Boye A, Kan H, Wu C, Jiang Y, Yang X, He S, Yang Y
Department of Pharmacology and Institute of Natural Medicine, Anhui Medical University, Hefei, 230032, China.
Tumour Biol. 2015 May;36(5):3643-51. doi: 10.1007/s13277-014-3002-x. Epub 2015 Jan 7.
The aim of this study was to investigate the mitogen-activated protein kinase (MAPK) pathway, which crosstalk with TGF-β/Smad signaling via linker phosphorylation of Smad2/3 to promote hepatocarcinogenesis. After DEN-induced hepatocellular carcinoma (HCC) in rats showed increased phosphorylation of JNK1/2, p38, and ERK1/2, we next antagonized TGF-β1-induced phosphorylation of JNK1/2, p38, ERK1/2, Smad2/3 signaling in HepG2 cells using SP600125, SB203580, and PD98059, respectively. Cell proliferation and invasion were assessed by MTT assay and transwell invasion chambers, respectively. Smad2/3, Smad4, and Smad7 expressions and PAI-1 messenger RNA (mRNA) transcription were measured by using immuno-precipitation/immuno-blotting and real-time RT-PCR, respectively. All the MAPK-specific inhibitors suppressed cell invasion, while all but PD98059 suppressed cell proliferation. Both SP600125 and SB203580 blocked pSmad2C/L and oncogenic pSmad3L. PD98059 blocked pSmad2L but had no effect on elevated pSmad2C and oncogenic pSmad3L. All but PD98059 blocked Smad2/3/4 complex formation and restored Smad7 expression, while all the three MAPK-Specific inhibitors repressed PAI-1 mRNA transcription. Both SP600125 and SB203580 inhibited HepG2 cells' proliferation and invasion by blocking oncogenic pSmad3L and Smad2/3/4 complex formation. PD98059 repressed PAI-1 mRNA by an unknown mechanism.
本研究的目的是探究丝裂原活化蛋白激酶（MAPK）通路，该通路通过Smad2/3的接头磷酸化与TGF-β/Smad信号传导相互作用，以促进肝癌发生。在二乙基亚硝胺（DEN）诱导的大鼠肝细胞癌（HCC）中JNK1/2、p38和ERK1/2的磷酸化增加后，接下来我们分别使用SP600125、SB203580和PD98059拮抗HepG2细胞中TGF-β1诱导的JNK1/2、p38、ERK1/2、Smad2/3信号传导的磷酸化。分别通过MTT法和Transwell侵袭小室评估细胞增殖和侵袭。分别使用免疫沉淀/免疫印迹和实时RT-PCR检测Smad2/3、Smad4和Smad7的表达以及PAI-1信使核糖核酸（mRNA）转录。所有MAPK特异性抑制剂均抑制细胞侵袭，而除PD98059外的所有抑制剂均抑制细胞增殖。SP600125和SB203580均阻断pSmad2C/L和致癌性pSmad3L。PD98059阻断pSmad2L，但对升高的pSmad2C和致癌性pSmad3L无影响。除PD98059外的所有抑制剂均阻断Smad2/3/4复合物形成并恢复Smad7表达，而所有三种MAPK特异性抑制剂均抑制PAI-1 mRNA转录。SP600125和SB203580均通过阻断致癌性pSmad3L和Smad2/3/4复合物形成来抑制HepG2细胞的增殖和侵袭。PD98059通过未知机制抑制PAI-1 mRNA。
