title: Grape seed extract inhibits EGF-induced and constitutively active mitogenic signaling but activates JNK in human prostate carcinoma DU145 cells: possible role in antiproliferation and apoptosis.
abstract: A loss of functional androgen receptor and an enhanced expression of growth factor receptors and associated ligands are causal genetic events in prostate cancer (PCA) progression. These genetic alterations lead to an epigenetic mechanism where a feedback autocrine loop between membrane receptor and ligand (e.g. EGFR-TGFalpha) results in a constitutive activation of MAPK-Elk1-AP1-mediated mitogenic signaling in human PCA at an advanced and androgen-independent stage. We rationalized that inhibiting these epigenetic events could be useful in controlling advanced PCA growth. Recently, we found that grape seed extract (GSE), a dietary supplement rich in flavonoid procyanidins, inhibits advanced and androgen-independent human PCA DU145 cell growth in culture and nude mice. Here, we performed detailed mechanistic studies to define the effect of GSE on EGFR-Shc-MAPK-Elk1-AP1-mediated mitogenic signaling in DU145 cells. Pretreatment of serum-starved cells with GSE resulted in 70% to almost complete inhibition of EGF-induced EGFR activation and 50% to complete inhibition of Shc activation, which corroborated with a comparable decrease in EGF-induced Shc binding to EGFR. Conversely, EGF-induced ERK1/2 phosphorylation was inhibited only by lower doses of GSE; in fact, higher doses showed an increase. Additional studies showed that GSE alone causes a dose- and time-dependent increase in ERK1/2 phosphorylation in starved DU145 cells that is inhibited by an MEK1 inhibitor PD98059. Independent of this increase in ERK1/2 phosphorylation, GSE showed a strong inhibition of ERK1/2 kinase activity to Elk1 in both cellular and cell-free systems. GSE treatment of cells also inhibited both EGF-induced and constitutively active Elk1 phosphorylation and AP1 activation. GSE treatment also showed DNA synthesis inhibition in starved and EGF-stimulated cells as well as loss of cell viability and apoptotic death that was further increased by adding MEK1 inhibitor. Since GSE strongly induced apoptosis independent of its affect on an increase in phospho-ERK1/2, we hypothesized that apoptotic effect of GSE could be by other mechanism(s) including its effect on stress-associated MAPK, the JNK. Indeed, GSE-treated cells showed a strong and sustained increase in phospho-JNK1/JNK2 levels, JNK activity and phospho-cJun levels. An inhibition of GSE-induced JNK activation by a novel JNK inhibitor SP600125 resulted in a significant reversal of GSE-induced apoptotic death suggesting the involvement of JNK activation by GSE in its apoptosis response. Together, these results suggest that anticancer effects of GSE in PCA be mediated via impairment of EGFR-ERK1/2-Elk1-AP1-mediated mitogenic signaling and activation of JNK causing growth inhibition and apoptosis, respectively.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver 80262, USA.
authors.forename: Alpana
authors.initials: A
authors.lastname: Tyagi
authors.forename: Rajesh
authors.initials: R
authors.lastname: Agarwal
authors.forename: Chapla
authors.initials: C
authors.lastname: Agarwal
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology*; D000873:Anthracenes / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D002474:Cell-Free System; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010936:Plant Extracts / Q000494:pharmacology*; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012639:Seeds / Q000737:chemistry; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000187:drug effects*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D027843:Vitis / Q000737:chemistry*; D050807:ets-Domain Protein Elk-1
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000873:Anthracenes; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; C494112:ELK1 protein, human; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009363:Neoplasm Proteins; D010936:Plant Extracts; D011506:Proteins; D011518:Proto-Oncogene Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; C432165:pyrazolanthrone; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D017346:Protein Serine-Threonine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one