吡咯烷二硫代氨基甲酸盐抑制NF-κB活化对HL-60细胞的化疗增敏作用

目的：探讨化疗诱导性核因子κB的活化机制及抗氧化抑制NF-κB活化对白血病细胞凋亡及化疗敏感性的影响。 方法： 采用间接免疫荧光方法和凝胶迁移率变动试验（EMSA）观察不同浓度的化疗药物单独或与抗氧化剂吡咯烷二硫代氨基甲酸盐（PDTC）联合作用于HL-60细胞后NF-κB的活化反应；运用流式细胞术和MTT试验比较NF-κB活化及活化抑制对HL-60细胞凋亡及化疗敏感性的影响。 结果： EMSA试验表明，柔红霉素（DNR）和足叶乙甙（VP-16）均呈剂量依赖性诱导NF-κB活化；RelA亚基位于细胞核进一步证实了NF-κB活化。PDTC呈剂量依赖性抑制诱导性NF-κB活化，当其浓度达到100 μmol/L时，NF-κB活化被完全抑制。比较PDTC干预前后细胞的凋亡反应，发现2.5 mg/L、5 mg/L、10 mg/L VP-16诱导的细胞凋亡指数分别由(5.34±0.62)%、(10.16±0.42)%、(17.32±1.15)%增至(8.97±0.81)%、(16.01±1.06)%、(22.96±1.33%)，且PDTC显著增强DNR及VP-16对HL-60细胞的生长抑制率(P<0.01)。 结论： 反应性氧中间产物介导化疗诱导性NF-κB活化，抗氧化抑制NF-κB活化可以增进HL-60细胞凋亡及化疗敏感性。

Inhibition of NF-κB activation by pvrrolidine dithiocarbamate increases sensitivity of HL-60 cells to cytotoxic drugs

AIM: To explore whether inhibition of NF-κB by antioxidant pvrrolidine dithiocarbamate (PDTC) sensitizes leukemia cells to cytotoxic drugs and its mechanism. METHODS: The indirect immunofluorescence method and electrophoretic mobility shift assay (EMSA) were used to measure the activation of NF-κB. The apoptotic cells were evaluated by flow cytometry (FCM) and the in vitro growth inhibitory effect was performed using a MTT assay. RESULTS: EMSA showed that NF-κB was activated by daunorubicin (DNR), VP-16 and then was inhibited by PDTC in a dose-dependent manner. NF-κB activation was further verified because of subunit RelA of NF-κB locating in the nuclei. FCM analysis showed that apoptotic index of HL-60 cells was up to (8.97±0.81)%, (16.01±1.06)%, (22.96±1.33)% from (5.34±0.62)%, (10.16±0.42)%, (17.32±1.15)% after exposure of HL-60 cells to 2.5-10 mg/L VP-16 combined with PDTC. VP-16 added with PDTC produced greater growth inhibitory effect to HL-60 cells than did VP-16 or DNR only (P<0.01). CONCLUSION: Reactive oxygen intermediates play an important role in inducible NF-κB activation. The inhibition of NF-κB by antioxidant PDTC sensitizes HL-60 cells to cytotoxic drugs.