Effect Of Arsenic Trioxide On Leukemia Cell Line HL-60 In Vitro

Objective Arsenic trioxide was one of power drug in treatment acute promyelocytic leukemia (APL), and clinical trial results showed that more than 85% patients with APL received complete remission after exposed to arsenic trioxide. To investigate the effect of arsenic trioxide on leukemic cells with HL-60, we studied the effect of arsenic trioxide on cell proliferation, apoptosis and differentiation in HL-60 cells.Methods 1. Growth curve of HL-60 cells exposed to delicious concentration of arsenic trioxide was detected by trypan blue exclusion assay;the effect of IC 50 value on arsenic trioxide in HL-60 cells was analysed by MTT. 2. The expression of CD11b, cell cycle and apoptosis were investigated by flow cytometry. 3. DNA(deoxyribonucleic acid) fragmentation was detected by agarose electrophresis. 4. Changes of cell nucleus stained by Hoecst 33258 in HL-60 cells treated by arsenic trioxide were studied by fluorescence microscope. 5. The mRNA expression of c-myc was showed by RT-PCR. 6. Protein expression (bcl-2, bcl-XL, Ki-67, bax, p21, p27) was performed by immunoblot analysis test. The results were analyzed with SPSS 10.0 analyzed software.Results Arsenic trioxide inhibited the proliferation of HL-60 cells, with time-,dose- depedent manners;IC₅₀ value was 13. 2 μ M, 6. 4 μ M and 1. 9 μ M treated by arsenic trioxide for 24, 48and 72h, respectively, as well arsenic trioxide inhibited the growth of HL-60 cells, arsenic trioxide down -regulated the protein expression of Ki-67, but up-regulated the proliferation negative protein of p21and p27 .DNA fragment appeared after HL-60 cells exposed to10 μ M arsenic trioxide from 24 to 48h: cell nucleus were concentrated and fragmented after HL-60 cells exposed to 10μM arsenic trioxide for 48h. Treated with 10 μ M arsenic trioxide for 12hours, 24hours and 48h, the apoptotic rate was (8. 5 ± 2. 1) %, (12. 8 ± 3. 4) %and(21. 4±5. 8) %, respectively, but the apoptotic rate in the negative control group only (1.5±0.5)%(P<0.05). In the course of apoptosis, we found caspase-3 and PARP were leaved to special activity fragments;the expression of anti-apoptosis proteins bcl-2 and bcl-XL were deceased, on the contrary, pro-apoptosis protein bax up-regulated by arsenic trioxide. Arsenic trioxide significantly increased the CDllb in HL-60 cells, expression rate of CDllb was increased from(14. 5 ± 2. 1) % to(35.4 ± 5. 7) % (P<0.05) after treated by 2.5 μ M arsenic trioxide for 90 hours. In the present study, we find 2.5 μ M arsenic trioxide increased percent of NBT positive, the NBT positive percent of HL-60cells treatment with 2.5 μ M arsenic trioxide for 90 hours, was (31.2 ± 3.4) %, but the percent in vehicle group only (10.0±2.1) %(P<0.05), mRNA expression ofc-myc was reduced in HL-60 cells, during the course of differentiation induced by arsenic trioxide.Conclusion Arsenic trioxide blocked cell proliferation, induced apoptosis and increased cells differentiation in HL-60 cells. Inhibition of cell proliferation mediated by Ki-67 protein;bcl-2 family proteins and c-myc gene took part in apoptosis and cells differentiation, respectively.Postgraduate student: Liu Yinghui(Hematology) Professor: Zhao Hongguo...