Abstract
Background/Aim: Obesity is correlated with an increased risk of developing malignancies, including prostate cancer. Adipocytokines, such as leptin and adiponectin, are a family of hormones derived from adipose tissue that are involved not only in metabolism, but also in the development and progression of various malignancies. However, little is known about their role in prostate cancer. This study aimed to determine how leptin, adiponectin, and their receptors impact the spread of prostate cancer. Materials and Methods: We first performed immunohistochemical analysis of prostate cancer tissue microarrays to detect leptin, leptin receptor (Ob-R), adiponectin, and adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2). Wound healing assays and western blot analysis were then performed in human prostate cancer cell lines. Results: Immunohistochemistry showed that prostate tissue was not significantly positive for adiponectin. However, its expression tended to decrease according to the International Society of Urological Pathology (ISUP) grade of prostate cancer (p=0.056). In prostate cancer cell lines, administration of the synthetic adiponectin AdipoRon suppressed cell migration as well as the expression of phospho-NF-
B and cyclooxygenase-2, whereas leptin stimulated these effects. Conclusion: Adiponectin expression tended to be suppressed according to ISUP grade in prostate cancer tissues. In vitro, tumor cell migration was induced by leptin but suppressed by adiponectin. Targeting adipocytokines could be a novel treatment strategy for prostate cancer.
Prostate cancer (PCa) is the second most common malignancy worldwide (1). Established risk factors include advanced age, Black race, genetic factors, and family history (2). Obesity is also a well-known risk factor for PCa (3-5), and a high body mass index has been linked to tumor aggressiveness and worse outcomes (6).
Adipocytokines are bioactive proteins secreted from adipose tissue and include hormones, such as leptin and adiponectin (7). Individuals with obesity or metabolic syndrome tend to have increased serum leptin and decreased adiponectin (8); these adipocytokines are involved in regulating cancer cell proliferation (9, 10).
Using bladder cancer cell lines, we have previously reported that down-regulation of adiponectin expression and up-regulation of leptin expression were independent predictors of cancer recurrence and progression. We also found that adiponectin conferred protection against bladder cancer spread, whereas leptin promoted it (11).
Patients with PCa have been reported to have reduced serum adiponectin concentrations compared with individuals with benign prostatic hyperplasia, and expression of adiponectin receptors in PCa tissue was also found to be reduced (12). In contrast, high serum leptin concentration is a risk factor for high-volume PCa (13). These results suggest that adipocytokines, particularly adiponectin and leptin, are involved in PCa development. However, the precise mechanisms are unknown.
In this study, we investigated the functional roles of adiponectin and adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) and those of leptin and leptin receptor (Ob-R) in the development and progression of PCa.
Materials and Methods
Antibodies and chemicals. Antibodies against COX-2 (160112) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Anti-leptin (sc-842), anti-NF-kB (sc-109), anti-Ob-R (sc-1834), anti-AdipoR1 (sc-46748), and anti-AdipoR2 (sc-46751) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-snail (#3879), anti-slug (#9585), and anti-phospho-NF-
B (#3033) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA), and anti-adiponectin (ab22554) antibody was obtained from Abcam (Cambridge, MA, USA). AdipoRon (ENZ-CHM101) was purchased from Enzo Life Science (Lausanne, Switzerland), and leptin (L4146) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Cell lines. Human PCa PC-3, LNCaP, 22Rv1, and DU145 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). PC-3 and DU145 cells were cultured in Minimum Essential Medium, whereas LNCaP and 22Rv1 cells were cultured in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific). The cell lines were maintained in an incubator with a 5% CO2 atmosphere at 37°C.
Immunohistochemistry. A prostate tissue microarray (TMA) was constructed after obtaining appropriate approval from our institutional review board. The TMA, which consisted of prostate tissue from 51 PCa cases, was constructed from formalin-fixed paraffin-embedded specimens obtained by radical prostatectomy performed at Yokohama City University Hospital (Yokohama, Japan), as described previously (14). The clinicopathologic characteristics of these 51 patients have also been described previously (14). Briefly, the median age of the patients was 68 years (mean±SD, 65.1±11.6 years); 10 patients (19.6%) had International Society of Urological Pathology (ISUP) grade 1 disease, 28 (54.9%) had grade 2, and 13 (25.5%) had grade ≥3. Thirty patients (58.2%) had pathological T stage 2 and 20 (39.2%) had grade ≥3.
Immunohistochemistry was performed on 5-μm sections from the prostate TMA, as described previously (11), using primary antibodies (diluted 1:100) against leptin, Ob-R, adiponectin, AdipoR1, and AdipoR2, and a broad spectrum secondary antibody (Invitrogen, Carlsbad, CA, USA). Each prostate stain was manually quantified by a single pathologist (Hiroshi Miyamoto) blinded to sample identity. German immunoreactive scores were calculated by multiplying the percentage of immunoreactive cells (0%=0; 1%-10%=1; 11%-50%=2; 51%-80%=3; 81%-100%=4) by the staining intensity (negative=0; weak=1; moderate=2; strong=3). The results were classified as negative (0; 0-1), weakly positive (1+; 2-4), moderately positive (2+; 6-8), or strongly positive (3+; 9-12).
Western blot. Western blot analyses were prepared as described previously (15). Briefly, equal quantities of proteins (30 μg) obtained from cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. The membranes were incubated with each appropriately diluted antibody and developed using a chemiluminescence protocol (GE Healthcare, Milwaukee, WI, USA). Images were obtained using an image analyzer (LAS-3000 mini; Fujifilm, Tokyo, Japan).
Cell migration. A scratch wound-healing assay was performed to evaluate cell migration. Cells in 12-well plates at a density of 90%-100% confluence were scratched manually with a sterile 200-μl plastic pipette tip. After culturing for 12 h, cells were fixed with methanol and stained with 0.1% crystal violet. The width of the wound area was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Statistical analysis. All statistical analyses were performed using JMP15.0 software (SAS Institute, Cary, NC, USA). Correlations between parameters were examined by χ2 test or Student’s t-test. p-Values <0.05 were considered significant.
Results
Expression of adipocytokines and their receptors in PCa specimens. We investigated the expression of adiponectin, AdipoR1, AdipoR2, leptin, and Ob-R by immunohistochemical staining in TMAs consisting of 51 different PCa tissue samples. Positive signals were detected predominantly in the cytoplasm of prostate cells (Figure 1).
Immunohistochemical analysis of leptin, leptin receptor (Ob-R), adiponectin, and adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) in prostate cancer specimens. Expression of (A) leptin, (B) Ob-R, (C) adiponectin, (D) AdipoR1, and (E) AdipoR2 (original magnification: ×100).
Table I summarizes the expression of each protein in PCa tissues. The rate of positive adiponectin expression (0 vs. 1+/2+/3+) was not significant but tended to be lower according to ISUP grade (p=0.056).
Correlations between leptin/Ob-R/adiponectin/AdipoR1/AdipoR2 expression and tumor grade/stage of prostate cancer.
Suppression of PCa cell migration by adiponectin. First, we confirmed that all four PCa cell lines (PC3, LNCaP, 22Rv1, and DU145) expressed Ob-R and AdipoR2, and that AdipoR1 was expressed only in LNCaP and DU145 cells (Figure 2A). We then investigated whether the synthetic adiponectin AdipoRon (16) had antitumor activity in these PCa cell lines.
Effects of AdipoRon on prostate cancer cells. (A) Expression of leptin receptor (Ob-R) and adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) in PC3, LNCaP, 22Rv1, and DU145 cells. (B) Representative images of wound healing assays in LNCaP cells. After the cells grew to confluence, monolayers were gently scratched, and the wound area was measured after a 12-h culture with ethanol (control) or AdipoRon (0.5 μM). (C) Migration as determined by the proportion of LNCaP or PC3 cells filling the wound area following AdipoRon treatment relative to the control treatment in each cell line (control=100%). Error bars represent standard deviation. Each experiment was performed in triplicate. (D) LNCaP, 22Rv1 and PC3 cells were cultured with the indicated concentrations of AdipoRon (μM) for 48 h; cell lysates (30 mg) were analyzed for phosphor-NF-
B (P-NF-
B), NF-
B, snail, slug, and cyclooxygenase-2 (COX-2) using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting with specific antibodies. β-actin was used as a loading control.
Using 2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, we found that the addition of 1-10 μM of AdipoRon to LNCaP and PC3 cells for up to 72 h did not significantly affect their viability (data not shown). However, wound healing assays showed that as little as 0.5 μM of AdipoRon significantly suppressed cell migration (Figure 2B and C). Next, we evaluated the effect of AdipoRon on the expression of proteins involved in epithelial-mesenchymal transition (EMT) in PCa cells (17, 18). Results showed that AdipoRon reduced the expression of phosphor-NF-kB and cyclooxygenase-2 (COX-2) dose-dependently in LNCaP, 22Rv1, and PC3 cells, while slug was reduced only in LNCaP cells (Figure 2D).
In contrast, leptin 5 μg/ml induced cell migration (Figure 3A and B). Leptin also up-regulated phosphor-NF-kB, NF-kB, snail, and COX-2 dose-dependently in LNCaP and 22Rv1 cells, whereas slug was up-regulated only in LNCaP cells (Figure 3C). MTT assay showed that treatment with 0.1-10 μg/ml of leptin did not affect cell viability in PCa cell lines (data not shown).
Effects of leptin on prostate cancer cells. (A) Representative images of wound healing assays in PC3 cells. After the cells grew to confluence, monolayers were gently scratched, and the wound area was measured after a 12-h culture with ethanol (control) or leptin (5 μg/ml). (B) Migration as determined by the proportion of LNCaP or PC3 cells filling the wound area following leptin treatment relative to control treatment in each line (control=100). Error bars represent standard deviation. Each experiment was performed in triplicate. (C) LNCaP and 22Rv1 cells were cultured with the indicated concentrations of leptin (μg/ml) for 48 h; cell lysates (30 mg) were analyzed for phosphor-NF-
B (P-NF-
B), NF-
B, snail, slug, and cyclooxygenase-2 (COX-2) using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting with specific antibodies. β-actin was used as a loading control.
Discussion
Adipocytokines, such as adiponectin and leptin, play an important role in cancer progression (9, 10, 19) and we have previously reported that they are important prognosticators in bladder cancer (11). Since the mechanisms of their functional role in PCa are unknown, we investigated whether adipocytokines are involved in PCa progression. Our in vitro data demonstrated that leptin promoted PCa cell migration, whereas synthetic adiponectin inhibited migration. Immunohistochemistry in PCa samples revealed that adiponectin expression was not significant; however, it tended to be inversely related to ISUP grade. These data suggest that adipocytokines play important roles in PCa metastasis and targeting them may represent a new PCa treatment strategy.
The adipocytokine adiponectin has a preventive role in diseases such as diabetes by modulating inflammation and regulating glucose and lipid metabolism via insulin-sensitizing effects (20-22). It has also been reported that adiponectin suppresses the development of certain obesity-associated cancers, such as breast, liver, pancreatic, ovarian, colorectal, and prostate cancers (23).
Many investigators have examined the relationship between adipocytokines and PCa. Adiponectin concentration was found to be inversely associated with PCa risk and histological grade (24), and expression of AdipoR1 and AdipoR2 were lower in PCa samples compared with benign prostate tissue (12). Our data demonstrated that adiponectin levels tended to decrease according to ISUP grade (Table I), suggesting that the adiponectin signaling axis may mediate cancer initiation and progression. Same situation was observed in colorectal cancer where serum adiponectin concentration was inversely correlated with tumor grade (25). However, a meta-analysis investigating the association between serum concentrations of adiponectin and leptin and PCa reported that neither adipocytokine was a useful indicator of PCa initiation or progression (26). Further research is warranted.
In contrast to adiponectin, leptin was highly expressed in PCa samples compared with benign prostatic hyperplasia. Moreover, it was also highly expressed in metastatic PCa compared to localized tumors, and in diseases with a high Gleason score (8-10) compared to a low score (≤6) (27). However, the study reported no difference in leptin receptor expression between PCa and benign prostatic hyperplasia.
In an in vitro study, leptin induced both PCa cell migration and EMT via the signal transducer and activator of transcription 3 (STAT3) pathway (28), prevented apoptosis via the extracellular signal-regulated kinase 1/2 pathway (29), and increased cellular angiogenesis via the vascular endothelial growth factor pathway (30). These results suggest that local leptin expression is an important mediator of PCa progression, and that targeting the leptin axis may have antitumor effects.
Adiponectin acts by binding its specific receptors AdipoR1 and AdipoR2. The adiponectin effect has been summarized as modulating NF-kB, STAT3, AMP-activated protein kinase, and glycogen synthase kinase-3β, and suppressing cell growth, survival, and proliferation (23). In addition, adiponectin suppresses COX-2 expression in colorectal cancer cell lines and may prevent invasion and metastasis by modulating EMT (31). EMT is a key mechanism of metastasis and is thought to induce the generation of cancer stem cells in some cancers; preventing EMT could thus be a novel therapeutic strategy for cancer (32). NF-kB and COX-2 are also known to be EMT mediators, and in this study, we showed that the synthetic adiponectin AdipoRon suppressed phosphor NF-kB and COX-2 protein expression, resulting in suppressed cell migration in PCa cell lines.
AdipoRon was the first orally active adiponectin receptor agonist to be identified (16) and it has been shown to affect metabolic conditions, such as obesity and diabetes, as well as diseases with ischemic features (33). Moreover, preclinical studies have shown that it had an antitumor effect in ovarian and breast cancers (34, 35). We could not show that AdipoRon suppressed PCa cell growth (data not shown) or expression of the EMT mediator snail; however, we did observe its ability to suppress migration. We have previously reported that AdipoRon suppressed snail and slug expression in bladder cancer cell lines (11). Together these results suggest that AdipoRon suppresses cell migration, but the mechanisms differ between cancer cell types. Based on these results, activating the adiponectin axis could be a novel strategy for suppressing PCa metastasis. Adiponectin could be used as an adjunctive treatment for PCa, along with other anti-androgen receptor drugs.
Conclusion
Adiponectin and leptin are involved in PCa cell migration. Targeting adipocytokines has potential as a novel therapeutic strategy for PCa.
Acknowledgements
The Authors thank Alison McTavish, MSc, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. This work was supported by JSPS KAKENHI Grant Number 22K09477.
Footnotes
Authors’ Contributions
Eiji Kashiwagi: Conceptualization, data curation, formal analysis, investigation, methodology, validation, project administration, writing–original draft, writing–review & editing. Takashi Kawahara: Investigation, methodology. Fumio Kinoshita: Data curation, formal analysis, investigation, methodology. Masaki Shiota: Data curation. Junichi Inokuchi: Data curation. Hiroshi Miyamoto: Investigation. Masatoshi Eto: Methodology.
Conflicts of Interest
The Authors have no conflicts of interest to declare in relation to this study.
- Received January 13, 2024.
- Revision received February 4, 2024.
- Accepted February 6, 2024.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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