Mirtazapine Inhibits Tumor Growth via Immune Response and Serotonergic System

<div><p>To study the tumor inhibition effect of mirtazapine, a drug for patients with depression, CT26/<em>luc</em> colon carcinoma-bearing animal model was used. BALB/c mice were randomly divided into six groups: two groups without tumors, i.e. <em>wild-type</em> (no drug) and <em>drug</em> (mirtazapine), and four groups with tumors, i.e. <em>never</em> (no drug), <em>always</em> (pre-drug, i.e. drug treatment before tumor inoculation and throughout the experiment), <em>concurrent</em> (simultaneously tumor inoculation and drug treatment throughout the experiment), and <em>after</em> (post-drug, i.e. drug treatment after tumor inoculation and throughout the experiment). The “psychiatric” conditions of mice were observed from the immobility time with tail suspension and spontaneous motor activity post tumor inoculation. Significant increase of serum interlukin-12 (sIL-12) and the inhibition of tumor growth were found in mirtazapine-treated mice (<em>always</em>, <em>concurrent</em>, and <em>after</em>) as compared with that of <em>never</em>. In addition, interferon-γ level and immunocompetent infiltrating CD4+/CD8+ T cells in the tumors of mirtazapine-treated, tumor-bearing mice were significantly higher as compared with that of <em>never.</em> Tumor necrosis factor-α (TNF-α) expressions, on the contrary, are decreased in the mirtazapine-treated, tumor-bearing mice as compared with that of <em>never</em>. Ex vivo autoradiography with [¹²³I]ADAM, a radiopharmaceutical for serotonin transporter, also confirms the similar results. Notably, better survival rates and intervals were also found in mirtazapine-treated mice. These findings, however, were not observed in the immunodeficient mice. Our results suggest that tumor growth inhibition by mirtazapine in CT26/<em>luc</em> colon carcinoma-bearing mice may be due to the alteration of the tumor microenvironment, which involves the activation of the immune response and the recovery of serotonin level.</p> </div

Experimental designs.

<p>(A) Tumor inoculation, mirtazapine treatment, and monitoring of tumor growth and survival. (B) On day 22, mice were assayed for behaviors, then sacrificed for the measurement of lymphocyte subsets and performed with <i>ex vivo</i> autoradiography.</p

Effects of mirtazapine (10 mg/kg/d) on behavior changes of normal and CT26/<i>luc</i> tumor-bearing mice.

<p>(A) Immobility time in the tail-suspension test. (B) Spontaneous motor activity. (n = 4, *<i>p</i><0.05, **<i>p<</i>0.01 vs. <i>wild-type</i>; ^#<i>p</i><0.05, ^##<i>p</i> <0.01 between two groups).</p

Mirtazapine inhibits tumor growth and prolongs the survival rate and interval in CT-26/<i>luc</i> tumor-bearing model.

<p>On day -14, only “Always” mice were inoculated with tumor cells and treated with mirtazapine throughout the experiment, the other three groups were treated with 0.05 ml of 0.9% NaCl plus 0.5% absolute ethanol up to day 0. On day 0, only “<i>concurrent</i>” mice were inoculated with tumor cells and treated with mirtazapine throughout the rest of the experiment, “<i>after</i>” mice were treated with 0.05 ml of 0.9% NaCl plus 0.5% absolute ethanol but without mirtazapine up to day 14, while “<i>never</i>” mice were treated with 0.05 ml of 0.9% NaCl plus 0.5% absolute ethanol and throughout the experiment. On day 14, “<i>after</i>” mice were inoculated with tumor cells and treated with mirtazapine throughout the rest of the experiment. (A) Tumor growth curves are monitored with digital caliper. (B) Left panel: tumor growth curves are monitored with noninvasive bioluminescence imaging (BLI). The value under each mouse is the tumor volume determined with a caliper. Right panel: quantification of the photon counts in ROIs from the left panel. (C) No antitumor effect of mirtazapine was found on immunodeficient SCID mice with CT26/<i>luc</i> tumors. Left panel: tumor growth curves for <i>always</i> and <i>never</i>. Right panel: quantification of the photon counts in ROIs from the left panel. (D) No significant body-weight change (within 20%) through the whole experiment was found among <i>wild-type</i>, <i>drug</i>, <i>never</i>, <i>always</i>, <i>concurrent</i>, and <i>after</i>. (E) Effects of mirtazapine on the survival rate and interval of CT26/<i>luc</i>-bearing mice. The mean survival times are 67, 64, 57, 43 days for <i>always</i>, <i>concurrent</i>, <i>after</i>, and <i>never</i>, respectively. (n = 10 per group, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001).</p

The CD4+ and CD8+ T cell subsets with or without mirtazapine treatments in BALB/c mice with or without CT26/<i>luc</i> tumors.

<p>n = 6, ^*<i>p</i><0.05, ^**<i>p</i><0.01, and ^***<i>p</i><0.001 as compared with that of <i>wild type</i>, ^#<i>p</i><0.05, ^##<i>p</i><0.01, and ^###<i>p</i><0.001 as compared with that of <i>never</i>, ⁺<i>p</i><0.05 as compared with that of <i>after</i>.</p

Effect of mirtazapine on IFN-γ levels in tumors^* of CT-26/<i>luc</i> tumor-bearing mice.

<p>n = 3/group, ^##<i>p</i><0.01 and ^#<i>p</i><0.05 as compared with that of <i>never</i>, ⁺⁺<i>p<</i>0.01 and ⁺<i>p<</i>0.05 as compared with that of <i>after</i>. Student’s <i>t</i> test was used for the analysis. Experiments were repeated twice.</p>*<p>The tumors were removed from the mice at 6 weeks post tumor cells inoculation.</p

Serotonin transporter determined with [¹²³I]ADAM/<i>ex vivo</i> autoradiography in the brain of CT26/<i>luc</i> tumor-bearing mice.

<p>The mouse brain obtained at 90 min post caudal vein injection of 1 mCi/ml [¹²³I]ADAM was performed with <i>ex vivo</i> autoradiography. The top and bottom rows in each subfigure are the anatomy and <i>ex vivo</i> autoradiography, respectively. The blue circle is the target, and the red square is the cortex. (OT  =  olfactory tubercle; LS  =  lateral septal nucleus; ThN  =  thalamic nuclei; SN  =  substantia nigra; HN  =  hypothalamic nuclei).</p

Specific binding ratios of [¹²³I]ADAM in brains of BALB/c mice with or without CT26/<i>luc</i> tumors determined with <i>ex vivo</i> autoradiography.

<p><i>Ex vivo</i> autoradiography was performed at 90 mins post i.v. injection of 1 mCi [¹²³I]ADAM/0.1 ml. Specific binding ratio  =  (target – cortex)/cortex. (OT  =  olfactory tubercle; LS  =  lateral septal nucleus; ThN  =  thalamic nuclei; SN  =  substantia nigra; HN  =  hypothalamic nuclei).</p>*<p><i>p</i><0.05, <b>^**</b><i>p</i><0.01 vs. <i>wild-type</i>; <b>^#</b><i>p</i><0.05, <b>^##</b><i>p</i><0.01, <b>^###</b><i>p</i><0.001 vs. <i>never</i>, <b>^{<>\scale30%\raster="rg2"<>}</b><i>p</i><0.05, <b>^{<>\scale30%\raster="rg2"<><>\scale30%\raster="rg2"<>}</b><i>p</i><0.01, <b>^{<>\scale30%\raster="rg2"<><>\scale30%\raster="rg2"<><>\scale30%\raster="rg2"<>}</b><i>p</i><0.001 vs. <i>after</i>. Data are means±S.E. n = 3/group. Experiments were repeated twice.</p

Tumor growth inhibition among <i>never, after, concurrent</i>, and <i>always</i> of mice.

a<p>Mean tumor growth time: the time at which the tumor volume reaches to 400 mm³.</p>b<p>Mean tumor growth delay time: the tumor growth time of the treated group minus that of the <i>Never</i>.</p>c<p>Mean growth inhibition rate: growth rate of treated group/ growth rate of <i>Never</i>.</p>d<p>NA: not available.</p

Immunohistostaining of infiltrating CD4+ and CD8+ T cells in tumor tissues of mirtazapine-treated, i.e. <i>concurrent</i> and <i>always</i>, and <i>never</i> mice.

<p>(A) CD4 and (B) CD8 in tumors were performed on day 42 post tumor inoculation. Magnification 200 ×. (C) Quantification of CD4+ and CD8+ T cells. (n = 3, **<i>p</i><0.01 and ***<i>p</i><0.001 as compared to those of <i>never</i>).</p