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Indoleamine 2,3-dioxygenase promotes peritoneal metastasis of ovarian cancer by inducing an immunosuppressive environment.

Tanizaki Y, Kobayashi A, Toujima S, Shiro M, Mizoguchi M, Mabuchi Y, Yagi S, Minami S, Takikawa O, Ino K - Cancer Sci. (2014)

Bottom Line: This tumor-progressive effect was coincident with significantly reduced numbers of CD8(+) T cells and natural killer cells within tumors as well as increased levels of transforming growth factor-β and interleukin-10 in ascites.Finally, treatment with the IDO inhibitor 1-methyl-tryptophan significantly suppressed tumor dissemination and ascites with reduced transforming growth factor-β secretion.These findings showed that tumor-derived IDO promotes the peritoneal dissemination of ovarian cancer through suppression of tumor-infiltrating effector T cell and natural killer cell recruitment and reciprocal enhancement of immunosuppressive cytokines in ascites, creating an immunotolerogenic environment within the peritoneal cavity.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Wakayama Medical University, Wakayama, Japan.

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(a) Western blot analyses of indoleamine 2,3-dioxygenase (IDO) expression in interferon-γ (IFN-γ)-treated mouse ovarian carcinoma OV2944-HM-1 (HM-1) cells. Treatment with IFN-γ enhanced IDO protein expression. (b) Establishment of IDO-overexpressing HM-1 cells. Western blot analyses of IDO expression in stable clones of HM-1 cells transfected with mouse IDO cDNA (HM-1-IDO). (c) Evaluation of IDO enzymatic activity by measuring the concentrations of tryptophan (Trp) and its main catabolite, kynurenine (Kyn), in the conditioned medium using HPLC. Representative results from three independent experiments are shown. (d) Morphological views of HM-1-parental, control vector-transfected (HM-1-mock), and HM-1-IDO cells. Bar = 50 μm. (e) In vitro cell proliferative activity of HM-1-parental, HM-1-mock, and HM-1-IDO cells. Mean ± SD from three independent experiments are shown. O.D., optical density. (f) In vitro cell migratory potential of HM-1-parental, HM-1-mock, and HM-1-IDO cells evaluated by wound healing assay after 24 h. Mean ± SD from three independent experiments are shown.
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fig01: (a) Western blot analyses of indoleamine 2,3-dioxygenase (IDO) expression in interferon-γ (IFN-γ)-treated mouse ovarian carcinoma OV2944-HM-1 (HM-1) cells. Treatment with IFN-γ enhanced IDO protein expression. (b) Establishment of IDO-overexpressing HM-1 cells. Western blot analyses of IDO expression in stable clones of HM-1 cells transfected with mouse IDO cDNA (HM-1-IDO). (c) Evaluation of IDO enzymatic activity by measuring the concentrations of tryptophan (Trp) and its main catabolite, kynurenine (Kyn), in the conditioned medium using HPLC. Representative results from three independent experiments are shown. (d) Morphological views of HM-1-parental, control vector-transfected (HM-1-mock), and HM-1-IDO cells. Bar = 50 μm. (e) In vitro cell proliferative activity of HM-1-parental, HM-1-mock, and HM-1-IDO cells. Mean ± SD from three independent experiments are shown. O.D., optical density. (f) In vitro cell migratory potential of HM-1-parental, HM-1-mock, and HM-1-IDO cells evaluated by wound healing assay after 24 h. Mean ± SD from three independent experiments are shown.

Mentions: Under steady-state conditions, HM-1 cells slightly expressed IDO protein, but it was markedly enhanced by the IFN-γ treatment (Fig. 1a), which is consistent with a previous study in other tumor cell lines.(25) In order to establish stable clones of IDO-overexpressing mouse ovarian cancer cells, we transfected mouse IDO cDNA into HM-1 cells and selected clones with high IDO expression (Fig. 1b). In the next step, we evaluated IDO enzymatic activity in these clones by measuring the concentrations of tryptophan and its main catabolite kynurenine in the conditioned medium. In clone 8, tryptophan was almost completely depleted, and reciprocally kynurenine was strongly produced after 48 h (Fig. 1c), suggesting that this clone had high tryptophan catabolizing activity. Thus, we used clone 8 as the IDO-overexpressing cells (HM-1-IDO) in the following experiments. Clones transfected with control vector (HM-1-mock) scarcely expressed IDO protein, and showed no enzymatic activity (Fig. 1b,c).


Indoleamine 2,3-dioxygenase promotes peritoneal metastasis of ovarian cancer by inducing an immunosuppressive environment.

Tanizaki Y, Kobayashi A, Toujima S, Shiro M, Mizoguchi M, Mabuchi Y, Yagi S, Minami S, Takikawa O, Ino K - Cancer Sci. (2014)

(a) Western blot analyses of indoleamine 2,3-dioxygenase (IDO) expression in interferon-γ (IFN-γ)-treated mouse ovarian carcinoma OV2944-HM-1 (HM-1) cells. Treatment with IFN-γ enhanced IDO protein expression. (b) Establishment of IDO-overexpressing HM-1 cells. Western blot analyses of IDO expression in stable clones of HM-1 cells transfected with mouse IDO cDNA (HM-1-IDO). (c) Evaluation of IDO enzymatic activity by measuring the concentrations of tryptophan (Trp) and its main catabolite, kynurenine (Kyn), in the conditioned medium using HPLC. Representative results from three independent experiments are shown. (d) Morphological views of HM-1-parental, control vector-transfected (HM-1-mock), and HM-1-IDO cells. Bar = 50 μm. (e) In vitro cell proliferative activity of HM-1-parental, HM-1-mock, and HM-1-IDO cells. Mean ± SD from three independent experiments are shown. O.D., optical density. (f) In vitro cell migratory potential of HM-1-parental, HM-1-mock, and HM-1-IDO cells evaluated by wound healing assay after 24 h. Mean ± SD from three independent experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317851&req=5

fig01: (a) Western blot analyses of indoleamine 2,3-dioxygenase (IDO) expression in interferon-γ (IFN-γ)-treated mouse ovarian carcinoma OV2944-HM-1 (HM-1) cells. Treatment with IFN-γ enhanced IDO protein expression. (b) Establishment of IDO-overexpressing HM-1 cells. Western blot analyses of IDO expression in stable clones of HM-1 cells transfected with mouse IDO cDNA (HM-1-IDO). (c) Evaluation of IDO enzymatic activity by measuring the concentrations of tryptophan (Trp) and its main catabolite, kynurenine (Kyn), in the conditioned medium using HPLC. Representative results from three independent experiments are shown. (d) Morphological views of HM-1-parental, control vector-transfected (HM-1-mock), and HM-1-IDO cells. Bar = 50 μm. (e) In vitro cell proliferative activity of HM-1-parental, HM-1-mock, and HM-1-IDO cells. Mean ± SD from three independent experiments are shown. O.D., optical density. (f) In vitro cell migratory potential of HM-1-parental, HM-1-mock, and HM-1-IDO cells evaluated by wound healing assay after 24 h. Mean ± SD from three independent experiments are shown.
Mentions: Under steady-state conditions, HM-1 cells slightly expressed IDO protein, but it was markedly enhanced by the IFN-γ treatment (Fig. 1a), which is consistent with a previous study in other tumor cell lines.(25) In order to establish stable clones of IDO-overexpressing mouse ovarian cancer cells, we transfected mouse IDO cDNA into HM-1 cells and selected clones with high IDO expression (Fig. 1b). In the next step, we evaluated IDO enzymatic activity in these clones by measuring the concentrations of tryptophan and its main catabolite kynurenine in the conditioned medium. In clone 8, tryptophan was almost completely depleted, and reciprocally kynurenine was strongly produced after 48 h (Fig. 1c), suggesting that this clone had high tryptophan catabolizing activity. Thus, we used clone 8 as the IDO-overexpressing cells (HM-1-IDO) in the following experiments. Clones transfected with control vector (HM-1-mock) scarcely expressed IDO protein, and showed no enzymatic activity (Fig. 1b,c).

Bottom Line: This tumor-progressive effect was coincident with significantly reduced numbers of CD8(+) T cells and natural killer cells within tumors as well as increased levels of transforming growth factor-β and interleukin-10 in ascites.Finally, treatment with the IDO inhibitor 1-methyl-tryptophan significantly suppressed tumor dissemination and ascites with reduced transforming growth factor-β secretion.These findings showed that tumor-derived IDO promotes the peritoneal dissemination of ovarian cancer through suppression of tumor-infiltrating effector T cell and natural killer cell recruitment and reciprocal enhancement of immunosuppressive cytokines in ascites, creating an immunotolerogenic environment within the peritoneal cavity.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Wakayama Medical University, Wakayama, Japan.

Show MeSH
Related in: MedlinePlus