d -Limonene modulates inflammation,oxidative stress and Ras-ERK pathway to inhibit murine skin tumorigenesis

Abstract

d-Limonene, a common monoterepene has been shown to have antiproliferative, apoptosis-inducing and chemopreventive effects. In the present study, we have investigated the effects of d-limonene on the growth of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin tumor development. We found that d-limonene (50 and 100 mg/kg body weight) treatments to the mouse skin significantly reduced the TPA-induced (a) edema and hyperplasia (p < 0.001); (b) expression of cyclooxygenase-2; (c) ornithine decarboxylase activity (p < 0.001); and (d) [³H] thymidine incorporation into DNA (p < 0.001). In addition, treatment of d-limonene effectively restored the level of reduced glutathione, glutathione peroxidase, glutathione reductase, glutathione S-transferase, catalase and malondialdehyde production in TPA-treated mouse skin. In a two-stage skin tumorigenesis study, d-limonene significantly reduced the tumor burden (p < 0.005) and tumor incidence as compared to DMBA/TPA-treated mice. d-Limonene treatment also extended the latency period of tumor development from 4 to 9 weeks. d-Limonene treatment decreased the expression level of Ras, Raf and phosphorylation of extracellular signal-regulated protein kinase 1/2 in DMBA/TPA-induced tumors. A decrease in the expression of Bcl-2 and an increase in Bax expression were also observed in tumor tissues of mice treated with d-limonene. Taken together, our findings suggest that d-limonene may exert its chemopreventive activity through the inhibition of inflammation, oxidative stress and Ras-signaling as well as the induction of pro-apoptotic state during TPA-mediated promotion of DMBA-induced skin cancer in mouse model.

Introduction

Cancer is a growing health problem due to the changes in lifestyle such as inappropriate diet, obesity, infections, radiation, stress, use of tobacco, lack of physical activity and exposure to the environmental pollutants. ¹ The risk of cancer can be reduced either by eliminating the identified carcinogens or by minimizing exposure to them, for instance, by dietary habits. Epidemiological studies indicated that many dietary agents are well-known chemopreventive agents. ² These agents have the ability to modulate at more than one critical pathway of carcinogenesis. So, the chemopreventive intervention using the naturally occurring dietary substance is extremely desirable to delay the process of carcinogenesis. ³

The development of skin cancer is a multistage process that involves initiation, promotion and progression. ^4,5 In the mouse model of skin cancer, tumor initiation can be caused by a single topical application of 7,12-dimethylbenz[a]anthracene (DMBA; a carcinogen that mutates H-Ras proto-oncogene). ⁶ Subsequently, the repeated applications of 12-O-tetradecanoylphorbol 13-acetate (TPA) trigger tumor promotion, involving clonal expansion of initiated cells. ⁶ The mutated Ras protein gets farnesylated by the enzyme farnesyl transferase (FTase). The farnesylated Ras protein then gets attached to the cell membrane which leads to the activation of Ras/Raf/extracellular signal-regulated protein kinase 1/2 (ERK1/2) cascade. ^7,8

Applications of TPA also induced the generation of reactive oxygen species (ROS) which induces DNA strand breaks and oxidation of DNA bases, leading to mutagenesis. ^5,9 Increased level of ROS damages almost all biomolecules such as DNA, RNA and proteins, thus playing a key role in the pathophysiology of a number of degenerative and chronic diseases, including cancer. ⁹ During oxidative stress, a reduction in the activities of antioxidative enzymes viz. catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPX), glutathione S-transferase (GST) and reduced glutathione (GSH) content has been found with an increase in the level of ROS in murine skin. ⁹ The status of intracellular redox is regulated by antioxidant enzymes (CAT, GR, GPX and GST) and nonenzymatic antioxidants (GSH and vitamin C). ¹⁰ They also activate proinflammatory genes such as in the expression of cyclooxygenase-2 (COX-2) and various transcription factors. ¹¹

d-Limonene (1-methyl-4-isopropyl-cyclohexene) is a natural product found in essential oils of orange, lemon, mandarin, lime, grapefruit and many other plants. d-Limonene has been reported to modulate the oxidative stress and to have the chemopreventive activity. ¹² The chemopreventive and chemotherapeutic efficacies of d-limonene have been reported against various chemically induced animal tumor models. ^{13 –15} d-Limonene has been shown (a) to induce phases I (cytochrome P450) and II (glutathione transferase and uridine diphosphate-glucuronyl transferase) carcinogen-detoxifying enzymes, during the initiation phase of carcinogenesis ¹⁶ ; (b) to inhibit farnesylation of Ras protein ¹⁷ ; and (c) to trigger apoptosis. ¹⁵ Phase I and phase II clinical trials of d-limonene have also been undertaken with advanced cancer and exhibited minimal toxicity in these studies. ¹⁸ However, its mechanism of antitumor activity is still unclear.

In the present study, we investigated the effect of d-limonene on TPA-induced edema, hyperplasia, expression of COX-2, oxidative stress enzymes, ornithine decarboxylase (ODC) activity and [³H] thymidine incorporation as early tumor promotion biomarkers in the skin of Swiss albino mice. We also investigated the effect of d-limonene on Ras/Raf/ERK1/2-signaling pathway and expression of Bax and Bcl-2 in DMBA/TPA-mediated skin tumorigenesis.

Materials and methods

Chemicals

The chemicals used in this study such as d-limonene, 5-5′-dithiobis-2-nitrobenzoic acid, GSH, GR, oxidized glutathione, reduced nicotinamide adenine dinucleotide phosphate, 1-chloro 2,4-dinitrobenzene, DMBA, TPA, aprotinin, leupeptin, pepstatin, 3,3′-diaminobenzidine (DAB), ethylenediamine tetra acetic acid (EDTA), pyridoxal phosphate, phenylmethylsulfonyl fluoride (PMSF), 2-mercaptoethanol, dithiothreitol, Tween-80 and Brij-35 were obtained from Sigma Chemical Co., Missouri, USA. [¹⁴C] ornithine and [³H] thymidine were purchased from Amersham (Little Chalfont, UK). Primary monoclonal antibodies against H-Ras, c-Raf, p-ERK1/2, COX-2 and β-actin were purchased from BD Biosciences Pharmingen (San Jose, California, USA). Bax and Bcl-2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, California, USA). Horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG) were obtained from Genei (Bangalore, India). All other chemicals and solvents were at highest purity available.

Animals

Female Swiss albino mice (6–8 weeks old; 20–25 g) were obtained from Central Animal House Facility of Hamdard University, New Delhi. The mice were housed in a well-ventilated room at 25 ± 2°C under 12-h light:12-h dark cycle. The mice were fed pellet diet (Hindustan Lever Ltd, Bombay, India) and drinking water ad libitum. The study protocols (CAHF file no. 283) were approved by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), India. The mice were treated and killed according to approved ethical guidelines. The dorsal skin of the mice was shaven with an electric clipper (Oster A2, USA) followed by the application of hair-removing cream (Anne French, Geoffery Manners, Bombay, India) at least 2 days before treatment.

Treatment schedule for investigating the effect of d-limonene on TPA-induced tumor promotion biomarkers in the skin

Mice were randomly divided into five groups of six each. Mice of group 1 received topical treatment of 200 μl acetone and served as vehicle control group. Mice of group 3 were topically applied with d-limonene (50 mg/kg body weight was dissolved in 200 μl acetone/mouse), and those on groups 4 and 5 were applied with d-limonene (100 mg/kg body weight in 200 μl acetone/mouse). Thirty minutes after these treatments, mice of groups 2, 3 and 4 received topical treatment of TPA (2 μg/200 μl acetone/mouse). The treatments were carried out for 3 days, once a day. Mice of all groups including vehicle control were killed by cervical dislocation 12 h after the last TPA treatment. The dorsal skin of each mouse was immediately removed and cleaned to remove the extraneous material with sharp scalpel blade in ice cold 0.8% NaCl. The skin was then processed for evaluation of edema, histopathology, immunohistochemistry and oxidative stress enzymes.

For estimation of ODC activity and [³H] thymidine incorporation, the treatment schedule and allotment of mice were exactly same as above. To measure the ODC activity, the animals of all groups were killed after 6 h of last TPA treatment. For the [³H] thymidine incorporation into epidermal DNA, mice of all the groups were injected intraperitoneally with [³H] thymidine (20 μCi/mouse/200 μl saline) 18 h after the last TPA treatment and were killed after 2 h of [³H] thymidine treatment.

Mouse skin edema and histopathological studies

The estimation of skin edema was done per the method described by Afaq et al. ⁴ A skin punch of 1 cm diameter was made fat-free and weighed quickly. The skin punch was dried for 24 h at 50°C and reweighed. The loss of water content was determined. The extent of edema was calculated as the difference in water gain between control and TPA-treated groups. The inhibitory effect of d-limonene was calculated as the difference between control and d-limonene-/TPA-treated groups.

For histopathological study, the dorsal skins were removed and fixed in 10% neutral buffered formalin. The skins were then embedded in paraffin wax as standard procedure and 5 µm sections were cut. The sections were stained with hematoxylin & eosin. The infiltrations of leukocytes in dermis were observed and epidermal thickness (as an indicator of hyperplasia) was measured at least in six different regions using microscope (Motic Digital Microscope).

Immunohistochemical analysis

The dorsal skins of mice were excised and fixed in 10% buffered formalin. The skins were embedded in paraffin wax. Five-micrometer thick sections were cut and mounted on silanized glass slides. The sections were deparaffinized in xylene and dehydrated in graded ethanol. For antigen retrieval, the sections were kept in 10 mM citrate buffer (pH 6.0) at 95°C for 6 min. Each section was treated with 3% H₂O₂ for 20 min and then with blocking solution (1% non-fat skimmed milk) for 30 min. Sections were incubated with monoclonal anti-COX-2 antibody at room temperature for 2 h. Thereafter, they were incubated with secondary antibody at room temperature for 1 h. In order to develop the color, DAB was applied on each section for 3–5 min. Sections were then rinsed with distilled water and counterstained with hematoxylin. Finally, they were microscopically (Motic Digital Microscope) analyzed at least in six different regions.

Tissue preparation and assays of nonenzymatic and enzymatic antioxidants

The dorsal skins of mice were minced into small pieces and homogenized (Kinematica Polytron PT 3100 Homogenizer, Switzerland) in chilled phosphate buffer (0.1 M, pH 7.4). The epidermal homogenates were centrifuged at 10,000g for 30 min to get the post-mitochondrial supernatant, which were used for the estimation of GSH, GPX, GR, GST and CAT. GSH was determined by the method of Jollow et al. ¹⁹ GR and GPX activities were measured by the method of Mohandas et al. ²⁰ GST activity was estimated by the method of Habig et al. ²¹ CAT activity was assayed per the method of Claiborne, ²² and the level of malondialdehyde (MDA) as an end product of lipid peroxidation was assessed according to the method of Wright et al. ²³

Estimation of ODC activity

The dorsal skins were minced into small pieces and were homogenized in ice-cold Tris-HCl buffer (50 mM, pH 7.5) containing 0.4 mM EDTA, 0.32 mM pyridoxal phosphate, 0.1 mM PMSF, 1 mM 2-merceptoethanol, 4 mM dithiothreitol and 0.1% Tween-80. The homogenates were then centrifuged at 10,000g for 30 min and a portion of supernatant was centrifuged at 105,000g for 60 min at 4°C to obtain the microsomal pellet and cytosol. The cytosol was used for the estimation of ODC activity per the method of Verma et al. ²⁴ In brief, the reaction mixture (495 μl) contained 400 μl of cytosolic enzyme solution and 95 μl of co-factor mixture containing 0.32 mM pyridoxal phosphate, 0.4 mM EDTA, 4 mM dithiothreitol, 0.4 mM ornithine, 0.02% Brij-35 and 0.05 μCi DL [¹⁴C] ornithine. After adding the enzyme solution and cofactor mixture, the test tubes were immediately closed with rubber stopper and the central well assemblies were containing 0.2 ml ethanolamine and methoxyethanol mixture in 2:1, v/v ratio. The reaction mixture was kept in water bath at 37°C for 1 h. The reaction was arrested by injecting 1.0 ml of 2.0 M citric acid solution along side of glass tubes and the solution was kept for 1 h to ensure complete absorption of CO₂. Finally, solution in central well was transferred to vial containing 2 ml ethanol and 10 ml scintillation fluid. Radioactivity was counted in scintillation counter (Beckman LS 6500 multipurpose). ODC activity was expressed as pmol ¹⁴ CO₂ released per hour per mg protein.

Assessment of [³H] thymidine incorporation

Assessment of incorporation of [³H] thymidine was done per the method of Smart et al. ²⁵ Skins were quickly removed and the extraneous materials including dermis were cleaned, and homogenates (10%, w/v) were prepared in ice-cold water. To the homogenate, an equal volume of ice-cold 10% TCA (trichloroacetic acid) was added and centrifuged at 2500g for 10 min. The supernatant was then discarded and the precipitate was washed with cold 5% TCA and centrifuged. The precipitate thus obtained was incubated with cold 10% perchloric acid (PCA) at 4°C overnight. The suspension was centrifuged, washed with 5% cold PCA and dissolved in warm 10% PCA, followed by incubation in a boiling water bath for 30 min. It was then filtered through Whatman 50 filter paper to get clear nucleotide solution, which was taken for DNA estimation by DPA (diphenylamine) method. A fixed volume of filtrate was used for [³H] thymidine counting in liquid scintillation counter (Beckman LS 6500 multipurpose). The amount of [³H] thymidine incorporated was expressed as dpm/μg DNA.

Two-stage mouse skin tumorigenesis

The mice were divided in four groups of 20 mice each. Mice of group 1 received 200 μl acetone only and served as vehicle control. The treatment of the dorsal skin of mice of groups 2, 3 and 4 was initiated by single topical application of 50 μg DMBA in 200 μl acetone. One week later, tumor growth was promoted by repeated topical applications of TPA (2 mg in 200 μl acetone). Mice of group 2 were treated with TPA alone and served as positive control. However, mice of groups 3 and 4 topically received d-limonene 50 and 100 mg/kg body weight in 200 μl acetone, respectively. Thirty minutes after this treatment, TPA was applied to mouse skin for tumor promotion. The treatments were performed thrice a week up to the termination of experiments (21 weeks). Skin tumors with a diameter of >1 mm were counted and recorded every week. The data are expressed as the percentage of mice with tumors (tumor incidence) and the number of tumors/mouse (tumor burden) are plotted as a function of weeks on test. On the completion of experiment, the mice were killed by cervical dislocation. The skin/tumors were harvested for Western blot analysis.

Preparation of skin/tumor lysates for western blot analysis

The excised dorsal skin/tumors were cleaned to remove the extraneous materials with a sharp scalpel blade in ice-cold phosphate-buffered saline and pooled in vial. The skin/tumor lysates were prepared as described by Kundu et al., ²⁶ with minor modifications. Briefly, the pooled skins/tumors were minced and homogenized in ice-cold lysis buffer (20 mM Tris-Cl, pH 7.5; 10 mM KCl, 0.1 mM EDTA, 2 mM MgCl₂, 1 mM PMSF, 1 mM DTT, 0.1% nonylphenol ethoxylate (NP)-40 and freshly prepared 1 mM each of pepstatin, leupeptin and aprotinin). Then, the homogenate was centrifuged at 10,000g for 30 min at 4°C, and the supernatant was collected as cytosol fraction and aliquoted in 1 ml tubes. The pellet was processed for membrane fraction, resuspended in buffer (20 mM Tris-Cl, pH 7.5; 0.1 mM PMSF, 0.1% sodium dodecyl sulfate (SDS) and 1% NP-40) and centrifuged at 14,000g for 10 min at 4°C. The supernatant was collected as membrane fraction and aliquoted in 1 ml tubes. All aliquots were stored at −80°C. The protein content in each sample was assayed according to the method of Bradford ²⁷ using bovine serum albumin as standard.

Western blot analysis

For Western blot analysis, 50–75 µg of the proteins were resolved by 10–12% SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membrane. The membranes containing transferred protein were blocked with 5% (w/v) non-fat dry milk in TTBS buffer (0.1% Tween-20, 20 mM Tris base and 137 mM NaCl, pH 7.6) for 1 h at room temperature followed by incubation with appropriate primary antibody (Ras, Raf, p-ERK1/2, Bax, Bcl-2 and β-actin) for 2 h at 4°C. After washing the membrane three times in TTBS for 10 min each, the membrane was incubated with horseradish peroxidase conjugated with anti-mouse IgG or anti-rabbit IgG secondary antibodies for 2 h. The membrane was washed 3 times with TTBS buffer for 10 min each and detected by DAB in 0.1 M Tris-Cl pH 7.5 in the presence of H₂O₂. To quantify equal loading, identical gels were run simultaneously and probed with β-actin antibody in the same manner as described above.

Statistical analysis

The difference between various groups was analyzed by Student-Newman-Keuls t test after the application of one-way analysis of variance by InStat software. Values of p < 0.05 were considered statistically significant.

Results

Effect of d-limonene on TPA-induced mouse skin edema and skin morphological changes

Earlier studies suggested that TPA treatment to mouse skin causes cutaneous edema, hyperplasia and infiltration of leukocytes, mainly neutrophils and monocytes, in the dermis ^4,8 which play an important role in tumor promotion. After 12 h of topical treatment of TPA, the skin punch weight was significantly increased upto 48%; p < 0.001 as compared to acetone treatment (Table 1). Whereas, the pretreatment with d-limonene at both the doses (50 and 100 mg/kg body weight), 30 min prior to TPA treatment resulted in only 27% (p < 0.05) and 9% (p < 0.001) increase in weight of skin punches. The topical treatment of d-limonene alone even at 100 mg/kg body weight dose did not evoke any sign of skin edema (Table 1). Thus, this observation indicates that topical application of d-limonene exhibits inhibitory effect against TPA-induced skin edema.

Table 1.

Effect of limonene on TPA-induced histological changes (epidermal thickness, number of epidermal layers and leukocyte infiltration) in mouse skin ^a

Group	Treatment	Edema (mg)	Epidermal thickness (µm)	No. of epidermal layers	Leukocyte infiltration
1	Control	21.10 ± 0.98	28.30 ± 2.60	2–3	−−−
2	TPA	31.20 ± 1.46 d	84.60 ± 10.40 d	6–7	++++
3	Limonene (50 mg/kg body weight) + TPA	26.80 ± 0.67 b	54.60 ± 4.60 c	5–6	++
4	Limonene (100 mg/kg body weight) + TPA	23.00 ± 0.89 d	34.30 ± 4.70 d	<3–4	+
5	Limonene (100 mg/kg body weight)	21.50 ± 1.13 e	31.70 ± 3.40 e	2–3	−−−

TPA: 12-O-tetradecanoylphorbol-13-acetate.

^aEach value represents the mean ± SE of six regions in the slides of each groups. Treatment protocols and dose regimen are described in Material and methods section. (++++) severe infiltrations; (++) moderate infiltrations; (+) mild infiltrations; (−−−) no infiltration. Groups 5 (limonene, 100 mg/kg body weight) and 2 (TPA-treated; 2 µg/200 µl acetone/mouse) are compared to the control group 1 (200 µl acetone/mouse). Groups 3 (limonene, 50 mg/kg body weight + TPA) and 4 (limonene, 100 mg/kg body weight + TPA) are compared to group 2 (TPA-treated).

^b p < 0.05.

^c p < 0.01.

^d p < 0.001.

^eNot significant.

The effect of d-limonene on TPA-induced hyperplasia (epidermal thickness) and infiltration of leukocytes was determined (Figure 1). As shown in Figure 1, 12 h after multiple TPA treatments, there was a significant increase in mean epidermal thickness (84.6 ± 10.4 µm) and mean vertical thickness of epidermal cell layers (6–7) compared with the acetone-treated normal mouse skin (28.3 ± 2.6 µm thick and 2–3 cell layers). Topical treatment of d-limonene at doses of 50 and 100 mg/kg body weight significantly reduced the TPA-induced skin epidermal thickness (54.6 ± 4.6 µm; p < 0.01 and 34.3 ± 4.7 µm; p < 0.001) and vertical thickness of epidermal cell layers hyperplasia to 5–6 and <3–4 number of epidermal layers, respectively (Table 1). In addition, application of d-limonene alone at a dose of 100 mg/kg body weight did not induce any sign of hyperplasia.

Figure 1.

Effect of pretreatment of d-limonene on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced hyperplasia in Swiss albino mice skin. TPA (2 µg/200 µl acetone/mouse) was applied to the shaved dorsal skin of Swiss albino mice and TPA was applied three times a day. To determine the protective effect of d-limonene, it was topically applied to dorsal skin of mouse as described in Materials and Methods section. The mice were killed after 12 h of TPA application and the dorsal skins were collected. Sections of dorsal skin treated with the vehicle acetone (control) (a), treated with TPA alone (b), d-limonene (50 mg/kg body weight) and TPA-treated skin (c) and d-limonene (100 mg/kg body weight) and TPA-treated skin (d). Epidermal hyperplasia and infiltration of lymphocytes can be seen in section (b), whereas sections (c) and (d) show less alteration in dorsal skin. Section (d) shows significant inhibition in the induction of hyperplasia than section (c). Application of d-limonene alone (100 mg/kg body weight) to the mouse skin did not show hyperplasia (e). Skin sections were processed for hematoxylin & eosin (H&E) staining. Details are given under Material and methods section. Magnification (a–e) ×10.

Effect of d-limonene on TPA-induced expression of COX-2 enzyme in mouse skin

TPA application induces COX-2 expression in epidermal cells which plays an important role in inflammation, cell proliferation and skin tumor promotion. ²⁸ By immunohistochemical analysis, we demonstrated the effect of pretreatment of d-limonene on TPA-induced COX-2 expression in mouse skin (Figure 2). Topical treatment of d-limonene at doses of 50 and 100 mg/kg body weight prior to TPA application significantly reduced COX-2-positive cells in epidermis. Thus, our data confirmed that d-limonene possesses anti-inflammatory activity.

Figure 2.

Effect of pretreatment of d-limonene on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of cyclooxygenase-2 (COX-2) enzyme in Swiss albino mice skin by immunohistochemistry. Paraffin-embedded skin samples from acetone/d-limonene/TPA-treated mice were immunoassayed for COX-2, as described in Materials and methods section. Section of skin treated with the vehicle acetone (control) (a), treated with TPA alone shows COX-2 expression as a brown-colored product (b), skin pretreated with d-limonene 50 and 100 mg/kg body weight, 30 min prior to TPA application (c and d) and treated with d-limonene alone (100 mg/kg body weight (e)) Magnification (a–e) ×40.

Effect of d-limonene on TPA-mediated decrease in CAT, GSH and its redox enzymes in mouse skin

Topical application of TPA to mouse skin has been reported to reduce the activity of CAT, GSH and its redox enzyme. ^9,29 As compared to acetone-treated mice, the activity of CAT in TPA-treated skin exhibited a decrease of 58% (p < 0.001). Whereas d-limonene-pretreated mouse skin at doses of 50 and 100 mg/kg body weight recovered the CAT activity up to 30% (p < 0.01) and 55% (p < 0.001), respectively (Table 2). A significant 51% decrease (p < 0.001) was observed in the GSH level in the mice skin treated with TPA when compared with acetone-treated mice. Whereas 38% (p > 0.05) and 19% (p < 0.01) decrease was observed in the GSH levels with d-limonene pretreatment at doses of 50 and 100 mg/kg body weight, respectively (Table 2). GR and GPX activities in the skin of TPA-treated mice exhibited a 50% and 37% (p < 0.001), decrease, respectively. GR and GPX activities in the skin of mice pretreated with 50 mg/kg body weight of d-limonene showed 28% (p < 0.01) and 25% (p < 0.05) decrease, respectively. However, with 100 mg/kg body weight of d-limonene showed 15% (p < 0.001) and 13% (p < 0.001) decrease, respectively, as compared to acetone-treated mice (Table 2). Similarly, 100 mg/kg body weight d-limonene-pretreated mice skin showed a restoration of 72% (p < 0.001) in the GST activity of TPA-treated mice skin. The TPA-induced level of MDA was also restored by 32% (p < 0.01) and 79% (p < 0.001) with the pretreatment of d-limonene at doses of 50 and 100 mg/kg body weight, respectively (Table 2).

Table 2.

Effect of pretreatment of limonene on TPA-induced alterations in cutaneous levels of reduced glutathione (GSH) and activities of antioxidant enzymes ^a

Group	Treatment groups	CAT (nmol H2O2 consumed/min per mg protein)	GSH (mmol GSH/mg tissue)	GR (nmol NADPH oxidized/min per mg protein)	GPx (nmol NADPH oxidized/min per mg protein)	GST (nmol CDNB conjugate formed/min per mg protein)	MDA (nmol MDA formed/h per mg tissue)
1	Control	95.63 ± 3.15	36.68 ± 3.37	71.16 ± 3.48	94.28 ± 3.88	73.61 ± 4.87	2.12 ± 0.15
2	TPA	39.34 ± 1.93 d	18.08 ± 1.24 d	35.57 ± 2.99 d	59.27 ± 3.33 d	40.43 ± 2.91 d	4.90 ± 0.21 d
3	Limonene (50 mg/kg body weight) + TPA	56.52 ± 3.36 c	22.69 ± 2.17 e	51.42 ± 3.58 c	70.79 ± 3.9 b	53.22 ± 3.51 b	3.99 ± 0.22 c
4	Limonene (100 mg/kg body weight) + TPA	70.43 ± 3.17 d	29.58 ± 2.71 c	60.54 ± 4.16 d	82.06 ± 4.4 d	64.27 ± 3.93 d	2.75 ± 0.14 d
5	Limonene (100 mg/kg body weight)	95.19 ± 5.4 e	34.05 ± 1.95 e	68.04 ± 3.05 e	94.78 ± 3.6 e	70.37 ± 3.5 e	2.29 ± 0.18 e

CAT: catalase; CDNB: 1-chloro 2,4-dinitrobenzene; GPx: glutathione peroxidase; GR: glutathione reductase; GSH: reduced glutathione; GST: glutathione S-transferase; MDA: malondialdehyde; NADPH: reduced nicotinamide adenine dinucleotide phosphate; TPA: 12-O-tetradecanoylphorbol-13-acetate.

^aEach value represents the mean ± SE of six animals. Treatment protocols and dose regimen are described in Material and methods section. Groups 5 (limonene, 100 mg/kg body weight) and 2 (TPA treated; 2 µg/200 µl acetone/mouse) are compared to the control group 1 (200 µl acetone/mouse). Groups 3 (limonene, 50 mg/kg body weight + TPA) and 4 (limonene, 100 mg/kg body weight + TPA) are compared to group 2 (TPA-treated).

^b p < 0.05.

^c p < 0.01.

^d p < 0.001.

^eNot significant.

Effect of d-limonene on TPA-induced epidermal ODC activity and [³H] thymidine incorporation

Studies suggested that TPA application to mouse skin results in overexpression of ODC enzyme. ^4,30 As shown in Figure 3(a), application of TPA to mouse skin significantly increased the ODC activity by 733% (p < 0.001) as compared to acetone-treated mice. However, the application of d-limonene (50 and 100 mg/kg body weight), 30 min prior to TPA application significantly decreased the epidermal ODC activity by 40% (p < 0.001) and 68% (p < 0.001), respectively, as compared to acetone-treated one. Further, we assessed the effect of 50 and 100 mg/kg body weight d-limonene on TPA-induced [³H] thymidine incorporation into epidermal DNA (Figure 3(b)). Pretreatment of 50 and 100 mg/kg body weight of d-limonene significantly decreased the TPA-mediated increase of [³H] thymidine incorporation, by 37% (p < 0.05) and 79% (p < 0.001), respectively, however mice treated with TPA resulted in 139% (p < 0.001) increase by [³H] thymidine incorporation as compared to [³H] thymidine uptake in the skin of acetone-treated (vehicle control) mice. Thus, it is clear that TPA-mediated cutaneous ODC activity and [³H] thymidine incorporation were reduced by treatment of d-limonene, prior to TPA treatment.

Figure 3.

Effect of pretreatment of d-limonene on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal ornithine decarboxylase (ODC) activity and [³H] thymidine incorporation. Results represent mean ± SE of six mice per group. Treatment protocols and dose regimen are described in Materials and methods section. The group 5 (d-limonene; 100 mg/kg body weight) and group 2 (TPA-treated) are compared with the group 1 (acetone-treated control). Groups 3 (d-limonene; 50 mg/kg body weight + TPA) and 4 (d-limonene; 100 mg/kg body weight + TPA) are compared with group 2 (TPA-treated). *p < 0.05 and ***p < 0.001 were considered as significant and ^#considered as not significant.

Effect of d-limonene on DMBA/TPA-mediated skin tumor promotion

The effect of d-limonene on DMBA/TPA-mediated skin tumorigenesis in Swiss albino mice is shown in Figure 4. In the present study, we observed that d-limonene at dose of 100 mg/kg body weight significantly reduced the tumor promoting effect of TPA when tumor burden (11.2 ± 1.15; p < 0.0026) were compared with DMBA/TPA-mediated mice group (20.6 ± 2.6). Whereas, the application of d-limonene at the dose of 50 mg/kg body weight, 30 min prior to TPA application reduced the tumor burden up to 15.1 ± 1.85, p < 0.0695 in DMBA/TPA-mediated mice group. Surprisingly, 100% tumor incidence was observed in all group treated with both the doses of d-limonene and DMBA/TPA. However, the latency period of tumor induction was enhanced from 4 to 9 weeks with pretreatment of d-limonene at dose of 100 mg/kg body weight. Tumor diameter was also significantly reduced in mice pretreated with d-limonene at dose of 100 mg/kg body weight (2.6–10.8 mm) as compared to TPA-treated mice (5–18 mm).

Figure 4.

Effect of pretreatment of d-limonene on 7,12-dimethylbenz[a]anthracene (DMBA)-initiated- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin tumorigenesis in Swiss albino mice. Mice skin were initiated by topical application with a single dose of DMBA (40 μg/100 μl/mouse) and promoted with TPA (2 μg/200 μl/mouse) by thrice weekly application for 21 weeks. Each value represents the mean of 20 mice and was plotted as a function of number of weeks on test. (a) Percentage of mice bearing tumors. (b) Average number of tumors per mouse. (c) Representative skin tumors of group (i) control, (ii) DMBA/TPA; (iii) d-limonene with 50 mg/kg body weight and (iv) d-limonene with 100 mg/kg body weight) were shown.

Effect of d-limonene on DMBA/TPA-induced expression of Ras, Raf and p-ERK1/2 in skin/tumors

To determine whether DMBA/TPA could induce the activation of Ras/Raf/ERK1/2 signaling pathway under in vivo condition, Western blot analysis was performed using Ras, Raf and phospho-specific ERK1/2 antibodies. The level of membrane-bound Ras protein in d-limonene-pretreated skin tumors was found to be reduced in comparison with DMBA/TPA-mediated skin tumor (Figure 5). Treatment of d-limonene at both the doses, prior to TPA application resulted in the increase in level of Ras protein in the cytosolic fraction as compared DMBA/TPA-mediated tumors. Pretreatment of d-limonene at 100 mg/kg body weight was more effective than that of at 50 mg/kg body weight. The normal mouse skin showed detectable levels of both cytosolic and membrane-bound Ras protein.

Figure 5.

Effect of pretreatment of d-limonene on expression of Ras, Raf and phosphorylation of extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA)-induced skin tumors of Swiss albino mice by Western blot analysis. The skin tumors were pooled. Cytosolic and membrane fractions were prepared, and protein expression was determined as described in Materials and methods section. (a) Western blotting showing the expression of Ras, Raf and p-ERK1/2. (b) Relative density was calculated using densitometric values of each individual band and expressed as mean ± SE of three individual values. Significance between two groups was determined using Student’s t test. Equal loading was confirmed by parallel running gel for β-actin. The immunoblots shown here are representative of three independent experiments with similar results. ***p < 0.001 was considered as significant.

Raf protein expression was analyzed in connection with Ras protein analysis. An elevated level of Raf protein was observed in DMBA/TPA-mediated skin tumor in comparison with acetone-treated skin (Figure 5). Whereas, pretreatment of d-limonene at both the doses (50 and 100 mg/kg body weight), prior to TPA application showed reserve effect in expression of Raf protein as compared to that of DMBA/TPA-treated group. To study the Ras-mediated signaling pathway, we next analyzed the phosphorylation of p-ERK1/2 as downstream effectors of Ras protein. Application of TPA in DMBA-initiated mice skin elevated the phosphorylation of ERK1/2 in comparison to acetone-treated. The phosphorylation of ERK1/2 in d-limonene-pretreated (50 and 100 mg/kg body weight) skin tumors were significantly inhibited in comparison to that of DMBA/TPA-treated tumors. So, d-limonene treatments, prior to TPA application significantly suppressed the Ras/Raf/ERK1/2-signaling pathway in DMBA-initiated skin tumor.

Effect of d-limonene on DMBA/TPA-induced expression of Bax and Bcl-2 in skin/tumors

To find out the role of Bax and Bcl-2 protein expression for apoptosis, we performed Western blot. It was observed that DMBA/TPA treatment downregulated Bax expression in comparison to acetone treatment. However, pretreatment with d-limonene at both the doses (50 and 100 mg/kg body weight) resulted in the upregulation of Bax in comparison to DMBA/TPA-mediated mouse skin (Figure 6(a)). Whereas the pretreatment with d-limonene at both the doses downregulated the expression of Bcl-2 induced by DMBA/TPA treatment. The Bax/Bcl-2 ratio was significantly increased in d-limonene treatment group (Figure 6(c)), which suggests the possibility that d-limonene-mediated tumor cell death occurs by an intrinsic apoptosis mechanism.

Figure 6.

Effect of pretreatment of d-limonene on the expression of Bax and Bcl-2 protein 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA)-induced skin tumors of Swiss albino mice by Western blot analysis. The pooled skin tumor lysates were prepared and protein expression was determined as described in Materials and methods section. (a and b) Western blotting showing the expression of pro-apoptotic Bax and anti-apoptotic Bcl-2. (c) Bax:Bcl-2 ratio was calculated using densitometric values of each individual band and expressed as mean ± SE of three individual values. Significance between two groups was determined using Student’s t test. Equal loading was confirmed by parallel running gel for β-actin. The immunoblots shown here are representative of three independent experiments with similar results. ***p < 0.001 was considered as significant.

Discussion

The promotion stage in multistep tumorigenesis offers an opportunity to delay the process of cancer development. It provides a realistic path to understand the mechanism of action of chemopreventive dietary agents. Multiple lines of studies showed that phorbol-type tumor promoters induce inflammatory responses like induction of edema, hyperplasia and COX-2 expression in epidermal cells leading to malignant transformation. ^4,8 Histological changes (edema and hyperplasia) of skin and infiltration of polymorphonuclear (PMN) leukocytes in dermis are responsible for increasing the risk of carcinogenesis. TPA-induced upregulation of COX-2 and subsequently production of prostaglandins (PGs) indicate its role in tumor promotion and in tumorigenesis. ³¹ Maier et al. ³² reported that interleukin-1a induces PG synthesis and primes leukocytes to enhance the production of reactive oxygen intermediates in epidermal keratinocytes; the latter have been implicated in the induction of tumor formation. However, our result showed that pretreatment with d-limonene attenuated the induction of COX-2 enzyme by TPA, suggesting that COX-2 is likely to be potential target for d-limonene.

Accumulating evidences showed that the tumor promoter induces infiltration and activation of leukocytes which induce the production of ROS in the cells. ^9,33 According to earlier observations, d-limonene alters the increased level of GPX, GST and GSH content, indicating the potency to diminish the oxidative stress response. ^29,34 We observed that pretreatment with d-limonene, prior to TPA treatment reduced the effect of ROS by elevating the activities of antioxidative enzymes CAT, GR, GPX, GST and GSH content. Our findings suggest that d-limonene reduces the TPA-induced oxidative stress in murine skin through the activation of antioxidative enzymes.

ODC is a rate-limiting enzyme of polyamines (putrescine, spermidine and spermine) biosynthesis that is essential for normal cell growth and differentiation. ³⁵ Elevation in the activity of ODC has been detected in transformed cell line and experimental animal tumors. ³⁰ Application of tumor promoter induced the activity of ODC and incorporation of thymidine into DNA, thereby enhancing the growth and differentiation of cell leading to development of cancer. Our data showed that d-limonene significantly inhibits TPA-mediated increase in epidermal ODC activity and thymidine incorporation.

We demonstrated that d-limonene suppressed the TPA-induced promotional changes, thus extending the latency period of tumor and reducing the tumor burden. In summary, d-limonene exhibited an inhibitory effect on DMBA/TPA-mediated tumorigenesis by reducing the TPA-mediated inflammation (including COX-2 induction), oxidative stress and hyperproliferation responses, at least in part. DMBA treatment produces carcinogen–DNA adducts which may induce G→A transition or A→T transversion. Such point mutations in codon 61 have been shown to be associated with overexpression of the H-Ras gene. ³⁶ Ras protein becomes activated and anchored to the inner cell membrane via farnesylation to transduce the cell proliferation signal. Since, d-limonene and other monoterpenes are known to inhibit FTase activity. ^7,17 In this context, we examined the Ras/Raf/ERK1/2 pathway in DMBA/TPA-induced skin tumors. In our study, d-limonene decreased the level of membrane-bound Ras protein and increased the level in the cytosol. Activation of Ras protein (anchored) leads to the activation of Raf protein (first step, the best accepted downstream Ras effector), which in turn activates the ERKs by phosphorylation. Activated ERKs target many proteins including transcription factors and other protein kinases resulting in transduction of signal to nucleus. ³⁶ Our analysis confirms that the application of d-limonene significantly suppresses the DMBA/TPA-mediated activation of Raf and phosphorylation of ERK1/2, leading to the suppression of Ras/Raf/ERK1/2 cascade during the promotion phase of murine skin tumorigenesis.

Inhibition of Ras pathway alters the expression level of many intermediate apoptotic proteins like Bim, Bax, Bcl-2 and so on, thereby leading to apoptosis. ³⁷ The Bcl-2 gene product is a negative regulator of apoptosis, which forms a heterodimer complex with Bax and neutralizes the effect of pro-apoptosis. Thus, the ratio of Bax-to-Bcl-2 protein plays a crucial role in apoptosis in DMBA/TPA-treated mouse skin. ³⁸ In the present study, we found that d-limonene treatment suppresses the DMBA/TPA-induced downregulation of Bax and upregulation of Bcl-2 during skin tumorigenesis. Therefore, d-limonene efficiently suppresses the formation of DMBA/TPA-mediated skin tumor through the induction of pro-apoptotic state. Ji et al. ³⁹ demonstrated that d-limonene treatment to human leukemia cells induce apoptosis through the mitochondrial death pathway with an increase in Bax protein expression and involvement of caspases. However, elevated level of ROS can also activate ERK1/2; and induction of apoptosis by triggering mitochondrial permeability transition pore opening, release of pro-apoptotic factors and activation of caspase-3 and -9 and subsequent cleavage of poly (ADP-ribosyl) polymerase. ⁴⁰ Recently, Chang et al. ³⁶ reported that TPA treatment activates the Ras/Raf/ERK1/2-signaling pathway, NF-κB and expression of COX-2. Additionally, many epithelial tumors exhibit elevated levels of ODC enzyme that can also lead to upregulation of the Ras/Raf/ERK1/2 pathway. ⁴¹ In a previous study, d-limonene exerts the antiproliferative mechanism through the production of H₂O₂ and ERK pathway activation and induces the apoptotic process. ¹²

In conclusion, the topical treatment of d-limonene inhibits Ras/Raf/ERK1/2 signaling pathway and promoted to pro-apoptotic state and delayed the DMBA/TPA-mediated murine skin tumorigenesis, by attenuating the skin inflammatory responses (edema, hyperplasia and COX-2 expression). Thus, d-limonene exhibited several potent chemopreventive effects on the reduction of skin tumors, providing protection from the development of this form of papilloma.