Serotonin Transporter Expression and DNA Methylation are Altered by Coffee Exposure in Caco-2 Cells

Serotonin Transporter Expression and DNA Methylation are Altered by Coffee Exposure in Caco-2 Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Serotonin Transporter Expression and DNA Methylation are Altered by Coffee Exposure in Caco-2 Cells Satoshi Kikkawa, Miki Bundo, Emi Kiyota, Serika Imamura, Kana Harada, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8214835/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Consumption of coffee is associated with a reduced risk of colorectal cancer (CRC); however, the underlying mechanisms are not fully understood. Gut serotonin (5-HT) plays a complex role in CRC development. Intestinal 5-HT levels are regulated by the serotonin transporter (SERT), in intestinal epithelial cells. Recent evidence suggests a correlation between SERT expression and DNA methylation of a functional CpG site (CpG3) in the promoter region of SLC6A4 , the SERT gene. This study investigated the effects of coffee on SERT expression in Caco-2 cells, an intestinal epithelial cell model. Exposure of Caco-2 cells to instant coffee solution 1-10% (v/v) for 48 h was found to result in a concentration-dependent decrease in SERT-mediated 5-HT uptake and SERT mRNA expression. This effect was observed for different instant coffee brands and coffee bean species, and were not reproduced by exposure to major coffee components such as caffeine and chlorogenic acid, or by extracts from unroasted green coffee beans. Pyrosequencing revealed that coffee exposure altered the DNA methylation of CpG3. This preliminary study suggests a novel mechanism by which coffee protects against CRC: suppression of SERT expression in intestinal epithelial cells, possibly via epigenetic modification of SLC6A4 . Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Genetics Biological sciences/Molecular biology Colon cancer serotonin transporter epigenetic regulation Caco-2 cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 New & Noteworthy Coffee consumption is linked to a reduced risk of colorectal cancer (CRC), but the mechanism involving intestinal serotonin is unclear. This study found that a 48-hour coffee exposure in intestinal epithelial cells reduced serotonin transporter (SERT) expression alongside epigenetic changes. This proposed mechanism may explain coffee's preventive effects against CRC and other associated diseases. Introduction Serotonin (5-HT), a neurotransmitter synthesized from tryptophan (Trp), plays diverse roles in both the central and peripheral nervous systems. The synthesis of 5-HT is mediated by two distinct rate-limiting enzymes, tryptophan hydroxylase (TPH) 1 and TPH 2. TPH2 regulates 5-HT synthesis in neurons of the central and enteric nervous systems, whereas TPH1 is responsible for 5-HT production by enterochromaffin (EC) cells in the intestinal mucosa 1 , 2 . Cellular uptake of 5-HT is primarily mediated by the Na + /Cl − -dependent serotonin transporter (SERT), which is expressed in intestinal epithelial cells and platelets 3 . Platelets transport 5-HT to the peripheral tissues, where they exert various physiological effects. Free 5-HT is known to influence intestinal motility 4 . SERT, particularly in intestinal epithelial cells, is considered a key regulator of 5-HT concentration in intestinal tissues, because intracellular 5-HT is inactivated by monoamine oxidase (MAO) 5 , 6 . Colorectal cancer (CRC) is the third most common type of cancer worldwide. The incidence and mortality rates of CRC are projected to increase, particularly in developing countries, making it crucial to understand its pathogenesis 7 , 8 . Intestinal 5-HT has been shown to have dual and opposing roles in CRC development 9 . A pioneering study using mice with intestinal epithelial cell-specific deletion of TPH1 demonstrated that 5-HT protects intestinal stem cells from DNA damage and early tumorigenesis but promotes the growth of established tumors 10 . This finding suggests that the modulation of intestinal SERT expression or activity could influence both the prevention and progression of CRC. SERT is encoded by the SLC6A4 gene located on chromosome 17 (17q11-17q12) 11 . Two mechanisms have been implicated in the regulation of SLC6A4 expression: a functional polymorphism in the promoter region, known as the serotonin transporter-linked polymorphic region (5-HTTLPR), and methylation of CpG sites near the SLC6A4 promoter 12 , 13 . The 5-HTTLPR comprises 14 or 16 repeats of a 20–23 bp highly homologous sequence, known as the short (S) and long (L) alleles, respectively 14 . The S allele is generally associated with a reduced transcriptional activity 15 . Furthermore, Sugawara et al. (2011) and Ikegame et al. (2020) identified a functional CpG site (chr17:30,235,246 − 30,235,247) on the CpG island shore of the SLC6A4 promoter and reported that DNA methylation at this site suppresses transcriptional activity 16 , 17 . However, most studies of these mechanisms have focused on psychiatric disorders, and their relevance to CRC remains largely unknown. Numerous environmental factors, including diet and lifestyle, contribute to and prevent CRC. Coffee is one such factor that has been extensively studied, owing to its status as one of the most consumed beverages worldwide. Several studies have suggested that coffee consumption is associated with a reduced risk of CRC, potentially through caffeine-independent mechanisms 18 – 21 . Indeed, a 2011 meta-analysis of 15 prospective studies demonstrated that high coffee consumption was associated with a lower risk of CRC than no or low consumption 22 . Studies reporting this protective effect have often linked it to the consumption of approximately ≥ 4 cups per day 23 . The relationship between coffee consumption and health extends beyond CRC. A J-shaped relationship between coffee consumption and mortality from various diseases has been consistently observed 24 – 27 . For example, a protective effect against cardiovascular disease development and mortality is evident at moderate consumption levels, whereas excessive intake may be counterproductive 28 . Other large-scale studies have consistently confirmed an inverse correlation between coffee consumption and all-cause mortality 29 . Despite these epidemiological findings, evidence regarding CRC remains inconclusive, and the underlying mechanisms are poorly understood. This variability may be influenced by differences in the study design 29 . Notably, both systemic and CRC-specific beneficial effects of coffee are widely attributed to components other than caffeine 29 , 30 . Coffee contains two major groups of bioactive compounds: phenolic compounds, primarily chlorogenic acid (CGA), and alkaloids such as caffeine and trigonelline. The profile of these phenolic antioxidants is significantly affected not only by roasting 31 but also by various processing methods 32 . Furthermore, coffee melanoidins, which possess high antioxidant activity 33 , may incorporate CGA derivatives during roasting 34 . Recent research has begun to explore the molecular impact of these components, revealing their involvement in the activation of the aryl hydrocarbon receptor (AhR) and suggesting that they may exert a global DNA methylation inhibitory effect while inducing site-specific DNA methylation changes 35 , 36 . Given that approximately 8 L of fluid is secreted and absorbed in the gastrointestinal lumen daily, and assuming the consumption of ≥ 4 cups of coffee per day (with each cup estimated at 227 mL, a standard coffee cup), it can be deduced that intestinal epithelial cells are exposed to coffee at an average concentration of more than 10% within the luminal fluid 37 . However, the specific roles of organic compounds in the physiological response to coffee consumption remain unclear 38 . Therefore, further research is required to clarify the pathways responsible for the observed reduction in CRC risk. We hypothesized that coffee may reduce CRC risk, in part, by decreasing SERT expression in intestinal epithelial cells, thereby increasing 5-HT availability in the gut. This study investigated the effects of coffee on SERT expression using Caco-2 cells, a model intestinal epithelial cell line that expresses SERT. We report that coffee exposure suppresses SERT expression, and that this effect is accompanied by changes in CpG3 methylation. Materials and methods Medium and buffer MEMα with L-Glutamine and Phenol Red (MEMα) was procured from FUJIFILM Wako Pure Chemical Industries, Ltd (Tokyo, Japan). Fetal Bovine Serum (FBS) was acquired from NICHIREI BIOSCIENCE, INC. (Tokyo, Japan). The 0.5 g/L-Trypsin/0.53 mmol/L-EDTA Solution (trypsin) and Penicillin-Streptomycin Mixed Solution (P/S) were obtained from Nacalai Tesque (Kyoto, Japan). The Krebs-Ringer-HEPES (KRH) buffer contained the following: 120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl 2 , 25 mM HEPES, 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , and 10 mM Glucose, adjusted to pH 7.4. The radioimmunoprecipitation assay (RIPA) buffer contained 10 mM Tris-HCl, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA, adjusted to pH 7.4. Reagents Chlorogenic acid (5-CQA), D(-)-Quinic acid (QA), and Trigonelline Hydrochloride (TRG) were procured from Sigma-Aldrich (St. Louis, MO, USA). Caffeic Acid (CafA) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Nicotinic Acid (NA) was acquired from Nacalai Tesque. These were dissolved in DMSO (Sigma-Aldrich) to a concentration of 100 mM and preserved at -30°C until just before use. D(-)-Mannitol (Mannitol) was purchased from Fujifilm. Caffeine was procured from Fujifilm. The p38-MAPK pathway inhibitor (SB203580) was purchased from Cayman Chemical (Ann Arbor, MI, USA). PKA inhibitor (KT5720) and AhR inhibitor (SR-1) were obtained from Selleck (Yokohama, Japan). Hydroxytryptamine (5-HT), 5-[1,2-3H(N)]-, 1mCi ([ 3 H] 5-HT) were acquired from PerkinElmer (Waltham, MA, USA). Fluvoxamine maleate (Fluvoxamine) was sourced from Tocris Bioscience (Bristol, UK). Instant coffee samples The manufacturer of the instant coffee utilized has been documented, and the specifications are listed in Table 1 . Table 1 List of instant coffee users Products Manufacturer Coffee Bean Type Place of origin Abbreviations NESCAFÉ Gold Blend* Nestlé Japan Limited Blend (unknown) Colombia, Honduras or Colombia, Indonesia Regular coffee/coffee NESCAFÉ Gold Blend Decaffeinated* Nestlé Japan Limited Blend (unknown) Vietnam, Colombia Decaf coffee Organic Mountain Colombian Coffee Growers Federation Arabica Colombia Arabica Robusta Freeze Dried Instant Coffee Helena., JSC Robusta Vietnam Robusta MAXIM® Ajinomoto AGF, Inc. Blend (unknown) Vietnam, Brazil, or other AGF The Blend 117 Instant Coffee UCC Ueshima Coffee Co., Ltd. Blend (unknown) Brazil, Papua New Guinea, or other UCC Doutor Fragrant Delicious Cup Instant Coffee Doutor Coffee Co., Ltd. Blend (unknown) Vietnam, Laos Doutor *For the NESCAFÉ Gold Blend series, "regular soluble coffee" is the official term used by Nestlé Japan. Coffee Preparation Instant coffee was dissolved directly in the room-temperature buffered solution. Following the manufacturer's instructions, a mixture of 2 g instant coffee and 140 mL KRH buffer was prepared. After centrifuging at 1,000 rpm for one minute, the supernatant was designated as 100% concentrated coffee (100%(v/v)), equivalent to the typical concentration of coffee used in common applications. For the treatment of cells with coffee, a coffee solution was added to the cell culture medium to achieve the appropriate concentration. KRH buffer alone was added to the control samples. Raw beans and roasted coffee bean samples Green (unroasted) Brazil Santos No.2 coffee beans, a blend of Arabica varieties ( Caturra, Bourbon, Catuai, and Mundo Novo) grown in Terra Rossa soil, were donated by the Nishinaya Coffee Corporation (Hiroshima, Japan). The coffee beans were roasted using a TORNADO KING roaster (I.C. Electronics Industry Co., Ltd.). Following the manufacturer's guidelines, beans were roasted to two different degrees: light roasted (Light) and Italian roasted (Italian). Green coffee beans and both roasts (Light and Italian) were ground using a manual mill and then brewed with 95°C distilled water using a French press coffee maker (Bodum, Inc.) according to the manufacturer's instructions (Supplemental Fig. 1). The resulting extracts were cryogenically preserved at -80°C and thawed immediately before experimentation. Cell Culture Caco-2 cells derived from human colon adenocarcinoma were acquired from Riken Cell Bank (Tsukuba, Japan) at passage 46. Cells between passages 61 and 76 (15th–30th passage after acquisition) were used for experiments. MEMα medium supplemented with 10% FBS and 1% P/S was used for cell cultivation. Cells were cultured and passaged in 100 mm Thermo Scientific™ Nunc™ EasYDishes. Cells were passaged using a conventional protocol involving washing with PBS and trypsin detachment. To minimize the influence of spontaneous differentiation on experimental outcomes, cells were passaged at 50% confluency (approximately every 3 days) according to Natoli et al. (2011) 39 and seeded at a density of 0.25 × 10 6 cells/dish. For experiments, cells were seeded in Costar® 24-well Clear TC-treated Multiple Well Plates (Corning, NY, USA), or 60 mm Thermo Scientific™ Nunc™ EasYDishes, at a density of 0.2 × 10 5 cells/cm 2 and cultured for predetermined periods. To ensure even cell distribution, cells were seeded in 24-well plates, left undisturbed for 20 min in an incubator, and then gently agitated back and forth and side to side. Two days later, uniform cell distribution was verified, and the cells were used for the experiments. The procedure for preparing the Caco-2 cells used in this analysis is shown in Fig. 1 . Briefly, cells were evenly spread and cultured for approximately 3 days until confluence was reached, followed by an additional 7 days of culture to promote spontaneous differentiation into intestinal epithelial-like cells. After two days of drug treatment, the cells were used for various analyses, including 5-HT uptake assays and RT-PCR. The culture medium was replaced every three days. 5-HT Uptake Assay The assay employed freshly prepared KRH buffer containing 100 µM pargyline, 100 µM L-ascorbic acid, and 100 nM [ 3 H]5-HT. KRH buffer containing 10 µM Fluvoxamine was used to measure non-specific 5-HT uptake. Caco-2 cells cultured for 10 d (7 d post-confluence) in 24-well plates were used for the assay. The medium in each well was removed and rinsed with 200 µL of KRH buffer. Subsequently, 200 µL of the radiolabeled KRH buffer was added to each well and incubated at 37°C for 15 min. Non-specific uptake was measured in wells containing KRH buffer with 10 µM Fluvoxamine. After incubation, wells were washed twice with 200 µL of KRH buffer containing 10 µM Fluvoxamine (wash buffer). After removing the wash buffer, 750 µL of RIPA buffer was added, followed by a 45-minute incubation at 37°C. Cells were solubilized in RIPA buffer by pipetting. For scintillation counting, 500 µL of the cell lysate was mixed with 4 mL of Clear-sol II (Nacalai Tesque) in scintillation tubes. The radioactivity was quantified using an AccuFLEX LSC-8000 liquid scintillation counter (Aloka Co., Ltd. Musashino, Japan). The protein concentration was determined using the Protein Assay BCA Kit (Nacalai Tesque) with bovine serum albumin standards (Thermo Fisher Scientific) according to the manufacturer's instructions. The absorbance was measured at 562 nm using a Model 680 microplate reader (Bio-Rad). The measured radioactivity was normalized to the protein concentration to calculate [ 3 H]5-HT uptake per unit protein mass. Quantitative Real-time PCR (qRT-PCR) Caco-2 cells were detached from a 60 mm dish, and total RNA was extracted using the RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Total RNA was reverse-transcribed into cDNA using the QuantiTect® Reverse Transcription Kit (QIAGEN). The expression of SLC6A4 mRNA (coding for SERT) was quantified using pre-designed TaqMan® FAM™-labeled MGB primer-probe sets (Hs00984349_m1; Thermo Fisher Scientific). GAPDH mRNA expression was quantified using PrimeTime qPCR assay (Integrated DNA Technologies). qPCR was performed using the THUNDERBIRD qPCR Mix (TOYOBO, Osaka, Japan). Target genes were amplified and quantified using a 7300 Real-Time PCR System (Applied Biosystems). The relative expression levels of SLC6A4 mRNA were calculated using the 2 −ΔΔCT method with GAPDH as the reference gene. DNA Methylation Analysis The procedures were primarily conducted using optimized methods based on previous reports 16 , 17 DNA Extraction and Bisulfite Modification Genomic DNA was extracted from Caco-2 cells detached from a 60 mm dish using the AllPrep DNA/RNA/Protein Mini Kit (QIAGEN) according to the manufacturer's instructions. The extracted DNA was bisulfite-converted using an EpiTect Fast Bisulfite Conversion Kit (QIAGEN). Bisulfite PCR Amplification The SLC6A4 CpG island shore (chr17:30,235,139 − 30,235,342 in GRCh38/hg38) was amplified by bisulfite PCR using the following primers: forward, 5'-TTTTAGTTGTTTGGTATTTGTGTTA-3'; reverse (5'-biotinylated), 5'-AAAACTTTACAACCTCTTAAAAACCC-3. ’ Bisulfite PCR amplification was performed in a total volume of 50 µL containing 5 µL of 10×PCR Amplification Buffer (Invitrogen), 10 µL of 5 M Betaine (Sigma Aldrich), 3 µL of 50 mM MgCl2 (Invitrogen), 1 µL of 10 mM dNTP (Invitrogen), 2 µL of each 10 µM primer, 2 ng of Single-Stranded DNA Binding Protein (Promega), 5 U of Platinum Taq DNA Polymerase (Invitrogen), and 1 µL of bisulfite-modified genomic DNA. The thermocycling conditions involved an initial incubation at 95°C for 3 min, followed by 40 cycles of 98°C for 10 s, 55°C for 30 s, and 72°C for 15 s. All PCR amplicons were verified by electrophoresis using a MultiNA System (SHIMAZU). Pyrosequencing Bisulfite PCR fragments were prepared for pyrosequencing using a PyroMark Q96 Vacuum Workstation (QIAGEN). A mixture consisting of 25 µL of bisulfite PCR product, 2 µL of streptavidin sepharose beads (Amersham Biosciences), and 40 µL of PyroMark Binding Buffer (QIAGEN) was prepared, and the plate was vortexed at 1400 RPM at room temperature for 10 min. Subsequently, the filter probe of the vacuum tool was inserted into the plate to capture PCR products and beads. The filter probe was then transferred into 100 mL of 70% ethanol for 5 s, then into Denaturation Solution (0.2 N NaOH) for 5 s, and finally into washing buffer for 10 s. The captured beads were transferred to a PSQ 96 Plate (QIAGEN) containing 48 µL of PyroMark Annealing Buffer (QIAGEN), 1 µL of 10 µM sequencing primer (5'-AATATAAATTATGGTTGAA-3'), and 1 µL of diluted single-stranded DNA-binding protein, gently stirring the tool in the well. This plate was heated at 90℃ for 3 min to denature the DNA and then cooled to room temperature to anneal the sequencing primer. Pyrosequencing was performed using a PSQ 96MA system (QIAGEN) according to the manufacturer's instructions. DNA methylation was measured at two CpG sites within the SLC6A4 CpG island shore: CpG3 (chr17:30,235,246 − 30,235,247) and CpG4 (chr17:30,235,271 − 30,235,272; GRCh38/hg38). The DNA methylation levels of each CpG were calculated using PSQ 96MA software (QIAGEN). Statistical Analysis Statistical analyses and figure creation were performed using R version 4.3.1. Specific statistical tests included the Dunnett’s test, two-sample t-test, and Tukey’s multiple comparison test. Statistical significance was defined as a p-value < 0.05. Results Expression of SERT in Caco-2 Cells SERT mRNA expression in Caco-2 cells cultured in 60 mm dishes was measured using quantitative real-time PCR (qRT-PCR), which showed that Caco-2 cells expressed SERT. SERT expression varied with the passage number (Supplemental Fig. 2). Effect of Coffee on SERT Uptake Activity The effect of coffee on SERT uptake activity in Caco-2 cells was examined. To investigate the effect of coffee on SERT activity, Caco-2 cells were exposed to various concentrations of instant coffee solution for 48 h. Both regular (NESCAFÉ Gold Blend) and decaffeinated coffee (NESCAFÉ Gold Blend Decaffeinated) decreased SERT uptake activity in a concentration-dependent manner (Fig. 2 A). Globally, coffee is primarily derived from two species: Coffae arabica L . and Coffae canephora var. robusta. There were discernible differences in the principal constituent concentrations between the two types of coffee produced from these species, Arabica and Robusta coffee, and in the presence of variety-specific components. Experiments were conducted using instant coffee manufactured solely from beans of each species. Arabica-derived instant coffee and robusta-derived instant coffee were administered at a concentration of 10% to CaCo-2 cells for 48 h, and both coffees significantly reduced SERT uptake activity (Fig. 2 B, left). To assess reproducibility, instant coffee samples from different manufacturers were tested. All the tested instant coffees showed decreased SERT uptake activity (Fig. 2 B, right). The mechanism through which coffee regulates SERT uptake was investigated. In Caco-2 cells, the cAMP/PKA pathway decreased SERT uptake via SERT phosphorylation (Latorre et al., 2016). To test the involvement of the cAMP/PKA pathway, a PKA inhibitor (KT5720, 1 µM) was used. PKA inhibition did not affect coffee-induced decrease in SERT uptake (Fig. 2 C). Coffee is a weakly acidic, hypotonic solution. When dissolved in PBS (pH 7.4), the resulting solution had a pH of approximately 6 and an osmolality of 50 mOsm/L. To test the effects of acidity and osmolarity, the cells with PBS adjusted to pH 6 with HCl and a medium containing 50 mM mannitol were tested. Neither acidity nor osmolarity affected SERT uptake activity (Supplemental Fig. 3). Effect of Coffee on SERT mRNA Expression Since coffee reduced SERT uptake activity, we hypothesized that coffee might also affect SERT expression levels. We measured the SERT mRNA expression in Caco-2 cells after 48-hour exposure to various concentrations of instant coffee solution. Both regular and decaffeinated coffee decreased the SERT expression in a concentration-dependent manner (Fig. 3 A). This decrease in SERT mRNA expression was observed in instant coffee from different varieties (Fig. 3 B), which is consistent with the decrease in SERT uptake activity. Furthermore, the effects of short-term (6-hour) coffee exposure on SERT mRNA expression were investigated. This short exposure significantly reduced SERT mRNA expression (Fig. 4 A). To assess the reversibility of the coffee-induced changes in SERT expression, Caco-2 cells were exposed to coffee for 48 h, washed twice with PBS, and then cultured in fresh MEMα. The SERT mRNA expression levels were measured every 24 h. SERT expression returned to the control levels within 24 h of coffee washout (Fig. 4 B). These results suggest that the effect of coffee on SERT mRNA expression occurs relatively early and is due to reversible transcriptional regulation. Role of Coffee Roasting and Components on SERT mRNA expression Since coffee composition varies with roasting 38 , we investigated the effects of extracts from green and roasted coffee beans (light and Italian roasts, refer to Supplemental Fig. 1) on SERT mRNA expression. Only the extracts from roasted coffee beans decreased SERT expression (Fig. 5 A). Moreover, the effects of the major coffee components on SERT mRNA expression were tested using the appropriate concentrations reported in the literature. 38,40–42 . Caffeine (1 or 5 mM) (Fig. 5 B), chlorogenic acid and its degradation products, and trigonelline and its degradation products did not affect SERT mRNA expression (Fig. 5 C). These results suggest that the effect of coffee on SERT mRNA expression is not due to caffeine or other well-known components of coffee but rather to substances produced during roasting. Signaling Pathways and Epigenetic Modifications Related to Coffee-Induced SERT mRNA Decrease Little is known about the signaling pathways that regulate SERT expression. In Caco-2 and intestinal epithelial cells, the p38 MAPK pathway may be involved in the reduction of SERT expression 43 , 44 . It has been known that coffee components are ligands for the aromatic hydrocarbon receptor (AhR), and a functional correlation exists between SERT activity and the AhR pathway 45 – 48 . To test the involvement of the p38 MAPK and AhR pathways, we used a p38 MAPK inhibitor (SB203580, 20 µM) and an AhR inhibitor (SR-1, 1 µM). Neither p38 MAPK nor AhR inhibition affected SERT mRNA expression (Fig. 6 A and 6 B, respectively). Because SERT expression is associated with psychiatric disorders, we hypothesized that epigenetic modifications, such as DNA methylation, might be involved in coffee-induced changes in SERT expression. Previous studies have identified functional methylation sites (CpG3 and CpG4) on the CpG island shore of SLC6A4 that are associated with bipolar disorder and reduced SERT expression 17 (Fig. 7 A). High CpG3 methylation has also been observed in schizophrenia, suggesting a role for CpG3 methylation in the regulation of SERT expression 16 . To investigate the effect of coffee on SLC6A4 methylation, Caco-2 cells were exposed to decaffeinated coffee for 48 h. Genomic DNA was extracted, bisulfite-converted, and analyzed by pyrosequencing to determine the methylation rates of CpG3 and CpG4. Coffee exposure significantly increased methylation at CpG3 by 3.7% (decaffeinated coffee) and showed a trend toward increased methylation at CpG3 by 2.8% (regular coffee) (Fig. 7 B, left). Exposure to coffee did not affect the methylation at CpG4 (Fig. 7 B, right panel). Discussion To the best of our knowledge, this study provides the first evidence that coffee exposure induces concentration-dependent suppression of SLC6A4 mRNA expression (Fig. 3 A) and an associated reduction in 5-HT uptake activity (Fig. 2 A) in a human intestinal epithelial cell model. Critically, this transcriptional repression was demonstrated to correlate with a significant increase in DNA methylation at a specific CpG site (CpG3) located on the CpG island shore of the SLC6A4 gene (Fig. 7 B). This SERT suppression represents a highly robust phenomenon rather than an artifact of a single coffee preparation. This effect was consistently observed across different brands of instant coffee, the major coffee species, Coffea arabica and Coffea robusta, and both caffeinated and decaffeinated coffee (Fig. 2 A, 2 B, 3 A, 3 B). Notably, the observation that decaffeinated coffee exhibited an effect comparable to that of caffeinated coffee strongly indicated that caffeine was not the principal causative agent of the observed action. Collectively, these observations indicate that the bioactive substance(s) responsible for suppressing SERT expression are components universally present in roasted coffee beans rather than specific to a particular cultivar or brand. Our findings strongly suggest that the active ingredient responsible for SERT suppression is not a major component originally present in green coffee beans but rather a substance generated or released during the thermal processing of roasting. First, the most common bioactive components in coffee were excluded based on our negative data. Caffeine (up to 5 mM), the primary alkaloid in coffee, did not affect SERT mRNA expression (Fig. 5 B). Similarly, 5-CQA, a major polyphenol abundant in green beans and its principal degradation products, caffeic acid and quinic acid, failed to suppress SERT expression (Fig. 5 C). Furthermore, trigonelline, another major alkaloid, and its primary metabolite nicotinic acid were inactive (Fig. 5 C). Although these compounds have been widely studied for their association with the health benefits of coffee, our findings demonstrated that they are not directly involved in the specific phenomenon of SERT suppression observed here. These data implied that the active component was a substance other than the major constituents. One of the most compelling pieces of evidence in this study is the stark contrast in the effects of roasted and unroasted green bean extracts. Only extracts prepared from light- and Italian-roasted beans significantly suppressed SERT mRNA expression, whereas extracts from green beans showed no such effect (Fig. 5 A). This result unequivocally indicates that a bioactive compound is generated during the thermal processing of coffee beans 49 . For example, coffee melanoidins, which possess high antioxidant activity, are degradation products of 5-CQA formed during roasting 50 . Therefore, it is highly plausible that the active component(s) that suppress SERT expression reside within these roasted products, which warrants further investigation. Coffee-Induced Epigenetic Modifications This study demonstrated that coffee exposure specifically increased the methylation level at the CpG3 site by approximately 3–4%, while it had no effect on the CpG4 site (Fig. 7 B). Although this absolute change appears modest, its biological significance is likely substantial. The CpG3 site is located in a region known as the "CpG island shore," adjacent to the conventional CpG island. Recent epigenomic studies revealed that tissue-specific or disease-associated methylation changes that strongly correlate with gene expression often occur in these shore regions rather than in the CpG islands themselves 51 . The specificity of the effect on CpG3, but not on CpG4, suggests that this phenomenon is likely a targeted regulatory event rather than a non-specific genome-wide methylation change. The coffee components may induce site-specific DNA methylation changes and exhibit general inhibitory effects on DNA methylation 35 , 36 . Another critical finding of this study was the rapid and reversible nature of SERT suppression. A significant decrease in SERT mRNA was observed after 6 h of coffee exposure (Fig. 4 A), and upon removal of coffee from the medium (washout), the expression levels returned to baseline within 24 h (Fig. 4 B). Although the precise mechanisms underlying CpG3 hypermethylation and its associated transcriptional repression remain to be elucidated, these results indicate that this transcriptional repression occurs swiftly and is likely reversible. Changes in DNA methylation during short-term coffee exposure have not been verified; however, DNA methylation alterations occurring within hours have been confirmed in vivo via fear conditioning and physical stimuli 52 , 53 . Whether coffee induced similar immediate changes remains to be determined. Considering the interaction between multiple epigenetic regulatory mechanisms 54 , further investigation is imperative to elucidate the causal relationship between the transcriptional repression of SERT mRNA and hypermethylation at CpG3 55 Relationship Between 5-HTTLPR and Transcription Levels The 5-HTTLPR in the Caco-2 cells was L A /L A . This is not surprising, given that it is a cell line derived from a Caucasian donor. The extent to which this genetic polymorphism affects intestinal SERT expression remains unclear. DNA methylation of SLC6A4 is 5-HTTLPR genotype-dependent 16 , 56 , and samples harboring the S allele may yield different results. However, pioneering detailed examinations of the SERT promoter region suggested a potential lack of a functional association between specific promoter regions regulating intestinal epithelial SERT transcription and 5-HTTLPR 57 . The CpG3 focused on in this study is located downstream of Exon1A on the island shore of CpG. The strong correlation between CpG3 hypermethylation and either promoter region remains a subject for future investigation. Limitations Our study has several limitations. First, this study was a basic investigation reporting only in vitro experimental results. Additional studies are required to determine whether the SERT expression-suppressing effect of coffee can be reproduced in living organisms. Further verification is needed to determine whether the observed decrease in SERT expression occurs in vivo , considering anatomical and physiological conditions. As mentioned above, orally ingested fluids come into contact with epithelial cells in the upper gastrointestinal tract for a short period and remain in the small intestine for several hours 58 . The extent and duration of coffee exposure to epithelial cells at these anatomical sites, particularly under conditions of excessive coffee intake, are issues for future research. When using Caco-2 cells derived from colon adenocarcinoma, it is necessary to keep in mind that they have a different transcriptional profile than that of normal cells 59 . Moreover, several factors such as passage number, number of replications, and culture conditions significantly affect the performance of Caco-2 cells 60 . It is crucial to recognize that Caco-2 cells exhibit phenotypes of both small intestinal epithelial cells (enterocytes) and large intestinal epithelial cells (colonocytes) 61 . Although T84 cells are considered an excellent model of the colonocyte epithelium 62 , Caco-2 cells were chosen for this study, specifically for stable SERT expression 63 . Furthermore, the CpG3 site that we focused on in this study is not present in rodents; therefore, it was necessary to use human-derived cells for the experiments. This study did not identify the components involved in the transcriptional repression of SERT. This is due to the large number of components in coffee. Nevertheless, our data provided some insights. SERT expression decreased in both regular and decaffeinated coffee, but this phenomenon was not reproduced by caffeine alone. Since reproducibility was observed with instant coffees sold by different beverage manufacturers, instant coffees made from Arabica and Robusta beans, and coffee made from roasted coffee beans, the involvement of universal components common to coffee is expected. The increase in reactions due to roasting suggests the influence of degradation products of green coffee components or newly generated substances. Conclusion This study demonstrates that coffee modulates SERT expression in a Caco-2 intestinal epithelial cell model. This finding offers a novel perspective, suggesting that the preventive effects of coffee against CRC may involve modulation of SERT-dependent intra- and extracellular 5-HT activities 9 . Declarations Conflict of interest The authors have no conflicts of interest regarding this study. Funding This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Sports, and Culture, Japan (JSPS KAKENHI Grant Numbers 19H03409, 21K06802, 22K06862, 22K20993, 23K06361, 23K19402, 25K18967, 25K10182). It was also supported by grants from the Takeda Science Foundation, the Uehara Memorial Foundation and Smoking Research Foundation. Author Contribution Satoshi Kikkawa: Conceived and designed research, Analyzed data, Performed experiments, Interpreted results of experiments, Prepared figures, Drafted manuscript, Edited and revised the manuscript, Approved final version of the manuscript. Miki Bundo: Conceived and designed research, Interpreted results of experiments, Edited and revised the manuscript. Emi Kiyota: Conceived and designed research, Interpreted results of experiments. Serika, Imamura: Conceived and designed research, Resources, Funding acquisition. Kana Harada: Interpreted results of experiments, Resources, Funding acquisition. Hiroko Shiraki: Interpreted results of experiments, Resources, Funding acquisition. Shigeru Tanaka: Interpreted results of experiments, Resources, Funding acquisition. Kazuya Iwamoto: Conceived and designed research, Interpreted results of experiments, Edited and revised the manuscript, Approved final version of the manuscript. Norio Sakai: Conceived and designed research, Analyzed data, Interpreted results of experiments, Prepared figures, Drafted manuscript, Edited and revised the manuscript, Approved final version of the manuscript Resources, Funding acquisition. Data Availability All relevant data are within the manuscript and its [Supporting information](https:/journals.plos.org/plosone/article?id=10.1371/journal.pone.0263395) files. References Li, D., Yang, Y., Li, Y. P., Zhu, X. H. & Li, Z. Q. Epigenetic regulation of gene expression in response to environmental exposures: From bench to model. Sci. 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1","display":"","copyAsset":false,"role":"figure","size":149434,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental procedures of the present study using Caco-2 cells.\u003c/p\u003e\n\u003cp\u003eCells were evenly spread and cultured for approximately 3 days until confluence was reached, followed by an additional 7 days of culture to promote spontaneous differentiation into intestinal epithelial-like cells. After two days of drug treatment, the cells were used for various analyses, including 5-HT uptake assays and RT-PCR. The culture medium was replaced every three days.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8214835/v1/485d1540de49d14e780421cf.png"},{"id":97666224,"identity":"dc031ff3-f202-42a4-a094-a5cbad14be4c","added_by":"auto","created_at":"2025-12-08 09:20:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96905,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 48-hour exposure to coffee on SERT uptake activity.\u003c/p\u003e\n\u003cp\u003eA: Changes in SERT Uptake Activity Due to Instant Coffee.\u003c/p\u003e\n\u003cp\u003eThe vertical axis represents SERT uptake activity relative to the control group. The horizontal axis represents the coffee concentration (v/v%). Regular coffee and Decaf coffee indicated NESCAFÉ Gold Blend and NESCAFÉ Gold Blend Decaffeinated, respectively, as described in Table 1. Three independent experiments, Mean ± SD, Dunnet test, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003eB: Effect of Coffee Varieties and Manufacturers' Products on SERT Uptake Activity.\u003c/p\u003e\n\u003cp\u003eThe vertical axis represents the relative SERT uptake activity in the control group at a coffee concentration of 10% (v/v). The left panel shows the results for Arabica and Robusta coffee varieties. The right panel shows the results using instant coffee products from three different manufacturers, labeled A, B, and C. Details of the instant coffee products used are presented in Table 1. Three independent experiments, Mean ± SD, Dunnet test, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003eC: Investigation of Post-Translational Modifications\u003c/p\u003e\n\u003cp\u003eThe vertical axis represents SERT uptake activity relative to the control group at a coffee concentration of 10 (v/v%). DMSO and KT5720(1 μM) were simultaneously treated with coffee for 48 h. Three independent experiments, Mean ± SD, Two Sample t-test, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8214835/v1/e5441decdcc027438ce7e860.png"},{"id":97418138,"identity":"36a09c88-054d-48c3-b892-5112290920a0","added_by":"auto","created_at":"2025-12-04 07:49:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":85014,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 48-hour exposure to coffee on SERT uptake activity.\u003c/p\u003e\n\u003cp\u003eA: Changes in SERT expression due to instant coffee\u003c/p\u003e\n\u003cp\u003eThe vertical axis represents the SERT expression relative to that in the control group. The horizontal axis represents the coffee concentration (v/v%). Regular coffee and Decaf coffee indicated NESCAFÉ Gold Blend and NESCAFÉ Gold Blend Decaffeinated, respectively, as described in Table 1. 4 independent experiments, Mean ± SD, Dunnet test, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003eB: Effect of Coffee varieties on SERT mRNA expression.\u003c/p\u003e\n\u003cp\u003eThe vertical axis represents the SERT expression relative to that in the control group at a coffee concentration of 10% (v/v). Arabica and Robusta are organic, mountainous, and robust freeze-dried instant coffee, respectively, as described in Table 1.Threeindependent experiments, Mean ± SD, Dunnet test, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8214835/v1/1f4ec9146b5c67f0564d1507.png"},{"id":97666627,"identity":"f0be7cb3-f330-4c57-9c09-0330e8e4e309","added_by":"auto","created_at":"2025-12-08 09:21:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":79275,"visible":true,"origin":"","legend":"\u003cp\u003eExamination of time-dependent effects of coffee on SERT mRNA expression.\u003c/p\u003e\n\u003cp\u003eA: The effect of short-term (6-hour) coffee exposure on SERT mRNA expression.\u003c/p\u003e\n\u003cp\u003eThe vertical axis represents the SERT expression relative to that in the control group at a coffee concentration of 10% (v/v). 3 independent experiments, Mean ± SD, Two Sample t-test, ***p \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003eB: SERT Expression variation after the washout of coffee.\u003c/p\u003e\n\u003cp\u003eLeft image: experimental protocol. Right image: The vertical axis represents the relative expression of SERT compared to the previous time point. The coffee concentration was 10% (v/v). Three independent experiments, Mean ± SD, Two Sample t-test, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8214835/v1/3eb7a13dbabd6793065cef67.png"},{"id":97666607,"identity":"8af1021e-793d-4d38-ba71-249bf94d93d7","added_by":"auto","created_at":"2025-12-08 09:21:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":88343,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of Coffee roasting degree and major coffee components on SERT expression\u003c/p\u003e\n\u003cp\u003eA: Impact of coffee roasting degree on SERT expression\u003c/p\u003e\n\u003cp\u003eCoffee samples with different levels of roasting were prepared as described in the Materials and Methods section and Supplemental figure 1. Three types of roasted coffee (raw, light, and Italian) were used in this study. The vertical axis represents the SERT expression relative to that in the control group at a coffee concentration of 10% (v/v). Three independent experiments, Mean ± SD, Dunnet test, **p \u0026lt; 0.01.\u003c/p\u003e\n\u003cp\u003eB: Effects of caffeine on SERT mRNA expression\u003c/p\u003e\n\u003cp\u003eThe cells were treated with caffeine (1 and 5 mM). The vertical axis represents the SERT expression relative to that in the control group. Three independent experiments, Mean ± SD, Dunnet test.\u003c/p\u003e\n\u003cp\u003eC: Effects of major coffee components on SERT Expression\u003c/p\u003e\n\u003cp\u003eIn the left and right graphs, all reagents were used at concentrations of 100 μM. Abbreviations are as follows: Chlorogenic acid (5-CQA), Caffeic Acid (CafA), D(-)-Quinic acid (QA), Trigonelline Hydrochloride (TRG), and Nicotinic Acid (NA). 3 independent experiments, Mean ± SD, Dunnet test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8214835/v1/92ed6d368761a135b492b90c.png"},{"id":97418144,"identity":"02e1668f-dc6b-4942-b785-3dca6bc88a21","added_by":"auto","created_at":"2025-12-04 07:49:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":73531,"visible":true,"origin":"","legend":"\u003cp\u003eSignaling pathway involved in \u0026nbsp;coffee-induced decrease of SERT ｍRNA expression\u003c/p\u003e\n\u003cp\u003eA: Effects of p38 MAPK inhibitor SB203580 on SERT mRNA expression. DMSO or SB203580 (20 μM) was simultaneously applied with 10% coffee or buffer (control). The vertical axis represents SERT expression relative to the control group. Three independent experiments, Mean ± SD, Tukey multiple comparisons of means. a: vs Ctl / DMSO, b: vs Ctl / SB203580, p \u0026lt; 0.01.\u003c/p\u003e\n\u003cp\u003eB: Effects of the AhR inhibitor SR-1 on SERT mRNA expression. DMSO or SR-1 (1 μΜ) was simultaneously applied with the coffee or buffer (control). The vertical axis represents the SERT expression relative to that in the control group. Three independent experiments, Mean ± SD, Tukey multiple comparisons of means. a: vs Ctl / DMSO, b: vs Ctl / SR-1, p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8214835/v1/9b66aa1259dfb64626e23849.png"},{"id":97666379,"identity":"949d3f0c-1872-4c31-939b-edcdc8a08262","added_by":"auto","created_at":"2025-12-08 09:21:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":103255,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of coffee on epigenomic modification of SERT CpG islands.\u003c/p\u003e\n\u003cp\u003eA. Location of CpG islands (CpG3 and CpG4) in the SERT promoter regions.\u003c/p\u003e\n\u003cp\u003eB: Effects of coffee on the methylation rates of CpG3 and CpG4. Caco-2 cells were exposed to a 10% concentration of coffee for 48 h, followed by bisulfite conversion and pyrosequencing to measure the methylation rate. The vertical axis represents the methylation rate (%) at each CpG site. The coffee concentration was 10% (v/v). 4 independent experiments, Mean ± SD, Dunnet test.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8214835/v1/b3251bf26d467936ed74a18d.png"},{"id":97677473,"identity":"9ce74c5c-fcb8-4059-a780-471e9b8c5045","added_by":"auto","created_at":"2025-12-08 09:53:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1597179,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8214835/v1/2767cc76-81bb-48be-b940-893dcff304b6.pdf"},{"id":97667127,"identity":"3d1eadb6-0399-454c-b28d-2f58dc3107c1","added_by":"auto","created_at":"2025-12-08 09:22:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2845063,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8214835/v1/b72062ded4058fda0acefefe.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Serotonin Transporter Expression and DNA Methylation are Altered by Coffee Exposure in Caco-2 Cells","fulltext":[{"header":"New \u0026 Noteworthy","content":"\u003cp\u003eCoffee consumption is linked to a reduced risk of colorectal cancer (CRC), but the mechanism involving intestinal serotonin is unclear. This study found that a 48-hour coffee exposure in intestinal epithelial cells reduced serotonin transporter (SERT) expression alongside epigenetic changes. This proposed mechanism may explain coffee\u0026apos;s preventive effects against CRC and other associated diseases.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eSerotonin (5-HT), a neurotransmitter synthesized from tryptophan (Trp), plays diverse roles in both the central and peripheral nervous systems. The synthesis of 5-HT is mediated by two distinct rate-limiting enzymes, tryptophan hydroxylase (TPH) 1 and TPH 2. TPH2 regulates 5-HT synthesis in neurons of the central and enteric nervous systems, whereas TPH1 is responsible for 5-HT production by enterochromaffin (EC) cells in the intestinal mucosa \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Cellular uptake of 5-HT is primarily mediated by the Na\u003csup\u003e+\u003c/sup\u003e/Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e-dependent serotonin transporter (SERT), which is expressed in intestinal epithelial cells and platelets \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Platelets transport 5-HT to the peripheral tissues, where they exert various physiological effects. Free 5-HT is known to influence intestinal motility \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. SERT, particularly in intestinal epithelial cells, is considered a key regulator of 5-HT concentration in intestinal tissues, because intracellular 5-HT is inactivated by monoamine oxidase (MAO) \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eColorectal cancer (CRC) is the third most common type of cancer worldwide. The incidence and mortality rates of CRC are projected to increase, particularly in developing countries, making it crucial to understand its pathogenesis \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Intestinal 5-HT has been shown to have dual and opposing roles in CRC development \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. A pioneering study using mice with intestinal epithelial cell-specific deletion of TPH1 demonstrated that 5-HT protects intestinal stem cells from DNA damage and early tumorigenesis but promotes the growth of established tumors \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This finding suggests that the modulation of intestinal SERT expression or activity could influence both the prevention and progression of CRC.\u003c/p\u003e\u003cp\u003eSERT is encoded by the \u003cem\u003eSLC6A4\u003c/em\u003e gene located on chromosome 17 (17q11-17q12) \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Two mechanisms have been implicated in the regulation of \u003cem\u003eSLC6A4\u003c/em\u003e expression: a functional polymorphism in the promoter region, known as the serotonin transporter-linked polymorphic region (5-HTTLPR), and methylation of CpG sites near the \u003cem\u003eSLC6A4\u003c/em\u003e promoter \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The 5-HTTLPR comprises 14 or 16 repeats of a 20\u0026ndash;23 bp highly homologous sequence, known as the short (S) and long (L) alleles, respectively \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The S allele is generally associated with a reduced transcriptional activity \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Furthermore, Sugawara et al. (2011) and Ikegame et al. (2020) identified a functional CpG site (chr17:30,235,246\u0026thinsp;\u0026minus;\u0026thinsp;30,235,247) on the CpG island shore of the \u003cem\u003eSLC6A4\u003c/em\u003e promoter and reported that DNA methylation at this site suppresses transcriptional activity \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, most studies of these mechanisms have focused on psychiatric disorders, and their relevance to CRC remains largely unknown.\u003c/p\u003e\u003cp\u003eNumerous environmental factors, including diet and lifestyle, contribute to and prevent CRC. Coffee is one such factor that has been extensively studied, owing to its status as one of the most consumed beverages worldwide. Several studies have suggested that coffee consumption is associated with a reduced risk of CRC, potentially through caffeine-independent mechanisms \u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Indeed, a 2011 meta-analysis of 15 prospective studies demonstrated that high coffee consumption was associated with a lower risk of CRC than no or low consumption \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Studies reporting this protective effect have often linked it to the consumption of approximately\u0026thinsp;\u0026ge;\u0026thinsp;4 cups per day \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe relationship between coffee consumption and health extends beyond CRC. A J-shaped relationship between coffee consumption and mortality from various diseases has been consistently observed \u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. For example, a protective effect against cardiovascular disease development and mortality is evident at moderate consumption levels, whereas excessive intake may be counterproductive \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Other large-scale studies have consistently confirmed an inverse correlation between coffee consumption and all-cause mortality \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite these epidemiological findings, evidence regarding CRC remains inconclusive, and the underlying mechanisms are poorly understood. This variability may be influenced by differences in the study design \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Notably, both systemic and CRC-specific beneficial effects of coffee are widely attributed to components other than caffeine \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Coffee contains two major groups of bioactive compounds: phenolic compounds, primarily chlorogenic acid (CGA), and alkaloids such as caffeine and trigonelline. The profile of these phenolic antioxidants is significantly affected not only by roasting \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e but also by various processing methods \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Furthermore, coffee melanoidins, which possess high antioxidant activity \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, may incorporate CGA derivatives during roasting \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRecent research has begun to explore the molecular impact of these components, revealing their involvement in the activation of the aryl hydrocarbon receptor (AhR) and suggesting that they may exert a global DNA methylation inhibitory effect while inducing site-specific DNA methylation changes \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Given that approximately 8 L of fluid is secreted and absorbed in the gastrointestinal lumen daily, and assuming the consumption of \u0026ge;\u0026thinsp;4 cups of coffee per day (with each cup estimated at 227 mL, a standard coffee cup), it can be deduced that intestinal epithelial cells are exposed to coffee at an average concentration of more than 10% within the luminal fluid \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. However, the specific roles of organic compounds in the physiological response to coffee consumption remain unclear \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Therefore, further research is required to clarify the pathways responsible for the observed reduction in CRC risk.\u003c/p\u003e\u003cp\u003eWe hypothesized that coffee may reduce CRC risk, in part, by decreasing SERT expression in intestinal epithelial cells, thereby increasing 5-HT availability in the gut. This study investigated the effects of coffee on SERT expression using Caco-2 cells, a model intestinal epithelial cell line that expresses SERT. We report that coffee exposure suppresses SERT expression, and that this effect is accompanied by changes in CpG3 methylation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMedium and buffer\u003c/h2\u003e\u003cp\u003eMEMα with L-Glutamine and Phenol Red (MEMα) was procured from FUJIFILM Wako Pure Chemical Industries, Ltd (Tokyo, Japan). Fetal Bovine Serum (FBS) was acquired from NICHIREI BIOSCIENCE, INC. (Tokyo, Japan). The 0.5 g/L-Trypsin/0.53 mmol/L-EDTA Solution (trypsin) and Penicillin-Streptomycin Mixed Solution (P/S) were obtained from Nacalai Tesque (Kyoto, Japan). The Krebs-Ringer-HEPES (KRH) buffer contained the following: 120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 25 mM HEPES, 1.2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 1.2 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, and 10 mM Glucose, adjusted to pH 7.4. The radioimmunoprecipitation assay (RIPA) buffer contained 10 mM Tris-HCl, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA, adjusted to pH 7.4.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eReagents\u003c/h3\u003e\n\u003cp\u003eChlorogenic acid (5-CQA), D(-)-Quinic acid (QA), and Trigonelline Hydrochloride (TRG) were procured from Sigma-Aldrich (St. Louis, MO, USA). Caffeic Acid (CafA) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Nicotinic Acid (NA) was acquired from Nacalai Tesque. These were dissolved in DMSO (Sigma-Aldrich) to a concentration of 100 mM and preserved at -30\u0026deg;C until just before use. D(-)-Mannitol (Mannitol) was purchased from Fujifilm. Caffeine was procured from Fujifilm. The p38-MAPK pathway inhibitor (SB203580) was purchased from Cayman Chemical (Ann Arbor, MI, USA). PKA inhibitor (KT5720) and AhR inhibitor (SR-1) were obtained from Selleck (Yokohama, Japan). Hydroxytryptamine (5-HT), 5-[1,2-3H(N)]-, 1mCi ([\u003csup\u003e3\u003c/sup\u003eH] 5-HT) were acquired from PerkinElmer (Waltham, MA, USA). Fluvoxamine maleate (Fluvoxamine) was sourced from Tocris Bioscience (Bristol, UK).\u003c/p\u003e\n\u003ch3\u003eInstant coffee samples\u003c/h3\u003e\n\u003cp\u003eThe manufacturer of the instant coffee utilized has been documented, and the specifications are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eList of instant coffee users\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProducts\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eManufacturer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoffee Bean Type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePlace of origin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAbbreviations\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNESCAF\u0026Eacute; Gold Blend*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNestl\u0026eacute; Japan Limited\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBlend (unknown)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eColombia, Honduras or Colombia, Indonesia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRegular coffee/coffee\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNESCAF\u0026Eacute; Gold Blend Decaffeinated*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNestl\u0026eacute; Japan Limited\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBlend (unknown)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVietnam, Colombia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDecaf coffee\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOrganic Mountain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColombian Coffee Growers Federation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eArabica\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eColombia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eArabica\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRobusta Freeze Dried Instant Coffee\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHelena., JSC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRobusta\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVietnam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRobusta\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMAXIM\u0026reg;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAjinomoto AGF, Inc.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBlend (unknown)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVietnam, Brazil, or other\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAGF\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThe Blend 117 Instant Coffee\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUCC Ueshima Coffee Co., Ltd.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBlend (unknown)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBrazil, Papua New Guinea, or other\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUCC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDoutor Fragrant Delicious Cup Instant Coffee\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDoutor Coffee Co., Ltd.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBlend (unknown)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVietnam, Laos\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDoutor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e*For the NESCAF\u0026Eacute; Gold Blend series, \"regular soluble coffee\" is the official term used by Nestl\u0026eacute; Japan.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eCoffee Preparation\u003c/h3\u003e\n\u003cp\u003eInstant coffee was dissolved directly in the room-temperature buffered solution. Following the manufacturer's instructions, a mixture of 2 g instant coffee and 140 mL KRH buffer was prepared. After centrifuging at 1,000 rpm for one minute, the supernatant was designated as 100% concentrated coffee (100%(v/v)), equivalent to the typical concentration of coffee used in common applications. For the treatment of cells with coffee, a coffee solution was added to the cell culture medium to achieve the appropriate concentration. KRH buffer alone was added to the control samples.\u003c/p\u003e\n\u003ch3\u003eRaw beans and roasted coffee bean samples\u003c/h3\u003e\n\u003cp\u003eGreen (unroasted) Brazil Santos No.2 coffee beans, a blend of Arabica varieties ( Caturra, Bourbon, Catuai, and Mundo Novo) grown in Terra Rossa soil, were donated by the Nishinaya Coffee Corporation (Hiroshima, Japan). The coffee beans were roasted using a TORNADO KING roaster (I.C. Electronics Industry Co., Ltd.). Following the manufacturer's guidelines, beans were roasted to two different degrees: light roasted (Light) and Italian roasted (Italian). Green coffee beans and both roasts (Light and Italian) were ground using a manual mill and then brewed with 95\u0026deg;C distilled water using a French press coffee maker (Bodum, Inc.) according to the manufacturer's instructions (Supplemental Fig.\u0026nbsp;1). The resulting extracts were cryogenically preserved at -80\u0026deg;C and thawed immediately before experimentation.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCell Culture\u003c/h2\u003e\u003cp\u003eCaco-2 cells derived from human colon adenocarcinoma were acquired from Riken Cell Bank (Tsukuba, Japan) at passage 46. Cells between passages 61 and 76 (15th\u0026ndash;30th passage after acquisition) were used for experiments. MEMα medium supplemented with 10% FBS and 1% P/S was used for cell cultivation. Cells were cultured and passaged in 100 mm Thermo Scientific\u0026trade; Nunc\u0026trade; EasYDishes. Cells were passaged using a conventional protocol involving washing with PBS and trypsin detachment.\u003c/p\u003e\u003cp\u003eTo minimize the influence of spontaneous differentiation on experimental outcomes, cells were passaged at 50% confluency (approximately every 3 days) according to Natoli et al. (2011) \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and seeded at a density of 0.25 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/dish. For experiments, cells were seeded in Costar\u0026reg; 24-well Clear TC-treated Multiple Well Plates (Corning, NY, USA), or 60 mm Thermo Scientific\u0026trade; Nunc\u0026trade; EasYDishes, at a density of 0.2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e and cultured for predetermined periods.\u003c/p\u003e\u003cp\u003eTo ensure even cell distribution, cells were seeded in 24-well plates, left undisturbed for 20 min in an incubator, and then gently agitated back and forth and side to side. Two days later, uniform cell distribution was verified, and the cells were used for the experiments. The procedure for preparing the Caco-2 cells used in this analysis is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Briefly, cells were evenly spread and cultured for approximately 3 days until confluence was reached, followed by an additional 7 days of culture to promote spontaneous differentiation into intestinal epithelial-like cells. After two days of drug treatment, the cells were used for various analyses, including 5-HT uptake assays and RT-PCR. The culture medium was replaced every three days.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e5-HT Uptake Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe assay employed freshly prepared KRH buffer containing 100 \u0026micro;M pargyline, 100 \u0026micro;M L-ascorbic acid, and 100 nM [\u003csup\u003e3\u003c/sup\u003eH]5-HT. KRH buffer containing 10 \u0026micro;M Fluvoxamine was used to measure non-specific 5-HT uptake.\u003c/p\u003e\u003cp\u003eCaco-2 cells cultured for 10 d (7 d post-confluence) in 24-well plates were used for the assay. The medium in each well was removed and rinsed with 200 \u0026micro;L of KRH buffer. Subsequently, 200 \u0026micro;L of the radiolabeled KRH buffer was added to each well and incubated at 37\u0026deg;C for 15 min. Non-specific uptake was measured in wells containing KRH buffer with 10 \u0026micro;M Fluvoxamine. After incubation, wells were washed twice with 200 \u0026micro;L of KRH buffer containing 10 \u0026micro;M Fluvoxamine (wash buffer). After removing the wash buffer, 750 \u0026micro;L of RIPA buffer was added, followed by a 45-minute incubation at 37\u0026deg;C. Cells were solubilized in RIPA buffer by pipetting. For scintillation counting, 500 \u0026micro;L of the cell lysate was mixed with 4 mL of Clear-sol II (Nacalai Tesque) in scintillation tubes. The radioactivity was quantified using an AccuFLEX LSC-8000 liquid scintillation counter (Aloka Co., Ltd. Musashino, Japan).\u003c/p\u003e\u003cp\u003eThe protein concentration was determined using the Protein Assay BCA Kit (Nacalai Tesque) with bovine serum albumin standards (Thermo Fisher Scientific) according to the manufacturer's instructions. The absorbance was measured at 562 nm using a Model 680 microplate reader (Bio-Rad).\u003c/p\u003e\u003cp\u003eThe measured radioactivity was normalized to the protein concentration to calculate [\u003csup\u003e3\u003c/sup\u003eH]5-HT uptake per unit protein mass.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eQuantitative Real-time PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eCaco-2 cells were detached from a 60 mm dish, and total RNA was extracted using the RNeasy\u0026reg; Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Total RNA was reverse-transcribed into cDNA using the QuantiTect\u0026reg; Reverse Transcription Kit (QIAGEN). The expression of \u003cem\u003eSLC6A4\u003c/em\u003e mRNA (coding for SERT) was quantified using pre-designed TaqMan\u0026reg; FAM\u0026trade;-labeled MGB primer-probe sets (Hs00984349_m1; Thermo Fisher Scientific). GAPDH mRNA expression was quantified using PrimeTime qPCR assay (Integrated DNA Technologies). qPCR was performed using the THUNDERBIRD qPCR Mix (TOYOBO, Osaka, Japan). Target genes were amplified and quantified using a 7300 Real-Time PCR System (Applied Biosystems). The relative expression levels of \u003cem\u003eSLC6A4\u003c/em\u003e mRNA were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method with GAPDH as the reference gene.\u003c/p\u003e\n\u003ch3\u003eDNA Methylation Analysis\u003c/h3\u003e\n\u003cp\u003eThe procedures were primarily conducted using optimized methods based on previous reports \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eDNA Extraction and Bisulfite Modification\u003c/h2\u003e\u003cp\u003eGenomic DNA was extracted from Caco-2 cells detached from a 60 mm dish using the AllPrep DNA/RNA/Protein Mini Kit (QIAGEN) according to the manufacturer's instructions. The extracted DNA was bisulfite-converted using an EpiTect Fast Bisulfite Conversion Kit (QIAGEN).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eBisulfite PCR Amplification\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eSLC6A4\u003c/em\u003e CpG island shore (chr17:30,235,139\u0026thinsp;\u0026minus;\u0026thinsp;30,235,342 in GRCh38/hg38) was amplified by bisulfite PCR using the following primers: forward, 5'-TTTTAGTTGTTTGGTATTTGTGTTA-3'; reverse (5'-biotinylated), 5'-AAAACTTTACAACCTCTTAAAAACCC-3. \u0026rsquo; Bisulfite PCR amplification was performed in a total volume of 50 \u0026micro;L containing 5 \u0026micro;L of 10\u0026times;PCR Amplification Buffer (Invitrogen), 10 \u0026micro;L of 5 M Betaine (Sigma Aldrich), 3 \u0026micro;L of 50 mM MgCl2 (Invitrogen), 1 \u0026micro;L of 10 mM dNTP (Invitrogen), 2 \u0026micro;L of each 10 \u0026micro;M primer, 2 ng of Single-Stranded DNA Binding Protein (Promega), 5 U of Platinum Taq DNA Polymerase (Invitrogen), and 1 \u0026micro;L of bisulfite-modified genomic DNA. The thermocycling conditions involved an initial incubation at 95\u0026deg;C for 3 min, followed by 40 cycles of 98\u0026deg;C for 10 s, 55\u0026deg;C for 30 s, and 72\u0026deg;C for 15 s. All PCR amplicons were verified by electrophoresis using a MultiNA System (SHIMAZU).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePyrosequencing\u003c/h2\u003e\u003cp\u003eBisulfite PCR fragments were prepared for pyrosequencing using a PyroMark Q96 Vacuum Workstation (QIAGEN). A mixture consisting of 25 \u0026micro;L of bisulfite PCR product, 2 \u0026micro;L of streptavidin sepharose beads (Amersham Biosciences), and 40 \u0026micro;L of PyroMark Binding Buffer (QIAGEN) was prepared, and the plate was vortexed at 1400 RPM at room temperature for 10 min. Subsequently, the filter probe of the vacuum tool was inserted into the plate to capture PCR products and beads. The filter probe was then transferred into 100 mL of 70% ethanol for 5 s, then into Denaturation Solution (0.2 N NaOH) for 5 s, and finally into washing buffer for 10 s. The captured beads were transferred to a PSQ 96 Plate (QIAGEN) containing 48 \u0026micro;L of PyroMark Annealing Buffer (QIAGEN), 1 \u0026micro;L of 10 \u0026micro;M sequencing primer (5'-AATATAAATTATGGTTGAA-3'), and 1 \u0026micro;L of diluted single-stranded DNA-binding protein, gently stirring the tool in the well. This plate was heated at 90℃ for 3 min to denature the DNA and then cooled to room temperature to anneal the sequencing primer. Pyrosequencing was performed using a PSQ 96MA system (QIAGEN) according to the manufacturer's instructions. DNA methylation was measured at two CpG sites within the \u003cem\u003eSLC6A4\u003c/em\u003e CpG island shore: CpG3 (chr17:30,235,246\u0026thinsp;\u0026minus;\u0026thinsp;30,235,247) and CpG4 (chr17:30,235,271\u0026thinsp;\u0026minus;\u0026thinsp;30,235,272; GRCh38/hg38). The DNA methylation levels of each CpG were calculated using PSQ 96MA software (QIAGEN).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses and figure creation were performed using R version 4.3.1. Specific statistical tests included the Dunnett\u0026rsquo;s test, two-sample t-test, and Tukey\u0026rsquo;s multiple comparison test. Statistical significance was defined as a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eExpression of SERT in Caco-2 Cells\u003c/h2\u003e\u003cp\u003eSERT mRNA expression in Caco-2 cells cultured in 60 mm dishes was measured using quantitative real-time PCR (qRT-PCR), which showed that Caco-2 cells expressed SERT. SERT expression varied with the passage number (Supplemental Fig.\u0026nbsp;2).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eEffect of Coffee on SERT Uptake Activity\u003c/h2\u003e\u003cp\u003eThe effect of coffee on SERT uptake activity in Caco-2 cells was examined. To investigate the effect of coffee on SERT activity, Caco-2 cells were exposed to various concentrations of instant coffee solution for 48 h. Both regular (NESCAF\u0026Eacute; Gold Blend) and decaffeinated coffee (NESCAF\u0026Eacute; Gold Blend Decaffeinated) decreased SERT uptake activity in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGlobally, coffee is primarily derived from two species: \u003cem\u003eCoffae arabica L\u003c/em\u003e. and \u003cem\u003eCoffae canephora var. robusta.\u003c/em\u003e There were discernible differences in the principal constituent concentrations between the two types of coffee produced from these species, Arabica and Robusta coffee, and in the presence of variety-specific components. Experiments were conducted using instant coffee manufactured solely from beans of each species. Arabica-derived instant coffee and robusta-derived instant coffee were administered at a concentration of 10% to CaCo-2 cells for 48 h, and both coffees significantly reduced SERT uptake activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, left).\u003c/p\u003e\u003cp\u003eTo assess reproducibility, instant coffee samples from different manufacturers were tested. All the tested instant coffees showed decreased SERT uptake activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, right).\u003c/p\u003e\u003cp\u003eThe mechanism through which coffee regulates SERT uptake was investigated. In Caco-2 cells, the cAMP/PKA pathway decreased SERT uptake via SERT phosphorylation (Latorre et al., 2016). To test the involvement of the cAMP/PKA pathway, a PKA inhibitor (KT5720, 1 \u0026micro;M) was used. PKA inhibition did not affect coffee-induced decrease in SERT uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eCoffee is a weakly acidic, hypotonic solution. When dissolved in PBS (pH 7.4), the resulting solution had a pH of approximately 6 and an osmolality of 50 mOsm/L. To test the effects of acidity and osmolarity, the cells with PBS adjusted to pH 6 with HCl and a medium containing 50 mM mannitol were tested. Neither acidity nor osmolarity affected SERT uptake activity (Supplemental Fig.\u0026nbsp;3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eEffect of Coffee on SERT mRNA Expression\u003c/h2\u003e\u003cp\u003eSince coffee reduced SERT uptake activity, we hypothesized that coffee might also affect SERT expression levels. We measured the SERT mRNA expression in Caco-2 cells after 48-hour exposure to various concentrations of instant coffee solution. Both regular and decaffeinated coffee decreased the SERT expression in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This decrease in SERT mRNA expression was observed in instant coffee from different varieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), which is consistent with the decrease in SERT uptake activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, the effects of short-term (6-hour) coffee exposure on SERT mRNA expression were investigated. This short exposure significantly reduced SERT mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess the reversibility of the coffee-induced changes in SERT expression, Caco-2 cells were exposed to coffee for 48 h, washed twice with PBS, and then cultured in fresh MEMα. The SERT mRNA expression levels were measured every 24 h. SERT expression returned to the control levels within 24 h of coffee washout (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results suggest that the effect of coffee on SERT mRNA expression occurs relatively early and is due to reversible transcriptional regulation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eRole of Coffee Roasting and Components on SERT mRNA expression\u003c/h2\u003e\u003cp\u003eSince coffee composition varies with roasting \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, we investigated the effects of extracts from green and roasted coffee beans (light and Italian roasts, refer to Supplemental Fig.\u0026nbsp;1) on SERT mRNA expression. Only the extracts from roasted coffee beans decreased SERT expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMoreover, the effects of the major coffee components on SERT mRNA expression were tested using the appropriate concentrations reported in the literature. \u003csup\u003e38,40\u0026ndash;42\u003c/sup\u003e. Caffeine (1 or 5 mM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), chlorogenic acid and its degradation products, and trigonelline and its degradation products did not affect SERT mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eThese results suggest that the effect of coffee on SERT mRNA expression is not due to caffeine or other well-known components of coffee but rather to substances produced during roasting.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eSignaling Pathways and Epigenetic Modifications Related to Coffee-Induced SERT mRNA Decrease\u003c/h2\u003e\u003cp\u003eLittle is known about the signaling pathways that regulate SERT expression. In Caco-2 and intestinal epithelial cells, the p38 MAPK pathway may be involved in the reduction of SERT expression \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. It has been known that coffee components are ligands for the aromatic hydrocarbon receptor (AhR), and a functional correlation exists between SERT activity and the AhR pathway \u003csup\u003e\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. To test the involvement of the p38 MAPK and AhR pathways, we used a p38 MAPK inhibitor (SB203580, 20 \u0026micro;M) and an AhR inhibitor (SR-1, 1 \u0026micro;M). Neither p38 MAPK nor AhR inhibition affected SERT mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, respectively).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBecause SERT expression is associated with psychiatric disorders, we hypothesized that epigenetic modifications, such as DNA methylation, might be involved in coffee-induced changes in SERT expression. Previous studies have identified functional methylation sites (CpG3 and CpG4) on the CpG island shore of \u003cem\u003eSLC6A4\u003c/em\u003e that are associated with bipolar disorder and reduced SERT expression \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). High CpG3 methylation has also been observed in schizophrenia, suggesting a role for CpG3 methylation in the regulation of SERT expression \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate the effect of coffee on \u003cem\u003eSLC6A4\u003c/em\u003e methylation, Caco-2 cells were exposed to decaffeinated coffee for 48 h. Genomic DNA was extracted, bisulfite-converted, and analyzed by pyrosequencing to determine the methylation rates of CpG3 and CpG4. Coffee exposure significantly increased methylation at CpG3 by 3.7% (decaffeinated coffee) and showed a trend toward increased methylation at CpG3 by 2.8% (regular coffee) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, left). Exposure to coffee did not affect the methylation at CpG4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, right panel).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo the best of our knowledge, this study provides the first evidence that coffee exposure induces concentration-dependent suppression of \u003cem\u003eSLC6A4\u003c/em\u003e mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and an associated reduction in 5-HT uptake activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) in a human intestinal epithelial cell model. Critically, this transcriptional repression was demonstrated to correlate with a significant increase in DNA methylation at a specific CpG site (CpG3) located on the CpG island shore of the \u003cem\u003eSLC6A4\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eThis SERT suppression represents a highly robust phenomenon rather than an artifact of a single coffee preparation. This effect was consistently observed across different brands of instant coffee, the major coffee species, Coffea arabica and Coffea robusta, and both caffeinated and decaffeinated coffee (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Notably, the observation that decaffeinated coffee exhibited an effect comparable to that of caffeinated coffee strongly indicated that caffeine was not the principal causative agent of the observed action. Collectively, these observations indicate that the bioactive substance(s) responsible for suppressing SERT expression are components universally present in roasted coffee beans rather than specific to a particular cultivar or brand.\u003c/p\u003e\u003cp\u003eOur findings strongly suggest that the active ingredient responsible for SERT suppression is not a major component originally present in green coffee beans but rather a substance generated or released during the thermal processing of roasting. First, the most common bioactive components in coffee were excluded based on our negative data. Caffeine (up to 5 mM), the primary alkaloid in coffee, did not affect SERT mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Similarly, 5-CQA, a major polyphenol abundant in green beans and its principal degradation products, caffeic acid and quinic acid, failed to suppress SERT expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Furthermore, trigonelline, another major alkaloid, and its primary metabolite nicotinic acid were inactive (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Although these compounds have been widely studied for their association with the health benefits of coffee, our findings demonstrated that they are not directly involved in the specific phenomenon of SERT suppression observed here. These data implied that the active component was a substance other than the major constituents.\u003c/p\u003e\u003cp\u003eOne of the most compelling pieces of evidence in this study is the stark contrast in the effects of roasted and unroasted green bean extracts. Only extracts prepared from light- and Italian-roasted beans significantly suppressed SERT mRNA expression, whereas extracts from green beans showed no such effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This result unequivocally indicates that a bioactive compound is generated during the thermal processing of coffee beans \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. For example, coffee melanoidins, which possess high antioxidant activity, are degradation products of 5-CQA formed during roasting \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Therefore, it is highly plausible that the active component(s) that suppress SERT expression reside within these roasted products, which warrants further investigation.\u003c/p\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eCoffee-Induced Epigenetic Modifications\u003c/h2\u003e\u003cp\u003eThis study demonstrated that coffee exposure specifically increased the methylation level at the CpG3 site by approximately 3\u0026ndash;4%, while it had no effect on the CpG4 site (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Although this absolute change appears modest, its biological significance is likely substantial. The CpG3 site is located in a region known as the \"CpG island shore,\" adjacent to the conventional CpG island. Recent epigenomic studies revealed that tissue-specific or disease-associated methylation changes that strongly correlate with gene expression often occur in these shore regions rather than in the CpG islands themselves \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The specificity of the effect on CpG3, but not on CpG4, suggests that this phenomenon is likely a targeted regulatory event rather than a non-specific genome-wide methylation change. The coffee components may induce site-specific DNA methylation changes and exhibit general inhibitory effects on DNA methylation \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAnother critical finding of this study was the rapid and reversible nature of SERT suppression. A significant decrease in SERT mRNA was observed after 6 h of coffee exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and upon removal of coffee from the medium (washout), the expression levels returned to baseline within 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Although the precise mechanisms underlying CpG3 hypermethylation and its associated transcriptional repression remain to be elucidated, these results indicate that this transcriptional repression occurs swiftly and is likely reversible. Changes in DNA methylation during short-term coffee exposure have not been verified; however, DNA methylation alterations occurring within hours have been confirmed \u003cem\u003ein vivo\u003c/em\u003e via fear conditioning and physical stimuli \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Whether coffee induced similar immediate changes remains to be determined. Considering the interaction between multiple epigenetic regulatory mechanisms \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, further investigation is imperative to elucidate the causal relationship between the transcriptional repression of SERT mRNA and hypermethylation at CpG3 \u003csup\u003e55\u003c/sup\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eRelationship Between 5-HTTLPR and Transcription Levels\u003c/h2\u003e\u003cp\u003eThe 5-HTTLPR in the Caco-2 cells was L\u003csub\u003eA\u003c/sub\u003e/L\u003csub\u003eA\u003c/sub\u003e. This is not surprising, given that it is a cell line derived from a Caucasian donor. The extent to which this genetic polymorphism affects intestinal SERT expression remains unclear. DNA methylation of \u003cem\u003eSLC6A4\u003c/em\u003e is 5-HTTLPR genotype-dependent \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, and samples harboring the S allele may yield different results. However, pioneering detailed examinations of the SERT promoter region suggested a potential lack of a functional association between specific promoter regions regulating intestinal epithelial SERT transcription and 5-HTTLPR \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The CpG3 focused on in this study is located downstream of Exon1A on the island shore of CpG. The strong correlation between CpG3 hypermethylation and either promoter region remains a subject for future investigation.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eLimitations\u003c/h2\u003e\u003cp\u003eOur study has several limitations. First, this study was a basic investigation reporting only \u003cem\u003ein vitro\u003c/em\u003e experimental results. Additional studies are required to determine whether the SERT expression-suppressing effect of coffee can be reproduced in living organisms. Further verification is needed to determine whether the observed decrease in SERT expression occurs \u003cem\u003ein vivo\u003c/em\u003e, considering anatomical and physiological conditions. As mentioned above, orally ingested fluids come into contact with epithelial cells in the upper gastrointestinal tract for a short period and remain in the small intestine for several hours \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The extent and duration of coffee exposure to epithelial cells at these anatomical sites, particularly under conditions of excessive coffee intake, are issues for future research.\u003c/p\u003e\u003cp\u003eWhen using Caco-2 cells derived from colon adenocarcinoma, it is necessary to keep in mind that they have a different transcriptional profile than that of normal cells \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Moreover, several factors such as passage number, number of replications, and culture conditions significantly affect the performance of Caco-2 cells \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. It is crucial to recognize that Caco-2 cells exhibit phenotypes of both small intestinal epithelial cells (enterocytes) and large intestinal epithelial cells (colonocytes) \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Although T84 cells are considered an excellent model of the colonocyte epithelium \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, Caco-2 cells were chosen for this study, specifically for stable SERT expression \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Furthermore, the CpG3 site that we focused on in this study is not present in rodents; therefore, it was necessary to use human-derived cells for the experiments.\u003c/p\u003e\u003cp\u003eThis study did not identify the components involved in the transcriptional repression of SERT. This is due to the large number of components in coffee. Nevertheless, our data provided some insights. SERT expression decreased in both regular and decaffeinated coffee, but this phenomenon was not reproduced by caffeine alone. Since reproducibility was observed with instant coffees sold by different beverage manufacturers, instant coffees made from Arabica and Robusta beans, and coffee made from roasted coffee beans, the involvement of universal components common to coffee is expected. The increase in reactions due to roasting suggests the influence of degradation products of green coffee components or newly generated substances.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that coffee modulates SERT expression in a Caco-2 intestinal epithelial cell model. This finding offers a novel perspective, suggesting that the preventive effects of coffee against CRC may involve modulation of SERT-dependent intra- and extracellular 5-HT activities \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThe authors have no conflicts of interest regarding this study.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Sports, and Culture, Japan (JSPS KAKENHI Grant Numbers 19H03409, 21K06802, 22K06862, 22K20993, 23K06361, 23K19402, 25K18967, 25K10182). It was also supported by grants from the Takeda Science Foundation, the Uehara Memorial Foundation and Smoking Research Foundation.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSatoshi Kikkawa: Conceived and designed research, Analyzed data, Performed experiments, Interpreted results of experiments, Prepared figures, Drafted manuscript, Edited and revised the manuscript, Approved final version of the manuscript. Miki Bundo: Conceived and designed research, Interpreted results of experiments, Edited and revised the manuscript. Emi Kiyota: Conceived and designed research, Interpreted results of experiments. Serika, Imamura: Conceived and designed research, Resources, Funding acquisition. Kana Harada: Interpreted results of experiments, Resources, Funding acquisition. Hiroko Shiraki: Interpreted results of experiments, Resources, Funding acquisition. Shigeru Tanaka: Interpreted results of experiments, Resources, Funding acquisition. Kazuya Iwamoto: Conceived and designed research, Interpreted results of experiments, Edited and revised the manuscript, Approved final version of the manuscript. Norio Sakai: Conceived and designed research, Analyzed data, Interpreted results of experiments, Prepared figures, Drafted manuscript, Edited and revised the manuscript, Approved final version of the manuscript Resources, Funding acquisition.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll relevant data are within the manuscript and its [Supporting information](https:/journals.plos.org/plosone/article?id=10.1371/journal.pone.0263395) files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi, D., Yang, Y., Li, Y. P., Zhu, X. H. \u0026amp; Li, Z. Q. Epigenetic regulation of gene expression in response to environmental exposures: From bench to model. \u003cem\u003eSci. 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Physiol-Cell Ph\u003c/em\u003e. \u003cb\u003e318\u003c/b\u003e, C1294\u0026ndash;C1304. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1152/ajpcell.00477.2019\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1152/ajpcell.00477.2019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Colon cancer, serotonin transporter, epigenetic regulation, Caco-2 cells","lastPublishedDoi":"10.21203/rs.3.rs-8214835/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8214835/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eConsumption of coffee is associated with a reduced risk of colorectal cancer (CRC); however, the underlying mechanisms are not fully understood. Gut serotonin (5-HT) plays a complex role in CRC development. Intestinal 5-HT levels are regulated by the serotonin transporter (SERT), in intestinal epithelial cells. Recent evidence suggests a correlation between SERT expression and DNA methylation of a functional CpG site (CpG3) in the promoter region of \u003cem\u003eSLC6A4\u003c/em\u003e, the SERT gene. This study investigated the effects of coffee on SERT expression in Caco-2 cells, an intestinal epithelial cell model. Exposure of Caco-2 cells to instant coffee solution 1-10% (v/v) for 48 h was found to result in a concentration-dependent decrease in SERT-mediated 5-HT uptake and SERT mRNA expression. This effect was observed for different instant coffee brands and coffee bean species, and were not reproduced by exposure to major coffee components such as caffeine and chlorogenic acid, or by extracts from unroasted green coffee beans. Pyrosequencing revealed that coffee exposure altered the DNA methylation of CpG3. This preliminary study suggests a novel mechanism by which coffee protects against CRC: suppression of SERT expression in intestinal epithelial cells, possibly via epigenetic modification of \u003cem\u003eSLC6A4\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Serotonin Transporter Expression and DNA Methylation are Altered by Coffee Exposure in Caco-2 Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 07:49:36","doi":"10.21203/rs.3.rs-8214835/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-07T16:08:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-06T14:16:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-03T16:33:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-19T18:46:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180725551864979772626696752961700296408","date":"2025-12-12T19:07:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152597734101755721154289021903011728302","date":"2025-12-12T13:50:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26819639283439552344493418474514239813","date":"2025-12-02T12:07:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T08:29:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-28T17:56:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-27T06:36:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-27T06:34:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-26T16:00:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"51e57bb9-a140-4f45-9dcb-c54ac73041c0","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":58957027,"name":"Biological sciences/Cancer"},{"id":58957028,"name":"Biological sciences/Cell biology"},{"id":58957029,"name":"Biological sciences/Genetics"},{"id":58957030,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-01-07T16:23:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 07:49:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8214835","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8214835","identity":"rs-8214835","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}