| OBJECTIVE:The multidrug resistance(MDR) of leukemia cells is always accompanied with the apoptosis resistance phenomenon, and leukemia stem cells (LSC) play an important role in drug resistance and relapse of leukemias. In this study, we will investigate and compare the apoptosis-inducing effect of realgar nanoparticle (nano-realgar) on drug-sensitive and drug-resistant leukemia cells and their leukemia stem cells, and explore the anti-leukemia efficacy and mechanism of nano-realgar to drug-resistant leukemia cells and leukemia stem cells.METHODS:The nano-realgar preparation was made by use of mechanical polishing method. The multidrug-resistant leukemia K562/ADM cell and its parental drug-sensitive K562 cell were used as the model cells. MTT assay was used to detect the proliferating activity of K562 cells and K562/ADM cells, and the cellular apoptosis was investigated with double staining of FITC-Annexin V and propidium iodide (PI) by flow cytometry. Flow cytometry (FCM) was employed to detect expression of intracellular Bax, Bcl-2, P-53 protein and the activity of Caspase-3. The content of LSC and the expression of P-gp and BCRP were analyzed by using FCM.RESULTS:The raw realgar was made to ultra-fine powder by ball milling, and the average diameter of the nanoparticle was (72.72±22.18) nm measured with electron microscopes. Nano-realgar significantly inhibited the proliferation of K562 cells and K562/ADM cells. Treated for 24,48 and 72 hours, the 50% inhibitory concentration (IC50) was 43.48μg/ml.20.52μg/ml,16.07μg/ml for K562 cells, and 56.53μg/ml, 35.19μg/ml and 24.75μg/ml for K562/ADM cells, respectively. After exposure to 20μg/ml and 50μg/ml nano-realgar for 48 hours, the apoptosis of K562/ADM cells and K562 cells detected by Annexin V/PI staining was increased, the apoptotic rate of K562/ADM cells was 13.35% and 72.70%, and that of K562 cells was 10.52% and 73.25%, respectively. Realgar in a time-dose-dependent manner inhibited K562 cells and K562/ADM cell proliferation and induced target cells to undergo apoptosis, but both of realgar was lower than the nano-realgar. Treated K562 cells and K562/ADM cells with realgar for 24h,48h and 72h, IC50 values of K562 cells were 36.51μg/ml,33.26μg/ml,29.59μg/ml, and that of K562/ADM cells were 54.17μg/ml,21.76μg/ml,40.78μg/ml. Treated cells with 20ug/ml and 50ug/ml realgar for 48h, AnnexinV/PI staining showed the apoptosis-rate significantly increased, and the apoptotic rate of K562/ADM cells was 3.82% and 20.04%, and that of K562 cells was 9.12% and 14.28%, respectively. After the target cells were treated with 20μg/ml and 50μg/ml nano-realgar for 24h and 48h, the expression of P53,Bax,Bcl-2 markedly increased in a time and dose-dependent manner. Furthermore, the value of Bax/Bcl-2 was correspondingly increased. K562 cells and K562/ADM cells were treated with 20μg/ml and 50μg/ml nano-realgar for 12h and 24h, the activity of Caspase-3 protein was significantly enhanced. Treated target cells with 20μg/ml and 50μg/ml nano-realgar for 24h, the content of CD34+CD38-CD123+(LSCs) cells in K562/ADM cells and K562 cells had obviously increased. At the same time, combined the immunophenotypes and Annexin-V labeling to analyze the apopotosis of the LSC, which relatively enriched for LSCs, the apoptotic rates of LSC were (31.35±3.75)% and (49.50±8.90)% in K562/ADM cells, (18.90±1.20)% and (54.80±3.20)% in K562 cells, respectively. After administration of 20ug/ml and 50ug/ml nano-realgar for 24h and 48h, the percentage of BCRP+, P-gp+and co-expressing P-gp and BCRP cell population in K562 cells incrased dramatically. In high concertration, the expression ratio of BCRP+, P-gp+and co-expressing P-gp and BCRP were (50.23±1.16)% and (81.85±0.35)%, (57.95±5.45)% and (59.11±1.84)%, (43.75±2.45)% and (55.35±1.55)%. Morever, only the expression ratio of BCRP+ and co-expressing P-gp and BCRP in K562/ADM cells enhanced after treated with 20μg/ml and 50μg/ml nano-realgar for 24h and 48h, the expression of P-gp had no obvious change. After treatment with 50μg/ml nano-realgar for 24h and 48h, the subset of BCRP+and P-gp+BCRP+in K562/ADM cell population was (44.45±2.30) % and (81.65±8.01)%, (44.35±2.25)%and (81.40±8.20)%, respectively.CONCLUSION:Nano-realgar significantly inhibits the proliferation of multidrug-resistant leukemia K562/ADM cells and its drug-sensitive parental K562 cells, and induces the cell population and their LSCs to undergo apoptosis via canonical caspase-dependent mitochondrial pathway. The proliferation-inhibiting and apoptosis-inducing effects of realgar nanoparticle are more powerful than that of raw realgar in both K562 cells and K562/ADM cells. The sensitivity of multidrug-resistant K562/ADM cells to nano-realgar is corresponding to that of drug-sensitive K562 cells as well as their own leukemia stem cells.
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