Geng Y J, Hellstrand K, Wennmalm A, Hansson G K
King Gustaf V Research Institute, Karolinska Institute, Stockholm, Sweden.
Cancer Res. 1996 Feb 15;56(4):866-74.
The host defense against tumor cells is in part based on the production of nitric oxide (NO) by activated macrophages. However, cells of the blood vessels can also participate in antitumor defense responses. They produce NO either constitutively [endothelial cells (ECs)] or after stimulation by proinflammatory cytokines (ECs and vascular smooth muscle cells). We have used a tumor cell-vascular cell coculture system to evaluate whether vascular cells can mediate cytotoxic effects on tumor cells. Treatment with IFN-gamma and tumor necrosis factor-alpha induced death of human erythroleukemic K562 cells cocultured with rodent vascular smooth muscle cells or ECs. The synergistic antitumor activity of the two cytokines depended on de novo gene expression of the inducible isoform of NO synthase and on synthesis of reactive nitrogen intermediates (RNIs) in the vascular cells. K562 cells did not produce any appreciable levels of NO, but they were targeted by RNIs released from the cytokine-stimulated vascular cells, as demonstrated by electron paramagnetic resonance spectrometry, which showed formation of nonheme iron-nitrosyl complexes in the tumor cells. Assays for mitochondrial respiration demonstrated that the tumor cells suffered from a block of the complexes I and II of the mitochondrial respiratory chain. Further analysis of the cytotoxic mechanism by fluorescent microscopy, flow cytometry, and DNA electrophoresis revealed that K562 cells attacked by NO-producing vascular cells underwent apoptosis with plasma membrane blebbing, cell volume reduction, condensation of cytoplasm and chromatin, and fragmentation of genomic DNA at internucleosomal sites. In contrast, only a few vascular cells exhibited these apoptotic changes, suggesting that these cells resist the RNI attack. Inhibition of NO production in vascular cells by NG-monomethyl-L-arginine, an inhibitor of NO synthases, significantly reduced the death of the K562 cells. These observations suggest that vascular cells induce apoptotic death of tumor cells by producing RNIs in response to cytokine stimulation.
机体对肿瘤细胞的防御部分基于活化巨噬细胞产生一氧化氮（NO）。然而，血管细胞也可参与抗肿瘤防御反应。它们或者组成性地产生NO（内皮细胞），或者在促炎细胞因子刺激后产生NO（内皮细胞和血管平滑肌细胞）。我们利用肿瘤细胞-血管细胞共培养系统来评估血管细胞是否能介导对肿瘤细胞的细胞毒性作用。用γ干扰素和肿瘤坏死因子-α处理可诱导与啮齿动物血管平滑肌细胞或内皮细胞共培养的人红白血病K562细胞死亡。这两种细胞因子的协同抗肿瘤活性依赖于诱导型一氧化氮合酶的从头基因表达以及血管细胞中活性氮中间体（RNI）的合成。K562细胞不产生任何可观水平的NO，但它们被细胞因子刺激的血管细胞释放的RNI靶向，电子顺磁共振光谱法证明了这一点，该方法显示肿瘤细胞中形成了非血红素铁-亚硝酰复合物。线粒体呼吸测定表明肿瘤细胞的线粒体呼吸链复合物I和II受阻。通过荧光显微镜、流式细胞术和DNA电泳对细胞毒性机制的进一步分析表明，被产生NO的血管细胞攻击的K562细胞发生凋亡，表现为质膜起泡、细胞体积减小、细胞质和染色质浓缩以及基因组DNA在核小体间位点断裂。相反，只有少数血管细胞表现出这些凋亡变化，表明这些细胞抵抗RNI攻击。一氧化氮合酶抑制剂NG-单甲基-L-精氨酸抑制血管细胞中NO的产生，显著降低了K562细胞的死亡。这些观察结果表明，血管细胞通过响应细胞因子刺激产生RNI来诱导肿瘤细胞凋亡死亡。
