Biological Effects of Prostagrandin E2-EP4 Antagonist (AAT-008): Enhancement of Immunoresponse to Radiotherapy and a Potential as a Radiosensitizer

Abstract

The cyclooxygenase-2 product prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on a family of four G protein-coupled receptors (EP1-4). Recently, an antagonist of PGE2-EP4 (AAT-008) has been newly developed for clinical use. The purpose of this study is to investigate the biological effects of AAT-008 as a radiosensitizer for the treatment of murine colon cancer (CT26WT) in vivo and explore the mechanism using flow cytometry.CT26WT tumor cells transplanted into the hind legs of BALB/c mice were used. Treatments were carried out when the mean diameter of the tumors reached approximately 10 mm for tumor growth delay assay and 5 mm for flow cytometry (day 0). AAT-008 (0, 3, 10, and 30 mg/kg/day) was administered orally twice a day from day 0 to day 18. The tumor was irradiated at 9 Gy on day 3 for the radiation treatment (RT) group. In the tumor growth assay, twelve mice were used for each group. The three dimensions of each tumor were measured every other day using a caliper and the tumor doubling time was calculated. For the first flow cytometry, the dose of AAT-008 was fixed to 10 mg/kg/day. Administration of AAT-008 and RT dose were the same as in the tumor growth assay. The population of cytotoxic T-cells (CD45+CD8+CD69+: CTL) was investigated using the tumor cells on day 19. For the second flow cytometry, the dose of AAT was fixed to 30 mg/kg/day. AAT-008 was administered from day 0 to day 12. The population of CTL and the ratio of CTL / regulatory T-cells (Foxp3+: Treg) were investigated using the tumor cells on day 13.In the tumor growth delay assay, the median tumor doubling times in the 0-, 3-, 10-, and 30-mg group without RT were 3.2, 4.4, 4.4, and 5.7 days, respectively. The times in those dose groups with RT were 6.1, 6.6, 17.3, and 19.8 days, respectively (P < 0.001 between the 0- and 10-mg/30-mg groups with RT). There was no significant difference between the 10- and 30-mg groups with RT (P = 0.56). In the first flow cytometry, the mean CTL proportion of the irradiated tumors in the 10 mg + RT (n = 4) and 0 mg + RT groups (n = 5) was 43% and 31%, respectively. Of note, 67% of CTL were observed in responded mice (n = 2) in the 10 mg + RT group. In the second flow cytometry, the mean Treg proportion and the CTL/ Treg ratio of the irradiated tumors in the 30 mg + RT (n = 5) and 0 mg + RT groups (n = 5) were 4.0% and 1.5%, respectively (P = 0.02) and 10 and 22, respectively (P = 0.02).AAT-008 had a potential of enhancing radiosensitivity in the treatment of colon cancer cells. It was assumed that the optimal dose of AAT-008 with RT was 10 mg/kg/day. It was suggested that AAT-008 stimulated the immune system against the cancer cells.