Induction of tryptophan hydroxylase in the liver of s.c. tumor model of prostate cancer

Abstract

Enhanced degradation of tryptophan (Trp) and thus decreased plasma Trp levels are common in several types of cancers. Although it is well known that Trp catabolism is induced in the tumor microenvironment by the enzymes expressed in cancer cells, immune cells, or both, few studies have examined systemic Trp catabolism in cancer pathophysiology. The present study aimed to evaluate Trp catabolism in both tumor and peripheral tissues using tumor-engrafted Copenhagen rats that were s.c. inoculated with AT-2 rat prostate cancer cells negative for expression of Trp catabolic enzymes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics showed significantly decreased plasma Trp levels in AT-2 engrafted rats, accompanied by increased kynurenine/Trp ratios in spleen and thymus and serotonin levels in liver and thymus. Quantitative PCR and enzymatic activity assays showed indoleamine-2, 3-dioxygenase, an inducible enzyme that catalyzes Trp to kynurenine, was increased in tumor tissues, whereas tryptophan-2,3-dioxygenase, a major Trp catabolic enzyme that regulates systemic level of Trp, tended to be increased in the liver of AT-2 engrafted rats. Furthermore, tryptophan hydroxylase-1 (TPH1), an enzyme that catalyzes the reaction of Trp to serotonin, was significantly increased in liver and spleen of AT-2 engrafted rats. Further histochemical analysis revealed that the induction of TPH1 in the liver could be attributed to infiltration of mast cells. A similar phenomenon was observed with nonneoplastic liver samples from colorectal cancer patients. These results suggested that Trp catabolism toward serotonin synthesis might be induced in peripheral remote tissues in cancer, which could have a pathophysiological effect on cancer.

Keywords: liver; mast cell; serotonin; tryptophan; tryptophan hydroxylase.

Figure 1

Tryptophan metabolism along kynurenine and serotonin pathways. IDO, indoleamine‐2, 3‐dioxygenase; TDO, tryptophan‐2, 3‐dioxygenase; TPH, tryptophan hydroxylase

Figure 2

Tryptophan (Trp) and its metabolite concentrations (conc.) in plasma. Plasma Trp (A), kynurenine (Kyn) (B), and 5‐hydroxytryptophan (5‐HTP) (C) concentrations, and Kyn / Trp (D) and 5‐HTP / Trp (E) ratios in control and AT‐2 engrafted rats were measured every week after tumor inoculation for 5 weeks. Data are the means ± SD (N = 6/group). *P < .05, **P < .01, ***P < .001 vs control by Student’s t test

Figure 3

Tryptophan‐2, 3‐dioxygenase (TDO) activities tend to be increased in livers of AT‐2 engrafted rats. A, B, mRNA (A) and protein levels of TDO2 (B) in the liver after 5 weeks of tumor inoculation. C, TDO activities in the liver. D, Correlation between TDO activities and kynurenine concentration (Kyn conc.) in liver. Pearson’s correlation coefficient (R) and P values are reported. E, NADPH levels in liver. PC, Positive control (HepG2 cell homogenates)

Figure 4

Indoleamine‐2, 3‐dioxygenase‐1 (IDO1) is induced in tumor but not in immune tissues of AT‐2 engrafted rats. A, mRNA levels of Ido1 in spleen, thymus, and tumor. B, IDO1 activities in spleen and tumor. Data are means ± SD (N = 6/group). *P < .05, calculated by Student’s t test. Kyn, kynurenine

Figure 5

Tryptophan hydroxylase‐1 (TPH1) expression and activity are significantly increased in liver and spleen of AT‐2 engrafted rats. A, Tph1 mRNA levels in investigated tissues. Results were normalized to the level in spleen of control that showed lowest expression. Data are means ± SD (N = 6/group). *P < .05, **P < .01 vs control by Student’s t test. B, TPH1 activities in liver, spleen, and tumor. Data are means ± SD (N = 6/group). ***P < .001 vs control by Student’s t test. C, Representative images of immunohistochemical staining of TPH1 in livers of control and AT‐2 rats. Brown staining indicates TPH1 protein. D, Representative images of in situ hybridization of TPH1 in livers of control and AT‐2 rats. Red staining indicates TPH1 expression. E, Representative images of toluidine blue staining of livers of control and AT‐2 rats. Red purple staining indicates the presence of mast cells. Scale bar  = 100 µm

Figure 6

Tryptophan hydroxylase‐1 (TPH1)‐positive cell infiltration into nonneoplastic livers of colorectal cancer (CRC) patients. Representative images of TPH1 immunohistochemical staining in the livers of 2 CRC patients and a healthy subject. Arrowheads in top panels indicate TPH1‐positive cells. Scale bar = 100 µm