Page 40 - Read Online

Bijnsdorp et al. Cancer Drug Resist 2021;4:719-27 Cancer
DOI: 10.20517/cdr.2021.21
Drug Resistance

Original Article Open Access

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

vitro

1,2
I.V. Bijnsdorp , Godefridus J. Peters 1,3
1
Department of Medical Oncology, Amsterdam UMC, location VUMC, 1007 MB Amsterdam, Netherlands.
2
Department of Urology, Amsterdam UMC, location VUMC, 1007 MB Amsterdam, Netherlands.
3
Department of Biochemistry, Medical University of Gdansk, 80-210 Gdansk, Poland.
Correspondence to: Prof. Dr. Godefridus J. Peters, Department of Medical Oncology, Amsterdam UMC, location VUMC, CCA
1.52, 1007 MB Amsterdam, Netherlands. E-mail: Gj.peters@amsterdamumc.nl
How to cite this article: Bijnsdorp IV, Peters GJ. Protective autophagy by thymidine causes resistance to rapamycin in colorectal
cancer cells in vitro. Cancer Drug Resist 2021;4:719-27. https://dx.doi.org/10.20517/cdr.2021.21
Received: 8 Mar 2021 First Decision: 8 Apr 2021 Revised: 6 May 2021 Accepted: 12 May 2021 Available online: 24 May 2021

Academic Editor: Eric Raymond Copy Editor: Yue-Yue Zhang Production Editor: Yue-Yue Zhang

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.

© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0
International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing,
adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as
long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and
indicate if changes were made.

www.cdrjournal.com