Abstract
Scope:
The objective of this study was to elucidate molecular mechanisms behind the antitumor activities of the isothiocyanate sulforaphane (SFN) in colorectal cancer cells.
Methods and results:
Cell growth was determined by BrdU incorporation and crystal violet staining. Protein levels were examined by Western blot analysis. Ornithine decarboxylase (ODC) activity was assayed radiometrically. Reverse transcriptase-PCR was used for measuring mRNA expression. For reporter gene assays plasmids were transfected into cells via lipofection and luciferase activity was measured luminometrically. Acetyl-histone H3 and H4 chromatin immunoprecipitation (ChIP) assays were performed followed by PCR with TGF-β-receptor II promoter specific primers. We could show that SFN-mediated cell growth inhibition closely correlates with a dose-dependent reduction of protein expression and enzymatic activity of ODC. This effect seems to be due to reduced protein levels and transactivation activity of transcription factor c-myc, a direct regulator of ODC expression, as a consequence of SFN-induced TGF-β/Smad signaling. The coherency of these results was further confirmed by using TGF-β receptor kinase inhibitor SB431542, which largely abolishes inhibitory effects of SFN on both, ODC activity and cell growth.
Conclusion:
Since elevated ODC enzyme activity is associated with enhanced tumor development, SFN may be a dietary phytochemical with potential to prevent carcinogenesis.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Anticarcinogenic Agents / pharmacology
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Caco-2 Cells
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Cell Proliferation / drug effects
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Colorectal Neoplasms / drug therapy*
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DNA-Binding Proteins / antagonists & inhibitors
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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Gene Expression Regulation, Neoplastic / drug effects
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Humans
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Isothiocyanates / pharmacology*
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Ornithine Decarboxylase / metabolism*
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Protein Kinase Inhibitors / pharmacology
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism
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RNA, Messenger / metabolism
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Receptor, Transforming Growth Factor-beta Type I
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Receptor, Transforming Growth Factor-beta Type II
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Receptors, Transforming Growth Factor beta / antagonists & inhibitors
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Receptors, Transforming Growth Factor beta / genetics
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Receptors, Transforming Growth Factor beta / metabolism
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Signal Transduction / drug effects*
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Smad Proteins / genetics
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Smad Proteins / metabolism*
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Smad3 Protein / genetics
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Smad3 Protein / metabolism
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Smad4 Protein / genetics
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Smad4 Protein / metabolism
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Sulfoxides
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Thiocyanates / pharmacology*
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Transforming Growth Factor beta / genetics
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Transforming Growth Factor beta / metabolism*
Substances
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Anticarcinogenic Agents
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DNA-Binding Proteins
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Isothiocyanates
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Protein Kinase Inhibitors
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RNA, Messenger
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Receptors, Transforming Growth Factor beta
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SMAD3 protein, human
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SMAD4 protein, human
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Smad Proteins
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Smad3 Protein
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Smad4 Protein
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Sulfoxides
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Thiocyanates
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Transforming Growth Factor beta
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Protein Serine-Threonine Kinases
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Receptor, Transforming Growth Factor-beta Type I
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Receptor, Transforming Growth Factor-beta Type II
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Ornithine Decarboxylase
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sulforaphane