Synergistic Cytotoxicity Effect Of Bortezomib In Combination With Obatoclax In Acute Lymphoblastic Leukemia

BACKGROUDAcute lymphoblastic leukaemia(ALL),is a kind of malignant cell proliferation of lymphatic grandmother blood disease.ALL seen in children and adults at the same time,common in children aged 2 to 5.As the further understanding of leukemia biology and promote high-tech treated equipment,the survival rate of ALL got a lot of ascension,especially children,which cure rate is as high as 90%.However,refractory/relapsed acute lymphoblastic leukaemia remains a problem with poor prognosis and short survival time.Therefore,the development of new treatment strategies and tumor molecular mechanism is particularly important.Bortezomib(Velcade),which a dipeptidyl boronic acid,the first proteasome inhibitor approved by the US Food and Drug Administration(FDA)for the treatment of multiple myeloma(MM)in 2003,subsequently used to treat Mantle cell lymphoma.Nowadays,as an effective inhibitor of proteasome,bortezomib is the most widely studied,and exhibited outstanding antitumor activity against the 60 NCI tumor cell line panel in vitro experiments.Meanwhile,it has good curative effect on various blood diseases,particularly the treatment of ALL now in clinical ?/? phase.B cell lymphoma-2(Bcl-2)is an oncogene,and the earlist study about proteins of Bcl-2 family is related to apoptosis.The Bcl-2 family proteins are divided into anti-apoptotic proteins(Bcl-2,Bcl-xl,Bcl-w,Mcl-1 and Al,etc.)and promoting apoptosis protein(Bak,Bax,Bok,Bik,Bim,etc.).The Bcl-2 family proteins are mainly located in the mitochondria,and the regulation of cell apoptosis through mitochondrial pathways.Studies found that most tumor cells over expressing the Bcl-2 protein,the researchers developed a series of Bcl-2 family protein inhibitors,including obatoclax,AT-101,ABT-199 and so on.OBJECTIVEAutophagy and ubiquitin proteasome,as the two pathways of protein degradation in cells,the former is mainly responsible for the degradation of long-lived proteins and damage organelles,the latter is responsible for the degradation of short-lived proteins.Bortezomib is a boric acid salt,also an effective 26 s proteasome inhibitors,but at the same time it can induce autophagy and cause drug resistance.Bortezomib alone can cause the accumulation of antiapoptotic proteins,such as Mcl-1.Therefore,in this study,we designed an experimental scheme that explored bortezomib combination with Bcl-2 inhibitors(obatoclax.AT-101K ABT-199)whether has synergistic antitumor effection on acute lymphatic leukemia cells.And our initial explanation of mechanism of synergistic antitumor by two drugs combined treatment,we hopes to solve the single drug in clinical research activity of low resistance,easy to provide a valuable reference for clinical drug application,and offers a new treatment options for ALL.METHODS AND RESULTS1.The cytotoxicity effect of drugs determined by MTT assay2.The effect of antiapoptotic proteins on bortezomib alone treatment by western blot 3.Using CompuSyn software to calculate drug Combination Index(Combination Index)and the concentration change before and after the Combination4.The effect on apoptosis after bortezomib combination with obatoclax1)Flow cytometry was to detected apoptosis of leukemia cells after bortezomib combination with obatoclax:2)Western Blotting detected the effect on expession of apoptosis related proteins,such as cleavage of PARP,ubiquitin protein accumulation and autophagy protein,endoplasmic reticulum stress related proteins and P38 protein.5.RT-PCR was used to detect the mRNA levels of genes association withendoplasmic reticulum stress response6.The effect on apoptosis of drug combination group cell after added ER stressinhibitors TUDC1)MTT assay:various concentrations bortezomib combination with obatoclax,and added the same concentration TUDC(200 ?M),cell viability of drug combination group was detected by MTT:2)Using Flow cytometrythe to detect the effect on apoptosis of TUDC put in the drug combination group cells.CONCLUSION1.Bortezomib?Obatoclax?AT-101 andABT-199 alone treament can significantlyinhibit the viability of human acute lymphoblastic leukemia cell lines Jurkat andNalm-6.2.Bortezomib alone caused anti-apoptotic protein accumulation on ALL cell lines.3.Bortezomib combined with Obatoclax can synergistic induce the apoptosis ofleukemia cells,but not including Bortezomib respectively combined with AT-101,ABT-199 by MTT assay.4.Bortezomib combined with Obatoclax both block autophagy and the ubiquitin-proteasome two major protein degradation pathways.5.Bortezomib combined with Obatoclax synergistic induce apoptosis mediated by endoplasmic reticulum stress response(ERS).6.The endoplasmic reticulum stress inhibitor(TUDC)reduces apoptosis mediated by ERS on drug combination group(bortezomib + obatoclax)cells.