Protective autophagy by thymidine causes resistance to rapamycin in colorectal cancer cells in vitro

Abstract

Aim: Thynidine phosphorylase (TP) acts as a proangiogenic growth factor which may regulate mammalian Target of Rapamycin (mTOR). We investigated whether the TP substrate thymidine and overexpression of TP affected mTOR signaling by comparing Colo320 (TP deficient) cells and its TP-transfected variant (Colo320TP1). Methods: Drug resistance was assessed with the sulforhodamine B assay, protein expression with Western blotting, cell cycle distribution and cell death with Fluorescence-activated cell sorting analysis, and autophagy with immunofluorescence. Results: Colo320 and Colo320TP1 cells had comparable levels of sensitivity to the mTOR inhibitor rapamycin. Thymidine treatment led to 13- and 50-fold resistance to rapamycin in Colo320 and Colo320TP1 cells, respectively. In Colo320TP1 cells, the thymidine phosphorylase inhibitor (TPI) reversed the thymidine induced resistance to rapamycin, but not in Colo320 cells, indicating a role for TP in the protection. Thymidine increased p70/S6k-phosphorylation (downstream of mTOR) in Colo320TP1, but it was not affected in Colo320. As a mechanism behind resistance, we studied the levels of autophagy and found that, in Colo320TP1 cells, autophagy was highly induced by thymidine-rapamycin, which was decreased by TPI. In addition, the autophagy inhibitor 3-methyl-adenine completely inhibited autophagy and its protection. Conclusion: Rapamycin resistance in TP-expressing cancer cells may therefore be related to thymidine-mediated autophagy activation.

Keywords: Thymidine phosphorylase; autophagy; mTOR; rapamycin; thymidine; thymidine phosphorylase inhibitor.

Figure 1

FACS analysis of Colo230 and Colo320TP1 cells after 72 h exposure to 20 nM rapamycin, combined with 100 µM thymidine (TdR), 10 µM TPI, or TdR + TPI: (A) cell distribution; and (B) the percentage of cells in the sub-G1-phase. Values represent means of at least four independent experiments ± SEM. Rapa: Rapamycin; TdR: thymidine; TPI: thymidine phosphorylase inhibitor.

Figure 2

Western blot of expression levels of intracellular protein kinases after 6 h exposure to 100 µM thymidine (TdR), 20 nM rapamycin, or 10 µM TPI. Blot is representative of three independent experiments. TPI: Thymidine phosphorylase inhibitor.

Figure 3

Immunofluorescent staining of LC3B in autophagic vesicles. Cells were exposed for 72 h to 20 nM rapamycin (rapa) and combinations with 100 µM TdR and/or 10 µM TPI. The figure is representative for three independent experiments. Rapa: Rapamycin; TdR: thymidine; TPI: thymidine phosphorylase inhibitor.