| Background and objectivesAlthough the therapeutic efficacy of acute leukemia has been greatly improved in the recent years, 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 was found protein degradation in eukaryotic cells mostly through the ubiquitin-proteasome pathway to complete, in human leukemia cells contain abnormally high levels of proteasome activity, and in the induction of terminal differentiation of leukemia cells when the protease body was quickly lowered, indicating that proteasome activity and malignant proliferation of leukemic cells is closely related, the proteasome may be a new target for leukemia treatment. The proteasome plays an essential role in the targeted degradation of many 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 ADRinistration (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 As2O3, the effect has been abviously improved. In this study, we assume to investigate the effection and mechanism of bortezomib in combination with arsenic trioxide on reverse multidrug resistance in HL60/ADR leukemia cells.MethodsHL60/ADR 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/ADR 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/ADR 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/ADR leukemia cells were detected by fluorescence microscopy (hoechst33342) and flow cytometry (AnnexinV-EGFP/PI). Intracellular concentration of Adriamycin(ADR) was determined by flow cytometry. And we used Western blot melthod detecting the expressions of IκBα,p65,bcl-2,bax,caspase-3,caspase-9,PARP proteins and RT-PCR detecting the expressions of PDCD7 and APP gene. We determine the optimal time of culture is 48h and use 0～40μM As2O3 to treat HL60/ADR leukemia cells for 48h respectively. The half maximal inhibitory concentration (IC50) of As2O3 was confirmed then(15μM). Bortezomib combined with IC50 concentration of As2O3 treating, the proliferation and apoptosis changes of HL60/ADR leukemia cells were detected by the same methods above. The differences of effect between groups were done by statistics anylysis.Results1. The multidrug resistance of HL60/ADR leukemia cellsFrom the ADR toxicity experiment which was detected by MTT assay, we found that the IC50 of ADR to HL60/ADR cell approximately is 16.223μg/ml, while the IC50 of ADR to HL60 is 0.091μg/ml; HL60/ADR is highly multidrug resistance(178.27 times).2. Effect of bortezomib on the multidrug resistance of HL60/ADR cellsCultured with 5nM bortezomib (24h cell surviving rate≥95% at this concentration), the 24h IC50 ADR of HL60/ADR cell dropped to 8.249μg/ml, almost nearly a half of primrate IC50, the difference was significant (P=0.005).3. Effect of bortezomib, arsenic trioxide or their combination on the proliferation of HL60/ADR cellsMTT assays showed that bortezomib can inhibited the proliferation of HL60/ADR cells. In 10～50nM bortezomib treated cells, inhibition rates enhanced in a time- and dose-dependently manner.50nM bortezomib showed the best efficiency to inhibit the proliferation of HL60/ADR cells when incubated for 48 hours. The 48h IC50 dose of bortezomib in HL60/ADR cells was 21.6nM. When we prolonged the incubated time to 60h, the inhibition rates had no significant enhancement (P=0.161). As2O3 could inhibit HL60/ADR cell proliferation independently. When cultured for 48 hours,2,4,10,20,30,40μM As2O3 brought inhibition rates of19.1%,29.7 %,36.1%,56.2%,63.5%,69.4%, respectively, with the IC50 dose as15μM. When cultured for 48 hours. 15μM As2O3 combined with 10-50nM of bortezomib could inhibit the proliferation of HL60/ADR, and the dual inhibition of bortezomib plus As2O3 was significant higher then that of either above. Fluorescence microscope showed that the apoptotic cells in both compounds combined groups were more than those in the single-agent groups.4. Effect of bortezomib, arsenic trioxide or their combination on apoptosis of HL60/ADR cellsNormal 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. There were more apoptosis cells with the increasing of concentration of botezomib, or at the combination of bortezomib and arsenic trioxide.After detecting the apoptosis of HL60/ADR cells with flow cytometry, there were apoptotic cells in lOnM or 20nM bortezomib treating groups.10nM bortezomib treating for 24h,48h provided a apoptosis rate 6.07%,9.24%.While 20nM treating group with 16.80% and 38.04% apoptosis rate respectively, which is significant higher than lOnM group and control group. As2O3 alone can also inhibit the proliferation of HL60/ADR. 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 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 ADR of HL60/ADR cells in the botezomib group is stronger than the bortezomib-free group(P< 0.05).10nM Bortezomib could significantly enhanced the intracellular accumulation of ADR in HL60/ADR cells which indicated that bortezomib might have the ability to reverse multidrug resistant.5. Effect of brotezomib, arsenic trioxide or their combination on the accumulation of ADRBortezomib can increase accumulation of ADR in HL60/ADR cells. 10nM,20nM bortezomib,15μM As2O3, and 10nM bortezomib,15μM As2O3 combination 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. while the inhibiton rate of 20nM bortezomib combined with 15μM As2O3 is significantly higher then those of either treating alone.(P<0.05)6. Effect of brotezomib, arsenic trioxide or their combination on the expression of MRP1Bortezomib can decrease the expression of MRP1 of HL60/ADR cells. After treating with 10nM,20nM bortezomib for 24h, the expression of MRP1 of HL60/ADR cells decreased from 94.82% to 92.47%,89.87%. When it's time to 48h, the expression of MRP1 decreased to 77.41%,66.09%. After treating with 15μM As2O3 for 24h and 48h, the expression of MRP1 of HL60/ADR cells was 91.36%, 72.58%,which lower significantly to control group. The expression of MRP1 of 10nM bortezomib and 15μM As2O3 combination group was significantly lower than those of either treating alone.(P<0.05) 7. Effect of bortezomib, arsenic trioxide or their combination on the expression of IκBα,p65 in HL60/ADR cellsThe expression of IKB-αin HL60/ADR cells treating with bortezomib for 48 hours was non-significant different with control group(P>0.05). While the expression of p65 in bortezomib group decreased significantly(P<0.05). After treating with 15μM arsenic trioxide, the expression of IKB-a decreased significantly, which was lower than combination group. The expression of p65 in combination group was much lower than that in group treating with bortezomib or arsenic trioxide alone.8. Effect of bortezomib, arsenic trioxide or their combination on the expression of bcl-2/bax,caspase cell signaling pathway in HL60/ADR cellsThe expression of bax which is pro-apoptosis in HL60/ADR cells treating with bortezomib for 48 hours was significantly increased(P<0.05). At the same time, the expression of bcl-2 which is anti- apoptosis decreased significantly(P<0.05). Compared to control, the expression of cleaved caspase-3, caspase-9 and PARP protein which are all involved in caspase cell signaling pathway were also increased significantly after treating with bortezomib,arsenic trioxide or their combination(P< 0.05). And the expression of these proteins in combination groups was significant than that of each drug alone group except for bax.Conclusiones1. Bortezomib has a dose- and time- dependent antiproliferation and proapoptotic effect on H60/ADR leukemia cells in vitro. With prolongation of time and increase of concentration of bortezomib, the effect has gradually increased.2. As2O3 also have a dose dependent antiproliferation and proapoptic effects on HL60/ADR 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 ADR in HL60/ADR cells which indicated that bortezomib might have the ability to reverse multidrug resistant.4. Bortezomib and As2O3 have a dose-dependent antiproliferation effect on relapsed/refractory AML primary cells in vitro. And the effect was obviously inferior to HL60/ADR leukemia cells.5. Bortezomib in combination with As2O3 has antiproliferation on relapsed/refractory AML primary cells in vitro. Compared with the effect of which drugs used alone, the effect of bortezomib in combination As2O3 group is superior to the effects of either group which drugs used alone. |
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