Inhibition of Grb2‑mediated activation of MAPK signal transduction suppresses NOR1/CB1954‑induced cytotoxicity in the HepG2 cell line

Gui,Rong; Li,Dengqing; Qi,Guannan; Suhad,Ali; Nie,Xinmin

doi:10.3892/ol.2012.774

Inhibition of Grb2‑mediated activation of MAPK signal transduction suppresses NOR1/CB1954‑induced cytotoxicity in the HepG2 cell line

Authors:
- Rong Gui
- Dengqing Li
- Guannan Qi
- Ali Suhad
- Xinmin Nie
View Affiliations

Affiliations: Clinical Laboratory Centre of the Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, P.R. China, Clinical Laboratory Centre of the Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, P.R. China, Hormones and Cancer Research Unit, Royal Victoria Hospital, McGill University, Quebec H3A 1A1, Canada
Published online on: June 27, 2012 https://doi.org/10.3892/ol.2012.774
Pages: 566-570

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

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 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.

View Figures

View References

1	Zhang B, Nie X, Xiao B, Xiang J, Shen S, Gong J, Zhou M, et al: Identification of tissue-specific genes in nasopharyngeal epithelial tissue and differentially expressed genes in nasopharyngeal carcinoma by suppression subtractive hybridization and cDNA microarray. Genes Chromosomes Cancer. 38:80–90. 2003. View Article : Google Scholar
2	Nie XM, Zhang BC, Li XL, Xiang JJ, Xiao BY, Ma J, Zhou M, et al: Cloning, expression and mutation analysis of NOR₁, a novel human gene down-regulated in HNE1 nasopharyngeal carcinoma cell line. J Cancer Res Clin Oncol. 129:410–414. 2003.PubMed/NCBI
3	Nie XM, Zhou M, Tang K, Zhang BC, Xiong W, Lu HB, Li XL, et al: Molecular cloning and characterization of a novel nitroreductase gene, NOR₁, possibly involved in chemical carcinogenesis of NPC. Chin J Biochem Mol Biol. 19:423–428. 2003.
4	Poirier S, Bouvier G, Malaveille C, Ohshima H, Shao YM, Hubert A, Zeng Y, et al: Volatile nitrosamine levels and genotoxicity of food samples from high-risk areas for nasopharyngeal carcinoma before and after nirosation. Int J Cancer. 44:1088–1094. 1989. View Article : Google Scholar : PubMed/NCBI
5	Wang Y, Ausman LM, Greenberg AS, Russell RM and Wang XD: Nonalcoholic steatohepatitis induced by a high-fat diet promotes diethylnitrosamine-initiate early hepatocarcinogenesis in rats. Int J Cancer. 124:540–546. 2009. View Article : Google Scholar
6	Liu C and Russell RM: Nutrition and gastric cancer risk: an update. Nutr Rev. 66:237–249. 2008. View Article : Google Scholar : PubMed/NCBI
7	Bartsch H, Pignatelli B, Calmels S and Ohshima H: Inhibition of nitrosation. Basic Life Sci. 61:27–44. 1993.
8	Lowenstein EJ, Daly RJ, Batzer AG, Li W, Margolis B, Lammers R, Ullrich A, et al: The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 70:431–442. 1992. View Article : Google Scholar : PubMed/NCBI
9	Rozakis-Adcock M, Fernley R, Wade J, Pawson T and Bowtell D: The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature. 363:83–85. 1993. View Article : Google Scholar : PubMed/NCBI
10	Skolnik EY, Batzer A, Li N, Lee CH, Lowenstein E, Mohammadi M, Margolis B and Schlessinger J: The function of GRB2 in linking the insulin receptor to Ras signaling pathways. Science. 260:1953–1955. 1993. View Article : Google Scholar : PubMed/NCBI
11	Daly RJ, Gu H, Parmar J, Malaney S, Lyons RJ, Kairouz R, Head DR, et al: The docking protein Gab2 is overexpressed and estrogen regulated in human breast cancer. Oncogene. 21:5175–5181. 2002. View Article : Google Scholar : PubMed/NCBI
12	Nguyen DH, Webb DJ, Catling AD, Song Q, Dhakephalkar A, Weber MJ, Ravichandran KS and Gonias SL: Urokinase-type plasminogen activator stimulates the Ras/extracellular signal-regulated kinase (ERK) signaling pathway and MCF-7 cell migration by a mechanism that requires focal adhesion kinase, Src, and Shc. Rapid dissociation of GRB2/Sps-Shc complex is associated with the transient phosphorylation of ERK in urokinase-treated cells. J Biol Chem. 275:19382–19388. 2000.
13	Normanno N, Campiglio M, Maiello MR, De Luca A, Mancino M, Gallo M, D'Alessio A and Menard S: Breast cancer cells with acquired resistance to the EGFR tyrosine kinase inhibitor gefitinib show persistent activation of MAPK signaling. Breast Cancer Res Treat. 112:25–33. 2008. View Article : Google Scholar : PubMed/NCBI
14	Martínez N, García-Domínguez CA, Domingo B, Oliva JL, Zarich N, Sánchez A, Gutiérrez-Eisman S, et al: Sprouty2 binds Grb2 at two different proline-rich regions, and the mechanism of ERK inhibition is independent of this interaction. Cell Signal. 19:2277–2285. 2007.PubMed/NCBI
15	Diwan BA, Ramakrishna G, Anderson LM and Ramljak D: Overexpression of Grb2 in inflammatory lesions and preneoplastic foci and tumors induced by N-nitrosodimethylamine in Helicobacter hepaticus-infected and -noninfected A/J mice. Toxicol Pathol. 28:548–554. 2000. View Article : Google Scholar : PubMed/NCBI
16	Li DQ, Tang H, Gui R and Nie XM: Changes in global gene expression induced by NOR₁ over expression in HepG2 cells. Prog Biochem Biophys. 35:457–464. 2008.
17	Dachs GU, Tupper J and Tozer GM: From bench to bedside for gene-directed enzyme prodrug therapy of cancer. Anticancer Drugs. 16:349–359. 2005. View Article : Google Scholar : PubMed/NCBI
18	Djeha HA, Todryk SM, Pelech S, Wrighton CJ, Irvine AS, Mountain A and Lipinski KS: Antitumor immune responses mediated by adenoviral GDEPT using nitroreductase/CB1954 is enhanced by high-level coexpression of heat shock protein 70. Cancer Gene Ther. 12:560–571. 2005. View Article : Google Scholar : PubMed/NCBI
19	Helsby NA, Ferry DM, Patterson AV, Pullen SM and Wilson WR: 2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT. Br J Cancer. 90:1084–1092. 2004. View Article : Google Scholar : PubMed/NCBI
20	Tang MH, Helsby NA, Wilson WR and Tingle MD: Aerobic 2- and 4-nitroreduction of CB 1954 by human liver. Toxicology. 216:129–139. 2005. View Article : Google Scholar : PubMed/NCBI
21	Clark SG, Stern MJ and Horvitz HR: C. elegans cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains. Nature. 356:340–344. 1992. View Article : Google Scholar