The Role of Indoleamine 2,3-Dioxygenase in Diethylnitrosamine-Induced Liver Carcinogenesis

Abstract

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing intracellular enzyme of the L-kynurenine pathway, causes preneoplastic cells and tumor cells to escape the immune system by inducing immune tolerance; this mechanism might be associated with the development and progression of human malignancies. In the present study, we investigated the role of IDO in diethylnitrosamine (DEN)-induced hepatocarcinogenesis by using IDO-knockout (KO) mice. To induce hepatocellular carcinoma (HCC), hepatic adenoma, and preneoplastic hepatocellular lesions termed foci of cellular alteration (FCA), male IDO-wild-type (WT) and IDO-KO mice with a C57BL/6J background received a single intraperitoneal injection of DEN at 2 weeks of age. The mice were sacrificed to evaluate the development of FCA and hepatocellular neoplasms. HCC overexpressed IDO and L-kynurenine compared to surrounding normal tissue in the DEN-treated IDO-WT mice. The number and cell proliferative activity of FCAs, and the incidence and multiplicity of HCC were significantly greater in the IDO-WT than in the IDO-KO mice. The expression levels of the IDO protein, of L-kynurenine, and of IFN-γ, COX-2, TNF-α, and Foxp3 mRNA were also significantly increased in the DEN-induced hepatic tumors that developed in the IDO-WT mice. The mRNA expression levels of CD8, perforin and granzyme B were markedly increased in hepatic tumors developed in IDO-KO mice. Moreover, Foxp3-positive inflammatory cells had infiltrated into the livers of DEN-treated IDO-WT mice, whereas fewer cells had infiltrated into the livers of IDO-KO mice. Induction of IDO and elevation of L-kynurenine might play a critical role in both the early and late phase of liver carcinogenesis. Our findings suggest that inhibition of IDO might offer a promising strategy for the prevention of liver cancer.

Fig 1. Analysis of pre-neoplastic lesions and nodules of DEN-treated mice.

Development of foci of cellular alteration (FCA), the proliferating cell nuclear antigen (PCNA)-labeling indices of the FCA, and the nodule frequency on the liver surface of DEN-treated experimental mice. (A) A representative photograph of a FCA that developed in DEN-treated WT mice at 32 weeks of age (H&E staining, left panel) and the average number of FCA in DEN-treated WT and IDO-/- mice at 20 and 32 weeks of age (right panel). (B) Representative photographs of PCNA-immunohistochemical analysis of the FCA that developed in DEN-treated WT mice and IDO-/- mice at 32 weeks of age (left panels). The PCNA-labeling indices of the FCA that developed in the experimental mice were determined by counting the number of PCNA-positive nuclei in the FCA (right panel). (C) Tumor nodules on the liver surface of DEN-treated WT and IDO-/- mice at 32 weeks. The white arrows indicate tumor nodules (left panel). The number of liver surface tumors in IDO-/- mice was significantly lower than that in wild-type mice (right panel). *, P<0.01 Data points represent the mean ± SD.

Fig 2. Representative immunohistochemical expression (IHC) of IDO and kynurenine (KYN).

Representative images of normal tissue (top panel) of a saline treated WT mouse, of HCC (second panels) and adenoma (third panels) of a WT mouse, and of adenoma of an IDO-/- mouse (bottom panels), at 32 weeks of DEN treatment that were stained with H&E (a-d), or were immunohistochemically stained for the IDO protein (e-h) or for L-KYN (i-l). The black line in the HCC tissues demarcates HCC from non-HCC tissue. (Bar = 50 μm)

Fig 3. Gene expression of IFN-γ, COX-2, and TNF-α in the liver.

Expression levels of (A) IFN-γ, (B) COX-2, and (C) TNF-α mRNA in hepatic tumors and in normal liver tissues of the indicated mice were measured using quantitative RT-PCR. Data points represent the mean ± SD. *, p<0.05.

Fig 4. Expression of Foxp3 analyzed by immunohistochemistry and RT-PCR.

(A) Gene expression of Foxp3 in control and DEN-treated non-tumorous liver tissues at 32 weeks. (B) Representative pictures of the immunohistochemical analysis of FCA for Foxp3 and (C) Foxp3 mRNA expression in hepatic tumors of DEN-treated mice. mRNA expression was determined using quantitative RT-PCR. Data points represent the mean ± SD. *, p<0.05.

Fig 5. Expression levels of CD8, FasL, perforin and granzyme B in non-tumorous liver tissues and hepatic tumors.

Expression levels of (A) CD8, (B) FasL, (C) perforin and (D) granzyme B mRNA in hepatic tumors and in normal liver tissues of the indicated mice were measured using quantitative RT-PCR. Data points represent the mean ± SD. *, p<0.05.

Fig 6. The serum L-kynurenine/L-tryptophan ratio and tissue gene expression of IDO and TDO.

A, The serum L-kynurenine/L-tryptophan ratio (Kyn/Trp) was determined at 32 weeks by measuring the concentrations of L-kynurenine and L-tryptophan using HPLC. (B, C) Liver tissues were separated into hepatic tumors and non-tumorous tissues. Total RNA was then extracted and the expression levels of IDO and TDO mRNA were measured using quantitative RT-PCR. Data points represent the mean ± SD. * p<0.05, ** p<0.01, N.S.: not significant