AIM:To investigate the effects of celecoxib on viability , apoptosis and autophagy in acute myeloid leukemia (AML) cell lines HL-60 and HL-60A.METHODS:The HL-60 cells and HL-60A cells were cultured with vari-ous concentrations (0, 20, 40, 60, 80 and 100μmol/L) of celecoxib.The inhibitory effect of celecoxib on the cell viabil-ity was evaluated by MTT assay .Apoptosis was analyzed by Annexin-V/PI staining.Apoptosis-related and autophagy-relat-ed proteins were determined by Western blot .RESULTS:IC50 of celecoxib were 49.4 μmol/L, 32.0 μmol/L and 25.1μmol/L for HL-60 cells treated with celecoxib for 24 h, 48 h and 72 h, respectively.For HL-60A cells, the corresponding IC50 were 69.1 μmol/L, 42.5 μmol/L and 29.6 μmol/L, respectively.The results of flow cytometry analysis showed the proportions of Annexin-Ⅴ+PI-, Annexin-Ⅴ+PI+and Annexin-Ⅴ-PI+cells were increased in the HL-60 cells, and those of Annexin-Ⅴ+PI-and Annexin-Ⅴ+PI+cells were increased in the HL-60A cells treated with celecoxib for 24 h. After treated with celecoxib , the induction of apoptosis was observed , the apoptosis-related proteins cleaved caspase-3 and cleaved PARP were upregulated , the autophagy-related proteins LC3 II and P62 were both increased , and mTOR, p-mTOR, 4-EBP and p-4-EBP were not changed , indicating that celecoxib inhibited autophagy in the AML cells without the mTOR pathway involvement .CONCLUSION:Celecoxib inhibits the viability of HL-60 cells and HL-60A cells in a time-and dose-dependent manner by its effects of inducing apoptosis and necrosis .Celecoxib inhibits mTOR-independent autoph-agy in AML cells, indicating a possible way of using celecoxib for enhancing the antitumor activity of therapeutic agents to induce cytoprotective autophagy in the AML cells .%目的：探讨塞来昔布对急性髓细胞白血病（AML） HL－60细胞和HL－60A细胞的活力、凋亡及自噬的影响。方法：用不同浓度的（0、20、40、60、80和100μmol／L）塞来昔布作用于HL－60细胞和HL－60A细胞，24 h、48 h和72 h后用MTT法检测细胞活力。用流式细胞术检测塞来昔布作用HL－60细胞和HL－60A细胞24 h后的凋亡率。用Western blot法检测凋亡相关蛋白cleaved caspase－3、cleaved PARP，自噬相关蛋白LC3、P62，以及mTOR信号途径相关蛋白。结果：塞来昔布作用于HL－60细胞24 h、48 h和72 h的 IC50分别为49．4μmol／L、32．0μmol／L和25．1μmol／L，对于HL－60A细胞，相应的IC50分别是69．1μmol／L、42．5μmol／L和29．6μmol／L。塞来昔布作用24 h后，流式细胞术检测显示HL－60细胞中Annexin－Ⅴ＋PI－、Annexin－Ⅴ＋PI＋及Annexin－Ⅴ－PI＋细胞的比例增多；HL－60A细胞中Annexin－Ⅴ＋PI－及Annexin－Ⅴ＋PI＋细胞的比例增多。 Western blot实验结果显示塞来昔布作用后， cleaved caspase－3和cleaved PARP的蛋白水平增高，提示该凋亡作用是通过caspase途径的。自噬相关蛋白LC3Ⅱ及P62的表达均增加，mTOR、p－mTOR以及下游的4－EBP、p－4－EBP的蛋白水平没有变化，说明塞来昔布能够抑制AML细胞自噬，该作用与mTOR途径无关。结论：塞来昔布对HL－60细胞和HL－60A细胞活力的抑制作用呈浓度以及时间依赖性，该作用与塞来昔布诱导细胞凋亡及坏死有关。塞来昔布能够通过非mTOR依赖途径抑制AML细胞自噬，有望联合应用于AML的治疗，有助于增强某些引起保护性自噬的化疗药物的细胞毒作用。
展开▼
