G. T. Bowden

Raf and MEK Protein Kinases Are Direct Molecular Targets for the Chemopreventive Effect of Quercetin, a Major Flavonol in Red Wine

Considerable attention has focused on the health-promoting effects of red wine and its nonflavonoid polyphenol compound resveratrol. However, the underlying molecular mechanisms and molecular target(s) of red wine or other potentially active ingredients in red wine remain unknown. Here, we report that red wine extract (RWE) or the red wine flavonoid quercetin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation of JB6 promotion-sensitive mouse skin epidermal (JB6 P+) cells. The activation of activator protein-1 and nuclear factor-kappaB induced by TPA was dose dependently inhibited by RWE or quercetin treatment. Western blot and kinase assay data revealed that RWE or quercetin inhibited mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) 1 and Raf1 kinase activities and subsequently attenuated TPA-induced phosphorylation of ERK/p90 ribosomal S6 kinase. Although either RWE or quercetin suppressed Raf1 kinase activity, they were more effective in inhibiting MEK1 activity. Importantly, quercetin exerted stronger inhibitory effects than PD098059, a well-known pharmacologic inhibitor of MEK. Resveratrol did not affect either MEK1 or Raf1 kinase activity. Pull-down assays revealed that RWE or quercetin (but not resveratrol) bound with either MEK1 or Raf1. RWE or quercetin also dose dependently suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are involved in the activation of MEK/ERK signaling. Docking data suggested that quercetin, but not resveratrol, formed a hydrogen bond with the backbone amide group of Ser(212), which is the key interaction for stabilizing the inactive conformation of the activation loop of MEK1.

Myricetin Suppresses UVB-Induced Skin Cancer by Targeting Fyn

Skin cancer is currently the most common type of human cancer in Americans. Myricetin, a naturally occurring phytochemical, has potent anticancer-promoting activity and contributes to the chemopreventive potential of several foods, including red wine. Here, we show that myricetin suppresses UVB-induced cyclooxygenase-2 (COX-2) expression in mouse skin epidermal JB6 P+ cells. The activation of activator protein-1 and nuclear factor-kappaB induced by UVB was dose-dependently inhibited by myricetin treatment. Western blot and kinase assay data revealed that myricetin inhibited Fyn kinase activity and subsequently attenuated UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays revealed that myricetin competitively bound with ATP to suppress Fyn kinase activity. Importantly, myricetin exerted similar inhibitory effects compared with 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a well-known pharmacologic inhibitor of Fyn. In vivo mouse skin data also revealed that myricetin inhibited Fyn kinase activity directly and subsequently attenuated UVB-induced COX-2 expression. Mouse skin tumorigenesis data clearly showed that pretreatment with myricetin significantly suppressed UVB-induced skin tumor incidence in a dose-dependent manner. Docking data suggest that myricetin is easily docked to the ATP-binding site of Fyn, which is located between the N and C lobes of the kinase domain. Overall, these results indicated that myricetin exerts potent chemopreventive activity mainly by targeting Fyn in skin carcinogenesis.

UVB Irradiation-induced Activator Protein-1 Activation Correlates with Increased c-fos Gene Expression in a Human Keratinocyte Cell Line

The effects of UVB irradiation on transcription factor activator protein-1 (AP-1) DNA binding and AP-1 transactivation were studied in a human keratinocyte cell line, HaCaT. UVB-induced AP-1 binding to a consensus AP-1 binding site was observed by gel shift assays with maximum stimulation at 12 h after UVB irradiation. A promoter region of the human collagenase-1 gene containing the same AP-1 binding sequence linked to a luciferase reporter gene was stably transfected into HaCaT cells. UVB irradiation significantly increased luciferase activity in these stably transfected cells, with maximum activity observed at 24 h after UVB irradiation. c-Fos and Jun D were identified by antibody clearing assays as the main components of the bound AP-1 complexes. Inhibition of transcription with actinomycin D and inhibition of protein synthesis with cycloheximide significantly abrogated the effect of UVB on AP-1 DNA binding, indicating that transcription and translation were required for AP-1 activation. Northern and Western analyses revealed a correlation between increased AP-1 activity and accumulation of c-fos mRNA and c-Fos protein after UVB irradiation. UVB irradiation increased c-fos transcription in HaCaT cells stably transfected with a plasmid containing the human c-fos promoter driving a luciferase reporter gene. These results suggest that increased c-fos expression may play an important role in UVB-induced AP-1 activation in HaCaT cells.

Extracellular Signal-regulated Kinase 1/2-mediated Phosphorylation of JunD and FosB Is Required for Okadaic Acid-induced Activator Protein 1 Activation

Previously, we reported that in papilloma-producing 308 mouse keratinocytes, the tumor promoter okadaic acid, a serine-threonine phosphatase inhibitor, increased binding of activator protein 1 (AP-1) to a consensus 12-O-tetradecanoylphorbol-13-acetate-responsive element (Rosenberger, S. F., and Bowden, G. T. (1996) Oncogene 12, 2301–2308). In this study, we investigated the correlation between AP-1 DNA binding and transactivation and examined molecular mechanisms involved in this process. Using a luciferase reporter driven by region −74 to +63 of the human collagenase gene, we demonstrated induction of AP-1-mediated transcription following okadaic acid treatment. By performing in vitro kinase assays, we found elevated activities of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase. The ERK-1/2-specific inhibitor PD 98059 completely abrogated okadaic acid-induced AP-1 transactivation without altering AP-1 expression, DNA binding, or complex composition. Phosphorylation analyses indicated that inhibition of ERK-1/2 decreased okadaic acid-elevated phosphorylation of JunD and FosB. To further examine the role of JunD and FosB in okadaic acid-induced AP-1 transactivation, we generated fusion proteins of the DNA-binding domain of the yeast transcription factor Gal4 and the transactivation domain of either JunD or FosB. Cotransfection experiments of these constructs with a Gal4-luciferase reporter demonstrated that both JunD and FosB are required for okadaic acid-induced transcription. Treatment with PD 98059 reduced JunD/FosB-dependent transactivation, suggesting that ERK-1/2-mediated phosphorylation is a critical component in this process.

Regulation of DNA binding and transactivation in p53 by nuclear localization and phosphorylation

Compelling evidence indicates that p53 acts as a transcription factor and that this activity is regulated by several factors including subcellular localization and phosphorylation status of the protein. To learn more about how these two processes determine whether p53 becomes activated, we studied the temperature sensitive murine p53, p53val135. At nonpermissive temperatures, p53val135 remains sequestered in the cytoplasm of cells which express it. Electrophoretic mobility shift assays demonstrated that, under these conditions, the protein lacked DNA binding activity. However, by shifting to the permissive temperature, p53val135 became concentrated in the nucleus, hyperphosphorylated, and had acquired the ability to bind DNA in a sequence specific manner. This was accompanied by the induction of two p53 regulated genes, mdm2 and p21waf1, which indicated that p53val135 had become an active transcription factor. Two dimensional gel electrophoresis and tryptic peptide mapping showed that entry into the nucleus resulted in the appearance of new phosphorylated isoforms and that the protein had become extensively phosphorylation at the N-terminus. Notably, phosphorylation at the N-terminus occurred only in the nucleus, whereas phosphorylation at the C-terminus could occur in both the cytoplasm and the nucleus. Based on these observations, we suggest that phosphorylation of p53s N-terminus is compartmentally restricted.

Interleukin-1β Secreted from Monocytic Cells Induces the Expression of Matrilysin in the Prostatic Cell Line LNCaP

Matrilysin is a matrix metalloprotease that is overexpressed in cancer cells of epithelial origin and in normal tissues during events involving matrix remodeling such as the cycling endometrium. We previously observed that inflamed ductule and acinar epithelia in the prostate also overexpress matrilysin. The presence of infiltrating macrophages in these areas prompted us to determine if factors secreted from monocytes could induce matrilysin expression in a human prostatic cell line. Conditioned media collected from the monocyte cell line THP-1 following lipopolysaccharide treatment substantially induced matrilysin protein and mRNA expression in LNCaP prostate carcinoma cells. Matrilysin expression in LNCaP cells was also induced by recombinant interleukin (IL)-1 (50 pm), but not by equimolar concentrations of recombinant tumor necrosis factor-α or IL-6. The matrilysin-inducing activity of THP-1 conditioned medium was completely abrogated by preincubation with a neutralizing antibody to IL-1β. Transient transfection analyses with a chimeric human matrilysin promoter-chloramphenicol acetyltransferase reporter construct demonstrated that IL-1β activates transcription through the matrilysin promoter in LNCaP cells. This is the first report of matrilysin induction by an inflammatory cytokine in a cell line of epithelial origin, and the results suggest a potential mechanism for the overexpression of matrilysin in inflamed ducts and glands of the prostate.

JunB negatively regulates AP-1 activity and cell proliferation of malignant mouse keratinocytes

Abstract Objective: Previously we have shown that a malignant mouse keratinocyte cell line, 10Gy5, has elevated AP-1 transactivation and reduced JunB protein levels compared to its parental benign cell line, 308, and that the tumorigenicity in the 10Gy5 cells could be blocked by a dominant negative c-Jun mutant protein. We wished to determine whether the change in JunB protein levels could account for the elevated AP-1 activity and whether re-expression of JunB in malignant 10Gy5 cells altered their proliferative capacity. Design: In the current study, we reduced JunB expression in benign 308 cells with antisense oligonucleotides and increased JunB expression in malignant 10Gy5 cells by stable transfection of a JunB expression vector. Results: Increased AP-1 activity was detected after treatment of the benign 308 cell line with JunB antisense oligonucleotides that reduced JunB protein levels. Stably JunB-transfected clones of malignant 10Gy5 cells showed decreased AP-1 activity, slowed in vitro cell proliferation and reduced tumor growth when xenografted to athymic nude mice. Conclusion: These findings suggest that expression of JunB protein has a negative effect on malignant tumor cell proliferation in part through its ability to inhibit AP-1 transactivation.

Aberrant expression of fibroblast growth factor receptor-1 in prostate epithelial cells allows induction of promatrilysin expression by fibroblast growth factors

Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins, and there is evidence that they play a role in tumor cell growth, invasion and metastasis. Matrilysin (MMP‐7) is over‐expressed in prostate cancer cells and increases prostate cancer cell invasion. Prostate stromal fibroblasts secrete a factor(s), including fibroblast growth factor‐1 (FGF‐1), which induces promatrilysin expression in the prostate carcinoma cell line LNCaP but not in normal prostate epithelial cells (PrECs). Since FGF‐1 is present in the prostate, an altered sensitivity to FGF‐1 might explain the up‐regulation of matrilysin expression in prostate cancer cells compared to normal prostate epithelium. FGF receptor‐1 (FGFR‐1) is not normally expressed by normal prostate epithelial cells; however, aberrant expression of this receptor has been reported in prostate cancer cells, including the LNCaP cell line. We hypothesized that aberrant expression of FGFR‐1 in PrECs would render them sensitive to induction of promatrilysin expression by recombinant FGF‐1. To test this hypothesis, we transiently transfected PrECs with an FGFR‐1 expression vector, which resulted in over‐expression of FGFR‐1 protein in approximately 40% of cells. FGF‐1 increased promatrilysin expression in FGFR‐1–transfected PrECs 4‐fold over mock‐transfected cells, and this induction was inhibited by a specific FGFR‐1 inhibitor, SU5402, and by co‐expression of a dominant negative FGFR‐1 protein. Our results demonstrate that aberrant FGFR‐1 expression, an epigenetic phenomenon that has been associated with prostate cancer progression, allows induction of promatrilysin expression by FGF‐1 in PrECs.

Inhibition of p38 MAP kinase increases okadaic acid mediated AP-1 expression and DNA binding but has no effect on TRE dependent transcription.

By performing in vitro kinase assays we found in papilloma producing 308 mouse keratinocytes that okadaic acid elevated activities of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinases (MAPKs). This okadaic acid mediated activation of MAP kinases correlated with increased AP-1 binding to a consensus TPA responsive element (TRE) and elevated TRE dependent transcription. To determine the role of p38 MAP kinases in these processes we employed the specific p38 MAP kinase inhibitor SB 203580. Using orthophosphate labeling we showed a decrease in phosphorylation of MAPK activated protein kinase-2 (MAPKAP-K2) indicating reduced activity of p38 MAPKs utilizing this kinase as substrate. In contrast, we found that SB 203580 raised activities of ERK-1/2 and JNKs. Electrophoretic mobility shift assays revealed an increase in TRE binding activity in response to SB 203580 most likely resulting from increased expression of the major TRE binding components JunD and FosB as indicated by Western blot analyses. Increased TRE DNA binding failed to lead to increased transactivation correlating with the inability of SB 203580 to increase phosphorylation of these AP-1 proteins. These data indicate that SB 203580 sensitive p38 MAP kinases are not involved in okadaic acid mediated increases in TRE DNA binding and transactivation.

Stability of sulforaphane for topical formulation.

Abstract Context: Sulforaphane (SFN) is a natural compound that has been investigated as a chemopreventive agent. SFN has been shown to inhibit the activator-protein-1 (AP-1) transcription factor and may be effective for inhibition of ultraviolet (UV) induced skin carcinogenesis. This study was designed to investigate the stability of SFN as a function of pH, temperature and in various solvents and formulations. Materials and methods: Stability was analyzed using high-performance liquid chromatography. A potential lead formulation was identified and evaluated in vivo. Results: SFN was determined to undergo apparent first-order degradation kinetics for the conditions explored. It was observed that SFN undergoes base catalyzed degradation. Buffer species and solvent type impacts stability as well. SFN was found to be very sensitive to temperature with degradation rate changing by a factor of nearly 3.1 for every 10 °C change in temperature (at pH 4.0). SFN completely degraded after 30 days in a conventional pharmaceutical cream formulation. Improved stability was observed in organic formulation components. Stability studies were conducted on two nonaqueous topical formulations: a polyethylene glycol (PEG) ointment base and an organic oleaginous base. Conclusion: Topically applied SFN in the PEG base formulation significantly reduced AP-1 activation after UV stimulation in the skin of a transgenic mouse model, indicating that SFN in this formulation retains efficacy in vivo.