【摘要】：目的:探討塞來(lái)昔布對(duì)急性髓細(xì)胞白血病(AML)HL-60細(xì)胞和HL-60A細(xì)胞的活力、凋亡及自噬的影響。方法:用不同濃度的(0、20、40、60、80和100μmol/L)塞來(lái)昔布作用于HL-60細(xì)胞和HL-60A細(xì)胞,24h、48h和72h后用MTT法檢測(cè)細(xì)胞活力。用流式細(xì)胞術(shù)檢測(cè)塞來(lái)昔布作用HL-60細(xì)胞和HL-60A細(xì)胞24 h后的凋亡率。用Western blot法檢測(cè)凋亡相關(guān)蛋白cleaved caspase-3、cleaved PARP,自噬相關(guān)蛋白LC3、P62,以及mTOR信號(hào)途徑相關(guān)蛋白。結(jié)果:塞來(lái)昔布作用于HL-60細(xì)胞24h、48h和72h的IC_(50)分別為49.4μmol/L、32.0μmol/L和25.1μmol/L,對(duì)于HL-60A細(xì)胞,相應(yīng)的IC_(50)分別是69.1μmol/L、42.5μmol/L和29.6μmol/L。塞來(lái)昔布作用24h后,流式細(xì)胞術(shù)檢測(cè)顯示HL-60細(xì)胞中Annexin-V~+PI~-、Annexin-V~+PI~+及Annexin-V~-PI~+細(xì)胞的比例增多;HL-60A細(xì)胞中Annexin-V~+PI~-及Annexin-V~+PI~+細(xì)胞的比例增多。Western blot實(shí)驗(yàn)結(jié)果顯示塞來(lái)昔布作用后,cleaved caspase-3和cleaved PARP的蛋白水平增高,提示該凋亡作用是通過(guò)caspase途徑的。自噬相關(guān)蛋白LC3Ⅱ及P62的表達(dá)均增加,mTOR、p-mTOR以及下游的4-EBP、p-4-EBP的蛋白水平?jīng)]有變化,說(shuō)明塞來(lái)昔布能夠抑制AML細(xì)胞自噬,該作用與mTOR途徑無(wú)關(guān)。結(jié)論:塞來(lái)昔布對(duì)HL-60細(xì)胞和HL-60A細(xì)胞活力的抑制作用呈濃度以及時(shí)間依賴(lài)性,該作用與塞來(lái)昔布誘導(dǎo)細(xì)胞凋亡及壞死有關(guān)。塞來(lái)昔布能夠通過(guò)非mTOR依賴(lài)途徑抑制AML細(xì)胞自噬,有望聯(lián)合應(yīng)用于AML的治療,有助于增強(qiáng)某些引起保護(hù)性自噬的化療藥物的細(xì)胞毒作用。
[Abstract]:AIM: To investigate the effect of celecoxib on the viability, apoptosis and autophagy of HL-60 cells and HL-60A cells in acute myeloid leukemia (AML). Methods: HL-60 cells and HL-60A cells were treated with celecoxib at different concentrations (0,20,40,60,80 and 100 micromol/L). Cell viability was measured by MTT at 24, 48 and 72 hours after treatment. The apoptosis rates of HL-60 cells and HL-60A cells were measured by Western blot. Apoptosis-related proteins cleaved caspase-3, cleaved PARP, autophagy-related proteins LC3, P62 and mTOR signaling pathway-related proteins were detected. Results: The IC_ (50) of HL-60 cells treated with celecoxib for 24 h, 48 h and 72 h were 49.4, 32.0 and 25.1 micromol/L, respectively. Flow cytometry showed that the proportion of Annexin-V~+PI~-, Annexin-V~+PI~+ and Annexin-V~+PI~+ cells increased in HL-60A cells, and the proportion of Annexin-V~+PI~- and Annexin-V~-PI~+ cells increased in HL-60A cells. The results showed that cleaved caspase-3 and cleaved PARP protein levels increased after celecoxib treatment, suggesting that the apoptosis was mediated by caspase pathway. The expressions of autophagy-associated proteins LC3 II and P62 increased, while the levels of mTOR, p-mTOR and downstream 4-EBP, p-4-EBP protein did not change, indicating that celecoxib could inhibit AML cells from autophagy. Conclusion: Celecoxib inhibits the activity of HL-60 cells and HL-60A cells in a concentration-and time-dependent manner, which is related to celecoxib-induced apoptosis and necrosis. Enhance the cytotoxicity of some chemotherapeutic drugs that cause protective autophagy.
【作者單位】： 中山大學(xué)附屬第三醫(yī)院輸血科;中山大學(xué)附屬第三醫(yī)院血液科;
【基金】：廣東省自然科學(xué)基金資助項(xiàng)目(No.2014A030313138);廣東省自然科學(xué)基金博士啟動(dòng)基金資助項(xiàng)目(No.2014A030310292)
【分類(lèi)號(hào)】：R733.7
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本文編號(hào)：2191558