Update your browser to view this website correctly. Update my browser now
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.
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.
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.
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).
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.
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.
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.
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).
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).
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).
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.
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.
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.
Animal experiments were performed with approval from the Baylor Scott & White Institutional Animal Care and Use Committee (protocol no: 2012-051).