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

I.V Bijnsdorp,Godefridus J Peters

doi:10.20517/cdr.2021.21

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.

Highlights

Thymidine phosphorylase (TP) is known as platelet derived-endothelial cell growth factor, which plays a role in angiogenesis[1]
Rapamycin resistance in TP-expressing cancer cells may be related to thymidine-mediated autophagy activation
thymidine phosphorylase inhibitor (TPI) addition to rapamycin hardly affected the sensitivity to rapamycin in both Colo320 and Colo320TP1 cells

Summary

Introduction

Thymidine phosphorylase (TP) is known as platelet derived-endothelial cell growth factor, which plays a role in angiogenesis[1]. TP is often overexpressed in cancer, including colorectal, breast and bladder cancer[2]. TP is related to a poor prognosis for the patient, induces metastasis, and leads to a higher microvessel density[3,4]. The enzymatic activity of TP has been demonstrated to be essential for stimulating angiogenesis in various studies[5]. Inhibitors of TP have been developed as antiangiogenesis therapy, including the thymidine phosphorylase inhibitor (TPI)[6]. TPI inhibits TP very potently and and has demonstrated anti-angiogenic activity. Its primary application is in the combination with trifluorothymidine (TFT) in TAS-102 (Lonsurf), which is registered as third line therapy in the treatment of advanced colorectal cancer[7,8]

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Conclusion