According to the World Health Organization, in 2014, approximately 1.9 billion adults were overweight, and at least 600 million adults were obese. In addition, 42 million children under the age of five were reported to be overweight or obese in 2013. Thus, obesity rates are expected to increase globally. Wolin and colleagues estimated that overweight and obesity cause approximately 20% of all cancer cases [1
]. Epidemiological studies indicated that obesity is associated with increased risks of several types of cancer, such as colon, breast, and hepatic cancer [2
Prostate cancer is the second leading cause of death from cancer in men in the U.S. [4
]. However, studies assessing the link between prostate cancer and obesity have generated puzzling results. A recent review of the epidemiological data linking prostate cancer and obesity indicates that obesity is associated with reduced risk of nonaggressive, indolent disease, and increased risk of more aggressive disease with increased local and distant invasion. Obesity is also associated with worse post-treatment results and increased risk of prostate cancer death [5
Similar to the results of epidemiological studies, there are inconsistent results among studies using mouse tumor models. Consumption of a Western-type diet (enriched in fat and cholesterol) accelerated tumor progression in the transgenic adenocarcinoma mouse prostate (TRAMP) model [6
], and lowering dietary fat (corn oil) delays prostate cancer development in the Hi-Myc transgenic mouse model [7
]. However, a recent study has shown that a high-fat diet (HFD) containing soybean oil increased tumor weight and tumor volume in TRAMP-C2 allograft tumor model but did not affect tumor development in the TRAMP model [8
]. Therefore, more studies are needed to clarify the relationship between obesity and prostate cancer, and to explore the underlying mechanisms.
Numerous studies have reported the biological mechanisms linking obesity to cancer, including: insulin and insulin-like growth factor, sex steroids, adipokines, inflammation, and obesity-induced hypoxia [9
]. Adipose tissue, which is made up of various cell types (including adipocytes, fibroblasts, macrophages, and blood vessels), serves as an important endocrine organ and secretes adipokines (leptin, tumor necrosis factor, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, vascular endothelial growth factor (VEGF), etc
.). The well-known adipokines leptin and adiponectin were associated with cancer [12
]. In addition, some adipokines (for example, leptin and MCP-1) contribute to the accumulation of macrophages in adipose tissue [14
]. Therefore, obesity is thought to induce a state of chronic low-grade inflammation. Because chronic inflammation has been linked to various steps involved in tumorigenesis, including initiation, promotion, malignant conversion, invasion, and metastasis [17
], obesity-induced inflammation and various adipokines may play an important role in the development and progression of prostate cancer via endocrine mechanisms. In addition, the results from recent studies suggested that cancer-associated adipocytes may also stimulate tumor progression via paracrine mechanisms [18
In the present study, we examined the effects of a high-fat diet (HFD) containing lard on tumor development and progression using the TRAMP and TRAMP-C2 allograft models. In TRAMP mice, prostate cancer spontaneously develops with subsequent progression to metastasis. We also attempted to examine the effects of HFD on the survival rate of TRAMP mice, and to explore the pro-tumorigenic roles of soluble factors released from the adipose tissue of HFD-fed mice. Since the major cause of obesity is considered to be consumption of a Western-type diet, rich in animal fat and saturated fat, an animal fat (lard)-enriched diet (Table 1
) was employed in the present study.
Compositions of experimental diets.
Compositions of experimental diets.
|Control Diet (10 kcal % Fat)||High-Fat Diet (60 kcal % Fat)|
|Casein, 80 Mesh||200||800||200||800|
|Mineral Mix S10026||10||0||10||0|
|Potassium Citrate, 1 H2O||16.5||0||16.5||0|
|Vitamin Mix V10001||10||40||10||40|
|FD & C Yellow Dye #5||0.05||0|
|FD & C Blue Dye #1||0.05||0|
2. Experimental Section
The following reagents were purchased from the indicated suppliers: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Sigma (St. Louis, MO, USA); Matrigel, BD Biosciences (San Jose, CA, USA); antibodies against proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase (CDK)2, CDK4, Cyclin A, CD31, VEGF-A, and VEGF-C, Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against Ki67, VEGF-D, and lymphatic vessel endothelial hyaluronan receptor (LYVE)-1, Abcam (Cambridge, MA, UK); anti-VEGF receptor (VEGFR)-2, Cell Signaling (Beverly, MA, USA); antibodies against CD45 and CXCR5, recombinant mouse MCP-1, CXCL1, CXCL2 and CXCL13 protein, R & D systems (Minneapolis, MN, USA).
2.2. Cell Culture
TRAMP-C2 cells (established from a prostate tumor of a TRAMP mouse) [20
] were purchased from the American Type Culture Collection (Manassas, MA, USA) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), 100,000 U/L penicillin, and 100 mg/L streptomycin. Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Walkersville, MD, USA) and maintained in Medium 199 (M199) containing 20% FBS, 100,000 U/L penicillin, 100 mg/L streptomycin, 1.5 μg/L epidermal growth factor, and 17 μg/L hydrocortisone. For all in vitro
experiments, the cells were subjected to no more than 10 cell passages.
2.3. Animal Studies
2.3.1. TRAMP-C2 Allograft Tumor Model
Four-week old, male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, USA) were fed a commercially semi-purified control diet (CD, 10 kcal % fat, catalogue #D12450B, Research Diets Inc., New Brunswick, NJ, USA) or a high-fat diet (HFD, 60 kcal % fat, D12492, Research Diets, Inc.) ad libitum (10 mice/group). Twenty-four weeks after the beginning of feeding, TRAMP-C2 cells (1 × 106 cells suspended in 0.1 mL Matrigel/PBS) were subcutaneously injected into the right rear flanks of the mice. All animals were killed 11 weeks after the TRAMP-C2 cell injection.
2.3.2. TRAMP Model
TRAMP mice (Jackson Laboratory) were bred and maintained under specific pathogen-free conditions at the animal facility of Hallym University (Chuncheon, Korea). After selection of male TRAMP mice by genotyping [21
], male TRAMP mice and their nontransgenic littermates at 4 weeks of age were fed the CD or the HFD ad libitum
. In order to evaluate the effects of HFD on the survival rate of TRAMP mice, mice were fed a CD (n
= 21) or a HFD (n
= 24) for up to 46 weeks (50 weeks of age). To avoid pain or distress to the mice, we followed the guidelines for the use of animals in the cancer research published in the British Journal of Cancer [22
]. When symptoms including severe body weight loss, persistent hypothermia, hunching behavior, etc.
, were noted in a mouse, the mouse was euthanized with CO2
asphyxiation. Euthanasia was then equated with death.
To evaluate the effects of HFD on prostate cancer development and metastasis, mice were sacrificed at 24 and 32 weeks of age, respectively. (24 weeks: CD- and HFD-fed nontransgenic littermates, n = 6 mice/group; CD- and HFD-fed TRAMP, n = 12 mice/group and 32 weeks: CD- and HFD-fed nontransgenic littermates, n = 8 mice/group; CD-fed TRAMP, n = 17 mice; HFD-fed TRAMP, n = 20 mice).
At the time of sacrifice, mice were anesthetized with an intraperitoneal injection of 2.5% Avertin, and blood was harvested by retro-orbital bleeding, and the serum was stored at −70 °C for further analysis. The tumor (from TRAMP-C2 allograft tumor model), genitourinary (GU) tract (from TRAMP model), liver, lung, and fat tissues were removed, weighed, and fixed in 4% paraformaldehyde. Hemoglobin contents in tumor tissues were determined using Drabkin’s solution and a cyanmethemoglobin standard solution (Sigma) as described previously [23
]. All animal experiments were approved by the Animal Care and Use Committee of Hallym University (Hallym2009-124 and Hallym2009-125).
2.4. Immunohistochemical (IHC) and Immunofluorescence (IF) Analysis
Fixed tissue samples were embedded in paraffin, and 5 μm sections were prepared. For the evaluation of pathologic grades in the dorsolateral lobes of the prostate, the paraffin-embedded sections were stained with hematoxylin and eosin (H & E). For IHC analyses, the paraffin-embedded sections were incubated with their relevant antibodies, and then developed using an LSAB+ kit (Dako, Carpinteria, CA, USA) in accordance with the manufacturer’s instructions.
For IF staining, the tumor sections were incubated with their relevant antibodies, and then incubated with corresponding secondary antibodies labeled with Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) or Cy3 (Rockland, Gilbertsville, PA, USA). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI).
2.6. Cytokine Array and Enzyme Linked Immunosorbent Assay (ELISA)
The levels of cytokines in pooled samples (serum (normal, 6 mice/group; TRAMP, 12 mice/group) or ATCM (5 mice/group)) were measured using Proteome Profiler™ (mouse cytokine array) in accordance with the manufacturer’s instructions (R&D Systems). The relative abundance of each dot to positive control spots (contained within the membrane) was quantified by densitometric scanning of the exposed film using Image J (NIH, Bethesda, MD, USA). The concentrations of MCP-1, IL-6, CXCL1, CXCL2, and CXCL13 were measured using ELISA kits in accordance with the manufacturer’s instructions (R & D Systems).
2.7. MTT Assay and Western Blot Analysis
TRAMP-C2 cells were plated in 24-well plates at 50,000 cells/well with DMEM containing 10% FBS. One day later, cells were serum-deprived for 24 h in DMEM containing 1% FBS. The cells were then treated with 0 or 50% ATCM for 24 h. Viable cell numbers were estimated by the MTT assay. Total cell lysates were prepared and Western blot analysis was performed as previously described [24
2.8. Transwell Migration Assay
Transwell migration assay was conducted as described [25
]. Briefly, TRAMP-C2 cells were added to the upper chamber, and the lower chamber of the well was filled with SFM in the absence or presence of 50% ATCM or 100 μg/L recombinant proteins (MCP-1, CXCL1, and CXCL2). Cells were then incubated for 1 h. Migrated cells into Type IV collagen-coated membrane were stained with H&E and counted. In order to examine the effect of CXCL13-CXCR5 axis on cell migration, TRAMP-C2 cells were pretreated with CXCR5 antibody (1 mg/L) for 30 min and CXCL13 (100 μg/L) was added to the lower chamber.
2.9. Tube Formation Assay and Aorta Ring Assay
HUVECs (50,000 cells) were plated in Matrigel-coated 24-well plates and incubated with SFM in the absence or presence of 5% ATCM. Tubular structures were photographed and tube length was quantified. For ex vivo aorta ring assay, aortic rings (1 mm thickness) taken from thoracic aortas of Sprague Dawley rats (24 weeks of age, Orient Bio Inc, Gapyung, Korea) were seeded in Matrigel-coated 48-well plates and treated with SFM in the absence or presence of 5% ATCM. Aortic rings were photographed using a light microscope.
2.10. Statistical Analysis
The results were expressed as means ± SEM. Differences between the two groups were assessed by Student’s t-test. When there were more than three groups, differences were tested by ANOVA followed by Duncan’s multiple range test, utilizing the SAS statistical software version 9.2 (SAS Institute, Cary, NC, USA). Survival rates were estimated from Kaplan-Meyer curves by log-rank test. Differences were considered significant at p < 0.05.
Epidemiological studies assessing the link between prostate cancer and obesity have generated puzzling results. For example, even though several prospective cohort studies observed that obesity was associated with increased risk of death due to prostate cancer [26
], large prospective cohort studies in the United States found that obesity was associated with a reduced diagnosis of prostate cancer [28
]. Obese men have lower prostate-specific antigen (PSA) values than non-obese men, possibly due to hemodilution with larger volume in the obese men [31
]. As a result, obese men have lower chances of having elevated PSA, receive less recommendation to undergo biopsy, and are less likely to be diagnosed with prostate cancer. In addition, obese men have larger prostates [32
], making cancer detection more difficult at biopsy. Freedland et al
., reported that obesity was associated with a 98% increased risk of prostate cancer after adjusting for the lower PSA levels and larger prostate size [33
]. Using TRAMP mice, Llaverias et al
., demonstrated that a Western-type diet containing high cholesterol and fat accelerates tumor progression [6
]. Additionally, similar results were obtained from the Hi-Myc transgenic mouse model [7
]. In the present study, we showed that feeding TRAMP mice with HFD containing lard induces body weight gain, and enhances tumor growth and progression, thereby leading to decrease in survival rate. Taken together, these results indicate that a HFD containing lard is detrimental for individuals who are genetically predisposed to prostate cancer, or have indolent prostate cancer.
In the present study, mice fed the HFD containing lard consumed more energy and also became obese. Epidemiological evidence indicates that obesity (elevated BMI) is positively correlated with prostate cancer-specific mortality [34
]. Therefore, increased body weight can be a contributing factor to the increased tumor growth and high mortality observed in the mice fed lard. Dietary fat itself can also stimulate tumor progression. It has been reported that LNCaP tumor growth was decreased in nude mice when their diet was switched from a high-corn oil diet to a low fat diet (LFD) with no differences in total energy intakes or body weight gains [36
]. Severe combined immunodeficient mice fed an isocaloric LFD diet displayed delayed LAPC-4 xenograft tumor growth and decreased blood PSA levels relative to mice fed a HFD containing corn oil [37
]. Additionally, in a similar mouse model, reduction in dietary corn oil without changes in calorie intakes slowed down the advancement of LAPC-4 xenografts from androgen dependency to independency [38
]. These results clearly indicate that dietary fat, without changes in energy intake or body weight, stimulates tumor promotion. The present study did not determine whether the dietary fat or obesity itself was responsible for the stimulation of tumor progression and reduction in survival rates in these animal models. However, in the allograft models, we noted that the body weights of the tumor-bearing mice started to decrease when the tumor volumes increased very rapidly, which was more pronounced in the HFD-fed mice (Figure 1
). These results indicate that more energy was stored in the HFD-fed mice, which was in turn used to support the rapid tumor growth.
In addition to amount of fat intake, the composition and quality of the fat also affect tumor progression. For example, it was reported that a diet containing 35% kcal fish oil (rich in ω-3 fatty acid) slowed tumor growth and improved survival in the LAPC-4 xenograft model, as compared to a diet containing the same amount of olive oil, corn oil, or lard/milk fat [39
]. Similarly, diets containing high levels of walnuts, fish oil, or docosahexaenoic acid and eicosapentaenoic acid were found to have beneficial effects on prostate cancer risk and tumorigenesis [39
]. On the other hand, normolipidic diets containing pork fat (7% lard) have cancer-promoting effects (increase in prostatic weight associated with epithelial hyperplasia and increased expression of AR and PPARγ) [43
]. These results indicate that the quality of the fat (e.g., the relative amounts of ω-3 fatty acid, ω-6/ω-3 ratio, and saturated fatty acid), as well as the quantity, is an important factor for prostate cancer progression. In addition to fat, the intake of protein is also associated with prostate cancer risk. A high intake of dairy protein was associated with an increased risk of human prostate cancer [44
], whereas restriction of dietary protein intake (7% kcal) inhibited tumor growth in mouse xenograft models of human prostate and breast cancer [45
]. In our allograft model, the daily protein intakes (g/day) were 0.53 ± 0.01 and 0.65 ± 0.01 in the CD and high lard groups, respectively. Thus, in addition to lard, the increased protein intake may have been a factor in increased tumor progression in the mice fed the HFD containing lard.
Increased levels of a variety of cytokines were observed in the blood and adipose tissues of HFD-fed mice, and the changes in these cytokines were found to be associated with the stimulation of tumor progression. It has been reported that different types of dietary fat exert different effects on inflammatory responses [46
] as well as cancer [48
]. For example, saturated fats and n-6 polyunsaturated fats have pro-inflammatory properties and are associated with increased risk of prostate cancer, while n-3 polyunsaturated fats and monounsaturated fats are known to have anti-inflammatory and anti-cancer properties [46
]. The HFD used in the present study contained more lard than the CD. Unfortunately, the present results cannot provide any information regarding whether the differences in cytokine production between the CD and HFD groups were due to the differences in lard intake in the diet, or simply due to the development of obesity itself. Future studies are needed to determine the effects of high-fat diet-induced obesity, as well as the effects of different types of fat on tumor progression and tumor-stimulating molecules, such as cytokine production.
Several hypotheses were proposed to explain the association between obesity and cancer. Of those, a recent study showed that adipocytes surrounding the tumor, which are referred to as cancer-associated adipocytes (CAAs), contribute to breast cancer invasion via modifying the cancer cell characteristics/phenotype [18
]. Moreover, Nieman and colleagues suggest that adipocytes provide adipokines to attract tumor cells and fatty acids for tumor growth [19
]. These studies suggest that adipose tissues provide fatty acids and soluble factors that stimulate tumor growth and metastasis via a paracrine mechanism. In addition, our previous results showed that adipocytes produce chemoattractants, which induce monocyte migration. We have also shown that the crosstalk between melanoma cells, adipocytes and macrophages increases chemoattractants for monocytes and angiogenic and lymphangiogenic factors [49
]. In the present study, the number of lipid vacuoles and CD45+
leukocytes was increased in the tumor tissues of HFD-fed mice injected with TRAMP-C2 prostate cancer cells (Figure 2
A,C). Taken together, these results indicate that HFD feeding increases CAAs that stimulate leukocyte infiltration into tumor tissues, and heterotypic interactions between CAAs, leukocytes, and tumor cells produce many growth factors, chemokines, and cytokines for tumor growth and progression.
In the present study, we observed that ATCM stimulated the growth and migration of prostate cancer cells and angiogenesis, which were further enhanced when ATCM were prepared from HFD-fed mice. Furthermore, ATCM from HFD-fed mice increased the expression of CDK2 and CDK4 in TRAMP-C2 cells (Figure 6
). Consistent with these observations, several in vitro
studies have shown that peri-prostatic adipose tissue explants secreted substances promoting proliferation and migration of prostate cancer cells [50
]. We also noted that Ki67+
proliferating cells and the expression of cell proliferation-related proteins (CDK2, CDK4, and Cyclin A) were increased in the tumor tissues of HFD-fed mice injected with TRAMP-C2 prostate cancer cells (Figure 2
) and in the DP of HFD-fed TRAMP mice (Figure 3
). Taken together, these results indicate that soluble factors leaked from adipose tissues of HFD-fed mice stimulate prostate tumor progression via paracrine and/or endocrine mechanisms.
Serum adipokine levels are altered in obese animals and humans [52
]. Consistent with this notion, in the blood of TRAMP mice, a variety of cytokines including MCP-1, CXCL1, CXCL10, CXCL13 etc.
were markedly increased by HFD feeding. Similar changes were noted in nontransgenic littermates but the changes in some cytokines due to HFD feeding were smaller in the normal littermates (e.g., CXCL1 and CXCL10) (Figure 5
A). We also noted that the levels of many of these cytokines were increased in the ATCM from HFD-fed nontransgenic C57BL/6J mice as compared to CD-fed mice (Figure 5
C). These increases in cytokine levels indicate that adipose tissues in HFD-fed mice release increased levels of cytokines, and this increase substantially contributes to increases in serum cytokine levels because the weights of fat tissues were also tremendously increased in HFD-fed mice (Table 2
and Table 3
In the present study, the levels of MCP-1, CXCL1, and CXCL2 were increased in the serum (Figure 5
A) and ATCM (Figure 5
C,D,F,G) of HFD-fed mice. TRAMP-C2 migration was increased by ATCM from HFD-fed mice as compared to that of CD-fed mice (Figure 6
C), and cytokines induced by HFD feeding (MCP-1, CXCL1, and CXCL2) stimulates TRAMP-C2 cell migration (Figure 6
D). It has been reported that MCP-1 levels both in the blood and in white adipose tissues are increased in HFD-fed mice and their blood levels correlate with changes in body weights [16
]. MCP-1 induces the infiltration of macrophages into adipose tissues [16
] as well as proliferation and invasion of prostate cancer cells [54
]. CXCL1 and CXCL2 produce their effects by signaling through CXCR2 [55
]. A study with human prostate biopsy reported that CXCR2 expression is correlated with advancing state of the disease [57
]. Serum CXCL13 was significantly higher in prostate cancer patients compared to patients with benign prostatic hyperplasia or high-grade prostatic intraepithelial neoplasia and normal healthy donors, suggesting that CXCL13 is a better predictor of prostate cancer than PSA [58
]. We observed that serum CXCL13 levels were increased in CD-fed TRAMP mice compared with those in CD-fed nontransgenic littermates, and the levels were further increased in HFD-fed TRAMP mice (Figure 5
B). It has been also reported that CXCL13 increased migration and invasion of LNCaP and PC3 prostate cancer cells [59
]. Expression of CXCR5, a corresponding receptor for CXCL13, was higher in prostate cancer cases, and the intensity of CXCR5 expression positively correlated with the Gleason score [60
]. We also observed that CXCL13 stimulated TRAMP-C2 cell migration, which was attenuated by a CXCR5 neutralizing antibody (Figure 6
E). Taken together, these results suggest that HFD-induced increases in the production of soluble factors (MCP-1, CXCL1, CXCL2, and CXCL13) play important roles in the stimulation of prostate tumor progression in mice.