SiRNA knockdown of mitochondrial thymidine kinase 2 (TK2) sensitizes human tumor cells to gemcitabine.

Christine Di Cresce,Mark D Vincent,James Koropatnick,Colin Way,Saman Maleki Vareki,Rene Figueredo,Peter J Ferguson,Mateusz Rytelewski

doi:10.18632/oncotarget.4272

Abstract

Nucleoside metabolism enzymes are determinants of chemotherapeutic drug activity. The nucleoside salvage enzyme deoxycytidine kinase (dCK) activates gemcitabine (2', 2'-difluoro-2'-deoxycytidine) and is negatively regulated by deoxycytidine triphosphate (dCTP). Reduction of dCTP in tumor cells could, therefore, enhance gemcitabine activity. Mitochondrial thymidine kinase 2 (TK2) phosphorylates deoxycytidine to generate dCTP. We hypothesized that: (1) TK2 modulates human tumor cell sensitivity to gemcitabine, and (2) antisense knockdown of TK2 would decrease dCTP and increase dCK activity and gemcitabine activation. siRNA downregulation of TK2 sensitized MCF7 and HeLa cells (high and moderate TK2) but not A549 cells (low TK2) to gemcitabine. Combined treatment with TK2 siRNA and gemcitabine increased dCK. We also hypothesized that TK2 siRNA-induced drug sensitization results in mitochondrial damage that enhances gemcitabine effectiveness. TK2 siRNA and gemcitabine decreased mitochondrial redox status, DNA content, and activity. This is the first demonstration of a direct role for TK2 in gemcitabine resistance, or any independent role in cancer drug resistance, and further distinguishes TK2 function from that of other dTMP-producing enzymes [cytosolic TK1 and thymidylate synthase (TS)]. siRNA knockdown of TK1 and/or TS did not sensitize cancer cells to gemcitabine indicating that, among the 3 enzymes, only TK2 is a candidate therapeutic target for combination with gemcitabine.

Highlights

Nucleoside-metabolizing enzymes are important determinants of the activity of antimetabolite chemo therapy drugs [1]
Antisense treatment decreased thymidine kinase 2 (TK2) protein by only about 25% in MCF7 and human cervical carcinoma (HeLa) cells (Figure 1B, 1C), it sensitized those cells to gemcitabine by as much as 50% (Figure 2A–2D)
An increase in deoxycytidine kinase (dCK) levels in human tumor cells was demonstrated for the first time in response to combined treatment with TK2 siRNA and gemcitabine (Figure 3B, 3D)

Summary

Introduction

Nucleoside-metabolizing enzymes are important determinants of the activity of antimetabolite chemo therapy drugs [1]. The nucleoside salvage enzyme deoxycytidine kinase (dCK) normally catalyzes phosphorylation of deoxycytidine to its monophosphorylated form [3]. DCK activates gemcitabine (2′, 2′-difluoro2′-deoxycytidine, dFdC; a deoxycytidine analog antimetabolite anticancer drug) by catalyzing production of gemcitabine monophosphate and subsequent active metabolites [6]. DCK is negatively regulated by deoxycytidine triphosphate (dCTP) and increased dCTP diminishes the net cytotoxic, anticancer activity of gemcitabine. TK2, but not TK1, is promiscuous with respect to substrate [4,5,6]: it can, in addition to phosphorylating thymidine, phosphorylate deoxycytidine to generate dCTP that, in turn, can reduce gemcitabine activity. TK2 in human tumor cells could reduce gemcitabine effectiveness

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