Journal of the National Cancer Institute Advance Access originally published online on July 28, 2009
JNCI Journal of the National Cancer Institute 2009 101(16):1141-1155; doi:10.1093/jnci/djp227
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© The Author 2009. Published by Oxford University Press.
ARTICLES |
MEK4 Function, Genistein Treatment, and Invasion of Human Prostate Cancer Cells
Affiliations of authors: Department of Medicine (LX, YD, KW, XH, RCB), Department of Urology (WJC), Department of Pathology (XJY), Department of Molecular Pharmacology (WFA), Department of Preventive Medicine (BJ), and Department of Chemistry (KAS), Robert H. Lurie Cancer Center and Center for Drug Discovery and Chemical Biology of Northwestern University, Chicago, IL; Institute for Drug Development, San Antonio, TX (JK); Department of Medicine, University of Washington School of Medicine, Seattle, WA (RBM)
Correspondence to: Raymond C. Bergan, MD, Department of Medicine, Northwestern University, Olson 8321, 710 N Fairbanks, Chicago, IL 60610 (e-mail: r-bergan{at}northwestern.edu).
Background: Dietary intake of genistein by patients with prostate cancer has been associated with decreased metastasis and mortality. Genistein blocks activation of p38 mitogen-activated protein kinase and thus inhibits matrix metalloproteinase-2 (MMP-2) expression and cell invasion in cultured cells and inhibits metastasis of human prostate cancer cells in mice. We investigated the target for genistein in prostate cancer cells.
Methods: Prostate cell lines PC3-M, PC3, 1532NPTX, 1542NPTX, 1532CPTX, and 1542CPTX were used. All cell lines were transiently transfected with a constitutively active mitogen-activated protein kinase kinase 4 (MEK4) expression vector (to increase MEK4 expression), small interfering RNA against MEK4 (to decrease MEK4 expression), or corresponding control constructs. Cell invasion was assessed by a Boyden chamber assay. Gene expression was assessed by a quantitative reverse transcription–polymerase chain reaction. Protein expression was assessed by Western blot analysis. Modeller and AutoDock programs were used for modeling of the structure of MEK4 protein and ligand docking, respectively. MMP-2 transcript levels were assessed in normal prostate epithelial cells from 24 patients with prostate cancer from a phase II randomized trial comparing genistein treatment with no treatment. Statistical significance required a P value of .050 or less. All statistical tests were two-sided.
Results: Overexpression of MEK4 increased MMP-2 expression and cell invasion in all six cell lines. Decreased MEK4 expression had the opposite effects. Modeling showed that genistein bound to the active site of MEK4. Genistein inhibited MEK4 kinase activity with a half maximal inhibitory concentration of 0.40 µM (95% confidence interval [CI] = 0.36 to 0.45 µM). The MMP-2 transcript level in normal prostate epithelial cells was statistically significantly higher in the untreated group (100%) than in the genistein-treated group (24%; difference = 76%, 95% CI = 38% to 115%; P = .045).
Conclusions: We identified MEK4 as a proinvasion protein in six human prostate cancer cell lines and the target for genistein. We showed, to our knowledge for the first time, that genistein treatment, compared with no treatment, was associated with decreased levels of MMP-2 transcripts in normal prostate cells from prostate cancer–containing tissue.
| CONTEXT AND CAVEATS Prior knowledge Consumption of foods with high levels of genistein has been associated with decreased metastasis and mortality among patients with prostate cancer. Genistein has been shown to inhibit matrix metalloproteinase-2 (MMP-2) expression and cell invasion. Study design Six prostate cell lines were used to study the effects of genistein treatment on the expression of mitogen-activated protein kinase kinase 4 (MEK4), a protein kinase, and various activities, including cell invasion. The structure of MEK4 protein was modeled, and genistein docking was studied. MMP-2 expression was assessed in normal prostate epithelial cells from 24 patients with prostate cancer from a phase II randomized trial comparing genistein treatment with no treatment. Contribution MEK4 expression was associated with MMP-2 expression and cell invasion in all six cell lines. Genistein appeared able to bind to the active site of MEK4 by computer modeling. Genistein inhibited MEK4 kinase activity. MMP-2 expression was statistically significantly higher in normal prostate epithelial cells from untreated patients than in those from genistein-treated patients. Implications MEK4 was identified as a proinvasion protein and a target for genistein. These results may indicate a mechanism to link high dietary consumption of genistein-containing foods with lower rates of prostate cancer metastasis and mortality. Limitations The possibility that genistein has at least one more target cannot be ruled out. MEK3 function was not investigated in all six cell lines. The target for genistein action has not yet been localized to a specific component in a signaling pathway in human tissue specimens. From the Editors
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Manuscript received August 11, 2008; revised June 2, 2009; accepted June 12, 2009.
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J Natl Cancer Inst 2009 101: 1101.