Gene Electrotransfer of Plasmid with Tissue Specific Promoter Encoding shRNA against Endoglin Exerts Antitumor Efficacy against Murine TS/A Tumors by Vascular Targeted Effects.

Monika Stimac,Tanja Dolinsek,Gregor Sersa,Maja Cemazar,Ursa Lampreht,Ilya Ulasov

doi:10.1371/journal.pone.0124913

Monika Stimac, Tanja Dolinsek + Show 4 more

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https://doi.org/10.1371/journal.pone.0124913

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Journal: PloS one	Publication Date: Apr 24, 2015
Citations: 18	License type: CC BY 4.0

Affiliation: Institute of Oncology Ljubljana

Abstract

Vascular targeted therapies, targeting specific endothelial cell markers, are promising approaches for the treatment of cancer. One of the targets is endoglin, transforming growth factor-β (TGF-β) co-receptor, which mediates proliferation, differentiation and migration of endothelial cells forming neovasculature. However, its specific, safe and long-lasting targeting remains the challenge. Therefore, in our study we evaluated the transfection efficacy, vascular targeted effects and therapeutic potential of the plasmid silencing endoglin with the tissue specific promoter, specific for endothelial cells marker endothelin-1 (ET) (TS plasmid), in comparison to the plasmid with constitutive promoter (CON plasmid), in vitro and in vivo. Tissue specificity of TS plasmid was demonstrated in vitro on several cell lines, and its antiangiogenic efficacy was demonstrated by reducing tube formation of 2H11 endothelial cells. In vivo, on a murine mammary TS/A tumor model, we demonstrated good antitumor effect of gene electrotransfer (GET) of either of both plasmids in treatment of smaller tumors still in avascular phase of growth, as well as on bigger tumors, already well vascularized. In support to the observations on predominantly vascular targeted effects of endoglin, histological analysis has demonstrated an increase in necrosis and a decrease in the number of blood vessels in therapeutic groups. A significant antitumor effect was observed in tumors in avascular and vascular phase of growth, possibly due to both, the antiangiogenic and the vascular disrupting effect. Furthermore, the study indicates on the potential use of TS plasmid in cancer gene therapy since the same efficacy as of CON plasmid was determined.

Highlights

Antiangiogenic therapies are promising approaches for the treatment of cancer [1]
To determine tissue specificity of TS plasmid in different cell lines, the number of cells expressing enhanced green fluorescent protein (EGFP) was assessed by flow cytometry, after gene electrotransfer (GET) of plasmid encoding EGFP
We demonstrated that TS plasmid, the one with the tissue specific promoter of endothelial cell marker endothelin-1, is tissue specific and has almost the same antiangiogenic efficacy on tube formation as CON plasmid

Summary

Introduction

Antiangiogenic therapies are promising approaches for the treatment of cancer [1]. Forty years ago, the study of Folkman et al [2], was the first associating tumor growth with angiogenesis, PLOS ONE | DOI:10.1371/journal.pone.0124913 April 24, 2015. The first demonstration of tumor regression when using GET of plasmid encoding Stat-3, was published by Niu et al in 1999 [26], followed by many other studies targeting different molecules such as HIV-1 Vpr [27], Plk 1 and Bcl-2 [28], VEGF [5], integrins [29] and others stimulating the immune response, for example IL-12 [30,31,32], IL-15 [33], IFNα [34] alone or in combination with other therapies, as for example chemotherapy and radiotherapy [35,36] Those preclinical studies have led to the fist clinical trials using GET of plasmid encoding human IL-12 (hIL-12) [37] and plasmid AMEP for binding on integrins for treatment of melanoma [38]. The effect was the same when using either of both plasmids silencing endoglin; CON and TS plasmid, indicating on the therapeutic potential of the latter

Materials and Methods

Ethics Statement

Results

Discussion