Carcinogenesis Advance Access originally published online on February 22, 2008
Carcinogenesis 2008 29(9):1701-1709; doi:10.1093/carcin/bgn055
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Requirement for Ras/Raf/ERK pathway in naringin-induced G1-cell-cycle arrest via p21WAF1 expression
Department of Food and Biotechnology, Chungju National University, Chungju, Chungbuk 380-702, South Korea
1 Department of Food and Nutrition, Brain Korea 21 Project, Yonsei University, Seoul 120-749, Korea
2 Department of Biotechnology, Juseong College, Chungbuk 363-794, Korea
3 Department of Urology, College of Medicine, Chungbuk National University, Cheongju, Chungbuk 361-763, South Korea
* To whom correspondence should be addressed. 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 anticancer 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 (the half maximal inhibitory concentration) in 5637 cells. In addition, naringin treatment strongly induced p21WAF1 expression, independent of the p53 pathway, and downregulated expression of cyclins and cyclin dependent kinases (CDKs). Moreover, treatment with naringin induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. 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 the therapeutic use of flavonoids to treat malignancies.
Abbreviations: CDK, cyclin dependent kinase; ERK, extracellular signal-regulated kinase; EV, empty vector; GTP, guanosine triphosphate; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA
These authors contributed equally to this work. Received October 9, 2007; revised February 5, 2008; accepted February 16, 2008.