| Objective: Mantle cell lymphoma(Mantle cell lymphoma,MCL)is a B cell non-hodgkin’s lymphoma(B-NHL)that shows high invasive and extremely poor prognosis,which is difficult to cure in clinical practice and so far there is no standard treatment.The selection of clinical treat ment is usually based on age,physical condition,risk stratification and s o on.Although the high dose intensive chemotherapy regimens(includin g R-CHOP plan and R-hyper-CVAD)are effective for most of patients.However,chemotherapy related side effects,treatment related mortality ar e very high,and most of patients eventually will relapse and/or resistan ce.So far,allogeneic hematopoietic stem cell transplantation(allo-HSCT)is the only way to cure for MCL,but,transplant related mortality is h igher,and HLA matching suitable donor is insufficiency,so it is particular ly important to improve the curative effect of treatment of MCL for loo king for new drugs and methods.Histone deacetylase inhibitor(Histone d eacetylase inhibitor,HDACi)Chidamide is a new type of drug that can play antitumor activity through regulating the expression of genes,increas ing cell histone acetylation levels,produces cell cycle arrest,induction of cells apoptosis in a variety of biological effects.Bortezomib is a type o f proteasome inhibitors that can play to inhibit the cell proliferation,spr ead of tumor cells and angiogenesis through reducing Cyclin D1 expressi on and inhibiting NF-Kb pathway,and it has less side effect,and no cro ss-resistance to conventional chemotherapy drugs,so focus clinical attenti on.In recent years,both in vivo and in vitro experiments have confirm ed that the proteasome inhibitor monotherapy or histone deacetylase mo notherapy has therapeutic effects on solid tumors(such as lung cancer,colorectal cancer,ovarian cancer,ect.)and hematological malignancies(such as leukemia,lymp-homa,ect.),and according to the relevant literature r eports,histone deacetylase inhibitors can potentiates bortezomib-induced antitumor effect by enhancing the inhibitory activity of NF-Kb.But so f ar,there has been no studies published about the effection of the chida mide combinded with bortezomib on human mantle lymphoma cells prol iferation and apoptosis.The purpose of this study is to investigate the influence of chidami de in combination with bortezomib on human MCL lines,explore the re lated mechanism,provide theory accordances and experiment datas for c linic.Methods:1 Cell culture and passage of Jeko-1 cells or Grant-519 cellsThe human MCL cell line Jeko-1 cells,Grant-519 cells were purcha sed from the cell bank of Chinese Academy of Science,Shang hai,maint ained in modified type RPMI 1640,containing 20% fetal bovine serum,100 units/ml penicillin and 100 ug/ml streptomycin.Jeko-1 cells or Grant-519 cells were cultured at 37℃ in a humidified of 5% CO2 atmosphere.The cells were passaged every two or three days.All experiments were using logarithmically growing cells,and cells′ viability>95% tested by typ an blue staining.2 The effect of chidamide and/or bortezomib on Jeko-1 cells or Grant-519 cells tested by CCK-8Jeko-1 cells or Grant-519 cells were randomly divided into blank c ontrol group,negative control group and different concentration of chida mide or bortezomib single drug group and chidamide and bortezomib gr oup,randomly.The proliferation inhibition of Jeko-1 cells and Grant-519 c ells were observed for 24 hours,48 hours or 72 hours,and calculated the combination of two drugs is to add,synergy or antagonism by Jin Zhen gjun calculation formula.3 Assessment of apoptosis and cell cycle with FCMThe apoptosis levels were evaluated by propidium iodide(PI)stainin g and Annexin V staining.Jeko-1 cells or Grant-519 cells were cultured in the presence or absence of chidamide and/or bortezomib for 48 hours,then followed by BD apoptosis kit.4 Test the m RNA expression levels of NF-Kb(p65),Bcl-2,Bax,CCND1,Caspase3/9 with Realtime-PCRJeko-1 cells or Grant-519 cells,which in exponential growth state,we re randomly divided into chidamide group,bortezomib group,chidamide w ith bortezomib group,control group for 48 hours.Total RNA were collect ed from each group,then reverse transcript into c DNA,detected the m RN A expression levels of NF-Kb(p65),Bcl-2,Bax,CCND1,Caspase3/9 relati ve to B-actin with RT-PCR.5 Test the protein levels of NF-Kb(p65),Bcl-2,Bax,CCND1,Caspase3/9with western blot.Jeko-1 cells or Grant-519 cells,which in exponential growth state,we re randomly divided into chidamide group,bortezomib group,chidamide w ith bortezomib group,control group for 48 hours.The total protein of cell s in each group were collected,50ug(or 20ug)protein from each group were loaded into SDS-PAGE electrophoresis.After bloting into film,protei n were detected with antibodies.Results:1 The effect of chidamide and/or bortezomib on Jeko-1 cells or Grant-519 cells tested by CCK-81.1 Human MCL Jeko-1 cells or Grant-519 cells were cultured with diff erent drug concentration(0.50,1.00,2.00,4.00,8.00,16.00μM),(4.00,8.00,16.00,32.00μM)of chidamide for 24 hours,48 hours,72 hours.Absorbance val ues were reduced in different degress by CCK-8 test.Under the same tre atment time,the inhibition rate of Jeko-1 cells or Grant-519 cells was gr adually increased with the increase of drug concentration,and under the same drug concentration,the inhibition rate of Jeko-1 cells or Grant-519 cells was also gradually increased with the increase of the time.Chidami de inhibited the proliferation of Jeko-1 cells or Grant-519 cells with tim e and concentration dependency under a centrain dose scope.The differe nce was statistically significant(P<0.05).1.2 Human MCL Jeko-1 cells or Grant-519 cells were cultured with diff erent drug concentration(0.25,0.50,1.00,2.00,4.00,6.00,8.00 n M),(0.01,0.10,1.00,10.00μM)of brotezomib for 24 hours,48 hours,72 hours.Absorbance v alues were reduced in different degress by CCK-8 test.Under the same t reatment time,the inhibition rate of Jeko-1 cells or Grant-519 cells was gradually increased with the increase of drug concentration,and under th e same drug concentration,the inhibition rate of Jeko-1 cells or Grant-519 cells was also gradually increased with the increase of the time.Brotez omib inhibited the proliferation of Jeko-1 cells or Grant-519 cells with t ime and concentration dependency under a centrain dose scope.The diff erence was statistically significant(P<0.05).1.3 Chidamide interacted synergistically with bortezomib on inhibiting th e proliferation of Jeko-1 cells or Grant-519 cells when compared with s ingle drug.For example,the Grant-519 cell inhibition ratio was(27.46±2.96)%,(6.58±1.10)% and(42.10±3.01)% for 4u M chidamide,0.01 u M bortezo mib and the combination of both drugs for 48 hours,respectively.The tw o drugs had synergistic effect by Jin Zhengjun calculation formula and t he Q value was 1.31.The difference was statistically significant compare d with the single drug group and control group(P<0.05).2 Assessment of apoptosis and cell cycle with FCMChidamide and bortezomib along can both induce Jeko-1 cells or Gr ant-519 cells apoptosis under a certain dose scope.And with the drug co ncentration increased,apoptosis rate increased,the combination of chidam ide and bortezomib induced more cell apoptosis.The difference was stati stically significant(P<0.05).3 Test the mRNA expression levels of NF-Kb(p65),Bcl-2,Bax,CCND1,Caspase3/9 with Realtime-PCRUnder a centrain does scope,chidamide group can down-regulate Bcl-2 and CCND1 m RNA expression,up-regulate Bax,Caspase3/9 m RNA ex pression in Jeko-1 cells and Grant-519 cells for 48 hours,and with the i ncrease of drug concentration,enhanced(P<0.05),while NF-Kb(p65)m R NA expression compared with control group had no significant differenc e(P>0.05).In bortezomib group,NF-Kb(p65),Bcl-2 and CCND1 m RNA e xpression were down-regulated and Bax,Caspase3/9 m RNA expression w ere up-regulated with the increase of drug concentration(P<0.05).The eff ect of the combination of chidamide and bortezomib is more significan t,and the difference was statistically significant compared with the single drug group and control group(P<0.05).4 Test the protein levels of NF-Kb(p65),Bcl-2,Bax,CCND1,Caspase3/9with western blotBoth chidamide and bortezomib can down-regulate Bcl-2 and CCN D1 protein expression,up-regulate Bax,Caspase3/9 protein expression in J eko-1 cells and Grant-519 cells,in addition,bortezomib can also down-re gulate NF-Kb(p65)protein expression.The effect of the combination of chidamide and bortezomib is more significant,which was consistent with the previous RT-PCR results.The difference was statistically significantc ompared with the control group(P<0.05).Conclusions:1 Under a certain concentration scope,chidamide and bortezomib alo ne or combined can inhibit the proliferation and induce the apoptosis of Jeko-1 cells or Grant-519 cells,with time and concentration dependency.The effect of the combination of two drugs can enhance.2 Under a certain concentration scope,chidamide and bortezomib co mbined can down-regulate Cyclin D1,NF-Kb(p65)and Bcl-2 gene and protein expression levels,and up-regulate Bax,Caspase3/9 gene and protein expression levels.The effect of the combination of chidamide and bortez omib is more significant.The possible mechanisms are down-regulate Cy clin D1,NF-Kb activity and triggered Caspase3 pathway. |
PDF Full Text Request