The Research Of Aminopeptidase Inhitors Ubenimex Induce Myeloma Cells Apoptosis

ForewordThe multiple myeloma is one kind of malignant tumor of the hemotoligical system.The same with other tumors,its generation and development has close relationship with the disorder toapoptosis.CD13 is one kind of glucoprotein of cell envelop under the name of aminopeptidase because of hydrolyzing residue-N of amino acids.CD13 is concluded of the surface marker of the normal or malignant pulpal cells early for the qualitation and typing of the leukemia and lymphoma cells.But CD13 dosen't express only on pulpal cells,many cells of other types also can express CD13 on some phases of growth cycle.Literature has indicated the detection of high expression on myeloma ceils,which offered the thereunder of molecular biology of CD13 inhibitors Ubenimex making targeted therapy to the muliple myeloma.CD13 has maked an important contribution on physiologic adjustment and signal teansmission with the organism rhagiocrine cells as hydrolyzing some cell factors of immune function which is not benefit of educing of normal immune function.With the profound research of CD13,more evidences indicate that CD13 has close relationship with the generation and development of tumors.CD13 can enhance cell infestation which is benefit to tumor metastasis and enhance tumor neogenesis angiopoiesis,and also can enhance tumor cell multiplication and inhibit tumor cenn apoptosis.CD13 inhibitor Ubenimex is a kind of micromolecule bipeptid compound with antibacterial action and immunologic enhancement which found in the culture solution of dictyo-olive streptomycete.Ubenimex can inhibit the aminopeptidaseB.N,and leucine aminopeptidase on tumor cells,and can induced tumor cell apoptosis and enhance the host's immune function with the antineoplasmic activity both direct and through intermediate of host.The antineoplasmic activity of Ubenimex is considered early of the implement with the non-specificity enhancement to tumor immune reaction in corpore.Subsequent study found that Ubenimex also can inhibit several tumor cells growth and enhance apoptosis without rhagiocrine cell and T cell.Ubenimex has antimultiplication and induction of apoptosis effect with CML cells K562.The antimultiplication effect is considered with the degression of expression of cycline D1,and the induction of apoptosis effect is implemented through the signal pathway of Caspase-3 and PI3K-GSK.There are a few repots on that Ubenimex has direct growth inhibiting effect on tumor cells and leukemic cells in vitro these years abroad.But,the repots on the stady of the effect on Ubenimex with myeloma cells are rare.This experiment has investigated the inhibitory action of Ubenimex with myeloma cells XG and RPMI8226 and has observated wether Uenimex can induce myeloma cells apoptosis or not with the hope of offering some clue of the therapeutic tools on multiple myeloma.Methods1.Experimental materialMyeloma cells XG and RPMI82262.Experimental methodsCell culture:The two cells use the conventional culture adding rhIL-6 of 5ng/ml to XG cells and exchange and passage once 3-4 days,and use the cells in log page growth in experiment.Setting the concentration of drug:According to the preliminary experiment and literature repot,we set the concentration of Ubenimex in MTT test with 30ug/ml,125ug/ml,500ug/ml,2000ug/ml and 1000ug/ml in both TUNEL and FCM.The detection of cell growth with MTT:The log phase growth cells were modulated to 1*10⁵/ml after fresh culture solution washing and were inoculated in 96-bore culture plank with 100ul per bore.At the same time,100ul drug of different concentration was also inoculated and each concentration for six bores with the isochoric RPMI1640 in blank control and the isochoric PBS in surrounding bores for decreasing the edge effect.Then put the plank in the cell culture incubator with 37℃,saturated humidity and 5%CO₂ for 24,48,72 hours.After adding 20ul MTT in each bore coculture for 4 hours,efference for 10 minutes and deduct soare fluid and add 150ul DMSO.Shake the plank to misce bene the pept and detect with the appearance of immunodetection and read the OD of 490nm to compute the inhibition ratio.The detection of apoptosis:TUNEL:Work as the description.Set the XG cells without Ubenimex as control and take a photograph after viewing with the microscope.There was some flava grains in the apoptosis cells after in situ end-labeling but not in the normal cells.Count the cells in three 400^* campus visualis and compute the apoptosis ratio.FCM:Collect 1*10⁶ RPMI8226 cells both control and experiment after 1000ug/ml Ubenimex effecting 48 hours.Add 1ml 70%alcohol for fication to stay overnight after washing twice with cold PBS.Then deduct spare fluid after washing twice with cold PBS and add 1ml PI for 30 minutes on 4℃and detect with FCM.Statistics analysis was performed using SPSS12.0 computer software.Result1.The growth inhibitory action of Ubenimex on myeloma cellsUbenimex had a growth inhibitory action on XG cells and RPMI8226 cells.There was different growth inhibitory action both ong different concentration and different time.Ubenimex of 30ug/ml had a patency growth inhibitory action on XG cells,compared with control group,t=8.73,P<0.01,and the IC₅₀of 24 hours is 1729.8ug/ml,48 hours 957.2ug/ml,72 hours 603.7ug/ml.But the growth inhibitory action on RPMI8226 was obviously weaker than XG.Ubenimex of 30ug/ml had a patency growth inhibitory action on RPMI8226 cells,compared with control group, t=23.28,P<0.01,and the IC₅₀of 24 hours is 2454.7ug/ml,48 hours 1636.8ug/ml,72 hours 1349.0ug/ml.The inhibitory ratio rised with time extended and concentration increased.Both inhibitory action had dose and time-dependent relation.2.The detection of apoptosis(1)The change of cell morphous and the apoptosis cells detected by DNA-frag in situ end-labeling:The cell nucleus of control group without Ubenimex was big and complete,the endochylema was full,the cell membrane was continuous and complete.But the cells changed to contract,caryoplasm pyknosis,caryotheca and chromatospherite chipped and dissolved,endochylema concentrated and appeared to typical apoptotic body after 1000ug/ml Ubenimex effected for 48 hours.(2)The effect of cell cycle with Ubenimex:In the DNA content figure,there was an apoptotic peak before G1-stage.The cells were blocked down on G1-stage manifested as the rate of S and G2-stage decreased,which was showed that the blockage on G1-stage was the main reason of the apoptosis of myeloma cells induced by Ubenimex.DiscussionThe biological activity of Ubenimex observed early was the inhibitory action on leucine aminopeptidase and aminopeptidase B.N.on cellular membrane,which modified the immunocell and elevated the immune function and manifested an antitumous effect on several transferred tumors through intermediate of host.Subsequent study found that Ubenimex also can inhibit several tumor cells growth and enhance apoptosis without rhagiocrine cell and T cell.The result of this experiment manifested that the inhibitory action of Ubenimex on XG cells and RPMI8226 cells had dose and time-dependent relation.Ubenimex of 30ug/ml had a patency growth inhibitory action on XG cells,compared with control group,t=8.73,P<0.01,and the IC₅₀of 24 hours is 1729.8ug/ml,48 hours 957.2ug/ml,72 hours 603.7ug/ml.But the growth inhibitory action on RPMI8226 was obviously weaker than XG.Ubenimex of 30ug/ml had a patency growth inhibitory action on RPMI8226 cells,compared with control group,t=23.28,P<0.01,and the IC₅₀of 24 hours is 2454.7ug/ml,48 hours 1636.8ug/ml,72 hours 1349.0ug/ml.Kazuhiko had found that Ubenimex had growth inhibitory action on chorionic carcinoma cells,which had direct correlation with the expression of aminopeptidase on cellular membrane.Therefore,they presumed that Ubenimex reacts through inhibiting the activity of aminopeptidase.We had also found that the sensibility of Ubenimex on XG cells was harder than RPMI8226 cells.The growth inhibitory action may have some dependability with the expression of aminopeptidase.The expression of CD13 on the two cells surface need further detection,Hitherto,cellular morphology is still a basic and capital technology to discriminate spoptosis and necrosis.We found that the myeloma cells had a typical change on morphology of apoptosis and the method of TUNEL had also detected the positive apoptotic cells.In the DNA content figue,there was an apoptotic peak before G1-stage,which showed that Ubenimex had a growth inhibitory action and can induce apoptosis.Therefore,inducing apoptosis was one of the most important antineoplastic mechanism of Ubenimex.The mechanism of inducing apoptosis of Ubenimex is still not clear.The analysis of the cell cycle of the myeloma cells effected with Ubenimex showed that the cells were blocked down on G1-stage,which is showed that the blockage on G1-stage is the main reason of the apoptosis of myeloma cells induced by Ubenimex.The pathway of inducing apoptosis and the signal transmission and control of Ubenimex need further study and research.ConclusionUbenimex can obviously inhibit the growth of XG cells and RPMI8226 cells,and the growth inhibitory action has dose and time-dependent relation.The XG cells have a typical change on morphology of apoptosis and the method of TUNEL have also detected the positive apoptosis cells.There is an apoptotic peak before G1-stage in the DNA content figure of RPMI8226 detected by FCM.These all confirm that Ubenimex can induce myeloma cells apoptosis.