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

Background and objectivesAlthough the therapeutic efficacy of acute leukemia has been greatly improved in the recent years, but there are still up to 15～30% patients present original drug resistance. And 60～80% relapsed after the first CR, some of them die soon after relapsed.With great progress in Molecular Biology, it has been found that the maintenance of homeostasis and the ability of cells to respond to their environment depend on the orderly degradation of key regulatory proteins and their inhibitors. The proteasome plays an essential role in the targeted degradation of such proteins and is therefore involved in the activation and inactivation of many cellular processes. In fact, studies using proteasome inhibitors have shown that the proteasome is responsible for the elimination of more than 80% of all cellular proteins. Specific targets of proteasome include cell-cycle proteins, tumor suppressors, and transcription factors, as well as mutant and damaged proteins. The proteasome has been identified as an excellent target for cancer therapy because of its critical metabolic function. In 2003, bortezomib became first-in-class proteasome inhibitor that received approval from the U.S. Food and Drug Administration (FDA) for the treatment of multiple myeloma patients who have received at least two prior therapies and have demonstrated disease progression on the last therapy. However, bortezomib has demonstrated significant activity against a broad range of human tumor cells. In the earlier stage of our research, we found that bortezomib has a dose- and time- dependent antiproliferation and proapoptotic effect on HL60 leukemia cells in vitro, when combined with IC₅₀ HT or IC₅₀ As₂O₃, the effect has been abviously improved. In this study, we assume to investigate the effect of bortezomib in combination with harringtonine or arsenic trioxide on proliferation and apoptosis of multidrug resistant leukemia cells.MethodsHL60/ADM leukemia cells were selected in this study and were cultured in the RPMI-1640 medium supplemented with 10% calf serum. First of all we design a MTT assay experiment to ensure that HL60/ADM is highly multidrug resistant(compare with HL60, with at least 30 multiples). Then , in order to determine the best concentration and time of experiment, 0-1000nM bortezomib was used to treat cells for 0-72h in vitro and proliferation activity of HL60/ADM leukemia cells was detected by trypan blue dye exclusion method. We chose 0-50nM bortezomib as the experiment concentration. The change of cell activity after treated by bortezomib was analyzed by MTT assay and apoptosis of HL60/ADM leukemia cells were detected by by fluorescence microscopy (hoechst33342) and flow cytometry (AnnexinV-FITC/PI). Intracellular concentration of Adriamycin(ADM) was determined by flow cytometry. We determine the optimal time of culture is 48h and use 0-752nM HT and 0-40μM As₂O₃ to treat HL60/ADM leukemia cells for 48h respectively. The half maximal inhibitory concentration (IC₅₀) of HT and As₂O₃ was confirmed then(752nM and 15μM). Bortezomib combined with IC₅₀ concentration of HT or As₂O₃ treating, the proliferation and apoptosis changes of HL60/ADM leukemia cells and primary cells which come from 9 patients were detected by the same methods above. The differences of effect between groups were done by statistics anylysis.Results1. The multidrug resistance of HL60/ADM leukemia cellsFrom the ADM toxicity experiment which was detected by MTT assay, we found that the IC₅₀ of ADM to HL60/ADM is 16.283μg/ml, while the IC₅₀ of ADM to HL60 is 0.087μg/ml; HL60/ADM is highly multidrug resistance (187 times).2. Effect of bortezomib alone on the proliferation and apoptosis of HL60/ADM cellsDates from trypan blue dye exclusion method reveal that there is no significant difference between 0-50nM bortezomib treating for 12 hours(P>0.05). after 24 h, the number of cells in different concentration has gradually decreased in a dose- and time- manner. Dates from MTT assays show that In 10-50nM bortezomib treated cells, inhibition rates enhanced in a time- and dose-dependently manner , 40nM bortezomib can best inhibit the proliferation activity of HL60/ADM cells when incubated for 48 hours, with the IC50 dose 20nM. When we raised the dose to 50nM or prolonged the incubated time to 60 h, the inhibition rates had no significant enhancement (P=0.478, P=0.161); Normal cells and apoptotic cells could be distinguished under the fluorescence microscope after staining with Hochest 33342. Normal nuclei kept intact and stained evenly. Apoptotic cells show strong chromatin condensation and nuclear fragmentation. apoptosis cells shows in 10nM,20nM or 50nM, and increased in dose maner.After detecting the apoptosis of HL60/ADM cells with flow cytometry, there were apoptotic cells in 10nM or 20nM bortezomib treating groups.10nM bortezomib treating for 24h provide a apoptosis rate 9.58%,while 20nM treating group with 16.45% apoptosis rate which is significant higher then 10nM group. Prolong the treating time to 48h, the apoptosis rate in 10nM or 20nM group increased to 17.22% and 73.46%, respectively, which were significant higher then those in the same concentration of 24h.3. Effect of bortezomib in combination with harringtonine or arsenic trioxide on the proliferation and apoptosis of HL60/ADM cellsAs₂O₃ alone can also inhibit the proliferation of HL60/ADM. When cultured for 48 hours, 2,4,10,20,30,40μM As₂O₃ brought inhibition rates of 25%,33%,40% > 58% , 64% , 69%, respectively, with the IC50 dose as 15μM. When cultured for 48 hours, 94,188,376,564,752 nM HT brought inhibition rates of 9%,20%,31 %,39%,52%, respectively, with the IC₅₀ dose as 752nM. 15μM As₂O₃ or 752nM HT which is combined with 10-50nM of bortezomib can inhibit the proliferation of HL60/ADM, and the dual inhibition of bortezomib plus As₂O₃ is significant higher then that of either above. While there is no different between bortezomib plus HT group and HT treating group. fluorescence microscope show that The apoptotic cells in both compounds combined groups are more than those in the single-agent groups. flow cytometry show that there were apoptotic cells in all groups. After they were cultured for 24 or 48 hours, the apoptotic rates of the agents combined groups were higher than the single-agent groups. After the cells had been cultured for 48 hours, the apoptotic rates of the bortezomib plus As₂O₃ , bortezomib plus HT group and the bortezomib group were 81.89% , 80.64% and 17.22%(P<0.01), respectively. And mostly, apoptosis in the combined group present in a late-stage. The intraceflular accumulation of ADM of HL60/ADM cells in the botezomib group is stronger than the bortezomib-free group(P<0.05 ).10nM Bortezomib could significantly enhance the intracellular accumulation of ADM in HL60/ADM cells which indicated that bortezomib might have the ability to reverse multidrug resistant.4. Effect of brotezomib in combination with harringtonine or arsenic trioxide on the proliferation of primary leukemia cells10-200nM bortezomib can inhibit proliferation of primary cells, inhibition rates increased in a dose-dependently manner; 10nM,20nM,40nM,80nM,100nM,200nM bortezomib treating for 48h provide inhibition rates as 8.7%,18.1%,26.8 %,39.2%,48.06%,51.44%, respectively. 100nM bortezomib could best inhibit the proliferation activity. Higher doses made no significant increase of the inhibition rate. 20nM bortezomib combined with 752nM HT treating for 48h, make no difference with either of above treating alone(P>0.05). while the inhibiton rate of 20nM bortezomib combined with 15μM As₂O₃ is significantly higher then those of either treating alone.(P<0.05)Conclusiones1. Bortezomib has a dose- and time- dependent antiproliferation and proapoptotic effect on H60/ADM leukemia cells in vitro. With prolongation of time and increase of concentration of bortezomib, the effect has gradually increased.2. HT and As₂O₃ also have a dose dependent antiproliferation and proapoptic effects on HL60/ADM cells, while combined with bortezomib, the effect is obviously improved when compared with bortezomib used alone.3. Bortezomib could significantly enhance the intracellular accumulation of ADM in HL60/ADM cells which indicated that bortezomib might have the ability to reverse multidrug resistant.4. Bortezomib,HT and As₂O₃ have a dose-dependent antiproliferation effect on relapsed/refractory AML primary cells in vitro. And the effect was obviously inferior to HL60/ADM leukemia cells.5. Bortezomib in combination with HT or As₂O₃ has antiproliferation on relapsed/refractory AML primary cells in vitro. Compared with the effect of which drugs used alone, the effect of bortezomib with HT group has no improved, while the effect of bortezomib in combination As₂O₃ group is superior to the effects of either group which drugs used alone.