Denlinger Chadrick E, Rundall Brian K, Keller Michael D, Jones David R
Department of Thoracic and Cardiovascular Surgery, University of Virginia, Charlottesville, Virginia 22908-0679, USA.
Ann Thorac Surg. 2004 Oct;78(4):1207-14; discussion 1207-14. doi: 10.1016/j.athoracsur.2004.04.029.
My colleagues and I have previously shown that chemotherapy activates the antiapoptotic transcription factor nuclear factor (NF)-kappaB in non-small-cell lung cancer (NSCLC). We hypothesized that inhibition of NF-kappaB by using the proteasome inhibitor bortezomib (Velcade) would sensitize NSCLC to gemcitabine-induced apoptosis.
Tumorigenic NSCLC cell lines (H157 and A549) were treated with nothing, gemcitabine, bortezomib, or both compounds. NF-kappaB activity was determined by nuclear p65 protein levels, electrophoretic mobility shift assays, and reverse transcription-polymerase chain reaction of the NF-kappaB-regulated genes interleukin-8, c-IAP2, and Bcl-xL. The p21 and p53 protein levels were determined in similarly treated cells. Cell-cycle dysregulation was assessed by fluorescence-activated cell sorting analysis. Cell death and apoptosis were quantified by clonogenic assays, caspase-3 activation, and DNA fragmentation. NSCLC A549 xenografts were generated and treated as noted previously. Tumor growth was assessed over a 4-week treatment period. Statistical analysis was performed with analysis of variance.
Gemcitabine enhanced nuclear p65 levels, NF-kappaB binding to DNA, and transcription of all NF-kappaB-regulated genes. Bortezomib inhibited each of these effects. Combined gemcitabine and bortezomib enhanced p21 and p53 expression and induced S-phase and G2/M cell-cycle arrests, respectively. Combined treatment killed 80% of the NSCLC cells and induced apoptosis, as determined by caspase-3 activation (p = 0.05) and DNA fragmentation (p = 0.02). NSCLC xenografts treated with combination therapy grew significantly slower than xenografts treated with gemcitabine alone (p = 0.02).
Bortezomib inhibits gemcitabine-induced activation of NF-kappaB and sensitizes NSCLC to death in vitro and in vivo. This combined treatment strategy warrants further investigation and may represent a reasonable treatment strategy for select patients with NSCLC given the current clinical availability of both drugs.
我和同事们之前已经表明，化疗可激活非小细胞肺癌（NSCLC）中的抗凋亡转录因子核因子（NF）-κB。我们假设，使用蛋白酶体抑制剂硼替佐米（万珂）抑制NF-κB会使NSCLC对吉西他滨诱导的凋亡敏感。
用空白对照、吉西他滨、硼替佐米或这两种化合物处理致瘤性NSCLC细胞系（H157和A549）。通过核p65蛋白水平、电泳迁移率变动分析以及对NF-κB调控基因白细胞介素-8、c-IAP2和Bcl-xL进行逆转录-聚合酶链反应来测定NF-κB活性。在同样处理的细胞中测定p21和p53蛋白水平。通过荧光激活细胞分选分析评估细胞周期失调情况。通过克隆形成试验、半胱天冬酶-3激活和DNA片段化来定量细胞死亡和凋亡。生成NSCLC A549异种移植瘤并按之前所述进行处理。在4周的治疗期内评估肿瘤生长情况。采用方差分析进行统计学分析。
吉西他滨提高了核p65水平、NF-κB与DNA的结合以及所有NF-κB调控基因的转录。硼替佐米抑制了这些效应中的每一种。联合使用吉西他滨和硼替佐米分别增强了p21和p53表达，并诱导了S期和G2/M期细胞周期阻滞。联合治疗杀死了80%的NSCLC细胞并诱导了凋亡，这通过半胱天冬酶-3激活（p = 0.05）和DNA片段化（p = 0.02）得以确定。联合治疗的NSCLC异种移植瘤生长明显慢于单独用吉西他滨治疗的异种移植瘤（p = 0.02）。
硼替佐米抑制吉西他滨诱导的NF-κB激活，并使NSCLC在体外和体内对死亡敏感。鉴于这两种药物目前在临床上均可获得，这种联合治疗策略值得进一步研究，可能代表了针对特定NSCLC患者的一种合理治疗策略。
