,K562 cells were treated with 0.4μg/mlADM and/or 1μg/ml BBD9 for 24h,and the results showed that the apoptosis rate in0.2μg/ml ADM group was 11.77±8.44%,the apoptosis rate in 1μg/ml BBD9 group was3.52±2.0%,the apoptosis rate in control group was 1.77±0.58%,the apoptosis rate in0.4μg/ml ADM combined with 1μg/ml BBD9 group was 2.93±0.48%,the difference between these groups were no statistical significance(P＞0.05).The results of measuringADM concentration in cells showed that after treated with BBD9 for lh,ADM positiveK562/A02 increased from 8.88±12.98% in 2μg/ml ADM group to 87.82±12.03% in2μg/ml ADM combined with 1μg/ml BBD9 group.After treatment with BBD9 for 24h,the mean fluorescence intensity of ADM in K562/A02 cells increased from39.78±3.64% in 2μg/ml ADM group to 54.4±-5.63% in 2μg/ml ADM group combinedwith 1μg/ml BBD9 group.In addition,BBD9 decreased neither the expression of mdr-1mRNA nor the membrane p-gp.Conclusion:(1)BBD9 increased the sensitivity to adriamycin in multi-drug resistant K562/A02cells but not in K562 cells.(2)BBD9 increased the sensitivity adriamycin in K562/A02 cells via raising ADMconcentration in cells.(3)The mechanisms of increasing sensitivity to adriamycin by BBD9 did notinvolved the alteration in p-gp protein,but the efflux-pumping function of p-gp proteinprobably was interfered by BBD9.Summary:BBD9 inhibited cell growth in MM cell lines and primary cells from patients withMM apoptosis was induced.Pro-apoptotic protein Bak,Bax and Bim were up-regulatedand anti-apoptotic protein Mcl-1 was down-regulated in BBD9 treated MM cells.Furthermore,BBD9 induced cell cycle arrested in G2/M phase through down-regulationof cyclin A and cuclin B1.In addition,BBD9 increased the expression of FOXO3a innuclear and decreased the expression of mir-17-5p,resulting in the increasement of Bimprotein in MM cells.Furthermore,BBD9 inhibited the production of IL-6 as well asIL-6R,and inhibited 2 important IL-6 signaling pathways:STAT3 and PI3K/AKT, indicating that the blockade of IL-6 signaling is one of the important mechanisms ofapoptosis induced by BBD9 in MM cells.In addition,our study showed that BBD9increased the sensitivity to adriamycin in multi-drug resistant K562/A02 cells but not inK562 cells.BBD9 increased the sensitivity to adriamycin in K562/A02 cells via raisingADM concentration in cells.As BBD9 decreased neither mdr-1 mRNA nor p-gp protein,we deduced that BBD9 increased sensitivity to adriamycin in K562/A02 cells throughinhibition of p-gp function.
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