Incorporation and Repair of Epigenetic Intermediates as Potential Chemotherapy Agents

TL;DR

This study explores how certain nucleoside analogs, including epigenetic intermediates, can be used as chemotherapy agents for glioblastoma by being incorporated into DNA and causing cell toxicity.

Contribution

The study identifies 5HmdU and TFT as cytotoxic agents with distinct DNA incorporation and repair mechanisms in glioblastoma cells.

Findings

5HmdU and TFT are efficiently incorporated into DNA and are cytotoxic to U87 glioblastoma cells.

5HmdC is cytotoxic but not directly incorporated into DNA; it is converted to 5HmdU, which is incorporated.

5HmdU is removed via BER, while TFT remains stably incorporated and is not excised by BER.

Abstract

The incorporation of nucleoside analogs into DNA by polymerases, followed by their removal through base excision repair (BER), represents a promising strategy for cancer chemotherapy. In this study, we investigated the incorporation and cytotoxic effects of several nucleoside analogs—some of which are epigenetic reprogramming intermediates—in the U87 glioblastoma cell line. We found that two analogs, 5-hydroxymethyl-2′-deoxyuridine (5HmdU) and trifluorothymidine (TFT), are both cytotoxic and are efficiently incorporated into genomic DNA. In contrast, the 5-carboxy analogs—5-carboxy-2′-deoxyuridine (5CadU) and 5-carboxycytidine (5CadC)—showed no cytotoxicity and were not incorporated into DNA. Interestingly, 5-hydroxymethyl-2′-deoxycytidine (5HmdC) was cytotoxic but was not directly incorporated into DNA. Instead, it was deaminated into 5HmdU, which was then incorporated and likely responsible for the observed toxicity. 5HmdU is actively removed from DNA through the BER pathways. In contrast, TFT remains stably incorporated and is neither excised by BER nor does it hydrolyze into 5CadU—a known substrate for the DNA glycosylase SMUG1. We also found that N6-benzyladenosine (BzAdo), an inhibitor of the enzyme 2′-deoxynucleoside 5′-phosphate N-hydrolase (DNPH1), enhances the cytotoxicity of 5HmdU. However, the thymidine phosphorylase inhibitor tipiracil hydrochloride (TPI) does not increase the cytotoxic effect of TFT in U87 cells. Together, these findings highlight 5HmdU and TFT as promising chemotherapeutic agents for glioblastoma, each with distinct mechanisms of action and cellular processing.

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