Osada Shinji, Imai Hisashi, Tomita Hiroyuki, Tokuyma Yasuharu, Okumura Naoki, Sakashita Fumio, Nonoka Kenichi, Sugiyama Yasuyuki
Surgical Oncology, Gifu University School of Medicine, 1-1 Yanagido, Gifu City 501-1194, Japan.
J Gastroenterol Hepatol. 2006 Jun;21(6):988-93. doi: 10.1111/j.1440-1746.2006.04223.x.
The aim of the present study was to examine coordination of the vascular endothelial growth factor (VEGF) and VEGF receptor (Flk-1) system and to study control of VEGF expression by oxidative stress, which is considered a model for chronic liver disease.
Cell viability was determined by test method with 3-[4, 5-dimethylthiazol-2-yl]-2, 5-dephenyl tetrazolium bromide (MTT). Expressions of cellular proteins were evaluated by western blot analysis.
The c-Met tyrosine phosphorylation in PLC/PRF/5 hepatoma cells was increased by treatment with 20 ng/mL hepatocyte growth factor (HGF), and extracellular signal-regulated kinase (ERK) was also activated. Although Flk-1 was phosphorylated in response to VEGF (>50 ng/mL), phosphorylated ERK was not detected at these concentrations. A total of 5.0 and 10 micromol/L hydrogen peroxide (H(2)O(2)) caused cell death in a dose-dependent manner after 24 h. On western blot analysis at 1 h with H(2)O(2), rapid phosphorylation of both ERK1/2 and c-Jun NH(2)-terminal kinase (JNK) was observed. In the first 6 h, H(2)O(2) induced cell death for 58.4 +/- 6.8%, whereas the presence of 100 ng/mL VEGF improved the survival rate to 77.2 +/- 4.2%. The VEGF significantly decreased H(2)O(2)-induced cell death after 12 h, whereas HGF (20 ng/mL) did not have a similar effect. When cells were incubated with 5 micromol/L H(2)O(2), expression of VEGF protein was detected. Furthermore, H(2)O(2)-induced phosphorylation of ERK and JNK was also reduced by VEGF (100 ng/mL). In contrast, HGF did not induce phosphorylation of ERK and JNK.
Hepatoma cells might be able to survive under continuous oxidative stress through expression of VEGF.
本研究旨在检测血管内皮生长因子（VEGF）和VEGF受体（Flk-1）系统的协调性，并研究氧化应激对VEGF表达的调控，氧化应激被视为慢性肝病的一种模型。
采用3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐（MTT）检测法测定细胞活力。通过蛋白质印迹分析评估细胞蛋白的表达。
用20 ng/mL肝细胞生长因子（HGF）处理后，PLC/PRF/5肝癌细胞中的c-Met酪氨酸磷酸化增加，细胞外信号调节激酶（ERK）也被激活。虽然Flk-1在VEGF（>50 ng/mL）作用下发生磷酸化，但在这些浓度下未检测到磷酸化的ERK。24小时后，5.0和10 μmol/L的过氧化氢（H₂O₂）以剂量依赖方式导致细胞死亡。在用H₂O₂处理1小时后的蛋白质印迹分析中，观察到ERK1/2和c-Jun氨基末端激酶（JNK）均迅速磷酸化。在最初6小时，H₂O₂诱导58.4±6.8%的细胞死亡，而100 ng/mL VEGF的存在将存活率提高到77.2±4.2%。12小时后，VEGF显著降低H₂O₂诱导的细胞死亡，而20 ng/mL HGF没有类似作用。当细胞与5 μmol/L H₂O₂孵育时，检测到VEGF蛋白的表达。此外，VEGF（100 ng/mL）也降低了H₂O₂诱导的ERK和JNK磷酸化。相反，HGF未诱导ERK和JNK磷酸化。
肝癌细胞可能通过VEGF的表达在持续氧化应激下存活。
