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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2013 Oct 29;94(6):362–372. doi: 10.1111/iep.12042

Protective effect of γ-tocopherol-enriched diet on N-methyl-N-nitrosourea-induced epithelial dysplasia in rat ventral prostate

Lucas D Sanches *, Sergio A A Santos †, Jaqueline R Carvalho †, Gabriela D M Jeronimo *, Wagner J Favaro ‡, Maria D G Reis *, Sérgio L Felisbino †, Luis A Justulin Jr *,†
PMCID: PMC3944448  PMID: 24205794

Abstract

Despite recent advances in understanding the biological basis of prostate cancer (PCa), the management of this disease remains a challenge. Chemoprotective agents have been used to protect against or eradicate prostate malignancies. Here, we investigated the protective effect of γ-tocopherol on N-methyl-N-nitrosourea (MNU)-induced epithelial dysplasia in the rat ventral prostate (VP). Thirty-two male Wistar rats were divided into four groups (n = 8): control (CT): healthy control animals fed a standard diet; control+γ-tocopherol (CT+γT): healthy control animals without intervention fed a γ-tocopherol-enriched diet (20 mg/kg); N-methyl-N-nitrosourea (MNU): rats that received a single dose of MNU (30 mg/kg) plus testosterone propionate (100 mg/kg) and were fed a standard diet; and MNU+γ-tocopherol (MNU+γT): rats that received the same treatment of MNU plus testosterone and were fed with a γ-tocopherol-enriched diet (20 mg/kg). After 4 months, the VPs were excised to evaluate morphology, cell proliferation and apoptosis, as well as cyclooxygenase-2 (Cox-2), glutathione-S-transferase-pi (GST-pi) and androgen receptor (AR) protein expression, and matrix metalloproteinase-9 (MMP-9) activity. An increase in the incidence of epithelial dysplasias, such as stratified epithelial hyperplasia and squamous metaplasia, in the MNU group was accompanied by augmented cell proliferation, GST-pi and Cox-2 immunoexpression and pro-MMP-9 activity. Stromal thickening and inflammatory foci were also observed. The administration of a γ-tocopherol-enriched diet significantly attenuated the adverse effects of MNU in the VP. The incidence of epithelial dysplasia decreased, along with the cell proliferation index, GST-pi and Cox-2 immunoexpression. The gelatinolytic activity of pro-MMP-9 returned to the levels observed for the CT group. These results suggest that γ-tocopherol acts as a protective agent against MNU-induced prostatic disorders in the rat ventral prostate.

Keywords: cell proliferation, epithelial dysplasia, MMP-9, N-methyl-N-nitrosourea, ventral prostate, γ-tocopherol

Prostate cancer (PCa) is the most common non-cutaneous malignant neoplasm in men in Western countries (Koh et al. 2010). Risk factors for prostate cancer include advanced age, race, family history and environmental factors such as diet and inflammation (Thapa & Ghosh 2012). Androgen ablation therapy is widely accepted and performed for prostate cancer because androgens are essential for the development and growth of normal prostate and prostate cancer cells (Cunha & Donjacour 1987). However, the outgrowth of hormone-independent cancer cells occurs and eventually leads to death in many cases (Takahashi et al. 2009).

Because elevated oxidative stress in the cellular microenvironment has been considered a common denominator in prostate cancer and ageing (Thapa & Ghosh 2012), chemoprevention against PCa has been considered an important area of research (Strope & Andriole 2010). Indeed, prostate carcinogenesis is considered an ideal candidate for chemoprevention due to the long latency period and slow disease progression, which provides an opportunity to intervene before the malignancy is established, thus delaying progression and improving quality of life of the patients (Syed et al. 2007; Stephenson et al. 2010). Many observational and intervention studies have been conducted using vitamins, phytochemicals and minerals as chemoprotective compounds (Griffiths et al. 2007). The use of vitamin E as a chemoprotective agent against PCa is based upon the ability of vitamin E to scavenge free radicals and prevent oxidative damage to the prostate epithelium (Campbell et al. 2011). α-tocopherol has been traditionally recognized as ‘the vitamin E’ because it is the most abundant isoform of vitamin E found in serum and dietary supplements. However, experimental and epidemiological studies have suggested that γ-tocopherol may be superior to the commonly tested α-tocopherol as a chemopreventive agent due to its more potent antinitrative and anti-inflammatory activities (Jiang et al. 2001; Campbell et al. 2003; Hensley et al. 2004).

To confirm the protective effect of γ-tocopherol in vivo, we performed animal experiments using MNU-plus-testosterone-induced epithelial dysplasia in the rat prostate, a model used for studying the biology of prostate carcinogenesis and for developing and evaluating cancer control strategies (Narayanan et al. 2009; Gonçalves et al. 2010; Banudevi et al. 2011). In this model, the additive effect of the MNU carcinogen with testosterone-induced cell proliferation maximizes the appearance of epithelial injuries when compared to MNU alone, accelerating the establishment of prostate disorders in a rodent model (Liao et al. 2002; Kaina et al. 2007; Gonçalves et al. 2010).

We observed a significant reduction in the occurrence of prostate epithelial dysplasia, a decrease in epithelial proliferation, GST-pi and Cox-2 immunoexpression and pro-MMP-9 gelatinolytic activity in the MNU group after the ingestion of a γ-tocopherol-enriched diet. These results indicate that γ-tocopherol acts as a chemoprotective agent against MNU-induced prostatic disorders in the rat ventral prostate.

Material and methods

Animals and experimental procedures

Thirty-two adult (90-day-old) male Wistar rats were conducted in accordance with institutional guidelines for animal treatment, and the experiment was approved by the Ethics Committee of Experimental Animals of Federal University of Triangulo Mineiro (protocol number: 165/2011). The animals were housed in plastic cages under conventional conditions (25 °C, 40–70% relative humidity, 12 h light/12 h dark), in pathogen-free conditions with a supply of water and balanced chow ad libitum. N-methyl-N-nitrosourea (MNU) (Sigma, St. Louis, MO, USA) was stored at −20 °C in the dark, and the MNU solution was freshly prepared and dissolved in physiological saline just before use.

After 7 days of acclimatization, the animals were randomly divided into four groups (n = 8 animals): control (CT): healthy control animals without intervention fed a normal diet; control+γ-tocopherol (CT+γT): healthy control animals without intervention fed a γ-tocopherol-enriched diet (20 mg/kg); N-methyl-N-nitrosourea (MNU): animals that received a single dose of MNU (30 mg/kg, 0.1 ml/application; subcutaneously injected) and a single dose of testosterone propionate diluted in corn oil (100 mg/kg, 0.1 ml/application; subcutaneously injected) and were fed a normal diet; and MNU+γ-tocopherol (MNU+γT): animals that received the same treatment as the MNU group and were fed a γ-tocopherol-enriched diet (20 mg/kg). Food consumption was determined using weekly measurements of the difference in the amount of pellet food provided at the beginning of the interval from that remaining at the end of the interval (Rinaldi et al. 2013).

Takahashi et al. (2009) demonstrated that the consumption of a γ-tocopherol-enriched diet (50 mg/kg) for 10 weeks decreased the incidence of neoplastic lesions in the ventral prostate from the transgenic rat for adenocarcinoma of prostate (TRAP) model. Our study was conducted to investigate whether a γ-tocopherol-enriched diet (20 mg/kg) administered for 16 weeks was sufficient to protect the ventral prostate against the development of epithelial disorders in MNU-treated Wistar rats.

After the treatment period, the animals were euthanized by CO2 inhalation followed by decapitation (Sarobo et al. 2012). The animals’ weights were determined prior to euthanasia. The ventral (VP) prostatic lobes were excised, weighted and processed for posterior analysis.

Histopathological analysis

For light microscopy, 12 ventral prostatic lobes per group were fixed for 1 h in methacarn (60% absolute methanol, 30% chloroform, 10% glacial acetic acid) and processed for embedding in Paraplast (Sigma Co) (six prostatic lobes) or in glycol methacrylate resin (Leica™ Historesin Embedding Kit, Nussloch, Germany) (six prostatic lobes).

Resin sections (3 μm) were stained with haematoxylin–eosin (HE) for morphological and morphometric analysis. Paraplast sections (5 μm) were stained with Picrosirius red (Junqueira et al. 1979) for collagen (type I and type III collagen fibres) detection followed by stereological analysis or analysed by immunohistochemistry reactions. The histopathological classification of prostate lesions observed in the ventral prostate was accomplished according to the Bar Harbor Classification System for the mouse prostate, developed by the National Cancer Institute's Mouse Models of Human Cancers Consortium, Prostate Steering Committee (Shappell et al. 2004).

Morphometric and stereological analysis

Morphometric analysis was performed using a Leica™ DMLB 80 microscope (Leica Microsystems, Nussloch, Germany) connected to a Leica™ DC300FX camera and Leica™ q-win software Version 3 for Windows™. Epithelial height measurements were evaluated in six rat ventral prostate lobes for each group. A total of 10 random microscopic fields (400× magnification) per prostatic lobe section from six different animals were acquired with 10 random interactive measurements per field (600 measurements per experimental group). To determine the collagen volume density, another 10 random microscopic fields (200× magnification) per prostatic lobe section from six different animals in each group were acquired. The Sirius red–stained area was determined by the automatic image detection of red colours, and the collagen volume density was calculated as a percentage of red-stained areas per total prostatic area (Sarobo et al. 2012). It has previously been determined that 1 mg of fresh rat ventral prostate tissue has a volume of approximately 1 mm3 (DeKlerk & Coffey 1978). Therefore, the volume density of collagen fibres was calculated by multiplying the mean collagen fibre volume by the mean prostatic weight. All measurements were performed in the intermediate and distal regions of the prostate lobe ducts, which represent the major portions of the prostatic lobes (Nemeth & Lee 1996).

Immunohistochemistry

The antibodies applied in this study were anti-Ki67 (rabbit polyclonal, clone ab66155, dilution 1:100, Abcam, Cambridge, MA, USA), anticyclooxygenase-2 (Cox-2 rabbit polyclonal, clone ab15191, dilution 1:100, Abcam), antiglutathione-S-transferase-pi (GST-pi rabbit polyclonal, clone ab106268, dilution 1:100, Abcam) and antiandrogen receptor (AR goat polyclonal, clone N-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

For the analysis, paraplast sections collected on previously silanized slides were deparaffinized and rehydrated through graded alcohol, and antigen retrieval was performed in 10 mM citrate buffer pH 6.0, at 97 °C for 20 min. Blocking of endogenous peroxidase was obtained by covering the slides with H2O2 (3% in methanol) for 20 min, and blocking of non-specific protein–protein interactions was achieved by incubating sections with 3% bovine serum albumin (BSA, Sigma). After pretreatment, the sections were incubated overnight at 4 °C with the antibodies described above diluted in 1% BSA. The slides were then incubated with peroxidase-conjugated secondary antibodies diluted 1:100 in 1% BSA. Chromogen colour development was accomplished with diaminobenzidine tetrahydrochloride (DAB; Sigma). Slides were counterstained with Harris's haematoxylin and digitalized with a Leica™ DMLB 80 microscope connected to a Leica™ DC300FX camera and Leica™ q-win software Version 3 for Windows™. As a negative control, the primary antibody was replaced with the corresponding normal isotype serum (data not shown).

In situ detection of cell apoptosis using the TUNEL assay

Apoptosis detection was based on a reaction for in situ terminal deoxynucleotidyl-transferase (TdT)-mediated biotinylated UTP nick-end labelling (TUNEL). This was essentially performed using the FragEL DNA kit (Calbiochem, La Jolla, CA, USA) according to the manufacturer's directions. Briefly, samples of the paraplast-embedded tissue were collected on silanized glass slides and treated with proteinase K (2 mg/ml) diluted 1:100 in 10 mM Tris-HCl, pH 8.0. The slides were then washed in Tris-buffered saline (TBS), and endogenous peroxidase was blocked for 5 min in 3% H2O2 diluted in methanol. After washes in TBS, sections were treated with 1× TdT equilibration buffer at room temperature for 30 min and were subsequently incubated with TdT labelling reaction mix (57 μl equilibration buffer plus 3 μl TdT enzyme) for 1.5 h at 37 °C. The slides were rinsed in TBS and incubated with blocking buffer for 10 min at room temperature, followed by 50× biotin-conjugated secondary antibody (diluted 1:50 in blocking buffer) for 30 min at room temperature. After three washes in TBS, the detection of TUNEL-positive cells was performed with DAB for 5 min. The specimens were counterstained with Harris's haematoxylin and mounted in Permount medium.

Androgen receptor, proliferation, apoptosis and GST-pi index determinations

The number of epithelial cells that showed positive immunolabelling for AR, Ki67, TUNEL and GST-pi was counted in 50 random fields at 400× magnification from six different VP lobes for each treatment and expressed as a percentage (%) (mean ± SD) of the total cells counted. All image acquisition and quantitative measurements were performed with the investigators blind to both the animal identity and experimental condition.

Zymography

Frozen VP lobes (n = 6/group) were mechanically homogenized in 50 mM of Tris buffer pH 7.5 plus 0.25% Triton-X 100 by Polytron for 30 s at 4 °C and centrifuged, and the protein was extracted from the supernatant and quantified using the Bradford (1976) method. Aliquots (25 μg protein) from ventral prostate extracts were subjected to electrophoresis in gelatin-containing polyacrylamide (8% acrylamide) gels in the presence of SDS under non-reducing conditions. The gelatin substrate was present at a final concentration of 0.1% in the gel. The gels (0.75 mm thick) were electrophoresed for 2 h at 100 V, 4 °C, in a Bio-Rad MiniProtean II system (Bio-Rad Laboratories Inc., Richmond, CA, USA). Following electrophoresis, the gels were gently shaken at room temperature with 2.5% Triton-X100 (two changes of 15 min). The gels were then incubated overnight (18 h) in 50 mM Tris-HCl (pH 8.4) containing 5 mM CaCl2 and 1 μM ZnCl2 at 37 °C. Following incubation, the gels were stained with Coomassie Blue. Areas of proteolysis appeared as clear zones against a blue background. Images of the gels were captured, and the bands corresponding to each enzyme form were quantified by densitometry as integrated optical density in an image master vds version 3.0 (Pharmacia Biotech, Piscataway, NJ, USA). The values were analysed statistically and plotted as a histogram.

Statistical analysis

Values are expressed as the mean ± SD. One-way analysis of variance was performed to determine whether differences existed among all groups, and the Tukey–Kramer post hoc test was employed to determine the significance of the differences. A P-value of <0.05 was considered significant. Statistical analyses were performed using instat version 3.0 software (GraphPad, San Diego, CA, USA).

Results

Morphological and histopathological analysis

None of the treatments influenced either the mean body weight or the absolute or relative ventral prostate weight in any of the groups significantly, although a tendency towards increased prostate weight was observed in the MNU group (Table 1). Food intake did not change throughout the experimental period in the age-matched experimental groups (Table 1). The VPs from the CT (Figure 1a,b) and CT+γT (Figure 1c,d) animals presented regular glandular structures lined with tall and columnar epithelial cells surrounded by a thin stroma. In contrast, marked morphological changes were observed in the VPs from the MNU group. In this group, 100% of the animals demonstrated morphological characteristics of glandular dysplasia (Table 1) classified as stratified epithelial hyperplasia (Figure 1e,f) and squamous metaplasia (Figure 1h,i), characterized by agglomerated epithelial cells with heterogeneous phenotypes and a change in epithelial cell morphology from columnar to low cuboidal/flattened aspect respectively. The presence of flattened cells may be responsible for the reduction in the epithelial cell height in the MNU group (Table 1). In this group, approximately 87% also showed inflammatory foci within the stromal compartment adjacent to the glandular epithelium (Figure 1g and Table 1). There were partial pathological responses to γ-tocopherol intake in the MNU+γT group, as demonstrated by significant reductions in the prostatic epithelial and stromal disorders. However, small foci of epithelial dysplasia and inflammatory cells still remained (Figure 1j,k and Table 1). Quantitative evaluation demonstrated a significant reduction in the number of animals presenting epithelial dysplasia (62% of animals) compared to 100% of the animals in the MNU group. The stromal inflammatory foci in the MNU+γT group also decreased compared to the MNU group (37.5% vs. 87.5% respectively) (Table 1).

Table 1.

γ-tocopherol effects on biometric and histopathological parameters of the animals and ventral prostate

Experimental groups
Parameters (at 28 weeks age) CT CT+γT MNU MNU+γT
Food intake (g/day) 28 ± 1.9 27 ± 2,4 26 ± 1,5 27 ± 2.2
Body weight (g) 590 ± 56.9 576 ± 19.04 620.5 ± 68.7 600 ± 52.1
VP absolute weight (mg) 580.7 ± 68 573.7 ± 86.5 650.2 ± 52.1 598.4 ± 93.6
VP relative weight 0.97 ± 0.26 0.98 ± 0.19 1.04 ± 0.3 0.99 ± 0.15
Epithelial cell height (μm) 26.69 ± 3.4a 27.43 ± 2.4a 19.49 ± 1.04b 25.95 ± 1.87a
Collagen volume density (mm3) 2.61 ± 1.23a 2.94 ± 0.56a 6.23 ± 1.98b 4.01 ± 0.84c
Epithelial dysplasia 0/8 (0%)a 0/8 (0%)a 8/8 (100%)b 5/8 (62.5%)c
Stromal inflammatory foci 0/8 (0%)a 0/8 (0%)a 7/8 (87,5%)b 3/8 (37.5%)c
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Values expressed as mean ± standard deviation. Different superscript letters indicate significant differences among experimental (P < 0.05). CT, control; MNU, N-methyl-N-nitrosourea administered group; MNU+γT, N-methyl-N-nitrosourea administered and enriched γ-tocopherol diet; VP, ventral prostate.

Figure 1.

Figure 1

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Haematoxylin–eosin (H&E) staining. (a–b) Histological sections of ventral prostate from CT group; (a) general aspect of the gland. Acini structures presented large lumen (l) lined by regular tall columnar epithelium (ep) surrounded by a thin stroma (s); (b) detail of the epithelium. (c–d) Histological sections from CT+γT group; (c) general aspect of the ventral prostate, revealing no structural difference from CT. Lumen (l), stroma (s); (d) detail of figure c; (e–i) histological sections from MNU group; (e) general aspect of the gland showing lumen (l), stroma (s) and stratified epithelial hyperplasia (asterisks); (f) detail of figure e showing epithelial hyperplasia; (g) note the inflammation focus in the prostate stroma (inf); (h) histological sections showing epithelial stratification (asterisks) and focus of squamous metaplasia (arrows). Lumen (l), stroma (s); (i) detail of squamous metaplasia; (j) histological section of MNU+γT group demonstrating improvement in glandular morphology and few stromal inflammatory cell (inf). Lumen (l), stroma (s). (k) Detail of figure j showing the epithelial stratification and inflammation (inf). Scale bar: a, c, e, g, h and j: 506 m; b, d, f, i and k: 106 m.

Picrosirius red staining showed that the CT and CT+γT groups had thin collagen fibres above the epithelium and around the smooth muscle cells in the VP (Figure 2a–d respectively). After MNU administration, collagen fibre density was increased above the epithelium, between the smooth muscle cell layers, around the blood vessels and in the axis of connective tissue supporting the epithelial folding, growing into the glandular lumen (Figure 2e,f). In the MNU+γT group, the volume of collagen fibres as well as the foci of epithelial hyperplasia was decreased (Figure 2g,h). However, the values observed in the MNU+γT group were still higher than those in the CT and CT+γT groups. The measurement of picrosirius-stained areas confirmed the observation of collagen volume changes in all experimental groups (Table 1).

Figure 2.

Figure 2

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Picrosirius-stained ventral prostate sections from CT (a,b), CT+γT(c,d), MNU (e,f) and MNU+γT (g,h). In a and b, collagen fibres (arrows) are observed around acini, mainly in the interstitial space. (c) In MNU group, prostate presented increased amount of collagen fibres in the interstitial compartment, around blood vessels (arrow) and in the axis of epithelial folding (arrowheads). (d) In MNU+γT, the collagen was detected under the epithelium (arrows) and in thin epithelial folding (arrowheads). l (lumen). (b) insert of figure a; d: insert of figure c; (f) insert of figure e; (h) insert of figure g. Scale Bars: a, c, e and g: 50/m; b, d, f and h: 10/m; I, j, k and l: 15/m.

To evaluate whether γ-tocopherol intake interfered with VP epithelial cell proliferation after MNU administration, we performed immunohistochemistry to detect Ki-67 expression. In the CT (Figure 3a,b) and CT+γT groups (Figure 3c,d), Ki-67 positive cells were observed; conversely, the number of positive nuclei was higher in the VP from the MNU group compared with the CT and CT+γT groups (Figures 3e,f and 4a). In the MNU+γT group (Figure 3g,h), the total number of epithelial cells also increased compared to the CT and CT+γT groups; however, the value was lower than that observed in the MNU group (Figure 4a), demonstrating the protective effect of γ-tocopherol on MNU-induced epithelial cell proliferation in the rat ventral prostate. The effect of γ-tocopherol treatment on epithelial cell apoptosis was also assessed by analysing the percentage of positive nuclei by the TUNEL reaction. This analysis revealed no significant change in the epithelial cell apoptotic index in all experimental groups (Figure 3i–l). These results were confirmed via a quantitative analysis of the TUNEL index (Figure 4b).

Figure 3.

Figure 3

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Ki67 immunohistochemistry from control group (a,b), CT+γT(c,d), MNU (e,f) and MNU+γT (g,h) and TUNEL reaction from CT (i), CT+γT (j), MNU (k) and MNU+γT (l) in rat ventral prostate. (a–h) Arrows indicated positive Ki67 proliferating epithelial cells. (i–l) Arrows pointed apoptotic cells marked by TUNEL reaction. (b) insert of figure a; (d) insert of figure c; (f) insert of figure e; (h) insert of figure g. Scale Bars: a, c, e and g: 503 m; b, d, f and h: 103 m; I, j, k and l: 153 m.

Figure 4.

Figure 4

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Proliferation (a) and apoptotic (b) indexes in rat ventral prostate from CT, CT+γT, MNU and MNU+γT groups. The results are expressed as mean ± SD. Different superscript letters means statically difference among experimental groups with P < 0.05.

Androgen receptor was detected mainly in the nuclei of columnar epithelial cells in the CT (Figure 5a,b) and CT+γT groups (Figure 5c,d). In the MNU group, an increase in the cells expressing AR was observed, mainly at areas of epithelial stratification (Figure 5c,d). γ-tocopherol intake decreased the MNU-induced epithelial hypercellularity and consequently, the number of AR-positive nuclei (Figure 5g,h). Although morphological differences were observed, the percentage of AR-positive nuclei was not statistically significant for any of the treated-experimental groups compared to the control group (Figure 5i). GST-pi expression was detected in the epithelial basal cells in the CT (Figure 6a,b) and CT+γT groups (Figure 6c,d). In the MNU group, an increased number of GST-pi positive basal cells was observed, mainly at areas of hyperplastic epithelial folding (Figure 6e,f,i) and in some luminal cells (Figure 6g,h). In the MNU+γT group (Figure 6g,h), GST-pi expression decreased compared to the MNU group, but it remained higher than that observed in the CT and CT+γT groups. Quantitative analysis of the immunohistochemistry for AR (Figure 5i) and GST-pi (Figure 6i) confirmed the results of the histopathological analysis. As observed for AR and GST-pi, γ-tocopherol attenuated the increased immunostaining of Cox-2 protein (Figure 7g,h) resulting from MNU treatment because more intense staining was observed in the MNU and MNU+γT groups than in the CT (Figure 7a,b) and CT+γT groups (Figure 7c,d).

Figure 5.

Figure 5

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Immunohistochemistry for androgen receptor (AR) from control group (a,b), CT+γT (c,d), MNU (e,f) and MNU+γT (g,h). Note the uniform intensity of the positive reaction in the nuclei (arrows) of the epithelial cells in ventral prostate in all experimental groups. (b) insert of figure a; (d) insert of figure c; (f) insert of figure e; (h) insert of figure g. Lumen (l), stroma (s). Scale Bars: a, c, e and g: 50 m; b, d, f and h: 10 m. (i) Semi-quantitative analysis of the nuclear AR immunoreactivity of the epithelial cells from ventral prostate CT, CT+γT, MNU and MNU+γT groups. The results are expressed as mean ± SD.

Figure 6.

Figure 6

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Immunohistochemistry for GST-pi from control group (a,b), CT+γT (c,d), MNU (e–i) and MNU+γT (j,k). Note few positive basal epithelial cells (arrows) in the CT (a,b) and CT+γT (c,d). In MNU group, an increased frequency of positive basal cells at hyperplasic epithelium (e,f,i) and luminal cells (g,h) is observed. In MNU+γT group, the number of positive basal cells decreases (j,k) but remained higher that CT ones. (b) insert of figure a; (d) insert of figure c; (f) insert of figure e; (h) insert of figure g. Scale Bars: a, c, e, g, 1 and j: 501 m; b, d, f, h and k: 101 m. l: Semi-quantitative analysis of the nuclear GST-pi immunoreactivity of the epithelial cells from ventral prostate CT, CT+γT, MNU and MNU+γT groups. The results are expressed as mean ± SD. Different superscript letters means statically difference among experimental groups with P < 0.05.

Figure 7.

Figure 7

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Immunohistochemical staining of cyclooxygenase-2 (Cox-2) in the ventral prostate of control (a,b), CT+γT (c,d), MNU (e,f) and MNU+γT (g,h). Note the slight nuclear (arrowhead) and cytoplasmic (asterisks) staining in CT (a,b) and CT+ γT (c,d). In MNU group (e,f), there is an evident increase in immunostaining, detected as brown colour in the nucleus (arrowhead) and cytoplasm (asterisks). The intensity of Cox-2 detection decreased in nuclei (arrowheads) and cytoplasm (asterisks) in MNU+γT group (e,f). (b) detail of figure a; (d) detail of figure c; (f) detail of e; (h) detail of figure g; s (stroma); l (lumen). Scale Bars: a, c, e and g: 60/m; b, d, f and h: 20/m.

Gelatin zymography analysis demonstrated an increase in pro-MMP-9 activity in the MNU group compared with the CT and CT+γT groups. γ-tocopherol intake restored the levels of pro-MMP-9 activity to those observed for the control groups. There was no change in the gelatinolytic activity of the active form of MMP-9 (Figure 8a). These results were confirmed by quantitative analysis of the representative zymography gels (Figure 8b).

Figure 8.

Figure 8

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(a) Representative gelatin zymography for ventral prostate MMP-9 from CT, CT+γT, MNU and MNU+γT. (b) Densitometric analyses of the clear bands expressed in IOD. *Means significantly difference with P < 0.05.

Discussion

In a large-scale (35,533 randomized men) study, the Selenium Vitamin E and Prostate Cancer Prevention Trial (SELECT) showed that selenium and α-tocopherol failed to prevent prostate carcinogenesis (Klein et al. 2011). Due to the disappointing results from this clinical trial on the effects of α-tocopherol, experimental and epidemiological studies have focused on other isoforms of vitamin E and suggested that γ-tocopherol may be superior to the more commonly tested isoform of vitamin E, α-tocopherol, as a cancer chemopreventive agent (Jiang et al. 2001; Campbell et al. 2003; Hensley et al. 2004).

There are few experimental studies focusing on the protective effects of a γ-tocopherol-enriched diet on the development of prostate disorders. Takahashi et al. (2009) demonstrated that consuming a γ-tocopherol-enriched diet (50 mg/kg) for 10 weeks decreased the incidence of neoplastic lesions in the transgenic rat for adenocarcinoma of prostate (TRAP) model. In our non-transgenic model, prostatic dysplasia was induced by the combination of the carcinogen MNU with a testosterone promoting agent, which maximizes the development of epithelial injuries and accelerates the establishment of prostate disorders in a rodent model (Liao et al. 2002; Kaina et al. 2007; Gonçalves et al. 2010). In this model, we checked the potential for a lower dose of a γ-tocopherol-enriched diet (20 mg/kg) administered for a prolonged time (16 weeks) to protect the rat ventral prostate against MNU plus testosterone-induced dysplasia.

It has been reported that Japanese men ingest an average of 12.2 ± 2.1 mg γ-tocopherol in their daily life (Yoshikawa et al. 2005). Although we have used a lower dosage of γ-tocopherol compared to Takahashi et al. (2009) (20 vs. 50 mg/kg), the amount of γ-tocopherol used in our study was 5–6 times higher than the human exposure levels and was equivalent to an intake of 950 mg/day by a 70 kg-sized human (Takahashi et al. 2009).

Vitamin E consists of a group of eight structurally related compounds: α-, β-, γ- and δ-tocopherols (α-, β-, γ- and δ-T) and α-, β-, γ- and δ-tocotrienols (α-, β-, γ- and δ-TT) (Traber 2007). α-tocopherol is the isoform found at the highest concentration in serum and dietary supplements. Although both α- and γ-tocopherol are potent antioxidants, γ-tocopherol has a unique function due to its different chemical structure that scavenges reactive nitrogen species that damage proteins, lipids and DNA (Takahashi et al. 2009).

Overall, our results demonstrated that γ-tocopherol attenuated the incidence of epithelial dysplasia and inflammatory foci after MNU administration. Recently, the protective effect of γ-tocopherol against carcinogenesis has been confirmed in different organs, such as breast (Smolarek et al. 2013), colon (Guan et al. 2012) and lung (Lu et al. 2010). In the prostate, Yang et al. (2012) demonstrated that a γ-tocopherol-enriched diet was effective in preventing tumours in a xenograft model derived from human lung and prostate cancer cells and attributed the results to the antinitrative and anti-inflammatory activities of γ-tocopherol. Barve et al. (2009) also described that the antioxidant properties of a γ-tocopherol-enriched diet suppressed the incidence of palpable tumours and prostate intraepithelial neoplasia (PIN) in the TRAMP model, corroborating the protective effect of γ-tocopherol observed in our study.

Picrosirius staining demonstrated an increase in stromal collagen density after MNU administration, leading to stromal hyperplasia. γ-tocopherol decreased the collagen volume observed in the MNU group, but the values were still higher than in the CT and CT+γT groups. Because γ-tocopherol intake decreased the percentage of inflammatory cells in the stromal compartment, we believe that the stromal reaction to MNU administration was attenuated, leading to a decrease in collagen volume. It has been proposed that α-tocopherol treatment attenuates the progression of renal fibrosis in the obstructed kidney (Tasanarong et al. 2011). Moreover, Jiang et al. (2011) demonstrated that α-tocopherol reduces oxidative stress, ameliorating inflammation and fibrosis by downregulating the mRNA expression of collagen-α1(I), leading to the alleviation of inflammation and fibrosis in a rat model of chronic pancreatitis. Thus, the effects of γ-tocopherol in improving inflammation may have some implications for the reduction in collagen volume and stromal thickening in MNU-treated rat ventral prostate.

Because we observed a reduction in epithelial dysplasia after γ-tocopherol intake, we analysed the effects on epithelial cell proliferation and apoptosis. The disruption of the molecular mechanisms that regulate these two processes may underlie the abnormal growth of the gland, leading to the development of prostatic diseases (Kyprianou et al. 1996; Xie et al. 2000). Immunohistochemical analysis revealed a lower proliferation index in the MNU+γT group compared to the MNU group. Torricelli et al. (2013) have shown that the potential of γ-tocopherol for reducing cell proliferation was associated with reduced levels of cyclin D1 and cyclin E in the human prostate PC-3 tumour cell line. In the same way, Campbell et al. (2009) demonstrated that γ-tocopherol induced a 40% growth arrest in PC-3 cells by upregulating the expression of peroxisome proliferator-activated receptor (PPAR) γ, a nuclear receptor involved in fatty acid metabolism, cell proliferation and differentiation. The molecular pathway by which γ-tocopherol decreases cell proliferation in MNU-injected rats is not completely clarified as yet, but our results highlight the importance of γ-tocopherol as a protective agent against MNU-induced proliferative disorders in the rat prostate.

The effect of γ-tocopherol on the apoptotic index was also investigated. Our results demonstrated that there was no difference in the apoptotic index among all experimental groups. There is evidence that antioxidants such as γ-tocopherol are able to induce epithelial cell apoptosis in the prostate. Jiang et al. (2004) also demonstrated that γ-tocopherol (50 μM) is able to induce apoptosis in vitro by interrupting the de novo sphingolipid pathway in the LNCaP prostate cancer cell line without affecting normal human prostate epithelial cells. Gunawardena et al. (2000) showed that α-tocopherol (0.078–2.5 μg/ml) stimulated apoptosis in actively dividing LNCaP cells in vitro, but not in confluent quiescent cells. In another study, Lindshield et al. (2010) demonstrated that γ-tocopherol (200 mg/kg diet) did not alter proliferation or apoptosis rates in a model of Dunning R3327-H rat prostate adenocarcinomas in male transplanted Copenhagen rats. These discrepant results could be attributed to different methodologies (in vivo vs. in vitro, cell linage and vitamin dosage). Moreover, the results described by Jiang et al. (2004) and Gunawardena et al. (2000) suggested that malignant cells are more sensitive to γ-tocopherol than normal prostatic epithelial cells.

It is widely known that inflammation and oxidative stress may play an important role in the aetiology and progression of prostate disorders such as epithelial dysplasias and prostate cancer (Kim et al. 2011), highlighting the concept that chronic inflammation may represent an important therapeutic target in prostate carcinogenesis (Koul et al. 2010). Because we demonstrated that γ-tocopherol decreased the incidence of inflammatory foci in the ventral prostate, we decided to investigate the expression of Cox-2 and GST-pi. Cox-2, an inducible enzyme, belongs to a family of myeloperoxidases located on the luminal side of the endoplasmic reticulum and nuclear membrane that catalyses the rate-limiting step of prostaglandin biosynthesis from arachidonic acid and whose expression is stimulated by growth factors, cytokines, inflammatory processes and tumour promoters (Kutchera et al. 1996; Chandrasekharan & Simmons 2004). Here, we also demonstrated the potential of γ-tocopherol in reducing MNU-inducible Cox-2 overexpression in the rat ventral prostate. The overexpression of Cox-2 has been implicated in the progression of most types of cancer (Liu et al. 1998; Lee et al. 2002; Pruthi et al. 2003). Conversely, the protective effect of vitamin E on the inhibition of Cox-2 activity has been demonstrated for a variety of age-associated diseases, including cancer, arthritis and cardiovascular disease (Beharka et al. 2002). As such, Jiang et al. (2000) demonstrated that γ-tocopherol, but not α-tocopherol, inhibited Cox-2 activity in macrophages and epithelial cells in vitro, acting as a competitive inhibitor of its substrate, arachidonic acid. These authors also observed a reduction in nitrite accumulation and the suppression of inducible nitric oxide synthase expression, indicating that γ-tocopherol may be important in human disease protection. Although there are no data on the γ-tocopherol inhibition of Cox-2 during the development of prostatic lesions in vivo, Katkoori et al. (2013) demonstrated in vitro that Celecoxib, a clinically available Cox-2 inhibitor, reduced the growth of LNCaP and PC-3 prostate cancer cells. Thus, the reduction in Cox-2 immunostaining after γ-tocopherol intake observed in our study demonstrates the protective effect of this form of vitamin E against inflammatory induced-tissue damage in prostate.

Glutathione-S-transferases (GSTs) are a group of isoenzymes that catalyse intracellular detoxification reactions by conjugating glutathione with electrophilic compounds including carcinogens, natural toxins and exogenous drugs. It has been proposed that increased GST-pi expression at areas of inflammatory foci may result from the presence of an ongoing oxidative insult to the tissue (Parsons et al. 2001). In the normal prostate, GST-pi is expressed mainly in prostate epithelial basal cells (Cookson et al. 1997; De Marzo et al. 1999), with no expression detected in the secretory luminal cells. In contrast, prostatic lesions demonstrate both basal and luminal epithelial cells with elevated levels of GST-pi protein detection (De Marzo et al. 1999). Here, we demonstrated an increase in the basal cell expression of GST-pi in the MNU group, mainly in areas of epithelial dysplasia associated with increased epithelial cell hyperproliferation. In these areas, we observed positive reactions in both the basal and luminal cells. This pattern of expression supports the theory that cells located in a microenvironment of tissue injury are affected by an increase in oxidative stress. γ-tocopherol attenuated the expression of GST-pi in basal cells, as demonstrated by immunohistochemistry. Although there are no previous reports on the effects of γ-tocopherol on GST-pi expression in the rat ventral prostate, van Haaften et al. (2002) demonstrated that α-tocopherol is able to inhibit GST-pi expression in human placenta in vitro. Because tissue damage leads to an increase in GST-pi expression, we believe that the reduction in GST-pi after γ-tocopherol intake may represent a protective effect of γ-tocopherol on the ventral prostate, as demonstrated by a reduction in inflammatory foci, epithelial dysplasia and cell proliferation in the MNU+γT group.

Regardless of the MMP-9 activity, we demonstrated an increase in the pro-form of this enzyme in the MNU group. This result can be linked to the evident inflammatory response of the prostate after MNU injection. Because inflammatory cells secrete and accumulate the pro-MMP-9 enzyme in cytoplasmic granules, the presence of inflammatory foci may be responsible for the increase in pro-MMP-9 gelatinolytic activity in the MNU group (Opdenakker et al. 2001; Wilson et al. 2004). Because γ-tocopherol reduced the inflammatory response in MNU-treated animals, the reduction in pro-MMP-9 activity can be attributed to the anti-inflammatory properties of this tocopherol. In an earlier study, Zhang et al. (2004) demonstrated that RRR-alpha-tocopheryl succinate, one of the vitamin E derivatives, inhibits human prostate cancer cell invasion through a reduction in secreted MMP-9 activity, suggesting the potential use of this drug as a protective agent and therapy for prostate cancer invasion in association with conventional chemotherapy. Together, these results corroborate the protective effect of γ-tocopherol on MNU-induced prostatic disorders.

In conclusion, our results confirm that γ-tocopherol can be helpful as a protective compound against tissue damage induced by MNU in the rat ventral prostate in vivo, highlighting the possible use of this form of vitamin E as a protective agent and therapy.

Acknowledgments

This study was supported by Minas Gerais State Research Foundation (FAPEMIG), grants no APQ-01275-10.

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