Affiliations of authors: P. Mukherjee, A. V. Sotnikov,Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; H. J. Mangian, W. J. Visek, Division of Nutritional Sciences, College of Medicine, University of Illinois, Urbana; J.-R. Zhou, Department of Surgery, Beth Israel-Deaconess Medical Center, Boston; S. K. Clinton, The Arthur G. James Cancer Hospital and Research Institute, The Ohio State University, Columbus.
Correspondence to: Steven K. Clinton, M.D., Ph.D., B402 Starling-Loving Hall, 320 West 10th St., The Ohio State University, Columbus, OH 43210.
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ABSTRACT |
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INTRODUCTION |
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We have focused our present investigations on the role of energy intake in rodent prostate carcinogenesis for several reasons. Increasing rates of obesity, reflecting a disequilibrium of energy balance, are characteristic of nations where prostate cancer rates are high (9-12). The relationships between prostate cancer and energy intake, physical activity, anthropometrics, and the many genetic and exogenous factors that modulate their interactions are just beginning to be investigated (4,5,13-15). In parallel with the increasing obesity seen in the U.S. population (16), laboratory rodents used in carcinogenesis studies have been selected in recent decades by commercial suppliers to grow faster and reach mature weight more quickly for economic reasons (17). Typical laboratory housing for rodents used in cancer studies provides limited physical activity and unrestricted access to food (ad libitum). These circumstances promote adiposity, higher risk of spontaneous cancer, diminished life span, and increased sensitivity to known carcinogens (17). The role of energy balance in prostate carcinogenesis thus far has received limited attention in laboratory models (8).
Laboratory animal models encompassing the diverse characteristics of human prostate tumorigenesis do not exist. We have selected two prostate tumor models that reflect several specific and relevant features for our investigations: the Dunning R3327-H adenocarcinoma in rats and the LNCaP human carcinoma in severe combined immunodeficient (SCID) mice. The androgen-dependent Dunning R3327-H transplantable prostate adenocarcinoma was originally derived from a spontaneous lesion in a Copenhagen rat. The tumor is composed of ducts and numerous proliferative acini, similar to those of moderately differentiated human prostate carcinoma (18). The slow-growing R3327-H tumor is very sensitive to androgens (19,20), as is true for most newly diagnosed human prostate cancers. We chose to substantiate our findings from the R3327-H model with the androgen-sensitive, poorly differentiated LNCaP human prostate carcinoma grown as xenografts in SCID mice (21). In addition to exploring the dose-dependent effects of diet restriction on tumor growth, we compared the growth of prostate tumors when energy restriction was accomplished by total diet restriction or the withdrawal of equivalent amounts of energy as carbohydrate or lipid.
The characterization of biomarkers sensitive to dietary interventions is critical for planning intervention studies designed to test dietary hypotheses concerning prostate cancer prevention and as an adjuvant to other therapeutic interventions. We hypothesize that energy restriction acts on hormonal, growth factor, and cytokine networks mediating interactions between the growing tumor and the vasculature. Folkman and colleagues (22) have proposed that progressive tumor growth requires angiogenesis, a process that ensures continued delivery of oxygen and nutrients and the removal of metabolic waste products. Furthermore, they (22-24) have suggested that tumor cell proliferation rates and apoptosis are inversely associated and intimately interrelated with the efficiency of the tumor vasculature. We therefore explored the effects of energy restriction on prostate tumor growth and the interrelationships between tumor proliferation, apoptosis, and angiogenesis. Although a growing array of mediators is being characterized as modulators of angiogenesis, we have focused on circulating insulin-like growth factor-I (IGF-I) (25-28) and tissue vascular endothelial growth factor (VEGF) (29-33) as potential mediators of dietary effects on prostate tumor growth.
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MATERIALS AND METHODS |
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Male Fisher x Copenhagen F1 rats were provided by Dr. Norman Altman of the Papanicolau Cancer Institute, University of Miami (Miami, FL). They were housed in individual stainless-steel, wire-bottomed cages in rooms maintained at 22 °C ± 1 °C with 14 hours of fluorescent lighting per 24-hour period. Male SCID mice (Taconic, Germantown, MD) at 10 weeks of age were maintained in plastic shoebox cages with autoclaved bedding and filtered air with 12 hours of darkness daily at an ambient air temperature of 22 °C ± 2 °C. The care and use of the laboratory animals followed guidelines set forth by the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals (revised September 1986). All animal procedures were reviewed and approved by the Dana-Farber Cancer Institute Animal Care and Use Committee or the University of Illinois Laboratory Animal Care Advisory Committee.
Prostate Adenocarcinoma Tumor Cell Lines
The Dunning R3327-H adenocarcinoma was transported from the Papanicolaou Cancer Research Institute as bilateral subcutaneous implants in the flank donor rats. The tumors were allowed to reach 2.5 cm in diameter and were harvested by sterile techniques. Tumor slices approximately 6 x 5 x 2 mm and weighing 73 mg ± 3 mg (mean ± standard deviation [SD]) were prepared. The slices were transplanted to bilateral subcutaneous sites in the flanks of the anesthetized recipient rats that were then randomly assigned to experimental groups. When the most rapidly growing tumors reached diameters of approximately 2.5 cm, the experiments were terminated, and the tumor tissue was harvested for histopathologic studies.
The LNCaP human prostate adenocarcinoma cell line obtained from the American Type Culture Collection (Manassas, VA) was maintained at 37 °C and in 5% CO2 with Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, penicillin (60 000 U/L), streptomycin (60 mg/L), and L-glutamine (2.4 mM). All cell culture materials were purchased from Sigma Chemical Co. (St. Louis, MO). LNCaP tumors were initially established in SCID mice by subcutaneous inoculation of 2 x 106 LNCaP cells into the mice, and they were subsequently maintained in the laboratory by serial transplantation. LNCaP tumors, approximately 1 cm in diameter, from donor mice were harvested under sterile conditions and dissociated with a Teflon homogenizer in sterile phosphate-buffered saline (PBS). Cells were washed, counted, and immediately inoculated into recipient mice at 2 x 106 viable cells (viability determined by trypan blue dye exclusion) in 0.1 mL of sterile PBS. Mice were then randomly assigned to dietary treatments. The study was terminated after 35 days, and the tumor tissue was harvested for histopathologic evaluation.
Diet Restriction and Dunning R3327-H Tumorigenesis in Rats
Rats were randomly assigned (10 per group) to one of the following four treatment groups: 1) ad libitum fed, hormonally intact, 2) total diet restriction of 20%, 3) total diet restriction of 40%, or 4) ad libitum fed and castrated at the time of tumor transplantation. All rats were fed the AIN76A diet (Research Diets, Inc., New Brunswick, NJ) (34,35). Total diet restriction is technically easy and maintains a constant ratio of nutrients to energy in all treatment groups. Diets were stored at 4 °C, and fresh food was provided daily. Dietary treatments began on the day after tumor transplantation and were continued throughout the experiment. All animals had continual access to distilled water. The average daily feed intake for the ad libitum fed rats was determined for each 7-day period, and the two restricted groups were provided 80% (20% restriction) and 60% (40% restriction) of that quantity on a daily basis. Calipers were used to quantify largest tumor diameter on a weekly basis. At 16 weeks after transplantation, all rats were killed and their tumors were weighed. Samples of each tumor were fixed in formalin for histologic evaluation.
Energy Restriction Versus Diet Restriction and Dunning R3327-H Tumorigenesis in Rats
The inhibition of tumor growth when total diet is restricted may be due to reduced energy
intake and may be accentuated by the lower intake of essential amino acids, lipids, or certain
vitamins and minerals. In contrast, restriction of energy alone by selective removal of fat or
carbohydrate calories from the diet allowed ad libitum fed controls and the restricted
animals to consume the same amounts of protein, vitamins, and minerals. In this experiment, rats
were randomly assigned (eight per group) to one of five treatment groups: 1) ad libitum
fed controls, 2) rats subjected to restriction of energy intake by 30% from lipid only, 3)
rats subjected to restriction of energy intake by 30% from carbohydrate only, 4) rats
subjected to total diet restriction of 30%, or 5) ad libitum fed rats castrated at the
time of tumor transplantation. Diets for studies comparing differing methods of restriction (Table
1) are based on the American Institute of Nutrition recommendations and
were prepared according to our formulations by Research Diets, Inc. (New Brunswick, NJ). At
16 weeks after transplantation, all rats were killed and their tumors were weighed. Samples of
each tumor were fixed in formalin for histologic evaluation.
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Adult male SCID mice (eight per group) were subcutaneously inoculated with tumor cells and were randomly assigned to one of the following four treatment groups: 1) ad libitum fed controls, 2) mice whose energy intake was restricted by 30% from lipid only, 3) mice whose energy intake was restricted by 30% from carbohydrate only, or 4) mice whose total diet was restricted by 30%. At 4 weeks after inoculation, all mice were killed and their tumors were weighed. Samples of each tumor were fixed in formalin for histologic evaluation.
Histology and Morphometry
Formalin-fixed tumor specimens were embedded in paraffin and sectioned at 5 µm thickness. Hematoxylin-eosin-stained slides were examined by light microscopy (Olympus BHTU, Tokyo, Japan) for characterization of tumor morphology and cytologic features. Low-power images of whole-tumor sections were digitized at 2700 dots per inch by use of a 35-mm scanner (Polaroid SprintScan 35; Polaroid Corp., Cambridge, MA) and PathScan Enabler (Meyer Instruments, Houston, TX). The digitized images were used to compute tumor density, a value representing the extent to which the tissue elements are compact. We determined tissue density by measuring total tumor area and quantitating the proportion (expressed as %) occupied by cells and compact matrix while digitally removing lumina of glands, ducts, blood vessels, and stromal elements composed of loose extracellular matrix or fluid (nonstaining areas). Many blood vessels were readily identifiable on the hematoxylin-eosin-stained sections because of the presence of eosin-stained red blood cells. We utilized this characteristic to compute indirectly the vascular density determined by measuring the area containing red blood cells to the total area of the tumor. These studies were completed by use of image analysis software (KS400; Kontron Elektronik, Munich, Germany).
Factor VIII Staining and Microvessel Quantitation
After deparaffinization, rehydration, and washing, sections were incubated with trypsin at 37 °C for 30 minutes, quenched with 0.3% H2O2-methanol for 30 minutes, and blocked with 10% normal goat serum in buffer (100 mL 0.01 M phosphate buffer and 0.9% sodium chloride [pH 7.4], with 1.0 g bovine serum albumin and 0.1 mL Tween 20 [PBA]). The sections were treated with a rabbit polyclonal antibody directed against human factor VIII-related antigen (Dako Corp., Carpinteria, CA; 1 : 100 dilution with PBA), followed by a biotinylated anti-rabbit immunoglobulin G (IgG) at 1 : 100 dilution (Vector Laboratories, Inc., Burlingame, CA). The sections were then treated with avidin-biotin complex followed by 3-3' diaminobenzidine as substrate for staining according to the manufacturer's directions (Vectastain Elite ABC Kit; Vector Laboratories, Inc.). The sections were then rinsed three times between all steps with PBS (100 mL 0.01 M phosphate buffer and 0.9% sodium chloride). Sections were counterstained with methyl green and mounted. We calculated microvessel density by counting microvessels on 200x fields under light microscopy at three representative sites without necrosis of each section. Branching structures were counted as a single vessel. Slides were evaluated without knowledge of treatment group.
Proliferation Index
To determine the proliferation index, we calculated the fraction of cells with proliferating cell nuclear antigen (PCNA) staining. After deparaffinization, rehydration, and washing, sections were soaked in 10 mM citrate buffer (pH 6.0), heated in a microwave oven, and cooled to room temperature to unmask the PCNA. Sections were then stained by following the procedures that were described for factor VIII staining; we used 10% horse serum for blocking and a PCNA mouse monoclonal antibody (Dako Corp.) as the primary antibody. Both PCNA-positive proliferating cells and total tumor cells were counted in three non-necrotic areas of each section by use of light microscopy at a 400-fold magnification.
In Situ Apoptotic Cell Detection
To detect apoptotic cells, we used the ApopTag in situ detection kit (Oncor, Inc., Gaithersburg, MD) and followed the manufacture's procedures with minor modifications (36); this procedure is also known as terminal deoxynucleotidyltransferase (TdT)-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL). In brief, after deparaffinization, rehydration, and washing in PBS, sections were treated with 20 µg/mL of proteinase K for 15 minutes at room temperature and washed again. Endogenous peroxidase activities in sections were quenched with 3% H2O2 in PBS for 5 minutes. The 3' hydroxy DNA strand breaks were enzymatically labeled with digoxygenin nucleotide via TdT and incubated for 1 hour at 37 °C, and the reaction was terminated with a stop buffer according to the manufacturer's directions (Oncor, Inc.). Sections were then incubated with anti-digoxygenin peroxidase for 30 minutes at room temperature, washed, stained with 3-3' diaminobenzidine substrate, counterstained with methyl green, and mounted. Positive and negative control slides were used for comparison. Substitution of TdT with distilled water served as a negative control. Three representative areas lacking necrosis were selected for each slide without knowledge of the treatment group, and both apoptotic cells and tumor cells were counted by use of light microscopy at a 400-fold magnification. The apoptotic index (AI) was expressed as percentage, AI (%) = A x 100/(A + C), where A = apoptotic cells and C = counterstained, unlabeled cells.
VEGF Immunohistochemistry
After deparaffinization, rehydration, and washing, slides were immersed in 0.01 M citrate buffer (pH 6.0), heated in a microwave oven to boiling for 5-6 minutes, and cooled in buffer at room temperature. After quenching was performed, the following steps were taken to determine VEGF immunoreactivity: (a) The sections were first blocked with 10% normal horse serum in PBA for 30 minutes; (b) the sections were treated with rabbit polyclonal VEGF antiserum (Calbiochem, La Jolla, CA) as primary antibody at a 1 : 20 dilution in blocking solution overnight at 4 °C; (c) the sections were then treated with biotinylated anti-rabbit IgG at a 1 : 100 dilution (Vectastain Elite ABC Kit) for 30 minutes; (d) the sections were treated for 30 minutes with avidin-biotin complex following the manufacturer's instruction (Vector Laboratories, Inc.); (e) VEGF immunoreactivity was then visualized by 3-3' diaminobenzidine tetrahydrochloride (Vector Laboratories, Inc.); and (f) after being washed with water, sections were counterstained with hematoxylin for 30 seconds. After dehydration and clearing in xylene, the slides were mounted. Unless otherwise stated, all incubations were done at room temperature and PBS washes were completed between each step.
We reviewed and digitized immunostained sections without knowledge of the treatment group. After linearization and 1-hour warm-up of the imaging workstation (Roche Pathology Workstation, RIAS, Elon College, SC), six high-power fields per tissue section were acquired by use of a digital camera (ProgRes 3012; Kontron Elektronics) and the light microscope (Olympus BHTU). Each high-power field was shadow corrected and minimally processed to eliminate compromised areas that could bias the analysis, such as artifacts generated during processing or staining. True-color image analysis was utilized to segment VEGF-labeled cells and counterstained cells for calculating labeling index based on the following formula: labeling index (%) = L x 100/(L + C), where L = labeled cells and C = counterstained, unlabeled cells. Nonspecific antibody staining in the lumina of tumor glands was digitally removed for preparation of representative figures.
IGF-I Analysis
Blood was collected into EDTA-coated tubes from the orbital venous plexus of individual mice anesthetized with metofane before the tumor was harvested. Mice were fasted for 3 hours before their blood was collected, and plasma was isolated by centrifugation at room temperature for 10 minutes at 375g and was stored at -70 °C until assayed. IGF-I concentrations were measured by radioimmunoassay (Nichols Institutes Diagnostics, Capistrano, CA) with purified plasma-derived IGF-I as the standard.
Statistical Analysis
Food intake, body weight, tumor size, and quantitative measurements based on tumor histologic sections were analyzed initially by analysis of variance (ANOVA) (37) followed by Scheffé's test (38) or Fisher's protected least-significant difference (37) to calculate two-sided pairwise comparisons among treatment groups by use of Statview 4.5 (Abacus Concepts, Inc., Berkeley, CA) and Power Macintosh computers (Apple Computer, Cupertino, CA).
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RESULTS |
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All data regarding food intake are summarized in Fig.
1. The ad libitum fed rats consumed between
55 and 65 kcal/day for the duration of the experiment (Fig. 1
, A). The
castrated rats showed a gradual and progressive reduction in energy
intake that was approximately equivalent to a 17% restriction over
the duration of the experiment (P<.001 versus ad
libitum fed rats). The final body weight (Fig. 1
, B) was reduced by
14% (P<.01) and 30% (P<.01) compared
with that of the ad libitum fed rats (400 g ± 37 g [mean
± SD]) for rats fed 20% (346 g ± 12 g) and 40% (281 g
± 10 g) total diet restriction, respectively. Castrated males
weighed 19% (324 g ± 20 g) less (P<.01) than ad
libitum fed, hormonally intact rats. The tumor diameter (Fig. 1
, C)
in hormonally intact rats (2.2 cm ± 0.4 cm, mean ± SD) was
reduced 74% in castrated rats (0.6 cm ± 0.1 cm;P<.001), 39% in rats restricted by 40% total diet
restriction (1.3 cm ± 0.5 cm; P<.01), and 24% in rats having
20% total diet restriction (1.7 cm ± 0.4 cm; P<.05).
No significant difference in tumor diameter was detected between the
two diet-restricted groups, although the tumor diameters of both groups
were different from the tumor diameter of the castrated group
(P<.001). The final tumor weight measured at necropsy was
8.9 g ± 2.3 g (mean ± SD) in ad libitum fed, hormonally
intact rats and was reduced by 98% with castration (0.2 g ± 0.1
g; P<.001), 76% by 40% energy restriction (3.4 g
± 2.4 g; P<.001), and 62% by 20% energy
restriction (2.1 g ± 1.2 g; P<.01). No significant
difference was detected in final tumor weights between rats fed 20%
restricted or 40% restricted diets, although the final tumor
weights in both groups were different from those of castrated animals
(P<.001 for each comparison). All of the above data were
analyzed initially by ANOVA (37) followed by Fisher's
protected least-significant difference (37) to calculate
two-sided pairwise comparisons among treatment groups.
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The average daily intake of energy, food, and selected nutrient
fractions for the rats fed the various diets is shown in Table
2. The hormonally intact and ad libitum fed
rats consumed between 61 and 69 kcal/day for the duration of the
experiment (Fig. 2,
A). Castrated males showed a
gradual reduction in energy intake during the experiment (49 kcal/day
by week 16) that averaged 16% less than hormonally intact, ad
libitum fed rats for the total experiment (P<.001).
Diets were provided to the three restricted groups each day and
consumed without waste. Castrated males (357 g ± 14 g, mean ± SD)
weighed 21% less than hormonally intact and ad libitum fed
rats (453 g ± 39 g) (Fig. 2
, B) by the end of the study
(P<.01). Each type of energy restriction produced nearly
identical changes in body weight (Fig. 2
, B). The final body weights
for rats restricted in fat (329 g ± 13 g), in carbohydrate (334 g
± 19 g), or in total diet (330 g ± 19 g) intake were 27%,
26%, and 27% less than those of ad libitum fed rats,
respectively (P<.001, for each comparison). The tumor
diameter (Fig. 2, C) in ad libitum fed control rats was 2.8 cm
± 0.8 cm and was reduced by 90% in castrated rats (0.2 cm ±
0.3 cm, mean ± SD; P<.0001), by 26% in rats
restricted by lipid calories (2.0 cm ± 0.6 cm; P<.05),
by 36% in rats restricted by carbohydrate calories (1.7 cm ± 0.9
cm; P<.008), and by 39% in rats having total diet
restriction (1.6 cm ± 0.6 cm; P<.005). No
statistically significant difference in tumor diameter was detected
between the three diet-restricted groups, although all restricted
groups exhibited tumor diameters that were different from those of
castrated rats (P<.0003). The final tumor weight in
castrated males (0.2 g ± 0.2 g, mean ± SD) was reduced
by 99% compared with that in hormonally intact and ad
libitum fed rats (18.8 g ± 9.0 g; P<.0001).
Restriction of energy intake as fat, carbohydrate, or total diet
resulted in final tumor weights that were reduced by 37% (11.8
g ± 7.6 g; P<.05), 62% (7.1 g ± 5.2 g;
P<.002), and 60% (7.5 g ± 5.9 g;
P<.002), respectively, compared with final tumor weights
in ad libitum fed rats. Among the groups subjected to the
three types of restriction, there was no statistically significant
difference, although the mean tumor diameter, calculated tumor volume,
or final tumor weight were consistently greater for the
lipid-restricted group than for the other two restricted groups. All of
the above data were analyzed initially by ANOVA (37) followed
by Fisher's protected least-significant difference (37) to
calculate two-sided pairwise comparisons among treatment groups.
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Energy intake averaged 15.4 kcal/day ± 1.1 kcal/day (mean ±
SD) for ad libitum fed mice and 11.1 kcal/day ± 0.0
kcal/day for the three restricted groups, representing an average
reduction in energy intake of 28.1% for the experiment (Fig.
3, A). The final body weights (Fig. 3
, B) for mice
restricted in fat (19.6 g ± 0.7 g, mean ± SD), carbohydrate (20.4
g ± 1.3 g), or total diet (19.6 g ± 0.9 g) were 17%, 13%,
and 12% lower, respectively, than those for the ad libitum
fed mice (23.4 g ± 1.8 g) (P<.001, for each comparison).
The tumor diameter at necropsy (Fig. 3
, C) was 2.6 cm ± 0.9 cm
(mean
± SD) in controls and was reduced by 31% in mice restricted in
lipid calories (1.8 cm ± 0.4 cm; P<.02), by 44% in
those restricted in carbohydrate calories (1.5 cm ± 0.5 cm;
P<.002), and by 48% in rats with total diet restriction
(1.4 cm ± 0.6 cm; P<.001). No significant difference in
tumor diameter was detected between the three diet-restricted groups.
The calculated tumor volumes were 2.1 cm3 ± 0.7
cm3 (mean ± SD) in ad libitum fed mice and 1.4
cm3 ± 0.3 cm3 in mice restricted in fat calories
(P<.02), 1.2 cm3 ± 0.4 cm3 in those
restricted in carbohydrate calories (P<.002), and 1.1
cm3 ± 0.5 cm3 in total diet-restricted mice
(P<.002). Tumor weights were not obtained because of the
invasion of the local tissues and precluded reliable dissection for
accurate quantitation. All of the above data were analyzed initially by
ANOVA (37) followed by Scheffé's test (38) to
calculate two-sided pairwise comparisons among treatment groups.
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The results of our histopathologic analysis showed no major
differences between animals restricted by 20% and those restricted
by 40% of energy or restricted by different methods of restriction.
Therefore, the following descriptive observations are relevant to all
restricted groups. The cross-sectional diameters of prostate tumors
from control rats were larger than those of the tumors from castrated
rats, whereas tumors from diet-restricted rats (all types of
restriction) had cross-sectional diameters intermediate in size (Fig.
4, A, B, and C). We initially used light microscopy
to examine hematoxylin-eosin-stained sections of the Dunning R3327-H
tumors (Fig. 4
, D, E, and F). Tumors from hormonally intact and ad
libitum fed rats resembled a moderately differentiated human
prostate adenocarcinoma exhibiting numerous glands and duct-like
structures of variable and irregular shape with large luminal volumes
containing secretions (Fig. 4
, D). The glands were surrounded by a
loose connective tissue defined by faint or nonstaining stromal
elements. A rich supply of vascular structures of varying size
containing red blood cells was readily apparent within the tumor stroma
of hematoxylin-eosin-stained sections from ad libitum fed
rats. These characteristics differed considerably in tumors from
castrated rats (Fig. 4
, F). The glandular elements were small and very
uniform in shape, with glands and ducts closely packed, back-to-back,
and separated by a layer of dense stromal matrix. The entire tumor from
a castrated rat was surrounded by a prominent fibrous connective tissue
capsule that was poorly developed in the tumors from hormonally intact
and ad libitum fed rats. Tumors from castrated rats also
typically had dense septa dividing the tumor into lobules. Vascular
elements were virtually undetectable under low-power light microscopy
within the tumors from the castrated animals and were observed only
within or near the surrounding capsule. Prostate tumors from rats
consuming restricted diets exhibited a tumor architecture that was
different from that of the ad libitum fed or castrated groups
(Fig. 4
, E). The glandular and ductal structures of the tumors from
rats consuming restricted diets were smaller and more homogeneous in
shape than those of the tumors from ad libitum fed rats.
Tumors from diet-restricted rats also had larger amounts of stromal
cells and matrix between the glandular structures. Vascular structures
were detected by light microscopy in tumors from diet-restricted rats
but were fewer and smaller than those in tumors from ad
libitum fed rats.
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We also used computerized image analysis to quantitate vascular channels containing red
blood cells identified in hematoxylin-eosin-stained sections, which we refer to as the blood
vessel density ratio. Representative images of tumor tissue from hormonally intact and ad
libitum fed rats, diet-restricted rats, and castrated rats containing a vascular space filled with
red blood cells are shown in Fig. 4, G, H, and I. The fraction of prostate
tumor tissue containing vascular elements was 2.6% ± 0.1% (mean
± SD) for ad libitum fed rats versus 0.3% ± 0.1% in the
diet-restricted groups (pooled). In contrast, it was not possible to accurately quantitate vessels in
tumors of castrated animals on the basis of red blood cell detection with the use of image
analysis, since the few vessels were small and below the satisfactory resolution
(<0.1%). All of the above data were analyzed initially by ANOVA (37) followed by Scheffé's test (38) to calculate
two-sided pairwise comparisons among treatment groups.
LNCaP Tumor Histology and Morphometry
We used light microscopy to examine the LNCaP tumors that appeared as a continuous sheet of anaplastic cells that would have been classified as high-grade or poorly differentiated tumors if observed in a human biopsy specimen (images not shown). The tumors showed virtually no evidence of gland formation. The LNCaP tumors showed some areas of necrosis, as is typically observed in rapidly growing transplantable tumors. Blood vessels were present but were smaller and more homogeneous in the LNCaP tumor than in the Dunning tumor. Little stroma or matrix was observed by light microscopy in the LNCaP tumor. All of the above data were analyzed initially by ANOVA (37) followed by Scheffé's test (38) to calculate two-sided pairwise comparisons among treatment groups.
Proliferation and Apoptosis
We quantitated the proliferation and apoptosis in R3327-H prostate
tumor samples from both experiments. The data in Table 3 are derived
from ad libitum fed rats, castrated rats, and rats restricted
at 20% or 40% of total energy. We pooled the data for the
restricted groups, since no significant differences in tumor
proliferation or apoptosis were observed between rats subjected to
different levels of energy restriction or different methods of caloric
restriction. The PCNA labeling index was reduced 54% in castrated
rats compared with ad libitum fed rats (P<.0001),
whereas no significant effect was noted with energy restriction. The
apoptotic index more than doubled with castration (P<.001)
or total dietary restriction (P<.001). The above results
were confirmed by the analysis of tumors obtained from the second
R3327-H experiment in which energy restriction was accomplished by
three different approaches (data not shown). LNCaP tumors in mice also
showed no changes in proliferation index with restriction, but the
increase in apoptotic index was more than twofold
(P<.0007). As we observed in the Dunning R3327-H rat study,
we detected no significant difference in the effect on apoptosis or the
proliferation index between the different methods of energy restriction
(data not shown). All of the above data were analyzed initially by
ANOVA (37) followed by Scheffé's test (38) to
calculate two-sided pairwise comparisons among treatment groups.
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Factor VIII staining and quantitation of vessel density (Table
3) in Dunning tumors revealed an approximately
62% reduction in vascularity from energy restriction compared with
that in ad libitum fed rats (P<.003). Vessel
density in castrated animals was profoundly reduced
(P<.0001), and occasional small vessels were noted within
the tumor and some larger vessels were observed in the periphery of the
tumor infiltrating from the dense stromal capsule. Similarly, we
observed an approximately 49% reduction in microvessel quantitation
as a result of energy restriction in the LNCaP tumor
(P<.04). All of the above data were analyzed initially by
ANOVA (37) followed by Scheffé's test (38) to
calculate two-sided pairwise comparisons among treatment groups.
VEGF staining in Dunning R3327-H tumors from ad libitum fed rats was
24.5% ± 7.5% (mean ± SD) of cells (Table 3). Staining was primarily intracellular and most intense in the glandular elements of
the tumor (Fig. 4
, J through O). VEGF staining was observed in only one
small cluster of glands in a slide from tumors obtained from energy-restricted rats (0.4%
± 0.8%; P<.0001 versus ad libitum fed rats). Furthermore, no
detectable staining was observed in Dunning tumors from castrated rats (0.0% ±
0.0%; P<.0001 versus ad libitum fed). Staining in consecutive
sections showed identical patterns, which further suggested specificity of detection for VEGF
expression that occurs in large patches of the Dunning tumor, as was observed in human
specimens (39-41). All of the above data were analyzed initially by
ANOVA (37) followed by Scheffé's test (38) to calculate two-sided pairwise comparisons among treatment groups.
VEGF expression in LNCaP tumors was very low with less than 5% of the tumor area from all rats showing positive staining and qualitatively very different from the Dunning tumor (images not shown). In contrast to the large areas of VEGF expression in the Dunning tumor, staining in the LNCaP tumor was by individual cells, approximately one in 30. The LNCaP cells expressing VEGF exhibited cytologic changes suggestive of cell death with vacuolated cytoplasm or dissolution of the cell. These observations suggest that LNCaP cells do not express VEGF under baseline conditions in vivo but may activate the gene during apoptosis or death from toxic effects. Changes in diet did not alter the low baseline VEGF expression in the LNCaP model. All of the above data were analyzed initially by ANOVA (37) followed by Scheffé's test (38) to calculate two-sided pairwise comparisons among treatment groups.
Plasma IGF-I
Blood samples were obtained from SCID mice bearing the LNCaP tumors at the time of necropsy, and IGF-I was evaluated by immunoassay. Circulating IGF-I concentrations (mean ± SD) in ad libitum fed mice were 339 ng/mL ± 59 ng/mL, in contrast to 125 ng/mL ± 44 ng/mL in mice having total diet restriction (P<.0001 versus ad libitum fed), 160 ng/mL ± 35 ng/mL in mice restricted in fat calories (P<.0001 versus ad libitum fed), and 114 ng/mL ± 26 ng/mL in mice restricted in carbohydrate calories (P<.0001 versus ad libitum fed, P<.05 versus fat restriction). All of the above data were analyzed initially by ANOVA (37) followed by Scheffé's test (38) to calculate two-sided pairwise comparisons among treatment groups.
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DISCUSSION |
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To our knowledge, these studies are the first to characterize the effects of energy intake on prostate tumorigenesis in experimental models. Restriction of total dietary consumption or the limitation of energy intake by selective reductions in the consumption of fat or carbohydrate calories has been shown to inhibit the incidence or growth of tumors having diverse histologic origins (42-44). Our studies report that energy intake ad libitum enhances the growth rate of rat and growth of transplantable human and rat prostate tumors illustrating diverse histologic, biologic, and molecular characteristics. The Dunning R3327-H adenocarcinoma, a slow-growing, well-differentiated to moderately differentiated, and androgen-dependent tumor, mimics many of the features observed in newly diagnosed, localized human prostate cancer. In contrast, the human LNCaP line is a rapidly growing anaplastic (poorly differentiated) prostate cancer with no evidence of glandular formation in vivo. Our studies with the Dunning model clearly showed that restriction of energy intake can significantly inhibit tumor growth without inducing malnutrition in the host. The adult rats with the highest degree of energy restriction (40%) continued to show a gradual increase in body weight. The main difference between the restricted and the ad libitum fed rats is the scarcity of carcass adipose tissue stores, which was readily apparent at necropsy (although body composition was not measured).
Our studies also provide insight into the interrelationship between energy intake, dietary fat concentration, and prostate tumorigenesis. Previous studies have shown that prostate tumor growth is inhibited by essential fatty acid deficiency (45) as well as by diets rich in omega-3 fatty acids (46), whereas it is stimulated by linoleic acid (47) or high-fat diets (48). Comparing different types of energy restriction required that we use a high-fat diet providing 40% of energy as the ad libitum fed treatment group. This afforded the opportunity to reduce energy intake by restricting either fat or carbohydrate calories by 30%. It is interesting that restriction of fat calories, carbohydrate calories, or total diet provided 15%, 57%, and 40% of energy from fat, respectively. However, all types of energy restriction produced a significant and similar reduction in prostate tumor growth. Our studies revealed no evidence that high-fat diets promote prostate tumor growth under energy-restricted conditions. Prior studies (48) showing that high-fat diets enhance prostate tumor growth in rodents were completed under conditions of ad libitum feeding. Our studies indicate that the effect of dietary fat concentration on prostate tumor growth may depend on total energy intake as was suggested in studies with other experimental tumor models (49-53).
An obstacle to translational research in nutrition and prostate cancer prevention or to the application of nutrition as an adjuvant to therapy is the lack of surrogate biomarkers relevant to both dietary intervention and prostate cancer progression. The biomarkers examined in our study may be useful individually but, more importantly, they may be useful as a pattern. Folkman and colleagues (22) have observed that biomarkers of tumor angiogenesis are predictably associated with a specific pattern of changes in proliferation and apoptosis. For example, antiangiogenic growth factors and cytokines typically reduce tumor microvessel density in parallel with increased apoptosis and little effect on proliferation rates (23,24,54,55). We observed a twofold to threefold reduction in microvessel density, increased apoptosis, and minimal change in PCNA labeling index with energy restriction in our studies with the Dunning or LNCaP tumor models. Our studies suggest that energy restriction produces a pattern of biomarker changes identical to that observed following institution of antiangiogenic therapy.
Our results are consistent with a hypothesis that energy restriction inhibits prostate tumor growth via a shift in the balance between angiogenic and antiangiogenic factors (22). Solid tumor growth beyond a volume of 2-3 mm3 cannot be sustained by diffusion and depends on establishment of a vascular network for supplying nutrients and removing metabolic waste products (56,57). Enhanced tumor angiogenesis reflects the summation of many signaling processes between the tumor cells, matrix, and host vascular cells. The critical mediators are the angiogenic and antiangiogenic growth factors or hormones found in the circulation and tumor microenvironment (57). We propose that nutritional status directly or indirectly influences interactions between tumor cells and local vasculature by changing the expression of angiogenic growth factors. We evaluated this hypothesis by immunohistochemically examining the expression of VEGF. VEGF is one of the most potent angiogenic factors known, enhancing endothelial cell proliferation and formation of new vessels (29). Furthermore, VEGF enhances vessel permeability, which may thereby provide an improved supply of nutrients to the tumor (29). VEGF expression by tumor cells in vitro is enhanced by glucose deficiency and hypoxia (29-32). With regard to the human prostate, immunoreactive VEGF or messenger RNA is constantly detected in the cancer specimens, whereas VEGF expression is variable in benign prostate hyperplasia and normal prostate tissue (39,41). VEGF expression has also been documented in prostate cancer cell lines, and the DU-145 line has been reported to exhibit the highest expression, the PC3 line intermediate expression, and the LNCaP line the lowest expression (33,39,40). Using an antibody against a VEGF epitope that is common to all isoforms, we examined VEGF expression in the Dunning and LNCaP tumors in vivo. In rats having ad libitum access to food, prostate tumors showed a high intracellular cytoplasmic expression of VEGF, particularly in the cells lining glandular and duct-like structures, as was reported for human prostate cancer (39,41). Furthermore, areas of positive staining occurred in a patchwork pattern throughout the tumors from ad libitum fed rats, a characteristic feature observed in human prostate cancer specimens suggesting that regulation may vary between specific microenvironments within the tumor, perhaps as a result of local changes in oxygenation or nutrient metabolism. We observed a striking inhibition of VEGF expression with diet and energy restriction in the Dunning model, with only a single glandular focus of staining detected in only one tumor specimen. A different picture emerged with the LNCaP tumor grown in vivo; this tumor exhibited little baseline VEGF staining, with image analysis detecting only 5% of the total area exhibiting positive staining. Furthermore, VEGF expression in the LNCaP tumor was associated with cells that appeared to be undergoing cytolysis or apoptosis. On the basis of these observations, we propose that VEGF is one of possibly many regulators of tumor neovascularization altered by diet restriction to change the balance from a proangiogenic to an antiangiogenic microenvironment. However, our studies also indicate that diet-induced alterations in angiogenesis in the LNCaP prostate tumor model are not dependent on VEGF and that there must be other contributory factors.
We propose that another factor linking dietary intake with prostate tumor angiogenesis is
IGF-I. We observed that circulating IGF-I is significantly reduced by diet or energy restriction in
rats bearing prostate tumors, as was previously reported in studies of leukemia, breast cancer, and
bladder cancer (52,58,59). A recent study of prospectively collected
blood samples in a human cohort (25) showed that higher concentrations
of circulating IGF-I were associated with increased risk of prostate cancer. IGF-I is mitogenic for
normal and malignant prostate tumor cells (26). IGF-I receptors are
present in prostate tissue, and expression is reduced by 5-reductase inhibitors (27). IGF-I is also considered to be an angiogenic growth factor, stimulating
endothelial cell proliferation, migration, and tube formation in vitro(28). IGF-I also has been implicated in promoting angiogenesis and
collateral neovascularization following ischemic injury (60). The
reduction in circulating IGF-I by dietary interventions represents a potential mechanism whereby
prostate tumor angiogenesis may be reduced with an attenuation of tumor growth and reduced
risk of metastases.
We used androgen deprivation by castration as an intervention known to have a potent antitumor response with the Dunning R3327-H tumor. We observed a 54% reduction in the proliferation index and more than a twofold increase in apoptosis in conjunction with an enormous reduction in tumor vascularity either by microvessel number or by blood vessel density ratio. The enhanced apoptosis with androgen ablation is consistent with findings reported in earlier studies (61-64). In contrast to dietary restriction, castration significantly reduced the proliferative index in the R3327-H tumor as was seen in other models (65). The profound inhibition of microvessel density estimated by factor VIII staining, blood vessel density index, and VEGF expression by androgen deprivation observed in the Dunning R3327-H prostate tumor suggests that testosterone is necessary for maximal prostate tumor angiogenesis. Our in vivo results are consistent with reports from other laboratories using a variety of complementary techniques (33). Since castration reduces food intake by approximately 20%, the effects of castration on tumor growth and biomarkers reflect a combination of both energy restriction and androgen deprivation. Investigators using animal models of prostate cancer should be aware of the possible confounding of results that may occur as a result of a depression of energy intake secondary to manipulation of the pituitary-androgen axis.
Prostate cancer is common in affluent nations and contributes significantly to the morbidity and mortality of aging males. Furthermore, the costs of screening, diagnosis, primary treatment, and management of metastatic disease are a major financial burden to the health care system. Dietary interventions capable of inhibiting the stepwise prostate cancer cascade or of slowing the progression of the disease, thereby shifting diagnosis to later decades of life, represents an attractive alternative. Our studies demonstrate suppression of prostate tumor neovascularization by dietary intervention and suggest that biomarkers of tumor angiogenesis may be useful in the identification of effective dietary strategies for reducing the burden of prostate cancer in our society.
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We acknowledge the support of Dr. Norman H. Altman and the Papanicolaou Cancer Research Institute (Miami, FL) for the Dunning R3327-H rat prostate carcinoma. We thank Dr. Toshihide Tanaka, Jikei University School of Medicine, Tokyo, Japan, for his assistance in establishing the histologic assays in our laboratory.
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Manuscript received March 24, 1998; revised December 24, 1998; accepted December 31, 1998.
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