The Study Of Bortezomib In Combination With Harringtonine Or Arsenic Trioxide On Proliferation And Apoptosis Of Acute Myeloid Leukemia Cells

Background and objective:Acute leukemia is a group of malignant clonal diseases originated fromhematopoietic stem cell disorder. With the high dose chemotherapy and synthetictherapy being widely used, the therapeutic efficacy of acute leukemia has beengreatly improved in the recent years, but there are still many patients converted intorefractory because of relapse or primary resistance. So it is necessary to look for newstrategies to cure this disease nowadays.With the great success of imatinib on treatment of CML, the research ofmolecular targeted drugs has gradually become a new heated topic in tumor therapyand among them the research of proteasome inhibitor is the most remarkable one. Ithas been documented that proteasome is a multicatalytic enzyme complex which isfounded in all eukaryotic cells. The proteasome has many important functions,including intracellular proteins degrading, MHC-â… antigen presenting and misfoldproteins repairing. Many proteins which participated in cell cycle regulation, cell signal transduction or cell apoptosis were degraded by proteasome. So if we inhibitthe function of proteasome, the level of these intracellular proteins will bedistinguished changed, which may induce cell apoptosis. As the first proteasomeinhibitor which has been approved by FDA, bortezomib (Bor) has demonstratedgreat success in patient with refractory or relapsed multiple myeloma. On the otherside of malignant hematological disease or solid tumors, many documents havereported that bortezomib alone or in combination with other chemical drugs has afavorable effect on Non-Hodgkin's lymphoma (NHL), Chronic LymphocyticLeukemia (CLL), adult T-cell leukemia, lung cancer and ovarian cancer. Up to now,the most research of bortezomib in the world has been limited in NHL, CLL andMM. There are seldom reports about the effect of bortezomib on acute leukemia,especially in acute myeloid leukemia, which is high incidence of intemal. So weassume to investigate the effect of bortezomib alone or in combination with HT orAs₂O₃, which were common used in leukemia therapy in intemal, on AML cells.Hopefully, our research will help us to look for a new method in AML therapy area.MethodsHL60 leukemia cells were selected in this study and were cultured in theRPMI-1640 medium supplemented with 10% calf serum. In order to determine thebest concentration and time of experiment. 0～1000nM bortezomib was used totreat cells for 0～48 hours in vitro and the proliferation activity of HL60 leukemiacells was detected by trypan blue dye exclusion method. According to the result, wechoose 0～50nM bortezomib as the experiment concentration. The change of cellactivity after treated by bortezomib was analyzed by MTT colorimetric assay andapoptosis of HL60 leukemia cells were detected by DNA gel electrophoresis,fluorescence microscope and flow cytometry. At the same. time, we determine the optimal time of culture is 24 hours and use 7.5n～75nM HT and 5～50μM As₂O₃ totreat HL60 leukemia cells for 24 hours, respectively. The half maximal inhibitoryconcentration (IC₅₀) of HT and As₂O₃ to the HL60 leukemia cells was based on theeffect which their used alone. Bortezomib combined with the IC₅₀ concentration ofHT or As₂O₃ treating, the proliferation and apoptosis changes of HL60 leukemiacells and primary leukemia cells which come from 8 AML patients were detected bythe same methods. The differences of effect between combination or not were doneby statistics analysis.Results1. Effect of bortezomib on the proliferation and apoptosis of HL60 leukemiacellsThe number of HL60 leukemia cells was no significant difference when 0～40nM bortezomib treating them for 6 hours detected by trypan blue dye exclusionmethod. Bortezomib incubating 12 hours, the number of HL60 leukemia cells indifferent concentration has gradually decreased in a dose-and-time dependentmanner. 30nM bortezomib can basic inhibit the proliferation of HL60 leukemia cellswhen it incubate them for 24 hours. MTT colorimetric assay detected, theproliferation activity of HL60 leukemia cells has gradually decreased in adose-and-time dependent manner when bortezomib treated them for 12～48 hours.10～50nM concentration treating 12 hours, the cell viability has decreased to 82%,75%,60%,50% and 40%, respectively. While when the culture time has prolongedto 24 hours, the cell viability has decreased to 77%,46%,31%,24% and 23%,respectively. Compared with the effect of bortezomib in 12 hours, the difference ofcell viability in every concentration between 12 hour and 24 hours has a statisticalsignificance(P＜0.05). However, there was no obviously different between the effect of bortezomib in 24 hours and 48 hours, (P＞0.05). The DNA Ladder can be observedwhen 10-50nM bortezomib treat cells for 12h by means of DNA gel electrophoresismethod, meanwhile accompany the DNA Ladder, the genome band can also beobserved in the 10nM bortezomib lane. Bortezomib culturing 24h, the DNA Ladderstill exist in every lane, but the genome band in 10nM concentration lane disappear.Fluorescence microscope detected, 10～50nM bortezomib can induce HL60leukemia cells apoptosis and the apoptosis cells in the high dose group are more thanthat in the low dose group at each visual fields. The rates of subGl phase cell were5.9% and 59% when 10nM and 30nM bortezomib treated HL60 leukemia cells for12h, detected by flow cytometry. Compared with the result of control, there was asignificant difference. When the culture time has prolonged to 24 hours, the rates ofsubG₁ phase cell has no change in control group, while the rates in 10nM and 30nMgroup have increased to 19% and 62%, respectively. Compared with their effect in12 hours, there was a significant difference between the effect of 10nM in 12 hoursand 24 hours, P＜0.05, but the effect of 30nM has not influenced by thetime(59%:62%).2. Effect of bortezomib in combination with harringtonine or arsenic trioxideon the proliferation and apoptosis of HL60 leukemia cellsHT and As₂O₃ also have an antiproliferative effect on HL60 leukemia cells in adoes-dependent manner, the half maximal inhibitory concentration (IC₅₀) of HT andAs₂O₃ to HL60 leukemia cells were 30nM and 15μM, respectively. Before themaximum inhibitory effect of treatment, the proliferation activity of 10nMbortezomib has obviously decreased to 40% and 33%, when 10nM bortezomibcombined with 30nM HT or 15μM As₂O₃. Compared with the effect of their usedalone, the difference has significant meaning, (P＜0.05). Fluorescence microscopedetected, HT and AS₂O₃ can induce HL60 leukemia cells apoptosis in a dose dependent manner and apoptosis Cells in bortezomib combined with HT or As₂O₃groups are more markedly increased than that in the bortezomib, HT or As₂O₃group. Flow cytometry detected, the rates of subG1 phase cell were 11% and 19%when 30nM HT and 15μM As₂O₃ treated HL60 leukemia cells for 24 hours. Comparedwith the effect of control group, there was a significant difference (P＜0.05). In thesame condition, the rates of subG1 phase cell has increased to 44% and 47% when10nM bortezomib in combination with 30nM HT or 15μM As₂O₃.Compared with theeffect that those drugs used alone, the difference also has a statistical significance(P＜0.05)3. Effect of bortezomib in combination with harringtonine or arsenic trioxideon the proliferation of primary leukemia cellsResults of bone marrow mononuclear cells which come from 8 patients beforetreatment have displayed: 10～500nM bortezomib has a dose dependentantiproliferative effect on acute myeloid leukemia primary cells. With the increase ofconcentration, the proliferation activity of primary leukemia cells has increasedgradually. However, except 500nM concentration, the difference of proliferationactivity between 10～100nM concentration has no statistical significance. HT andAs₂O₃ also have a dose dependent antiproliferative effect on primary leukemia cells.But the activity of primary leukemia cells was much higher than that of HL60leukemia cells when 30nM HT treated each of them for 24 hours. While, the similareffect was not found when 15μM As₂O₃ treated each cells for the same time.Compared with the effect of those drugs used alone, the cell activity of primaryleukemia cells decreased, when 20nM bortezomib combined with 30nM HT or 15μMAs₂O₃ treated for 24 hours. Though there is no statistical significance between theeffect of combination or not, the effect of bortezomib in combination with As₂O₃group is superior to the effect of As₂O₃ group in fact(0.51: 0.61,P=0.07). Conclusions1. Bortezomib has a dose-and-time dependent antiproliferative and proapoptoticeffects on HL60 leukemia cells in vitro. Along with prolongation of the time ofdrug action and increase of its concentration, the effect has gradually increased.2. HT and As₂O₃ also have a dose dependent antiproliferative and proapoptoticeffects on HL60 leukemia cells in vitro. But compared with the effect whichdrugs used alone, the antiproliferative and proapoptosis effect has been obviouslyimproved when bortezomib in combination with the IC₅₀ concentration of HT orAs₂O₃.3. Bortezomib, HT and As₂O₃ also have a dose dependent antiproliferative effect onacute myeloid leukemia primary cells in vitro. While the effect ofbortezomib orHT on primary leukemia cells was obviously inferior to HL60 leukemia cells.But the effect of As₂O₃ on HL60 leukemia cells or acute myeloid primaryleukemia cells has no significant difference.4. Bortezomib in combination with HT or As₂O₃ has an antiproliferative effect onacute myeloid leukemia primary cells in vitro. Compared with the effect whichdrugs used alone, the effect of bortezomib in combination with HT group has noimproved, but the effect of bortezomib in combination with As₂O₃ group maybesuperior to the effect of As₂O₃ group(P=0.07).