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Carcinogenesis Advance Access originally published online on October 24, 2007
Carcinogenesis 2007 28(12):2581-2588; doi:10.1093/carcin/bgm231
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Inhibitory effect of citrus 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone on 12-O-tetradecanoylphorbol 13-acetate-induced skin inflammation and tumor promotion in mice

Ching-Shu Lai1,6, Shiming Li2, Chee-Yin Chai3, Chih-Yu Lo4, Chi-Tang Ho2,5, Ying-Jan Wang1 and Min-Hsiung Pan6,*

1 Department of Environmental and Occupational Health, National Cheng Kung University Medical College, 138 Sheng-Li Road, Tainan 70428, Taiwan
2 Department of Food Science, Rutgers University, New Brunswick, NJ 08901-8520, USA
3 Department of Pathology, Kaohsiung Medical University, Kaohsiung 160, Taiwan
4 Department of Food Science, National Chiayi University, Chiayi 807, Taiwan
5 Graduate Institute of Food Science and Technology, National Taiwan University, Taipei 600, Taiwan
6 Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Road, Nan-Tzu, Kaohsiung 811, Taiwan

* To whom correspondence should be addressed. Tel: +886 7 361 7141; Fax: +886 7 361 1261; Email: mhpan{at}mail.nkmu.edu.tw Correspondence may also be addressed to Ying-Jan Wang. Tel: +886 6 235 3535 ext. 5804; Fax: +886 6 2752484; Email: yjwang{at}mail.ncku.edu.tw


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
5-Hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF), a polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. Herein, we report the first investigation of the inhibitory effects of 5-OH-HxMF on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in mouse skin. We found that the topical application of 5-OH-HxMF can effectively inhibit the transcriptional activation of iNOS and COX-2 mRNA and protein in mouse skin stimulated by TPA. Pre-treatment with 5-OH-HxMF resulted in the reduction of TPA-induced nuclear translocation of nuclear factor-{kappa}B (NF-{kappa}B) subunit and DNA binding by blocking phosphorylation of inhibitor {kappa}B (I{kappa}B) {alpha} and p65 and subsequent degradation of I{kappa}B{alpha}. In addition, 5-OH-HxMF can inhibit TPA-induced phosphorylation and nuclear translocation of the signal transducer and activator of transcription-3. Moreover, 5-OH-HxMF can suppress TPA-induced activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt, which are upstream of NF-{kappa}B. We also found that 5-OH-HxMF significantly inhibited TPA-induced mouse skin inflammation by decreasing inflammatory parameters. Furthermore, 5-OH-HxMF significantly inhibited 7,12-dimethylbenz[a]anthracene/TPA-induced skin tumor formation by reducing the tumor incidence and tumor multiplicity of papillomas at 20 weeks. Therefore, all these results revealed for the first time that 5-OH-HxMF is an effective antitumor agent and its inhibitory effect is through the down-regulation of inflammatory iNOS and COX-2 gene expression in mouse skin, suggesting that 5-OH-HxMF is a novel functional agent capable of preventing inflammation-associated tumorigenesis.

Abbreviations: COX-2, cyclooxygenase-2; DMBA, 7,12-dimethylbenz[a]anthracene; ERK, extracellular signal-regulated kinase; I{kappa}B, inhibitor {kappa}B; iNOS, nitric oxide synthase; ICR, institute of cancer research; MAPK, mitogen-activated protein kinase; NF-{kappa}B, nuclear factor-{kappa}B; 5-OH-HxMF, 5-Hydroxy-3,6,7,8,3',4'-hexamethoxyflavone; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; PI3K, phosphatidylinositol 3-kinase; STAT, signal transducer and activator of transcription; TPA, tetradecanoylphorbol-13-acetate; VEGF, vascular endothelial growth factor


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Polymethoxyflavones, which are found exclusively in the Citrus genus, particularly in the peel of sweet oranges (Citrus sinensis) and mandarin oranges (Citrus reticulate), have a broad spectrum of biological activity including anti-carcinogenic, anti-inflammatory and antitumor activities (1–4). The intake of citrus fruit has been suggested to prevent the development of certain human cancers. It is also commonly recognized that cancer induction can be prevented by ingestion of certain food phytochemicals, and flavonoids in Citrus fruits and juices are one of the most prominent cancer-preventing agents (5,6). Compared with polyhydroxylated flavonoids, polymethoxyflavones have higher permeability through the small intestine and are readily absorbed into the human blood circulatory system (7,8). The recent isolation of 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) from sweet orange peel extract (7) and the reported biological activities of other polymethoxyflavones promoted us to study its anti-inflammatory and antitumor activities. We have estimated that the amount of 5-OH-HxMF in dried orange peel is ~10 p.p.m. (10 mg/kg of dried orange peel).

It has been known that inflammation is causally linked to carcinogenesis and acts as a driving force in premalignant and malignant transformation of cells (9,10). Topical application of tetradecanoylphorbol-13-acetate (TPA) to 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mice leads to edema and papilloma formation by enhancing inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression. Specific iNOS and COX-2 inhibitors are able to counteract the biological events (11,12). Recent studies revealed that constitutive activation of signal transducers and activators of transcriptions (STATs), particularly STAT3, is found in a number of primary human epithelial tumors and cancer cell lines. Persistently active STAT3 induces tumor angiogenesis by up-regulation of vascular endothelial growth factor (VEGF) and its immune evasion (13,14). Activated nuclear factor-{kappa}B (NF-{kappa}B) often facilitates transcription of numerous genes, including iNOS and COX-2, resulting in inflammation and tumorigenesis. Activation of NF-{kappa}B by TPA is induced by a cascade of events leading to the activation of inhibitor {kappa}B (I{kappa}B) kinases, which in turn phosphorylates I{kappa}B. The subsequent ubiquitination and proteasomal degradation of I{kappa}B lead NF-{kappa}B free to translocate to the nucleus (15). These kinases can be activated through phosphorylation by upstream kinases, including NF-{kappa}B-inducing kinase, mitogen-activated protein kinase (MAPK) and protein kinase C (16,17). In addition, many studies have confirmed the cytokine function in the induction of transcription activity of NF-{kappa}B through extracellular signal-regulated kinase (ERK)1/2 (p42/44), p38 MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways (18–21). More importantly, iNOS has been shown to be involved in regulating COX-2, which plays a pivotal role in colon tumorigenesis (22). These observations suggest that iNOS may exacerbate turmorigenesis.

Our recent studies have shown that 5-OH-HxMF inhibits cell growth and induced apoptosis in human leukemia cells (23); however, the exact molecular mechanisms underlying the chemopreventive effect of 5-OH-HxMF remain largely unresolved. In this research, we have studied the effect of 5-OH-HxMF on the TPA-induced iNOS and COX-2 expression in mouse skin, explored underlying molecular mechanisms, tested the anti-inflammatory activity of 5-OH-HxMF in mouse skin following TPA application and investigated the inhibitory effect of 5-OH-HxMF on mouse skin tumor promotion using a two-stage carcinogenesis model including tumor incidence, multiplicity and volume.


   Materials and methods
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 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Chemicals and animals
5-OH-HxMF was isolated and purified from commercial orange peel extract (Florida Flavor Co., Lakeland, FL) as described previously. The purity of the compound is over 99.5% according to High-performance liquid chromatography analysis (7). TPA and DMBA were purchased from Sigma Chemical Co. (St Louis, MO). All other chemicals used were in the purest form available commercially. Female Institute of Cancer Research (ICR) mice at 5–6 weeks old were supplied from the BioLASCO Experimental Animal Center (Taiwan Co., Ltd, BioLASCO, Taipei, Taiwan). All animals were housed in a controlled atmosphere (25 ± 1°C at 50% relative humidity) and with a 12-h light/12-h dark cycle. The dorsal skin of each mouse was shaved with surgical clippers before the application of tested compound. 5-OH-HxMF and TPA were dissolved in 200 µl of acetone and applied topically to the shaved area of each mouse.

Western blot analysis
The female ICR mice were topically treated on their shaved backs with 5-OH-HxMF in 200 µl of acetone and 30 min prior to 10 nmol TPA treatment. The mice were killed by cervical dislocation at indicated time. For protein isolation from mouse skin, the dorsal skins of mice derived from different experiments were excised. After the fat from the dorsal skin was removed on ice, the skin samples were immediately placed in liquid nitrogen. The epidermal protein was homogenized on ice for 15 s with a polytron tissue homogenizer and lysed in 0.5 ml ice-cold lysis buffer [50 mM Tris–HCl, pH 7.4, 1 mM NaF, 150 mM NaCl, 1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 1 mM phenylmethanesulfonyl fluoride, 1% Nonident P-40 and 10 µg/ml leupeptin] for 30 min followed by centrifugation at 10 000g for 30 min at 4°C. The cytosolic fraction (supernatant) proteins were measured by Bio-Rad protein assay (Bio-Rad Laboratories, Munich, Germany). The samples (50 µg of protein) were mixed with 5x sample buffer containing 0.3 M Tris–HCl (pH 6.8), 25% 2-mercaptoethanol, 12% sodium dodecyl sulfate, 25 mM ethylenediaminetetraacetic acid, 20% glycerol and 0.1% bromophenol blue. The mixtures were boiled at 100°C for 5 min and were subjected to stacking gel then resolved by 12% sodium dodecyl sulfate–polyacrylamide minigels at a constant current of 20 mA. Subsequently, electrophoresis was carried out on sodium dodecyl sulfate–polyacrylamide gels. For western blot analysis, proteins on the gel were electrotransferred onto the 45 µm immobile membrane (polyvinylidene difluoride; Millipore Corp., Bedford, MA) with transfer buffer composed of 25 mM Tris–HCl (pH 8.9), 192 mM glycine and 20% methanol. The membranes were blocked with blocking solution (20 mM Tris–HCl pH 7.4, 0.2% Tween 20, 1% bovine serum albumin and 0.1% sodium azide). The membrane was further incubated with respective specific antibodies at appropriate dilution (1:1000) using blocking solution with the primary antibody of iNOS, I{kappa}B{alpha}, p50, p65 and phospho-PI3K(Tyr508) polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); COX-2 monoclonal antibodies (BD Transduction Laboratories, Lexington, KY); phospho-p65 (Ser536), phospho-STAT3 (Ser727), phospho-STAT3 (Tyr705), phospho-p38 (Thr180/Tyr182), phospho-ERK1/2 (Thr202/Tyr204), STAT3, ERK and p38 polyclonal antibodies (Cell Signaling Technology, Beverly, MA) and phospho-I{kappa}B{alpha} (Ser32/Ser36), phospho-Akt (Ser473) and Akt polyclonal antibody (Upstate Biotechnology, Lake Placid, NY) overnight at 4°C. The membranes were subsequently probed with anti-mouse or anti-rabbit immunoglobulin G antibody conjugated to horseradish peroxidase (BD Transduction Laboratories, Lexington, KY) and visualized using enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK). The densities of the bands were quantitated with a computer densitometer (AlphaImagerTM 2200 System Alpha Innotech Corporation, San Leandro, CA). All the membranes were stripped and reprobed for β-actin (Sigma Chemical Co.) as loading control.

Measurement of epidermal hyperplasia
In the epidermal thickness study, skin samples from different treatment groups were fixed in 10% formalin and embedded in paraffin for histological examinations. Sections (4 µm in thickness) of the skin samples were cut and mounted on polylysin-coated slides. Each section was deparaffinized in xylene, rehydrated through a series of graded alcohols and subjected to stain with hematoxylin and eosin. The thickness of the epidermis (µm) was measured using a Nikon light microscope (Japan) equipped with an ocular micrometer by the magnification (400x) in 15 fields per section. The number of dermal infiltrating leukocytes was determined by counting the stained cells at five different areas.

Proliferating cell nuclear antigen immunohistochemistry
For indirect proliferating cell nuclear antigen (PCNA) immunochemistry, the deparaffinized skin sections (4 µm) were incubated with 1.2% H2O2 in phosphate-buffered saline to quench the endogenous peroxidase activity. The primary antibody of PCNA (Santa Cruz Biotechnology) was diluted 100 times then applied to each section overnight at 4°C. After washing with phosphate-buffered saline, the sections were incubated with a biotin-conjugated horseradish peroxidase antibody (1:200; Vector Laboratories, Burlingames, CA) for 1 h at room temperature. Finally, the peroxidase was detected using the 3,3-diaminobenzidine tetrahydrochloride reaction, which produced the brown label in the epidermal. The numbers of PCNA-positive staining cells were counted in six different fields (200x) at both ends as well as in the middle for each section. The PCNA index was expressed as the average number of stained cells per field divided by the total number of dermal cells and multiplied by 100.

Quantitative real-time reverse transcription–polymerase chain reaction
Total RNA of epidermal skin was extracted using TRZOL reagent according to the supplier's protocol. The concentration of RNA content was determined by measuring the UV absorbance at 260 and 280 nm and the RNA was stored at –70°C until real-time polymerase chain reaction (PCR) analysis. Total of 2 µg RNA was transcribed into complementary DNA using SuperScript II RNAse H–reverse transcriptase (Invitrogen, Renfrewshire, United Kingdom) in a final volume of 20 µl. Reverse transcription reactions were performed at 42°C for 50 min and 99°C for 5 min in Gene Cycler thermal cycler (Bio-Rad). Negative controls were simultaneously performed by including all the components except reverse transcription.

In the real-time PCR analysis, specific primers and a fluorogenic probes were designed to target the conserved regions of various genes using the LightCycler probe design software (Roche Applied Science, Indianapolis, IN) according to the manufacturer's guidelines for the design of PCR primers and TaqMan probes. The PCR primers and TaqMan probes used in this experiment are shown in Table I.


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  		Table I. Characteristics of primers and probes used for quantitative real-time reverse transcription-polymerase chain reaction

 
All TaqMan PCR primers were located in two different exons of each gene to avoid amplification of any contaminating genomic DNA. All PCRs were performed using the LightCycler System (Roche Diagnostics. Inc., Rotkreuz, Switzerland) in a total volume of 20 µl containing 1x Taq polymerase buffer, 5 mmol/l MgCl2, 200 µmol/l deoxynucleotides, 300 nmol/l each primer, 150 nmol/l probe, 1 U Taq polymerase and 20 ng complementary DNA. Water instead of complementary DNA template was used for the negative controls. The gene amplification was done in duplicate for each sample. The thermal cycling conditions are 5 min at 94°C followed by 45 cycles, in which each cycle was at 94°C for 15 s and at 60°C for 1 min. The relative expression level of the gene in samples was calculated with the LightCycler software, normalized with housekeeping control (β-actin).

Preparation of cytosolic and nuclear extracts from mouse skin
Cytosolic and nuclear protein extraction was performed as described previously. In brief, the skins were washed with cold water and the epidermal cells from the dorsal skin of mice were stripped off. The epidermal samples were extracted by homogenization in 0.5 ml of ice-cold hypotonic buffer A containing 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid and 0.1 mM phenylmethylsulfonyl fluoride and then homogenized in a polytron for 1 min. The homogenates were incubated on ice under gentle shaking for 15 min. After centrifuged at 1000 r.p.m. for 5 min to remove tissue debris, the supernatant constituted the cytosolic fraction. The pellet was re-suspended in buffer A composed of 50 µl of 10% Nonident P-40 and vortex and followed by centrifugation for 2 min at 14 000 r.p.m. The nuclear pellet was re-suspended in 200 µl of high salt extraction buffer C [50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride and 10% glycerol] and kept on ice for 30 min and then centrifuged at 14 000 r.p.m. for 5 min. The supernatant containing nuclear proteins was transferred into a new tube and stored at –70°C after determination of protein concentration by using a protein assay kit (Bio-Rad, São Paulo, Brazil).

Electrophoretic mobility shift assay
Nuclear protein extraction was described as above. The electrophoretic mobility shift assay analysis was performed by using a non-radioactive (biotin label) gel shift assay according to the manufacturer's protocol. The NF-{kappa}B consensus oligonucleotide probe (5'-AGTTGAGGGGACTTTCCCAGGC-3') was end labeled with biotin (Pierce, Rockford, IL) with terminal deoxynucleotidyl transferase. For binding reaction, 6 µg of nuclear extract protein was incubated in a total volume of 20 µl with binding buffer containing 50 fmol of biotin end-labeled oligonucleotide. The mixture was further incubated at room temperature for 20 min. The specificity was determined by adding a 100-fold excess of unlabeled double-stranded consensus oligonucleotide to the reaction mixture to act as a competition reaction. Following the addition of 5 µl of sample buffer, the DNA–protein complexes were resolved on a 6% non-denaturing polyacrylamide gel in a 0.5x TBE buffer at 100 V for 2 h and then transferred to nylon membrane. Finally, the biotin-labeled DNA was detected by chemiluminescence using the LightShift Chemiluminescent EMSA kit (Pierce) and exposed to X-ray film.

Two-stage tumorigenesis in mouse skin
The antitumor-promoting activity of 5-OH-HxMF was examined by a standard initiation–promotion with DMBA and TPA, as reported previously (24). One group was composed of 12 female ICR mice. These mice were given commercial rodent pellets and fresh tap water ad libitum, both of which were changed twice a week. The dorsal region of each mouse was shaved with an electric clipper 2 days before initiation. Mice at 6 weeks old were started on 200 nmol DMBA in 200 µl acetone and control mice received 200 µl acetone alone. One week after initiation, the mice were topically treated with 200 µl acetone or promoted with TPA (5 nmol in 200 µl acetone) twice a week for 20 weeks. For the other two groups, the mice were treated with 5-OH-HxMF (1 and 3 µmol in 200 µl acetone) 30 min before each TPA treatment. Tumors of at least 1 mm of diameter in an electronic digital caliper were counted and recorded twice every week and the diameters of skin tumors were measured at the same time. The results were expressed as the average number of tumors per mouse, percentage of tumor-bearing mice and size distribution per mouse.

Statistical analysis
Data are presented as means ± standard errors for the indicated number of independently performed experiments. One-way Student's t-test was used to assess the statistical significance between the TPA- and 5-OH-HxMF plus TPA-treated groups. A P value< 0.05 was considered statistically significant.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Inhibitory effects of 5-OH-HxMF on TPA-induced iNOS and COX-2 expression in mouse skin
The anti-inflammatory activity of 5-OH-HxMF can be demonstrated by its effect on iNOS and COX-2 expression in TPA-stimulated mouse skin. When TPA was applied topically on the shaved area (backs) of female ICR mice, the levels of iNOS and COX-2 proteins were increased with maximal expression observed at 2 and 4 h, respectively (Figure 1A). As shown in Figure 1B, topical application of 5-OH-HxMF, 30 min prior to TPA treatment, resulted in a dramatic reduction in the levels of iNOS and COX-2 proteins in a dose-dependent manner in mouse skin. In our study, after the topical application of 10 nmol TPA to the shaved backs of female ICR mice, the iNOS mRNA (β-actin as control gene) expression reached the peak level in 1 h based on real-time PCR analysis. Under the same experimental conditions, the level of COX-2 gene expression was increased within tested time period (Figure 1C). To investigate whether or not 5-OH-HxMF has any influence on TPA-induced iNOS and COX-2 gene expression, we applied 5-OH-HxMF at 1 or 3 µmol, 30 min prior to TPA treatment. From the result of this experiment, we have found that there is a statistically significant (P < 0.01) suppression of iNOS and COX-2 gene expression in a dose-dependent manner in mouse skin (Figure 1D).


Figure 1
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  		Fig. 1. Inhibitory effects of 5-OH-HxMF on phorbol ester-induced iNOS and COX-2 protein expression. (A) Time course for iNOS, COX-2 and COX-1 protein expression on topical application of TPA in mouse skin. Female ICR mice were treated topically with acetone alone or 10 nmol TPA on dorsal skins for indicated time periods. (B) Mice were treated topically with 0.2 ml acetone or 5-OH-HxMF (1 and 3 µmol) in the same volume of acetone 30 min prior to 10 nmol TPA, and animals were killed 2 and 4 h, respectively, after the TPA treatment. The epidermal proteins were analyzed for iNOS, COX-2 and COX-1 by western blotting analysis. The western blot is representative of at least three independent experiments. Quantification of iNOS and COX-2 expression was normalized to β-actin using a densitometer. (C) Effect of iNOS and COX-2 gene expression on topical application of TPA in mouse skin. Female ICR mice were treated topically with acetone alone or 10 nmol TPA on dorsal skins for indicated time periods. (D) Mice were treated topically with 0.2 ml acetone or 5-OH-HxMF (1 and 3 µmol) in the same volume of acetone 30 min prior to 10 nmol TPA, and animals were killed 1 and 2 h, respectively, after the TPA treatment. A total of 2 µg of complementary DNA were subject to real-time PCR. The mRNA expressions of iNOS and COX-2 gene were performed using the LightCycler System and TaqMan probe real-time PCR. Data are mean ± standard error. *, P < 0.05; **, P < 0.01 (for iNOS gene) and ##, P < 0.01 (for COX-2 gene) were verse TPA alone.

 
Inhibitory effect of 5-OH-HxMF on TPA-induced activation of NF-{kappa}B and phosphorylation and degradation of I{kappa}B{alpha} protein expression
Since iNOS and COX-2 are frequently regulated by activating NF-{kappa}B signaling pathway (12,25) and NF-{kappa}B activation and nuclear translocation are preceded by the phosphorylation and proteolytic degradation of I{kappa}B{alpha} (26), it is of importance to investigate the inhibitory activity of 5-OH-HxMF against the activation and nuclear translocation of p65 and p50, the functional active subunit of NF-{kappa}B in mouse skin. By the topical application of 5-OH-HxMF onto mouse skin (prior to TPA application), we found that TPA-induced NF-{kappa}B nuclear translocation was inhibited in a dose-dependent manner as shown by western blot analysis (Figure 2A). The phosphorylation of p65/RelA at serine 536 was also inhibited by the 5-OH-HxMF pre-treated animal groups in a dose-dependent manner (Figure 2A). In this experiment, Poly (ADP-ribose) polymerase, a nuclear protein, and β-actin, a cytosolic protein, were used as controls to confirm that there was no contamination during extraction of each fraction. We also determined the phosphorylation and cytoplasmic levels of I{kappa}B{alpha} protein expression by immunoblot analysis to differentiate the potential attribution due to the inhibitory effect of 5-OH-HxMF and its effect on I{kappa}B{alpha} degradation. By topical application of 5-OH-HxMF, 30 min prior to TPA treatment, we found that the inhibition of TPA-induced phosphorylation and degradation of I{kappa}B{alpha} protein exhibited a dose-dependent manner (Figure 2B). The effect of 5-OH-HxMF treatment on TPA-induced NF-{kappa}B-DNA-binding activity was evaluated using electrophoretic mobility shift assay (Figure 2C), and it was found that 5-OH-HxMF suppressed TPA-induced NF-{kappa}B-DNA-binding activity in mouse skin. Addition of cold consensus NF-{kappa}B oligonucleotide (100-fold) abolished the mobility shift band, demonstrating the specificity of protein–DNA interaction. Supershift analysis has shown that induced NF-{kappa}B was a p65/p50 heterodimer. We also found that the induction of NF-{kappa}B-DNA-binding activity coincided with the degradation of I{kappa}B{alpha} and nuclear translocation of both p50 and p65/RelA.


Figure 2
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  		Fig. 2. Effect of 5-OH-HxMF on phorbol ester-induced NF-{kappa}B and STAT3 activation. (A) Mice were treated topically with 0.2 ml acetone or 5-OH-HxMF (1 and 3 µmol) in the same volume of acetone 30 min prior to 10 nmol TPA. All mice were killed 1 h after the TPA treatment, and nuclear and cytosolic extracts from mouse skin were assayed for p-p65 (Ser 536), p65, p50, Poly (ADP-ribose) polymerase, β-actin, (B) p-I{kappa}B and I{kappa}B and by western blotting analysis. (C) Electrophoretic mobility shift assay analysis was performed by equal 6 µg of nuclear extracts from mouse epidermis with a biotin-labeled (non-radioactive) NF-{kappa}B probe. All analyses were representative of at least three independent experiments. The values under each lane indicate relative density of the band normalized to β-actin. (D) Mice were treated topically with 0.2 ml acetone or 5-OH-HxMF (1 and 3 µmol) in the same volume of acetone 30 min prior to 10 nmol TPA. Mice were killed 1 h after the TPA treatment, and nuclear extracts from mouse skin were assayed for p-STAT3 (Ser727 and Tyr705) and total STAT3 by western blotting analysis. The values under each lane indicate relative density of the band normalized to Poly (ADP-ribose) polymerase.

 
5-OH-HxMF inhibits STAT3 (Ser727 and Tyr705) phosphorylation and its nuclear translocation in TPA-induced skin inflammation
Owing to our observation of iNOS and COX-2 being up-regulated by TPA in mouse skin and the subsequent decrease of its efficacy against TPA-induced inflammation caused by 5-OH-HxMF, we examined STAT3 status and discovered a possible role for this transcription factor in the inflammation and tumor promotion stage of epithelial carcinogenesis (27). Western blot analysis has shown that TPA induced a dramatic increase in the phosphorylation level of STAT3 at Ser727 and Tyr705 and its nuclear translocation, which is necessary for STAT3 transcriptional activity. Topical application of 5-OH-HxMF prior to TPA application dose-dependently inhibited TPA-induced phosphorylation and translocation of STAT3 (Figure 2D). These results also suggest that TPA activates STAT3 signaling in mouse skin inflammation that could be effectively targeted by 5-OH-HxMF in its chemopreventive effects.

Inhibitory effect of 5-OH-HxMF on TPA-induced epidermal MAPK and PI3K and phosphorylation of Akt
Studies have shown that p38, ERK (p44/42) MAPK and PI3K/Akt signaling pathways are involved in the TPA-mediated induction of iNOS and COX-2 by diverse mechanisms including the modulation of signaling via NF-{kappa}B, AP-1, STATs in mouse skin (11,14,28). Therefore, we investigated the effects of 5-OH-HxMF on the TPA-induced phosphorylation of p38, ERK MAPK and PI3K/Akt in mouse skin. Western blot analysis revealed that topical application of TPA alone caused significant increase in the phosphorylation of p38 (6.4-fold increase) and ERK MAPK (3.6-fold) in mouse skin as compared with vehicle-treated controls. However, pre-treatment of 5-OH-HxMF retarded the phosphorylation of p38 and ERK MAPK in TPA-treated mouse skin in a dose-dependent manner (Figure 3A). We also assessed whether or not PI3K/Akt signaling is involved in cellular responses to TPA by performing western blot analysis with antibody in phosphorylated form of PI3K and Akt. Densitometric analysis of blots revealed significant increase in the phosphorylation of PI3K and Akt in mouse skin treated with a single topical application of TPA. We observed that pre-application of 5-OH-HxMF prior to TPA treatment attenuated TPA-induced phosphorylation of PI3K and Akt in mouse skin (Figure 3B). More importantly, no changes were observed in the total epidermal Akt content in mice treated with both TPA and 5-OH-HxMF as compared with vehicle-treated control. These results of our immunoblot analyses suggest that the inhibition of iNOS and COX-2 expression by 5-OH-HxMF might block TPA-induced NF-{kappa}B and STAT3 activation by inhibiting p38, ERK MAPK and PI3K/Akt/I{kappa}B kinase pathways, which interrupts the degradation of I{kappa}B{alpha}. In addition, in the skin tumors, DMBA initiated and promoted by TPA for 20 weeks, 5-OH-HxMF treatment showed strongly reduced iNOS, COX-2 and VEGF levels, but slighted suppressed matrix metalloproteinase-9 level in skin tumor (Figure 3D).


Figure 3
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  		Fig. 3. Effects of 5-OH-HxMF on TPA-induced activation of MAPK, PI3K/Akt and I{kappa}B kinases in mouse skin. Mice were treated topically with 0.2 ml acetone or 5-OH-HxMF (1 and 3 µmol) in the same volume of acetone 30 min prior to 10 nmol TPA. All mice were killed 1 h after the TPA treatment, and expression of (A) MAPK and (B) p-PI3K, p-Akt and Akt was measured by western blotting analysis. The values under each lane indicate relative density of the band normalized to β-actin. Data are representative of at least independent experiments, which showed a similar result. (C) Inhibitory effects of 5-OH-HxMF on phorbol ester induction of PCNA-positive cells in mouse skin. Three mice were used in one experimental group and the mice were treated as described in Table II legend. All mice were killed at 24 h after the TPA treatment for PCNA immunostaining as described in Materials and methods. PCNA-labeling index (%) was counted from six different fields (200x) from each mouse. (D) Effect of 5-OH-HxMF on angiogenesis in skin tumors. iNOS, COX-2, VEGF and matrix metalloproteinase-2 were measured by western blotting analysis using specific antibodies.

 
Anti-inflammatory activities of 5-OH-HxMF in mouse skin
We performed inhibition study of 5-OH-HxMF against TPA-stimulated inflammation. As shown in Table II, TPA application, at a dose of 10 nmol, led to marked edema and an increase in epidermal thickness was observed (8.9 ± 0.8 µm in group 1 versus 21.2 ± 3.4 µm in group 2; P < 0.001). Pre-treatment with 5-OH-HxMF prior to TPA application dramatically suppressed epidermal thickness in a dose-dependent manner (13.7 ± 1.8 µm in group 3 and 11.8 ± 1.7 µm in group 4; P < 0.01). As shown in Table II, a greater number of leukocytes were found to have infiltrated the dermis by TPA application as compared with the control (22.0 ± 2.8/mm2 in group 1 versus 131.0 ± 8.5/mm2 in group 2; P < 0.001). Pre-treatment with 1 and 3 µmol of 5-OH-HxMF inhibited leukocyte infiltration by 45.9 and 68.3%, respectively. The PCNA-labeling index, a marker for cell proliferation, in the epidermis of the group 2 mice increased by 2.7-fold over that of group 1 (24.7 ± 2.4% in group 1 versus 67.5 ± 3.4% in group 2; P < 0.001). Pre-treatment with 1 and 3 µmol of 5-OH-HxMF prior to TPA application significantly reduced PCNA-labeling indices by 59.8 and 67.1%, respectively (Table II and Figure 3C).


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  		Table II. Inhibitory effects of 5-OH-HxMF on TPA-induced skin thickness, infiltrated leukocytes and PCNA in mouse skin

 
Antitumor-promoting activity of 5-OH-HxMF in mouse skin
Up-regulation of iNOS and COX-2 occurs in many pathological conditions, such as in tumorigenesis. Since application of 1 and 3 µmol of 5-OH-HxMF to mouse skin significantly inhibited various molecular targets that play significant roles in the progression of skin tumors, we selected this dose for assessing the antitumor-promoting potential of 5-OH-HxMF in DMBA-initiated mouse skin. As shown in Figure 4, with DMBA and TPA treatment, the tumor incidence in this positive control group was 100% 20 weeks after promotion. In contrast, administration of DMBA followed by repeated application of acetone produced no tumors. Throughout the experiment, there was no noticeable difference in weight gain between the mice treated with two doses of 5-OH-HxMF and with those not treated, indicating that the topical application of 5-OH-HxMF did not cause any toxicity. When 5-OH-HxMF (3 µmol)-pre-treated groups (30 min before TPA), the incidence was reduced by 27% (Figure 4B). The average number of tumors per mouse in the control was 17.0 at the end of the 20-week experiment. In the treated groups, pre-treatment with 5-OH-HxMF dose-dependently reduced the number of tumors per mouse by 36.5 and 52.9% at 1 µmol dose and 3 µmol dose, respectively (Figure 4A). The tumor promotion data were analyzed in terms of size distribution of papillomas and compared with the positive control group. The number of papilloma (1 to <3 mm in diameter) per mouse was dose-dependently inhibited in the 5-OH-HxMF-treated group (Table III). In addition, 5-OH-HxMF treatment showed a significant inhibition in tumors at sizes of 3 to <5 mm and ≥5 mm as is evident in a significant reduction (67 and 57%, 75 and 61%, P < 0.05, Student's t-test) in tumor size in 1 and 3 µmol of 5-OH-HxMF-treated groups of mice, respectively, compared with the positive control group. The animals started on DMBA and treated twice weekly with 3 µmol of 5-OH-HxMF were devoid of any skin tumors throughout the experiment (data not shown), suggesting that 5-OH-HxMF itself is not a tumor promoter.


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  		Table III. Effects of 5-OH-HxMF on tumor diameter (mm2) in DMBA/TPA-induced skin tumorigenesis

 


Figure 4
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  		Fig. 4. Antitumor-promoting effects of 5-OH-HxMF on DMBA/TPA-induced skin tumorigenesis in ICR mice. Tumor promotion in all mice was initiated with DMBA (200 nmol) and promoted with TPA (5 nmol) twice weekly, starting 1 week after initiation. 5-OH-HxMF (1 and 3 µmol) was dissolved in 0.2 ml acetone and topically applied 30 min prior to each TPA treatment. Tumors of at least 1 mm in diameter were counted and recorded weekly, as described in Materials and methods. (A) Average number of tumors per mouse (tumor multiplicity). (B) Percentage of tumor-bearing mice (tumor incidence). *P < 0.05 and **P < 0.01 indicate statistically significant differences from the TPA-treated group. Statistical analysis was done by Student's t-test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
COX-2 and iNOS are important enzymes involved in mediating the inflammation process, cell proliferation and skin tumor promotion (28,29). In the current study, our data clearly demonstrated that pre-application of 5-OH-HxMF before TPA treatment affords significant inhibition of TPA-induced iNOS and COX-2 mRNA and protein expression in a dose-dependent manner (Figure 1).

The inhibition of 5-OH-HxMF against TPA-induced biological and histological phenotype related to inflammation is summarized in Table II. On one hand, TPA has recently been reported to produce Nitric oxide and VEGF in human polymorphonuclear leukocytes, whereas over-expressed VEGF leads to induction of vascular hyperpermeability (11). On the other hand, Prostaglandin E2 is well known to increase vascular permeability. In this study, we showed the significant inhibitory effects of 5-OH-HxMF against TPA-stimulated induction of epidermal iNOS and COX-2 expression in ICR mouse (Figure 1). As a result, we conclude that the inhibition by 5-OH-HxMF of edema formation and the reduction of epidermal thickness and cell growth (Table II and Figure 3C) may be partly attributable to the suppression of Nitric oxide, VEGF and Prostaglandin E2 production (Figure 3D). Our examination of the effects of 5-OH-HxMF on vascular permeability is in progress. These inhibitory effects of 5-OH-HxMF in the suppression of TPA-mediated responses in mouse skin suggest that the primary effect of 5-OH-HxMF may be against inflammatory responses, which may then result in the inhibition of tumor promotion.

Activation of NF-{kappa}B is necessary for TPA induction of the iNOS and COX-2 promoter (28). In our study, 5-OH-HxMF was found to inhibit TPA-induced DNA-binding activity of NF-{kappa}B by suppressing phosphorylation of I{kappa}B{alpha} and p65 and subsequent nuclear translocation of p50 and p65/RelA subunits of NF-{kappa}B (Figure 2). Moreover, a critical role of STAT3 in promoting tumor cell survival, proliferation, angiogenesis and immune evasion has been shown in various pathophysiological conditions, including carcinogenesis (30). Our study has also shown that topical application of 5-OH-HxMF prior to TPA application to mouse skin resulted in the reduction in the TPA-induced phosphorylation of STAT3 and translocation to the nucleus (Figure 2D). These findings imply that STAT3 is a novel potential target for the prevention of skin carcinogenesis.

Both NF-{kappa}B and PI3K/Akt signaling pathways have emerged as promising molecular targets in the prevention of cancers. Many signaling pathways, including PI3K/Akt and MAPK, have been proposed to respond to TPA stimulation (31). PI3K activation leads to phosphorylation of phosphatidylinositides, which then activates the downstream main target, Akt, which appears to play various important roles in regulating cellular growth, differentiation, adhesion and the inflammatory reaction (32). Since 5-OH-HxMF can significantly inhibit the induction of iNOS and COX genes and proteins, we have investigated whether or not 5-OH-HxMF exerts any influence on or interferes with the signaling molecules, in turn regulating them. In this study, we clearly demonstrated that topical application of TPA resulted in the activation of p38, ERK and PI3K/Akt. Topical application of 5-OH-HxMF prior to TPA application to mouse skin resulted in the reduction of TPA induction phosphorylation of p38, ERK MAPK and PI3K/Akt in mouse skin (Figure 3).

As predicted by the suppressive efficacies of biochemical markers related to inflammation, topical application of 5-OH-HxMF at doses of 1 and 3 µmol, before TPA treatment during the tumor promotion process, significantly lowered the number and size of papillomas. 5-OH-HxMF inhibited TPA-induced formation of the average number of skin tumors per mouse in a dose-dependent manner. The possible mechanism is that 5-OH-HxMF down-regulates inflammatory iNOS and COX-2 gene expression in mouse skin by inhibiting the activation of NF-{kappa}B and by interfering with the activation of PI3K/Akt and MAPK. 5-OH-HxMF seems to act as a modulating agent in multiple signaling pathways, thus proving it as an excellent and novel example of being an ideal chemopreventive agent. Further thorough evaluation of 5-OH-HxMF is needed to verify this proposed mechanism. Angiogenesis is the process of forming new blood vessels from pre-existing vessels and is an essential process for solid tumor growth, invasion and metastasis. For the first time, we also observed 5-OH-HxMF suppressed the expression of VEGF and matrix metalloproteinase-9 in skin tumor (Figure 3D). Here we suggest that 5-OH-HxMF suppresses the tumor growth by inhibiting the tumor angiogenesis.

Furthermore, our data suggest that 5-OH-HxMF could block the activation and infiltration of macrophage in the skin tumor (Table II), because a greater number of leukocytes were found to have infiltrated the dermis by TPA application as compared with the control. However, pre-treatment with 5-OH-HxMF can inhibit leukocyte infiltration and iNOS and COX-2 expression (Figure 1B). Based on our findings, we suggest that the 5-OH-HxMF promotes a strong protective effect against TPA-mediated epithelial carcinogenesis via down-regulation of inflammation and proliferation, involving PI3K/Akt/I{kappa}B kinase and MAPK signaling pathways, STAT3 and NF-{kappa}B transcription factors, as well as iNOS and COX-2 (Figure 5). Therefore, we conclude that 5-OH-HxMF has great potential as a novel chemopreventive agent to be used in the treatment of inflammation-associated tumorigenesis, especially in the prevention and treatment of epithelial skin cancer.


Figure 5
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  		Fig. 5. A schematic representation of suppression of TPA-induced NF-{kappa}B and STAT3 activation and iNOS and COX-2 expression by 5-OH-HxMF in mouse skin. Topical application of TPA activates p38, p44/42 MAPK, I{kappa}B kinases and PI3K/Akt, which, in turn, phosphorylation I{kappa}B{alpha} and p65 thereby contributing to the activation of NF-{kappa}B and subsequent induction of iNOS and COX-2. Each of these events can be blocked by 5-OH-HxMF. The inhibition of TPA-induced STAT3 phosphorylation by 5-OH-HxMF may also contribute to suppression of skin tumorigenesis. Ub, ubiquitin and {perp}, site of inhibition.

 

   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Science Council (95-2313-B-022-003-MY3 and 95-2321-B-022-001).


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received July 29, 2007; revised October 10, 2007; accepted October 15, 2007.


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C.-S. Lai, S. Li, C.-Y. Chai, C.-Y. Lo, S. Dushenkov, C.-T. Ho, M.-H. Pan, and Y.-J. Wang
Anti-inflammatory and antitumor promotional effects of a novel urinary metabolite, 3',4'-didemethylnobiletin, derived from nobiletin
Carcinogenesis, December 1, 2008; 29(12): 2415 - 2424.
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