过氧化物酶1对人肝癌细胞HepG2细胞皮下移植瘤生长的影响

Effect of Prx-1 on growth of hepatocellular carcinoma xenografts

摘要:

目的  探讨过氧化物酶1（Prx 1）在肝癌形成和恶性进展中的作用。方法  蛋白质印迹法（Western blot）检测远癌肝组织和近癌肝组织中Prx 1、pAkt及Akt表达差异。肝细胞饥饿培养24 h后，10 ng/ml表皮生长因子（EGF）处理10 min，提取总蛋白，Western blot检测Prx 1、pAkt、不同位点磷酸化表皮生长因子受体（EGFR）和总EGFR。Western blot检测检测未处理组、转染无义shRNA组及转染Prx 1 shRNA组HepG2细胞中Prx 1蛋白的表达。DCFH-DA法检测3种HepG2细胞中活性氧簇总量。转染无义shRNA组及转染Prx 1 shRNA组HepG2细胞皮下注射至SCID小鼠，注射后每周测量1次皮下肿瘤体积。Western blot检测两组小鼠皮下肿瘤组织中Prx 1和pAkt的表达情况。结果  与正常肝细胞比较，Prx 1在肝转化细胞中表达升高。Prx 1增强了EGF介导的EGFR和Akt激活。Prx 1表达沉默抑制了HepG2体内成瘤能力。减少HepG2皮下移植瘤的肝转移。Prx 1表达沉默还抑制了Akt体内激活。结论  Prx 1具有调控人肝细胞中EGFR介导信号通路的潜在作用。Prx 1能够通过EGFR-Akt信号通路介导细胞正常至异常转化、肿瘤形成及转移

Abstract:

Objectives To investigate the role of Prx 1 in hepatocellular carcinoma. Methods The expression of Prx-1, pAkt and Akt in distal hepatocellular carcinoma and para- carcinoma tissue was detected by Western blot. Human liver cells were treated with 10 ng/ml EGF for 10 min after 24 hrs after serum starvation. Total proteins were extracted immediately after 10-min EGF treatment and used for Western blot. Equal amount of protein (20 μg) was separated by SDS-PAGE and probed with antibodies specific to Prx 1, pAkt, various phosphorylated forms of EGFR and total EGFR. Prx 1 protein levels in parental (untransfected), scramble, and Prx-1shRNA transfected HepG2 cells were measured by Western blot. Reactive oxygen species (ROS) levels determined from HepG2 sublines by using probe DCFH-DA. HepG2/Prx 1Sc and HepG2/Prx-1KD cells were injected into the dorsal flank of male SCID mice. Tumor size was measured every week after injection. Protein extracted from HepG2/Prx 1Sc and HepG2/Prx 1KD tumor tissues was subjected to Western blot analysis. Equal amount of protein (20 μg) was separated by SDS-PAGE and probed with antibodies specific to Prx-1and pAkt. Results Increased expression of Prx 1 was observed in abnormal (pre-malignant) liver cells compared to their paired normal liver cells. Prx 1 enhances EGF-mediated EGFR and Akt activation. Prx-1 knock-down inhibits tumor growth of HepG2 xenografts in vivo. Prx 1 knock-down reduces liver metastasis of HepG2 xenografts in vivo. Prx-1 knock-down inhibits Akt activation in vivo. Conclusions A potential role for Prx-1 in regulation of EGFR mediated signaling pathways in human liver cells. Prx-1 in mediates normal-to-abnormal cell transition, tumorigenesis, and metastasis through EGFR-Akt signaling pathways.