Vrije Universiteit Brussel, Cancer Research Unit, Brussels, Belgium.
Int J Radiat Oncol Biol Phys. 2010 Apr;76(5):1520-7. doi: 10.1016/j.ijrobp.2009.10.047.
Nitric oxide (NO), synthesized by the inducible nitric oxide synthase (iNOS), is known to inhibit metabolic oxygen consumption because of interference with mitochondrial respiratory activity. This study examined whether activation of iNOS (a) directly in tumor cells or (b) in bystander macrophages may improve radioresponse through sparing of oxygen.
EMT-6 tumor cells and RAW 264.7 macrophages were exposed to bacterial lipopolysaccharide plus interferon-gamma, and examined for iNOS expression by reverse transcription polymerase chain reaction, Western blotting and enzymatic activity. Tumor cells alone, or combined with macrophages were subjected to metabolic hypoxia and analyzed for radiosensitivity by clonogenic assay, and for oxygen consumption by electron paramagnetic resonance and a Clark-type electrode.
Both tumor cells and macrophages displayed a coherent picture of iNOS induction at transcriptional/translational levels and NO/nitrite production, whereas macrophages showed also co-induction of the inducible heme oxygenase-1, which is associated with carbon monoxide (CO) and bilirubin production. Activation of iNOS in tumor cells resulted in a profound oxygen sparing and a 2.3-fold radiosensitization. Bystander NO-producing, but not CO-producing, macrophages were able to block oxygen consumption by 1.9-fold and to radiosensitize tumor cells by 2.2-fold. Both effects could be neutralized by aminoguanidine, a metabolic iNOS inhibitor. An improved radioresponse was clearly observed at macrophages to tumor cells ratios ranging between 1:16 to 1:1.
Our study is the first, as far as we are aware, to provide evidence that iNOS may induce radiosensitization through oxygen sparing, and illuminates NO-producing macrophages as a novel determinant of tumor cell radioresponse within the hypoxic tumor microenvironment.
诱导型一氧化氮合酶（iNOS）合成的一氧化氮（NO）已知会通过干扰线粒体呼吸活动来抑制代谢耗氧量。本研究旨在检验 iNOS 的激活（a）是否直接在肿瘤细胞中，或（b）在旁观者巨噬细胞中，通过节省氧气来改善放射反应。
EMT-6 肿瘤细胞和 RAW 264.7 巨噬细胞暴露于细菌脂多糖和干扰素-γ，通过逆转录聚合酶链反应、Western 印迹和酶活性检测 iNOS 的表达。单独的肿瘤细胞或与巨噬细胞一起进行代谢缺氧，并通过集落形成试验分析放射敏感性，通过电子顺磁共振和克拉克型电极分析耗氧量。
肿瘤细胞和巨噬细胞在转录/翻译水平和 NO/亚硝酸盐产生方面均表现出一致的 iNOS 诱导，而巨噬细胞还表现出诱导型血红素加氧酶-1的共同诱导，该酶与一氧化碳（CO）和胆红素产生相关。肿瘤细胞中 iNOS 的激活导致明显的氧气节省和 2.3 倍的放射增敏作用。产生旁观者 NO、但不产生 CO 的巨噬细胞能够将耗氧量降低 1.9 倍，并将肿瘤细胞的放射增敏作用提高 2.2 倍。这两种作用都可以被代谢型 iNOS 抑制剂氨基胍中和。在巨噬细胞与肿瘤细胞的比例为 1:16 到 1:1 之间时，明显观察到改善的放射反应。
我们的研究首次提供证据表明，iNOS 可能通过氧气节省诱导放射增敏，并阐明产生 NO 的巨噬细胞是缺氧肿瘤微环境中肿瘤细胞放射反应的一个新决定因素。
