Enhancement of radiation therapy by indoleamine 2,3 dioxygenase 1 inhibition through multimodal mechanisms

Abstract

Background: Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme that converts tryptophan to kynurenine. IDO1 expression is found not only in tumor cells but also in immune cells and is associated with tumor proliferation and immune responses. IDO1 inhibitors and radiation may cooperatively suppress tumor proliferation through the alterations in the Wnt/β-catenin pathway, cell cycle, and immune response. We investigated the antitumor effects of combination therapy of an IDO1 inhibitor, 1-methyl tryptophan (1-MT), and radiation on colorectal cancer.

Methods: In vitro experiments were conducted using human and murine colon cancer cell lines (HCT116, HT-29, and Colon26). Cell growth inhibition was assessed using a MTS assay and Clonogenic assay. Cells were cultured for 48 h with or without 500 µM 1-MT after exposure to radiation (4 Gy). Cell cycle effects and modulation of Wnt/β-catenin pathway were evaluated using western blot analysis, flow cytometry, RT-PCR. Subcutaneous Colon26 tumors in BALB/c mice were treated by oral 1-MT (6 mg/mL) for 2 weeks and/or local radiation (10 Gy/10 fr). Bromodeoxyuridine (BrdU) incorporation in tumor cells and expression of differentiation markers of immune cells were evaluated using immunohistochemistry.

Results: 1-MT and a small interfering RNA against IDO1 suppressed proliferation of all cell lines, which was rescued by kynurenine. Clonogenic assay showed that administration of 1-MT improved radiosensitivity by suppressing the Wnt/β-catenin pathway activated by radiation and enhancing cell cycle arrest induced by radiation. Combination therapy showed a further reduction in tumor burden compared with monotherapies or untreated control, inducing the highest numbers of intratumoral CD3 + and CD8 + T cells and the lowest numbers of Foxp3 + and BrdU-positive tumor cells.

Conclusions: The combination of 1-MT and radiation suppressed colon cancer cells in vitro and in vivo via multiple mechanisms.

Keywords: Colorectal cancer; Indoleamine 2,3 dioxygenase 1; Radiation therapy; Wnt/β-catenin pathway.

Fig. 1

IDO1 expression in colorectal cancer cell lines. IDO1 was stained by green fluorescence, and the nuclei were visualized blue with DAPI. Bars, 20 µm

Fig. 2

Effects of IDO1 inhibition on the proliferation of colorectal cancer cells in vitro. Inhibition by gene silencing of IDO1 in HCT116 (a) and HT-29 cells (b). Inhibition by 1-methyl tryptophan (1-MT) in HCT116 cells (c,d), HT-29 (e) and Colon26 (f). Results after 48-h treatment are shown as absorbances measured at 490 nm by the MTS assay. Concentration of siRNA,10 nM; concentration of kynurenine (Kyn), 100 µM. Bars indicate standard deviations. * p < 0.001

Fig. 3

Clonogenic assay of colorectal cancer cells after exposure to various doses of radiation with or without 1-MT. HCT116 (upper left panel), HT-29 (upper right panel), and Colon26 (lower left panel). Bars indicate standard deviations. * p < 0.05 in comparison between cells treated with and without 1-MT (500 µM)

Fig. 4

Axin2 mRNA expression in colorectal cancer cells treated by 1-MT and/or radiation by quantitative reverse-transcription polymerase chain reaction. HCT116 (upper left panel), HT-29 (upper right panel), and Colon26 (lower left panel). RT: radiation. Results 48 h after administration of 1-MT (500 µM) and/or radiation (4 Gy) are shown. Bars indicate standard deviations. * p < 0.0083

Fig. 5

Cell cycle analysis of colorectal cancer cells treated by 1-MT and/or radiation by flow cytometry. HCT116 (upper left panel), HT-29 (upper right panel), and Colon26 (lower left panel). RT: radiation. Results 48 h after administration of 1-MT (500 µM) and/or radiation (4 Gy) are shown. Bars indicate standard deviations. * p < 0.0083

Fig. 6

Effects of 1-MT and/or radiation on subcutaneous Colon26 tumors. Temporal change in tumor size over treatment course was shown for each group. RT: radiation. Bars indicate standard deviations. * p < 0.001

Fig. 7

BrdU incorporation in tumor cells and expression of differentiation markers of immune cells in subcutaneous Colon26 tumors. a BrdU-positive tumor cells, b CD3 + T cells, c CD8 + T cells, d Foxp3 + T cells. RT: radiation. Treatments are shown as horizontal axis labels of the graph. Bars in graphs indicate standard deviations. * p < 0.001

Fig. 8

Schematic diagrams illustrating the mechanisms for tumor-suppression by IDO1 blockade and radiation. a Schematic model for IDO1-induced sequential activations of kynurenine-PI3K-Akt axis and Wnt/β-catenin pathway in tumor cells, b Presumable mechanisms for activation of antitumor immune response by IDO1 blockade and radiation therapy. 1-MT: 1-methyl tryptophan, RT: radiation, Trp: tryptophan, IDO1: indoleamine 2,3 dioxygenase 1, Kyn: kynurenine, KPM: kynurenine pathway metabolites, LAT1: L-amino acid transporter 1, CTLA-4: cytotoxic T lymphocyte-associated antigen-4, GCN2: general control non-depressive 2 kinase, AHR: aryl hydrocarbon receptor