Carcinogenesis Advance Access published online on February 22, 2008
Carcinogenesis, doi:10.1093/carcin/bgn055
Requirement for Ras/Raf/ERK pathway in naringin-induced G1-cell cycle arrest via p21WAF1 expression
1 Department of Food and Biotechnology, Chungju National University, Chungju, Chungbuk 380-702, South Korea
2 Department of Food and Nutrition, Brain Korea 21 Project, Yonsei University, Seoul 120-749, Korea
3 Department of Biotechnology, Juseong College, Chungbuk 363-794, Korea
4 Department of Urology, Chungbuk National University College of Medicine, Cheongju, Chungbuk 361-763, South Korea
** Corresponding author: Sung-Kwon Moon, Department of Food and Biotechnology, Chungju National University, 123 Geomdan-ri Iryu-myeon, Chungju, Chungbuk, 380-702, Korea, (Tel: 82-43-841-5250, Fax: 82-43-841-5240, Email: sumoon66{at}dreamwiz.com)
Naringin, an active flavonoid found in citrus fruit extracts, has pharmacological utility. The present study identified a novel mechanism of the anti-cancer effects of naringin in urinary bladder cancer cells. Naringin treatment resulted in significant dose-dependent growth-inhibition together with G1-phase cell cycle arrest at a dose of 100 µM (IC50) in 5637 cells. In addition, naringin treatment strongly induced p21WAF1 expression, independent of the p53 pathway, and down-regulated expression of cyclins and CDKs. Moreover, treatment with naringin induced phosphorylation of ERK, p38 MAP kinase, and JNK. Among the pathways examined, only PD98059, an ERK-specific inhibitor, blocked naringin-dependent p21WAF1 expression. Consistently, blockade of ERK function reversed naringin-mediated inhibition of cell proliferation and decreased cell cycle proteins. Furthermore, naringin treatment increased both Ras and Raf activation. Transfection of cells with dominant negative Ras (RasN17) and Raf (RafS621A) mutant genes suppressed naringin-induced ERK activity and p21WAF1 expression. Finally, the naringin-induced reduction in cell proliferation and cell cycle proteins also was abolished in the presence of RasN17 and RafS621A mutant genes. These data demonstrate that the Ras/Raf/ERK pathway participates in p21WAF1 induction, subsequently leading to a decrease in the levels of cyclin D1/CDK4 and cyclin E/CDK2 complexes and naringin-dependent inhibition of cell growth. Overall, these unexpected findings concerning the molecular mechanisms of naringin in 5637 cancer cells provide a theoretical basis for therapeutic use of flavonoids to treat malignancies.
Key Words: Naringin • bladder cancer 5637 cells • ERK • G1-phase cell cycle arrest • p21WAF1 • Ras • Raf
* These authors contributed equally to this work.
Received October 9, 2007; revised February 5, 2008; accepted February 16, 2008.