Tumor radiosensitization by gene therapy against endoglin.

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.

MicroRNAs targeting mutant K-ras by electrotransfer inhibit human colorectal adenocarcinoma cell growth in vitro and in vivo.

Mutations of K-ras have been found in 30-60% of colorectal carcinomas and are believed to be associated with tumor initiation, tumor progression and metastasis formation. Therefore, silencing of mutant K-ras expression has become an attractive therapeutic strategy for colorectal cancer treatment. The aim of our study was to investigate the effect of microRNA (miRNA) molecules directed against K-ras (miRNA-K-ras) on K-ras expression level and the growth of colorectal carcinoma cell line LoVo in vitro and in vivo. In addition, we evaluated electroporation as a gene delivery method for transfection of LoVo cells and tumors with plasmid DNA encoding miRNA-K-ras (pmiRNA-K-ras). Results of our study indicated that miRNAs targeting K-ras efficiently reduced K-ras expression and cell survival after in vitro electrotransfection of LoVo cells with pmiRNA-K-ras. In vivo, electroporation has proven to be a simple and efficient delivery method for local administration of pmiRNA-K-ras molecules into LoVo tumors. This therapy shows pronounced antitumor effectiveness and has no side effects. The obtained results demonstrate that electrogene therapy with miRNA-K-ras molecules can be potential therapeutic strategy for treatment of colorectal cancers harboring K-ras mutations.

Vascular disrupting action of electroporation and electrochemotherapy with bleomycin in murine sarcoma.

Electrochemotherapy has a direct cytotoxic effect on tumour cells, and presumably, a vascular disrupting effect. In this study, on the basis of the prediction of the mathematical model, histological evaluation and physiological measurements of the tumours were carried out to confirm that electroporation and electrochemotherapy of tumours have a vascular disrupting action. In the study, SA-1 solid subcutaneous sarcoma tumours in A/J mice were treated by bleomycin (BLM) given intravenously (1 mg kg(-1)), application of electric pulses (8 pulses, 1040 V, 100 micros, 1 Hz) or a combination of both - electrochemotherapy. The vascular effect was determined by laser Doppler flowmetry, power Doppler ultrasonographic imaging and Patent blue staining. The extent of tumour hypoxia was determined immunohistochemically by hypoxia marker pimonidazole and partial pressure of oxygen (pO(2)) in tumours by electron paramagnetic resonance oximetry. Electrochemotherapy with BLM induced good antitumour effect with 22 days, tumour growth delay and 38% tumour cures. The application of electric pulses to the tumours induced instant but transient tumour blood flow reduction (for 70%) that was recovered in 24 h. During this tumour blood flow reduction, we determined an increase in hypoxic tumour area for up to 30%, which was also reflected in reduced tumour oxygenation (for 70%). According to the described mathematical model, endothelial cells lining in tumour blood vessels are exposed to a approximately 40% higher electric field than the surrounding tumour cells, and therefore easily electroporated, allowing access of high BLM concentration to the cytosol. Consequently, electrochemotherapy has, besides the immediate vascular disrupting action, also a delayed one (after 24 h), as a consequence of endothelial cell swelling and apoptosis demonstrated by extensive tumour necrosis, tumour hypoxia, prolonged reduction of tumour blood flow and significant tumour growth delay, and tumour cures. Our results demonstrate that in addition to the well-established direct cytotoxic effect on tumour cells, electrochemotherapy also has an indirect vascular disrupting action resulting altogether in extensive tumour cell necrosis leading to complete regression of tumours.

The effect of the histological properties of tumors on transfection efficiency of electrically assisted gene delivery to solid tumors in mice.

Uniform DNA distribution in tumors is a prerequisite step for high transfection efficiency in solid tumors. To improve the transfection efficiency of electrically assisted gene delivery to solid tumors in vivo, we explored how tumor histological properties affected transfection efficiency. In four different tumor types (B16F1, EAT, SA-1 and LPB), proteoglycan and collagen content was morphometrically analyzed, and cell size and cell density were determined in paraffin-embedded tumor sections under a transmission microscope. To demonstrate the influence of the histological properties of solid tumors on electrically assisted gene delivery, the correlation between histological properties and transfection efficiency with regard to the time interval between DNA injection and electroporation was determined. Our data demonstrate that soft tumors with larger spherical cells, low proteoglycan and collagen content, and low cell density are more effectively transfected (B16F1 and EAT) than rigid tumors with high proteoglycan and collagen content, small spindle-shaped cells and high cell density (LPB and SA-1). Furthermore, an optimal time interval for increased transfection exists only in soft tumors, this being in the range of 5-15 min. Therefore, knowledge about the histology of tumors is important in planning electrogene therapy with respect to the time interval between DNA injection and electroporation.

Reduced blood flow and oxygenation in SA-1 tumours after electrochemotherapy with cisplatin.

Electrochemotherapy is an antitumour treatment that utilises locally delivered electric pulses to increase cytotoxicity of chemotherapeutic drugs. Besides increased drug delivery, application of electric pulses affects tumour blood flow. The aim of this study was to determine tumour blood flow modifying effects of electrochemotherapy with cisplatin, its effects on tumour oxygenation and to determine their relation to antitumour effectiveness. Electrochemotherapy of SA-1 subcutaneous tumours was performed by application of electric pulses to the tumours, following administration of cisplatin. Tumour blood flow modifying effects of electrochemotherapy were determined by measurement of tumour perfusion using the Patent blue staining technique, determination of tumour blood volume, and microvascular permeability using contrast enhanced magnetic resonance imaging, and tumour oxygenation using electron paramagnetic resonance oximetry. Antitumour effectiveness was determined by tumour growth delay and the extent of tumour necrosis and apoptosis. Tumour treatment by electrochemotherapy induced 9.4 days tumour growth delay. Tumour blood flow was reduced instantaneously and persisted for several days. This reduction in tumour blood flow was reflected in reduced tumour oxygenation. The maximal reduction in partial oxygen pressure (pO2) levels was observed at 2 h after the treatment, with steady recovery to the pretreatment level within 48 h. The reduced tumour blood flow and oxygenation correlated well with the extent of tumour necrosis and tumour cells apoptosis induced by electrochemotherapy with cisplatin. Therefore, the data indicate that antitumour effectiveness of electrochemotherapy is not only due to increased cytotoxicity of cisplatin due to electroporation of tumour cells, but also due to anti-vascular effect of electrochemotherapy, which resulted in reduced tumour blood flow and oxygenation.