Synergistic Activity Of The Histone Deacetylase Inhibitor LBH589and Zoledronic Acid Against Multiple Myeloma In Vitro

Objective: Multiple myeloma(MM) is a malignant clonal disorder ofterminally differentiated B cells. The epidemiology evidence of MM isabout1/100000in our country,but the incidence of MM is increasing withaging of the population.While advances in treatment,the use of high-dosechemotherapy and novel drugs such as thalidomide, lenalidomide andbortezomib have improved patient outcomes,drug resistance and relapsesinvariably occur, indicating a need for continued investigation of novel agents.Zoledronic acid,a third generation bisphosphonates,has been used to treatand prevent bone disease of MM at home and abroad. Some sesearch showszoledronic acid induces the apoptosis of myeloma cells by inhibiting theenzyme farnesyl pyrophosphate synthase in myeloma cells, thereby preventingthe prenylation of small GTPase signaling proteins. Histone deacetylaseinhibitors (HDACi) induce a highly acetylation of histones in the target cells,to start specific gene expression, thus blocking the growth of tumor cells,byinhibiting histone deacetylase activity. LBH-589is a hydroxamate HDACi,showing anti-tumor activity in pre-clinical trials for solid tumors andhematological neoplasms, including potent anti-myeloma activity. Signaltransduction unit CD130(gp130) is the most important part of IL-6receptorcomplex, IL-6plays an important role in MM cell growth and survival,thereducing of CD130expression can affect the growth-promoting effect on MMcells.This experiment explores zoledronic acid combined with LBH589hassynergistic anti-tumor effect on human myeloma cell line KM3cells and theimpact on the surface markers CD130, provideing a theoretical basis andevidence for the future of these two drug combination used in clinical trials.Method:1Cell culture of KM3 The human MM cell line KM3cells were maintained inRPMI1640,containing10%fetal bovine serum,100units/ml penicillin and100μg/ml streptomycin. KM3cells were cultured at37℃in a humidified of5%CO2atmosphere. The cells were passaged every two or three days. Allexperiments were using logarithmically growing cells,and cells' viability≥95%tested by trypan blue staining.2Assessment of KM3cells' viabilityMTT assay was used to test the proliferation of KM3cells at differenttimes by different concentrations(0.01,0.05,0.1,0.5,1μmol/L)of zoledronicacid, different concentrations(5,10,15,20nmol/L)of LBH589and differentconcentrations of the two drugs combined.3Assessment of apoptosisDifferent concentrations (0.01,0.05,0.1μmol/L) of zoledronic acid,LBH589(5and10nmol/L) and the two drugs combined treatment of KM3cells after48h, samples were analyzed by FCM to detect cell apoptosis rate.4Assessment of cell cycleDifferent concentrations (0.01,0.05,0.1μmol/L) of zoledronic acid,LBH589(5and10nmol/L) and the two drugs combined treatment of KM3cells after48h,samples were analyzed by FCM to detect cell cycle.5Test the expression of CD130Different concentrations (0.01,0.05,0.1μmol/L) of zoledronic acid, LBH589(5and10nmol/L) and the two drugs combined treatment of KM3cells after48h, using FCM to test the expression of CD130.Results:1KM3cell proliferation inhibition assayed by MTT: KM3cells weretreated by0.01,0.05,0.1,0.5,1μmol/L concentration of zoledronic acid alone,5,10,15,20nmol/L concentrations of of LBH589alone and two drugscombination of the above-mentioned concentrations for24h,48h,72h,inhibition ratio of KM3cells increased differently and the inhibition ratioincreased with the increasing of drug concentration and time, the differencewas significant (P＜0.05). Groups of the same concentration were compared at different times, the difference was statistically significant (P＜0.05),groups of the different concentrations were compared at the same time, thedifference was statistically significant (P＜0.05). Zoledronic acid andLBH589can inhibit the growth of the KM3cell in a dose-dependent and time-dependent effect. Combination index(CI) ranged from0.535to1.180betweenthe two drugs, while CI was less than0.9when the concentration of the twodrugs is relatively small,which showing synergistic effects on the inhibitionof KM3cells.The most synergistic group is0.1μmol/L concentrationzoledronic acid combined with5nmol/L concentration LBH589.2Flow cytometric analysis of apoptosis of KM3cells: According to theMTT results,zoledronic acid and LBH589treatment concentration and timewas adjusted,KM3cells were treated by zoledronic acid0.01,0.05,0.1μmol/Land LBH5895,10nmol/L and the two drugs above the concentrationcombination of two for48h, there were hypodiploid cell peak before G0/G1peak in all groups, showing cell apoptosis appears in all groups.Comparedwith zoledronic acid combined with LBH589and the two drugs alone,anincrease of apoptosis in KM3cells appeared, the difference was statisticallysignificant(P＜0.05).3Flow cytometric analysis of cell cycle of KM3cells：Treated withdifferent concentrations of zoledronic acid, LBH589and the two-drugcombination for48h,(1) zoledronic acid alone group, with increasingconcentration, percentage of S-phase increased and G0/G1phase decreased, inwhich0.05and0.1μmol/L concentration group compared with the controlgroup, the difference was statistically significant (P＜0.05), the G2/Mpercentage of cells did not change significantly,(2) the LBH589alone group,with increasing concentration, percentage of G0/G1phase increased and Sphase decreased, compared with the control group,the difference wasstatistically significant (P＜0.05), G2/M phase did not change significantly,(3) the combined group, with increasing concentration, percentage of G2/Mphase decreased, in which0.05,0.1μmol/L concentration zoledronic acidcombined with5,10nmol/L concentration LBH589groups compared with the control group, the difference was statistically significant (P＜0.05), and therewas no difference of G0/G1and S phase compared with the control group.4Flow cytometric analysis of CD130expression of KM3cells:(1)zoledronic acid alone group, with increasing concentration, CD130-positivecells gradually decreased in KM3cells, and cell surface CD130meanfluorescence intensity decreased too, in which0.05and0.1μmol/L groupcompared with the control group, the difference was statistically significant (P＜0.05),(2) LBH589alone group, with increasing concentration, CD130-positive cell number decreased in KM3cells, CD130mean fluorescenceintensity gradually weakened on cell surface, compared with the control group,the difference was statistically significant (P＜0.05),(3) the combined groupcompared with the control group, with the increase of the concentration of thetwo drugs, CD130-positive cells were significantly reduced and CD130meanfluorescence intensity was significantly reduced, compared with the controlgroup, the difference was statistical significance(P＜0.05).Conclusions:1At a certain concentration range, zoledronic acid and LBH589caninhibit the proliferation of human MM cell line KM3,showing time andconcentration dependent,furthermore, zoledronic acid and LBH589cansynergistically inhibit the proliferation of KM3cells.2Zoledronic acid and LBH589can induce apoptosis of KM3cells.Zoledronic acid Induces KM3cells arrested in S phase, LBH589Induce aincrease on KM3cells in G0/G1phase,interfere with the cell cycle may beone of the anti-myeloma mechanism of action of the two drugs.3Zoledronic acid and LBH589caused a reduction in the number ofCD130positive cells and weakness of CD130mean fluorescence intensity ofcell surface, thus down-regulation of CD130expression may be one of theanti-myeloma mechanism of action of the two drugs.