Nobuhiko Nakamura1, Takeshi Hara1, Yuhei Shibata1, Takuro Matsumoto2, Ryoko Mabuchi1, Hiroshi Nakamura1, Junichi Kitagawa1, Nobuhiro Kanemura1, Naoe Goto1, Masahito Shimizu1, Hisashi Tsurumi1, Hisataka Moriwaki1
1First Department of Internal Medicine, Gifu University Graduate School of Medicine, Gifu, Japan
2Japanese Red Cross Takayama Hospital, Takayama, Japan
Tóm tắt
Background
Indoleamine 2,3-dioxygenase (IDO) catalyzes the rate-limiting step in the catabolism of tryptophan along the kynurenine pathway. In tumors, increased IDO activity inhibits T cell and natural killer cell proliferation and induces apoptosis. We have previously reported that IDO expression in lymphoma cells and serum concentration of kynurenine in diffuse large B-cell lymphoma are useful prognostic factors. Preclinical studies in rodents have demonstrated that IDO inhibitors, such as 1-methyl-D-tryptophan (D-1MT), are therapeutically beneficial, especially when combined with different types of cytotoxic chemotherapeutic agents. We investigated the therapeutic potential of IDO inhibitor D-1MT with cyclophosphamide (CY) by using an IDO-positive B-cell lymphoma model mouse.
Materials and Methods
To establish tumors, 1x106 A20 cells (BALB/c B-cell lymphoma cell line) were subcutaneously injected into the lower back of naïve syngeneic BALB/c mice. Seven days later, 20 BALB/c mice were treated with D-1MT (Sigma-Aldrich, Saint Louis, MO, USA) and/or CY. The mice were fed with D-1MT-supplemented water (5mg/mL), which was replaced every two to three days. CY was administered at 80 mg/kg i.p. on day 10. To examine the effect of D-1MT on A20 tumors and tumor-draining lymph nodes (TDLNs), mice were sacrificed on day 28. Serum concentration of kynurenine and tryptophan were measured by high-performance liquid chromatography. CD4+CD25+Foxp3+ regulatory T cells (Tregs) were counted by flow cytometry with a FACSCantoII flow cytometer (BD Biosciences, San Jose, CA, USA), and data were analyzed with FACSDiva 6.1 software (BD Biosciences). Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed with specific primer/probe sets that amplify IDO1, Foxp3, IFN-γ, cyclooxygenase-2 (COX-2), and GAPDH genes (TaqMan Gene Expression Assays; Applied Biosystems, Foster City, CA).
Results
The mean body and spleen weights of the mice did not differ significantly among the groups. The mean drinking water intakes (mL/day/mouse) of the D-1MT, CY, and D-1MT+CY groups were significantly less than that of the control group (p< 0.05). All mice implanted with A20 cells developed tumors that became palpable after day 7. On day 28, the tumor volume in the D-1MT+CY group was significantly less than that in the control group (p< 0.05), and there were no significant differences among the other treatment groups (Figure 1). There were no significant differences in the serum kynurenine-to-tryptophan ratio and the IDO1 expression level in the tumors among any treatment groups. The expression levels of IFN-γ and COX-2 mRNA in TDLNs were found to be significantly up-regulated in the CY and D-1MT+CY groups. By quantitative RT-PCR, the expression levels of IFN-γ and COX-2 mRNA in TDLNs were found to be significantly up-regulated in the CY and D-1MT+CY groups. The Foxp3 expression level and number of Tregs in TDLNs in the CY and D-1MT+CY groups were significantly higher than in the control group on day 28. The number of Tregs in TDLNs in the D-1MT+CY group was significantly lower than those in CY groups on day 17 (p< 0.05) (Figure 2).
Conclusion
Our results suggest that D-1MT in combination with CY is an effective treatment for IDO-positive lymphoma in a model mouse by reducing Tregs and breaking tumor tolerance. This antitumor effect might be induced by the suppression of Tregs by D-1MT. Our findings suggest that inhibition of IDO might offer a promising treatment strategy for lymphoma.
Disclosures:
No relevant conflicts of interest to declare.
