Isocytosine deaminase Vcz as a novel tool for the prodrug cancer therapy

Arunas Kazlauskas,Jaunius Urbonavičius,Rolandas Meškys,Adas Darinskas,Arimantas Tamašauskas

doi:10.1186/s12885-019-5409-7

Abstract

BackgroundThe cytosine deaminase (CD)/5-fluorocytosine (5-FC) system is among the best explored enzyme/prodrug systems in the field of the suicide gene therapy. Recently, by the screening of the environmental metagenomic libraries we identified a novel isocytosine deaminase (ICD), termed Vcz, which is able of specifically converting a prodrug 5-fluoroisocytosine (5-FIC) into toxic drug 5-fluorouracil (5-FU). The aim of this study is to test the applicability of the ICD Vcz / 5-FIC pair as a potential suicide gene therapy tool.MethodsVcz-expressing human glioblastoma U87 and epithelial colorectal adenocarcinoma Caco-2 cells were treated with 5-FIC, and the Vcz-mediated cytotoxicity was evaluated by performing an MTT assay. In order to examine anti-tumor effects of the Vcz/5-FIC system in vivo, murine bone marrow-derived mesenchymal stem cells (MSC) were transduced with the Vcz-coding lentivirus and co-injected with 5-FIC or control reagents into subcutaneous GL261 tumors evoked in C57/BL6 mice.Results5-FIC alone showed no significant toxic effects on U87 and Caco-2 cells at 100 μM concentration, whereas the number of cells of both cell lines that express Vcz cytosine deaminase gene decreased by approximately 60% in the presence of 5-FIC. The cytotoxic effects on cells were also induced by media collected from Vcz-expressing cells pre-treated with 5-FIC. The co-injection of the Vcz-transduced mesenchymal stem cells and 5-FIC have been shown to augment tumor necrosis and increase longevity of tumorized mice by 50% in comparison with control group animals.ConclusionsWe have confirmed that the novel ICD Vcz together with the non-toxic prodrug 5-FIC has a potential of being a new enzyme/prodrug system for suicide gene therapy.

Highlights

The cytosine deaminase (CD)/5-fluorocytosine (5-FC) system is among the best explored enzyme/ prodrug systems in the field of the suicide gene therapy
Expression of Vcz in the mammalian cells Identification and biochemical characterization of the bacterial isocytosine deaminase Vcz, which converts isocytosine into uracil and 5-FIC into 5-FU, was recently described in our recent publication [23]
The majority of in vitro experiments were conducted on glioblastoma cell line U87MG, whereas the murine glioblastoma tumor model GL261 was used for in vivo experiments. 5-FU, as a product of the isocytosine deaminase (ICD) Vcz/5-FIC activity, has been tested previously in a considerable number of glioma studies using mice and rat models, which subsequently lead to the initial clinical trials showing promising results and prospects towards phase II trials [25, 26]

Summary

Introduction

The cytosine deaminase (CD)/5-fluorocytosine (5-FC) system is among the best explored enzyme/ prodrug systems in the field of the suicide gene therapy. By the screening of the environmental metagenomic libraries we identified a novel isocytosine deaminase (ICD), termed Vcz, which is able of converting a prodrug 5-fluoroisocytosine (5-FIC) into toxic drug 5-fluorouracil (5-FU). Among the most promising approaches to solve this problem is the use of adenoviruses [17, 18] or tumor-tropic cells, such as mesenchymal stem/stromal cells (MSC) [19, 20] or neural stem cells [21], as carriers of the enzyme / prodrug system to tumor sites There is yet another problem, which persists with the CD/5-FC system, is that 5-FC is not an entirely harmless molecule as would be ideally expected for the cancer therapy. The new enzyme/prodrug pair, ICD/5-FIC, would alleviate the toxic side effects, which persist in the CD/5-FC system, and could be used as a novel enzyme-prodrug pair in the cancer therapy

Objectives

Methods

Results

Discussion

Conclusion