Department of Respiratory Diseases, Xiangya Hospital, Central South University, Changsha, Hunan 410007, China.
Chin Med J (Engl). 2013;126(17):3276-82.
Tumor hypoxia, one of the features of solid tumors, is associated with chemo-resistance. Recently, nuclear factor-κB (NF-κB) was found to be activated during hypoxia. However, the impact of NF-κB activation on chemo-resistance during hypoxia remains unknown.
Human lung adenocarcinoma A549 cells were transfected with NF-κB p65siRNA and treated with cobalt chloride (CoCl2) to mimic hypoxia in the presence or absence of cisplatin. NF-κB expression was measured by Western blotting, immune-fluorescence and real-time PCR. Hypoxia-inducible factor-1α (HIF-1α) and Bcl-2 expression were determined by Western blotting. Cell apoptosis and survival with half-maximum inhibitory concentration (IC50) of cisplatin were determined by Annexin V-FITC/PI and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), respectively.
Exposure of A549 cells to CoCl2 increased nuclear HIF-1a protein expression, and enhanced NF-κB p65 protein nuclear accumulation (the mark of NF-κB activation) in a time and dose dependant manner. CoCl2 did not promote apoptosis in A549 cells; on the contrary, it reduced cisplatin-induced apoptosis and increased the IC50 of cisplatin. However, when we inhibited CoCl2-induced activation of NF-κB through NF-κB p65siRNA, cisplatin-induced apoptosis was increased and IC50 of cisplatin was reduced to levels similar to those in control cells. Meanwhile, CoCl2-induced Bcl-2 overexpression was down-regulated in the presence of cisplatin when NF-κB activity was inhibited.
Up-regulating Bcl-2 might be involved in NF-κB activation induced resistance to cisplatin in A549 cells under CoCl2-induced chemical hypoxia.
肿瘤缺氧是实体瘤的特征之一，与化疗耐药有关。最近发现，核因子-κB（NF-κB）在缺氧时被激活。然而，NF-κB 激活对缺氧时化疗耐药的影响尚不清楚。
用人肺腺癌细胞 A549 转染 NF-κB p65siRNA，并在有或没有顺铂的情况下用氯化钴（CoCl2）模拟缺氧。用 Western blot、免疫荧光和实时 PCR 测定 NF-κB 的表达。用 Western blot 测定缺氧诱导因子-1α（HIF-1α）和 Bcl-2 的表达。用 Annexin V-FITC/PI 和 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）分别测定半最大抑制浓度（IC50）顺铂的细胞凋亡和存活。
CoCl2 暴露使 A549 细胞核 HIF-1a 蛋白表达增加，并以时间和剂量依赖的方式增强 NF-κB p65 蛋白核积累（NF-κB 激活的标志）。CoCl2 不会促进 A549 细胞凋亡；相反，它减少了顺铂诱导的凋亡，并增加了顺铂的 IC50。然而，当我们通过 NF-κB p65siRNA 抑制 CoCl2 诱导的 NF-κB 激活时，顺铂诱导的凋亡增加，顺铂的 IC50 降低至与对照细胞相似的水平。同时，当 NF-κB 活性被抑制时，CoCl2 诱导的 Bcl-2 过表达在顺铂存在下被下调。
在 CoCl2 诱导的化学缺氧下，上调 Bcl-2 可能参与了 NF-κB 激活诱导的 A549 细胞对顺铂的耐药。
