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Discipline
Medical, Biological
Keywords
Antidepressants
Biliary Cancer
Tumor Growth
Observation Type
Standalone
Nature
Standard Data
Submitted
Apr 29th, 2017
Published
Sep 14th, 2017
  • Abstract

    Cholangiocarcinoma is an aggressive cancer of the bile ducts with limited treatment options and a high mortality rate. Patients with cancer have over a twofold higher rate of depression than the general population and are prescribed selective serotonin reuptake inhibitors (SSRIs) to manage their depression. However, serotonin has been demonstrated to promote the proliferation of cancer cells. In this mouse xenograft study, we provide evidence that the SSRIs sertraline and fluoxetine lead to increased serotonin bioavailability that results in a proliferation of cholangiocarcinoma cells. Cholangiocarcinoma cell proliferation was not observed in vitro, indicating that the effects of SSRIs are due to increased serotonin levels in noncancerous cells of the body. These novel findings on the effect of SSRIs in promoting the growth of cholangiocarcinoma support the notion that these antidepressants should be used cautiously in this patient population.

  • Figure
  • Introduction

    Cancer of the bile ducts, or cholangiocarcinoma, is an extremely aggressive tumor that has very poor prognosis and limited treatment options. Cholangiocarcinomas accounts for around 15% of all liver cancers and causes 2% of all cancer deaths worldwide. Despite aggressive treatment, survival rates are low, generally only 6 months from diagnosis, as 90% of patients are not eligible for surgery-, and the cancer is relatively resistant to chemotherapy. The high mortality rate from cholangiocarcinoma is due in part to the late diagnosis, as the clinical manifestations of this cancer (such as abdominal pain, pruritus, weight loss, etc.) occur after the cancer is quite advanced.

    Serotonin, or 5-hydroxytryptamine (5-HT), is synthesized via the decarboxylation and hydroxylation of tryptophan by the enzymes tryptophan hydroxylase and decarboxylase. Serotonin can then generate a wide variety of intracellular effects as there are 16 receptor subtypes, with all but one being G protein–coupled receptors that lead to the activation of secondary messenger systems. The uptake of serotonin from the extracellular space into the cell via serotonin transporters terminates serotonin receptor-mediated signaling. Predominantly, it is the enzyme monoamine oxidase A that degrades serotonin once it is inside the cell. Serotonin has been classified as a growth factor for several different nontumorigenic cell types as well as in a variety of cancer cells, including small cell lung carcinoma, choriocarcinoma, bladder cancer, prostate cancer, hepatocellular carcinoma, and breast cancer. Similarly, cholangiocarcinoma produces increased amounts of serotonin, which exerts growth-promoting effects in vitro and in vivo. This increased biogenic amine production is due to the coordinated increase in synthesis and the epigenetic silencing of monoamine oxidase A.

    Cancer patients have a variety of stressors (physiological, monetary, and psychological) that result in clinical depression at rates much higher than the general population. The incidence of depression in the general population has been reported to be between 5% and 6%, whereas in cancer patients this jumps to 12.9%, with another 16.5% displaying subclinical depressive symptoms. The use of SSRIs for the management of depressive symptoms has become more common in recent years with antidepressant use increasing nearly 400% in the time period of 2005–2008 when compared to 1988–1994. As SSRI antidepressants work through inhibiting serotonin transporter activity, their use leads to increased serotonin bioavailability. The effects of SSRI use on cholangiocarcinoma growth have not yet been determined.

  • Objective

    The objective of this study was to assess the effects of the SSRIs sertraline and fluoxetine on cholangiocarcinoma growth using a xenograft tumor model in mice.

  • Results & Discussion

    The tumors from mice treated with sertraline or fluoxetine were significantly larger than those treated with vehicle and grew quicker over time (Fig. 1A and B). Immunohistochemistry for the cholangiocyte marker CK-19 was performed to determine whether the resulting tumors had similar cellular makeup and tumor architecture. Across all treatments, the majority of cells in the tumors were CK-19 positive tumor cells with similar morphology and a similar degree of non-tumor cells (Fig. 1C). However, in tumors from mice treated with sertraline or fluoxetine, there were increased numbers of PCNA-positive cells (Fig. 1C) and an increased expression of PCNA mRNA (Fig. 1D), indicating a greater degree of xenograft cell proliferation in response to SSRI treatment. Interestingly, when cholangiocarcinoma cells were treated with fluoxetine and sertraline in vitro, there was no effect on cell proliferation (Fig. 1E) or cell cycle progression (Fig. 1F), suggesting that the proliferative effects observed in vivo are more likely due to indirect action on other cells that make up the tumor microenvironment or even other organs in the body rather than direct action of these SSRIs on serotonin reuptake on the tumor cells themselves. In support of this, treatment of mice with the selected SSRIs significantly increased the serotonin levels in the serum (Fig. 1G).

  • Conclusions

    The data presented here suggest that treatment of cholangiocarcinoma xenograft-bearing mice with sertraline or fluoxetine increased tumor growth. These data may indicate that the use of SSRI antidepressants may be contraindicated for patients with cholangiocarcinoma.

  • Limitations

    The limitations of these observations may include the following:

    1. Not all SSRIs are equal: Our study only assessed two of the main SSRIs; however, there are many alternative SSRIs and antidepressants that work through other mechanisms, such as monoamine oxidase inhibitors or tricyclic antidepressants, which may have differing effects on cholangiocarcinoma growth. Further characterization of these antidepressants should be assessed.

    2. Not all species are equal: In this study, mice were given sertraline and fluoxetine 3 times per week at concentrations of 20 mg/kg and 10 mg/kg, respectively. These were the concentrations used in previous studies to assess their effects on depression-like behaviors in mice. These dosages are in excess of the recommended daily dose in humans (Fluoxetine; 20–80 mg per day; assuming an 80 kg person, corresponds to approximately 0.25–1 mg/kg/day. Sertraline; 50–200 mg/day, corresponds to approximately 0.625–2.5 mg/kg/day). However, it is difficult to apply similar doses to humans and mice, as the relative pharmacokinetics of these antidepressants in each species is different.

    3. The molecular or cellular target of the SSRIs is not identified: In this study, the effects of SSRIs on cholangiocarcinoma cell proliferation in vitro was negligible, leading to the suggestion that the effects of SSRIs on tumor growth in vivo are likely due to effects either on other cells that may contribute to the tumor microenvironment or perhaps other organs that serve to bring about an increase in systemic serotonin levels. This study did not identify the precise cellular target of these SSRIs, and this topic is one that merits ongoing investigation.

  • Methods

    Xenograft model of cholangiocarcinoma

    Human Mz-ChA-1 cholangiocarcinoma cells were resuspended in extracellular matrix (Sigma Aldrich, MO) and injected into male BALB/c nu/nu mice (2 million cells per tumor, subcutaneous in the flank). Once palpable tumors were present (~7 days), the mice were treated with vehicle (10% dimethyl sulfoxide (DMSO) in saline; ip, n = 6), fluoxetine (10 mg/kg in 10% DMSO; ip, n = 6) or sertraline (20 mg/kg in 10% DMSO; ip, n = 6) 3 times per week.

    The tumors were measured using electronic calipers and tumor volume was approximated by using the following equation:

    Tumor volume (mm3) = 0.5 × [length (mm) × width (mm) × height (mm)].

    After 24 days, the mice were humanely euthanized and tumors and serum were collected. All animal experiments were performed with approval from our Institutional Animal Care and Use Committee.

    Immunohistochemistry

    Excised tumors were put into 10% neutral-buffered formalin for 3 days and subsequently processed and embedded in paraffin. Sections were stained using standard immunohistochemistry procedures, using primary antibodies for the cholangiocyte marker CK-19 (Abcam AB52625, Cambridge, MA, 1:400) or the marker of proliferating cells, PCNA (Abcam AB29, 1:100). Sections were counterstained with Hematoxylin QS (Vector Laboratories, Burlingame, CA), and photomicrograph images were taken on a Leica SCN400 slide scanner (Buffalo Grove, IL).

    Real-time PCR

    Total RNA was extracted from tumor tissue using an RNeasy minikit (Qiagen, Germantown, MD) following the manufacturer's instructions. Synthesis of cDNA from total RNA was achieved using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Real time PCR was performed with commercially available and validated PCR primers (Qiagen, Germantown, MD) against human PCNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using an MX3005P real-time PCR machine (Agilent, Santa Clara, CA). A delta delta CT analysis was performed using the untreated cells or the H69 cholangiocyte cell line as the control samples, where appropriate. Data are expressed as relative mRNA levels ± SEM (n = 4).

    MTS cell viability assay

    Cholangiocarcinoma growth was evaluated in vitro by 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) proliferation assay in the Mz-ChA-1 cell line. After trypsinization, the cells were seeded into 96 well plates (10,000 cells per well) and allowed to adhere overnight. Cells were then stimulated for 48 h with various concentrations of fluoxetine or sertraline (0.1 nM to 1 μM). Cell proliferation was assessed using a colorimetric cell proliferation assay (CellTiter 96AQueous, Promega Corp., Madison, WI), and absorbance was measured at 490 nm by a microplate spectrophotometer (VersaMax, Molecular Devices, Sunnyvale, CA). Data are expressed as the relative proliferation index, which is the average fold change in absorbance compared to vehicle-treated cells (n = 7 per group).

    Analysis of cell cycle progression

    Mz-ChA-1 cells were stimulated for 24 h with various concentrations of fluoxetine or sertraline (100 nM to 1 mM). The cells were collected by detaching with TrypLE (Invitrogen, Carlsbad, CA) and resuspending in culture medium. Cells were fixed and stained according to the Muse Cell Cycle Kit manufacturer's instructions (EMD Millipore, Billerica, MA) followed by analysis on the Muse Cell Analyzer (EMD Millipore).

    Serotonin EIA

    Serum was collected from BALB/c nu/nu mice containing xenograft cholangiocarcinoma tumors and treated with sertraline or fluoxetine as described above. Serotonin levels were assessed using a commercially available competitive ELISA kit following the manufacturer's instructions (Enzo Life Sciences Inc, Farmingdale, NY). Data are expressed as average serotonin levels (ng/mL) ± SEM (n = 4).

    Statistics

    All data are expressed as avg ± SEM. Statistical significance was analyzed by ANOVA followed by an appropriate post hoc test. For in vivo tumor volume measurements, a two-way ANOVA was used to determine significance. In each case, a p value of <0.05 was used to indicate statistical significance.

  • Funding statement

    This study was supported by an American Cancer Society Research Scholar award (RSC118760), an NIH R01 award (DK082435), and a VA Merit award (BX002638) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. DeMorrow. This study was also funded by a VA Career Development award (BX003486) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. McMillin.

  • Acknowledgements

    This work was completed with support from the Veterans Health Administration and with resources and the use of facilities at the Central Texas Veterans Health Care System, Temple, Texas. The contents do not represent the views of the United States Department of Veterans Affairs or the United States Government.

  • Ethics statement

    Animal experiments were performed with approval from the Baylor Scott & White Institutional Animal Care and Use Committee (protocol no: 2012-051).

  • References
  • 1
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    Matters12.5/20

    The specific serotonin reuptake inhibitors sertraline and fluoxetine promote tumor growth in a mouse xenograft model of cholangiocarcinoma

    Affiliation listing not available.
    Abstractlink

    Cholangiocarcinoma is an aggressive cancer of the bile ducts with limited treatment options and a high mortality rate. Patients with cancer have over a twofold higher rate of depression than the general population and are prescribed selective serotonin reuptake inhibitors (SSRIs) to manage their depression. However, serotonin has been demonstrated to promote the proliferation of cancer cells. In this mouse xenograft study, we provide evidence that the SSRIs sertraline and fluoxetine lead to increased serotonin bioavailability that results in a proliferation of cholangiocarcinoma cells. Cholangiocarcinoma cell proliferation was not observed in vitro, indicating that the effects of SSRIs are due to increased serotonin levels in noncancerous cells of the body. These novel findings on the effect of SSRIs in promoting the growth of cholangiocarcinoma support the notion that these antidepressants should be used cautiously in this patient population.

    Figurelink

    Figure 1.

    (A) Nude mice were injected with the Mz-ChA-1 cholangiocarcinoma cell line, and a xenograft tumor was established. These mice were then treated with sertraline (20 mg/kg/day) or fluoxetine (10 mg/kg/day) 3 times per week, and the tumor volume was measured using digital calipers. Data are expressed as avg ± SEM of the tumor volume.

    (B) Representative images of Mz-ChA-1 tumors excised at 24 days following xenograft tumor establishment from vehicle-, sertraline-, and fluoxetine-treated mice.

    (C) Xenograft tumor sections were stained with the cholangiocyte marker cytokeratin 19 (CK-19) or proliferating cellular nuclear antigen (PCNA) as a marker of proliferation.

    (D) PCNA mRNA expression was assessed in total RNA extracted from xenograft tumors by real-time PCR. Data are expressed as avg ± SEM, * denotes p <0.05 compared to vehicle.

    (E) The effects of sertraline and fluoxetine on cholangiocarcinoma cell proliferation were assessed in vitro. Cells were treated with various concentrations of sertraline or fluoxetine for 48 h, and viability was assessed using MTS assays. Data are expressed as fold change in proliferation over vehicle-treated cells (relative proliferative index; avg ± SEM).

    (F) The effects of sertraline and fluoxetine on cell cycle progression were assessed in vitro. Cells were treated with various concentrations of sertraline or fluoxetine for 24 h, and the percentage of cells in the G2/M phase or G0/G1 phase of the cell cycle was assessed using the Muse® cell cycle assay kit, following the vendor's instructions. Data are expressed as avg ± SEM percentage of cells in each phase of the cell cycle.

    (G) Nude mice were treated with sertraline (20 mg/kg/day) or fluoxetine (10 mg/kg/day) 3 times per week, and serotonin levels were assessed in serum using a commercially available EIA kit. Data are expressed as avg ± SEM, * denotes p <0.05 compared to vehicle.

    Introductionlink

    Cancer of the bile ducts, or cholangiocarcinoma, is an extremely aggressive tumor that has very poor prognosis and limited treatment options[1][2]. Cholangiocarcinomas accounts for around 15% of all liver cancers and causes 2% of all cancer deaths worldwide[3]. Despite aggressive treatment, survival rates are low, generally only 6 months from diagnosis, as 90% of patients are not eligible for surgery-, and the cancer is relatively resistant to chemotherapy. The high mortality rate from cholangiocarcinoma is due in part to the late diagnosis, as the clinical manifestations of this cancer (such as abdominal pain, pruritus, weight loss, etc.) occur after the cancer is quite advanced[4].

    Serotonin, or 5-hydroxytryptamine (5-HT), is synthesized via the decarboxylation and hydroxylation of tryptophan by the enzymes tryptophan hydroxylase and decarboxylase[5]. Serotonin can then generate a wide variety of intracellular effects as there are 16 receptor subtypes, with all but one being G protein–coupled receptors that lead to the activation of secondary messenger systems[6]. The uptake of serotonin from the extracellular space into the cell via serotonin transporters terminates serotonin receptor-mediated signaling[7]. Predominantly, it is the enzyme monoamine oxidase A that degrades serotonin once it is inside the cell[8]. Serotonin has been classified as a growth factor for several different nontumorigenic cell types as well as in a variety of cancer cells, including small cell lung carcinoma, choriocarcinoma, bladder cancer, prostate cancer, hepatocellular carcinoma, and breast cancer[9][10][11][12][13][14][15]. Similarly, cholangiocarcinoma produces increased amounts of serotonin,[16] which exerts growth-promoting effects in vitro and in vivo. This increased biogenic amine production is due to the coordinated increase in synthesis and the epigenetic silencing of monoamine oxidase A[16][17][18].

    Cancer patients have a variety of stressors (physiological, monetary, and psychological) that result in clinical depression at rates much higher than the general population[19]. The incidence of depression in the general population has been reported to be between 5% and 6%, whereas in cancer patients this jumps to 12.9%, with another 16.5% displaying subclinical depressive symptoms[20][21]. The use of SSRIs for the management of depressive symptoms has become more common in recent years with antidepressant use increasing nearly 400% in the time period of 2005–2008 when compared to 1988–1994[22]. As SSRI antidepressants work through inhibiting serotonin transporter activity, their use leads to increased serotonin bioavailability. The effects of SSRI use on cholangiocarcinoma growth have not yet been determined.

    Objectivelink

    The objective of this study was to assess the effects of the SSRIs sertraline and fluoxetine on cholangiocarcinoma growth using a xenograft tumor model in mice.

    Results & Discussionlink

    The tumors from mice treated with sertraline or fluoxetine were significantly larger than those treated with vehicle and grew quicker over time (Fig. 1A and B). Immunohistochemistry for the cholangiocyte marker CK-19 was performed to determine whether the resulting tumors had similar cellular makeup and tumor architecture. Across all treatments, the majority of cells in the tumors were CK-19 positive tumor cells with similar morphology and a similar degree of non-tumor cells (Fig. 1C). However, in tumors from mice treated with sertraline or fluoxetine, there were increased numbers of PCNA-positive cells (Fig. 1C) and an increased expression of PCNA mRNA (Fig. 1D), indicating a greater degree of xenograft cell proliferation in response to SSRI treatment. Interestingly, when cholangiocarcinoma cells were treated with fluoxetine and sertraline in vitro, there was no effect on cell proliferation (Fig. 1E) or cell cycle progression (Fig. 1F), suggesting that the proliferative effects observed in vivo are more likely due to indirect action on other cells that make up the tumor microenvironment or even other organs in the body rather than direct action of these SSRIs on serotonin reuptake on the tumor cells themselves. In support of this, treatment of mice with the selected SSRIs significantly increased the serotonin levels in the serum (Fig. 1G).

    Conclusionslink

    The data presented here suggest that treatment of cholangiocarcinoma xenograft-bearing mice with sertraline or fluoxetine increased tumor growth. These data may indicate that the use of SSRI antidepressants may be contraindicated for patients with cholangiocarcinoma.

    Limitationslink

    The limitations of these observations may include the following:

    1. Not all SSRIs are equal: Our study only assessed two of the main SSRIs; however, there are many alternative SSRIs and antidepressants that work through other mechanisms, such as monoamine oxidase inhibitors or tricyclic antidepressants, which may have differing effects on cholangiocarcinoma growth. Further characterization of these antidepressants should be assessed.

    2. Not all species are equal: In this study, mice were given sertraline and fluoxetine 3 times per week at concentrations of 20 mg/kg and 10 mg/kg, respectively. These were the concentrations used in previous studies to assess their effects on depression-like behaviors in mice[23][24]. These dosages are in excess of the recommended daily dose in humans (Fluoxetine; 20–80 mg per day; assuming an 80 kg person, corresponds to approximately 0.25–1 mg/kg/day. Sertraline; 50–200 mg/day, corresponds to approximately 0.625–2.5 mg/kg/day). However, it is difficult to apply similar doses to humans and mice, as the relative pharmacokinetics of these antidepressants in each species is different.

    3. The molecular or cellular target of the SSRIs is not identified: In this study, the effects of SSRIs on cholangiocarcinoma cell proliferation in vitro was negligible, leading to the suggestion that the effects of SSRIs on tumor growth in vivo are likely due to effects either on other cells that may contribute to the tumor microenvironment or perhaps other organs that serve to bring about an increase in systemic serotonin levels. This study did not identify the precise cellular target of these SSRIs, and this topic is one that merits ongoing investigation.

    Methodslink

    Xenograft model of cholangiocarcinoma

    Human Mz-ChA-1 cholangiocarcinoma cells were resuspended in extracellular matrix (Sigma Aldrich, MO) and injected into male BALB/c nu/nu mice (2 million cells per tumor, subcutaneous in the flank). Once palpable tumors were present (~7 days), the mice were treated with vehicle (10% dimethyl sulfoxide (DMSO) in saline; ip, n = 6), fluoxetine (10 mg/kg in 10% DMSO; ip, n = 6) or sertraline (20 mg/kg in 10% DMSO; ip, n = 6) 3 times per week.

    The tumors were measured using electronic calipers and tumor volume was approximated by using the following equation:

    Tumor volume (mm3) = 0.5 × [length (mm) × width (mm) × height (mm)].

    After 24 days, the mice were humanely euthanized and tumors and serum were collected. All animal experiments were performed with approval from our Institutional Animal Care and Use Committee.

    Immunohistochemistry

    Excised tumors were put into 10% neutral-buffered formalin for 3 days and subsequently processed and embedded in paraffin. Sections were stained using standard immunohistochemistry procedures, using primary antibodies for the cholangiocyte marker CK-19 (Abcam AB52625, Cambridge, MA, 1:400) or the marker of proliferating cells, PCNA (Abcam AB29, 1:100). Sections were counterstained with Hematoxylin QS (Vector Laboratories, Burlingame, CA), and photomicrograph images were taken on a Leica SCN400 slide scanner (Buffalo Grove, IL).

    Real-time PCR

    Total RNA was extracted from tumor tissue using an RNeasy minikit (Qiagen, Germantown, MD) following the manufacturer's instructions. Synthesis of cDNA from total RNA was achieved using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Real time PCR was performed with commercially available and validated PCR primers (Qiagen, Germantown, MD) against human PCNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using an MX3005P real-time PCR machine (Agilent, Santa Clara, CA). A delta delta CT analysis was performed using the untreated cells or the H69 cholangiocyte cell line as the control samples, where appropriate[25]. Data are expressed as relative mRNA levels ± SEM (n = 4).

    MTS cell viability assay

    Cholangiocarcinoma growth was evaluated in vitro by 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) proliferation assay in the Mz-ChA-1 cell line. After trypsinization, the cells were seeded into 96 well plates (10,000 cells per well) and allowed to adhere overnight. Cells were then stimulated for 48 h with various concentrations of fluoxetine or sertraline (0.1 nM to 1 μM). Cell proliferation was assessed using a colorimetric cell proliferation assay (CellTiter 96AQueous, Promega Corp., Madison, WI), and absorbance was measured at 490 nm by a microplate spectrophotometer (VersaMax, Molecular Devices, Sunnyvale, CA). Data are expressed as the relative proliferation index, which is the average fold change in absorbance compared to vehicle-treated cells (n = 7 per group).

    Analysis of cell cycle progression

    Mz-ChA-1 cells were stimulated for 24 h with various concentrations of fluoxetine or sertraline (100 nM to 1 mM). The cells were collected by detaching with TrypLE (Invitrogen, Carlsbad, CA) and resuspending in culture medium. Cells were fixed and stained according to the Muse Cell Cycle Kit manufacturer's instructions (EMD Millipore, Billerica, MA) followed by analysis on the Muse Cell Analyzer (EMD Millipore).

    Serotonin EIA

    Serum was collected from BALB/c nu/nu mice containing xenograft cholangiocarcinoma tumors and treated with sertraline or fluoxetine as described above. Serotonin levels were assessed using a commercially available competitive ELISA kit following the manufacturer's instructions (Enzo Life Sciences Inc, Farmingdale, NY). Data are expressed as average serotonin levels (ng/mL) ± SEM (n = 4).

    Statistics

    All data are expressed as avg ± SEM. Statistical significance was analyzed by ANOVA followed by an appropriate post hoc test. For in vivo tumor volume measurements, a two-way ANOVA was used to determine significance. In each case, a p value of <0.05 was used to indicate statistical significance.

    Funding Statementlink

    This study was supported by an American Cancer Society Research Scholar award (RSC118760), an NIH R01 award (DK082435), and a VA Merit award (BX002638) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. DeMorrow. This study was also funded by a VA Career Development award (BX003486) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. McMillin.

    Acknowledgementslink

    This work was completed with support from the Veterans Health Administration and with resources and the use of facilities at the Central Texas Veterans Health Care System, Temple, Texas. The contents do not represent the views of the United States Department of Veterans Affairs or the United States Government.

    Conflict of interestlink

    The authors declare no conflicts of interest.

    Ethics Statementlink

    Animal experiments were performed with approval from the Baylor Scott & White Institutional Animal Care and Use Committee (protocol no: 2012-051).

    No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.

    Referenceslink
    1. Alpini, Gianfranco, McGill, James M., Larusso, Nicholas F.
      The pathobiology of biliary epithelia
      Hepatology, 35/2002, page 1256–68 chrome_reader_mode
    2. Sirica, Alphonse E.
      Cholangiocarcinoma: Molecular targeting strategies for chemoprevention and therapy
      Hepatology, 41/2005, page 5–15 chrome_reader_mode
    3. Shaib, Yasser, El-Serag, Hashem
      The prevalence and risk factors of functional dyspepsia in a multiethnic population in the United States
      The American Journal of Gastroenterology, 99/2004, page 2210–6 chrome_reader_mode
    4. Mitsugi, Shimoda, Kubota, Keiichi
      Multi-disciplinary treatment for cholangiocellular carcinoma
      World Journal of Gastroenterology, 13/2007, page 1500–4 chrome_reader_mode
    5. Diksic, Mirko, Young, Simon N.
      Study of the brain serotonergic system with labeled α-methyl-l-tryptophan
      Journal of Neurochemistry, 78/2001, page 1185–200 chrome_reader_mode
    6. Kroeze, W.K., Kristiansen, K., Roth, B.L.
      Molecular biology of serotonin receptors – Structure and function at the molecular level
      Current Topics in Medicinal Chemistry, 2/2002, page 507–28 chrome_reader_mode
    7. Martel, F.
      Recent advances on the importance of the serotonin transporter SERT in the rat intestine
      Pharmacological Research, 54/2006, pages 73-6 chrome_reader_mode
    8. Ekstedt, B.
      Substrate specificity of monoamine oxidase in pig liver mitochondria
      Medical Biology, 57/1979, page 220–23 chrome_reader_mode
    9. Nocito, Antonio, Georgiev, Panco, Dahm, Felix,more_horiz, Clavien, Pierre-Alain
      Platelets and platelet-derived serotonin promote tissue repair after normothermic hepatic ischemia in mice
      Hepatology, 45/2007, page 369–76 chrome_reader_mode
    10. Yang, Mo, Li, Karen, Ng, Pak Cheung,more_horiz, Fok, Tai Fai
      Promoting effects of serotonin on hematopoiesis: Ex Vivo expansion of cord blood CD34+ stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis
      Stem Cells, 25/2007, page 1800–1806 chrome_reader_mode
    11. Vicentini, Lucia M., Cattaneo, Maria G., Fesce, Riccardo
      Evidence for receptor subtype cross-talk in the mitogenic action of serotonin on human small-cell lung carcinoma cells
      European Journal of Pharmacology, 318/1996, page 497–504 chrome_reader_mode
    12. Sonier, Brigitte, Arseneault, Madeleine, Lavigne, Carole,more_horiz, Vaillancourt, Cathy
      The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin
      Biochemical and Biophysical Research Communications, 343/2006, page 1053–59 chrome_reader_mode
    13. Siddiqui, Emad J., Shabbir, Majid, Mikhailidis, Dimitri P.,more_horiz, Mumtaz, Faiz H
      The role of serotonin (5-Hydroxytryptamine1A and 1B) receptors in prostate cancer cell proliferation
      The Journal of Urology, 176/2006, page 1648–53 chrome_reader_mode
    14. Sonier, B., Lavigne, C., Arseneault, M.,more_horiz, Vaillancourt, C.
      Expression of the 5-HT2A serotoninergic receptor in human placenta and choriocarcinoma cells: mitogenic implications of serotonin
      Placenta, 26/2005, page 484–90 chrome_reader_mode
    15. Soll, Christopher, Jang, Jae Hwi, Riener, Marc-Oliver,more_horiz, Clavien, Pierre-Alain
      Serotonin promotes tumor growth in human hepatocellular cancer
      Hepatology, 51/2010, page 1244–54 chrome_reader_mode
    16. Alpini, Gianfranco, Invernizzi, Pietro, Gaudio, Eugenio,more_horiz, Demorrow, Sharon
      Serotonin metabolism is dysregulated in cholangiocarcinoma, which has implications for tumor growth
      Cancer Research, 68/2008, page 9184–93 chrome_reader_mode
    17. Huang, Li, Frampton, Gabriel, Rao, Arundhati,more_horiz, Demorrow, Sharon
      Monoamine oxidase A expression is suppressed in human cholangiocarcinoma via coordinated epigenetic and IL-6-driven events
      Laboratory Investigation, 92/2012, page 1451–1460 chrome_reader_mode
    18. Coufal, Monique, Invernizzi, Pietro, Gaudio, Eugenio,more_horiz, Demorrow, Sharon
      Increased local dopamine secretion has growth-promoting effects in cholangiocarcinoma
      International Journal of Cancer, 126/2010, page 2112–22 chrome_reader_mode
    19. McDaniel, J., Musselman, D. L., Porter, M. R.,more_horiz, Nemeroff, C. B.
      Depression in patients with cancer: Diagnosis, biology, and treatment
      Archives of General Psychiatry, 52/1995, page 89–99 chrome_reader_mode
    20. Bromet, Evelyn, Andrade, Laura Helena, Hwang, Irving,more_horiz, Kessler, Ronald C.
      Cross-national epidemiology of DSM-IV major depressive episode
      BMC Medicine, 9/2011, page 90 chrome_reader_mode
    21. Linden, Wolfgang, Vodermaier, Andrea, Mackenzie, Regina, Greig, Duncan
      Anxiety and depression after cancer diagnosis: Prevalence rates by cancer type, gender, and age
      Journal of Affective Disorders, 141/2012, page 343–51 chrome_reader_mode
    22. Pratt, Laura A., Brody, Debra J., Gu, Qiuping
      Antidepressant use in persons aged 12 and over: United States, 2005–2008
      NCHS Data Brief, 76/2011 chrome_reader_mode
    23. Roni, Monzurul Amin, Rahman, Shafiqur
      Effects of lobeline and reboxetine, fluoxetine, or bupropion combination on depression-like behaviors in mice
      Pharmacology Biochemistry and Behavior, 139/2015, page 1–6 chrome_reader_mode
    24. Dhingra, D., Goyal, P. K.
      Evidences for the involvement of monoaminergic and GABAergic systems in antidepressant-like activity of Tinospora cordifolia in mice.
      Indian Journal of Pharmaceutical Sciences, 70/2008, page 761–67 chrome_reader_mode
    25. Livak, Kenneth J., Schmittgen, Thomas D.
      Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method
      Methods, 25/2001, page 402–8 chrome_reader_mode
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