Thymidine phosphorylase expressed in monocyte-macrophages enhanced anticancer effect of 5'-deoxy-5-fluorouridine on colorectal carcinoma cells

Abstract

To detect the thymidine phosphorylase (dThdPase) expression in colorectal carcinoma tissue, and clarify whether dThdPase expressed in macrophage-like cell lines, and monocytes from human peripheral blood can modulate the anticancer effect of 5'-deoxy-5-fluorouridine (5'-DFUR) on colorectal carcinoma cells. Forty specimens resected from 40 patients with colorectal carcinoma were immunohistochemically stained by the monoclonal antibodies 654-1 (anti-dThdPase) and PG-M1 (anti-macrophage marker CD68). Then morphometrical analysis and positive cell counting were performed. In 27 of 40 specimens, dThdPase activity analysis was assayed by HPLC. The dThdPase level were also measured by ELISA in 4 colorectal cancer cell lines, LS174T, Clone A, Colo320, MIP101, and 2 macrophage-like cell lines, THP-1, U937. After estimated the drug sensitivities of each colorectal carcinoma cell both to 5'-DFUR and 5-Fu by MTT assay, THP-1, U937, or monocytes isolated from human blood were incubated in the medium containing 5-Fu or 5'-DFUR for 24 hours, respectively. Then the supernatant was collected and 2-fold serially diluted with the medium, in which the macrophage-like cells, or monocytes were also cultivated for 24 h without anticancer agents. Using the serially diluted medium, MTT assay were also carried out on 4 colorectal carcinoma cell lines. Each experiment was repeated six times and the means of IC50 value +/- standard errors (mean +/- S.E.M.) were calculated. Finally, THP-1 or U937 cells were cultured in medium containing 400 micro mol/L of 5'-DFUR for 24 hours, then the supernatants were collected and the amount of generated 5-Fu was measured by HPLC. dThdPase activities was significantly increased (139.7 micro g x 5-Fu x h(-1) x ml(-1) +/- 61.5 micro g x 5-Fu x h(-1) x ml(-1)) in colorectal carcinoma tissues compared with adjacent normal tissues (42.2 micro g x 5-Fu x h(-1) x ml(-1) +/- 21.4 micro g x 5-Fu x h(-1) x ml(-1)), P < 0.001. In immunohistochemical analysis, it was confirmed that most cells expressed dThdPase-positive cells were the stromal cells, especially macrophages, which surrounding cancer nests, or along the invasive margin of cancer. The distribution patterns of dThdPase-positive stromal cells are similar to that of the CD68-positive cells. The number of dThdPase positive cells was correlated with the number of macrophages in the cancerous tissues, r = 0.76. The dThdPase protein were detected at the levels of 18.2 unit/mg in THP-1, 19.3 unit/mg in U937, and 0.5 unit/mg in LS174T, however, not detected in other 3 colorectal carcinoma cells. The values of IC50 of 5'-DFUR on the 4 colorectal carcinoma cell lines were 11.5 approximately 84.8 times higher than those of 5-Fu (all P < 0.01). It was showed that no inhibiting effect for 5'-DFUR on the growth rates of THP-1, U937, and monocyte cells. After incubated with THP-1 or U937, 5'-DFUR expressed a significant enhanced antitumor effect (P < 0.0001), while almost no change is observed to 5-Fu (P > 0.05). Same conclusion was also demonstrated in using the monocytes instead of THP-1 or U937. 40.2 micro mol/L and 29.5 micro mol/L of 5-Fu were detected in the medium containing 400 micro mol/L of 5'-DFUR treated with THP-1 and U937 cells, respectively. 5'-DFUR cannot be converted into 5-Fu in colorectal carcinoma cells in vitro because no dThdPase is expressed in those cells. After being incubated with macrophage-like cell, THP-1, U937, or human monocytes, the anticancer effect of 5'-DFUR is significantly increased due to the activation by dThdPase expressed in above cells.