Histone Deacetylase Inhibitors And Zoledronic Acid For Multiple Myeloma Tumor-burdened Mice Antitumor Function And Mechanism Research

Objective：Multiple myeloma(MM) occurred in the B lymphocytemalignant plasma cells malignant tumor， accounted for1%of allmalignant tumor，the blood system malignant tumor of10%.In Europeand the United States has become the second only to non-hodgkin’slymphoma of the second most common malignant tumor of the bloodsystem. Happens in the elderly，are still not completely cured. In recentyears，stem cell transplantation and the application of new drugs such asVelcade although to a certain extent，improved the prognosis of patientswith MM. But still exist transplantation related toxic and drug resistance，and many other problems. Therefore，searching for a new effective drugsto slow disease progression and prolong remission become a hot researchtopic at present. Zoledronic acid(ZOL) as a new generation of doublephosphonic acid salts drugs， by inhibiting osteoclast activity andinhibition of bone resorption，and reversible bone dissolve the progress ofthe disease. At present has been applied in malignant tumor dissolvedosseous bone cause bone pain，hypercalcemia，multiple myeloma andlung cancer，etc. Domestic and international numerous preclinical studiessuggest，ZOL through the direct mechanism (tumor cell adhesion，invasion，apoptosis) and indirect mechanism (angiogenesis，γ δ T cells)play antitumor effect. Histone deacetylase inhibitors (HDACi) can makethe tumor cells histone acetylation level improved，induced specific geneactivation expression， cause tumor cell apoptosis.LBH-589is ahydroxamate HDACi. In the clinical trials，to a variety of solid tumor andblood system tumor cells showed inhibition of proliferation and inducethe apoptosis of action. And for multiple myeloma strong activity (IC50＜40nmol/L). Bate2-microglobulin(β2-MG)is human HLA antigen a subunit，and main expression in B cell system and mononuclear cellsystem. The patients of multiple myeloma with a large number ofsyntheticβ2-MG and into the blood circulation，it can be used as aclinical judgment MM illness，treatment effect and prognostic indicators.This study probes into the histone deacetylase inhibitors and zoledronicacid in different ways combined application of multiple myelomatumor-burdened rat role. And through the determination of serum IL-6and β2-MG levels in evaluation of the effect of multiple myeloma，inorder to provide more clinical treatment means and methods.Methods:1Cell culture and transfer of cultureThe human MM cell line KM3cells were maintained in RPMI1640，containing10%fetal bovine serum，100units/ml penicillin and100μg/mlstreptomycin. 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.2Establish KM3tumor-burdened nude mouse modelWill be in logarithmic phase of KM3cells in serum-free medium ofRPMI1640washing2times，heavy suspension in1:1blending serum freemedium of RPMI1640，adjust the cell density of1x108/ml，each nudemouse right fore lateral subcutaneous inoculation0.2ml (cell number2×107)，a total of16nude mouses.3Observation the tumor rate of nude mouseThe nude mouse to vaccination tumor cells after15days of in thelimit，nude mouse right fore visible outside can be used to measure themass as the standard，tumor rate=tumor/total x100%.4Laboratory animal groupSixteen tumor nude mouses，randomly divided into4groups (eachgroup of four)：a、blank group；b、zoledronic acid alone group；c、LBH-589alone group；d、the combined group. Each group begins with a 16days after inoculated with tumor cells，not medication group a，groupb and group c each concentration of nude mice subcutaneouslyrespectively for0.1umol/L ZOL and the concentration of5nmol/LLBH5890.2ml each， group d each successively only nude micesubcutaneously concentration of0.1umol/L ZOL and the concentration of5nmol/L LBH5890.2ml；Every2d injection time，injection of5times(Dose groups in terms of the above with reference to our departmentdetermine preliminary in vitro experiment).5Antitumor effect observation5.1Observe the general situation and tumor-burdened mice，the shape ofthe tumor volume change.5.1.1Observe the general situation of the tumor-burdened mice：includingmental status，skin gloss，activity，food，drinking water and weightchange.5.1.2Every two days measuring the length diameter and short diameter oftumor，and according to the calculation formula of the tumor size andtumor growth inhibition rate (IR)： V=(length diameter×shortdiameter2)/2，IR=(V0-Vn)/V0×l00％(V0for untreatment group of tumorsize，Vn for treatment group of tumor size)，The nude mouse tumorvolume are take its mean value.5.2Assessment of apoptosisDepolymerization of strips of solid tumor tissues，fragmented andpreparation into tissue homogenate, detected using flow cytometryinstrument to single-agent Zoledronic group，LBH-589single druggroup and KM3cells apoptosis rate of the two drugs combination group.5.3Application of radioimmunoassay method was used for determinationof β2-MG level.Results:1People KM3cell nude mouse subcutaneous transplantation tumorto tumor： about7-9days after vaccination visible injection site，namelyright fore lateral appear can be used to measure the mass，and along with the time increasing mass，the rate of vaccination into tumor is65.38％(17/26). Transplantation into tumor after15days， the tumor shortdiameter basic is in5mm above，from which the cele form，size close16nude mouse used for experiment group.2Experimental animal general situation：Group A of nude mouse inthe late growing failure，listlessness，dark skin，eating water quantitysignificantly reduce weight，reduce obviously. Group b and c of nudemouse spirit post-communist activity slightly difference，eating anddrinking water quantity slightly reduced. Group d nude mouse mentalstatus，activity，skin，diet water quantity and weight were not see obviouschanges.3The change of tumor form and volume：Group a with the timeextension，tumor volume increase obviously. Group b and c group oftumor volume increased slightly. Group d along with the time extensiontumor size even slightly narrow.4The experimental animal after four weeks therapy calculationtumor growth inhibition rate，the results：group b，c，d in turn for71.97％、69.16%、94.27%。The difference between the treatment group andcontrol group were significantly，there is statistical significance (P <0.05).Between single drug group and combination group with significantdifference (P <0.05).The nude mouse tumor growth and four weeks aftertreatment of tumor growth inhibition rate calculation results show thatzoledronic acid and LBH-589inhibits multiple myeloma KM3tumor-burdened nude mouse transplantation tumor growth，and twomedicine has synergistic inhibition effect.5Flow cytometric analysis of apoptosis of KM3cells：zoledronicacid alone group、LBH-589alone group and the combined group appliedto KM3tumor-burdened nude mouse after48hours， there werehypodiploid cell peak before G0/G1peak in all groups, showing cellapoptosis appears in all groups.Compared with zoledronic acid combinedwith LBH589and the two drugs alone,an increase of apoptosis in KM3 cells appeared，the difference was statistically significant（P＜0.05）.6Remove eye blood method，each a tumor-burdened group micecollected venous blood of about0.6-1ml，and application of radiationimmunoassay (RIA) β2-MG level. Zoledronic acid group，LBH-589group and combination group of β2-MG levels are decreased in theblank group，and the two drugs in combination with a single drug groupsdecreased significantly，more statistically significant difference (P <0.05).Conclusions:1Zoledronic acid and LBH-589all can inhibit multiple myelomaKM3tumor-burdened mouse transplantation tumor growth，and thesynergistic combination therapy.2Zoledronic acid and LBH589can induce apoptosis of KM3cells.And their combination has a synergistic effect.3Zoledronic acid and LBH589all can cause serumβ2-MG decline，and the combined synergistic.