title: Inhibition of Grb2-mediated activation of MAPK signal transduction suppresses NOR1/CB1954-induced cytotoxicity in the HepG2 cell line.
abstract: 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 NOR1overexpression 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 NOR1increases 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 NOR1increased Grb2 protein expression by 3-fold in HepG2 cells. Additionally, we demonstrated that NOR1enhanced 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 NOR1gene enhances CB1954-mediated cell cytotoxicity through the upregulation of Grb2 expression and the activation of MAPK signal transduction in the HepG2 cell line.
authors.affiliation: Clinical Laboratory Centre of the Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, P.R. China.
authors.forename: Rong
authors.initials: R
authors.lastname: Gui
authors.forename: Dengqing
authors.initials: D
authors.lastname: Li
authors.forename: Guannan
authors.initials: G
authors.lastname: Qi
authors.forename: Ali
authors.initials: A
authors.lastname: Suhad
authors.forename: Xinmin
authors.initials: X
authors.lastname: Nie
keywords: Grb2; MAPK; NOR1; siRNA