NOR1 Enhanced CB1954 Induced Cell Cytotoxicity in HepG2 Is Dependent on Grb2 Mediated MAPK Pathway

Nitroreductase gene NOR₁ is possibly involved in the chemical carcinogenesis of hepatic cancer and nasopharyngeal carcinoma. We have demonstrted that NOR₁ overexpression could convert monofunction alalkylating agent, CB1954 ([5-aziridin-1-yl]-2,4-dinitrobenzamide) into a toxic form by reduction of the 4-nitro group of CB1954 and enhancing cell killing in nasopharyngeal carcinoma(NPC) cell line CNE₁. However, the mechanisms are not known.Using cDNA microarrys and quantitative Real-time PCR, we previously found that NOR₁ increased the expression of Grb2 mRNA by 4.8 fold in hepatic cancer cell HepG2. In the present study, we found that NOR1 increased the Grb2 protein expression by 3 fold and enhanced the CB1954 induced cell killing in HepG2 cells ,and the cell cytotoxicity could be inhibited by adding tyrosine kinase inhibitor genestein or by stable transfection of Grb2 shRNA (pU6⁺²⁷-shGrb2) to silence the expression of Grb2. Western blot analysis showed that Grb2 downexpression could reduce the activity of MAPK. Moreover, inhibiting the activatiton of MAPK by MEK inhibtor PD98059 suppressed CB1954 induced cell killing. These results suggested that NOR1 gene enhanced CB1954 cell cytotoxicity through upregulating expression of Grb2 and activiation of MAPK signal transduction in HepG2 cells.