Departments of Radiation Oncology, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Mol Carcinog. 2013 Jul;52(7):555-67. doi: 10.1002/mc.21891. Epub 2012 Mar 5.
K-ras mutations have been identified in up to 95% of pancreatic cancers, implying their critical role in the molecular pathogenesis. Expression of K-ras oncogene in an immortalized human pancreatic ductal epithelial cell line, originally derived from normal pancreas (H6c7), induced the formation of carcinoma in mice. We hypothesized that K-ras oncogene correlates with increased non-mitochondrial-generated superoxide (O 2.-), which could be involved in regulating cell growth contributing to tumor progression. In the H6c7 cell line and its derivatives, H6c7er-Kras+ (H6c7 cells expressing K-ras oncogene), and H6c7eR-KrasT (tumorigenic H6c7 cells expressing K-ras oncogene), there was an increase in hydroethidine fluorescence in cell lines that express K-ras. Western blots and activity assays for the antioxidant enzymes that detoxify O 2.- were similar in these cell lines suggesting that the increase in hydroethidine fluorescence was not due to decreased antioxidant capacity. To determine a possible non-mitochondrial source of the increased levels of O 2.-, Western analysis demonstrated the absence of NADPH oxidase-2 (NOX2) in H6c7 cells but present in the H6c7 cell lines expressing K-ras and other pancreatic cancer cell lines. Inhibition of NOX2 decreased hydroethidine fluorescence and clonogenic survival. Furthermore, in the cell lines with the K-ras oncogene, overexpression of superoxide dismutases that detoxify non-mitochondrial sources of O 2.-, and treatment with the small molecule O 2.- scavenger Tempol, also decreased hydroethidine fluorescence, inhibited clonogenic survival and inhibited growth of tumor xenografts. Thus, O 2.- produced by NOX2 in pancreatic cancer cells with K-ras, may regulate pancreatic cancer cell growth.
K-ras 突变已在高达 95%的胰腺癌中被鉴定出来，这表明其在分子发病机制中起关键作用。在源自正常胰腺的永生化人胰腺导管上皮细胞系（最初源自正常胰腺（H6c7））中表达 K-ras 癌基因可诱导小鼠发生癌。我们假设 K-ras 癌基因与增加的非线粒体产生的超氧化物（O₂.-）相关，这可能参与调节细胞生长并促进肿瘤进展。在 H6c7 细胞系及其衍生物 H6c7er-Kras+（表达 K-ras 癌基因的 H6c7 细胞）和 H6c7eR-KrasT（表达 K-ras 癌基因的致瘤性 H6c7 细胞）中，表达 K-ras 的细胞系中超氧化物荧光增强。这些细胞系中的抗氧化酶的 Western 印迹和活性测定表明，超氧化物荧光的增加不是由于抗氧化能力降低所致。为了确定 O₂.- 增加的可能非线粒体来源，Western 分析表明 H6c7 细胞中不存在 NADPH 氧化酶-2（NOX2），但在表达 K-ras 和其他胰腺癌细胞系的 H6c7 细胞系中存在。NOX2 的抑制降低了超氧化物荧光和集落形成的存活率。此外，在具有 K-ras 癌基因的细胞系中，过表达解毒非线粒体来源 O₂.- 的超氧化物歧化酶，以及用小分子 O₂.- 清除剂 Tempol 处理，也降低了超氧化物荧光，抑制了集落形成的存活率并抑制了肿瘤异种移植物的生长。因此，具有 K-ras 的胰腺癌细胞中由 NOX2 产生的 O₂.- 可能调节胰腺癌细胞的生长。
