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. 2011;6(5):e19823.
doi: 10.1371/journal.pone.0019823. Epub 2011 May 20.

The indoleamine-2,3-dioxygenase (IDO) inhibitor 1-methyl-D-tryptophan upregulates IDO1 in human cancer cells

Christiane A Opitz  1 , Ulrike M Litzenburger, Uta Opitz, Felix Sahm, Katharina Ochs, Christian Lutz, Wolfgang Wick, Michael Platten
Affiliations

The indoleamine-2,3-dioxygenase (IDO) inhibitor 1-methyl-D-tryptophan upregulates IDO1 in human cancer cells

Christiane A Opitz et al. PLoS One. 2011.

Abstract

1-methyl-D-tryptophan (1-D-MT) is currently being used in clinical trials in patients with relapsed or refractory solid tumors with the aim of inhibiting indoleamine-2,3-dioxygenase (IDO)-mediated tumor immune escape. IDO is expressed in tumors and tumor-draining lymph nodes and degrades tryptophan (trp) to create an immunsuppressive micromilieu both by depleting trp and by accumulating immunosuppressive metabolites of the kynurenine (kyn) pathway. Here we show that proliferation of alloreactive T-cells cocultured with IDO1-positive human cancer cells paradoxically was inhibited by 1-D-MT. Surprisingly incubation with 1-D-MT increased kyn production of human cancer cells. Cell-free assays revealed that 1-D-MT did not alter IDO1 enzymatic activity. Instead, 1-D-MT induced IDO1 mRNA and protein expression through pathways involving p38 MAPK and JNK signalling. Treatment of cancer patients with 1-D-MT has transcriptional effects that may promote rather than suppress anti-tumor immune escape by increasing IDO1 in the cancer cells. These off-target effects should be carefully analyzed in the ongoing clinical trials with 1-D-MT.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist. Uta Opitz and Christian Lutz are employees of Heidelberg Pharma. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. SKOV-3 ovarian carcinoma cells constitutively degrade trp via indoleamine-2,3-dioxygenase-1 (IDO1).
(A) Relative mRNA expression of the three trp-degrading enzymes IDO1, IDO2 and tryptophan-2,3-dioxygenase (TDO) (white bars) and kyn production (black bar) of SKOV-3 cells, measured by quantitative RT-PCR and high performance liquid chromatography (HPLC). (B) Knockdown of IDO1 mRNA by siRNA measured by qRT-PCR. (C) Western blot analysis showing IDO1 protein expression in SKOV-3 cells with siRNA mediated knockdown of IDO1 in comparison to controls. (D) Immunocytochemistry (red, IDO1 staining; blue, DAPI nuclear staining) of control SKOV-3 cells and SKOV-3 cells with IDO1 knockdown. (E) Kyn release of SKOV-3 cells after knockdown of IDO1 in comparison to controls. Experiments were performed at least in triplicate. Data are mean ± SEM. * (p<0.05).
Figure 2
Figure 2. 1-D-MT reduces T cell proliferation in cocultures of SKOV-3 cells with mixed leukocyte reactions.
(A) Alloreactive T cell proliferation after addition of 25 µM kyn to mixed leukocyte reactions (MLR). (B) Alloreactive T cell proliferation in the presence of 6000 SKOV-3 cells. (C) T cell proliferation in MLR cocultured with 2000 control SKOV-3 cells (white bar) or 2000 SKOV-3 cells with a knockdown of IDO1 (black bar). (D) T cell proliferation in cocultures of MLR with 2000 SKOV-3 cells after addition of increasing concentrations of 1-L-MT. (E) T cell proliferation in cocultures of MLR with 2000 SKOV-3 cells after addition of increasing concentrations of 1-D-MT. (F) Representative result of MLR/SKOV-3 coculture experiments with PBMC from five different donors and 2000 or 6000 SKOV-3 cells. Cells were treated with or without 1 mM 1-D-MT in combination with or without 250 µM trp. Proliferation was measured by 3[H] methylthymidine uptake. Experiments were performed at least in triplicate. Data are mean ± SEM. * (p<0.05).
Figure 3
Figure 3. 1-D-MT does not inhibit the proliferation or cell cycle progression of SKOV-3 cells.
(A) 3[H] methylthymidine incorporation of SKOV-3 cells treated with 1 mM 1-D-MT (black bar) or vehicle (white bar) for 6 days. (B) Cell cycle analysis of SKOV-3 cells treated with 1 mM 1-D-MT or vehicle for 48 h. (C) Proliferation analysis of CFSE-stained lymphocytes from 6 day cocultures of MLR with 2000 SKOV-3 cells, treated with indicated concentrations of 1-D-MT (upper panel). Plot of the cell numbers in each generation of the above experiment (lower panel).
Figure 4
Figure 4. Increased kyn release of SKOV-3 cells upon 1-D-MT treatment.
(A) Kyn concentrations released by SKOV-3 cells after treatment with 1-D-MT (white circles), 1-L-MT (black circles) and the racemic mixture of 1-MT (black triangles) measured after 48 h by HPLC. (B) Kyn release of SKOV-3 cells in response to 500 µM 1-D-MT in the presence of increasing trp concentrations. (C) Kyn release of SKOV-3 cells treated with different concentrations of trp alone (open circles) or in combination with 1 mM 1-D-MT (filled circles) measured after 48 h by HPLC. (D) Kyn production in IDO1 enzymatic assays performed in the presence of 100 µM trp in combination with increasing 1-D-MT concentrations. (E) Kyn production of IDO1 enzyme in the presence of increasing concentrations of trp alone (open circles) or in combination with 1-D-MT (filled circles). Experiments were performed in triplicate. Data are mean ± SEM. * (p<0.05).
Figure 5
Figure 5. Upregulation of IDO1 mRNA by 1-D-MT in SKOV-3 cells.
(A) Left panel: mRNA expression of IDO1, IDO2 and TDO in SKOV-3 cells after treatment with 1 mM 1-D-MT, analyzed after 24 h by qRT-PCR. Right panel: Western Blot analysis of IDO1 expression in SKOV-3 cells performed after 48 h 1 mM 1-D-MT. GAPDH served as loading control. (B) IDO1 mRNA expression in response to increasing concentrations of 1-D-MT measured after 24 h by qRT-PCR. (C) Time course analysis of IDO1 mRNA induction by 1 mM 1-D-MT, analyzed by qRT-PCR. Experiments were performed in triplicate. Data are mean ± SEM. * (p<0.05).
Figure 6
Figure 6. 1-D-MT upregulates IDO1 expression and kyn release induced by different concentrations of IFN-γ in diverse cancer cells.
(A) Representative HPLC graphs of kyn production of HeLa cells, which were either untreated, treated with 1 mM 1-D-MT and/or 1000 U IFN-γ for 72 h. Absorption of kyn was measured at 365 nm. (B) In untreated HeLa cells 1 mM 1-D-MT did not induce de novo IDO1 mRNA, but increased IDO1 mRNA after its induction by 1000 U IFN-γ mRNA expression was analyzed by qRT-PCR 24 h after treatment. (C) Representative example of the effect of different IFN-γ concentrations on IDO1 mRNA induction by 1-D-MT, shown in U251 glioma cells. (D) IDO1 mRNA expression of indicated cell lines that were stimulated for 24 h with appropriate concentrations of IFN-γ alone (white bars) or in combination with 1 mM 1-D-MT (black bars). (E) Representative example of IDO1 mRNA induction by 200 µM 1-D-MT in IFN-γ-stimulated T98G glioma cells. (F) Kyn release of indicated cell lines that were stimulated for 72 h with appropriate concentrations of IFN-γ alone (white bars) or in combination with 1 mM 1-D-MT (black bars), measured by HPLC. Experiments were performed in triplicate. Data are mean ± SEM. * (p<0.05).
Figure 7
Figure 7. STAT1 signaling is not involved in the IDO1 upregulation in response to 1-D-MT.
(A) Knockdown of STAT1 mRNA by si-RNA (white bars) did not affect kyn release (black bars) of 1-D-MT (1 mM) treated SKOV-3 cells. (B) Analysis of IFN-γ and IFN-β mRNA expression in SKOV-3 cells after stimulation with 1 mM 1-D-MT for 24 h.
Figure 8
Figure 8. p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways participate in the 1-D-MT mediated upregulation of IDO1.
IDO1 mRNA (left panel) and kyn release (right panel) of SKOV-3 cells treated with (A) 50 µM of the MEK1 inhibitor PD98059, (B) 20 µM of the p38 MAPK inhibitor SB203580 and (C) 20 µM of the JNK inhibitor SP600125 or control 1 h before addition of 1 mM 1-D-MT analyzed by qRT-PCR after 24 h and HPLC after 48 h, respectively. Experiments were performed in triplicate. Data are mean ± SEM. * (p<0.05).
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