The Prostate

Dietary Methionine Restriction Inhibits Prostatic Intraepithelial Neoplasia in TRAMP Mice Raghu Sinha,1* Timothy K. Cooper,2 Connie J. Rogers,3 Indu Sinha,1 William J. Turbitt,3 Ana Calcagnotto,4 Carmen E. Perrone,5 and John P. Richie Jr.4** 1

Department of Biochemistryand Molecular Biology, Pennsylvania State University College of Medicine, Hershey Medical Center, Hershey, Pennsylvania 2 Department of Comparative Medicine, Pennsylvania State University College of Medicine, Hershey Medical Center, Hershey, Pennsylvania 3 Department of Nutritional Sciences, Pennsylvania State University,University Park, Pennsylvania 4 Department of Public Health Sciences, Pennsylvania State University College of Medicine, Hershey Medical Center, Hershey, Pennsylvania 5 Orentreich Foundation for the Advancement of Science, Inc.,Cold Spring-on-Hudson, NewYork

BACKGROUND. Prostate cancer (PCa) is a major aging-related disease for which little progress has been made in developing preventive strategies. Over the past several years, methionine restriction (MR), the feeding of a diet low in methionine (Met), has been identified as an intervention which significantly extends lifespan and reduces the onset of chronic diseases, including cancer, in laboratory animals. We, therefore, hypothesized that MR may be an effective strategy for inhibiting PCa. METHODS. Control (0.86% Met) or MR (0.12% Met) diets were fed to 5-week old TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, a well-characterized model for PCa. The mice were sacrificed at 16 weeks of age and prostate and other tissues were harvested for histological and biochemical analyses. RESULTS. As previously reported, MR was associated with a decrease in body weight which was not associated with lowered food intake. MR led to significant reductions in the development of Prostatic Intraepithelial Neoplasia (PIN) lesions, specifically in the anterior and dorsal lobes of the prostate where the incidence of high-grade PIN was reduced by
50% (P < 0.02). The reduction in PIN severity was associated with 46–64% reductions in cell proliferation rates (P < 0.02) and plasma IGF-1 levels (P < 0.0001), which might, in part, explain the effects on carcinogenesis. Additionally, no adverse consequences of MR on immune function were observed in the TRAMP mice. CONCLUSIONS. Overall, these findings indicate that MR is associated with a reduction in prostate cancer development in the TRAMP model and supports the continued development of MR as a potential PCa prevention strategy. Prostate # 2014 Wiley Periodicals, Inc. KEY WORDS: methionine restriction; sulfur amino acids; prostatic intraepithelial neoplasia; prostate cancer; TRAMP

Grant sponsor: Intercollegiate Graduate Degree Program in Physiology at Pennsylvania State University. Disclosure: None. 

Correspondence to: Raghu Sinha, PhD, Penn State Hershey Cancer Institute CH76, Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17033. E-mail: [email protected]  Correspondence to: John P. Richie, Jr., PhD, Penn State Hershey

ß 2014 Wiley Periodicals, Inc.

Cancer Institute H069, Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17033. E-mail: [email protected] Received 5 June 2014; Accepted 5 August 2014 DOI 10.1002/pros.22884 Published online in Wiley Online Library (wileyonlinelibrary.com).

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Sinha et al. INTRODUCTION

Prostate cancer (PCa) represents a major public health problem among middle age and older men for which very little progress has been made in the development of strategies for its control or prevention. While the etiology of PCa remains elusive, aging is considered a major risk factor and studies suggest that cellular and molecular changes occurring during aging are likely to be involved in the promotion of PCa. Thus, a potentially effective approach for PCa prevention could be the utilization of anti-aging strategies that could also target PCa-related pathways. One such intervention is calorie restriction (CR), which over the last 70 years has been proven effective at delaying aging and the development of aging-related diseases including cancer [1]. CR is effective at inhibiting cancer development including PCa in a number of transgenic and carcinogen-induced models [2,3]. While CR can modulate metabolic parameters and growth factors important in cancer prevention (e.g., insulin, insulin like growth factor-1 [IGF-1]) [4], its mechanisms of action remain under investigation. Further, recent studies have indicated that CR may have a negative impact on immune function leading to increased susceptibility to specific pathogens [5,6]. These, together with problems associated with the translation of CR to human populations, have hampered the development of clinically relevant disease prevention strategies based on CR. Studies over the past several years have pointed to the importance of dietary methionine (Met) in the modulation of aging and aging-related diseases. In laboratory animals, diets low in Met dramatically increase longevity and reduce the onset of agingrelated impairments and chronic diseases [7–11], whereas, diets high in Met have been associated with signs of toxicity [12,13]. In addition to enhancements in lifespan, lifelong dietary Met restriction (MR), the feeding of a diet low in Met (20–35% of control) as the sole source of sulfur amino acids, has been shown to have numerous additional beneficial effects in both mice and rats including reductions in adiposity and blood levels of lipids, glucose, IGF-1, and leptin and increases in blood levels of adiponectin and FGF21 levels [9,14–18], reductions in oxidative stress and mitochondrial free radical production [19–22], and delayed onset of numerous aging impairments including reductions in immune function and lens turbidity [9]. MR was also effective at enhancing lifespan even when initiated in fully-grown adult mice suggesting that the anti-aging effect was not simply driven by a delay in growth and development [23]. As a result of these findings, MR has been recognized as an important new approach to study mechanisms of aging and The Prostate

aging related diseases [24]. In addition, due to the feasibility of feeding MR diets, this intervention may have important clinical implications in disease prevention. There is little known on the effects of MR on cancer development. In a previous study, we demonstrated that MR was effective at inhibiting the development of preneoplastic colonic lesions in a chemically-induced model of colon cancer [10]. For the current investigation, we hypothesized that MR could be an effective intervention to control PCa development and progression. To test this, we examined the effect of MR on lobe-specific prostate tumorigenesis, primarily in the Prostatic Intraepithelial Neoplasia (PIN) stage, in the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. The transgenic mice were generated by using the prostate-specific rat probasin promoter for driving expression of simian virus 40 (SV40) large T antigen-coding region [25]. TRAMP mice expressing high levels of the transgene exhibit progressive forms of prostatic disease that histologically resemble human prostate cancer. These lesions can range from low grade PIN to poorly differentiated carcinoma. The TRAMP model is a well-characterized model for PCa development, prevention and therapy studies. Because IGF-1 plays a crucial role in PCa progression [26,27] and levels of IGF-1 are significantly reduced by MR [9,28], the effects of MR on lobe-specific proliferation as well as plasma IGF-1 levels were examined. Finally, given the importance of inflammation and immune surveillance in the development and prevention of prostate cancer [29], we examined the effects of MR on a variety of splenic immune function markers. MATERIALS AND METHODS TRAMP Model and Diets TRAMP mice on the C57BL/6J background were purchased from The Jackson Laboratories (C57BL/ 6-Tg(TRAMP)8247Ng/J, stock number 003135), bred, and maintained under specific pathogen free conditions in the animal vivarium at the Penn State College of Medicine. Briefly, transgene positive TRAMP males were bred with female C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) to generate hemizygous offspring. After weaning (4 weeks of age), male pups were genotyped by PCR for transformation related protein 53 (Trp53) and SV40 large T antigen using DNA from a tail biopsy as described previously [30]. The genetically confirmed male TRAMP mice were randomized into control (0.86% Met; n ¼ 9) or MR (0.12% MR; n ¼ 11) groups at 5 weeks of age and fed the corresponding diets ad lib for 11 weeks. The level of Met in the MR diet was selected based on previous

Methionine Intake and Prostate Cancer studies in mice where feeding 0.12% Met diets resulted in numerous beneficial effects including enhanced lifespan [14,16,23]. The composition of the control and MR diets, described in detail in Supplemental Table SI, was based upon previous studies of MR in mice [16,23]. Body weights and food intake were measured weekly. The mice were sacrificed at 16 weeks of age and blood, prostates, spleens, liver, kidneys, and testes were removed and processed for biochemical, histopathologic, and/or immune analysis. Pathological Investigation of Prostates and Seminal Vesicles and Lesion Scoring System Pelvic organs, including prostate lobes (anterior prostate, AP; dorsal prostate, DP; ventral prostate, VP; lateral prostate, LP), seminal vesicle, and urinary bladder were trimmed, placed into cassettes and fixed in 10% buffered formalin. Additionally, sections of liver, kidney, and testes were fixed for histology to examine for possible signs of toxicity. The tissues were processed in an automated Tissue-Tek VIP processor and paraffin-embedded with a Tissue-Tek TEC embedding station. Sections (5 mm) were stained with hematoxylin and eosin (H & E) and analyzed by a board certified veterinary pathologist who was blinded to the treatment. The proliferative lesions in the prostate and seminal vesicle were scored according to the Berman–Booty scheme that includes a numerical scoring system that accounts for both the most severe and most common histopathological lesions in each of the lobes of the prostate and their distributions [31]. According to this revised scheme, each of the four lobes (dorsal, ventral, lateral, anterior) of the TRAMP prostates are assessed individually and assigned two grade scores each, ranging from 0 to 7. The lesion grades for the refined scheme are as follows. Grade 0 is normal prostate. Grades 1, 2, and 3 represent low-, moderate-, and high-grade PIN, respectively. Grade 4 includes phyllodes-like lesions. Grades 5 and 6 represent well and moderately differentiated adenocarcinomas, respectively. Grade 7 is poorly differentiated carcinoma, which may have neuroendocrine features. The first score represents the grade for the most severe lesion within that lobe, while the second score reports the grade for the most common or widespread lesion. By assessing both the most severe lesion and most common lesion, a more complete picture of disease status was obtained. In addition, by accounting for both of these lesions, the refined grading scheme is a close approximation of the Gleason system used to grade prostate cancer in men [32]. Adjusting the lesion grades to include an indication of distribution provides two adjusted scores, one for the most severe lesion and the other for the most common, ranging

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from 0 (normal) to 21 (diffuse, poorly differentiated carcinoma) [31]. Body Composition Measurements Prior to sacrifice, changes in body composition were tracked noninvasively in conscious control fed and MR TRAMP mice using a 1H-NMR analyzer (Bruker LF90 proton-NMR Minispec; Bruker Optics, Woodlands, TX) for rapid measurement of total body fluid, lean and adipose tissue mass according to the manufacturer’s instructions. Cell Proliferation (Immunohistochemistry for Ki67) The percent of Ki67 positive cells counted in triplicates in high power fields (400) was used as a measure of proliferating cells. Briefly, formalin-fixed prostate sections were hydrated, subjected to antigen retrieval and incubated with anti-Ki67 antibody (M7249, Dako North America Inc, Carpinteria, CA). Slides were developed with a Vector ELITE ABC rat kit (PK-6104, Burlingame, CA) and visualized with DAB chromagen followed by hematoxylin counterstain. IGF-1Assay IGF-1 levels were measured in plasma from controland MR-fed mice using an ELISA kit (Quantikine, R & D Systems, Minneapolis, MN) according to manufacturer’s instructions. IGF-1 levels were quantified in a SpectraMAX 384 Plus Microplate Reader (Molecular Devices, Sunnyvale, CA). Each assay was performed in duplicate. Immune Assays Isolation of immune cells. Spleens were harvested via gross dissection and single cell suspensions of splenocytes were prepared for each mouse by mechanical dispersion, as described previously [33]. Briefly, harvested spleens were mechanically disrupted with a syringe plunger and passed through a 70 mm nylon mesh strainer (BD Biosciences; Bedford, MA), erythrocytes were lysed with ACK lysing buffer (Lonza; Basel, Switzerland), the splenocytes were then washed twice in cold PBS, counted and their viability determined via trypan blue exclusion (Mediatech; Manassas, VA). Flow Cytometric Analyses Single cell suspensions of splenocytes were washed twice in PBS at 4°C and 1  106 cells were stained with saturating concentrations of conjugated anti-mouse The Prostate

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monoclonal antibodies (against CD19 PE (1D3), CD3 FITC (145-2C11), CD4 FITC (GK1.5), Gr-1 FITC (RB68C5), F4/80 FITC (BM8), CD3 PE (145-2C11), CD8 PE (53-6.7), NK1.1 PE (PK136), CD11b PE (M1/70), CD11c PE (HL3) and appropriate isotype control for Hamster IgG1 FITC (A19-3), Rat IgG2b FITC (RTK4530), Rat IgG2a PE (RTK2758), and Mouse IgG2a PE (MOPC173), obtained from BD Biosciences; Bedford, MA and Biolegend; San Diego, CA) for 30 min at 4°C, as described previously [33]. Following incubation with the conjugated antibodies, the cells were washed twice in PBS and fixed in 1% paraformaldehyde for flow cytometric analyses. Lymphoid and myeloid cells were gated on forward vs. side scatter, and a total of 20,000 events were analyzed on a Beckman Coulter FC500 flow cytometer (Beckman Coulter; Indianapolis, IN) [5]. Histograms of flow cytometric analyses were plotted and analyzed using Flow Jo software (Tree Star; Ashland, OR). Immune Cell Proliferation Assays As previously described [34], 2  105 splenocytes were incubated in flat-bottomed, 96-well plates (Greiner Bio-One; Monroe, NC) in the presence of increasing concentrations of lipopolysaccharide (LPS) (Sigma-Aldrich; St. Louis, MI) or increasing concentrations of anti-CD3 antibody (BD Biosciences; Bedford, MA) for 72 hr. Proliferation data was analyzed by the MTT assay (Promega; Madison, WI) and quantified on an Epoch Microplate Spectrophotometer (Biotek; Winooski, VT). Each assay was performed in triplicate. Cytokine Production Assays Supernatants were harvested after 4 hr of incubation with 10 mg/mL LPS and 48 hr incubation with 1 mg/mL anti-CD3 and stored at 80°C. Tumor necrosis factor-a (TNF-a) and interferon-g (IFN-g) were measured using Legend Max ELISA kits (Biolegend; San Diego, CA) according to the manufacturer’s instructions. Cytokines were quantified on an Epoch Microplate Spectrophotometer (Biotek; Winooski, VT). Each assay was performed in duplicate. Statistical Analysis Summary statistics are provided for the outcome measurements for different experimental conditions defined by diet groups, MR versus control fed, and over time. The effects of experimental conditions on body weight and biological measures were analyzed by ANOVA or Student’s t-test where appropriate. The impact of MR on lesion incidence was assessed using Fisher’s Exact test. The pathological grading score was The Prostate

analyzed using the Mann-Whitney U-test. All tests are two sided. All analyses were done in SAS version 9.2 or GraphPad Prism 5. RESULTS MR Effects on Body Composition, Body and OrganWeights, and Food Intake As observed previously [7,16,23], body weight was significantly reduced by MR, without a reduction in food intake (Fig. 1A). No differences in diet consumption per mouse was observed throughout the experiment and food intake on a per g body weight basis was slightly greater in MR mice, and this trend became significant after 3 weeks on the diets. At sacrifice, most organs and tissues in MR mice weighed significantly less than those in control mice, except for the spleen (Fig. 1B). The weights of urogenital organs (urinary bladder, 4 mm of proximal urethra, seminal vesicles, anterior, ventral, dorsal and lateral prostate lobes, ampullary glands,
5 mm of both vas deferens,
5 mm of both ureters) and adipose tissues were significantly reduced by MR when expressed as a unit of body weight. Nuclear magnetic resonance in live mice revealed significant reductions in fat content, lean mass and fluid volume in MR as compared to control fed mice (Fig. 1C). Diets devoid in Met have been associated with signs of hepatic and renal toxicity including the development of steatosis and hepatocellular carcinoma [35,36]. While we do not expect that the MR diet used here would have similar effects, we conducted a comprehensive histopathologic analysis of numerous organs and tissues including liver, kidney, testis, and epididymis from TRAMP mice fed the control or MR diets to confirm the lack of a toxic effect. After 11 weeks, no signs of toxicity or abnormal pathology were observed and sperm production was not affected. Micrographs of representative H & E stained liver, kidney, testis, and epididymis sections from control and MR TRAMP mice are shown in Supplemental Figure 1.

MR Effects on Prostate Lesion Formation Preneoplastic lesions categorized as low-grade, moderate-grade and high-grade PIN were observed in all lobes of the prostate in the 16 week old TRAMP mice. Representative images of PIN lesions in different prostate lobes from control and MR mice are shown in Figure 2A. In order to describe and compare the changes in lesions, we utilized a recently described scoring system which takes into account both the severity of the lesions (Fig. 2A, 1st digit in score) and the prevalence of the most common lesions (Fig. 2A,

Methionine Intake and Prostate Cancer

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Fig. 1. Effects of methionine restriction on body and organ weights, food intake and body composition in TRAMP mice. TRAMP mice were fed control (n ¼ 9) or MR (n ¼11) diets beginning at 5 weeks of age.Body weights and food intake were measured weekly (A).Mice were sacrificed after11weeks and organ weights were measured (B). Prior to sacrifice1H-NMR analyses were performed on live animals (C). In A and C, error barsreflect SD values.

2nd digit in score) in each prostate lobe [31]. Particular attention was given to the anterior and dorsal lobes, as these are thought to be the primary sites for PCa development. Consistent with this, we found that the most severe lesions in both control and MR mice were observed in these two lobes (Fig. 2B, upper panel). The incidence profiles for the most severe and most common lesions were examined in a lobe specific manner by diet group (Fig. 2B). Lesion data were also examined using the above described grading system (Table I). Lesion incidence in control mice is consistent with that observed previously [31], with high grade PIN being most prevalent in the anterior prostate (AP) followed by the dorsal prostate (DP). MR reduced the incidence of high grade PIN on both lobes by 40–50% (Fig. 2B, upper panel) and in the severity scores of lesions in the AP and DP lobes by 42% and 22%, respectively (P < 0.02) (Table I). While the majority of the anterior lobe tissue was normal in both MR and control mice, there was a clear shift in the most common PIN lesions in the dorsal lobe from moderate grade to lower grade PIN (Fig. 2B, lower panel) and a

35% reduction in the most common lesion score, although this was not statistically significant (Table I). No MR effects were observed in other prostate lobes. The seminal vesicles in MR mice however, showed a significant decrease in the lesion severity scores (P < 0.05) (data not shown). In addition, there were three tumors observed in the TRAMP mice, two in the control group, which were traceable to the ventral prostate (VP), and one in the MR group, which was traceable to AP. These were reported as grade 19 (adjusted score) neuroendocrine tumors. About 20% incidence of neuroendocrine tumors is expected within the lifetime of C57BL/6J background TRAMP mice [37]. MR Effects on Prostate Cell Proliferation The effects of MR on prostate cell proliferation rates were examined by immunohistochemical analysis of Ki67 positive cells (Fig. 3). Significant reductions in cell proliferation of 64% and 46% were observed in both AP and DP, respectively, from MR mice as compared to control-fed mice (P < 0.02). The Prostate

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Fig. 2. Effect of methionine restriction on prostate lesions in TRAMP mice. TRAMP mice were fed control (n ¼ 9) or MR (n ¼11) diets beginning at 5 weeks of age and prostate lesions were assessed after11weeks. (A) Representative images of PIN lesions in different prostate lobes from control-fed and MR mice along with their associated lesions scores as described in Materials and Methods. (B) Percent of animals bearing PIN lesionsbylesion severity (upper panel) andbymostcommon lesion (lower panel) in differentlobes of theprostate.

MR Effects on IGF-1Levels IGF-1 levels in plasma samples from MR-fed 16 weeks old TRAMP mice (mean  SD: 288  36.8 ng/ml) were significantly reduced by
50% when compared to control-fed mice (145  40.0 ng/ml) (P < 0.0001).

MR Effects on Immune Cell Distribution and Function Although MR did not significantly reduce spleen weight (Fig. 1B), the total splenocyte number was significantly reduced in MR mice (Table II; P ¼ 0.003),

TABLE I. Effect of MR on PINLesion Scores in Different Prostate Lobes of TRAMP Mice Most severe lesion scorea

Most common lesion scorea

Lobe

Control

MR

AP VP LP DP

7.8  0.4 3.8  1.3 4.4  1.3 7.6  1.1

4.6  3.1 2.8  0.9 4.0  1.3 5.9  1.8b

Values are mean  SD (n ¼ 9–11 per group). Significantly different from control, P < 0.02.

a

b

The Prostate

b

Combined lesion scorea

Control

MR

Control

MR

0.4  0.9 3.0  1.6 3.9  1.5 5.0  2.6

0.2  0.6 1.8  1.5 2.9  1.4 3.3  2.8

8.2  0.8 6.8  2.6 8.3  2.4 12.6  3.1

4.7  3.1b 4.6  2.2 7.0  2.8 9.2  4.3b

Methionine Intake and Prostate Cancer

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counts than control mice. In contrast, when the spleens of mice were analyzed for the distribution of immune cell subsets, few differences were detected among MR and control mice. MR increased the percentage of total T cells (CD3þ; P ¼ 0.007), and this increase in CD3þ cells was due to a significant increase in the percentage of CD4þ helper T cells (P ¼ 0.018). However, overall there were little differences in the proportion of immune subsets in MR and control mice. Despite a reduction in splenocyte number, MR did not significantly reduce the proliferation of T (Fig. 4A) or B cells (Fig. 4B) in response to anti-CD3 or LPS stimulation, respectively. MR also did not significantly reduce cytokine production, specifically IFN-g (Fig. 4C) or TNF-a (Fig. 4D) production in response to anti-CD3 or LPS stimulation.

Fig. 3. Effect of methionine restriction on cell proliferation in prostate tissues fromTRAMP mice.TRAMP mice were fed control (n ¼ 9) or MR (n ¼11) diets beginning at 5 weeks of age. After 11weeks mice were sacrificed, prostate tissues were removed, and Ki67 positive cells were detected by immunohistochemistry as describedin Materials and Methods.Error barsreflect SD values.

DISCUSSION MR has been identified as a novel dietary manipulation for enhanced longevity and disease prevention in laboratory animals [7,9,24]. Based upon both in vitro and in vivo findings, Met reduction has been proposed as an important anti-cancer strategy [38] and we previously observed that MR significantly inhibited chemically-induced colon carcinogenesis in the rat [10]. Based on these previous findings, we hypothesized that MR could inhibit prostate carcinogenesis in the TRAMP mouse model. Our findings support this hypothesis and indicate that dietary MR reduces the severity of PIN lesions as compared to the control fed diet. The most significant effects were observed in the anterior and dorsal lobes of the prostate where the most severe lesions were located. These are also the lobes primarily involved in the development of prostate cancer in this model [31]. The inhibitory effect of MR on PIN lesion development in this highly aggressive TRAMP model reflects

and was proportional to the reduction in body weight observed in MR mice. To further characterize the effects of MR on immune endpoints, we explored the absolute number and the percentage of leukocyte subsets in the spleen among MR and control mice (Table II). MR reduced the total number of B cells (CD19þ; P ¼ 0.011) and T cells (CD3þ; P ¼ 0.038). Within the T-cell compartment, MR significantly reduced the number of cytolytic T cells (CD3þCD8þ; P ¼ 0.026) and helper T cells (CD3þCD4þ); however, the latter effect did not reach statistical significance. MR also significantly reduced the number of natural killer (NK) cells (NK1.1þ; P ¼ 0.011) and granulocytes (Gr-1þ; P ¼ 0.001) in the spleen. Thus, the reduction in splenocyte number was due to a reduction in all leukocyte subtypes, and was due to the fact the MR mice weighed less and had lower total splenocyte

TABLE II. Effect of MR on the Percentage and Number of Leukocytes in the Spleen of TRAMP Mice

Splenocytes (106) Immune Cells B cells (CD19þ) Total T cells (CD3þ) Helper T cells (CD3þ/CD4þ) Cytolytic T cells (CD3þ/CD8þ) NK cells (NK1.1þ) Granulocytes (Gr-1þ) Macrophages (CD11bþ)

Controla

MRa

65.04  14.81

43.33  8.57b

Percentage (%)

Number (106)

Percentage (%)

Number (106)

32.17  6.27 50.97  5.59 28.57  4.49 23.60  4.18 11.69  3.51 12.16  1.48 8.35  3.96

22.29  5.05 35.74  8.87 19.85  4.71 16.50  4.05 8.07  2.61 8.43  1.68 5.84  2.98

26.24  6.20 61.20  4.40b 35.42  2.32b 26.06  3.15 9.95  1.82 11.47  1.58 6.42  3.73

12.70  4.99b 28.42  2.87b 16.48  1.90 12.04  1.21b 4.67  1.11b 5.33  0.82b 3.12  2.08

Values are mean  SD (n ¼ 9–11 per group). Significantly different from control, P < 0.05.

a

b

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Fig. 4. Effect of methionine restriction on leukocyte proliferation and cytokine production. TRAMP mice were fed control (n ¼ 9) or MR (n ¼11) diets beginning at 5 weeks of age. After 11weeks, mice were sacrificed and spleens removed.Resulting splenocytes (2  105/well) were incubatedin the presence of increasing concentrations of anti-CD3 (A) or LPS (B) for 72 hr and analyzed by MTT assay. IFN-g in response to anti-CD3 stimulation (C) and TNF-a in response to LPS stimulation (D) was quantified as detailed in Materials and Methods and are shown as mean  SEM for each group (n ¼ 9^11per group).

favorably upon the potential use of MR in the prevention of prostate cancer. The use of the new PIN and prostate tumor grading system allowed for the quantitative assessment of lesion development based on both lesion severity and most common lesion distribution in a lobe-specific manner. Our results suggest that MR significantly inhibited the progression of severe PIN lesions in the anterior and dorsal prostate lobes. The TRAMP model has been used to examine the efficacy of several chemopreventive agents including green tea polyphenols, NSAIDS, vitamin E analog, epigallocatechin-3-gallate and genistein [39], as well as dietary restriction [40,41] and an energy restriction mimetic [42]. Most of these agents were effective when administered at earlier stages (5–16 weeks of age) of the prostate disease. A 20% dietary restriction for 13 weeks showed about 10% and 22% reductions in combined adjusted lesion scores in AP and DP lobes, respectively [40]. By contrast, in our study, the feeding of MR diet for 11 weeks was associated with a better inhibition in combined score for the AP and similar for DP lobes as compared to CR. The mechanism by which MR inhibits the development of PIN lesions in this model is not known, but The Prostate

may involve an inhibition of cell proliferation. Results of Ki67 immunohistochemistry analyses suggest that rates of proliferation are reduced by approximately 50% specifically in those prostate lobes showing inhibition of PIN formation by MR. However, it is also possible that a reduction in proliferation rates may simply be a reflection of the reduction in lesion severity. As observed previously in both rats and mice, MR was associated with significant decreases in serum levels of IGF-1 in the TRAMP mice [23,28]. This finding may indicate that MR is having a significant inhibitory effect on the insulin/IGF-1 axis known to be important in the development of prostate cancer. Indeed, similar reductions in cell proliferation and IGF-1 levels were observed following dietary restriction [41] and energy-restriction mimetic [42] in TRAMP mice. The prostate produces massive amounts of polyamines for export in the reproductive fluids [43]. In vitro and in vivo studies suggest that inhibition of polyamine synthesis inhibits the progression of prostate cancer. Considering the major role played by Met in polyamine synthesis via the methyl donor, S-adenosylmethionine, a possible mechanism for MR inhibiting development of PIN lesions may be due to reduced polyamine synthesis and will be explored in future studies in TRAMP model. MR reduced the total number of cells in the spleen, which was comprised of a reduction in all major subtypes of cells including T and B cells, NK cells and granulocytes. Similar results were observed in an aging mouse model, where MR reduced the accumulation of T cell subsets in the blood [9], suggesting that MR may be influencing hematopoiesis and/or the accumulation of immune cells in secondary lymphoid organs in proportion to the effects of MR on body weight. Despite changes in the number of splenocytes, subset proportions in the spleen, and their functional capacity on a per cell basis were not significantly different between the control and MR mice. There was no effect of MR on the proliferative capacity of T or B cells, both of which are important components of adaptive immune responses. These results are surprising given the important role of Met in protein synthesis, a necessary cellular event preceding lymphocyte proliferation. Protein turnover may be occurring in MR animals, thus releasing endogenous Met, which may be preferentially utilized by immune cells to maintain their function in the face of dietary MR. Alternatively, in vitro assay conditions may have normalized any endogenous differences in T and B cell proliferation between control and MR animals during the proliferation assays. Additional studies are needed to explore the role of MR on T and B cell responses to immunologic challenges (e.g., vaccination) to determine the functional capabilities of these cells in vivo.

Methionine Intake and Prostate Cancer Although there was a trend for MR animals to have reduced cytokine production, MR did not significantly reduce the production of IFN-g in response to anti-CD3 stimulation and TNF-a in response to LPS stimulation. Cysteine that can be produced through the metabolism of Met has been shown to modulate the activity of NF-kB [44,45]. NF-kB can induce the expression of many genes and activate many pathways important in the inflammatory response [46]. Thus, Met may indirectly impact the cytokine secretion in response to inflammatory stimuli via modulation of the NF-kB pathway. Although there is a theoretical role of sulfur amino acids in cytokine secretion, studies demonstrating the role of Met and cysteine on NF-kB activation or other signaling pathways important for cytokine release (e.g., ERK [Extracellular Signal Regulated Kinase], JNK [c-Jun N-terminal Kinase]), and NFAT (Nuclear Factor of Activated T-Cells) are needed. The inhibitory effects of MR on PCa development are similar to those observed with CR, which has previously been found to inhibit prostate cancer development in numerous models including the TRAMP mouse [2]. Despite the similarity of effects including reductions in growth and inhibition of carcinogenesis, it is important to note MR was not associated with a reduction in energy intake, consistent with results from previous studies [7,24,28]. Food intake data suggest that there are no major differences in caloric intake between control and MR diet groups, regardless of whether intake is expressed on a per mouse or on a per gram body weight basis. Another important difference between MR and CR is their impact on immune function. Previously, CR was found to have a negative impact on immune function whereas we have observed that although MR is associated with a decrease in the overall number of splenic leukocytes, there were no observed effects on splenic immune function on a per cell basis. Thus, MR may be an effective therapy to reduce the onset of prostate lesions while maintaining adequate T-cell proliferation and cytokine production, which is an important component of anti-tumor immunity [47]. Previous studies have demonstrated that diets devoid in Met are associated with signs of hepatic and renal toxicity including the development of steatosis and hepatocellular carcinoma [35,36]. While MR has been associated with reductions in growth of mice and rats, no signs of toxicity or nutritional deficiency have been observed. Indeed, MR animals appear to be healthier and have a longer life span [7–9,24]. The reductions in growth are more likely a direct result of adaptive changes in energetic pathways and overall activity levels rather than to any specific toxic event [48]. It should be noted that which reductions in body weight have been observed in this and other studies in young

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and growing animals, this is primarily due to a reduction in growth. When MR was initiated in adult, fully grown rats, no reductions in body weight were observed, while free radical production and oxidative stress biomarkers were significantly reduced [20]. In the present study, we also elected to examine for potential signs of toxicity to ensure that our experimental diets were not deficient in Met levels. To this end, we conducted a comprehensive pathologic analysis of liver, kidney and testes after 11 weeks of MR and no signs of toxicity or abnormal pathology were observed. CONCLUSIONS Altogether, these data suggest that MR is protective against PCa development in the TRAMP model. These encouraging results in an aggressive cancer model warrant further investigation of MR in a more aging relevant prostate cancer model such as the Lobund– Wistar model [49] prior to testing in humans. The potential translation of MR diets for disease prevention in humans is promising, particularly since it is not associated with a reduction in caloric intake. In the clinical studies that have been performed to date, the feeding of reduced methionine diets was found to be both safe and feasible for potential use in the prevention or treatment of aging related diseases [50,51]. Further, vegan diets that are consumed by many individuals are naturally low in Met as they are based on vegetable-derived proteins, which are relatively poor sources of Met [52]. This supports the possibility of designing Met-restricted human diets which are based vegan food sources for use in the prevention of aging-associated diseases including prostate cancer. ACKNOWLEDGEMENTS The authors are thankful to Dr. Todd Schell for providing genotyped TRAMP male mice. The authors also thank Weifang Lin for processing and sectioning tissue blocks for histopathological evaluation, and Qing Zhong for immunohistochemical staining of prostate tissue sections. Authors would like to thank Dr. Samina Alam for transporting spleens to Dr. Rogers’s laboratory at University Park Campus. William J Turbitt is supported by the Intercollegiate Graduate Degree Program in Physiology at Pennsylvania State University. REFERENCES 1. Omodei D, Fontana L. Calorie restriction and prevention of ageassociated chronic disease. FEBS lett 2011;585:1537–1542. 2. Bonorden MJ, Rogozina OP, Kluczny CM, Grossmann ME, Grambsch PL, Grande JP, Perkins S, Lokshin A, Cleary MP. The Prostate

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