Skip Navigation


Carcinogenesis Advance Access originally published online on December 6, 2006
Carcinogenesis 2007 28(6):1188-1196; doi:10.1093/carcin/bgl241
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
28/6/1188    most recent
bgl241v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (13)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Nonn, L.
Right arrow Articles by Peehl, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nonn, L.
Right arrow Articles by Peehl, D. M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Chemopreventive anti-inflammatory activities of curcumin and other phytochemicals mediated by MAP kinase phosphatase-5 in prostate cells

Larisa Nonn, David Duong and Donna M. Peehl*

Department of Urology, Stanford University, Stanford, CA 94305-5118, USA

* To whom correspondence should be addressed. Tel: +1 650 725 5531; Fax: +1 650 723 4200; Email: dpeehl{at}stanford.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
As inflammation emerges as a risk factor for prostate cancer (PCa), there is potential for chemoprevention by anti-inflammatory agents. Dietary phytochemicals have been shown to have chemopreventive properties which may include anti-inflammatory activities. In this study, we demonstrate a role for mitogen-activated protein kinase phosphatase-5 (MKP5) in mediating anti-inflammatory activities of the phytochemicals curcumin, resveratrol and [6]-gingerol. We utilized the cytokines tumor necrosis factor-{alpha} (TNF{alpha}) and interleukin (IL)-1ß to increase p38-dependent nuclear factor kappa-B (NF{kappa}B) activation and expression of pro-inflammatory genes cyclooxygenase-2 (COX-2), IL-6 and IL-8 in normal prostatic epithelial cells. MKP5 over-expression decreased cytokine-induced NF{kappa}B activation, COX-2, IL-6 and IL-8 in normal prostatic epithelial cells, suggesting potent anti-inflammatory activity of MKP5. Pretreatment of cells with a p38 inhibitor mimicked the results observed with MKP5 over-expression, further implicating p38 inhibition as the main activity of MKP5. Curcumin, the phytochemical found in turmeric, up-regulated MKP5, subsequently decreasing cytokine-induced p38-dependent pro-inflammatory changes in normal prostatic epithelial cells. Resveratrol and [6]-gingerol, phytochemicals present in red wine and ginger, respectively, also up-regulated MKP5 in normal prostate epithelial cells. Moreover, we found that PCa cell lines DU 145, PC-3, LNCaP and LAPC-4 retained the ability to up-regulate MKP5 following curcumin, resveratrol and [6]-gingerol exposure, suggesting utility of these phytochemicals in PCa treatment. In summary, our findings show direct anti-inflammatory activity of MKP5 in prostate cells and suggest that up-regulation of MKP5 by phytochemicals may contribute to their chemopreventive actions by decreasing prostatic inflammation.

Abbreviations: COX-2, cyclooxygenase-2; 1,25D1, 25-dihydroxyvitamin D3; DMSO, dimethyl sulfoxide; E-PZ, epithelial cells derived from the normal peripheral zone; IL, interleukin; JNK, jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MKP5, mitogen-activated protein kinase phosphatase 5; mRNA, messenger RNA; NF{kappa}B, nuclear factor kappa B; Pca, prostate cancer; siRNA, small interference RNA; TNF{alpha}, tumor necrosis factor {alpha}; VDR, vitamin D receptor; T +I, TNF{alpha} and IL-1ß


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
As the carcinogenic processes that ultimately lead to prostate cancer (PCa) are being revealed, inflammation has emerged as a significant risk factor for PCa development (1). Tissue inflammation can contribute to cancer development by inducing oxidative damage and promoting cell growth (2). Inflammation has been shown to play a causal role in the initiation and/or progression of other cancers, including liver, bladder and gastric cancers (2). Evidence linking inflammation to PCa includes (i) regular administration of non-steroidal anti-inflammatory drugs significantly decreased PCa risk in older men by 60–80% (3,4); (ii) men with chronic and/or acute inflammation of the prostate have an increased risk of developing PCa (1,5) and (iii) inflammatory cytokines are over-expressed in serum and prostate tissue of PCa patients. Furthermore, Nelson et al. (1) have characterized and identified areas within the prostate of proliferative inflammatory atrophy, a possible precursor lesion to adenocarcinoma, suggesting a multi-step process of PCa development.

The long latency observed in PCa is unique among malignancies and should provide a lengthy window of opportunity for intervention by chemopreventive agents. The hope of PCa prevention by ‘natural’ dietary products, also known as phytochemicals, has become increasingly popular and concordantly the mechanism of action of phytochemicals has become an active area of research. With the accumulation of evidence linking inflammation to PCa, anti-inflammatory activities may be a significant mechanism by which phytochemicals decrease PCa risk.

We have previously characterized mitogen-activated protein kinase phosphatase-5 (MKP5) as a mediator of anti-inflammatory activities of vitamin D [1,25-dihydroxyvitamin D3 (1,25D)] in the prostate (6). Although not typically considered a phytochemical, vitamin D is an essential part of the diet and has been implicated in the prevention of PCa by epidemiologic and laboratory studies (7). cDNA microarray experiments originally identified MKP5 as a gene up-regulated by vitamin D in prostate (8), skin (9), colon (10) and ovarian cells (11). MKP5 is a member of the dual specificity MKP family of proteins that dephosphorylate mitogen-activated protein kinases (MAPKs). MKP5 specifically dephosphorylates the stress-activated protein kinases p38 and jun N-terminal kinase (JNK) (12,13). The MKP5 knockout mice revealed that MKP5 regulates both innate and adaptive immune responses in vivo (14).

The ability of MKP5 to inhibit p38 is of interest to PCa prevention since p38 signaling mediates pro-inflammatory responses in cells (15), potentially contributing to PCa development (1). Downstream anti-inflammatory effects of p38 inhibition can include decreased activation of nuclear factor kappa B (NF{kappa}B), reduced cyclooxygenase-2 (COX-2) expression and decreased production of pro-inflammatory cytokines. Consistent with p38 inhibition, we found that up-regulation of MKP5 by 1,25D decreased p38 phosphorylation and subsequently reduced interleukin (IL)-6 production in prostatic epithelial cells derived from normal tissues and localized adenocarcinomas (6). 1,25D also decreased COX-2 expression in prostate cells (16) and NF{kappa}B activity (17) in melanoma cell lines, effects potentially mediated by MKP5.

The anticancer activity of curcumin (diferuloylmethane), a polyphenol from turmeric (Curcuma longa Linn.), has been extensively studied and is well established in vitro and in vivo (18). In PCa cell lines, curcumin and analogs of curcumin inhibit growth, induce apoptosis and decrease androgen receptor signaling (18). Anti-inflammatory properties of curcumin have been shown in keratinocytes in which curcumin inhibited p38 signaling and NF{kappa}B activation (19). Furthermore, Yan et al. (20) identified MKP5 as a gene up-regulated by curcumin in MDA-1986 squamous cell carcinoma cells. Since curcumin has been shown to have anti-inflammatory activity and inflammation may be a risk factor for PCa, some of the chemopreventive activities attributed to curcumin may be a result of MKP5-mediated anti-inflammatory activity.

The aim of these experiments was to investigate the potentially chemopreventive anti-inflammatory activities of curcumin and other phytochemicals mediated by MKP5 in prostate cells. Using a pro-inflammatory cytokine stress, we demonstrated an anti-inflammatory role of MKP5 in normal prostatic epithelial cells by exogenous modulation of MKP5 levels with an expression vector and with small interference RNA (siRNA). Curcumin up-regulated MKP5 and inhibited pro-inflammatory signaling in both normal prostatic epithelial cells and PCa cell lines. Lastly, we found that MKP5 was also up-regulated by the phytochemicals resveratrol and [6]-gingerol in normal prostatic epithelial cells and PCa cell lines.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture and reagents
Human primary prostatic epithelial cells were derived from radical prostatectomy specimens. The patients did not have prior chemical, hormonal or radiation therapy. Histological characterization of the tissue of origin and culture of the prostate cells was as described previously (21). Epithelial cells derived from the normal peripheral zone of the prostate (E-PZ) were cultured in supplemented MCDB 105 (Sigma–Aldrich, St Louis, MO) or PFMR-4A as described previously (21). Human PCa cell lines LNCaP, PC-3 and DU 145 were acquired from American Type Culture Collection (Manassas, VA). LNCaP cells were cultured in MCDB 105/10% fetal bovine serum, and PC-3 and DU 145 in DMEM (Invitrogen, Carlsbad, CA)/10% fetal bovine serum. LAPC-4 cells (provided by Dr Charles Sawyers, UCLA, Los Angeles, CA) were cultured in phenol red-free RPMI 1640 (Life Technologies, Rockville, MD)/10% fetal bovine serum. All chemicals were obtained from Sigma–Aldrich unless otherwise noted. SB202190 was obtained from Calbiochem (San Diego, CA), reconstituted in dimethyl sulfoxide (DMSO) and stored at –20°C. Curcumin and gingerol (Wako Pure Chemicals, Osaka, Japan) were reconstituted in 5% DMSO/95% ethanol and stored at –20°C. 1,25D (Biomol International, Plymouth Meeting, PA) and resveratrol (Calbiochem, La Jolla, CA) were reconstituted in 100% ethanol and stored at –20°C.

RNA isolation and real-time quantitative reverse transcription–polymerase chain reaction
RNA was isolated from cells by lysis in TrizolTM (Invitrogen) followed by chloroform extraction. The aqueous phase was then precipitated in 100% isopropanol and the pellet washed in 75% ethanol before re-suspension in water. RNA concentration and quality were determined by absorbance ratio at 260:280 nm using a UV spectrophotometer. Total RNA (2 µg) was reverse transcribed using Stratascript Reverse Transcriptase (Stratagene, La Jolla, CA). Resulting cDNA was used for quantitative polymerase chain reaction amplification with gene-specific primers and the Brilliant Sybr Green kit (Stratagene) in the MX3005-Pro thermocycler (Stratagene). Polymerase chain reaction conditions for all primer sets were optimized and have similar amplification efficiency under the following thermocycler conditions: 95°C, 5 min; 34x (95°C, 30 s; 58°C, 30 s and 72°C, 60 s) and 72°C, 5 min. Relative messenger RNA (mRNA) levels were calculated from the point where each curve crossed the threshold line (Ct) using the equation: relative value = 2-[Ct (control) – Ct (test)] test gene/2-[Ct (control) – Ct (test)] housekeeping gene (22). Reactions were performed in triplicate and the values were normalized to the expression of the housekeeping gene TATA box-binding protein (23). Primer sets were TATA box-binding protein: 5'-tgctgagaagagtgtgctggag-3' and 5'-tctgaataggctgtggggtc-3'; MKP5: 5'-atcttgcccttcctgttcct-3' and 5'-attggtcgtttgcctttgac-3'; IL-6: 5'-gaagattccaaagatgtagccg-3' and 5'-tgttttctgccagtgcctc-3'; IL-8: 5'-agccttcctgatttctgcagctct-3' and 5'-aaacttctccacaaccctctgcac-3'; COX-2: 5'-gatactcaggcagagatgatctaccc-3' and 5'-agaccaggcaccagaccaaaga-3'; tumor necrosis factor-{alpha} (TNF{alpha}): 5'-aggcggtgcttgttcctcag-3' and 5'-tacaggcttgtcactcgggg-3' and IL-1ß: 5'-ggcttattacagtggcaatgag-3' and 5'-tagtggtggtcggagattcgtagc-3'.

Cell lysate preparation and immunoblot
Cells were lysed in ice-cold 1x cell lysis buffer (Cell Signaling, Beverly, MA) containing 1 µM phenylmethylsulfonyl fluoride and 100 nM okadaic acid. Cells were disrupted by sonication and insoluble cell debris removed by centrifugation at 15 000g, 4°C. Protein concentrations of the cell lysates were quantified using the Bio-Rad Protein Dye (Bio-Rad, Hercules, CA). Fresh cell lysates were used for analysis of phosphorylated proteins. Cell lysates (10–30 µg) were mixed with laurel dodecyl sulfate NuPAGE sample buffer, separated by electrophoresis through 10% NuPAGE Bis–Tris Gels (Invitrogen) and transferred onto polyvinylidene fluoride membrane. Membranes were probed with the following primary antibodies: anti-phospho-p38 rabbit polyclonal and anti-p38 rabbit polyclonal from Cell Signaling, monoclonal anti-COX-2 (Cayman Chemical, Ann Arbor, MI) and monoclonal anti-actin from Santa Cruz Biotech (Santa Cruz, CA). Following primary antibody incubation overnight at 4°C, the blots were incubated with appropriate secondary horseradish peroxidase-conjugated antibodies (Cell Signaling) and developed with HyGlo ECL reagent (Denville Scientific, Metuchen, NJ).

siRNA transfection
Cells at 75% confluency in 60 mm collagen-coated dishes were transfected with 10 nmol of negative control (Ambion, Austin, TX) or MKP5-specific siRNA (Ambion) using 7 µl of siPORT NeoFX (Ambion) per dish, layering transfection mixture on top of the cells. Transfection reagent was not removed from the cells and cells were used for experiments after transfection as indicated in Results and figure legends.

MKP5 over-expression
Full-length MKP5 cDNA (GenBank accession no. BC063826 [GenBank] ) was cloned into pcDNA3.1 (Invitrogen) using primers 5'-tgaatgtgcagagtccatagc-3' and 5'-gttagcagggcaggtggtag-3' and confirmed by sequence analysis. pcDNA3.1-MKP5 or pcDNA3.1-LacZ were transiently transfected into 50% confluent E-PZ cells using siPORT NeoFX transfection reagent (Ambion). In Figure 2A, B and D, 1 µg of plasmid DNA and 7 µl of siPORT NeoFX transfection reagent were used with 75% confluent 60 mm collagen-coated dishes.

NF{kappa}B-luciferase reporter activity assay
NF{kappa}B-luciferase construct (Clonetech, Mountain View, CA) containing four tandem copies of the NF{kappa}B consensus sequence driving luciferase expression and pRL-null-Renilla (Promega, Madison, WI) were transiently transfected into E-PZ cells using siPORT NeoFX reagent. Cells (4.5 x 104 cells per well) were transfected in suspension with 10 ng of plasmid and 1 µl of NeoFX and plated onto 24-well collagen-coated plates. In Figure 2C, 250 and 500 ng of pcDNA3.1 plasmid were included in the transfection. Eight hours after transfection, cells were treated as indicated in figure legends. Luciferase activity was measured using the Dual-Luciferase Assay Kit (Promega). The ratio of luciferase to Renilla luciferase was determined to correct for transfection efficiency.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Characterization of TNF{alpha} and IL-1ß as inducers of p38-dependent NF{kappa}B activation, COX-2 expression and pro-inflammatory cytokine production in normal prostatic E-PZ cells
To evaluate the role of MKP5 in the inflammatory response of E-PZ, it was necessary to identify a pro-inflammatory stimulus that signaled through p38. TNF{alpha} and IL-1ß are potent pro-inflammatory cytokines secreted by macrophages to initiate inflammation by stimulating the expression of pro-inflammatory genes such as IL-6, IL-8 and COX-2 (24). The pro-inflammatory cytokines IL-6 and IL-8 are of particular interest to PCa prevention because they are characteristically over-expressed in PCa tissue and patient serum (25). COX-2 functions to increase prostaglandin secretion from prostate cells, which can further facilitate inflammatory cell recruitment as well as increase cell proliferation (16). IL-1ß and TNF{alpha} also induce their own expression causing an amplification loop of the inflammatory response (24). Importantly, TNF{alpha} and IL-1ß have been shown to signal through both the p38 MAPK and the NF{kappa}B pathways (24,26).

E-PZ cells were stimulated with a combination of 1 ng/ml TNF{alpha} and 5 ng/ml IL-1ß (T + I) for 15 min and 1, 3, 6 and 24 h. Cells were co-treated because TNF{alpha} and IL-1ß showed a synergistic effect on p38 phosphorylation, COX-2 and inflammatory cytokine expression in the prostate cells (data not shown), which is similar to other published results (27,28). Cell lysates and RNAs were collected at each time point. Immunoblot of cell lysates showed that phosphorylation of p38 occurred within 15 min of T + I treatment (Figure 1A). COX-2 mRNA and protein was increased after 6 h of T + I (Figure 1A and B). JNK phosphorylation was not stimulated by T + I in the E-PZ cells (data not shown). Since NF{kappa}B has been implicated in both TNF{alpha} and IL-1ß signaling, NF{kappa}B activation by T + I was determined by luciferase enzyme activity of a transiently transfected NF{kappa}B-luc reporter construct. Twenty-four hours following transfection with NF{kappa}B-luc and pRL-null, E-PZ cells were treated with T + I for 8 h and luciferase activity was measured. T + I stimulation increased NF{kappa}B-luc activity 10-fold (Figure 1C). NF{kappa}B p65 nuclear translocation and activity are dependent upon phosphorylation and subsequent degradation of the inhibitory protein I{kappa}B{alpha} (29). This is a point at which the p38 and NF{kappa}B pathways converge via p38-mediated phosphorylation of I{kappa}B{alpha} (30). To test whether T + I-stimulated NF{kappa}B activity was dependent upon p38 signaling in the E-PZ cells, SB202190, a small molecule inhibitor of p38, was used. The results showed that in E-PZ cells, T + I-stimulated NF{kappa}B activity was dependent upon p38 signaling as 1 h pretreatment with 10 µM SB202190 completely blocked the increase in NF{kappa}B-luc activity (Figure 1C).


Figure 1
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Characterization of TNF{alpha} and IL-1ß as inducers of p38-dependent NF{kappa}B activation, COX-2 expression and pro-inflammatory cytokine production in normal prostatic E-PZ cells. (A) Immunoblot of E-PZ cell lysates following a time course of T + I treatment. Blot was probed with antibodies to phospho-p38, total p38, COX-2 and actin. (B) Real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) measurement of COX-2 mRNA following T + I treatment. (C) Transactivation of NF{kappa}B-luc reporter construct 8 h following T + I treatment of E-PZ cells pretreated for 30 min with vehicle (0.01% DMSO) or 10 µM SB202190 (SB). Data are representative of three independent experiments, and error bars represent standard deviation of triplicate samples. (D) Real-time qRT–PCR measurement of IL-6, IL-8, IL-1ß and TNF{alpha} mRNA in E-PZ cells after 1, 3, and 6 h of T + I treatment. (E) Real-time qRT–PCR measurement of IL-6, IL-8, IL-1ß and TNF{alpha} mRNA following 3 h of T + I treatment in E-PZ cells pretreated for 30 min with vehicle (0.01% DMSO) or 10 µM SB202190 (SB). Dashed line represents basal mRNA levels for cytokines. For real-time qRT–PCR experiments, mRNA levels are shown relative to untreated control and normalized to expression of the housekeeping gene TATA box-binding protein. Data are representative of three independent experiments, and error bars represent standard deviation of duplicate or triplicate samples.

 
Increased mRNA accumulation of the pro-inflammatory cytokines IL-6, IL-8, IL-1ß and TNF{alpha} can occur by two mechanisms as follows: (i) increased p38-mediated mRNA stability and protein translation (31) and (ii) increased NF{kappa}B-mediated gene transcription (32). In E-PZ cells, we observed increased mRNA accumulation of IL-6, IL-8, IL-1ß and TNF{alpha} at 1 h following T + I with maximum mRNA levels at 6 h (Figure 1D). Levels of IL-1ß and TNF{alpha} mRNA were measured as positive controls, because IL-1ß and TNF{alpha} stimulate their own gene expression (24). SB202190 was used to test whether cytokine mRNA stabilization was dependent upon p38 signaling. A 1 h pretreatment with 10 µM SB202190 completely blocked the increase in cytokine mRNA by T + I (Figure 1E). MKP5 mRNA levels were unchanged by T + I (data not shown).

Therefore, T + I stimulation was used in all subsequent experiments because it caused a robust increase in cytokine mRNA, COX-2 mRNA and protein, and NF{kappa}B activity in E-PZ cells, all of which could be blocked by SB202190 and hence were p38 dependent.

MKP5 inhibited TNF{alpha}- and IL-1ß-stimulated p38 phosphorylation, COX-2 expression and cytokine production in E-PZ cells
Our next goal was to assess whether MKP5, as a p38 MKP, could inhibit or decrease the powerful pro-inflammatory molecular changes induced by T + I stimulation in E-PZ cells. We showed previously that up-regulation of MKP5 by 1,25D attenuated UV irradiation- and TNF{alpha}-induced p38 activation and IL-6 secretion in E-PZ cells (6).

To determine the role of MKP5 in the T + I response, the intracellular levels of MKP5 were up-regulated by an expression plasmid and down-regulated with siRNA. E-PZ cells were transiently transfected with pcDNA3.1-LacZ or pcDNA3.1-MKP5. Transfection with pcDNA3.1-MKP5 increased MKP5 mRNA levels 10- to 50-fold at 24 h (data not shown). Twenty-four hours following transfection, cells were stimulated with T + I for 15 min and p38 phosphorylation was measured by immunoblot of cell lysates. The cells transfected with pcDNA3.1-MKP5 had attenuated levels of phospho-p38 compared with pcDNA3.1-LacZ, showing that the exogenous MKP5 was active (Figure 2A). Exogenous MKP5 was able to block T + I-stimulated COX-2 expression as pcDNA3.1-MKP5-transfected E-PZ cells showed less COX-2 protein by immunoblot analysis (Figure 2B). As a control, SB202190 was used and blocked basal and T + I-stimulated COX-2 protein expression (Figure 2B). Conversely, when MKP5 levels were decreased in E-PZ cells by siRNA, basal and T + I-induced COX-2 protein expression was increased compared with negative control siRNA (Figure 2B). MKP5-siRNA decreased MKP5 mRNA levels to 20% of NEG-siRNA (data shown in Figure 3C). Since T + I-induced NF{kappa}B activation was dependent upon p38 signaling, the effect of exogenous MKP5 on NF{kappa}B-luc was measured. The luciferase assay showed that transfection of E-PZ cells with pcDNA3.1-MKP5 inhibited NF{kappa}B-luc activity in a dose-dependent manner (Figure 2C). Consistent with decreased p38 signaling and NF{kappa}B activity, the levels of T + I-induced IL-6 and IL-8 were reduced >10-fold in E-PZ cells transfected with pcDNA3.1-MKP5 compared with control vector (Figure 2D).


Figure 2
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. MKP5 inhibited TNF{alpha}- and IL-1ß-stimulated p38 phosphorylation, COX-2 expression and cytokine production in E-PZ cells. (A) Immunoblot of E-PZ cell lysates following 30 min of T + I treatment. Cells were transfected with 1 µg pcDNA3.1 or pcDNA3.1-MKP5 24 h prior to cytokine treatment. Blot was probed with antibodies to phospho-p38 and total p38. (B) Immunoblot of E-PZ cell lysates following 24 h of T + I treatment. Prior to cytokine treatment, cells were either transfected with 1 µg pcDNA3.1 or pcDNA3.1-MKP5 for 24 h, treated with 10 µM SB202190 for 30 min or transfected with 10 nmol of siRNA-NEG or siRNA-MKP5. Blot was probed with antibodies to COX-2 and actin. (C) Transactivation of NF{kappa}B-luc reporter construct in E-PZ cells 8 h following T + I treatment. Cells were transfected 24 h prior to cytokine treatment cells with NF{kappa}B-luc (10 ng), prL-null (10 ng) and pcDNA3.1 or pcDNA3.1-MKP5 (100 and 250 ng). Data are representative of two independent experiments, and error bars represent standard deviation of triplicate samples. (D) Real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) measurement of IL-6 and IL-8 mRNA in E-PZ cells after 3 h of T + I treatment. Cells were transfected with pcDNA3.1 or pcDNA3.1-MKP5 24 h prior to cytokine treatment. For real-time qRT–PCR experiments, mRNA levels are shown relative to untreated control and normalized to expression of the housekeeping gene TATA box-binding protein. Data are representative of three independent experiments, and error bars represent standard deviation of duplicate or triplicate samples.

 


Figure 3
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. MKP5 was up-regulated by curcumin and inhibited TNF{alpha}- and IL-1ß-stimulated p38 phosphorylation, COX-2 up-regulation, NF{kappa}B activation and cytokine production in E-PZ cells. (A) Real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) measurement of MKP5 mRNA in E-PZ cells 17 h after treatment with vehicle (0.01% EtOH), 10 and 50 µM curcumin. (B) Immunoblot of E-PZ cell lysates 30 min following T + I treatment. Blot was probed with antibodies to phospho-p38 and total p38. (C) Immunoblot of E-PZ cell lysates 24 h after T + I treatment in cells pretreated with vehicle (0.01% EtOH) or 50 µM curcumin. Four hours prior to pretreatment, cells were transfected with 10 nmol of siRNA-NEG or siRNA-MKP5. Blot was probed with antibodies to COX-2 and actin. qRT–PCR measurement of MKP5 mRNA to show knock-down with siRNA-MKP5 relative to siRNA-NEG. (D). Transactivation of NF{kappa}B-luc reporter construct in E-PZ cells, pretreated for 17 h with vehicle (0.01% EtOH), 10 and 50 µM curcumin, 8 h following T + I treatment. Data are representative of two independent experiments, and error bars represent standard deviation of triplicate samples. (E) Real-time qRT–PCR measurement of IL-6 and IL-8 mRNA after 3 h of T + I treatment in E-PZ cells, pretreated for 17 h with vehicle (0.01% EtOH) or 50 µM curcumin. Dashed line represents basal mRNA levels for cytokines. For real-time qRT–PCR experiments, mRNA levels are shown relative to untreated control and normalized to expression of the housekeeping gene TATA box-binding protein. Data are representative of three independent experiments, and error bars represent standard deviation of duplicate or triplicate samples.

 
These results reveal that MKP5 has a significant role in reducing T + I-stimulated inflammatory signaling by inhibiting p38-mediated pro-inflammatory processes in normal prostatic E-PZ cells.

MKP5 was up-regulated by curcumin and inhibited TNF{alpha}- and IL-1ß-stimulated p38 phosphorylation, COX-2 up-regulation, NF{kappa}B activation and cytokine production in E-PZ cells
Curcumin is the polyphenolic compound present in the spice turmeric. Curcumin has been shown to have significant anti-inflammatory effects in a variety of cell types (19). Interestingly, curcumin was found to up-regulate MKP5 mRNA in an MDA-1986 squamous cell carcinoma microarray study (20). We speculated that curcumin would have similar anti-inflammatory effects in the normal prostatic E-PZ cells and that these effects would be mediated by MKP5.

In agreement with our hypothesis, curcumin dose dependently up-regulated MKP5 mRNA (Figure 3A) and 17 h pretreatment with 50 µM curcumin inhibited T + I-stimulated p38 phosphorylation in E-PZ cells (Figure 3B). Curcumin did not affect mRNA stability of MKP5 as mRNA half-life following actinomycin D treatment was not different between vehicle- and curcumin-treated cells (L.Nonn, unpublished results). Curcumin pretreatment also blocked T + I-induced COX-2 protein expression and the effect was partially attenuated by MKP5-siRNA (Figure 3C). MKP5-siRNA attenuated MKP5 up-regulation by curcumin to 20% of NEG-siRNA (Figure 3C). Downstream of p38 signaling by T + I, NF{kappa}B-luc activity and cytokine mRNA accumulation were blocked by 17 h pretreatment with 50 µM curcumin (Figure 3D and E).

These results show that MKP5 is up-regulated by curcumin and mediates anti-inflammatory activities of curcumin in normal prostatic E-PZ cells.

PCa cell lines DU 145, PC-3, LNCaP and LAPC-4 retain ability to up-regulate MKP5 in response to curcumin
In our previous study, we found that 1,25D up-regulated MKP5 only in primary E-PZ cells and cells cultured from primary adenocarcinomas of the prostate, but not in the PCa cell lines DU 145, PC-3 and LNCaP (6). 1,25D typically alters gene expression through binding to the vitamin D receptor (VDR). VDR is a classical steroid receptor that translocates to the nucleus and binds to vitamin D response elements in the promoter regions of genes. This is the likely mechanism for regulation of MKP5 by 1,25D because the MKP5 promoter contains a putative vitamin D response element and up-regulation of MKP5 by 1,25D was dependent upon VDR (6). We did not find that curcumin up-regulated any of the classic VDR-regulated genes (data not shown) and this photochemical has not been reported to alter gene expression through VDR activation. The mechanism for MKP5 up-regulation by curcumin is probably distinct from that of 1,25D, and therefore MKP5 regulation by curcumin was examined in the immortal PCa cell lines DU 145, PC-3, LNCaP and LAPC-4.

It is well established that curcumin can induce cell death in PCa cell lines at high concentrations (33). After initially testing a range of curcumin concentrations (1–50 µM), we found that 25 µM curcumin was able to significantly up-regulate MKP5 mRNA in DU 145, PC-3, LNCaP and LAPC-4 cells (Figure 4A) without causing visible cell death [consistent with published data (33) therefore not shown]. When stimulated with T + I, only the DU 145 cells responded with increased p38 phosphorylation and COX-2 expression. In the DU 145 cells, pretreatment with 25 µM curcumin was able to block T + I-induced p38 phosphorylation and COX-2 protein expression (Figure 4B). Pretreatment of DU 145 cells with curcumin decreased COX-2 mRNA similarly to SB202190 (Figure 4B), showing COX-2 expression was p38 dependent. T + I-induced NF{kappa}B-luc activity was not decreased by pretreatment of DU 145 cells with either 10 µM SB202190 or 25 µM curcumin (Figure 4C). This finding correlates with published data showing that DU 145 cells have constitutively elevated NF{kappa}B activity due to increased activity of I{kappa}B kinase (34), which is p38 independent (30). However, IL-6 and IL-8 cytokine mRNA levels were decreased by curcumin or SB202190 pretreatment (Figure 4D), suggesting that T + I-induced expression of IL-6 and IL-8 is mediated by p38 rather than NF{kappa}B in DU 145 cells.


Figure 4
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. PCa cell lines DU 145, PC-3, LNCaP and LAPC-4 retain ability to up-regulate MKP5 in response to curcumin. (A) Real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) measurement of MKP5 mRNA in DU 145, PC-3, LNCaP and LAPC-4 following 17 h treatment with vehicle (0.01% EtOH) or 25 µM curcumin. (B) Immunoblot of DU 145 cell lysates (10 µg) following 30 min and 24 h of T + I treatment. Blots were probed with antibodies to phospho-p38, total p38, COX-2 and actin. Real-time qRT–PCR measurement of COX-2 mRNA after 6 h of T + I treatment in DU 145 cells, pretreated for 17 h with vehicle (0.01% DMSO) or 25 µM curcumin or 1 h with 10 µM SB202190. (C) Transactivation of NF{kappa}B-luc reporter construct in DU 145 cells 8 h following T + I treatment. Cells were pretreated for either 17 h with vehicle (0.01% EtOH) or 25 µM curcumin or 1 h with 10 µM SB202190. Data are representative of two independent experiments, and error bars represent standard deviation of triplicate samples. (D) Real-time qRT–PCR measurement of IL-6 and IL-8 mRNA after 3 h of T + I treatment in DU 145 cells, pretreated for 17 h with vehicle (0.01% EtOH) or 25 µM curcumin or 1 h with 10 µM SB202190. For qRT–PCR experiments, mRNA levels are shown relative to untreated control and normalized to expression of the housekeeping gene TATA box-binding protein. Data are representative of three independent experiments, and error bars represent standard deviation of duplicate or triplicate samples.

 
These results show that the PCa cell lines DU 145, PC-3, LNCaP and LAPC-4 retain the ability to up-regulate MKP5 in response to curcumin and that curcumin reduced p38-mediated, but not NF{kappa}B-mediated, pro-inflammatory signaling by T + I in DU 145 cells.

MKP5 is up-regulated by the phytochemicals resveratrol and gingerol in normal prostatic E-PZ cells and PCa cell lines
Two other phytochemicals, resveratrol and [6]-gingerol, have been shown to inhibit p38 phosphorylation and NF{kappa}B activation in mouse skin (35,36). Since MKP5 was up-regulated by 1,25D and curcumin, by presumably different mechanisms, we wondered if MKP5 could be up-regulated and play a role in the anti-inflammatory activities of resveratrol and [6]-gingerol in the prostate.

The ability of resveratrol and [6]-gingerol to regulate MKP5 expression in normal prostatic E-PZ cells and in PCa cell lines was determined. In E-PZ cells, we found that both 50 µM resveratrol and 50 µM [6]-gingerol increased MKP5 mRNA to levels similar to those induced by 1,25D and curcumin (Figure 5A). With the exception of resveratrol in LNCaP cells, 50 µM resveratrol or 50 µM [6]-gingerol was also able to up-regulate MKP5 in the PCa cell lines DU 145, PC-3, LNCaP and LAPC-4 (Figure 5B).


Figure 5
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. MKP5 was up-regulated by the phytochemicals resveratrol and gingerol in normal prostatic E-PZ cells and PCa cell lines. (A) Real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) measurement of MKP5 mRNA in E-PZ cells following 17 h treatment with vehicle (0.01 % EtOH), 50 nM 1,25D, 50 µM curcumin, 25 µM resveratrol or 25 µM gingerol. (B) Real-time qRT–PCR measurement of MKP5 mRNA in DU 145, PC-3, LNCaP and LAPC-4 following 17 h treatment with vehicle (0.01 % EtOH), 25 µM curcumin, 25 µM resveratrol or 25 µM gingerol. For real-time qRT–PCR experiments, mRNA levels are shown relative to untreated control and normalized to expression of the housekeeping gene TATA box-binding protein. Data are representative of three independent experiments, and error bars represent standard deviation of duplicate or triplicate samples.

 
These findings show that MKP5 is a common target of 1,25D and the phytochemicals curcumin, resveratrol and [6]-gingerol in normal prostatic E-PZ cells, whereas in the PCa cell lines, MKP5 is up-regulated by curcumin, resveratrol and [6]-gingerol but not by 1,25D.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We previously identified MKP5 as a mediator of inhibition of p38 and suppression of IL-6 by 1,25D in normal prostatic epithelial cells (6). The current study expands on the inhibitory role of MKP5 in pro-inflammatory pathways in prostatic cells. Here, a combination of T + I was chosen as the pro-inflammatory stimulus in E-PZ cells because it rapidly increased COX-2 protein levels, NF{kappa}B activity and cytokine mRNA accumulation via a p38-dependent pathway. JNK activation was not involved in T + I-stimulated inflammatory signaling as JNK phophorylation was not detected. TNF{alpha} and IL-1ß are also physiologically relevant to prostatic inflammation since they are secreted by resident macrophages and function as ‘alarm cytokines’ to initiate inflammatory cell recruitment by stimulating the expression of pro-inflammatory genes (24) (Figure 6A).


Figure 6
View larger version (45K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. Diagram of hypothesized anti-inflammatory changes induced by phytochemicals and 1,25D in prostatic epithelial cells. (A) Pro-inflammatory signaling in the normal prostate. TNF{alpha} and IL-1ß bind their surface receptors, TNF receptor (TNFR), IL-1 receptors (IL-1Rs) and Toll-like receptors (TLRs), which signal through p38 MAPK and NF{kappa}B to increase expression of pro-inflammatory genes to facilitate inflammatory cell recruitment (adapted from http://www.cellsignal.com with incorporation of data from this study). Solid lines represent our findings and dashed lines represent known pathways that were not shown in our study. (B) Model for disruption of pro-inflammatory signaling by phytochemicals and 1,25D via MKP5 in normal prostate.

 
We focused on T + I-induced expression of the pro-inflammatory genes for COX-2 and the cytokines IL-6 and IL-8. COX-2 is the inducible form of COX, the rate-limiting enzyme in prostaglandin synthesis (16). COX-2 expression results in prostaglandin secretion, which can increase cell proliferation and inflammatory cell recruitment (16). The precise nature of COX-2 expression changes during PCa development and progression remain under debate. However, high COX-2 levels at radical prostatectomy are an independent predictor of PCa recurrence (37). IL-6 and IL-8 are both found to be over-expressed in the tissues and serum of PCa patients (25,38,39). Elevated serum levels of IL-6 are associated with aggressive pathology and decreased survival of PCa patients (40). IL-8 has pro-angiogenic activity and is expressed by prostatic epithelial cells (41). In vitro, all the PCa-derived cell lines DU 145, PC-3 and LNCaP express the IL-6 receptor and are responsive to exogenous IL-6. However, DU 145 and PC-3 also constitutively over-express IL-6 whereas the LNCaP cell line does not express any IL-6 (39). Monoclonal antibody inhibition of IL-6 in LuCaP xenograft-bearing mice blocked conversion to androgen independence following castration (42). Therefore, inhibition of IL-6 may prove to be a powerful tool in PCa treatment.

We showed that when MKP5 levels were increased in normal prostatic E-PZ cells by an MKP5 expression plasmid, pro-inflammatory effects of T + I stimulation were diminished. MKP5 decreased the levels of phosphorylated p38 protein, COX-2 protein, NF{kappa}B activation and cytokine (IL-6, IL-8, TNF{alpha} and IL-1ß) mRNA accumulation. Pretreatment of the cells with SB202190, a small molecule inhibitor of p38, mimicked the anti-inflammatory effects of MKP5 expression and verified p38 dependence of T + I-induced COX-2, NF{kappa}B activation and cytokine mRNA. Conversely, when levels of MKP5 were decreased by siRNA, the pro-inflammatory effects of T + I were exaggerated. These results reveal that MKP5 has a significant role in reducing T + I-stimulated inflammatory signaling in normal prostatic epithelial cells.

Curcumin, the phytochemical present in turmeric that is believed to have cancer preventive or therapeutic capabilities, had been shown to up-regulate MKP5 mRNA in MDA-1986 squamous carcinoma cells (20). We showed that, as in MDA cells, curcumin was able to up-regulate MKP5 mRNA and inhibit p38 phosphorylation in normal prostatic E-PZ cells. Concordantly, the T + I-stimulated pro-inflammatory response in E-PZ cells was blunted by curcumin pretreatment. When siRNA was used to block the up-regulation of MKP5 by curcumin, COX-2 suppression by curcumin was attenuated, further implicating MKP5 as a mediator of curcumin's activity. T + I-stimulated NF{kappa}B activity was also blocked by curcumin. NF{kappa}B is a pro-inflammatory transcription factor whose regulation can be complex, involving many pathways. In the E-PZ cells, NF{kappa}B activation by T + I was completely p38 dependent and thus was inhibited by curcumin via MKP5. P38-dependent NF{kappa}B activation is not unique to the stimulus that we used, but also occurs during UV irradiation and is I{kappa}B kinase independent (30). These results show that MKP5 mediates anti-inflammatory activities of curcumin in normal prostatic E-PZ cells.

In our previous study, we observed that up-regulation of MKP5 by 1,25D was unique to prostate cells derived from normal tissues or localized adenocarcinomas (6). In the metastases-derived PCa cell lines, MKP5 basal levels were lower and were unchanged by 1,25D treatment (6). In our current study, we found that curcumin, in contrast to 1,25D, up-regulated MKP5 in normal prostatic E-PZ cells as well as in the metastases-derived PCa cell lines DU 145, PC-3, LNCaP and LAPC-4. In order to explore the relevance of induction of MKP5 to anti-inflammatory activities of curcumin in the PCa cell lines, we evaluated the response of the cell lines to T + I. Stimulation of the PCa cell lines by T + I selectively stimulated p38 phosphorylation only in DU 145 cells. This was not unanticipated since both TNF{alpha} and IL-1ß signal through receptor-mediated pathways that do not necessarily involve p38 MAPK (30). In the DU 145 cells, curcumin decreased T + I-stimulated p38 phosphorylation, COX-2 protein expression and IL-6 and IL-8 mRNA levels. Although DU 145 cells have constitutively activated NF{kappa}B (43), we found that T + I treatment further increased NF{kappa}B activity. However, activation of NF{kappa}B by T + I in the DU 145 cells, in contrast to E-PZ cells, was p38 independent as it was not blocked by curcumin or SB202190. Thus, although MKP5 is up-regulated by curcumin in the PCa cell lines, its activity is limited to p38-mediated effects on cytokine and COX-2 expression as NF{kappa}B activity is p38 independent in those cells.

Given the involvement of MKP5 in anti-inflammatory activities of two chemopreventive compounds, vitamin D and curcumin, we investigated several other phytochemicals suggested to have anticancer properties. Resveratrol (trans-3,5,4'-trihydroxystilbene) is a polyphenol present in grape skin and red wine (44). Jang et al. (44) have demonstrated chemopreventive activity of resveratrol in the initiation, promotion and progression stages of carcinogenesis. In PCa cell lines, resveratrol inhibits cell growth and decreases androgen receptor signaling (45). [6]-Gingerol, the major pungent phenolic found in ginger (Zingiber officinale Roscoe, Zingiberaceae), has been utilized extensively in oriental medicine for alleviation of inflammation and gastrointestinal ailments (46). However, prostate-specific anticancer activities of [6]-gingerol have not yet been studied. Both resveratrol and [6]-gingerol inhibit phorbol ester-induced COX-2 expression and NF{kappa}B activation (35,36), activities we have shown can be mediated by MKP5. We found that resveratrol and [6]-gingerol, like vitamin D and curcumin, up-regulated MKP5 in normal prostatic E-PZ cells and in the PCa cell lines, with the exception of resveratrol in the LNCaP cells. In general, we found that LNCaP cells had limited ability to up-regulate MKP5 in response to any of the phytochemicals, suggesting the presence of genomic or epigenomic suppression. MKP5 mRNA up-regulation by 1,25D and the phytochemicals is presumably via different mechanisms, as 1,25D altered MKP5 gene expression through the VDR (6) and the other phytochemicals do not. Although our studies focused on mRNA regulation of MKP5, the possibility of further post-translational regulation of MKP5 activity cannot be excluded. Since MKP5 is a common target of these and potentially other phytochemicals, it is possible that combination dosing strategies could amplify MKP5 up-regulation, perhaps increasing anti-inflammatory activities while decreasing dosage of individual phytochemicals, thus decreasing side effects.

The fact that the PCa cell lines retain the ability to up-regulate MKP5 in response to phytochemicals is relevant to PCa therapy. It shows that not only can phytochemicals ingested in the diet and via supplements play a role in PCa prevention but also that phytochemicals can be exploited for use in PCa treatment. P38 MAPK has diverse biological properties and activation can mediate apoptosis or cell survival, depending upon the type of stress, cell background and activities of the other MAPKs (15). Therefore, whereas inhibition of the p38 pathway in normal tissue is an anti-inflammatory chemopreventive activity, p38 inhibition in PCa could affect cell survival pathways leading to increased sensitivity to chemotherapies. In fact, all the phytochemicals we analyzed are currently under heavy investigation for their utility in cancer treatment and analogs with enhanced antitumor activity are being tested.

In conclusion, these experiments show that (i) MKP5 is a potent inhibitor of pro-inflammatory signaling in prostate cells and (ii) MKP5 may be a common mediator of phytochemical anti-inflammatory activities. Our data suggest that, in the prostate, TNF{alpha}- and IL-1ß-induced inflammatory cell recruitment could theoretically be decreased by phytochemical-induced MKP5 (Figure 6B), leading to a reduction in prostatic inflammation. Also, in contrast to 1,25D, which does not up-regulate MKP5 in PCa cell lines, curcumin, resveratrol and gingerol were able to up-regulate MKP5 and inhibit the p38 pathway in the PCa cell lines, implicating potential utility in management of early or advanced PCa. Although we believe MKP5 to be an important mediator of inflammation in prostate cells, we are not downplaying other molecular effects of the phytochemicals. Ultimately, phytochemicals have the ability to affect many pathways and a greater understanding of their mechanisms of action will facilitate exploitation of these naturally occurring ‘drugs’ for cancer prevention and therapy.


   Acknowledgments
 
The authors thank Srilatha Swami, Aruna Krishnan and Bryan Husbeck for insightful conversations about the manuscript and Krishnan Larry Hooser for providing invaluable cell culture assistance. L.N. was supported by a postdoctoral grant from the Department of Defense Congressionally Directed Medical Research Programs (PC040616).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Nelson WG, et al. The role of inflammation in the pathogenesis of prostate cancer. J. Urol. (2004) 172:S6–S11. discussion S11–S12.[CrossRef][Medline]
  2. Coussens LM, et al. Inflammation and cancer. Nature (2002) 420:860–867.[CrossRef][Medline]
  3. Palapattu GS, et al. Prostate carcinogenesis and inflammation: emerging insights. Carcinogenesis (2004) 26:1170–1181.[CrossRef][Web of Science][Medline]
  4. Platz EA, et al. Nonsteroidal anti-inflammatory drugs and risk of prostate cancer in the Baltimore Longitudinal Study of Aging. Cancer Epidemiol. Biomarkers Prev. (2005) 14:390–396.[Abstract/Free Full Text]
  5. Fernandez L, et al. Sexual behaviour, history of sexually transmitted diseases, and the risk of prostate cancer: a case-control study in Cuba. Int. J. Epidemiol. (2005) 34:193–197.[Abstract/Free Full Text]
  6. Nonn L, et al. Inhibition of p38 by vitamin D reduces interleukin-6 production in normal prostate cells via mitogen-activated protein kinase phosphatase 5: implications for prostate cancer prevention by vitamin D. Cancer Res. (2006) 66:4516–4524.[Abstract/Free Full Text]
  7. Krishnan AV, et al. Vitamin D and prostate cancer. In: Vitamin D—Glorieux FH, ed. (2005) Vol. 2. San Diego: Elsevier Academic Press. 1679–1707.
  8. Peehl DM, et al. Molecular activity of 1,25-dihydroxyvitamin D(3) in primary cultures of human prostatic epithelial cells revealed by cDNA microarray analysis. J. Steroid Biochem. Mol. Biol. (2004) 92:131–141.[CrossRef][Web of Science][Medline]
  9. Huang X, et al. Genistein inhibits p38 map kinase activation, matrix metalloproteinase type 2, and cell invasion in human prostate epithelial cells. Cancer Res. (2005) 65:3470–3478.[Abstract/Free Full Text]
  10. Palmer HG, et al. Genetic signatures of differentiation induced by 1alpha,25-dihydroxyvitamin D3 in human colon cancer cells. Cancer Res. (2003) 63:7799–7806.[Abstract/Free Full Text]
  11. Zhang X, et al. Suppression of death receptor-mediated apoptosis by 1,25-dihydroxyvitamin D3 revealed by microarray analysis. J. Biol. Chem. (2005) 280:35458–35468.[Abstract/Free Full Text]
  12. Tanoue T, et al. Molecular cloning and characterization of a novel dual specificity phosphatase, MKP-5. J. Biol. Chem. (1999) 274:19949–19956.[Abstract/Free Full Text]
  13. Theodosiou A, et al. MKP5, a new member of the MAP kinase phosphatase family, which selectively dephosphorylates stress-activated kinases. Oncogene (1999) 18:6981–6988.[CrossRef][Web of Science][Medline]
  14. Zhang Y, et al. Regulation of innate and adaptive immune responses by MAP kinase phosphatase 5. Nature (2004) 430:793–797.[CrossRef][Medline]
  15. Roux PP, et al. ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol. Mol. Biol. Rev. (2004) 68:320–344.[Abstract/Free Full Text]
  16. Moreno J, et al. Regulation of prostaglandin metabolism by calcitriol attenuates growth stimulation in prostate cancer cells. Cancer Res. (2005) 65:7917–7925.[Abstract/Free Full Text]
  17. Harant H, et al. 1alpha,25-dihydroxyvitamin D3 and a variety of its natural metabolites transcriptionally repress nuclear-factor-kappaB-mediated interleukin-8 gene expression. Eur. J. Biochem. (1997) 250:63–71.[Web of Science][Medline]
  18. Nakamura K, et al. Curcumin down-regulates AR gene expression and activation in prostate cancer cell lines. Int. J. Oncol. (2002) 21:825–830.[Web of Science][Medline]
  19. Cho JW, et al. Curcumin inhibits the expression of COX-2 in UVB-irradiated human keratinocytes (HaCaT) by inhibiting activation of AP-1: p38 MAP kinase and JNK as potential upstream targets. Exp. Mol. Med. (2005) 37:186–192.[Web of Science][Medline]
  20. Yan C, et al. Gene expression profiling identifies activating transcription factor 3 as a novel contributor to the proapoptotic effect of curcumin. Mol. Cancer Ther. (2005) 4:233–241.[Abstract/Free Full Text]
  21. Peehl DM. Growth or prostatic epithelial and stromal cells in vitro. In: Prostate Cancer Methods and Protocols—Russell PJ, Kingsley EA, eds. (2003) Totowa, NJ: Human Press. 41–57.
  22. Livak KJ, et al. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (2001) 25:402–408.[CrossRef][Web of Science][Medline]
  23. Radonic A, et al. Guideline to reference gene selection for quantitative real-time PCR. Biochem. Biophys. Res. Commun. (2004) 313:856–862.[CrossRef][Web of Science][Medline]
  24. Apte RN, et al. Interleukin-1–a major pleiotropic cytokine in tumor-host interactions. Semin. Cancer Biol. (2002) 12:277–290.[CrossRef][Web of Science][Medline]
  25. Veltri RW, et al. Interleukin-8 serum levels in patients with benign prostatic hyperplasia and prostate cancer. Urology (1999) 53:139–147.[CrossRef][Web of Science][Medline]
  26. Brinkman BM, et al. Engagement of tumor necrosis factor (TNF) receptor 1 leads to ATF-2- and p38 mitogen-activated protein kinase-dependent TNF-alpha gene expression. J. Biol. Chem. (1999) 274:30882–30886.[Abstract/Free Full Text]
  27. Kuldo JM, et al. Differential effects of NF-{kappa}B and p38 MAPK inhibitors and combinations thereof on TNF-{alpha}- and IL-1{beta}-induced proinflammatory status of endothelial cells in vitro. Am. J. Physiol. Cell Physiol. (2005) 289:C1229–C1239.[Abstract/Free Full Text]
  28. Said FA, et al. TNF-alpha, inefficient by itself, potentiates IL-1beta-induced PGHS-2 expression in human pulmonary microvascular endothelial cells: requirement of NF-kappaB and p38 MAPK pathways. Br. J. Pharmacol. (2002) 136:1005–1014.[CrossRef][Web of Science][Medline]
  29. Karin M. How NF-kappaB is activated: the role of the IkappaB kinase (IKK) complex. Oncogene (1999) 18:6867–6874.[CrossRef][Web of Science][Medline]
  30. Kato T Jr, et al. CK2 is a C-terminal IkappaB kinase responsible for NF-kappaB activation during the UV response. Mol. Cell (2003) 12:829–839.[CrossRef][Web of Science][Medline]
  31. Winzen R, et al. The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J (1999) 18:4969–4980.[CrossRef][Web of Science][Medline]
  32. Craig R, et al. p38 MAPK and NF-kappa B collaborate to induce interleukin-6 gene expression and release. Evidence for a cytoprotective autocrine signaling pathway in a cardiac myocyte model system. J. Biol. Chem. (2000) 275:23814–23824.[Abstract/Free Full Text]
  33. Mukhopadhyay A, et al. Curcumin downregulates cell survival mechanisms in human prostate cancer cell lines. Oncogene (2001) 20:7597–7609.[CrossRef][Web of Science][Medline]
  34. Gasparian AV, et al. The role of IKK in constitutive activation of NF-kappaB transcription factor in prostate carcinoma cells. J. Cell Sci. (2002) 115:141–151.[Abstract/Free Full Text]
  35. Kundu JK, et al. Resveratrol inhibits phorbol ester-induced cyclooxygenase-2 expression in mouse skin: MAPKs and AP-1 as potential molecular targets. Biofactors (2004) 21:33–39.[Web of Science][Medline]
  36. Kim SO, et al. [6]-Gingerol inhibits COX-2 expression by blocking the activation of p38 MAP kinase and NF-kappaB in phorbol ester-stimulated mouse skin. Oncogene (2005) 24:2558–2567.[CrossRef][Web of Science][Medline]
  37. Cohen BL, et al. Cyclooxygenase-2 (cox-2) expression is an independent predictor of prostate cancer recurrence. Int. J. Cancer (2006) 119:1082–1087.[CrossRef][Web of Science][Medline]
  38. Ricote M, et al. Pro-apoptotic tumor necrosis factor-alpha transduction pathway in normal prostate, benign prostatic hyperplasia and prostatic carcinoma. J. Urol. (2003) 170:787–790.[CrossRef][Web of Science][Medline]
  39. Corcoran NM, et al. Interleukin-6: minor player or starring role in the development of hormone-refractory prostate cancer? BJU Int. (2003) 91:545–553.[CrossRef][Web of Science][Medline]
  40. Michalaki V, et al. Serum levels of IL-6 and TNF-alpha correlate with clinicopathological features and patient survival in patients with prostate cancer. Br. J. Cancer (2004) 91:1227.[CrossRef][Web of Science]
  41. Bao BY, et al. 1{alpha}, 25-dihydroxyvitamin D3 suppresses interleukin-8-mediated prostate cancer cell angiogenesis. Carcinogenesis (2006) 27:1883–1893.[Abstract/Free Full Text]
  42. Wallner L, et al. Inhibition of interleukin-6 with CNTO328, an anti-interleukin-6 monoclonal antibody, inhibits conversion of androgen-dependent prostate cancer to an androgen-independent phenotype in orchiectomized mice. Cancer Res. (2006) 66:3087–3095.[Abstract/Free Full Text]
  43. Palayoor ST. Constitutive activation of IkappaB kinase alpha and NF-kappaB in prostate cancer cells is inhibited by ibuprofen. Oncogene (1999) 18:7389–7394.[CrossRef][Web of Science][Medline]
  44. Jang M, et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science (1997) 275:218–220.[Abstract/Free Full Text]
  45. Hsieh TC, et al. Differential effects on growth, cell cycle arrest, and induction of apoptosis by resveratrol in human prostate cancer cell lines. Exp. Cell Res. (1999) 249:109–115.[CrossRef][Web of Science][Medline]
  46. Newall CA, et al. Herbal Medicines: A guide for health-care professionals (1996) London: Pharmaceutical Press.
Received August 2, 2006; revised August 31, 2006; accepted November 27, 2006.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
P. Rafiee, V. M. Nelson, S. Manley, M. Wellner, M. Floer, D. G. Binion, and R. Shaker
Effect of curcumin on acidic pH-induced expression of IL-6 and IL-8 in human esophageal epithelial cells (HET-1A): role of PKC, MAPKs, and NF-{kappa}B
Am J Physiol Gastrointest Liver Physiol, February 1, 2009; 296(2): G388 - G398.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
S. Yu, G. Shen, T. O. Khor, J.-H. Kim, and A.-N. T. Kong
Curcumin inhibits Akt/mammalian target of rapamycin signaling through protein phosphatase-dependent mechanism
Mol. Cancer Ther., September 1, 2008; 7(9): 2609 - 2620.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
S. M. Zick, Z. Djuric, M. T. Ruffin, A. J. Litzinger, D. P. Normolle, S. Alrawi, M. R. Feng, and D. E. Brenner
Pharmacokinetics of 6-Gingerol, 8-Gingerol, 10-Gingerol, and 6-Shogaol and Conjugate Metabolites in Healthy Human Subjects
Cancer Epidemiol. Biomarkers Prev., August 1, 2008; 17(8): 1930 - 1936.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
28/6/1188    most recent
bgl241v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (13)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Nonn, L.
Right arrow Articles by Peehl, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nonn, L.
Right arrow Articles by Peehl, D. M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?