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Abstract
Purpose
Cyclooxygenase-2 (COX-2) and its metabolite, PGE2 affect multiple tumorigenesis, including angiogenesis, invasion, and tumor-induced immune suppression. Their overexpression is association with impaired immune cell function in many tumors. Indoleamine 2,3-dioxygenase (IDO) is an emerging immunoregulatory enzyme that can catalyze the initial rate-limiting step in tryptophan catabolism, by causing tryptophan depletion can block T lymphocyte activation, and thus, enable tumor cells to escape from immune system. Although the potential of immunosuppression associated with tumor-produced COX-2 has been suggested, the mechanism of immunosuppression in tumor immunology is not yet well defined. Thus, we hypothesized that the tumor immunity of COX-2 could be partly due to IDO-dependent immune tolerance. To test this hypothesis, we evaluated IDO expression in cancer cells treated with selective COX-2 inhibitor.
Materials and Methods
The A549 human adenocarcinoma cell line, murine Lewis lung carcinoma (LLC) cell line and C57Bl/6 mice were used for in vitro and in vivo studies. In vitro studies, A549 cells were treated with various concentrations of COX-2 inhibitor (PTPBS) or PGE2. IDO enzyme activity and protein expression were checked by IDO enzyme activity assay and Western blotting. In vivo study, the 20 mice were randomized into normal control, LLC inoculated control, and low and high selective COX-2 inhibitor (celecoxib 25 or 250 mg/kg/day) treated LLL inoculated mice groups (n=5 per group). At one month, mice were sacrificed and tumor mass was isolated for quantification of IDO expression by immunohistochemical stain and western blotting.
Results
In vitro studies, PTPBS treated A549 cells showed a significant decreased in IDO enzyme activity and expression but PGE2 treated A549 cells showed increased in IDO expression. In vivo studies, the tumor mass and lung metastasis were attenuated by celecoxib (respectively, p＜0.05, p＜0.01). Compared with the LLC inoculated control group, mice treated with celecoxib had significant reductions in IDO expression of tumor mass (IDO immunohistochemical stain and western blotting).
Conclusion
The present study reveals that COX-2 inhibitor serves to restore the tumor-induced IDO expression and promotes antitumor reactivity in an immunocompetent murine lung cancer model. These findings further support the suggestion that COX-2 inhibitor is a potential pharmacological immunotherapy in cancer.
References
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Fig. 1.
IDO expression in A549 cells stimulated with IFN-γ. (A) After incubating A549 cells with IFN-γ for 24 h, IDO expression was measured by Western blotting. (B) IDO activities were determined by measuring kynurenine formation and were calculated as percentages with respect to control. Data are means± SEM of three experiments (*p＜0.01 vs. control, † p ＜0.05 vs. IFN-γ treated group).
Fig. 2.
Effect of COX-2 inhibitor (PTPBS) on the activity of IDO produced by A549 cells in response to IFN-γ. (A) IDO activities were determined by measuring kynurenine formation and were calculated as percentages versus control. Data are means± SEM of three experiments (*p＜0.01 vs. IFN-γ 1,000 U/ml, † p＜0.05 vs. IFN-γ 1,000 U/ml+PTPBS 1μ M). (B) IDO expressions in A549 cells treated with IFN-γ plus PTPBS.
Fig. 3.
The growth curve of LLC tumors, in vivo. Tumors were measured in mice using a caliper in two perpendicular dia-meters, including the longest diameter and the shortest perpendicular diameter. A significant differences in tumor size was observed between the untreated LLC control group and celecoxib treated mice (*p＜0.05 vs. celecoxib treated mice).
Fig. 4.
Histopathology of LLC induced lung metastasis (H&E stain, A: 40×, B: 100×). LLC cells were injected into C57Bl/6 mice as described in Methods, and mice were sacrificed 4 weeks after injection. Results were analyzed using the Mann-Whitney U test. (A) H&E staining revealed the pre-sence of LLC cells in metastatic sites. "T" indicates tumor tissue. The arrow indicates microscopic metastasis close to a blood vessel. (B) Lungs were analyzed microscopically for metastasis. Mice treated with celecoxib showed a significant decrease in the number of metastatic tumors (*p＜0.01 vs. the untreated LLC control group).
Fig. 5.
Immunohistochemical staining for IDO. Celecoxib treated mice showed significantly lower levels of IDO expression in tumor cells and tumor-infiltrating inflammatory cells. (A) Paraffin sections of isolated primary tumors were stained with anti-IDO antibody. Slides were counterstained with hematoxylin and visualized by light microscopy (at ×40 and ×200 magnification). Positively-stained cells are arrowed. (B) Photomicrograph of a tumor (H&E staining), showing original LLC cells in a tumor mass at its injection site. (C) IDO expression was determined by calculating the IDO stained area per microscopic field. IDO expression was significantly lower in celecoxib treatment group (*p＜0.01 vs. the untreated LLC control group). However, no significant difference was observed between the celecoxib 25 mg and 250 mg groups.
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