Inhibition of Grb2-mediated activation of MAPK signal transduction suppresses NOR1/CB1954-induced cytotoxicity in the HepG2 cell line
Inhibition of Grb2-mediated activation of MAPK signal transduction suppresses NOR1/CB1954-induced cytotoxicity in the HepG2 cell line
复制标题
抑制 Grb2 介导的 MAPK 信号转导激活可抑制 NOR1/CB1954 诱导的 HepG2 细胞系细胞毒性
DOI：
10.3892/ol.2012.774
复制
发表时间：
2012-09-01
期刊：
影响因子：
2.9
通讯作者：
Nie, Xinmin
中科院分区：
文献类型：
--
作者：
Gui, Rong;Li, Dengqing;Nie, Xinmin
NOR 1基因可能参与了肝癌和鼻咽癌的化学致癌过程。我们先前已经证明，NOR 1过表达能够通过还原CB 1954的4-硝基将单功能烷化剂5-（氮丙啶-1-基）-2，4-二硝基苯甲酰胺（CB 1954）转化为毒性形式。毒性CB 1954能够增强NPC细胞系CNE 1中的细胞杀伤;然而，潜在的机制仍然未知。使用cDNA微阵列和定量实时PCR，我们以前发现，NOR 1增加生长因子受体结合蛋白2（Grb 2）mRNA的表达在人肝癌细胞系HepG 2的4.8倍。在本研究中，我们发现NOR 1在HepG 2细胞中使Grb 2蛋白表达增加3倍。此外，我们证明NOR 1增强CB 1954诱导的HepG 2细胞杀伤，酪氨酸激酶抑制剂genistein或稳定转染Grb 2小发夹RNA（shRNA）pU 6（+27）-shGrb 2以沉默Grb 2的表达抑制细胞毒性。Western blot分析表明，Grb 2下调可能会降低丝裂原活化蛋白激酶（MAPK）的活性。使用甲基乙基酮（MEK）阻断剂PD 98059抑制MAPK的活化可抑制CB 1954诱导的细胞杀伤。这些结果表明，NOR 1基因增强CB 1954介导的细胞毒性通过上调Grb 2表达和激活MAPK信号转导在HepG 2细胞系。
The nitroreductase oxidored-nitro domain containing protein 1 (NOR1) gene may be involved in the chemical carcinogenesis of hepatic cancer and nasopharyngeal carcinoma (NPC). We have previously demonstrated that NOR1 overexpression is capable of converting the monofunctional alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) into a toxic form by reducing the 4-nitro group of CB1954. Toxic CB1954 is able to enhance cell killing in the NPC cell line CNE1; however, the underlying mechanisms remain unknown. Using cDNA microarrays and quantitative real-time PCR, we previously discovered that NOR1 increases the expression of growth factor receptor-bound protein 2 (Grb2) mRNA by 4.8-fold in the human hepatocellular carcinoma cell line HepG2. In the present study, we revealed that NOR1 increased Grb2 protein expression by 3-fold in HepG2 cells. Additionally, we demonstrated that NOR1 enhanced CB1954-induced cell killing in HepG2 cells, and cell cytotoxicity was inhibited with the tyrosine kinase inhibitor genistein, or by stable transfection of Grb2 small hairpin RNA (shRNA) pU6(+27)-shGrb2 to silence the expression of Grb2. Western blot analysis revealed that Grb2 downregulation may reduce the activity of the mitogen-activated protein kinase (MAPK). Inhibiting the activation of MAPK using the methyl ethyl ketone (MEK) inhibtor PD98059 suppressed CB1954-induced cell killing. These results suggested that the NOR1 gene enhances CB1954-mediated cell cytotoxicity through the upregulation of Grb2 expression and the activation of MAPK signal transduction in the HepG2 cell line.