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Quantitative Proteomic Analysis Reveals Caffeine-Perturbed Proteomic Profiles in Normal Bladder Epithelial Cells

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Quantitative Proteomic Analysis Reveals Caffeine-Perturbed Proteomic Profiles in Normal Bladder Epithelial Cells HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Proteomics Manuscript Author . Author manuscript; Manuscript Author available in PMC 2019 October 11. Published in final edited form as: Proteomics. 2018 October ; 18(20): e1800190. doi:10.1002/pmic.201800190. Quantitative Proteomic Analysis Reveals Caffeine-Perturbed Proteomic Profiles in Normal Bladder Epithelial Cells Muhammad Shahid1, Minhyung Kim1, Austin Yeon1, Allen M. Andres2,3, Sungyong You1,4, and Jayoung Kim1,4,5,6,¶ 1Departments of Surgery and Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA 2Department of Medicine, Cedars Sinai Medical Center, Los Angeles, CA, USA 3Cedars-Sinai Heart Institute, Los Angeles, California, CA, USA 4Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA 5University of California Los Angeles, CA, USA 6Department of Urology, Ga Cheon University College of Medicine, Incheon, South Korea Abstract Lower urinary tract symptoms (LUTS) are highly prevalent among the elderly and negatively impact quality-of-life. Since caffeinated beverages are enjoyed worldwide and the relationship between LUTS and caffeine is still not fully understood, it would be of particular interest to examine the underlying mechanisms that drive caffeine’s influence on LUTS development and progression. The aim of this study was to characterize the effects of caffeine on hTert immortalized normal bladder epithelial cells by investigating whether exposure to caffeine can cause potential changes in the bladder proteome and/or biological pathways. Labeled LC-MS/MS proteomic analysis found 57 proteins as being differentially expressed in caffeine-treated bladder epithelial cells, compared to controls; this included 32 upregulated and 25 downregulated proteins. Further functional gene enrichment analysis revealed that caffeine affected major biological pathways, including those for “muscle contraction” and “chromatin assembly”. These findings provide new scientific insights that may be useful in future studies investigating the role of caffeine in bladder dysfunctions. ¶Correspondence: Jayoung Kim, PhD., Departments of Surgery and Biomedical Sciences, Cedars-Sinai Medical Center, Davis 5071, 8700 Beverly Blvd., Los Angeles, CA 90048, Tel: +1-310-423-7168, Fax:+1-310-967-3809, [email protected]. Author contributions MS and AY involved and performed most of the experiments. SY and MK performed proteomics analysis and helped to draft paper. AA performed metabolic and biochemical analyses in vitro. JK designed and conceived the study and wrote the paper. All authors discussed the results and commented on the manuscript at all stages, and read and approved the manuscript. Conflicts of Interests The authors declare that there are no conflicts of interest regarding the publication of this paper. Shahid et al. Page 2 Author ManuscriptAuthor Keywords Manuscript Author Manuscript Author Manuscript Author Lower urinary tract symptoms; caffeine; global proteome; bladder epithelial cells; biological network INTRODUCTION It is estimated that by the end of 2018, 2.3 billion individuals will be affected by at least one lower urinary tract symptom (LUTS)[1]. These symptoms include urinary storage problems, such as urgency, frequency, nocturia, or voiding problems. LUTS also weighs heavily on overall quality of life; patients report significantly higher mental health issues, lower work productivity, and diminished general health status[2]. The current standard treatment for LUTS involves α-blockers, 5α-reductase inhibitors, and antimuscarinics[3]. However, these are mainly palliative and require consistent maintenance. Consequently, there is a substantial economic burden associated with LUTS[4]. Previous reports have demonstrated that diet and stress play important factors in the development and progression of LUTS[5]. In particular, caffeine, a naturally occurring compound, has been reported to be a potential dietary risk factor in developing LUTS[6]. Caffeine is ubiquitously found in many plants, including cocoa beans, tea leaves, and coffee beans. It is a stimulatory drug that is widely used to prevent sleepiness and can be found in over-the-counter medications, such as some pain remedies. It has also been observed that caffeine may aggravate or worsen urinary symptoms in patients who already have some form of LUTS[7]. In recent years, caffeinated drinks have become a staple of the average diet; more than 85% of adults in the U.S. regularly consume caffeine[8]. A longitudinal study of caffeine intake in young healthy volunteers found that subjects who regularly drank coffee had significant increases in urinary urgency and frequency[9]. Additionally, a separate study observed that greater coffee intake raised the odds of LUTS progression in men and women more than carbonated or citrus beverages[10]. The effects of caffeine have also been studied in the context of bladder cancer (BC); however, rather than finding a negative risk, there have been reported benefits. A case-control study of BC patients in Italy found no causal relationship between caffeine and BC[11]. A separate study found that caffeine may actually benefit BC patients by making cells more susceptible to chemotherapy and apoptosis[12]. Mechanistically, this has been reported to be mediated through caffeine’s effects on the tumor suppressor protein, p53[13]. Despite the potential link between caffeine and LUTS, research into causative mechanisms and functions is lacking. One prior study suggested that caffeine may be facilitating bladder instability and frequent urination by enhancing the activation of neuronal micturition centers through increased expression of transcription factor c-Fos and nerve growth factor[14]. While informative, this study focused primarily on the bladder muscles with little attention on the bladder epithelium, which is more anatomically exposed to urine and its biological/chemical contents. A separate study using a mouse model found that oral caffeine administration resulted in detrusor overactivity and increased bladder sensory signaling[15]. Further studies Proteomics. Author manuscript; available in PMC 2019 October 11. Shahid et al. Page 3 found similar effects on the detrusor muscle in humans[16]. However, the direct effects of Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author caffeine, its delivery into cells, and its mechanisms remain unestablished. Our present study sought to examine the cellular effects of caffeine on the bladder epithelium without any pathological conditions. Quantitative and global proteomics analysis found that caffeine can perturb the whole proteome, possibly through the regulation of chromatin assembly in normal bladder epithelial cells. MATERIALS AND METHODS Cell culture and cell proliferation assay. Immortalized normal human bladder epithelial cells, TRT-HU1, were maintained as described previously[17]. The TRT-HU1 cell line was constructed and extensively characterized in previously published papers. The passage number of each cell line was below 10, and mycoplasma contamination was tested for monthly via PCR analysis. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal bovine serum (FBS, Invitrogen), 1% penicillin/streptomycin, and 1% L-glutamine (Sigma- Aldrich Corp., St. Louis, MO, USA) in a 37 °C humidified incubator with 5% CO2. To test cell growth during exposure to caffeine, TRT-HU1 cells were seeded onto 6-well plates with a density of 5 × 104 cells/well. Cells were then incubated with standard growth medium with varying doses of caffeine at 0, 0.005 mM, 0.05 mM, or 0.1 mM (C6035, Sigma-Aldrich Corp.) or vehicle for 24, 48, or 72 hrs. Cell proliferation was measured by manually counting cells using a hemocytometer. The averages of each count were used as the total density of the well after each time point. For crystal violet staining, the culture medium was removed, the cells were fixed with 4% paraformaldehyde at room temperature for 5 min and then stained with 0.05% crystal violet for 15 min. The cells were then washed with tap water, after which the water was removed and the cells were dried out on filter paper, and the cell plates were scanned and quantified as described previously[18]. All experiments were run in triplicate for each cell line, and the data are representative of three independent trials. Antibodies and reagents. Antibodies against various proteins were obtained from the following sources: ACTG2 (ab189385, Abcam), ACTA2 (ab5694, Abcam), MYH2 (ab124937, Abcam), MYH7B (ab172967, Abcam), HISTH2B (ab52599, Abcam), HIST1H2BM (SAB1301739, Sigma), and β-actin (A1978, Sigma). Commercially available horseradish peroxidase (HRP)- conjugated secondary antibodies (7074, 7076) were obtained from Cell Signaling Technology. All other chemical reagents were procured from Sigma Chemical Corp. Quantitative proteomics. Tandem mass tagging (TMT)-based quantitative proteomics was performed as described[19]. Briefly, cellular protein was extracted from caffeine-treated and control cells using 4% SDS- containing buffer. The protein concentration was measured using the Pierce 660nm Assay Kit. From each sample, 60 μg of protein was digested with trypsin using filter-aided sample preparation (FASP) and labeled with TMT6plex reagents in parallel. After TMT labeling, the peptides were merged, desalted with C18 spin columns (Thermo Scientific), and Proteomics. Author manuscript; available in PMC 2019 October
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