New targets and therapeutic approaches for vascular targeted strategies in oncology are continuously explored. Endoglin, a co-receptor of TGF-%, is a known target, however, its silencing with vector-based RNA interference technology has not been evaluated yet. Therefore, in our study, we assembled plasmid DNA coding for shRNA against endoglin, and used gene electrotransfer as a delivery method to determine its antitumor and vascular targeted effects. In vitro and in vivo data provide evidence of vascular targeted effects of endoglin silencing. The vascular targeted action of endoglin silencing could be described as a result of two separated effect; antiangiogenic and vascular disrupting effect. This was first supported by in vitro data; predominantly by reduction of proliferation and tube formation of endothelial cells. In the TS/A murine mammary carcinoma model, in which the tumor cells do not express endoglin, reduced tumor growth and number of vessels were observed. Quick destruction of existing activated blood vessels at the site of tumor cells' injection and sustained growth of tumors afterwards was observed in tumors that were growing in dorsal window chamber by intravital microscopy. This observation supports both vascular disrupting and antiangiogenic action. In conclusion, the results of our study provide evidence of endoglin as a valid target for cancer therapy and support further development of plasmid shRNA delivery, which have prolonged antitumor effect, especially in combined schedules.
COBISS.SI-ID: 2010235
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-% (TGF-%) 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.
COBISS.SI-ID: 2009979
Tumor irradiation combined with adjuvant treatments, either vascular targeted or immunomodulatory, is under intense investigation. Gene electrotransfer of therapeutic genes is one of these approaches. The aim of this study was to determine, whether gene electrotransfer of plasmid encoding shRNA for silencing endoglin, with vascular targeted effectiveness, can radiosensitize melanoma B16F10 tumors. Materials and methods. The murine melanoma B16F10 tumors, growing on the back of C57Bl/6 mice, were treated by triple gene electrotransfer and irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice. Furthermore, histological analysis of tumors (necrosis, apoptosis, proliferation, vascularization, presence of hypoxia and infiltration of immune cells,) was used to evaluate the therapeutic mechanisms. Results. Gene electrotransfer of plasmid silencing endoglin predominantly indicated vascular targeted effects of the therapy, since significant tumor growth delay and 44% of tumor free mice were obtained. In addition, irradiation had minor effects on radioresistant melanoma, with 11% of mice tumor free. The combined treatment resulted in excellent effectiveness with 88% of mice tumor free, with more than half resistant to secondary tumor challenge, which was observed also with the plasmid devoid of the therapeutic gene. Histological analysis of tumors in the combined treatment group, demonstrated similar mode of action of the gene electrotransfer of plasmid encoding shRNA for silencing endoglin and devoid of it, both through the induction of an immune response. Conclusions. The results of this study indicate that irradiation can in radioresistant melanoma tumors, by release of tumor associated antigens, serve as activator of the immune response, besides directly affecting tumor cells and vasculature. The primed antitumor immune response can be further boosted by gene electrotransfer of plasmid, regardless of presence of the therapeutic gene, which was confirmed by the high radiosensitization, resulting in prolonged tumor growth delay and 89% of tumor free mice that were up to 63% resistant to secondary challenge of tumor. In addition, gene electrotransfer of therapeutic plasmid for silencing endoglin has also a direct effect on tumor vasculature and tumors cells; however in combination with radiotherapy this effect was masked by pronounced immune response.
COBISS.SI-ID: 2609019
Endoglin (CD105), a transforming growth factor (TGF)-% coreceptor, and endothelin-1, a vasoconstrictor peptide, are both overexpressed in tumor endothelial and melanoma cells. Their targeting is therefore a promising therapeutic approach for melanoma tumors. The aim of our study was to construct a eukaryotic expression plasmid encoding the shRNA molecules against CD105 under the control of endothelin-1 promoter and to evaluate its therapeutic potential both in vitro in murine B16F10-luc melanoma and SVEC4-10 endothelial cells and in vivo in mice bearing highly metastatic B16F10-luc tumors. Plasmid encoding shRNA against CD105 under the control of the constitutive U6 promoter was used as a control. We demonstrated the antiproliferative and antiangiogenic effects of both plasmids in SVEC4-10 cells, as well as a moderate antitumor and pronounced antimetastatic effect in B16F10-luc tumors in vivo. Our results provide evidence that targeting melanoma with shRNA molecules against CD105 under the control of endothelin-1 promoter is a feasible and effective treatment, especially for the reduction of metastatic spread.
COBISS.SI-ID: 1537399748
Gene electrotransfer of plasmid encoding shRNA against endoglin exerts antitumor efficacy, predominantly by vascular targeted effect. As vascular targeting therapies can promote radiosensitization, the aim of this study was to explore this gene therapy approach with single and split dose of irradiation in an endoglin non-expressing TS/A mammary adenocarcinoma tumor model to specifically study the vascular effects. Intratumoral gene electrotransfer of plasmids encoding shRNA against endoglin, under the control of a constitutive or tissue-specific promoter for endothelial cells, combined with a single or three split doses of irradiations was evaluated for the antitumor efficacy and histologically. Both plasmids proved to be equally effective in tumor radiosensitization with 40-47% of tumor cures. The combined treatment induced a significant decrease in the number of blood vessels and proliferating cells, and an increase in levels of necrosis, apoptosis and hypoxia; therefore, the antitumor efficacy was ascribed to the interaction of vascular targeted effect of gene therapy with irradiation. Endoglin silencing by the shRNA technology, combined with electrotransfer and the use of a tissue-specific promoter for endothelial cells, proved to be a feasible and effective therapeutic approach that can be used in combined treatment with tumor irradiation.
COBISS.SI-ID: 2331003