Conditioned media from human macrophages of M1 phenotype attenuate the cytotoxic effect of 5­fluorouracil on the HT­29 colon cancer cell line.

ABSTRACT

Resistance of tumor cells to chemotherapy, such as 5­fluorouracil (5­FU), is an obstacle for successful treatment of cancer. As a follow­up of a previous study we have investigated the effect of conditioned media (CM) from macrophages of M1 or M2 phenotypes on 5­FU cytotoxicity on the colon cancer cell lines HT­29 and CACO­2. HT­29 cells, but not CACO­2 cells, having been treated with a combination of M1 CM and 5­FU recovered their cell growth to a much larger extent compared to cells having been treated with 5­FU alone when further cultured for 7 days in fresh media. M1 CM treatment of HT­29, but not CACO­2 cells, induced cell cycle arrest in the G0/G1 and G2/M phases. 5­FU treatment induced accumulation of cells in S­phase in both HT­29 and CACO­2 cells. This accumulation of cells in S­phase was attenuated by combined M1 CM and 5­FU treatment in HT­29 cells, but not in CACO­2 cells. The mRNA expression of cell cycle regulatory proteins and 5­FU metabolic enzymes were analyzed in an attempt to find possible mechanisms for the M1 CM induced attenuation of 5­FU cytotoxicity in HT­29. Thymidylate synthetase (TS) and thymidine phosphorylase (TP) were found to be substantially downregulated and upregulated, respectively, in HT­29 cells treated with M1 CM, making them unlikely as mediators of reduced 5­FU cytotoxicity. Among cell cycle regulating proteins, p21 was induced in HT­29 cells, but not in CACO­2 cells, in response to M1 CM treatment. However, small interfering RNA (siRNA) knockdown of p21 had no effect on the M1 CM induced cell cycle arrest seen in HT­29 and neither did it change the growth recovery after combined treatment of HT­29 cells with M1 CM and 5­FU. In conclusion, treatment of HT­29 cells with M1 CM reduces the cytotoxic effect of 5­FU and this is mediated by a M1 CM induced cell cycle arrest in the G0/G1 and G2/M phases. So far, we lack an explanation why this action is absent in the CACO­2 cells. The current findings may be important for optimization of chemotherapy in colon cancer.