过氧化物上调人结肠癌细胞血管内皮生长因子的表达

Upregulation of vascular endothelial growth factor by peroxide in human colon cancer

目的 探讨过氧化氢等活性氧类对于大肠癌的进一步发展可能存在的影响。方法 以低浓度过氧化氢 (10 -5mol/L、10 -7mol/L及 10 -9mol/L)作用人结肠癌细胞系LS174T和HCT8共 2 4h ,与人血管内皮细胞系ECV 30 4共培养 ,观察过氧化氢对结肠癌细胞诱导的内皮细胞迁移的影响。以逆转录 聚合酶链反应、酶联免疫吸附分析检测相对定量过氧化氢作用下癌细胞血管内皮生长因子的表达强度 ,以放线菌素D(1 5 μg/ml)抑制癌细胞的转录观察过氧化氢产生作用的机制 ,同时以激光扫描细胞仪直接定量肿瘤细胞NF KappaB活性的变化。结果 外源性低浓度过氧化氢促进两种结肠癌细胞诱导的内皮细胞迁移 ,其中在 10 -5mol/L过氧化氢的作用下 ,LS174T诱导的迁移数为 2 0 2 6±70 2 (个 ) (与对照组比t=3 4 75 1,P =0 0 0 83) ,HCT8诱导的迁移数为 14 5 4± 6 5 3(个 ) (与对照组比t=4 4 183,P =0 0 0 84 ) ,高于同组其余各浓度引起的迁移数目。同时癌细胞血管内皮生长因子的表达也见增强 ,放线菌素D能阻断过氧化氢的这种作用。半衰期分析提示过氧化氢未影响血管内皮生长因子mRNA的稳定性。两种癌细胞NF KappaB的活性在 2 4h的过氧化氢作用后获得提高 ,这与血管内皮生长因子的增强表达情形相仿。

Objective To clarify the possible effect of reactive oxygen species such as hydrogen peroxide on progression of human colorectal cancer. Method Human colon carcinoma cell lines, L174T and HCT8, were treated with low concentration of hydrogen peroxide (10 -5 mol/L?10 -7 mol/L and 10 -9 mol/L, possessed no effect on cancer cell growth) for 24 hours before being co cultured with human endothelial cell line ECV 304. The migration of ECV 304 inuced by cancer cells was calculated and the expression of vascular endothelial growth factor (VEGF) in cancer cells was determined using RT PCR and ELISA. Dactinomycin (1.5 μg/ml) was applied to block the transcription of cancer cell so as to obseve the effect site of cell. Finally the activity of NF KappaB of cancr cells was estimated using laser scanning cytometry. Results Exogenouse hydrogen peroxide of low concentration promoted the migration of endothelial cell induced by colon cancer cell. When the concentration of H 2O 2 was 10 -5 mol/L, the average number of migration of endothelial cell induced by LS174T was 202.6±70.2, higher than the value of 154.6±37.9 when no H 2O 2 was given ( t =3.4751, P =0.0083). When the concentration of H 2O 2 was 10 -5 mol/L, the average number of migration of endothelial cell induced by HCT8 was 145.4±65.3, higher than the value of 113±72.7 when no H 2O 2 was given ( t =4.4183, P =0.0084). The expression of vascular endothelial growth factor in cancer cells was enhanced to a certain degree, which could be blocked by Dactinomycin. And the half time of VEGF mRNA was not prolonged by treatment with hydrogen peroxide. The activity of NF KappaB of both colon caecinoma cell lines was at a higher level after being treated with hydrogen peroxide for 24 hours. Conclusion Hydrogen peroxide increases vascular endothelial growth factor expression in colon cancer cell, and it is possible that such reactive oxygen species as hydrogen peroxide facilitates development of colon cancer.