The Effects Of Bortezomib And Its Combination With Other Chemotherapy Drugs On Cells Of The Cell Line KM₃ From Multiple Myeloma In Vitro

Multiple myeloma(MM)is a clonal plasma cell disorder.It is characterized by the proliferation and the accumulation of malignant plasma cells predominantly in the bone marrow,secretion of a monoclonal protein and normal immunoglobulins secretion inhibited,resulting in widespread destructive bone lesions,infection,hemorrhage,anemia,renal insufficiency,hyperviscosity syndrome and so on.Although conventional chemotherapy and autologous stem cell transplantation have resulted in prolongation of survival,most patients eventually relapse and are refractory.MM is incurable.The ubiquitin-proteasome pathway(UPP)is an important intracellular proteolytic system which regulates the degradation of a broad spectrum of intracellular proteins in eukaryotic cells.Blocking UPP is a hot spot of tumor targeted therapy.Proteasome inhibitor bortezomib(commodity name velcade)is representative of a class of peptide boronate proteasome inhibitors that highly selected inhibit 26S proteasome activity,induces marked antitumor activity against humam tumor cell lines included MM clinically.It is reported that a combination of bortezomib plus dexamethasone,bortezomib plus dexamethasone plus adriamycin have revealed remarkable antitumor activity on MM patients,complete remission and near complete remission were 30%,improved remission rate and the life quality of patients.At present,there were more reports on bortezomib treated refractory myeloma but few reports on basic reseaech.The aim of this paper is to investigate the effects of bortezomib and its combination with dexamethasone,daunorubicin,especially with arsenic trioxide (As₂O₃)on cells of the cell line KM₃ from multiple myeloma in vitro,make an inquiry into its effect mechanism initially,hope to provide reliable basis for clinical utilization,so as to develop new treatment scheme for clinical.Using MTT assay we investigated the effect of bortezomib,As₂O₃,dexamethasone,daunorubicin on KM₃ cell growth.The result showed that KM₃ cell growth was inhibited by bortezomib,As₂O₃,dexamethasone,daunorubicin significantly,in comparison with the blank group(P＜0.05),this effect is in dose-dependent fashion,the proliferation of KM₃ cells was inhibited by bortezomib,As₂O₃,dexamethasone in a time-dependent fashion.when the concentration of bortezomib was 0.1μmol/L,the inhibition rate was 18.61%and concentration 5.0μmol/L,the inhibition rate 80.87%.The IC₅₀of bortezomib was 0.27μmol/L,only little lower than daunorubicin(0.16μmol/L),but much lower than dexamethasone(8.01μmol/L)and As₂O₃(3.10μmol/L).The results of the research confirmed that the proliferation of cells of the cell line KM₃ from multiple myeloma was inhibited strongly by bortezomib.It was worth to notice that the inhibiting effect of As₂O₃ was stronger than dexamethasone's.In the research,the inhibiting rate on KM₃ cells of bortezomib puls daunorubicin was much higher than bortezomib's(13.18±1.29%vs 46.93±5.33%,P＜0.05=.The inhibiting rate on KM₃ cells of bortezomib puls dexamethasone plus As₂O₃ was much higher than bortezomib plus dexamethasone's or As₂O₃'s(34.51±0.51%vs 23.80±0.78%;34.51±0.51% vs25.39±0.90%).Bortezomib puls daunorubicin was the strongest inhibiting agent in the all combinations.It suggested that bortezomib and daunorubicin had synergy,bortezomib may have something to do with enhancing sensitivity.These results were worth to consideration in designing clinical treatment programmes.To explore the possible mechanism of bortezomib on KM₃ cells,further investigation was carried out.The morphology of KM₃ cells was observed with or without bortezomib treatment under a light microscope(using Wright-Giemsa stain)and electric microscope.The result displayed that a series of typical morphological features of apoptosis were observed follow treatment with bortezomib,such as vacuolization of cell plasma,shrinkage of cell and nucleus,condensation of nuclear chromatin,marginated against the nuclear membranes,karyorrhexis and convolution of nuclear,and mambraned-bound apoptosis bodies(Fig5,6).In order to exactly analyze the cell apoptosis quantitatively and qualitatively,the agarose gel electrophoresis and the flow cytometry were used to evaluate the DNA content and translocation of phosphatidyl serine(PS).It showed that following treatment with 0.25μmol/L bortezomib for 24h,a typical DNA ladder was observed.With the help of flow cytometry follow annexinV-PI staining,exposured to 0.125μmol/L-0.25μmol/Lbortezomib caused KM₃ cells apoptosis.To explore the possible mechanism of apoptosis of bortezomib,the flow cytometry were used to detect mitochondria membrane potential(ΔΨm).It showed that following treatment with 0.125μmol/L-0.5μmol/L bortezomib causedΔΨm reduced.To sm up,the effects of bortezomib,As₂O₃,dexamethasone,daunorubicin on MM cell line KM₃ are summarized as follows.(1)Bortezomib,As₂O₃,dexamethasone,daunorubicin can inhibit the growth of MM cell line KM₃ cells in dose and time-dependent fasion,their IC₅₀were 0.27μmol/L,3.10μmol/L,8.01μmol/L,0.16μmol/L respectively.It was demonstrated that the inhibiting effect on MM cells of bortezomib was lower than daunorubicin's,the inhibiting effect of As₂O₃ was higher than dexamethasone's.(2)Comparing with bortezomib,its combination with daunorubicin had the highest inhibiting rate on cells proliferation all the time.(3)The inhibiting rate of bortezomib puls As₂O₃ puls dexamethasone was higher than bortezomib puls dexamethasone's all the time.It suggested that added As₂O₃ to bortezomib puls dexamethasone may help to improve survival. (4)Treatment with 0.125μmol/L-0.25μmol/L bortezomib for some time can induce KM₃ cells apoptosis in dose-dependent manners.Its combination with other drugs resulted in synergy.The apoptosis rate of bortezomib puls As₂O₃ puls dexamethasone was higher than bortezomib puls dexamethasone's(60.13±2.51%vs 48.66±4.31%,P＜0.05).(5)Treatment with 0.125μmol/L-0.5μmol/L bortezomib for some time can reduce mitochondria membrane potential of KM₃ cells.