Rosmarinic acid inhibits epidermal inflammatory responses: anticarcinogenic effect of Perilla frutescens extract in the murine two-stage skin model
Naomi Osakabe1,3,
Akiko Yasuda1,
Midori Natsume1 and
Toshikazu Yoshikawa2
1 Health and Bioscience Laboratories, Meiji Seika Kaisha Ltd, 5-3-1, Chiyoda Sakado 350-0289, Japan and 2 First Department of Medicine, Kyoto Prefectural University of Medicine, Kamigyoku, Kyoto 602-8566, Japan
 |
Abstract
|
---|
Perilla frutescens extract showed marked reduction on tumorigenesis in a murine, two-stage skin carcinogenesis model. In this model, cancer is initiated by application of 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by application of 12-tetradecanoylphorbol 13-acetate (TPA). Following tumor initiation with DMBA, topical application of a perilla-derived fraction (PF) at doses of 2 mg/mouse/application resulted in significant inhibition of tumorigenesis. The efficacy of each fraction was correlated with rosmarinic acid (RA) and luteolin concentration. Topical application of perilla extract (PE) that contained 68% RA or an equivalent amount of commercially available RA showed nearly identical antiinflammatory activity 5 h after TPA treatment. Application of luteolin had less anti-inflammatory activity. Marked neutrophil infiltration was observed in TPA-challenged skin by histological examination using hematoxylineosin. This change was greatly reduced by pre-treatment with PE or RA. Myeloperoxidase activity, a marker of neutrophil recruitment, was also increased in TPA-challenged skin and was significantly decreased in the PE and RA treated groups. Intercellular adhesion molecule 1 and vascular cell adhesion molecule-1 mRNA expression levels were reduced by pre-treatment with PE or RA. TPA-induced increases in synthesis of the chemokines KC and macrophage inflammatory protein-2 were significantly decreased by pre-treatment with PE or RA. Prostaglandin E2 and leukotriene B4 levels were slightly increased 5 h after TPA treatment. These levels were only numerically decreased in the PE and RA treated groups. However, induction of cyclooxygenase-2 mRNA expression was obviously reduced by pre-treatment with PE or RA. Reactive oxygen radical production, detected as thiobarbituric acid reactive substance and lipid peroxide, by double treatment of TPA was reduced by pre-treatment with PE or RA. Production of 8-hydroxy-2'deoxyguanosine, which was detected immunohistochemically, was also induced by double treatment with TPA. This adduct was barely visible in PE or RA treated mice. Thus, we conclude that part of the anticarcinogenic effects of P.frutescens extract is due to RA via two independent mechanisms: inhibition of the inflammatory response and scavenging of reactive oxygen radicals.
Abbreviations: COX-2, cyclooxygenase-2; DMBA, 7,12-dimethyl benz[a]anthracene; ICAM-1, intercellular adhesion molecule 1; LPO, lipid peroxide; LTB4, leukotriene B4; MIP-2, macrophage inflammatory protein-2; MPO, myeloperoxidase; NF
B, nuclear factor
B; 8OH-dG, 8-hydroxy-2'deoxyguanosine; PE, perilla extract; PF, perilla fraction; PGE2, prostaglandin E2; PKC, protein kinase C; RA, rosmarinic acid; TBARS, thiobarbituric acid reactive substance; TFA, trifluoroacetic acid; TPA, 12-tetradecanoylphorbol 13-acetate; VCAM-1, vascular cell adhesion molecule-1
 |
Introduction
|
---|
Results of epidemiological studies have shown that frequent consumption of plant-derived foods such as vegetables, fruits, tea and herbs, is associated with a lower risk of a variety of cancers (13). This suggests that cancer preventive substances exist in plant-derived foods. Many reports have suggested that plant-derived phytochemicals prevent cancer in experimental animal models (46). Specifically, antioxidants such as flavonoids, phenolic acid and proanthocyanidins have shown potent anticancer activity in two-stage skin papilloma models using 7,12-dimethylbenz[a]anthracene (DMBA) and 12-tetradecanoylphorbol 13-acetate (TPA) (79).
Rosmarinic acid (RA) is found in many medicinal species of the plant Lamiaceae (Figure 1) including basil, sage, rosemary, mint (10) and Perilla frutescens, one of the most popular garnishes in Japan. Extract of perilla appears to be a strong anti-inflammatory agent as it inhibits mast cell release of histamine (11), inhibits lipoxygenase activity (12) and is an antioxidant (1315). Recently, we reported that perilla extract (PE) and RA reduce liver injury induced by D-galactosamine and LPS in mouse, due to scavenging of superoxide molecules produced by Kupffer cells and inhibition of peroxynitrite formation induced by inducible nitric oxide synthase (iNOS) (16).
Previous reports suggest that the body's response to cancer has many parallels with the inflammatory response (17,18). For example, inflammatory cytokine networks may influence survival, growth, mutation, proliferation, differentiation and movement of both tumor and stromal cells (18). In addition, cyclooxygenase-2 (COX-2), a key enzyme in the inflammatory response, is thought to affect carcinogenesis indirectly via its modulatory effect on the inflammatory response and the immune system (19,20).
In the present study, we examined the effects of PE in a two-stage, mouse skin papilloma model system. To further elucidate these effects, we also compared the effects of PE, RA and luteolin on edema formation induced TPA.
 |
Materials and methods
|
---|
Mice
Seven- to nine-week-old male, BALB/c mice purchased from Crea Japan (Tokyo, Japan) were used for this study. Mice were kept in a controlled environment at 23°C and 55% humidity under a 12 h light/dark cycle. The Animal Committee of Meiji Seika Kaisha Health and Bioscience Laboratory approved this study and mice received care under guidelines provided by this committee.
Chemicals
RA was obtained from Extrasynthase (Genay, France). TPA and DMBA were obtained from Sigma (St Louis, MO). Anti-8-hydroxy-2'deoxyguanosine (8OH-dG) antibody was obtained from the Japanese Institute for the Control of Aging (Shizuoka, Japan). All other chemicals were of reagent grade.
The perilla fraction (PF) was prepared as follows. Fresh perilla leaves were extracted with 1% w/v citric acid at 90°C for 30 min. The extract was concentrated and applied to a Diaion HP2MG open column (Mitsubishi Kasei Co., Tokyo, Japan). The column was step-wise eluted by 0.1% w/v trifluoroacetic acid (TFA) in distilled water and methanol. Fractions eluted by 0.1% w/v TFA containing 20, 50 or 80% v/v methanol were collected, freeze-dried and used for experiment 1. The concentration of polyphenols, including caffeic acid, protocatecuic acid, RA and luteolin, was determined by HPLC using a Develosil HG-5 column (Nomura Chemical Co., Aichi, Japan) with solvents A (0.1% v/v TFA in distilled water) and B (0.1% TFA v/v in acetonitrile), under the following conditions: 1050% linear gradient of A in B; flow rate 0.8 ml/min; detection 280 nm. The concentration of polyphenols in each fraction is shown in Table I.
The PE used in experiments 2 and 3 was prepared as follows. The extract derived from fresh perilla, as described above, was concentrated and then mixed with n-butanol. The resulting n-butanol layer was dried and the residue was dissolved in distilled water. This solution was applied onto a Diaion HP2MG column and washed with distilled water. The fraction eluted by 0.1% w/v TFA containing 60% v/v methanol was collected and freeze-dried. The RA concentration in this fraction was 68% w/w. Concentration of luteolin, luteolin-7-O-glucoside, protocatechuic acid and caffeic acid were all under the detectable limit (>50 ng/ml).
Experimental procedure
All test chemicals and TPA were dissolved in ethanol, and 20 µl of this solvent was topically applied to each animal.
Experiment 1
Eighty mice were divided into four groups as shown in Figure 2a. All mice were treated topically on dorsal, saved skin with 50 µg/20 µl/mouse DMBA dissolved in acetone. One week later, animals were treated topically with PF 1, 2 or 3 at a dose of 2 mg/mouse or control vehicle 30 min before treatment with 2 nmol TPA/mouse. These treatments were repeated twice weekly until termination of the experiment at 20 weeks after DMBA treatment. Skin tumor formation was recorded weekly and tumors >1 mm in diameter were included in the cumulative total if they persisted for 2 or more weeks.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 2. Experimental procedure of murine two-stage skin papilloma model and TPA induced ear edema inflammation model. (a) Filled triangle, DMBA 50 µg/mouse; downwards arrow, TPA 2 nmol/mouse; empty triangle, TPA with PF-1, 2 mg/mouse; empty square, TPA with PF-2, 2 mg/mouse; filled square, TPA with PF-3, 2 mg/mouse topical application. (b) Filled triangle, ethanol 20 µl/mouse; downwards arrow, TPA 2 nmol/mouse; empty triangle, PE 2 mg/mouse; empty square, rosmarinic acid 0.25, 0.5, 1.0 or 1.35 mg/mouse; filled square, luteolin 0.25 or 0.5 mg/mouse topical application. (c) Filled triangle, ethanol 20 µl/mouse; downwards arrow, TPA 2 nmol/mouse; empty triangle, PE 2 mg/mouse; empty square, rosmarinic acid 1.35 mg/mouse topical application.
|
|
Experiment 2
Mice were divided into nine groups: vehicle, TPA-challenged control or TPA-challenged and treated with 2.0 mg/mouse PE or 0.25 or 0.5 mg/mouse luteolin, which is the maximum dissolved dosage, or 0.25, 0.5, 1.0 or 1.35 mg/mouse RA (Figure 2b). RA (1.35 mg/mouse) was administered in ethanol at an amount equivalent to 2.0 mg/mouse of PE. PE, RA and luteolin treatments were applied 30 min before treatment with 2 nmol TPA/mouse. Animals were killed under anesthesia 1 or 5 h after TPA application. A mouse ear punch was obtained with a 5 mm cork bowler and was subsequently weighed. Punches were kept at -80°C until analysis.
Experiment 3
Mice were divided into nine groups: vehicle, TPA single challenged, TPA double challenged, TPA double challenged and double treated with PE (2.0 mg/mouse) or RA (1.35 mg/mouse), TPA double challenged and treated with PE or RA 30 min prior to the first or second TPA treatment (Figure 2c). TPA, PE and RA treatments were performed as described above. The second TPA treatment was performed 24 h after the first TPA treatment. Animals were killed under anesthesia 1 h after the second TPA application.
Histological evaluation
The harvested ears were fixed in 3% buffered formalin and embedded in paraffin. Five-micrometer sections were affixed to slides, deparaffinized and stained with hematoxylin and eosin to assess morphological changes. The production of 8OH-dG was detected by immunohistochemical localization using an anti-8OH-dG monoclonal antibody. Briefly, deparaffinized slides were placed in blocking reagent containing H2O2 for 45 s to quench endogenous peroxidase. The sections were then blocked with 10% normal goat serum for 1 h. After blocking, 1 µg/ml 8OH-dG was incubated with the sections for 1 h at room temperature in a moist chamber. Incubation was followed by the addition of a biotinylated secondary antibody and streptavidinperoxidase conjugate. The slides were incubated with 3,3-diaminobenzidine tetrahydrochloride to detect 8OH-dG production, and were then counterstained with hematoxylin.
Analyses
Myeloperoxidase (MPO) activity was determined according to the method of Bradley et al. (21). Tissue thiobarbituric acid reactive substance (TBARS) levels were determined according to the method of Ohkawa et al. (22) using tetramethoxypropane (Wako Pure Chemical Industries, Tokyo, Japan). Lipid peroxide (LPO) content was measured spectrophotometrically using a commercially available kit (Determiner LPO, Kyowa Medics Co., Tokyo, Japan). KC, macrophage inflammatory protein-2 (MIP-2), prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) concentrations were analyzed by ELISA using a kit purchased from R&D Systems (Minneapolis, MN). Semi-quantitative analysis of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and COX-2 was performed using RTPCR. Total RNA was extracted from mice livers using TRIzol (Invitrogen, Carlsbad, CA). cDNA synthesis was performed using the Reverse Transcription System (Promega, Madison) according to the manufacturer's instructions. PCR primers (Sawady Technology, Tokyo, Japan) were as follows: ICAM-1 sense primer, 5'-TCG GAG GAT CAC AAA CGA AGC-3' corresponding anti-sense primer, 5'-AAC ATA AGA GGC TGC CAT CAC G-3'; VCAM-1 sense primer, 5'-CCT CAC TTG CAG CAC TAC GGG CT-3' and the corresponding anti-sense primer, 5'-TTT TCC AAT ATC CTC AAT GAC GGG-3'; COX-2 sense primer, 5'-ACT CAC TCA GTT TGT TGA GTC ATT C-3' and the corresponding anti-sense primer, 5'-TTT GAT TAG TAC TGT AGG GTT AAT G-3'; ß-actin sense primer, 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3' and the corresponding anti-sense primer, 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3'. PCR conditions for ICAM-1and VCAM-1 were 35 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 30 s and extension at 72°C for 45 s. PCR conditions for COX-2 were 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 60 s and extension at 72°C for 90 s. PCR conditions for ß-actin were 30 cycles for denaturation at 95°C for 30 s, annealing at 60°C for 1 min and extension at 72°C for 2 min. A final extension reaction was then carried out at 72°C for 5 min. The predicted size of ICAM-1, VCAM-1, COX-2 and ß-actin were 431, 441, 583 and 348 bp, respectively. PCR products were electrophoresed on 2.0% agarose gels. Ethidium bromide-stained gels were visualized under UV light (Bio-Rad, Hercules, CA) and each product was normalized to give an equivalent signal from the ß-actin.
Statistical analysis
The data were expressed as the mean plus or minus the standard error. Analyses were performed using SPSS statistical software. When ANOVA revealed P < 0.05, the data were further analyzed by Sheffe's multiple range test. Differences were considered statistically significant at P < 0.05.
 |
Results
|
---|
Antitumor-promoting activity of PF
Topical application of PF prior to TPA application resulted in significant inhibition of TPA tumor promotion in DMBA-initiated BALB/c mouse skin (Figure 3). The preventive effect of these agents was evident as a reduction in tumor incidence (Figure 3a) and tumor multiplicity (Figure 3b). The extent of tumor reduction was correlated with RA and luteolin concentrations.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3. Inhibitory effect of polyphenolic fractions derived from P.frutescens on TPA tumor promotion in DMBA-initiated mouse skin. One week after topical application of DMBA (50 µg/mouse), 2 nmolTPA/mouse was applied twice weekly with or without (filled circle) prior application of 2 mg/mouse of each fraction (filled triangle, fraction 1; empty circle, fraction 2; empty triangle, fraction 3). The percentage of mice with tumors (a) and the number of tumors per mouse (b) are shown.
|
|
Anti-inflammatory effects of PE and RA
As shown in Figure 4, ear weight was markedly increased by TPA treatment. This change was suppressed by pre-treatment with 2.0 mg PE/mouse. RA also reduced ear weight in a dose-dependent manner. However, luteolin did not reduce such ear weight gain of 0.25 and 0.5 mg/mouse. Treatment with 1.35 mg RA/mouse, a dose equivalent to 2.0 mg PE/mouse, decreased ear weight to a similar degree as PE treatment. Histological examination of ear sections from TPA-challenged animals revealed skin edema with massive neutrophil infiltration (Figure 5). In the PE (2.0 mg/mouse) and RA (1.35 mg/mouse) groups, slight edema was present with only a relatively small amount of neutrophil infiltration. Myeloperoxidase (MPO) activity in the ear was correlated with the histological changes shown in Figure 6. Specifically, TPA treatment increased MPO activity and this activity was significantly reduced in the PE and RA groups.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 4. Effects of prior application of PE, RA or luteolin on TPA-induced ear edema. Significant differences from TPA (treatment) are indicated; *P < 0.05; **P < 0.01 by ANOVA and Sheffe's multiple range test.
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 5. Histological effects of prior application of 2.0 mg PE/mouse or 1.35 mg RA/mouse on TPA-induced ear edema.
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 6. Effects of prior application 2.0 mg PE/mouse and/or 1.35 mg RA/mouse on MPO activity. Significant differences from TPA (treatment) are indicated; *P < 0.05; **P < 0.01 by ANOVA and Sheffe's multiple range test.
|
|
Induction of adhesion molecule mRNA in mouse ears
mRNA expression in mice ears was analyzed by RTPCR 1 and 5 h after TPA treatment. PCR products with lengths of 431 bp corresponding to ICAM-1 were detected in TPA-treated mice as shown in Figure 7. ICAM mRNA expression was slightly reduced by PE (2.0 mg/mouse) and RA (1.35 mg/mouse) 1 and 5 h after treatment. As shown in Figure 7, VCAM-1 mRNA expression was markedly increased by TPA treatment. VCAM-1 expression was barely detectable in these conditions, suggesting that PE and RA treatment blocked this induction. The effect was strongest 1 h after TPA treatment.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 7. Effect of prior application of 2.0 mg PE/mouse and/or 1.35 mg RA/mouse on VCAM-1 and ICAM-1 mRNA expression in mouse ears 1 and 5 h after topical application of TPA.
|
|
Chemokine expression in mouse ears
Chemokine concentrations are shown in Figure 8. KC was markedly increased by TPA challenge (Figure 8a). This elevation was significantly reduced in the PE (2.0 mg/mouse) and RA (1.35 mg/mouse) groups. Similar data were obtained for MIP-2 (Figure 8b).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 8. Effect of prior application of 2.0 mg PE/mouse and/or 1.35 mg RA/mouse on TPA induced chemokine expression. KC (a) and MIP-2 (b) levels in mouse ears 5 h after topical application of TPA were determined by ELISA. Significant differences from TPA (treatment) are indicated; *P < 0.05; **P < 0.01 by ANOVA and Sheffe's multiple range test.
|
|
Eicosanoid concentration and induction of cyclooxygenase mRNA in mouse ears
PGE2 and LTB4 levels are shown in Figure 9a and b. TPA challenge appeared to increase the concentration of both eicosanoids relative to the vehicle group and PE and RA treatment appeared to block this induction. However, these differences did not reach statistical significance. A PCR product that was 583 bp in length and corresponded to COX-2 was detectable in the control group 5 h after TPA challenge but was not detectable in mice pre-treated with PE or RA.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 9. Effect of prior application of 2.0 mg PE/mouse and/or 1.35 mg RA/mouse on TPA-induced PGE2 (a) and LTB4 (b) protein levels and COX-2 mRNA levels. Mouse ears were collected 5 h after topical application of TPA. PGE2 and LTB4 levels were determined by ELISA. COX-2 mRNA expression was detected by RTPCR.
|
|
TBARS and LPO concentration in mouse ears
While a single TPA challenge had no effect on TBARS and LPO levels, a double TPA challenge resulted in a significant elevation of these markers of oxidative stress (Figure 10a and b). PE or RA treatment significantly inhibited this elevation. The expression levels of TBARS and LPO were also reduced by both PE and RAeven when mice received only one treatment with these agents (prior to the first or second application of TPA).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 10. Effect of prior application of PE or RA on double TPA treatment-induced production of TBARS (a) and LPO (b). Vehicle (TPA untreated and RA/PE untreated), TPA single challenged and untreated (TPA single), TPA double challenged and untreated (TPA double), TPA double challenged and double treated (+/+), TPA double challenged and RA/PE treated prior to first TPA treatment (+/-), TPA double challenged and RA/PE treated prior to the second TPA (-/+) treatment with 2.0 mg PE/mouse or 1.35 mg RA/mouse. The second TPA treatment was performed 24 h after the first TPA treatment. Animals were killed under anesthesia 1 h after the second TPA application. Significant differences from control are indicated; *P < 0.05; **P < 0.01 by ANOVA and Sheffe's multiple range test.
|
|
8OH-dG expression in mouse ears
Figure 11 shows immunohistochemical staining for 8OH-dG (light brown staining). No 8OH-dG was detectable in the unchallenged group. However, 8OH-dG adducts were observed in the cell nuclei from TPA double-challenged but not TPA single-challenged mice. No staining was present in PE or RA treated mice, indicating that PE and RA blocked 8OH-dG expression.

View larger version (159K):
[in this window]
[in a new window]
|
Fig. 11. Detection of 8OH-dG adduct in ears of mice treated with TPA. 8OH-dG appears as light brown staining. Vehicle (TPA untreated and RA/PE untreated), TPA single challenged and untreated (TPA single treatment), TPA double challenged and untreated (TPA double treatment), TPA double challenged and double treated (+/+), TPA double challenged and RA/PE treated prior to first TPA treatment (+/-), TPA double challenged and RA/PE treated prior to the second TPA (-/+) treatment with 2.0 mg PE/mouse or 1.35 mg RA/mouse. The second TPA treatment was performed 24 h after the first TPA treatment. Animals were killed under anesthesia 1 h after the second TPA application.
|
|
 |
Discussion
|
---|
In this study, we evaluated the effects of Perilla frutescens extract in the mouse two-stage skin carcinogenesis model. This extract has been reported to have potent anti-inflammatory effects (11,12). However, the identity of the anti-inflammatory component of PE is unclear, because the purple leaf of the plant contains numerous components including anthocyanins (23,24), flavonoids and phenolic acids (25). We observed anticarcinogenic activity in all PF, although there was a difference in efficacy between the fractions that correlated with RA and luteolin concentrations. We also compared the effects of PF containing commercially available, reagent grade RA and luteolin on TPA-induced mouse ear edema. RA is more effective than luteolin in inhibiting the TPA-induced inflammatory response, as shown in Figure 3.
We show that pre-treatment with PE and RA reduced TPA-induced neutrophil infiltration and elevated MPO activity. To investigate the mechanism that is responsible for inhibiting neutrophil infiltration, we analyzed mRNA expression of the adhesion molecule ICAM-1, which is primarily involved in interactions with ß2 integrins during inflammatory responses (26). It has been reported that ICAM-1 protein is localized to epidermal keratinocytes and vascular endothelium in TPA-treated skin (27). In our study, TPA induced ICAM-1 mRNA was reduced in PE and RA pre-treated mice. It has been reported that VCAM-1, a cytokine-inducible member of the immunogloblin superfamily that binds to the integrin VLA-4, is also induced by TPA treatment in endothelial cells (28,29). Here, reduction of VCAM-1 mRNA expression was observed following pre-treatment with PE or RA.
The TPA induced increased levels of the pro-inflammatory chemokine KC, a homolog of IL-8, was significantly reduced by pre-treatment with PE or RA. Many studies have shown that IL-8 is an important endothelial (30) and epithelial (31,32) derived inflammatory mediator that induces neutrophil chemotaxis and stimulates neutrophil transmigration. Protein kinase C (PKC) induces up-regulation of IL-8 mRNA and protein expression (33,34). We also found that expression of MIP-2, a homolog of the human CXC chemokine growth related oncogene (GRO) (35), was also increased by TPA treatment. Mouse MIP-2 has been characterized as a potent chemoattractant and is functionally equivalent to human IL-8 (36,37). Other investigators have shown that TPA-induced ear hyperplasia accompanied by MIP-2 up-regulation is clearly present in transgenic mice that overexpress PKC-
in the epidermis (38). A significant reduction in MIP-2 elevation was observed in both the PE and RA groups.
Epidemiological and experimental studies indicate that prostaglandins play a role in carcinogenesis (39,40). We found that treatment with PE and RA tended to decrease the TPA-induced increase in PGE2 synthesis. Previous studies have shown that PGE2 promotes cell proliferation (41) and prevents apoptotic cell death (42), activities that enhance cancer promotion. Furthermore, non-steroidal anti-inflammatory drugs, which decrease the production of prostaglandins, reduce the incidence and progression of tumors in both animal models and in cancer patients (4345). In this study, COX-2 mRNA expression induced by TPA was eliminated when animals were pre-treated with PE or RA. Thus, reduction in prostaglandin synthesis may be a mechanism by which perilla and one of its effective components, RA, inhibit carcinogenesis. On the other hand, lipoxygenase-catalyzed products also have a profound influence on the development and progression of cancers (46). LTB4, a biologically active metabolite of arachidonic acid, has been implicated in inflammatory disease and is one of the most potent chemoattractants and leukocyte activators (47). Moreover, RA, and its methylester, derived from perilla seed reportedly inhibit 5-lipoxygenase and 12-lipoxygenase activity (48,49) in vitro. In this study, LTB4 appeared to be increased by TPA stimulation, and this small elevation was blocked by PE or RA treatment; however, these changes did not reach statistical significance.
Several previous studies have suggested an anti-inflammatory effect of PE. Yamazaki et al. (50) reported that PE administration reduced increases in plasma TNF
induced by i.v. injection of muramyldipeptide in mice. It has also been reported that induction of iNOS and NO production by cultured murine mesangial cells induced by interferon-
and TNF
was reduced by the addition of PE (51). In our experiments, RA and PE reduced expression of cell adhesion molecules and COX-2 and inhibited the synthesis of CXC chemokines. The anti-inflammatory effect of RA in perilla is probably due to inhibition of these various inflammatory mediators.
Previous reports have shown that the transcription factor nuclear factor
B (NF-
B) is a central mediator of the immune response as it regulates the expression of pro-inflammatory enzymes, cytokine receptors and cell adhesion molecules. In this study we showed that RA inhibits the expression of important inflammatory mediators including ICAM-1, VCAM-1, KC, MIP-2 and COX-2. Many reports have indicated that regulation of gene expression of these mediators occurs via NF-
B (5257). In contrast, PKC, which is induced by various agents that stimulate the immune response, is a strong activator of NF-
B (58). Several reports suggest that the properties of plant-derived phenolic compounds such as quercetin, myricetin and resvertorol may be mediated through downregulation of the NF-
B pathway (59,60). In addition, the novel antioxidant
-tocopherol inhibits PKC in various cell types (61). According to the results of this study, it seems that RA reduces TPA induced inflammatory responses by inhibiting the PKC/NF-
B pathway. Further study is needed to elucidate the molecular mechanism of the anti-inflammatory effects of RA.
It has been reported that double treatment with phorbol esters induces oxidative stress in mouse skin (62,63). PKC activation of recruited neutrophils in the inflamed area induced by the second application of TPA could have activated NADPH oxidase and acted as a major source of reactive oxygen in this model (64). Oxidized DNA levels in skin are also reportedly elevated by this treatment (65,66). In this study, pre-treatment with RA or PE in TPA application groups significantly reduced TBARS and LPO levels. In addition, immunohistochemical detection of 8OH-dG, one of the major markers of oxidative DNA damage, was inhibited in all RA and PE treatment groups. Consistent ith this finding, Nakamura et al. (67) reported that RA shows potent superoxide scavenging activity. We confirmed this ability using electron spin resonance in vitro (data not shown). In addition, we have shown recently that RA has antioxidative activity in vivo in an LPS- and D-galactosamine-induced liver injury model (16).
We conclude that RA plays an anticarcinogenic role in P.frutescens extract by two independent effects: anti-inflammatory activities such as inhibition of adhesion molecule, chemokine and eicosanoid synthesis, and antioxidative activity such as inhibition of oxidative DNA injury.
Conflict of interest statement
N.Osakabe is employed by Meiji Seika Kaisha Ltd, the manufacturers of foods and medicines, and is currently conducting research sponsored by this company.
 |
Notes
|
---|
3 To whom correspondence should be addressed Email: naomi_osakabe{at}meiji.co.jp 
 |
References
|
---|
- Howe,G.R. and Burch,J.D. (1996) Nutrition and pancreatic cancer. Cancer Causes Control, 7, 6982.[ISI][Medline]
- Gandini,S., Merzenich,H., Robertson,C. and Boyle,P. (2000) Meta-analysis of studies on breast cancer risk and diet: the role of fruit and vegetable consumption and the intake of associated micronutrients. Eur. J. Cancer, 36, 636646.[CrossRef][ISI][Medline]
- Hertog,M.G.L., Feskens,E.J.M., Hollman,P.C.H., Katan,M.B. and Kromhout,D. (1994) Dietary flavonoids and cancer risk in the Zutphen Elderly Study. Nutr. Cancer, 22, 175184.[ISI]
- Hirose,M., Hasegawa,R., Kimura,J. et al. (1995) Inhibitory effects of 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), green tea catechins and other antioxidants on 2-amino-6-methyldipyrido [1,2-a:3',2'-d]imidazole (Glu-P-1)-induced rat hepatocarcinogenesis and dose-dependent inhibition by HTHQ of lesion induction by Glu-P-1 or 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx). Carcinogenesis, 16, 30493055.[Abstract]
- So,F.V., Guthrie,N., Chambers,A.F., Moussa,M. and Carroll,K.K. (1996) Inhibition of human breast cancer cell proliferation and delay of mammary tumogenesis by flavonoids and citrus juices. Nutr. Cancer, 26, 167181.[ISI][Medline]
- Yamagishi,M., Natsume,M., Osakabe,N., Nakamura,H., Furukawa,F., Imazawa,T., Nishikawa,A. and Hirose,M. (2002) Effects of cacao liquor proanthocyanidins on PhIP-induced mutagenesis in vitro and in vivo mammary and pancreatic tumorigenesis in female SpragueDawley rats. Cancer Lett., 185, 123130.[CrossRef][ISI][Medline]
- Murakami,A., Nakamura,Y., Torikai,K. et al. (2000) Inhibitory effect of citrus nobiletin on phorbol ester-induced skin inflammation, oxidative stress and tumor promotion in mice. Cancer Res., 60, 50595066.[Abstract/Free Full Text]
- Huang,M.T., Smart,R.C., Wong,C.Q. and Conney,A.H. (1988) Inhibitory effect of curcumin, chlorogenic acid, caffeic acid and ferulic acid on tumor promotion in mouse skin by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res., 48, 59415946.[Abstract]
- Zhao,J., Wang,J., Chen,Y. and Agarwal,R. (1999) Anti-tumor-promoting activity of a polyphenolic fraction isolated from grape seeds in the mouse skin two-stage initiation-promotion protocol and identification of procyanidin B5-3'-gallate as the most effective antioxidant constituent. Carcinogenesis, 20, 17371745.[Abstract/Free Full Text]
- Lamaison,J.L., Petitjean-Freytet,C. and Carnat,A. (1990) Rosmarinic acid, total hydroxycinnamic derivatives and antioxidant activity of Apiaceae, Borraginaceae and Lamiceae medicinals. Ann. Pharm. Fr., 48, 103108.[Medline]
- Simpol,L.R., Otsuka,H., Otani,K., Kasai,R. and Yamasaki,K. (1994) Nitril glucosides and rosmarinic acid, the histamine inhibitor from Ehretia philippinensis. Phytochemistry, 36, 9195.[CrossRef][ISI]
- Yamamoto,H., Sakakibara,J., Nagatsu,A. and Sekiya,K. (1998) Inhibitors of arachidonate lipoxygenase from defatted perilla seed. J. Agric. Food Chem., 46, 862865.[CrossRef][ISI]
- Lamaison,J.L., Petitjean-Freytet,C. and Carnat,A. (1990) Rosmarinic acid, total hydroxycinnamic derivatives and antioxidant activity of Apiaceae, Borraginaceae and Lamiceae medicinals. Ann. Pharm. Fr., 48, 103108.[Medline]
- Frankel,E.N., Huang,S.W., Aeschbach,R. and Prior,E. (1996) Antioxidant activity of a rosmary extract and its constituents, carnosic acid, carnosol and rosmarinic acid in bulk oil and oil-in-water emulsion. J. Agric. Food Chem., 44, 131135.[CrossRef][ISI]
- Tada,M., Matsumoto,R., Yanmaguchi,H. and Chiba,K. (1996) Novel antioxidants isolated from Perilla flutescens britton var. crispa (Thunb). Biosci. Biotech. Biochem., 60, 10931095.[ISI]
- Osakabe,N., Yasuda,A., Natsume,M., Kato,Y., Osawa,T. and Yosikawa,T. (2002) Rosmarinic acid, a major polyphenolic component of Perilla frutescens, reduces lipopolysaccharide (LPS)-induced liver injury in D-galactosamine (D-GalN)-sensitized mice. Free Radic. Biol. Med., 33, 798806.[CrossRef][ISI][Medline]
- David,H. (1988) Rudolf Virchow and modern aspects of tumor pathology. Pathol. Res. Pract., 183, 356364.[ISI][Medline]
- Balkwill,F. and Mantovani,A. (2001) Inflammation and cancer: back to Virchow? Lancet, 357, 539545.[CrossRef][ISI][Medline]
- Xu,X.C. (2002) COX-2 inhibitors in cancer treatment and prevention, a recent development. Anticancer Drugs, 13, 127137.[CrossRef][ISI][Medline]
- Cao,Y. and Prescott,S.M. (2002) Many actions of cyclooxygenase-2 in cellular dynamics and in cancer. J. Cell Physiol., 190, 279286.[CrossRef][ISI][Medline]
- Bradley,P.P., Priebat,D.A., Christensen,R.D. and Rothstein,G. (1982) Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J. Invest. Dermatol., 78, 206209.[Abstract]
- Ohkawa,H., Ohishi,N. and Yagi,K. (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem., 95, 351358.[ISI][Medline]
- Kondo,T., Tamura,H., Yoshida,K. and Goto,T. (1989) Structure of malonylshisonin, a genuine pigment in purple leaves of perilla ocimoides L.var. crispa Benth. Agric. Biol. Chem., 53, 797800.[ISI]
- Yoshida,K., Kondo,T., Kameda,K. and Goto,T. (1990) Structure of anthocyanins isolated from purple leaves of Perilla ocimoides L. var. crispa benth and their isomerization of light. Agric. Biol. Chem., 54, 17451751.[ISI]
- Ishikura,N. (1981) Anthocyanins and flavons in leaves and seeds of perilla plant. Agric. Biol. Chem., 45, 18551860.[ISI]
- Staunton,D.E., Marlin,S.D., Stratowa,C., Dustin,M.L. and Springer,T.A. (1988) Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families. Cell, 52, 925933.[ISI][Medline]
- Oberyszyn,T.M., Conti,C.J., Ross,M.S., Oberyszyn,A.S., Tober,K.L., Rackoff,A.I. and Robertson,F.M. (1998) ß2 integrin/ICAM-1 adhesion molecule interactions in cutaneous inflammation and tumor promotion. Carcinogenesis, 19, 445455.[Abstract]
- Mason,J.C., Yarwood,H., Sugars,K. and Haskard,D.O. (1997) Human umbilical vein and dermal microvascular endothelial cells show heterogeneity in response to PKC activation. Am. J. Physiol., 273, C12331240.[ISI][Medline]
- Deisher,T.A., Haddix,T.L., Montgomery,K.F., Pohlman,T.H., Kaushansky,K. and Harlan,J.M. (1993) The role of protein kinase C in the induction of VCAM-1 expression on human umbilical vein endothelial cells. FEBS Lett., 331, 285290.[CrossRef][ISI][Medline]
- Huber,A.R., Kunkel,S.L., Todd,R.F.,3rd and Weiss,S.J. (1991) Regulation of transendothelial neutrophil migration by endogenous interleukin-8. Science, 254, 99102.[ISI][Medline]
- Ina,K., Kusugami,K., Yamaguchi,T., Imada,A., Hosokawa,T., Ohsuga,M., Shinoda,M., Ando,T., Ito,K. and Yokoyama,Y. (1997) Mucosal interleukin-8 is involved in neutrophil migration and binding to extracellular matrix in inflammatory bowel disease. Am. J. Gastroenterol., 92, 13421346.[ISI][Medline]
- Cole,A.T., Pilkington,B.J., McLaughlan,J., Smith,C., Balsitis,M. and Hawkey,C.J. (1996) Mucosal factors inducing neutrophil movement in ulcerative colitis: the role of interleukin 8 and leukotriene B4. Gut, 39, 248254.[Abstract]
- Graness,A., Chwieralski,C.E., Reinhold,D., Thim,L. and Hoffmann,W. (2002) Protein kinase C and ERK activation are required for TFF-peptide-stimulated bronchial epithelial cell migration and tumor necrosis factor-alpha-induced interleukin-6 (IL-6) and IL-8 secretion. J. Biol. Chem., 277, 1844018446.[Abstract/Free Full Text]
- Graca-Souza,A.V., Arruda,M.A., de Freitas,M.S., Barja-Fidalgo,C. and Oliveira,P.L. (2002) Neutrophil activation by heme: implications for inflammatory processes. Blood, 99, 41604165.[Abstract/Free Full Text]
- Baggiolini,M., Dewald,B. and Moser,B. (1994) Interleukin-8 and related chemotacticCXC and CC chemokines. Adv. Immunol., 55, 97179.[ISI][Medline]
- Wolpe,S.D., Sherry,B., Juers,D., Davatelis,G., Yurt,R.W. and Cerami,A. (1989) Identification and characterization of macrophage inflammatory protein 2. Proc. Natl Acad. Sci. USA, 86, 612616.[Abstract]
- Tekamp-Olson,P., Gallegos,C., Bauer,D., McClain,J., Sherry,B., Fabre,M., van Deventer,S. and Cerami,A. (1990) Cloning and characterization of cDNAs for murine macrophage inflammatory protein 2 and its human homologues. J. Exp. Med., 172, 911919.[Abstract]
- Wang,H.Q. and Smart,R.C. (1999) Overexpression of protein kinase C-alpha in the epidermis of transgenic mice results in striking alterations in phorbol ester-induced inflammation and COX-2, MIP-2 and TNF-alpha expression but not tumor promotion. J. Cell Sci., 112, 34973506.[Abstract/Free Full Text]
- Young,M.R. (1994) Eicosanoids and the immunology of cancer. Cancer Metast. Rev., 13, 337348.[ISI][Medline]
- Xu,X.C. (2002) COX-2 inhibitors in cancer treatment and prevention, a recent development. Anticancer Drugs, 13, 127137.[CrossRef][ISI][Medline]
- Kimura,M., Osumi,S. and Ogihara,M. (2000) Stimulation of DNA synthesis and proliferation by prostaglandins in primary cultures of adult rat hepatocytes. Eur. J. Pharmacol., 404, 259271.[CrossRef][ISI][Medline]
- Chan,T.A., Morin,P.J., Vogelstein,B. and Kinzler,K.W. (1998) Mechanisms underlying nonsteroidal antiinflammatory drug-mediated apoptosis. Proc. Natl Acad. Sci. USA, 95, 681686.[Abstract/Free Full Text]
- Subbaramaiah,K., Zakim,D., Weksler,B.B. and Dannenberg,A.J. (1997) Inhibition of cyclooxygenase: a novel approach to cancer prevention. Proc. Soc. Exp. Biol. Med., 216, 201210.[Abstract]
- Taketo,M.M. (1998) Cyclooxygenase-2 inhibitors in tumorigenesis (Part II). J. Natl Cancer Inst., 90, 16091620.[Abstract/Free Full Text]
- Gupta,R.A. and DuBois,R.N. (2000) Translational studies on Cox-2 inhibitors in the prevention and treatment of colon cancer. Ann. N.Y. Acad. Sci., 910, S196204.
- Steele,V.E., Holmes,C.A., Hawk,E.T., Kopelovich,L., Lubet,R.A., Crowell,J.A., Sigman,C.C. and Kelloff,G.J. (1999) Lipoxygenase inhibitors as potential cancer chemopreventives. Cancer Epidemiol. Biomarkers Prev., 8, 467483.[Abstract/Free Full Text]
- Yokomizo,T., Izumi,T. and Shimizu,T. (2001) Leukotriene B4: metabolism and signal transduction. Arch. Biochem. Biophys., 385, 231241.[CrossRef][ISI][Medline]
- Yamamoto,H., Sakakibara,J., Nagatsu,A. and Sekiya,K. (1998) Inhibitors of arachidonate lipoxygenase from defatted perilla seed. J. Agric. Food Chem., 46, 862865.[CrossRef][ISI]
- Puignero,V. and Queralt,J. (1997) Effect of topically applied cyclooxygenase-2-selective inhibitors on arachidonic acid- and tetradecanoylphorbol acetate-induced dermal inflammation in the mouse. Inflammation, 21, 431442.[CrossRef][ISI][Medline]
- Yamazaki,M., Ueda,H. and Du,D. (1992) Inhibition by perilla juice of tumor necrosis factor production. Biosci. Biotech. Biochem., 56, 152.[ISI][Medline]
- Makino,T., Ono,T., Ito,M., Muso,E. and Honda,G. (1999) Effect of Perilla frutescens extract on nitric oxide production by cultured murine mesangial cells. Biol. Pharm. Bull., 22, 476480.[ISI][Medline]
- Kim,I., Moon,S.O., Kim,S.H., Kim,H.J., Koh,Y.S. and Koh,G.Y. (2001) Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin through nuclear factor-kappa B activation in endothelial cells. J. Biol. Chem., 276, 76147620.[Abstract/Free Full Text]
- Chang,Y.J., Holtzman,M.J. and Chen,C.C. (2002) Interferon-gamma-induced epithelial ICAM-1 expression and monocyte adhesion. Involvement of protein kinase C-dependent c-Src tyrosine kinase activation pathway. J. Biol. Chem., 277, 71187126.[Abstract/Free Full Text]
- Friedland,J.S., Constantin,D., Shaw,T.C. and Stylianou,E. (2001) Regulation of interleukin-8 gene expression after phagocytosis of zymosan by human monocytic cells. J. Leukoc. Biol., 70, 447454.[Abstract/Free Full Text]
- Driscoll,K.E. (2000) TNFalpha and MIP-2: role in particle-induced inflammation and regulation by oxidative stress. Toxicol Lett., 112/113, 177183.[CrossRef]
- Mestre,J.R., Mackrell,P.J., Rivadeneira,D.E., Stapleton,P.P., Tanabe,T. and Daly,J.M. (2001) Redundancy in the signaling pathways and promoter elements regulating cyclooxygenase-2 gene expression in endotoxin-treated macrophage/monocytic cells. J. Biol. Chem., 276, 39773982.[Abstract/Free Full Text]
- Tegeder,I., Pfeilschifter,J. and Geisslinger,G. (2001) Cyclooxygenase-independent actions of cyclooxygenase inhibitors. FASEB J., 15, 20572072.[Abstract/Free Full Text]
- Ghosh,S. and Baltimore,D. (1990) Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B. Nature, 344, 678682.[CrossRef][ISI][Medline]
- Tsai,S.H., Lin-Shiau,S.Y. and Lin,J.K. (1999) Suppression of nitric oxide synthase and the down-regulation of the activation of NFkappaB in macrophages by resveratrol. Br. J. Pharmacol., 126, 673680.[Abstract/Free Full Text]
- Holmes-McNary,M. and Baldwin,A.S.,Jr (2000) Chemopreventive properties of trans-resveratrol are associated with inhibition of activation of the IkappaB kinase. Cancer Res., 60, 34773483.[Abstract/Free Full Text]
- Azzi,A., Ricciarelli,R. and Zingg,J.M. (2002) Non-antioxidant molecular functions of alpha-tocopherol (vitamin E). FEBS Lett., 519, 810.[CrossRef][ISI][Medline]
- Ji,C. and Marnett,L.J. (1992) Oxygen radical-dependent epoxidation of (7S,8S)-dihydroxy-7,8-dihydrobenzo[a]pyrene in mouse skin in vivo. Stimulation by phorbol esters and inhibition by antiinflammatory steroids. J. Biol. Chem., 267, 1784217848.[Abstract/Free Full Text]
- Kensler,T.W., Egner,P.A., Moore,K.G., Taffe,B.G., Twerdok,L.E. and Trush,M.A. (1987) Role of inflammatory cells in the metabolic activation of polycyclic aromatic hydrocarbons in mouse skin. Toxicol. Appl. Pharmacol., 90, 337346.[ISI][Medline]
- Mortensen,R.F. and Zhong,W. (2000) Regulation of phagocytic leukocyte activities by C-reactive protein. J. Leukoc. Biol., 67, 495500.[Abstract]
- Wei,H. and Frenkel,K. (1993) Relationship of oxidative events and DNA oxidation in SENCAR mice to in vivo promoting activity of phorbol ester-type tumor promoters. Carcinogenesis, 14, 11951201.[Abstract]
- Wei,H. and Frenkel,K. (1992) Suppression of tumor promoter-induced oxidative events and DNA damage in vivo by sarcophytol A: a possible mechanism of antipromotion. Cancer Res., 52, 22982303.[Abstract]
- Nakamura,Y., Ohto,Y., Murakami,A. and Ohigashi,H. (1998) Superoxide scavenging activity of rosmarinic acid from Perilla frutescens britton var. acuta f. viridis. J. Agric. Food Chem., 46, 45454550.[CrossRef][ISI]
Received June 25, 2003;
revised October 16, 2003;
accepted November 13, 2003.