Requirement for Ras/Raf/ERK pathway in naringin-induced G1-cell-cycle arrest via p21WAF1 expression.
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 G(1)-phase cell-cycle arrest at a dose of 100 microM (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.
Kim
Dong-Il
DI
Department of Food and Biotechnology, Chungju National University, Chungju, Chungbuk 380-702, South Korea.
Lee
Se-Jung
SJ
Lee
Soo-Bok
SB
Park
Keerang
K
Kim
Wun-Jae
WJ
Moon
Sung-Kwon
SK
D049109:Cell Proliferation; D002478:Cells, Cultured; D056741:Cyclin D; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D044950:Flavanones / Q000494:pharmacology*; D016193:G1 Phase / Q000187:drug effects*; D005799:Genes, Dominant; D006801:Humans; D047468:Immunoprecipitation; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D015513:Oncogene Proteins / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C475820:CCNE1 protein, human; D056741:Cyclin D; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D044950:Flavanones; D015513:Oncogene Proteins; D034741:RNA, Small Interfering; D019908:Proto-Oncogene Proteins c-raf; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D016283:Proto-Oncogene Proteins p21(ras); C005274:naringin