Abstract
Background/Aim: The efficacy of melatonin and its biological significance in human bladder cancer remain poorly understood. This study aimed to investigate the functional role of melatonin in urothelial carcinogenesis. Materials and Methods: In human normal urothelial SVHUC cells with exposure to the chemical carcinogen 3-methylcholanthrene, we assessed the effects of melatonin on the neoplastic/malignant transformation. Results: In the in vitro system with carcinogen challenge, melatonin significantly prevented the neoplastic transformation of SV-HUC-1 cells. In addition, melatonin treatment resulted in increased expression of SIRT1, Rb1, and E-cadherin, and decreased expression of N-cadherin and FGFR3 in SV-HUC-1 cells. Furthermore, publicly available datasets from GSE3167 revealed that the expression of melatonin receptor 1 and melatonin receptor 2 was significantly down-regulated in bladder urothelial carcinoma tissues, compared with adjacent normal urothelial tissues. Conclusion: These findings indicate that melatonin serves as a suppressor for urothelial tumorigenesis. To the best of our knowledge, this is the first preclinical study demonstrating the impact of melatonin on the development of urothelial cancer.
Urinary bladder cancer, mostly urothelial carcinoma, has been a common malignancy, especially in males, in various countries (1, 2). Moreover, the numbers of new bladder cancer cases and deaths from bladder cancer worldwide have even increased from 429,800 and 165,100 in 2012 (1) to 573,278 and 212,536 in 2020 (2), respectively. Approximately 75% of patients with bladder cancer present with non-muscle-invasive disease, which is typically managed with local therapy including transurethral resection of bladder tumor followed by risk adjusted intravesical treatments. However, a substantial proportion of these patients have recurrence(s) occasionally with invasive disease. Notably, muscle-invasive bladder cancer ultimately develops metastatic disease where the 5-year overall survival rate is still dismal [e.g., 8.3% (3)]. Further identification of key molecules, signaling pathways, or genes responsible for the development of urothelial carcinoma is thus anticipated to offer therapeutic options that more effectively prevent the occurrence and/or recurrence of bladder cancer.
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that was first isolated from the pineal glands and was later revealed to be present or synthesized in other organs or cells, such as the gastrointestinal tract, Harderian gland, lymphocyte, retina, and testis (4). In humans and other mammals, there are two G protein-coupled receptors that melatonin binds and activates, the melatonin receptor (MT) 1 (or MT1A) and MT2 (or MT1B) (4). Melatonin is a functionally diverse molecule and is best known as the regulation of circadian rhythm. In addition, it has a broad spectrum of biological effects particularly on oncostatic properties, such as those involving cell proliferation, migration, and invasion, as well as apoptosis, angiogenesis, inflammation, and immunoregulation (5).
Emerging evidence indicates that melatonin may inhibit the development, progression, and metastasis of several types of malignancies (6-8). In bladder cancer, limited preclinical studies have suggested suppressive effects of melatonin on tumor progression (9-12), while no clinical studies have demonstrated its therapeutic value in patients with urothelial carcinoma. Additionally, to the best of our knowledge, no preclinical or clinical studies have implicated the role of melatonin in the development of urothelial cancer. Hence, in the present study, we evaluated the impact of melatonin on urothelial carcinogenesis, using an in vitro model which is not subject to a range of potential confounders linked to sleep and hormone secretion in vivo (13).
Materials and Methods
Cell culture and chemicals. The immortalized human benign urothelial line SV-HUC-1 and the human urothelial carcinoma line TCCSUP were purchased from the American Type Culture Collection. All experiments were conducted within 20 passages. SV-HUC-1 and TCCSUP were cultured in Ham’s F-12K (Wako, Osaka, Japan) and high-glucose Dulbecco’s modified Eagle’s medium (Wako), respectively, containing 10% heat-inactivated fetal bovine serum (FBS), and penicillin (100 units/ml)-streptomycin (100 μg/ml). Cells were maintained in an incubator at 37°C in a humidified atmosphere of 5% CO2. We purchased melatonin from ICN Biochemicals (Aurora, OH, USA).
In vitro transformation. An in vitro neoplastic/malignant transformation method, using SV-HUC-1 cells with 3-methylcholanthrene (MCA) challenge, was applied, as originally described by Reznikoff and colleagues (14), with minor modifications (MCA-SV-HUC-1) (15). Briefly, cells (2×107/10-cm culture dish incubated for 48 h) were cultured in serum-free medium containing 5 μg/ml MCA (Sigma-Aldrich, St. Louis, MO, USA). After 24 h of MCA exposure, 1% FBS was added to the medium. After an additional 24 h, the cells were cultured in medium containing 5% FBS without MCA until near confluence. Subcultured cells (1:3 split ratio) were incubated in the presence of MCA for two additional 48-h exposure periods, using the above protocol. These MCA-exposed cells were then subcultured for 6 weeks in the presence or absence of melatonin in Ham’s F-12K supplemented with normal FBS and thereafter utilized for further assays.
Cell proliferation. The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenylte-trazolium bromide (MTT; Sigma-Aldrich) assay was utilized to assess the cell viability. The method used to perform MTT assay has been described previously (15).
Plate colony formation. The clonogenic assay was performed to evaluate the clonogenic potential, as described previously (15). Specifically, cells (1×103/well) seeded in 6-well tissue culture plates were incubated until colonies in the control well were easily distinguishable. The cells/colonies were then fixed with methanol and stained with 0.1% crystal violet. The number of colonies in photographed images was quantitated using ImageJ (National Institutes of Health, Bethesda, MD, USA).
Cell migration. The scratch wound-healing assay was adapted to evaluate the ability of cell migration, as described previously (15). Cells at a density of ≥90% confluence in 6-well tissue culture plates were scratched manually with a sterile 200 μl plastic pipette tip. The wounded monolayers of the cells were incubated in serum-free medium for 24 h, fixed with methanol, and stained with 0.1% crystal violet, and the width of the wound area was monitored with an inverted microscope. The normalized cell-free area in photographed pictures (24 h/0 h) was then quantitated using ImageJ software.
Western blotting. Proteins were extracted with RIPA buffer containing a protease and phosphatase inhibitor cocktail (Halt Protease and Phosphatase Inhibitor cocktail; Thermo Scientific, Rockford, IL, USA). We used Simple Western™ system to detect protein signals (16), as previously described (15). Signals were visualized using a Wes system with the Compass software (version 6.1.0) (ProteinSimple, Minneapolis, MN, USA). The primary antibodies against MT1/MT2 (clone B-8, dilution 1:50; Santa Cruz Biotechnology, Dallas, TX, USA), sirtuin1 (SIRT1; clone B-10, dilution 1:50; Santa Cruz Biotechnology), retinoblastoma 1 (clone Rb1, dilution 1:25, Santa Cruz Biotechnology), E-cadherin (clone G-10, dilution 1:250, Santa Cruz Biotechnology), N-cadherin (ab12221, dilution 1:250; Abcam, Cambridge, UK), fibroblast growth factor receptor 3 (FGFR3; clone B-9, dilution 1:25, Santa Cruz Biotechnology), and GAPDH (clone G-9, dilution 1:1,000, Santa Cruz Biotechnology) were used. The corresponding band areas of the targets were normalized by those of GAPDH.
Public database analysis. The R2 Genomics Analysis and Visualization Platform (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi, accessed on January 21, 2024) was utilized to assess the expression of MT1 and MT2 in bladder cancer and corresponding non-neoplastic bladder tissues. The datasets were obtained from the Gene Expression Omnibus repository (GSE3167) (17).
Statistical analysis. The student’s t-test was applied to assess differences in continuous variables between two groups. One-way ANOVA with Tukey’s post hoc test was used for comparison of three groups. p-Values of less than 0.05 were considered statistically significant.
Results
Expression of MT1/MT2 in human urothelial cells and urothelial carcinoma cells. We first investigated the status of MT1/MT2 protein expression in benign urothelial cells and urothelial carcinoma cells. Western blotting showed that MT1/MT2 signals were detected in both an immortalized human non-neoplastic urothelial line SV-HUC-1 and a human bladder cancer line TCCSUP, and their levels were higher in SV-HUC-1 than in TCCSUP (Figure 1). In addition, the levels of MT1/MT2 in SV-HUC-1 were considerably reduced upon MCA-mediated malignant transformation.
MT1/MT2 expression in non-neoplastic urothelial cells without and with malignant transformation and urothelial carcinoma cells. Western blotting (Simple Western™ system) of MT1/MT2 in SV-HUC-1 without MCA exposure, SV-HUC-1 undergoing malignant transformation induced by MCA, and TCCSUP. GAPDH served as an internal control. Representative images for each protein are shown. Densitometry values for MT1/MT2 were standardized by GAPDH that is relative to those of SV-HUC-1. Each value represents the mean±standard deviation (SD) from three independent experiments. Differences were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. *p<0.05 (vs. SV-HUC-1).
Impact of melatonin treatment on the neoplastic transformation of urothelial cells. We employed an established in vitro method where benign SV-HUC-1 cells could undergo the MCA-induced neoplastic/malignant transformation during the course of 6 weeks of culture (14, 15). First, SV-HUC-1 cells were exposed to the chemical carcinogen MCA for 48 h for three times. The MCA-SV-HUC-1 cells were then subcultured in the presence or absence of melatonin for 6 weeks during the transformation process. Oncogenic activity in the transformed cells was then evaluated by subsequent assays for cell viability (via the MTT assay; Figure 2A), colony forming capacity (via the clonogenic assay; Figure 2B), and cell migration ability (via the scratch wound-healing assay; Figure 2C) without further melatonin/mock treatment that could directly affect their outcomes. We thus compared the degree of neoplastic transformation in urothelial cells induced by MCA but did not aim to simply evaluate the effects of melatonin on the growth of SV-HUC-1-derived cells. Melatonin treatment in MCA-SV-HUC-1 cells significantly inhibited their malignant transformation in all assays in a dose-dependent manner.
Effects of melatonin on the neoplastic transformation of urothelial cells. SV-HUC-1 with MCA challenge subsequently cultured in the presence of dimethyl sulfoxide (mock) or Mel (melatonin; 10 μM or 100 μM) for 6 weeks, were seeded for MTT assay (A, cultured for 72 h), clonogenic assay (B, cultured for 2 weeks), or scratch wound-healing assay (C, cultured for 24 h) with no further melatonin treatment. Cell viability, colony number (≥20 cells), or width of the wound area presented relative to that of mock-treated cells. Each value represents the mean±SD from three independent experiments. The scale bars under the images indicate 200 μm. Differences were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. *p<0.05 (vs. mock treatment). #p<0.05 (vs. 10 μM melatonin treatment).
Efficacy of melatonin in oncogenic proteins in urothelial cells. We next assessed the impact of melatonin on the expression of several molecules associated with urothelial carcinogenesis, including Rb1 (18, 19), E-cadherin (15, 20), N-cadherin (15, 21), and FGFR3 (19, 22). We also evaluated the effect of melatonin on the expression of SIRT1 which could reduce reactive oxygen species (ROS) production in non-neoplastic cells (23, 24). Melatonin treatment induced the expression of SIRT1, Rb1, and E-cadherin, and reduced that of N-cadherin and FGFR3 in SV-HUC-1 cells (Figure 3).
Effects of melatonin on the expression of oncogenic molecules in urothelial cells. Western blotting (Simple Western™ system) of SIRT1, Rb1, E-cadherin, N-cadherin, and FGFR3 in SV-HUC-1 cells cultured with dimethyl sulfoxide (mock) vs. melatonin (10 μM) for 48 h. The compound-containing media was renewed every 24 h. GAPDH served as an internal control. Representative images for each protein are shown. Densitometry values for target proteins were standardized by GAPDH that is relative to those of mock treatment. Each value represents the mean±SD from three independent experiments. Differences were analyzed using the Student’s t-test. *p<0.05 (vs. mock treatment).
Expression of MT1/MT2 in bladder cancer tissues. Finally, a publicly available database was used to analyze the expression of MT1 and MT2 genes in bladder urothelial carcinomas vs. normal urothelial tissues. Data obtained from GSE3167 (17) showed that the expression levels of MT1 (Figure 4A) and MT2 (Figure 4B) were significantly lower in bladder cancer tissues than in corresponding normal urothelial tissues.
Expression levels of MT1 (A) and MT2 (B) genes in normal bladder tissues (n=9) versus bladder cancer tissues (n=40). The Gene Expression Omnibus repository datasets (GSE3167) were obtained from the R2 Genomics Analysis and Visualization Platform. Differences were analyzed using the Student’s t-test.
Discussion
There is growing evidence to suggest the inhibitory effects of melatonin on the development and progression of several types of cancers (6-8). In bladder cancer, suppression of its progression by melatonin has been documented in limited preclinical studies (9-12). However, no preclinical or clinical studies have indicated the involvement of melatonin in urothelial tumorigenesis which is generally considered to be a process distinct from tumor progression. In the present study, we therefore investigated the functional role of melatonin in the development of urothelial carcinoma.
Using a carcinogen-induced in vitro transformation model, we evaluated oncogenic activity (via cell viability, colony forming capacity, and cell migration ability), which revealed that melatonin inhibited the malignant transformation of urothelial cells in a dose-dependent manner. We then investigated the potential downstream targets linked to urothelial carcinogenesis (15, 16, 18-22) and demonstrated that melatonin treatment in benign urothelial cells increased the expression of Rb1 and E-cadherin which is frequently lost during neoplastic transformation of urothelial cells (15, 20), and reduced that of oncogenic molecules, including N-cadherin and FGFR3. Furthermore, the expression of MT1/MT2 genes and proteins was found to be down-regulated in urothelial carcinoma, compared with non-neoplastic urothelium. These findings suggested that melatonin could prevent urothelial carcinogenesis. To the best of our knowledge, this is the first study to demonstrate the impact of melatonin on the development of urothelial carcinoma.
Rb1 is encoded by RB1 tumor suppressor gene whose mutation is commonly detected in patients with bladder cancer (18, 19). FGFR3 is encoded by FGFR3 oncogenic gene whose genomic alterations are potent drivers in bladder cancer and represent predictive biomarkers of response to FGFR inhibitors, such as erdafitinib (25). Strikingly, of the molecular subtypes of muscle-invasive bladder cancer, the luminal papillary subtype was mainly enriched in FGFR3 mutations (26). Indeed, activating FGFR3 mutations have been described in 74% of non-invasive papillary urothelial carcinoma of the bladder (27). An immunohistochemical study in surgical specimens has demonstrated that FGFR3 overexpression is observed in 46.2% of muscle-invasive bladder urothelial carcinoma (28). In accordance with these genomic landscapes of bladder cancer, our observations suggest that melatonin up-regulates the expression of Rb1 and/or down-regulates that of FGFR3 in urothelial cells and thereby inhibits their malignant transformation.
Notably, epithelial-mesenchymal transition plays a critical role in various aspects of tumor progression, including tumor invasion, metastasis, and therapeutic resistance, and cadherin switching, from the expression of E-cadherin to that of N-cadherin, is its hallmark (29). An in vitro study demonstrated that melatonin inhibited cadherin switching in bladder cancer cells (30). In the present study, we demonstrated that melatonin reduced cadherin switching in SV-HUC-1 cells, further suggesting that melatonin could retard urothelial cancer development. Meanwhile, melatonin, as the main hormone involved in the control of the sleep-wake cycle, modulates clock genes through SIRT1 which is a conserved nicotinamide adenine dinucleotide-dependent deacetylase (23). Melatonin up-regulates SIRT1 expression, resulting in a reduction in ROS production and the regulation of cell homeostasis (23, 24). ROS induces DNA damage and cancer development; thus, melatonin could prevent urothelial carcinogenesis partially via SIRT1 up-regulation.
Nevertheless, further studies, particularly those in in vivo models, are needed to validate our results. In addition, the molecular mechanisms responsible for melatonin-mediated suppression of urothelial carcinogenesis need to be further elucidated. More specifically, it is generally accepted that melatonin exhibits its anticancer effect either via an interaction with its specific receptors or in a receptor-independent manner (6). Of note, melatonin has also been reported to functionally interact with steroid hormone receptors, especially the androgen receptor (5) which is known to, by itself, affect the development of bladder cancer (15, 31, 32).
Conclusion
The present results indicate that melatonin acts as a suppressor for urothelial carcinogenesis, which may thus provide a potential preventive strategy against not only the development of bladder cancer in high-risk patients but also the recurrence of superficial tumor following transurethral resection. Further assessment of melatonin, along with mechanistic details underlying its suppressive effects on tumor development, is required to determine the biological significance of melatonin and its receptors in urothelial carcinogenesis.
Acknowledgements
The authors would like to thank Miyako Shijo (the Shared-Use Research Center, University of Occupational and Environmental Health) for her technical assistance provided.
Footnotes
Conflicts of Interest
The Authors declare that they have no competing interests in relation to this study.
Authors’ Contributions
Conceptualization, Y.N.; Data curation, Y. N., N. T. Q., H. A., and H. M.; Formal analysis, Y. N., N. T. Q., H. A., K. H., H. M., and N. F; Writing – original draft, Y. N.; writing – review and editing, Y. N., K. H., H. M., and N. F. All Authors have read and agreed to the manuscript.
- Received January 21, 2024.
- Revision received May 15, 2024.
- Accepted May 24, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
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).
