安徽医科大学学报

目的 研究低氧环境对培美曲塞(PE)介导的非小细胞肺癌细胞凋亡的影响。方法 将A549细胞置于21%O₂或1%O₂中，给予8μmol/L PE处理48 h，分为常氧对照组、常氧溶剂组、常氧加药组、低氧对照组、低氧溶剂组、低氧加药组。台盼蓝染色法检测低氧环境与常氧环境下的细胞给予PE处理后的死亡情况；流式细胞术检测PE作用后常氧和低氧环境下细胞的凋亡率；细胞划痕实验检测PE作用后常氧和低氧环境下细胞的迁移距离；Western blot检测MRP-1、Cleaved Caspase-3、E-Cadherin、N-Cadherin表达。结果 台盼蓝染色结果显示，与常氧加药组相比，低氧环境下PE作用后的细胞死亡率下降(P<0.001)。流式细胞术结果显示，低氧和常氧环境下PE均导致细胞凋亡，但常氧加药组凋亡率显著高于低氧加药组(P<0.000 1)。细胞划痕实验结果显示，常氧和低氧环境下PE处理24 h和48 h后，A549细胞的迁移距离均小于对照组细胞(P<0.05)，但常氧加药组和低氧加药组细胞的迁移距离无显著性差异。Western blot结果显示，低氧环境下PE处理的细胞中MRP-1表达明显高于常氧环境下PE处理的细胞(P<0.000 1)。与对照组比较，常氧条件下，PE作用于A549细胞后，Cleaved Caspase-3的表达上升(P<0.05)，但低氧条件下，PE处理的细胞中Cleaved Caspase-3表达无明显变化。低氧和常氧环境下，PE处理的细胞中E-Cadherin表达下降(P<0.000 1、P<0.001),N-Cadherin表达降低(P<0.05、P<0.000 1)，但低氧加药处理的细胞中E-Cadherin和NCadherin表达明显低于常氧加药组(均P<0.000 1)。结论 低氧可显著削弱PE对A549细胞的促凋亡效应，并阻断Cleaved Caspase-3激活；其机制可能与低氧诱导MRP-1高表达、减少药物蓄积有关。干预MRP-1或恢复凋亡通路活性可能成为克服低氧耐药的新策略。

Objective To investigate the effect of hypoxia on pemetrexed(PE)-mediated apoptosis in non-small cell lung cancer cells.Methods A549 cells were cultured under 21% O₂(normoxia) or 1% O₂(hypoxia), and treated with 8 μmol/L PE for 48 h. The cells were divided into normoxic control group, normoxic vehicle group, normoxic drug-treated group, hypoxic control group, hypoxic vehicle group and hypoxic drug-treated group. Trypan blue staining was used to observe cell death after PE intervention under normoxia and hypoxia. Flow cytometry was adopted to detect cell apoptosis rate. Wound healing assay was used to assess cell migration. Western blot was performed to detect the protein levels of MRP-1, Cleaved Caspase-3, E-Cadherin and N-Cadherin.Results Trypan blue staining results showed that the cell death rate was reduced in PE-treated cells under hypoxia compared with the normoxic PE group(P<0. 001). Flow cytometry results indicated that PE induced cell apoptosis under both normoxic and hypoxic conditions. However, the apoptosis rate of the normoxic PE group was significantly higher than that of the hypoxic PE group(P<0. 000 1). Wound healing assay showed that the migration distance of A549 cells decreased after PE treatment for 24 h and 48 h under normoxia and hypoxia when compared with the control group(P<0. 05), while no significant difference was found between the normoxic PE group and the hypoxic PE group. Western blot results revealed that MRP-1 expression was markedly higher in hypoxic PE-treated cells than in normoxic PE-treated cells(P<0. 000 1). Compared with the control group, Cleaved Caspase-3 expression was upregulated in A549 cells following PE intervention under normoxia(P<0. 05), whereas no obvious alteration in Cleaved Caspase-3 expression was detected in hypoxic PE-treated cells. Under both normoxic and hypoxic conditions, PE treatment downregulated the expression of E-Cadherin(P<0. 000 1,P<0. 001) and N-Cadherin(P<0. 05,P<0. 000 1). In addition, the expression levels of E-Cadherin and N-Cadherin in the hypoxic PE group were significantly lower than those in the normoxic PE group(all P<0. 000 1).Conclusion Hypoxia significantly attenuates the pro-apoptotic effect of PE on A549 cells and blocks Cleaved Caspase-3 activation. This mechanism may be related to hypoxia-induced MRP-1 overexpression and reduced drug accumulation. Interfering with MRP-1 or restoring the activity of the apoptosis pathway may serve as a novel strategy to overcome hypoxia-induced drug resistance.