In JoVE (2)

Other Publications (105)

Articles by Gregor Sersa in JoVE

 JoVE Cancer Research

Operating Procedures of the Electrochemotherapy for Treatment of Tumor in Dogs and Cats

1Clinic for Surgery and Small Animals, Veterinary Faculty, University of Ljubljana, 2Department of Experimental Oncology, Institute of Oncology Ljubljana, 3Faculty of Health Sciences, University of Primorska, 4IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS, 5IPBS (Institut de Pharmacologie et de Biologie Structurale), Université de Toulouse

JoVE 54760

Other articles by Gregor Sersa on PubMed

Effective Gene Transfer to Solid Tumors Using Different Nonviral Gene Delivery Techniques: Electroporation, Liposomes, and Integrin-targeted Vector

Cancer Gene Therapy. Apr, 2002  |  Pubmed ID: 11960291

In this study, we measured transfection efficiency in vitro and in vivo using the following nonviral approaches of gene delivery: injection of plasmid DNA, electroporation-assisted, liposome-enhanced, and integrin-targeted gene delivery, as well as the combination of these methods. Four histologically different tumor models were transfected with a plasmid encoding the green fluorescent protein (GFP) (B16 mouse melanoma, P22 rat carcinosarcoma, SaF mouse sarcoma, and T24 human bladder carcinoma) using adherent cells, dense cell suspensions, and solid tumors. Emphasis was placed on different electroporation conditions to optimise the duration and amplitude of the electric pulses, as well as on different DNA concentrations for effective gene delivery. In addition, transfection efficiency was correlated with cell density of the tumors. The major in vivo findings were: (a) electroporation-assisted gene delivery with plasmid DNA, employing long electric pulses with low amplitude, yielded significantly better GFP expression than short electric pulses with high amplitude; (b) electroporation combined with liposome-DNA complexes yielded the highest percentage of transfected tumor area in B16F1 tumor (6%); (c) transfection efficiency of electroporation-assisted plasmid DNA delivery was dependent on tumor type; (d) integrin-targeted vector, alone or combined with electroporation, was largely ineffective. In conclusion, our results demonstrate that some nonviral methods of gene delivery are feasible and efficient in transfecting solid tumors. Therefore, this makes nonviral methods attractive for further development.

Schedule-dependent Interaction Between Vinblastine and Cisplatin in Ehrlich Ascites Tumors in Mice

The Journal of Pharmacology and Experimental Therapeutics. Jul, 2002  |  Pubmed ID: 12065735

Information on the in vivo antitumor efficiency of the combination of Vinca alkaloids in animal tumor models, especially vinblastine (VLB) with cisplatin [cis-diamminedichloroplatinum(II); CDDP] is very limited. Therefore, the aim of our study was to explore whether antitumor schedule dependence exists for the combination of CDDP and VLB on i.p. Ehrlich ascites tumors in mice. Animals were treated 3 days after tumor transplantation with VLB (0.006 mg/kg) or CDDP (0.05 mg/kg) alone, VLB followed by CDDP, and CDDP followed by VLB. The time interval between i.p. injections of the drugs was 24 h. Cell number was measured by counting viable cells using the trypan blue exclusion assay, cell platinum content by electrothermal atomic absorption spectrometry, DNA distribution pattern using flow cytometry, apoptosis by flow-cytometric terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and cell morphology. Combination of CDDP and VLB resulted in additive interaction when VLB preceded CDDP as determined from cell survival data 24 h after completion of the therapy and in increased platinum content (two times) compared with the same combination in a reverse schedule (CDDP given before VLB), which resulted in antagonism. None of the treatment combinations induced apoptosis. We propose that the observed increase in antitumor effectiveness is mainly due to higher platinum accumulation in tumor cells, which we unambiguously demonstrated by measurement of platinum content in the tumor cells, leading to increased cytotoxicity as well as to cell cycle-dependent effects of VLB and CDDP.

Optimisation of Pulse Parameters in Vitro for in Vivo Electrochemotherapy

Anticancer Research. May-Jun, 2002  |  Pubmed ID: 12168862

The aim of our study was to optimise electric pulse parameters for electrochemotherapy by sampling the space of pulse parameter variables using systematic in vitro experiments. For this purpose we defined parameters that describe the effectiveness of different sets of electric pulse parameters in vitro and combined them into an objective function that characterises requirements for successful electrochemotherapy. The objective function values were calculated for all the sets of electric pulse parameters included in in vitro experiments. Similar values were grouped together by hierarchical clustering. The 'electrochemotherapeutic' effectiveness of two sets of pulse parameters (8 pulses, 100 micros, 1 Hz and 1 pulse, 1000 micros, 1 Hz), which belong to the most efficient cluster, and one set of pulse parameters (16 pulses, 20 ms, 1 Hz), which belongs to the least efficient cluster, was tested in vivo on a murine tumor model. The sets of pulse parameters from the most efficient cluster had comparable effects in vivo, while the electrochemotherapy with the set of pulse parameters from the least efficient cluster was less effective. Our results demonstrated that electric pulse parameters for effective in vivo application can be determined from in vitro experiments considering application specifications.

Oxygenation and Blood Flow in Tumors Treated with Hydralazine: Evaluation with a Novel Luminescence-based Fiber-optic Sensor

Technology and Health Care : Official Journal of the European Society for Engineering and Medicine. 2002  |  Pubmed ID: 12368557

Two optical methods were used simultaneously to evaluate the effect of a vasoactive drug hydralazine on oxygenation and blood flow in SA-1 tumors in A/J mice. A novel luminescence-based optical sensor (OxyLite instrument, Oxford Optronix, UK) was used to monitor partial pressure of oxygen (pO2) in tumors. Laser Doppler flowmetry was used to assess relative blood perfusion in tumors. Measurements were performed continuously on anesthetized mice before and after i.v. injection of hydralazine at a dose of 2.5 mg/kg. Hydralazine reduced pO2 on average by 80% minutes after injection. Our results indicate that hydralazine could be used to improve the antitumor effectiveness of hypoxic cell-specific therapies. We demonstrated that both optical methods can be used successfully to detect changes in blood perfusion and oxygenation in tumors after blood flow-modifying treatments. Some aspects of pO2 measurement with the new luminescence-based method require further investigation.

Effect of Hydralazine on Blood Flow, Oxygenation, and Interstitial Fluid Pressure in Subcutaneous Tumors

Advances in Experimental Medicine and Biology. 2003  |  Pubmed ID: 12580400

Perturbation of Blood Flow As a Mechanism of Anti-tumour Action of Direct Current Electrotherapy

Physiological Measurement. Feb, 2003  |  Pubmed ID: 12636188

Anti-tumour effects of direct current electrotherapy are attributed to different mechanisms depending on the electrode configuration and on the parameters of electric current. The effects mostly arise from the electrochemical products of electrolysis. Direct toxicity of these products to tumour tissue is, however, not a plausible explanation for the observed tumour growth retardation in the case when the electrodes are placed into healthy tissue surrounding the tumour and not into the tumour itself. The hypothesis that the anti-tumour effectiveness of electrotherapy could result from disturbed blood flow in tumours was tested by the measurement of changes in blood perfusion and oxygenation in tumours with three different methods (in vivo tissue staining with Patent Blue Violet dye, polarographic oximetry, near-infrared spectroscopy). The effects induced by electrotherapy were evaluated in two experimental tumour models: Sa-1 fibrosarcoma in A/J mice and LPB fibrosarcoma in C57B1/6 mice. We found that perfusion and oxygenation were significantly decreased after electrotherapy. Good agreement between the results of different methods was observed. The effect of electrotherapy on local perfusion of tumours is probably the prevalent mechanism of anti-tumour action for the particular type of electrotherapy used in the study. The importance of this effect should be considered for the optimization of electrotherapy protocols in experimental and clinical trials. The non-invasive technique of near-infrared spectroscopy proved to be a reliable method for detecting perfusion and oxygenation changes in small solid tumours.

Electroporation of LPB Sarcoma Cells in Vitro and Tumors in Vivo Increases the Radiosensitizing Effect of Cisplatin

Anticancer Research. Jan-Feb, 2003  |  Pubmed ID: 12680224

Cisplatin is a cytotoxic drug with radiosensitizing effect. In this study a physical drug delivery system, electroporation, was used to facilitate cisplatin delivery into the cells and tumors with the aim of increasing radiation response.

Effects of Electrogenetherapy with P53wt Combined with Cisplatin on Curvival of Human Tumor Cell Lines with Different P53 Status

DNA and Cell Biology. Dec, 2003  |  Pubmed ID: 14683587

The aim of our study was to evaluate electrogenetherapy with p53wt alone or combined with cisplatin on two colorectal (HT-29 and LoVo) and two prostatic (PC-3 and Du145) carcinoma cell lines with different p53 status. In addition, the feasibility of electrogenetherapy with p53wt was tested also in vivo on PC-3 prostatic cancer xenografts. Electrogenetherapy with p53wt was dependent on the p53 status of the cell lines used. Electrogenetherapy was the most effective on the PC-3 (p53 null) and Du145 (p53mt) cells, and to the much lesser extent in LoVo cells (p53wt). The exception was the HT-29 cell line with overexpressed mutated p53, where electrogenetherapy with p53wt was the least effective. Sensitivity of the cell lines to cisplatin was independent of the p53 status. Furthermore, the presence of exogenous p53 due to electrogenetherapy did not enhance cisplatin cytotoxicity, since the combination of these therapies resulted in additive cytotoxic effect. The effectiveness of electrogenetherapy with p53wt was also demonstrated in vivo by successful treatment of subcutaneous PC-3 tumors in mice. In conclusion, our study shows that electrogenetherapy with p53wt is feasible, and resulted in comparable cytotoxic and antitumor effectiveness to viral-mediated p53wt gene therapy. This therapy was effective and dependent on the p53 status of the tumor cell lines. Combination of electrogenetherapy and cisplatin resulted in additional cell kill by cisplatin, and was not dependent on the p53 status.

EPR Oximetry of Tumors in Vivo in Cancer Therapy

Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy. May, 2004  |  Pubmed ID: 15134738

The partial oxygen pressure (pO2) in tumors is considered to be one of important factors that affect the response of tumors to different treatment. Therefore, we anticipate that the information about the variation of oxygen concentration in tumors can be used as a guide for individualizing radiotherapy, chemotherapy, and especially the combined therapies. There is thus a need to obtain quantitative data on the effects of different therapies on tumor oxygenation under in vivo conditions. One of the methods, which enable these measurements is EPR oximetry. In this work basic principles of the method will be described as well as some examples of tumor oxygenation changes after application of chemotherapeutic drugs (vinblastine, cisplatin, bleomycin) or electric pulses in combination with cisplatin or bleomycin to fibrosarcoma SA-1 tumors in mice. A paramagnetic probe, a char of Bubinga tree, was implanted into the tumor (center and periphery) and in the muscle or subcutis. EPR spectra line-width, which is proportional to oxygen concentration, was measured with time after the treatments. Tumor oxygenation was reduced for 58% of pretreatment value 1 h after intraperitoneal injection of 2.5 mg kg(-1) VLB and returned to pretreatment level within 24 h. Reduction in oxygenation of muscle and subcutis was much smaller and returned to pretreatment value faster as in tumors. With cisplatin (4 mg kg(-1)) and bleomicyn (1 mg kg(-1)) the reduction was less than 15%, but increases in combined therapy to 70%. Similar reduction was observed also with electric pulses alone (eight pulses, 1300 V cm(-1), 100 micros, 1 Hz) with fast recovery of 8h. After electrochemotherapy the recovery was slower and occurs only after 48 h. This study demonstrates that EPR oximetry is a sensitive method for monitoring changes in tissue oxygenation after different treatments, which may have implications in controlling side effects of therapy and in the planning of combined treatments.

Electrochemotherapy with Cisplatin of Cutaneous Tumor Lesions in Breast Cancer

Anti-cancer Drugs. Jul, 2004  |  Pubmed ID: 15205602

We have evaluated the efficiency of electrochemotherapy with cisplatin on cutaneous tumor lesions of breast cancer and have compared its efficiency with the efficiency of intratumoral (i.t.) administration of cisplatin alone. The study was performed on six breast cancer patients with 26 cutaneous lesions in whom all standard treatment modalities were exhausted. Of 26 lesions, 12 were treated by electrochemotherapy, six by i.t. cisplatin application, while eight were controls. In all 12 lesions treated by electrochemotherapy and followed-up for up to 26 weeks at the most, the objective response was obtained [complete response in 33% with mean duration of 10 weeks and partial response (PR) in 67% with a mean duration of 5 weeks]. In six lesions treated with i.t. application of cisplatin and followed-up for up to 12 weeks at the most, objective response was obtained in 83% of lesions; none of these responses were complete, the mean duration of PR was 5 weeks. During electrochemotherapy, only minimal local side-effects were observed, whereas no systemic side-effects of the treatment were noticed. We conclude that electrochemotherapy with i.t. cisplatin application is effective in local treatment of cutaneous tumor lesions of breast cancer.

Antitumour Effectiveness of Hyperthermia is Potentiated by Local Application of Electric Pulses to LPB Tumours in Mice

Anticancer Research. Jul-Aug, 2004  |  Pubmed ID: 15330182

The aim of our study was to determine whether local application of electric pulses to tumours, which induce transient reduction of tumour perfusion, could potentiate the antitumour effectiveness of hyperthermia.

Direct Visualization of Electroporation-assisted in Vivo Gene Delivery to Tumors Using Intravital Microscopy - Spatial and Time Dependent Distribution

BMC Cancer. Nov, 2004  |  Pubmed ID: 15546484

Electroporation is currently receiving much attention as a way to increase drug and DNA delivery. Recent studies demonstrated the feasibility of electrogene therapy using a range of therapeutic genes for the treatment of experimental tumors. However, the transfection efficiency of electroporation-assisted DNA delivery is still low compared to viral methods and there is a clear need to optimize this approach. In order to optimize treatment, knowledge about spatial and time dependency of gene expression following delivery is of utmost importance in order to improve gene delivery. Intravital microscopy of tumors growing in dorsal skin fold window chambers is a useful method for monitoring gene transfection, since it allows non-invasive dynamic monitoring of gene expression in tumors in a live animal.

Successful Sphincter-saving Treatment of Anorectal Malignant Melanoma with Electrochemotherapy, Local Excision and Adjuvant Brachytherapy

Anti-cancer Drugs. Mar, 2005  |  Pubmed ID: 15711188

Anorectal malignant melanoma is a rare tumor and there is no consensus on whether aggressive or local management is more appropriate. Local sphincter-saving excision has been shown to have a higher recurrence rate than abdominoperineal resection, although there is no long-term survival difference between the two approaches. Therefore, new adjuvant treatment strategies to permit local sphincter-saving excisions are warranted. In our case, a large anorectal malignant melanoma was successfully treated preoperatively by electrochemotherapy with cisplatin that, by reducing the tumor size, enabled sphincter-saving local excision. Brachytherapy was postoperatively delivered to the excision site. Fourteen months after the beginning of treatment, the patient is without signs of local recurrence and is continent.

The Effect of High Frequency Electric Pulses on Muscle Contractions and Antitumor Efficiency in Vivo for a Potential Use in Clinical Electrochemotherapy

Bioelectrochemistry (Amsterdam, Netherlands). Feb, 2005  |  Pubmed ID: 15713562

Muscle contractions present the main source of unpleasant sensations for patients undergoing electrochemotherapy. The contractions are a consequence of high voltage pulse delivery. Relatively low repetition frequency of these pulses (1 Hz) results in separate muscle contractions associated with each single pulse that is delivered. It would be possible to reduce the number of unpleasant sensations by increasing the frequency of electric pulses above the frequency of tetanic contraction, provided that the antitumor efficiency of electrochemotherapy remains the same. These assumptions were investigated in the present paper by measuring the muscle torque at different pulse repetition frequencies and at two different pulse amplitudes in rats and studying the antitumor efficiency of electrochemotherapy at different pulse repetition frequencies on tumors in mice. Measurements of muscle torque confirmed that pulse frequencies above the frequency of tetanic contraction (>100 Hz) reduce the number of individual contractions to a single muscle contraction. Regardless of the pulse amplitude, with increasing pulse frequency muscle torque increases up to the frequency of 100 or 200 Hz and then decreases to a value similar to that after application of a 1 Hz pulse train. Electrochemotherapy in vivo with higher repetition frequencies inhibits tumor growth and is efficient at all pulse frequencies examined (1 Hz-5 kHz). These results suggest that there is a considerable potential for clinical use of high frequency pulses in electrochemotherapy.

Effective Treatment of Perianal Tumors in Dogs with Electrochemotherapy

Anticancer Research. Mar-Apr, 2005  |  Pubmed ID: 15868917

Electrochemotherapy is an antitumor therapy that utilizes locally-delivered, short intense direct current electric pulse to the tumor nodule plus chemotherapy. The aim of the present study was to evaluate the electrochemotherapy treatment of perianal tumors of different sizes in dogs.

Radiosensitising Effect of Electrochemotherapy with Bleomycin in LPB Sarcoma Cells and Tumors in Mice

BMC Cancer. 2005  |  Pubmed ID: 16168056

Bleomycin is poorly permeant but potent cytotoxic and radiosensitizing drug. The aim of the study was to evaluate whether a physical drug delivery system - electroporation can increase radiosensitising effect of bleomycin in vitro and in vivo.

Sequence and Time Dependence of Transfection Efficiency of Electrically-assisted Gene Delivery to Tumors in Mice

Current Drug Delivery. Jan, 2006  |  Pubmed ID: 16472096

Electrically-assisted gene delivery is a non-viral gene delivery technique, using application of square wave electric pulses to facilitate uptake of plasmid DNA into the cells. Feasibility and effectiveness of this method in vivo was already demonstrated, elaborating on pulse parameters and plasmid construction. However, there were no studies performed on sequencing and timing of plasmid DNA injection into the tumors and application of electric pulses. For this purpose we measured luciferase expression in two tumor models (LPB fibrosarcoma, B16F1 melanoma) after electrically-assisted gene delivery at varying time intervals between the pCMV-Luc plasmid injection and electroporation. Expression of luciferase was determined by measurement of its activity using luminometer. The results demonstrated that pCMV-Luc plasmid has to be injected before the application of electric pulses, since no measurable expression was detected in the tumors when pCMV-Luc plasmid was injected after electroporation of tumors. In both tumor models the highest transfection efficiency was obtained when pCMV-Luc plasmid was injected not less than 5 minutes but also not more than 30 minutes before the application of electric pulses. The results also demonstrated variability in the transfection efficiency depending on the tumor model. High expression was obtained in B16F1 tumor model (approximately 5500 pg luc/mg tumor) and lower in LPB fibrosarcoma (approximately 200 pg luc/mg tumor). In conclusion, our results demonstrate that regardless of the susceptibility of the tumors to electrically-assisted gene delivery, the best timing for pCMV-Luc plasmid is between 30 to 5 minutes prior to the application of electric pulses to the tumors.

Cytotoxicity of Different Platinum (II) Analogues to Human Tumour Cell Lines in Vitro and Murine Tumour in Vivo Alone or Combined with Electroporation

Anticancer Research. May-Jun, 2006  |  Pubmed ID: 16827135

The in vitro cytotoxic activity of two new platinum(II) complexes (3P-SK and PtAMP) in comparison with cisplatin (CDDP), oxaliplatin (OXA) and carboplatin (CARBO) was determined in four different human tumour cell lines. The in vivo efficiencies of CDDP and 3P-SK in MCA mammary carcinoma tumours induced in CBA mice were compared.

In Vivo Imaging of Tumor Growth After Electrochemotherapy with Cisplatin

Biochemical and Biophysical Research Communications. Sep, 2006  |  Pubmed ID: 16899227

Imaging methods can give both temporal and spatial dimensions to characterize the processes in progression of and/or treatment of specific disease Subcutaneous tumors can be cured after electrochemotherapy (ECT). Growth and reduction of tumors as a result of cytotoxic therapy can be followed by fluorescence video imaging directly on the same animal after treatment. Imaging of tumors should bring more information on the cellular effects of ECT. Green fluorescent protein (eGFP) expressing B16F10 and LPB tumors implanted in C57Bl/6 mice were treated with ECT with cisplatin. The growth or regression of the tumors was monitored either classically by using a caliper or by a manual definition of the region of interest where critical fluorescence levels were detected on the animals. A very good correlation between the two methods was observed. The eGFP mean fluorescence emission was only slightly affected by ECT with intravenously injected cisplatin. Ex vivo observations under a fluorescence microscope showed that eGFP was only detected on the outer layer of the tumor. No fluorescence was detected in the central part of the tumors, which were necrotic.

The Endothelial Cytoskeleton As a Target of Electroporation-based Therapies

Molecular Cancer Therapeutics. Dec, 2006  |  Pubmed ID: 17172418

Electroporation-based therapies, such as electrochemotherapy and electrogene therapy, result in the disruption of blood vessel networks in vivo and cause changes in blood flow and vascular permeability. The effects of electroporation on the cytoskeleton of cultured primary endothelial cells and on endothelial monolayer permeability were investigated to elucidate possible mechanisms involved. Human umbilical vein endothelial cells (HUVECs) were electroporated in situ and then immunofluorescence staining for filamentous actin, beta-tubulin, vimentin, and VE-cadherin as well as Western blotting analysis of levels of phosphorylated myosin light chain and cytoskeletal proteins were performed. Endothelial permeability was determined by monitoring the passage of FITC-coupled dextran through endothelial monolayers. Exposure of endothelial cells to electric pulses resulted in a profound disruption of microfilament and microtubule cytoskeletal networks, loss of contractility, and loss of vascular endothelial cadherin from cell-to-cell junctions immediately after electroporation. These effects were voltage dependent and reversible because cytoskeletal structures recovered within 60 min of electroporation with up to 40 V, without any significant loss of cell viability. The cytoskeletal effects of electroporation were paralleled by a rapid increase in endothelial monolayer permeability. These results suggest that the remodeling of the endothelial cytoskeleton and changes in endothelial barrier function could contribute to the vascular disrupting actions of electroporation-based therapies and provide an insight into putative mechanisms responsible for the observed increase in permeability and cessation of blood flow in vivo.

Electrogene Therapy with P53 of Murine Sarcomas Alone or Combined with Electrochemotherapy Using Cisplatin

DNA and Cell Biology. Dec, 2006  |  Pubmed ID: 17233116

The aim of our study was to evaluate feasibility and therapeutic potential of electrogene therapy with p53 alone or combined with electrochemotherapy using cisplatin on two murine sarcomas with different p53 status. Antitumor effectiveness of three consecutive electrogene treatments with p53 was more effective in wild-type LPB tumors than mutated SA-1 tumors, resulting in 21.4% of tumor cures in LPB tumors and 12.5% in SA-1 tumors. Pretreatment of tumors with electrogene therapy with p53 enhanced chemosensitivity of both tumor models treated by electrochemotherapy with cisplatin. After only one application of this treatment combination in the LPB tumor model, specific tumor growth delay was prolonged in the combined treatment group compared to electrogene therapy with p53 or electrochemotherapy with cisplatin alone, whereas in SA-1 tumors this treatment combination resulted in 31.6% of cured animals. Results of our study show that electrogene therapy with p53 alone or combined with electrochemotherapy is feasible and effective treatment of tumors. The combination of electrogene therapy and electrochemotherapy after only one application resulted in complete regression of tumors.

Effective Treatment of Multiple Unresectable Skin Melanoma Metastases by Electrochemotherapy

Croatian Medical Journal. Jun, 2007  |  Pubmed ID: 17589984

Multiple unresectable melanoma skin metastases pose a treatment problem, especially in centers where isolated limb perfusion is not available. We report the case of a 59-year-old woman who developed multiple small unresectable cutaneous melanoma metastases on the thigh after her lower limb was amputated. Electrochemotherapy with bleomycin resulted in good local control of the disease, with a complete response of the treated melanoma nodules (224 tumor nodules) after 4 treatment sessions. Comparison between electrochemotherapy using repetition frequency of the applied electric pulses of 1 Hz and 5 kHz demonstrated equal antitumor effectiveness. Electrochemotherapy with intravenous bleomycin can also be used as a treatment of choice for local control of multiple unresectable cutaneous melanoma skin metastases.

Electrotransfer of Therapeutic Molecules into Tissues

Current Opinion in Molecular Therapeutics. Dec, 2007  |  Pubmed ID: 18041666

Electroporation is a physical method for the delivery of various molecules into cells by application of controlled external electrical fields that transiently increase permeability of the cell membrane. This technique is now widely used as an alternative to viral gene delivery for transfection of therapeutic genes into different tissues. Gene electrotransfer holds great potential for clinical application due to the ease of preparation of large quantities of endotoxin-free plasmid DNA, the control and reproducibility of this method, and the development of electric pulse generators approved for clinical use. Electroporation has been utilized mainly for DNA vaccination against infectious diseases and cancer. It has also been used for the delivery of other therapeutic genes, mainly cytokines, used in the treatment of various diseases, including cancer, arthritis, multiple sclerosis and inflammation, following organ transplantation. Electroporation as a delivery system for chemotherapeutic drugs, termed antitumor electrochemotherapy, is already at the clinical stage and is being used routinely in several oncology centers in Europe. In addition, the first clinical trials for electrogene therapy of cancer are ongoing. Therefore, it can be presumed that electrotransfer of therapeutic genes into tissues will soon form a validated alternative to viral delivery systems in a clinical setting.

Dynamic Contrast Enhanced MRI of Mouse Fibrosarcoma Using Small-molecular and Novel Macromolecular Contrast Agents

Physica Medica : PM : an International Journal Devoted to the Applications of Physics to Medicine and Biology : Official Journal of the Italian Association of Biomedical Physics (AIFB). Dec, 2007  |  Pubmed ID: 18061121

The aim of the study was a comparison of 2 novel macromolecular contrast agents, Gadomer-17 and Polylysine-Gd-DTPA, with commercially available Gd-DTPA in determining the quality of tumor microvasculature by dynamic contrast enhanced MRI. Three groups of 5 mice with SA-1 tumors were studied. To each group of animals one contrast agent was administered; i.e. the first group got Gd-DTPA, the second group Gadomer-17 and the third group Polylysine-Gd-DTPA. To perform dynamic contrast enhanced MRI a standard keyhole approach was used by which consecutive signal intensity change due to contrast agent accumulation in the tumor was measured. From the obtained data, tissue permeability surface area product PS and fractional blood volume BV were calculated on a pixel-by-pixel basis. PS and BV values were calculated for each contrast agent. Based on the values, contrast agents were classified according to their performance in characterizing tumor microvasculature. Results of our study suggest that Gadomer-17 and Polylysine-Gd-DTPA are significantly superior to Gd-DTPA in characterizing tumor microvasculature.

Gene Electrotransfer into Murine Skeletal Muscle: a Systematic Analysis of Parameters for Long-term Gene Expression

Technology in Cancer Research & Treatment. Apr, 2008  |  Pubmed ID: 18345697

Skeletal muscle is an attractive target tissue for delivery of therapeutic genes, since it is well vascularized, easily accessible, and has a high capacity for protein synthesis. For efficient transfection in skeletal muscle, several protocols have been described, including delivery of low voltage electric pulses and a combination of high and low voltage electric pulses. The aim of this study was to determine the influence of different parameters of electrotransfection on short-term and long-term transfection efficiency in murine skeletal muscle, and to evaluate histological changes in the treated tissue. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses, and different doses of plasmid DNA were tested for electrotransfection of tibialis cranialis muscle of C57Bl/6 mice using DNA plasmid encoding green fluorescent protein (GFP). Transfection efficiency was assessed on frozen tissue sections one week after electrotransfection using a fluorescence microscope and also noninvasively, followed by an in vivo imaging system using a fluorescence stereo microscope over a period of several months. Histological changes in muscle were evaluated immediately or several months after electrotransfection by determining infiltration of inflammatory mononuclear cells and presence of necrotic muscle fibers. The most efficient electrotransfection into skeletal muscle of C57Bl/6 mice in our experiments was achieved when one high voltage (HV) and four low voltage (LV) electric pulses were applied 5 seconds after the injection of 30 microg of plasmid DNA. This protocol resulted in the highest short-term as well as long-term transfection. The fluorescence intensity of the transfected area declined after 2-3 weeks, but GFP fluorescence was still detectable 18 months after electrotransfection. Extensive inflammatory mononuclear cell infiltration was observed immediately after the electrotransfection procedure using the described parameters, but no necrosis or late tissue damage was observed. This study showed that electric pulse parameters, time lag between the injection of DNA and application of electric pulses, and dose of plasmid DNA affected the duration of transgene expression in murine skeletal muscle. Therefore, transgene expression in muscle can be controlled by appropriate selection of electrotransfection protocol.

The Effect of Electroporation Pulses on Functioning of the Heart

Medical & Biological Engineering & Computing. Aug, 2008  |  Pubmed ID: 18415132

Electrochemotherapy is an effective antitumor treatment currently applied to cutaneous and subcutaneous tumors. Electrochemotherapy of tumors located close to the heart could lead to adverse effects, especially if electroporation pulses were delivered within the vulnerable period of the heart or if they coincided with arrhythmias of some types. We examined the influence of electroporation pulses on functioning of the heart of human patients by analyzing the electrocardiogram. We found no pathological morphological changes in the electrocardiogram; however, we demonstrated a transient RR interval decrease after application of electroporation pulses. Although no adverse effects due to electroporation have been reported so far, the probability for complications could increase in treatment of internal tumors, in tumor ablation by irreversible electroporation, and when using pulses of longer durations. We evaluated the performance of our algorithm for synchronization of electroporation pulse delivery with electrocardiogram. The application of this algorithm in clinical electroporation would increase the level of safety for the patient and suitability of electroporation for use in anatomical locations presently not accessible to existing electroporation devices and electrodes.

Electrode Commutation Sequence for Honeycomb Arrangement of Electrodes in Electrochemotherapy and Corresponding Electric Field Distribution

Bioelectrochemistry (Amsterdam, Netherlands). Nov, 2008  |  Pubmed ID: 18424240

Electrochemotherapy is a treatment based on combination of chemotherapeutic drug and electroporation. It is used in clinics for treatment of solid tumours. For electrochemotherapy of larger tumours multiple needle electrodes were already suggested. We developed and tested electrode commutation circuit, which controls up to 19 electrodes independently. Each electrode can be in one of three possible states: on positive or negative potential or in the state of high impedance. In addition, we tested a pulse sequence using seven electrodes for which we also calculated electric field distribution in tumour tissue by means of finite-elements method. Electrochemotherapy, performed by multiple needle electrodes and tested pulse sequence on large subcutaneous murine tumour model resulted in tumour growth delay and 57% complete responses, thus demonstrating that the tested electrode commutation sequence is efficient.

Electrochemotherapy Compared to Surgery for Treatment of Canine Mast Cell Tumours

In Vivo (Athens, Greece). Jan-Feb, 2009  |  Pubmed ID: 19368125

The aim of this study was to evaluate the effectiveness of local treatment electrochemotherapy (ECT) with cisplatin and to compare it with effectiveness of surgery for treatment of mast cell tumours (MCT) in dogs.

Limb Sparing Treatment of Bleeding Melanoma Recurrence by Electrochemotherapy

Tumori. May-Jun, 2009  |  Pubmed ID: 19688986

Electrochemotherapy is an effective local treatment for tumors that combines administration of a chemotherapeutic drug with the subsequent application of electric pulses to the tumor. In addition, it was also found to have a vascular-disrupting effect. We report a case of limb-sparing treatment of bleeding melanoma recurrence by electrochemotherapy

Electric Pulses Used in Electrochemotherapy and Electrogene Therapy Do Not Significantly Change the Expression Profile of Genes Involved in the Development of Cancer in Malignant Melanoma Cells

BMC Cancer. 2009  |  Pubmed ID: 19709437

Electroporation is a versatile method for in vitro or in vivo delivery of different molecules into cells. However, no study so far has analysed the effects of electric pulses used in electrochemotherapy (ECT pulses) or electric pulses used in electrogene therapy (EGT pulses) on malignant cells. We studied the effect of ECT and EGT pulses on human malignant melanoma cells in vitro in order to understand and predict the possible effect of electric pulses on gene expression and their possible effect on cell behaviour.

Local and Systemic Antitumor Effect of Intratumoral and Peritumoral IL-12 Electrogene Therapy on Murine Sarcoma

Cancer Biology & Therapy. Nov, 2009  |  Pubmed ID: 19755854

Soft tissue sarcomas pose a challenge for successful treatment with conventional therapeutic methods, therefore newer therapeutic approaches are considered. In this study, we evaluated the antitumor effect of IL-12 electrogene therapy (EGT) on murine SA-1 fibrosarcoma. The therapeutic plasmid was injected either intratumorally into subcutaneous SA-1 nodules or intradermally into the peritumoral region. We achieved a remarkable local and systemic antitumor effect with both approaches after single plasmid DNA application, with significant intratumoral and systemic production of IL-12 and IFNgamma. Intratumoral IL-12 EGT resulted in over 90% complete response rate of the treated tumors with 60% of cured mice being resistant to challenge with SA-1 tumor cells. Peritumoral EGT resulted in a lower complete response rate (16%), with significant growth delay of remaining tumors. Both therapies also resulted in significant inhibition of growth of untreated tumors, growing simultaneously at a distant site. These data suggest that IL-12 EGT may be useful in the treatment of soft tissue sarcomas, exerting a local and systemic antitumor effect.

Controlled Systemic Release of Interleukin-12 After Gene Electrotransfer to Muscle for Cancer Gene Therapy Alone or in Combination with Ionizing Radiation in Murine Sarcomas

The Journal of Gene Medicine. Dec, 2009  |  Pubmed ID: 19777440

The present study aimed to evaluate the antitumor effectiveness of systemic interleukin (IL)-12 gene therapy in murine sarcoma models, and to evaluate its interaction with the irradiation of tumors and metastases. To avoid toxic side-effects of IL-12 gene therapy, the objective was to achieve the controlled release of IL-12 after intramuscular gene electrotransfer.

Radiosensitizing Effect of Electrochemotherapy in a Fractionated Radiation Regimen in Radiosensitive Murine Sarcoma and Radioresistant Adenocarcinoma Tumor Model

Radiation Research. Dec, 2009  |  Pubmed ID: 19929414

Electrochemotherapy can potentiate the radiosensitizing effect of bleomycin, as shown in our previous studies. To bring this treatment closer to use in clinical practice, we evaluated the interaction between electrochemotherapy with bleomycin and single-dose or fractionated radiation in two murine tumor models with different histology and radiosensitivity. Radiosensitive sarcoma SA-1 and radioresistant adenocarcinoma CaNT subcutaneous tumors grown in A/J and CBA mice, respectively, were used. The anti-tumor effect and skin damage around the treated tumors were evaluated after electrochemotherapy with bleomycin alone or combined with single-dose radiation or a fractionated radiation regimen. The anti-tumor effectiveness of electrochemotherapy was more pronounced in SA-1 than CaNT tumors. In both tumor models, the tumor response to radiation was not significantly influenced by bleomycin alone or by electroporation alone. However, electrochemotherapy before the first tumor irradiation potentiated the response to a single-dose or fractionated radiation regimen in both tumors. For the fractionated radiation regimen, normal skin around the treated tumors was damaged fourfold less than for the single-dose regimen. Electrochemotherapy prior to single-dose irradiation induced more damage to the skin around the treated tumors and greater loss of body weight compared to other irradiated groups, whereas electrochemotherapy combined with the fractionated radiation regimen did not. Electrochemotherapy with low doses of bleomycin can also be used safely for radiosensitization of different types of tumors in a fractionated radiation regimen, resulting in a good anti-tumor effect and no major potentiating effect on radiation-induced skin damage.

Towards Treatment Planning and Treatment of Deep-seated Solid Tumors by Electrochemotherapy

Biomedical Engineering Online. 2010  |  Pubmed ID: 20178589

Electrochemotherapy treats tumors by combining specific chemotherapeutic drugs with an intracellular target and electric pulses, which increases drug uptake into the tumor cells. Electrochemotherapy has been successfully used for treatment of easily accessible superficial tumor nodules. In this paper, we present the first case of deep-seated tumor electrochemotherapy based on numerical treatment planning.

Antivascular Effects of Electrochemotherapy: Implications in Treatment of Bleeding Metastases

Expert Review of Anticancer Therapy. May, 2010  |  Pubmed ID: 20470005

Solid tumors of various etiologies can be treated efficiently by electrochemotherapy (ECT), a combined use of electroporation (EP) and chemotherapeutic drugs, such as bleomycin and cisplatin. EP alone and ECT in particular, induce a profound reduction in tumor blood flow, which contributes to the antitumor effect. After EP and ECT, the time course of blood flow changes and follows the same two-phase pattern. The first rapid and short-lived vasoconstriction phase is followed by the second much longer-lived phase resulting from disrupted cytoskeletal structures and a compromised barrier function of the microvascular endothelium. In the case of ECT, however, tumor vascular endothelial cells are also affected by the chemotherapeutic drug, which leads to irrecoverable damage to tumor vessels and to a further decrease in tumor blood flow within hours after application of ECT. Tumor cells surviving the direct effects of ECT are consequently exposed to lack of oxygen and nutrients and are pushed into the secondary cascade of induced cell death. Clinically, the antitumor effectiveness of ECT has been proven extensively in the treatment of melanoma metastases, with 70-80% complete responses. The antivascular effects of ECT were also exploited for palliative treatment of bleeding melanoma metastases, with immediate cessation of bleeding and very good antitumor effectiveness. The antivascular effect of ECT is of utmost importance for translation of ECT into the treatment of deep-seated tumors, especially in well vascularized organs, such as the liver, where it prevents bleeding of the treated area.

Cancer Electrogene Therapy with Interleukin-12

Current Gene Therapy. Aug, 2010  |  Pubmed ID: 20560875

Electrogene therapy combines administration of plasmid DNA into tissue followed by local application of electric pulses. In electrogene therapy with interleukin-12 (IL-12), different routes of administration, different doses of plasmid DNA and different protocols for delivery of electric pulses were evaluated in numerous preclinical studies. Antitumor effectiveness was tested in different types of primary tumors, distantly growing tumors and induced metastases. Intratumoral IL-12 electrogene therapy has been proved to be very effective in local tumor control, having also a systemic effect. Intramuscular and peritumoral IL-12 electrogene therapy had also a pronounced systemic effect and when combined with other treatment strategies resulted in tumor cures. Antitumor effectiveness of IL-12 electrogene therapy is due to the induction of adaptive immunity and innate resistance and anti-angiogenic action. Translation of preclinical studies into clinical trials in human and veterinary oncology has started with encouraging results that would hopefully lead to further investigation of this therapy, also in combination with other cancer treatment modalities.

Robustness of Treatment Planning for Electrochemotherapy of Deep-seated Tumors

The Journal of Membrane Biology. Jul, 2010  |  Pubmed ID: 20596859

Treatment of cutaneous and subcutaneous tumors with electrochemotherapy has become a regular clinical method, while treatment of deep-seated tumors is still at an early stage of development. We present a method for preparing a dedicated patient-specific, computer-optimized treatment plan for electrochemotherapy of deep-seated tumors based on medical images. The treatment plan takes into account the patient's anatomy, tissue conductivity changes during electroporation and the constraints of the pulse generator. Analysis of the robustness of a treatment plan made with this method shows that the effectiveness of the treatment is not affected significantly by small single errors in electrode positioning. However, when many errors occur simultaneously, the resulting drop in effectiveness is larger, which means that it is necessary to be as accurate as possible in electrode positioning. The largest effect on treatment effectiveness stems from uncertainties in dielectric properties and electroporation thresholds of treated tumors and surrounding tissues, which emphasizes the need for more accurate measurements and more research. The presented methods for treatment planning and robustness analysis allow quantification of the treatment reproducibility and enable the setting of suitable safety margins to improve the likelihood of successful treatment of deep-seated tumors by electrochemotherapy.

Increased Cellular Uptake of Biocompatible Superparamagnetic Iron Oxide Nanoparticles into Malignant Cells by an External Magnetic Field

The Journal of Membrane Biology. Jul, 2010  |  Pubmed ID: 20602230

Superparamagnetic iron oxide nanoparticles (SPIONs) are used as delivery systems for different therapeutics including nucleic acids for magnetofection-mediated gene therapy. The aim of our study was to evaluate physicochemical properties, biocompatibility, cellular uptake and trafficking pathways of the custom-synthesized SPIONs for their potential use in magnetofection. Custom-synthesized SPIONs were tested for size, shape, crystalline composition and magnetic behavior using a transmission electron microscope, X-ray diffractometer and magnetometer. SPIONs were dispersed in different aqueous media to obtain ferrofluids, which were tested for pH and stability using a pH meter and zetameter. Cytotoxicity was determined using the MTS and clonogenic assays. Cellular uptake and trafficking pathways were qualitatively evaluated by transmission electron microscopy and quantitatively by inductively coupled plasma atomic emission spectrometry. SPIONs were composed of an iron oxide core with a diameter of 8-9 nm, coated with a 2-nm-thick layer of silica. SPIONs, dispersed in 0.9% NaCl solution, resulted in a stable ferrofluid at physiological pH for several months. SPIONs were not cytotoxic in a broad range of concentrations and were readily internalized into different cells by endocytosis. Exposure to neodymium-iron-boron magnets significantly increased the cellular uptake of SPIONs, predominantly into malignant cells. The prepared SPIONs displayed adequate physicochemical and biomedical properties for potential use in magnetofection. Their cellular uptake was dependent on the cell type, and their accumulation within the cells was dependent on the duration of exposure to an external magnetic field.

The Influence of Electroporation on Cytotoxicity of Anticancer Ruthenium(III) Complex KP1339 in Vitro and in Vivo

Anticancer Research. Jun, 2010  |  Pubmed ID: 20651351

In our study, the ruthenium-based anticancer agent KP1339 was tested in combination with electroporation for its cytotoxic effect on CHO and SA-1 cell lines in vitro and on SA-1 murine tumour model in vivo. Cells were treated with different doses of KP1339 for 15 or 60 min with or without electroporation in vitro. Cell viability was measured with the MTS assay. In vivo, mice bearing SA-1 tumours were treated with different doses of KP1339 with or without electroporation. Tumour growth was measured at various time points after treatment. Intratumoural ruthenium content was analysed as a measure of KP1339 accumulation to correlate it with antitumour effectiveness. Our results show that electroporation does not potentiate the cytotoxicity of KP1339 in vitro, but significantly potentiates antitumour effectiveness in vivo. Electroporation enhanced ruthenium uptake immediately after treatment, consequently causing persistently higher intratumoural ruthenium content throughout the whole observation period (48 h). In addition, ruthenium content rose continuously in electroporated and intact tumours throughout the whole observation period. The observed antitumour effectiveness is the result of both the direct cytotoxicity of KP1339 and an antivascular effect of electroporation.

Development of Human Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-induced Gene Expression

Radiology and Oncology. Mar, 2010  |  Pubmed ID: 22933890

Human exposure to genotoxic agents in the environment and everyday life represents a serious health threat. Fast and reliable assessment of genotoxicity of chemicals is of main importance in the fields of new chemicals and drug development as well as in environmental monitoring. The tumor suppressor gene p21, the major downstream target gene of activated p53 which is responsible for cell cycle arrest following DNA damage, has been shown to be specifically up-regulated by genotoxic carcinogens. The aim of our study was to develop a human cell-based biosensor system for simple and fast detection of genotoxic agents.

Radiotherapy in Combination with Vascular-targeted Therapies

Radiology and Oncology. Jun, 2010  |  Pubmed ID: 22933894

Given the critical role of tumor vasculature in tumor development, considerable efforts have been spent on developing therapeutic strategies targeting the tumor vascular network. A variety of agents have been developed, with two general approaches being pursued. Antiangiogenic agents (AAs) aim to interfere with the process of angiogenesis, preventing new tumor blood vessel formation. Vascular-disrupting agents (VDAs) target existing tumor vessels causing tumor ischemia and necrosis. Despite their great therapeutic potential, it has become clear that their greatest clinical utility may lie in combination with conventional anticancer therapies. Radiotherapy is a widely used treatment modality for cancer with its distinct therapeutic challenges. Thus, combining the two approaches seems reasonable.

Study of Radiation Induced Changes of Phosphorus Metabolism in Mice by (31)P NMR Spectroscopy

Radiology and Oncology. Sep, 2010  |  Pubmed ID: 22933912

The aim of this study was to examine whether (31)P NMR can efficiently detect X-ray radiation induced changes of energy metabolism in mice. Exposure to ionizing radiation causes changes in energy supply that are associated with the tissue damage because of oxidative stress and uncoupled oxidative phosphorylation. This has as a consequence decreased phosphocreatine to adenosine triphosphate ratio (Pcr/ATP) as well as increased creatine kinase (CK) and liver enzymes (transaminases AST and ALT) levels in serum.

Metastatic Potential of Melanoma Cells is Not Affected by Electrochemotherapy

Melanoma Research. Jun, 2011  |  Pubmed ID: 20216102

Electrochemotherapy is a local treatment combining chemotherapy and application of electric pulses to the tumour. Electrochemotherapy with bleomycin and cisplatin has shown its effectiveness in controlling local tumour growth in the treatment of malignant melanoma. However, the effect of electrochemotherapy on the metastatic potential of tumour cells is not known. Prevention of metastasis is an important aspect of successful treatment; however, it is known that metastasis can be induced by different treatment modalities. Therefore, the aim of this study was to evaluate the effect of electrochemotherapy with cisplatin on the metastatic potential of human malignant melanoma cells. Cells treated by electrochemotherapy with cisplatin were tested for their ability to migrate and invade through Matrigel-coated porous membrane. In addition, RNA was isolated from cells after treatment and differentially expressed genes were investigated by microarray analysis to evaluate the effect of electrochemotherapy with cisplatin on gene expression. There were no significant changes observed in cell migration and invasion of melanoma cells after electrochemotherapy. In addition, there were no changes observed in cell adhesion on Matrigel. Gene expression analysis showed that a very low number of genes were differentially expressed after electrochemotherapy with cisplatin. Two genes, LAMB3 and CD63 involved in cell migration, were both downregulated after electrochemotherapy with cisplatin and the expression of metastasis promoting genes was not increased after electrochemotherapy. Our data suggest that electrochemotherapy does not increase the metastatic behaviour of human melanoma cells.

Irradiation, Cisplatin, and 5-azacytidine Upregulate Cytomegalovirus Promoter in Tumors and Muscles: Implementation of Non-invasive Fluorescence Imaging

Molecular Imaging and Biology : MIB : the Official Publication of the Academy of Molecular Imaging. Feb, 2011  |  Pubmed ID: 20396957

The cytomegalovirus (CMV) promoter is one of the most commonly used promoters for expression of transgenes in mammalian cells. The aim of our study was to evaluate the role of methylation and upregulation of the CMV promoter by irradiation and the chemotherapeutic agent cisplatin in vivo using non-invasive fluorescence in vivo imaging.

Electrochemotherapy of Chest Wall Breast Cancer Recurrence

Cancer Treatment Reviews. Aug, 2011  |  Pubmed ID: 21856080

Chest wall breast cancer recurrence after mastectomy is a disease difficult to treat. Its incidence varies between 5% and 30% in different subset of patients. When possible, radical surgical therapy represents the main treatment approach, however when the disease progresses and/or treatments are not successful, ulceration, bleeding, lymphedema and psychological distress of progressive disease significantly decrease the quality of the remaining life of a patient. When surgical excision of chest wall recurrence is not possible, other local treatments such as radiotherapy, radiotherapy with hyperthermia, topical chemotherapy and electrochemotherapy might be taken into account. Electrochemotherapy provides safe, efficient and non-invasive locoregional treatment approach for chest wall breast cancer recurrence. Several clinical studies have demonstrated high efficacy and a good safety profile of electrochemotherapy applied in single or multiple consecutive sessions, till clinical response was reached. Electrochemotherapy can be performed either with cisplatin injected intratumorally or with bleomycin given intratumorally or intravenously. Furthermore, it can be effectively used in heavily pre-treated areas, after surgery, radiotherapy or systemic chemotherapy. These are the advantages that might demand its use especially in patients with pre-treated extensive disease and in frail elderly patients. With development of the technology electrochemotherapy could even be suggested as a primary local therapy in patients not suitable for surgical removal of the primary tumor.

Magnetic Nanoparticles As Targeted Delivery Systems in Oncology

Radiology and Oncology. Mar, 2011  |  Pubmed ID: 22933928

Many different types of nanoparticles, magnetic nanoparticles being just a category among them, offer exciting opportunities for technologies at the interfaces between chemistry, physics and biology. Some magnetic nanoparticles have already been utilized in clinical practice as contrast enhancing agents for magnetic resonance imaging (MRI). However, their physicochemical properties are constantly being improved upon also for other biological applications, such as magnetically-guided delivery systems for different therapeutics. By exposure of magnetic nanoparticles with attached therapeutics to an external magnetic field with appropriate characteristics, they are concentrated and retained at the preferred site which enables the targeted delivery of therapeutics to the desired spot.

Electrogene Therapy with Interleukin-12 in Canine Mast Cell Tumors

Radiology and Oncology. Mar, 2011  |  Pubmed ID: 22933932

Mast cell tumors (MCT) are the most common malignant cutaneous tumors in dogs with extremely variable biological behaviour. Different treatment approaches can be used in canine cutaneous MCT, with surgical excision being the treatment of choice. In this study, electrogene therapy (EGT) as a new therapeutic approach to canine MCTs, was established. MATERIALS AND METHODS.: Eight dogs with a total of eleven cutaneous MCTs were treated with intratumoral EGT using DNA plasmid encoding human interleukin-12 (IL-12). The local response to the therapy was evaluated by repeated measurements of tumor size and histological examination of treated tumors. A possible systemic response was assessed by determination of IL-12 and interferon- γ (IFN-γ) in patients' sera. The occurence of side effects was monitored with weekly clinical examinations of treated animals and by performing basic bloodwork, consisting of the complete bloodcount and determination of selected biochemistry parameters.

Hyaluronidase and Collagenase Increase the Transfection Efficiency of Gene Electrotransfer in Various Murine Tumors

Human Gene Therapy. Jan, 2012  |  Pubmed ID: 21797718

Abstract One of the applications of electroporation/electropulsation in biomedicine is gene electrotransfer, the wider use of which is hindered by low transfection efficiency in vivo compared with viral vectors. The aim of our study was to determine whether modulation of the extracellular matrix in solid tumors, using collagenase and hyaluronidase, could increase the transfection efficiency of gene electrotransfer in histologically different solid subcutaneous tumors in mice. Tumors were treated with enzymes before electrotransfer of plasmid DNA encoding either green fluorescent protein or luciferase. Transfection efficiency was determined 3, 9, and 15 days posttransfection. We demonstrated that pretreatment of tumors with a combination of enzymes significantly increased the transfection efficiency of electrotransfer in tumors with a high extracellular matrix area (LPB fibrosarcoma). In tumors with a smaller extracellular matrix area and less organized collagen lattice, the increase was not so pronounced (SA-1 fibrosarcoma and EAT carcinoma), whereas in B16 melanoma, in which only traces of collagen are present, pretreatment of tumors with hyaluronidase alone was more efficient than pretreatment with both enzymes. In conclusion, our results suggest that modification of the extracellular matrix could improve distribution of plasmid DNA in solid subcutaneous tumors, demonstrated by an increase in transfection efficiency, and thus have important clinical implications for electrogene therapy.

Patient-specific Treatment Planning of Electrochemotherapy: Procedure Design and Possible Pitfalls

Bioelectrochemistry (Amsterdam, Netherlands). Oct, 2012  |  Pubmed ID: 22341626

Electrochemotherapy uses electroporation for enhancing chemotherapy. Electrochemotherapy can be performed using standard operating procedures with predefined electrode geometries, or using patient-specific treatment planning to predict electroporation. The latter relies on realistic computer models to provide optimal results (i.e. electric field distribution as well as electrodes' position and number) and is suitable for treatment of deep-seated tumors. Since treatment planning for deep-seated tumors has been used in radiotherapy, we expose parallelisms with radiotherapy in order to establish the procedure for electrochemotherapy of deep-seated tumors. We partitioned electrochemotherapy in the following phases: the mathematical model of electroporation, treatment planning, set-up verification, treatment delivery and monitoring, and response assessment. We developed a conceptual treatment planning software that incorporates mathematical models of electroporation. Preprocessing and segmentation of the patient's medical images are performed, and a 3D model is constructed which allows placement of electrodes and implementation of the mathematical model of electroporation. We demonstrated the feasibility of electrochemotherapy of deep-seated tumors treatment planning within a clinical study where treatment planning contributed to the effective electrochemotherapy treatment of deep-seated colorectal metastases in the liver. The described procedure can provide medical practitioners with information on using electrochemotherapy in the clinical setting. The main aims of this paper are: 1) to present the procedure for treating deep-seated tumors by electrochemotherapy based on patient-specific treatment planning, and 2) to identify gaps in knowledge and possible pitfalls of such procedure.

Surface Modified Magnetic Nanoparticles for Immuno-gene Therapy of Murine Mammary Adenocarcinoma

Biomaterials. Jun, 2012  |  Pubmed ID: 22429983

Cancer immuno-gene therapy is an introduction of nucleic acids encoding immunostimulatory proteins, such as cytokine interleukin 12 (IL-12), into somatic cells to stimulate an immune response against a tumor. Various methods can be used for the introduction of nucleic acids into cells; magnetofection involves binding of nucleic acids to magnetic nanoparticles with subsequent exposure to an external magnetic field. Here we show that surface modified superparamagnetic iron oxide nanoparticles (SPIONs) with a combination of polyacrylic acid (PAA) and polyethylenimine (PEI) (SPIONs-PAA-PEI) proved to be safe and effective for magnetofection of cells and tumors in mice. Magnetofection of cells with plasmid DNA encoding reporter gene using SPIONs-PAA-PEI was superior in transfection efficiency to commercially available SPIONs. Magnetofection of murine mammary adenocarcinoma with plasmid DNA encoding IL-12 using SPIONs-PAA-PEI resulted in significant antitumor effect and could be further refined for cancer immuno-gene therapy.

In Vivo Molecular Imaging and Histological Analysis of Changes Induced by Electric Pulses Used for Plasmid DNA Electrotransfer to the Skin: a Study in a Dorsal Window Chamber in Mice

The Journal of Membrane Biology. Sep, 2012  |  Pubmed ID: 22644389

Electropermeabilization/electroporation (EP) is a physical method that by application of electric pulses to cells increases cell membrane permeability and enables the introduction of molecules into the cells. One of the uses of EP in vivo is plasmid DNA electrotransfer to the skin for DNA vaccination. EP of tissues induces reduction of blood flow and, in combination with plasmid DNA, induction of an immune response. One of the EP protocols for plasmid DNA electrotransfer to the skin is a combination of high-voltage (HV) and low-voltage (LV) pulses. However, the effects of this pulse combination on skin-vessel blood flow are not known. Therefore, using intravital microscopy in a dorsal window chamber in mice and fluorescently labeled dextrans, the effects of one HV and eight LV pulses on skin vasculature were investigated. In addition, a detailed histological analysis was performed. Image analysis of fluorescence intensity changes demonstrated that EP induces a transient constriction and increased permeability of blood vessels as well as a "vascular lock." Histological analysis revealed rounding up of endothelial cells and stacking up of erythrocytes at 1 h after EP. In addition, extravasation of erythrocytes and leukocyte infiltration accompanied by edema were determined up to 24 h after EP. In conclusion, our results show that blood flow modifying effects of EP in skin contribute to the infiltration of immune cells in the exposed area. When combined with plasmid DNA for vaccination, this could enable the initial and prolonged contact of immune cells with encoded therapeutic proteins.

Assessment of the Tumourigenic and Metastatic Properties of SK-MEL28 Melanoma Cells Surviving Electrochemotherapy with Bleomycin

Radiology and Oncology. Mar, 2012  |  Pubmed ID: 22933978

Electrochemotherapy is a local treatment combining chemotherapy and electroporation and is highly effective treatment approach for subcutaneous tumours of various histologies. Contrary to surgery and radiation, the effect of electrochemotherapy on metastatic potential of tumour cells has not been extensively studied. The aim of the study was to evaluate the effect of electrochemotherapy with bleomycin on the metastatic potential of human melanoma cells in vitro.

Intravital Microscopy at the Single Vessel Level Brings New Insights of Vascular Modification Mechanisms Induced by Electropermeabilization

Journal of Controlled Release : Official Journal of the Controlled Release Society. Nov, 2012  |  Pubmed ID: 23017380

Electroporation/electropermeabilization, i.e. the result of the application of electric pulses to tissues, is a physical method for delivery of exogenous molecules into cells. It is effective particularly for compounds with limited transmembrane transport. In vivo, electropermeabilization facilitates the delivery of chemotherapeutic drugs into tumor cells that is the basic mechanism of the antitumor effectiveness of electrochemotherapy. This therapy has also blood flow modifying effects in tissues. The aim of our present study was to understand and explain the effects of electropermeabilization on the dynamics (vasomotricity, permeability and recovery) of subcutaneous blood vessels towards different size of molecules. These features were measured in C57Bl/6 mice via a dorsal skin fold window chamber, using fluorescently labeled dextrans of different sizes, intravital fluorescence microscopy imaging and specific image analysis. Application of electric pulses on the skin in vivo resulted in a rapid increase in vascular permeability that gradually recovered to basal levels at different times post-treatment, depending on dextran size. Simultaneously, the immediate constriction of the blood vessels occurred that was more pronounced for arterioles compared to venules. This vasoconstriction of arterioles results in a transient "vascular lock". The increased permeability of small vessels walls whatever the dextran size associated with delayed perfusion explains the improved delivery of the intravenous injected molecules (i.e. drugs, gene delivery) into the tissues induced by electropermeabilization in vivo.

Contrasting Effect of Recombinant Human Erythropoietin on Breast Cancer Cell Response to Cisplatin Induced Cytotoxicity

Radiology and Oncology. Sep, 2012  |  Pubmed ID: 23077460

Human recombinant erythropoietin (rHuEpo) that is used for the treatment of the chemotherapy-induced anaemia in cancer patients was shown to cause detrimental effects on the course of disease due to increased adverse events inflicting patient's survival, potentially related to rHuEpo-induced cancer progression. In this study, we elucidate the effect of rHuEpo administration on breast cancer cell proliferation and gene expression after cisplatin (cDDP) induced cytotoxicity.

IL-12 Based Gene Therapy in Veterinary Medicine

Journal of Translational Medicine. Nov, 2012  |  Pubmed ID: 23171444

The use of large animals as an experimental model for novel treatment techniques has many advantages over the use of laboratory animals, so veterinary medicine is becoming an increasingly important translational bridge between preclinical studies and human medicine. The results of preclinical studies show that gene therapy with therapeutic gene encoding interleukin-12 (IL-12) displays pronounced antitumor effects in various tumor models. A number of different studies employing this therapeutic plasmid, delivered by either viral or non-viral methods, have also been undertaken in veterinary oncology. In cats, adenoviral delivery into soft tissue sarcomas has been employed. In horses, naked plasmid DNA has been delivered by direct intratumoral injection into nodules of metastatic melanoma. In dogs, various types of tumors have been treated with either local or systemic IL-12 electrogene therapy. The results of these studies show that IL-12 based gene therapy elicits a good antitumor effect on spontaneously occurring tumors in large animals, while being safe and well tolerated by the animals. Hopefully, such results will lead to further investigation of this therapy in veterinary medicine and successful translation into human clinical trials.

Potentiation of Electrochemotherapy by Intramuscular IL-12 Gene Electrotransfer in Murine Sarcoma and Carcinoma with Different Immunogenicity

Radiology and Oncology. Dec, 2012  |  Pubmed ID: 23412658

BACKGROUND.: Electrochemotherapy provides good local tumor control but requires adjuvant treatment for increased local response and action on distant metastasis. In relation to this, intramuscular interleukin-12 (IL-12) gene electro-transfer, which provides systemic shedding of IL-12, was combined with local electrochemotherapy with cisplatin. Furthermore, the dependence on tumor immunogenicity and immunocompetence of the host on combined treatment response was evaluated. MATERIALS AND METHODS.: Sensitivity of SA-1 sarcoma and TS/A carcinoma cells to electrochemotherapy with cisplatin was tested in vitro. In vivo, intratumoral electrochemotherapy with cisplatin (day 1) was combined with a single (day 0) or multiple (days 0, 2, 4) intramuscular murine IL-12 (mIL-12) gene electrotransfer. The antitumor effectiveness of combined treatment was evaluated on immunogenic murine SA-1 sarcoma in A/J mice and moderately immunogenic murine TS/A carcinoma, in immunocompetent BALB/c and immunodeficient SCID mice. RESULTS.: Electrochemotherapy in vitro resulted in a similar IC(50) values for both sarcoma and carcinoma cell lines. However, in vivo electrochemotherapy was more effective in the treatment of sarcoma, the more immunogenic of the tumors, resulting in a higher log cell kill, longer specific tumor growth delay, and also 17% tumor cures compared to carcinoma where no tumor cures were observed. Adjuvant intramuscular mIL-12 gene electrotransfer increased the log cell kill in both tumor models, potentiating the specific tumor growth delay by a factor of 1.8-2 and increasing tumor cure rate by approximately 20%. In sarcoma tumors, the potentiation of the response by intramuscular mIL-12 gene electrotransfer was dose-dependent and also resulted in a faster onset of tumor cures. Comparison of the carcinoma response to the combined treatment modality in immunocompetent and immunodeficient mice demonstrated that the immune system is needed both for increased cell kill and for attaining tumor cures. CONCLUSIONS.: Based on the comparison of the antitumor effectiveness of electrochemotherapy to intratumoral cisplatin administration, we can conclude that the fraction of cells killed and the tumor cure rate are higher in immunogenic sarcoma tumor compared to moderately immunogenic carcinoma tumor. The tumor cell kill and cure rate depend on the immune response elicited by the destroyed tumor cells, which might depend on the tumor immunogenicity. The effect of adjuvant intramuscular mIL-12 gene electrotransfer is dependent on the amount of IL-12 in the system and the immune competence of the host, as demonstrated by the dose-dependent increase in the cure rate of SA-1 tumors after multiple intramuscular mIL-12 gene electrotransfer and in the differential cure rate of TS/A tumors growing in immunocompetent and immunodeficient mice.

Radiosensitizing Effect of Intratumoral Interleukin-12 Gene Electrotransfer in Murine Sarcoma

BMC Cancer. Jan, 2013  |  Pubmed ID: 23360213

Interleukin-12 (IL-12) based radiosensitization is an effective way of tumor treatment. Local cytokine production, without systemic shedding, might provide clinical benefit in radiation treatment of sarcomas. Therefore, the aim was to stimulate intratumoral IL-12 production by gene electrotransfer of plasmid coding for mouse IL-12 (mIL-12) into the tumors, in order to explore its radiosensitizing effect after single or multiple intratumoral gene electrotransfer.

Tumor Size and Effectiveness of Electrochemotherapy

Radiology and Oncology. Mar, 2013  |  Pubmed ID: 23450195

Electrochemotherapy (ECT) is an effective and safe method for local treatment of tumors. However, relatively large variability in effectiveness of ECT has been observed, which likely results from different treatment conditions and tumor characteristics. The aim of this study was to investigate the relationship between tumor size and effectiveness of a single-session ECT.

Differential Mechanisms Associated with Vascular Disrupting Action of Electrochemotherapy: Intravital Microscopy on the Level of Single Normal and Tumor Blood Vessels

PloS One. 2013  |  Pubmed ID: 23555705

Electropermeabilization/electroporation (EP) provides a tool for the introduction of molecules into cells and tissues. In electrochemotherapy (ECT), cytotoxic drugs are introduced into cells in tumors, and nucleic acids are introduced into cells in gene electrotransfer. The normal and tumor tissue blood flow modifying effects of EP and the vascular disrupting effect of ECT in tumors have already been determined. However, differential effects between normal vs. tumor vessels, to ensure safety in the clinical application of ECT, have not been determined yet. Therefore, the aim of our study was to determine the effects of EP and ECT with bleomycin on the HT-29 human colon carcinoma tumor model and its surrounding blood vessels. The response of blood vessels to EP and ECT was monitored in real time, directly at the single blood vessel level, by in vivo optical imaging in a dorsal window chamber in SCID mice with 70 kDa fluorescently labeled dextrans. The response of tumor blood vessels to EP and ECT started to differ within the first hour. Both therapies induced a vascular lock, decreased functional vascular density (FVD) and increased the diameter of functional blood vessels within the tumor. The effects were more pronounced for ECT, which destroyed the tumor blood vessels within 24 h. Although the vasculature surrounding the tumor was affected by EP and ECT, it remained functional. The study confirms the current model of tumor blood flow modifying effects of EP and provides conclusive evidence that ECT is a vascular disrupting therapy with a specific effect on the tumor blood vessels.

Multiple Delivery of SiRNA Against Endoglin into Murine Mammary Adenocarcinoma Prevents Angiogenesis and Delays Tumor Growth

PloS One. 2013  |  Pubmed ID: 23593103

Endoglin is a transforming growth factor-β (TGF- β) co-receptor that participates in the activation of a signaling pathway that mediates endothelial cell proliferation and migration in angiogenic tumor vasculature. Therefore, silencing of endoglin expression is an attractive approach for antiangiogenic therapy of tumors. The aim of our study was to evaluate the therapeutic potential of small interfering RNA (siRNA) molecules against endoglin in vitro and in vivo. Therapeutic potential in vitro was assessed in human and murine endothelial cells (HMEC-1, 2H11) by determining endoglin expression level, cell proliferation and tube formation. In vivo, the therapeutic potential of siRNA molecules was evaluated in TS/A mammary adenocarcinoma growing in BALB/c mice. Results of our study showed that siRNA molecules against endoglin have a good antiangiogenic therapeutic potential in vitro, as expression of endoglin mRNA and protein levels in mouse and human microvascular endothelial cells after lipofection were efficiently reduced, which resulted in the inhibition of endothelial cell proliferation and tube formation. In vivo, silencing of endoglin with triple electrotransfer of siRNA molecules into TS/A mammary adenocarcinoma also significantly reduced the mRNA levels, number of tumor blood vessels and the growth of tumors. The obtained results demonstrate that silencing of endoglin is a promising antiangiogenic therapy of tumors that could not be used as single treatment, but as an adjunct to the established cytotoxic treatment approaches.

Biological Properties of Melanoma and Endothelial Cells After Plasmid AMEP Gene Electrotransfer Depend on Integrin Quantity on Cells

The Journal of Membrane Biology. Nov, 2013  |  Pubmed ID: 23649038

The data on the biological responsiveness of melanoma and endothelial cells that are targeted by Antiangiogenic MEtargidin Peptide (AMEP) are limited; therefore, the antiproliferative, antimetastatic and antiangiogenic effects of AMEP were investigated in murine melanoma and human endothelial cells after plasmid AMEP gene electrotransfer into the cells in vitro. Plasmid AMEP, a plasmid coding for the disintegrin domain of metargidin targeting specific integrins, had cytotoxic and antiproliferative effects on murine melanoma and human endothelial cells. Among the metastatic properties of cells, migration, invasion and adhesion were investigated. Plasmid AMEP strongly affected the migration of murine melanoma and human endothelial cell lines and also affected the invasion of highly metastatic murine melanoma B16F10 and human endothelial cell lines. There was no effect on cell adhesion on Matrigel(TM) or fibronectin in all cell lines. The antiangiogenic effect was shown with tube formation assay, where human microvascular endothelial cell line (HMEC-1) proved to be more sensitive to plasmid AMEP gene electrotransfer than the human umbilical vein endothelial cell line (HUVEC). The study indicates that antiproliferative and antimetastatic biological responses to gene electrotransfer of plasmid AMEP in murine melanoma cells were dependent on the integrin quantity on melanoma cells and not on the expression level of AMEP. The strong antiangiogenic effect expressed in human endothelial cell lines was only partly dependent on the quantity of integrins and seemed to be plasmid AMEP dose dependent.

Planning of Electroporation-based Treatments Using Web-based Treatment-planning Software

The Journal of Membrane Biology. Nov, 2013  |  Pubmed ID: 23780414

Electroporation-based treatment combining high-voltage electric pulses and poorly permanent cytotoxic drugs, i.e., electrochemotherapy (ECT), is currently used for treating superficial tumor nodules by following standard operating procedures. Besides ECT, another electroporation-based treatment, nonthermal irreversible electroporation (N-TIRE), is also efficient at ablating deep-seated tumors. To perform ECT or N-TIRE of deep-seated tumors, following standard operating procedures is not sufficient and patient-specific treatment planning is required for successful treatment. Treatment planning is required because of the use of individual long-needle electrodes and the diverse shape, size and location of deep-seated tumors. Many institutions that already perform ECT of superficial metastases could benefit from treatment-planning software that would enable the preparation of patient-specific treatment plans. To this end, we have developed a Web-based treatment-planning software for planning electroporation-based treatments that does not require prior engineering knowledge from the user (e.g., the clinician). The software includes algorithms for automatic tissue segmentation and, after segmentation, generation of a 3D model of the tissue. The procedure allows the user to define how the electrodes will be inserted. Finally, electric field distribution is computed, the position of electrodes and the voltage to be applied are optimized using the 3D model and a downloadable treatment plan is made available to the user.

Magnetofection: a Reproducible Method for Gene Delivery to Melanoma Cells

BioMed Research International. 2013  |  Pubmed ID: 23862136

Magnetofection is a nanoparticle-mediated approach for transfection of cells, tissues, and tumors. Specific interest is in using superparamagnetic iron oxide nanoparticles (SPIONs) as delivery system of therapeutic genes. Magnetofection has already been described in some proof-of-principle studies; however, fine tuning of the synthesis of SPIONs is necessary for its broader application. Physicochemical properties of SPIONs, synthesized by the co-precipitation in an alkaline aqueous medium, were tested after varying different parameters of the synthesis procedure. The storage time of iron(II) sulfate salt, the type of purified water, and the synthesis temperature did not affect physicochemical properties of SPIONs. Also, varying the parameters of the synthesis procedure did not influence magnetofection efficacy. However, for the pronounced gene expression encoded by plasmid DNA it was crucial to functionalize poly(acrylic) acid-stabilized SPIONs (SPIONs-PAA) with polyethyleneimine (PEI) without the adjustment of its elementary alkaline pH water solution to the physiological pH. In conclusion, the co-precipitation of iron(II) and iron(III) sulfate salts with subsequent PAA stabilization, PEI functionalization, and plasmid DNA binding is a robust method resulting in a reproducible and efficient magnetofection. To achieve high gene expression is important, however, the pH of PEI water solution for SPIONs-PAA functionalization, which should be in the alkaline range.

Electroporation-based Treatment Planning for Deep-seated Tumors Based on Automatic Liver Segmentation of MRI Images

PloS One. 2013  |  Pubmed ID: 23936315

Electroporation is the phenomenon that occurs when a cell is exposed to a high electric field, which causes transient cell membrane permeabilization. A paramount electroporation-based application is electrochemotherapy, which is performed by delivering high-voltage electric pulses that enable the chemotherapeutic drug to more effectively destroy the tumor cells. Electrochemotherapy can be used for treating deep-seated metastases (e.g. in the liver, bone, brain, soft tissue) using variable-geometry long-needle electrodes. To treat deep-seated tumors, patient-specific treatment planning of the electroporation-based treatment is required. Treatment planning is based on generating a 3D model of the organ and target tissue subject to electroporation (i.e. tumor nodules). The generation of the 3D model is done by segmentation algorithms. We implemented and evaluated three automatic liver segmentation algorithms: region growing, adaptive threshold, and active contours (snakes). The algorithms were optimized using a seven-case dataset manually segmented by the radiologist as a training set, and finally validated using an additional four-case dataset that was previously not included in the optimization dataset. The presented results demonstrate that patient's medical images that were not included in the training set can be successfully segmented using our three algorithms. Besides electroporation-based treatments, these algorithms can be used in applications where automatic liver segmentation is required.

Gene Electrotransfer of Plasmid Antiangiogenic Metargidin Peptide (AMEP) in Disseminated Melanoma: Safety and Efficacy Results of a Phase I First-in-man Study

Human Gene Therapy. Clinical Development. Sep, 2013  |  Pubmed ID: 23980876

Antiangiogenic metargidin peptide (AMEP) is a novel anticancer agent exerting antiproliferative and antiangiogenic effects by binding to αvβ3 and α5β1 integrins. Electrotransfer designates the use of electric pulses (electroporation) to transfer plasmid DNA into tissues. This first-in-man phase I study investigated safety and tolerability of intratumoral plasmid AMEP electrotransfer into cutaneous metastatic melanoma. Secondary objectives were efficacy and pharmacokinetics. Five patients with disseminated melanoma without further treatment options were treated at two dose levels (1 and 2 mg DNA). In each patient, two cutaneous lesions were identified (one treated and one control). At day 1 and day 8, plasmid AMEP was injected intratumorally followed by electrotransfer. Patients were monitored weekly until day 29, and at day 64. Local efficacy was assessed at day 29 by direct measurement, and posttreatment biopsies for AMEP mRNA levels were evaluated by reverse transcriptase quantitative polymerase chain reaction. Plasmid copy number in blood and urine was determined by quantitative polymerase chain reaction. Minimal systemic toxicity was observed, including transient fever and transitory increase in C-reactive protein. No related serious adverse events occurred. Plasmid AMEP was detected in plasma but not in urine. AMEP mRNA was found in three of five treated lesions and none of the control lesions. At day 29, all five treated lesions were stable in diameter, whereas four of five control lesions increased more than 20%. No response occurred in distant lesions. This first-in-man study on electrotransfer of plasmid AMEP into cutaneous melanoma shows that the procedure and drug are safe and that local transfection was obtained.

In Vivo Real-time Monitoring System of Electroporation Mediated Control of Transdermal and Topical Drug Delivery

Journal of Controlled Release : Official Journal of the Controlled Release Society. Dec, 2013  |  Pubmed ID: 24113487

Electroporation (EP) is a physical method for the delivery of molecules into cells and tissues, including the skin. In this study, in order to control the degree of transdermal and topical drug delivery, EP at different amplitudes of electric pulses was evaluated. A new in vivo real-time monitoring system based on fluorescently labeled molecules was developed, for the quantification of transdermal and topical drug delivery. EP of the mouse skin was performed with new non-invasive multi-array electrodes, delivering different amplitudes of electric pulses ranging from 70 to 570 V, between the electrode pin pairs. Patches, soaked with 4 kDa fluorescein-isothiocyanate labeled dextran (FD), doxorubicin (DOX) or fentanyl (FEN), were applied to the skin before and after EP. The new monitoring system was developed based on the delivery of FD to and through the skin. FD relative quantity was determined with fluorescence microscopy imaging, in the treated region of the skin for topical delivery and in a segment of the mouse tail for transdermal delivery. The application of electric pulses for FD delivery resulted in enhanced transdermal delivery. Depending on the amplitude of electric pulses, it increased up to the amplitude of 360 V, and decreased at higher amplitudes (460 and 570 V). Topical delivery steadily enhanced with increasing the amplitude of the delivered electric pulses, being even higher than after tape stripping used as a positive control. The non-invasive monitoring of the delivery of DOX, a fluorescent chemotherapeutic drug, qualitatively and quantitatively confirmed the effects of EP at 360 and 570 V pulse amplitudes on topical and transdermal drug delivery. Delivery of FEN at 360 and 570 V pulse amplitudes verified the observed effects as obtained with FD and DOX, by the measured physiological responses of the mice as well as FEN plasma concentration. This study demonstrates that with the newly developed non-invasive multi-array electrodes and with the varying electric pulse amplitude, the amount of topical and transdermal drug delivery to the skin can be controlled. Furthermore, the newly developed monitoring system provides a tool for rapid real-time determination of both, transdermal and topical delivery, when the delivered molecule is fluorescent.

A Novel Method for Speciation of Pt in Human Serum Incubated with Cisplatin, Oxaliplatin and Carboplatin by Conjoint Liquid Chromatography on Monolithic Disks with UV and ICP-MS Detection

Talanta. Nov, 2013  |  Pubmed ID: 24148385

Conjoint liquid chromatography (CLC) on monolithic convective interaction media (CIM) disks coupled on-line to UV and inductively coupled plasma mass spectrometry (ICP-MS) detectors was used for the first time in speciation analysis of Pt in human serum spiked with Pt-based chemotherapeutics. CIM Protein G and CIM DEAE disks were assembled together in a single housing forming a CLC monolithic column. Such a set-up allows rapid two-dimensional separation by affinity and ion-exchange (IE) modes to be carried out in a single chromatographic run. By applying isocratic elution with Tris-HCl-NaHCO3 buffer (pH 7.4) in the first minute, followed by gradient elution with 1 mol L(-1) NH4Cl (pH 7.4) in the next 9 min, immunoglobulins (IgG) were retained by the Protein G disk enabling subsequent separation of unbound Pt from Pt bound to transferrin (Tf) and albumin (HSA) on the CIM DEAE disk. Further elution with acetic acid (AcOH) in the next 3 min allowed separation of Pt associated with IgG. Separated Pt species were quantified by post-column isotope dilution-ICP-MS. Pt recovery on the CLC column was close to 100%. In comparison to commonly applied procedures that involve separation of protein peaks by size-exclusion chromatography (SEC) followed by IE separation of metal-based chemotherapeutic fractions bound to serum proteins, the CLC method developed is much faster and simpler. Its sensitivity (LOQs adequate for quantification of all separated Pt species, lower than 2.4 ng Pt mL(-1)), good selectivity and method repeatability (RSD±3%) enabled investigation of the kinetics of interaction of Pt-based chemotherapeutics with serum proteins and the distribution of Pt species in spiked human serum. Pt species present in spiked serum were bound preferentially to HSA. The proportion of Pt associated with IgG and Tf was lower than 13%. Cisplatin and especially oxaliplatin react rapidly with serum proteins, while carboplatin much less. The method developed may be reliably applied in preclinical and clinical studies of the kinetics of the interaction and distribution of different metallodrugs with proteins in blood serum.

Determination of Vinblastine in Tumour Tissue with Liquid Chromatography-high Resolution Mass Spectrometry

Talanta. Nov, 2013  |  Pubmed ID: 24148490

There are virtually no analytical methods that describe determination of vinblastine and other vinca alkaloids in tumour tissue, albeit quantitative data on tumour drug amount is essential for maximal benefit of a particular anticancer treatment. The analytical method presented herein uses state-of-the-art sample preparation, separation and detection techniques to allow sensitive and selective determination of vinblastine in tumour tissue. After cryogenic grinding and sonication, tumour suspensions were extracted by Oasis MAX solid phase extraction and analysed for vinblastine with ultra-high performance liquid chromatography coupled to positive electrospray ionisation-high resolution mass spectrometric detection. The developed analytical method quantifies vinblastine down to 23 ng/g tumour tissue and shows satisfactory linearity (r(2)>0.99), precision (1.1-8.2%), accuracy (98%) and high selectivity with almost complete absence of matrix effects. The proposed method was found suitable to follow vinblastine levels in mice tumours and could be used to support preclinical pharmacologic studies.

Evaluation of P21 Promoter for Interleukin 12 Radiation Induced Transcriptional Targeting in a Mouse Tumor Model

Molecular Cancer. Nov, 2013  |  Pubmed ID: 24219565

Radiation induced transcriptional targeting is a gene therapy approach that takes advantage of the targeting abilities of radiotherapy by using radio inducible promoters to spatially and temporally limit the transgene expression. Cyclin dependent kinase inhibitor 1 (CDKN1A), also known as p21, is a crucial regulator of the cell cycle, mediating G1 phase arrest in response to a variety of stress stimuli, including DNA damaging agents like irradiation. The aim of the study was to evaluate the suitability of the p21 promoter for radiation induced transcriptional targeting with the objective to test the therapeutic effectiveness of the combined radio-gene therapy with p21 promoter driven therapeutic gene interleukin 12.

Electrochemotherapy with Intravenous Bleomycin Injection: an Observational Study in Superficial Squamous Cell Carcinoma in Cats

Journal of Feline Medicine and Surgery. Apr, 2014  |  Pubmed ID: 24127456

The aim of this study was to evaluate the efficacy and safety of electrochemotherapy (ECT) with bleomycin for treatment of squamous cell carcinoma (SCC) in cats. Between March 2008 and October 2011, 11 cats with 17 superficial SCC nodules in different clinical stages (ranging from Tis to T4), located on nasal planum (6/11), pinnae (3/11) and both locations (2/11), were included in a prospective non-randomised study. Sixteen of 17 SCC nodules were treated with ECT (15/16 with single session and in one case with two sessions); one nodule was surgically removed. Altogether, complete response (CR) was achieved for 81.8% (9/11) cats and 87.5% (14/16) nodules, lasting from 2 months up to longer than 3 years. Only 2/9 cats in which CR was initially observed, had recurrence 2 and 8 months after the ECT procedure. In the remaining two cats with highly infiltrative spread into adjacent tissues, progression of the disease was observed, despite ECT, and both were euthanased 4 and 5 months after the procedure. ECT in cats was well tolerated and no evident local or systemic side effects were observed. The results of this study suggest that ECT is a highly effective and safe method of local tumour control of feline cutaneous SCCs. It should be considered as an alternative treatment option, especially when other treatment approaches are not acceptable by the owners, owing to their invasiveness, mutilation or high cost.

Changing Electrode Orientation, but Not Pulse Polarity, Increases the Efficacy of Gene Electrotransfer to Tumors in Vivo

Bioelectrochemistry (Amsterdam, Netherlands). Dec, 2014  |  Pubmed ID: 24411306

Gene electrotransfer is an established method for plasmid delivery into different tissues. Contrary to extensive in vitro studies demonstrating increased gene electrotransfer by changing the electric field direction during the pulse delivery, little is known about the efficiency of both polarities pulses in vivo. Therefore the aim of our study was to evaluate the effect of pulse polarity and orientation on the efficacy of gene electrotransfer in the murine fibrosarcoma tumor model by using the luciferase and GFP reporter gene expression plasmids. Our results demonstrated no significant difference in luciferase activity, GFP transfected area or fluorescence intensity between different sets of electric pulses. Inversion of the pulse polarity did not result in the increase of gene transfer, but non-significant enhancement up to 7-fold was detected by changing the electric field orientation in perpendicular direction. Also, transfection of surrounding skin tissue was observed, meaning that intratumoral gene electrotransfer could also result in a systemic effect. Our data indicate that tested modifications of electric pulses are not significantly affecting the efficiency of gene electrotransfer to LPB tumors. Biological factors, like tissue composition, might be of greater importance and should therefore also be taken into account when selecting the electric pulse parameters for specific tissues.

Schedule-dependent Interaction Between Vinblastine and Irradiation in Experimental Sarcoma

Strahlentherapie Und Onkologie : Organ Der Deutschen Rontgengesellschaft ... [et Al]. Jul, 2014  |  Pubmed ID: 24615190

Prolonged vinblastine (VLB) infusion and irradiation (IR) lead to favourable results in certain tumours types; however the underlying biological mechanisms of interaction are not well known. The aim of our study was to evaluate the dose- and time-dependent interactions between split-dose VLB treatment (mimicking prolonged infusion) and IR of sarcoma SA-1 tumours in A/J mice.

Electrochemotherapy: from the Drawing Board into Medical Practice

Biomedical Engineering Online. Mar, 2014  |  Pubmed ID: 24621079

Electrochemotherapy is a local treatment of cancer employing electric pulses to improve transmembrane transfer of cytotoxic drugs. In this paper we discuss electrochemotherapy from the perspective of biomedical engineering and review the steps needed to move such a treatment from initial prototypes into clinical practice. In the paper also basic theory of electrochemotherapy and preclinical studies in vitro and in vivo are briefly reviewed. Following this we present a short review of recent clinical publications and discuss implementation of electrochemotherapy into standard of care for treatment of skin tumors, and use of electrochemotherapy for other targets such as head and neck cancer, deep-seated tumors in the liver and intestinal tract, and brain metastases. Electrodes used in these specific cases are presented with their typical voltage amplitudes used in electrochemotherapy. Finally, key points on what should be investigated in the future are presented and discussed.

Intraoperative Electrochemotherapy of Colorectal Liver Metastases

Journal of Surgical Oncology. Sep, 2014  |  Pubmed ID: 24782355

Electrochemotherapy is effective in treatment of various cutaneous tumors and could be translated into treatment of deep-seated tumors. With this aim a prospective pilot study was conducted to evaluate feasibility, safety, and efficacy of intraoperative electrochemotherapy in the treatment of colorectal liver metastases.

A Cell-based Biosensor System HepG2CDKN1A-DsRed for Rapid and Simple Detection of Genotoxic Agents

Biosensors & Bioelectronics. Nov, 2014  |  Pubmed ID: 24861570

The regulatory requirements for genotoxicity testing rely on a battery of genotoxicity tests, which generally consist of bacterial and mammalian cell assays for detection of gene mutations and chromosomal aberrations. However, for rapid screening, these methods are not appropriate. We have developed a new cell-based biosensor system that provides rapid and simple detection of genotoxic substances. This is based on stable transfection of human hepatoma HepG2 cells with a plasmid that encodes the red fluorescent protein DsRed2 under the control of the CDKN1A promoter (HepG2CDKN1A-DsRed cells). As the major downstream target gene of activated TP53, the tumour-suppressor gene CDKN1A is responsible for cell-cycle arrest following DNA damage, and it has been shown to be specifically up-regulated by genotoxic carcinogens. The assay is optimised for a 96-well microplate format and spectrofluorimetric quantification of induced DsRed expression. The assay was evaluated by testing direct-acting and indirect-acting genotoxic compounds with different mechanisms of action, along with non-genotoxic compounds. Out of 25 compounds that are known to be genotoxic in vitro and in vivo, 21 (84%) are detected as positive at non-cytotoxic doses, whereas of 12 compounds not considered genotoxic, 11 (92%) are negative. These data indicate the high sensitivity and specificity of our biosensor system. Based on its simplicity and sensitivity, this biosensor developed with HepG2CDKN1A-DsRed cells has the potential to become a valuable tool for genotoxicity screening for chemical safety evaluation, as well as for environmental and occupational monitoring of exposure to genotoxic agents and their complex mixtures.

Electroporation-based Technologies for Medicine: Principles, Applications, and Challenges

Annual Review of Biomedical Engineering. Jul, 2014  |  Pubmed ID: 24905876

When high-amplitude, short-duration pulsed electric fields are applied to cells and tissues, the permeability of the cell membranes and tissue is increased. This increase in permeability is currently explained by the temporary appearance of aqueous pores within the cell membrane, a phenomenon termed electroporation. During the past four decades, advances in fundamental and experimental electroporation research have allowed for the translation of electroporation-based technologies to the clinic. In this review, we describe the theory and current applications of electroporation in medicine and then discuss current challenges in electroporation research and barriers to a more extensive spread of these clinical applications.

Modulation of Activity of Known Cytotoxic Ruthenium(III) Compound (KP418) with Hampered Transmembrane Transport in Electrochemotherapy in Vitro and in Vivo

The Journal of Membrane Biology. Dec, 2014  |  Pubmed ID: 24957014

To increase electrochemotherapy (ECT) applicability, the effectiveness of new drugs is being tested in combination with electroporation. Among them two ruthenium(III) compounds, (imH)[trans-RuCl4(im)(DMSO-S)] (NAMI-A) and Na[trans-RuCl4(ind)2] (KP1339), proved to possess increased antitumor effectiveness when combined with electroporation. The objective of our experimental work was to determine influence of electroporation on the cytotoxic and antitumor effect of a ruthenium(III) compound with hampered transmembrane transport, (imH)[trans-RuCl4(im)2] (KP418) in vitro and in vivo and to determine changes in metastatic potential of cells after ECT with KP418 in vitro. In addition, platinum compound cisplatin (CDDP) and ruthenium(III) compound NAMI-A were included in the experiments as reference compounds. Our results show that electroporation leads to increased cellular accumulation and cytotoxicity of KP418 in murine melanoma cell lines with low and high metastatic potential, B16-F1 and B16-F10, but not in murine fibrosarcoma cell line SA-1 in vitro which is probably due to variable effectiveness of ECT in different cell lines and tumors. Electroporation does not potentiate the cytotoxicity of KP418 as prominently as the cytotoxicity of CDDP. We also showed that the metastatic potential of cells which survived ECT with KP418 or NAMI-A does not change in vitro: resistance to detachment, invasiveness, and re-adhesion of cells after ECT is not affected. Experiments in murine tumor models B16-F1 and SA-1 showed that ECT with KP418 does not have any antitumor effect while ECT with CDDP induces significant dose-dependent tumor growth delay in the two tumor models used in vivo.

Electrochemotherapy in Non-melanoma Head and Neck Cancers: a Retrospective Analysis of the Treated Cases

The British Journal of Oral & Maxillofacial Surgery. Dec, 2014  |  Pubmed ID: 25183266

Electrochemotherapy increases the permeability of tumours to drugs by electric voltages applied locally. Its value in tumours of the head and neck is unknown. We retrospectively reviewed a 2-centre database, and found 39 patients with squamous cell carcinoma (SCC) of the oral cavity or oropharynx (n=12) or non-melanoma skin tumours (n=27) who had been treated with bleomycin electrochemotherapy with needle electrodes. A further 3 patients were given cisplatin electrochemotherapy (n=2), or bleomycin electrochemotherapy by plate electrodes (n=1). Local toxicity was mild. The complete response rate was 38% and was associated with whether the tumour was primary or recurrent (p<0.001), its size (p=0.02), and the route by which the drug was given (p=0.02). We did not study enough patients with basal cell carcinomas to say whether the response was significantly better or not (p=0.07). Skin tumours and SCC of the oral cavity or oropharynx showed comparable complete responses (41% and 33%, p=0.73) and local control (1-year local progression-free survival, 51% compared with 59%, p=0.89), particularly if they were small (p=0.001), primary (p=0.002), chemonaive (p=0.03). Patients treated with cisplatin were unresponsive. Electrochemotherapy with bleomycin is an effective option for skin tumours of the head and neck and is a feasible alternative in highly selected (small, primary, and not previously treated by chemotherapy) SCC of the oral cavity and oropharynx.

Mcam Silencing With RNA Interference Using Magnetofection Has Antitumor Effect in Murine Melanoma

Molecular Therapy. Nucleic Acids. Oct, 2014  |  Pubmed ID: 25350580

The melanoma cell adhesion molecule (MCAM) is involved in melanoma development and its progression, including invasiveness, metastatic potential and angiogenesis. Therefore, MCAM represents a potential target for gene therapy of melanoma, whose expression could be hindered with posttranscriptional specific gene silencing with RNA interference technology. In this study, we constructed a plasmid DNA encoding short hairpin RNA against MCAM (pMCAM) to explore the antitumor and antiangiogenic effects. The experiments were performed in vitro on murine melanoma and endothelial cells, as well as in vivo on melanoma tumors in mice. The antiproliferative, antimigratory, antiangiogenic and antitumor effects were examined after gene therapy with pMCAM. Gene delivery was performed by magnetofection, and its efficacy compared to gene electrotransfer. Gene therapy with pMCAM has proved to be an effective approach in reducing the proliferation and migration of melanoma cells, as well as having antiangiogenic effect in endothelial cells and antitumor effect on melanoma tumors. Magnetofection as a developing nonviral gene delivery system was effective in the transfection of melanoma cells and tumors with pMCAM, but less efficient than gene electrotransfer in in vivo tumor gene therapy due to the lack of antiangiogenic effect after silencing Mcam by magnetofection.

Inhibitor of Endocytosis Impairs Gene Electrotransfer to Mouse Muscle in Vivo

Bioelectrochemistry (Amsterdam, Netherlands). Jun, 2015  |  Pubmed ID: 25200990

Application of electric pulses (electroporation/electropermeabilization) is an effective method for gene transfer (i.e. gene electrotransfer (GET)) in vitro and in vivo. Currently, the mechanisms by which the DNA enters the cell are not yet fully understood. Experimental evidence is building up that endocytosis is the main mechanism by which the DNA, which is later expressed, enters the cell. Therefore the aim of our study was to elucidate whether inhibitors of endocytosis, methyl-β-cyclodextrin (MβCD), Concanavalin A (ConA) and Dynasore, can impair the transfection efficacy of GET in vitro in B16F1 murine melanoma and in vivo in m. tibialis cranialis in mice. We show that MβCD--general inhibitor of endocytosis--can almost prevent GET of EGFP-N1 plasmid in vitro, that ConA--inhibitor of clathrin mediated endocytosis--also abrogates GET but to a lesser extent, and when using Dynasore--reversible inhibitor of dynamin--there is no effect on GET efficacy, if endocytosis is blocked for only 5 min after GET. Moreover, MβCD also reduced GET efficacy in vivo in m. tibialis cranialis and this effect was long lasting. The results of this study show that endocytosis is probably the main mechanism of entrance of DNA after GET in vitro and also in vivo.

Endoglin Silencing Has Significant Antitumor Effect on Murine Mammary Adenocarcinoma Mediated by Vascular Targeted Effect

Current Gene Therapy. 2015  |  Pubmed ID: 25619888

New targets and therapeutic approaches for vascular targeted strategies in oncology are continuously explored. Endoglin, a co-receptor of TGF-β, 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.

Adjuvant TNF-α Therapy to Electrochemotherapy with Intravenous Cisplatin in Murine Sarcoma Exerts Synergistic Antitumor Effectiveness

Radiology and Oncology. Mar, 2015  |  Pubmed ID: 25810699

Electrochemotherapy is a tumour ablation modality, based on electroporation of the cell membrane, allowing non-permeant anticancer drugs to enter the cell, thus augmenting their cytotoxicity by orders of magnitude. In preclinical studies, bleomycin and cisplatin proved to be the most suitable for clinical use. Intravenous administration of cisplatin for electrochemotherapy is still not widely accepted in the clinics, presumably due to its lower antitumor effectiveness, but adjuvant therapy by immunomodulatory or vascular-targeting agents could provide a way for its potentiation. Hence, the aim of the present study was to explore the possibility of adjuvant tumour necrosis factor α (TNF-α) therapy to potentiate antitumor effectiveness of electrochemotherapy with intravenous cisplatin administration in murine sarcoma.

Improved Specificity of Gene Electrotransfer to Skin Using PDNA Under the Control of Collagen Tissue-Specific Promoter

The Journal of Membrane Biology. Oct, 2015  |  Pubmed ID: 25840832

In order to ensure safe, efficient and controlled gene delivery to skin, the improvement of delivery methods together with proper design of DNA is required. Non-viral delivery methods, such as gene electrotransfer, and the design of tissue-specific promoters are promising tools to ensure the safety of gene delivery to the skin. In the scope of our study, we evaluated a novel skin-specific plasmid DNA with collagen (COL) promoter, delivered to skin cells and skin tissue by gene electrotransfer. In vitro, we determined the specificity of the COL promoter in fibroblast cells. The specific expression under the control of COL promoter was obtained for the reporter gene DsRed as well as for therapeutic gene encoding cytokine IL-12. In vivo, the plasmid with COL promoter encoding the reporter gene DsRed was efficiently transfected to mouse skin. It resulted in the notable and controlled manner, however, in lower and shorter expression, compared to that obtained with ubiquitous promoter. The concentration of the IL-12 in the skin after the in vivo transfection of plasmid with COL promoter was in the same range as after the treatment in control conditions (injection of distilled water followed by the application of electric pulses). Furthermore, this gene delivery was local, restricted to the skin, without any evident systemic shedding of IL-12. Such specific targeting of skin cells, observed with tissue-specific COL promoter, would improve the effectiveness and safety of cutaneous gene therapies and DNA vaccines.

Gene Electrotransfer of Canine Interleukin 12 into Canine Melanoma Cell Lines

The Journal of Membrane Biology. Oct, 2015  |  Pubmed ID: 25840833

A gene electrotransfer (GET) of interleukin 12 (IL-12) had already given good results when treating tumors in human and veterinary clinical trials. So far, plasmids used in veterinary clinical studies encoded a human or a feline IL-12 and an ampicillin resistance gene, which is not recommended by the regulatory agencies to be used in clinical trials. Therefore, the aim of the current study was to construct the plasmid encoding a canine IL-12 with kanamycin antibiotic resistance gene that could be used in veterinary clinical oncology. The validation of the newly constructed plasmid was carried out on canine malignant melanoma cells, which have not been used in GET studies so far, and on human malignant melanoma cells. Canine and human malignant melanoma cell lines were transfected with plasmid encoding enhanced green fluorescence protein at different pulse parameter conditions to determine the transfection efficiency and cell survival. The IL-12 expression of the most suitable conditions for GET of the plasmid encoding canine IL-12 was determined at mRNA level by the qRT-PCR and at protein level with the ELISpot assay. The obtained results showed that the newly constructed plasmid encoding canine IL-12 had similar or even higher expression capacity than the plasmid encoding human IL-12. Therefore, it represents a promising therapeutic plasmid for further IL-12 gene therapy in clinical studies for spontaneous canine tumors. Additionally, it also meets the main regulatory agencies' (FDA and EMA) criteria.

Nitroxoline Impairs Tumor Progression in Vitro and in Vivo by Regulating Cathepsin B Activity

Oncotarget. Aug, 2015  |  Pubmed ID: 25848918

Cathepsin B is a ubiquitously expressed lysosomal cysteine protease that participates in protein turnover within lysosomes. However, its protein and activity levels have been shown to be increased in cancer. Cathepsin B endopeptidase activity is involved in the degradation of extracellular matrix, a process that promotes tumor invasion, metastasis and angiogenesis. Previously, we reported an established antibiotic nitroxoline as a potent and selective inhibitor of cathepsin B. In the present study, we elucidated its anti-tumor properties in in vitro and in vivo tumor models. Tumor and endothelial cell lines with high levels of active cathepsin B were selected for functional analysis of nitroxoline in vitro. Nitroxoline significantly reduced extracellular DQ-collagen IV degradation by all evaluated cancer cell lines using spectrofluorimetry. Nitroxoline also markedly decreased tumor cell invasion monitored in real time and reduced the invasive growth of multicellular tumor spheroids, used as a 3D in vitro model of tumor invasion. Additionally, endothelial tube formation was significantly reduced by nitroxoline in an in vitro angiogenesis assay. Finally, nitroxoline significantly abrogated tumor growth, angiogenesis and metastasis in vivo in LPB fibrosarcoma and MMTV-PyMT breast cancer mouse models. Overall, our results designate nitroxoline as a promising drug candidate for anti-cancer treatment.

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

PloS One. 2015  |  Pubmed ID: 25909447

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.

Endoglin (CD105) Silencing Mediated by ShRNA Under the Control of Endothelin-1 Promoter for Targeted Gene Therapy of Melanoma

Molecular Therapy. Nucleic Acids. May, 2015  |  Pubmed ID: 25942402

Endoglin (CD105), a transforming growth factor (TGF)-β 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.

Electrochemotherapy of Tumors As in Situ Vaccination Boosted by Immunogene Electrotransfer

Cancer Immunology, Immunotherapy : CII. Oct, 2015  |  Pubmed ID: 26067277

Electroporation is a platform technology for drug and gene delivery. When applied to cell in vitro or tissues in vivo, it leads to an increase in membrane permeability for molecules which otherwise cannot enter the cell (e.g., siRNA, plasmid DNA, and some chemotherapeutic drugs). The therapeutic effectiveness of delivered chemotherapeutics or nucleic acids depends greatly on their successful and efficient delivery to the target tissue. Therefore, the understanding of different principles of drug and gene delivery is necessary and needs to be taken into account according to the specificity of their delivery to tumors and/or normal tissues. Based on the current knowledge, electrochemotherapy (a combination of drug and electric pulses) is used for tumor treatment and has shown great potential. Its local effectiveness is up to 80 % of local tumor control, however, without noticeable effect on metastases. In an attempt to increase systemic antitumor effectiveness of electrochemotherapy, electrotransfer of genes with immunomodulatory effect (immunogene electrotransfer) could be used as adjuvant treatment. Since electrochemotherapy can induce immunogenic cell death, adjuvant immunogene electrotransfer to peritumoral tissue could lead to locoregional effect as well as the abscopal effect on distant untreated metastases. Therefore, we propose a combination of electrochemotherapy with peritumoral IL-12 electrotransfer, as a proof of principle, using electrochemotherapy boosted with immunogene electrotransfer as in situ vaccination for successful tumor treatment.

Coupling Treatment Planning with Navigation System: a New Technological Approach in Treatment of Head and Neck Tumors by Electrochemotherapy

Biomedical Engineering Online. 2015  |  Pubmed ID: 26355773

Electrochemotherapy provides highly effective local treatment for a variety of tumors. In deep-seated tumors of the head and neck, due to complex anatomy of the region or inability to cover the whole tumor with standard electrodes, the use of long single needle electrodes is mandatory. In such cases, a treatment plan provides the information on the optimal configuration of the electrodes to adequately cover the tumor with electric field, while the accurate placement of the electrodes in the surgical room in patients can remain a problem. Therefore, during electrochemotherapy of two head and neck lymph-node metastases of squamous cell carcinoma origin, a navigation system for placement of electrodes was used.

Electrochemotherapy of Colorectal Liver Metastases--an Observational Study of Its Effects on the Electrocardiogram

Biomedical Engineering Online. 2015  |  Pubmed ID: 26356120

Electrochemotherapy (ECT) is a combined treatment in which high voltage electroporation (EP) pulses are used to facilitate the uptake of a chemotherapeutic drug into tumor cells, thus increasing antitumor effectiveness of the drug. The effect of ECT of deep-seated tumors located close to the heart on functioning of the heart has not been previously investigated. In this study, we investigate the effects of intra-abdominal ECT of colorectal liver metastases on functioning of the heart during the early post-operative care period.

Variation in Dielectric Properties Due to Pathological Changes in Human Liver

Bioelectromagnetics. Dec, 2015  |  Pubmed ID: 26508012

Dielectric properties of freshly excised human liver tissues (in vitro) with several pathological conditions including cancer were obtained in frequency range 100 MHz-5 GHz. Differences in dielectric behavior of normal and pathological tissues at microwave frequencies are discussed based on histological information for each tissue. Data presented are useful for many medical applications, in particular nanosecond pulsed electroporation techniques. Knowledge of dielectric properties is vital for mathematical calculations of local electric field distribution inside electroporated tissues and can be used to optimize the process of electroporation for treatment planning procedures.

Electrotransfer of Plasmid DNA Encoding an Anti-Mouse Endoglin (CD105) ShRNA to B16 Melanoma Tumors with Low and High Metastatic Potential Results in Pronounced Anti-Tumor Effects

Cancers. Dec, 2015  |  Pubmed ID: 26712792

Endoglin overexpression is associated with highly proliferative tumor endothelium and also with some tumors, including melanoma. Its targeting has anti-tumor effectiveness, which can also be obtained by RNA interference. The aim of our study was to explore the anti-tumor effectiveness of endoglin silencing by electrotransfer of plasmid DNA encoding short hairpin RNA against endoglin in two murine B16 melanoma variants with different metastatic potential on cells, spheroids and subcutaneous tumors in mice. The results demonstrate that endoglin silencing with gene electrotransfer reduces the proliferation, survival and migration of melanoma cells and also has anti-tumor effectiveness, as the therapy resulted in a high percentage of tumor cures (23% and 58% on B16F1 and B16F10 tumors, respectively). The effectiveness of the therapy correlated with endoglin expression in melanoma cells; in vitro the effects were more pronounced in B16F1 cells, which express more endoglin than B16F10. However, the opposite was observed in vivo in tumors, where there was a higher expression of endoglin and better anti-tumor effectiveness in the B16F10 tumor. In conclusion, targeting endoglin for the treatment of melanoma seems to be a concept worthy of further exploration due to the increased therapeutic effect of the therapy based on simultaneous vascular targeting and its direct effect on tumor cells.

Clinical Potential of Electroporation for Gene Therapy and DNA Vaccine Delivery

Expert Opinion on Drug Delivery. 2016  |  Pubmed ID: 26578324

Electroporation allows efficient delivery of DNA into cells and tissues, thereby improving the expression of therapeutic or immunogenic proteins that are encoded by plasmid DNA. This simple and versatile method holds a great potential and could address unmet medical needs such as the prevention or treatment of many cancers or infectious diseases.

Radioactivity of Cigarettes and the Importance of (210)Po and Thorium Isotopes for Radiation Dose Assessment Due to Smoking

Journal of Environmental Radioactivity. May, 2016  |  Pubmed ID: 26942842

Tobacco and tobacco smoke are very complex mixtures. In addition to various chemical and organic compounds they also contain natural radioactive elements (radionuclides). In this work, the natural radionuclide activity concentrations ((234)U, (238)U, (228)Th, (230)Th, (232)Th, (226)Ra, (210)Pb and (210)Po) of nine different cigarette samples available on the Slovenian market are reported. In addition to (210)Po, the transfer of thorium isotopes from a cigarette to a smoker's body and lungs have been determined for the first time. Cigarette smoke and exhaled air from smokers' lungs were collected from volunteer smokers (C-4 brand) to determinate what quantity of (210)Po and thorium isotopes is transferred from the tobacco to the smoker's lungs. Cigarette ash and smoked filters were also collected and analysed. Among the determined isotopes, (210)Pb and (210)Po showed the highest activity concentrations. During the smoking of one cigarette approximately 22% of (210)Po (and presumably its predecessor (210)Pb), 0.6% of (228)Th, 24% of (230)Th, and 31% of (232)Th are transferred from the cigarette and retained in the smoker's body. The estimated annual effective dose for smokers is 61 μSv/year from (210)Po; 9 μSv/year from (210)Pb; 6 μSv/year from (228)Th; 47 μSv/year from (230)Th, and 37 μSv/year from (232)Th. These results show the importance of thorium isotopes in contributing to the annual effective dose for smoking.

Bleomycin Pharmacokinetics of Bolus Bleomycin Dose in Elderly Cancer Patients Treated with Electrochemotherapy

Cancer Chemotherapy and Pharmacology. May, 2016  |  Pubmed ID: 26992379

With the aim to determine effective therapeutic window of electrochemotherapy, we analyzed bleomycin pharmacokinetic parameters in elderly patients.

Magnetic Field Contributes to the Cellular Uptake for Effective Therapy with Magnetofection Using Plasmid DNA Encoding Against Mcam in B16F10 Melanoma in Vivo

Nanomedicine (London, England). Mar, 2016  |  Pubmed ID: 27021639

We explored the distribution and cellular uptake of intratumorally injected SPIONs-PAA-PEI-pDNA (magnetofection complexes), and antitumor effectiveness of magnetofection with plasmid DNA encoding short hairpin RNA (shRNA) against Mcam (pDNA(anti-MCAM)).

Recommendations for Improving the Quality of Reporting Clinical Electrochemotherapy Studies Based on Qualitative Systematic Review

Radiology and Oncology. Mar, 2016  |  Pubmed ID: 27069444

Electrochemotherapy is becoming a well-established treatment for malignancies of skin and non-skin origin and its use is widening across Europe. The technique was developed and optimized from solid experimental and clinical evidence. A consensus document is now warranted to formalize reporting results, which should strengthen evidence-based practice recommendations. This consensus should be derived from high quality clinical data collection, clinical expertise and summarizing patient feedback. The first step, which is addressed in this paper, aims to critically analyze the quality of published studies and to provide the recommendations for reporting clinical trials on electrochemotherapy.

Effectiveness of Electrochemotherapy After IFN-α Adjuvant Therapy of Melanoma Patients

Radiology and Oncology. Mar, 2016  |  Pubmed ID: 27069446

The combination of electrochemotherapy with immuno-modulatory treatments has already been explored and proven effective. However, the role of interferon alpha (IFN-α) adjuvant therapy of melanoma patients and implication on electrochemotherapy effectiveness has not been explored yet. Therefore, the aim of the study was to retrospectively evaluate the effectiveness and safety of electrochemotherapy after the previous adjuvant treatment with IFN-α in melanoma patients.

Electrochemotherapy by Pulsed Electromagnetic Field Treatment (PEMF) in Mouse Melanoma B16F10 in Vivo

Radiology and Oncology. Mar, 2016  |  Pubmed ID: 27069448

Pulsed electromagnetic field (PEMF) induces pulsed electric field, which presumably increases membrane permeabilization of the exposed cells, similar to the conventional electroporation. Thus, contactless PEMF could represent a promising approach for drug delivery.

Cytotoxic Trans-platinum(II) Complex with 3-hydroxymethylpyridine: Synthesis, X-ray Structure and Biological Activity Evaluation

Journal of Inorganic Biochemistry. Aug, 2016  |  Pubmed ID: 27189143

To assess the potential cytostatic properties of Pt(II) complexes with 3-hydroxymethylpyridine (3-hmpy) as the only carrier ligand, novel cis-[PtCl2(3-hmpy)2] (1) and trans-[PtCl2(3-hmpy)2] (2) have been prepared. Elemental analysis, FTIR spectroscopy, multinuclear NMR spectroscopy and X-ray crystallography were used to determine their structures. Based on the results obtained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and clonogenic assay on T24 human bladder carcinoma cells (T24), the most potent compound 2 was further tested for cytotoxicity in human ovarian carcinoma cell lines - cisplatin sensitive (IGROV 1) and its resistant subclone (IGROV 1/RDDP). The cytotoxicity of compound 2 in IGROV 1/RDDP is comparable to cisplatin. Furthermore, compound 2 induced severe conformational changes in plasmid DNA, which resulted in a delayed onset of apoptosis in T24 cells, and higher amounts of Pt in tumours and serum compared to cisplatin. In addition, in vivo antitumour effectiveness was comparable to that of cisplatin with a smaller reduction of animals' body weight, thus demonstrating that it is a promising transplatin analogue which deserves further studies.

European Research on Electrochemotherapy in Head and Neck Cancer (EURECA) Project: Results of the Treatment of Skin Cancer

European Journal of Cancer (Oxford, England : 1990). Aug, 2016  |  Pubmed ID: 27267144

Electrochemotherapy is an effective and safe method for local treatment of cutaneous and subcutaneous tumours, where electric pulses cause increased permeability of cell membranes in the tumour mass, enabling dramatically enhanced effectiveness of bleomycin and other hydrophilic drugs. Here, we report results of a European multi-institutional prospective study of the effectiveness of electrochemotherapy in the treatment of skin cancer of the head and neck (HN) area, where standard treatments had either failed or were not deemed suitable or declined by the patient. A total of 105 patients affected by primary or recurrent skin cancer of the HN area were enrolled; of these, 99 were eligible for evaluation of tumour response. By far, the majority (82%) were treated only once, and 18% of patients had a second treatment. The objective response was highest for basal cell carcinoma (97%) and for other histologies was 74%. Small, primary, and treatment-naive carcinomas responded significantly better (p < 0.05), as investigated by univariate analysis. Electrochemotherapy was well tolerated and led to a significant improvement of quality of life, estimated by the European Organisation for Research and Treatment of Cancer quality of life questionnaires. At 1-year follow-up, the percentages of overall and disease-free survival were 76% and 89%, respectively. Electrochemotherapy is an effective option for skin cancers of the HN area and can be considered a feasible alternative to standard treatments when such an alternative is appropriate. The precise role for electrochemotherapy in the treatment algorithm for non-melanoma skin cancer of the HN region requires data from future randomised controlled studies. (ISRCTN registry N. 30427).

Bystander Effect Induced by Electroporation is Possibly Mediated by Microvesicles and Dependent on Pulse Amplitude, Repetition Frequency and Cell Type

The Journal of Membrane Biology. Oct, 2016  |  Pubmed ID: 27371159

Bystander effect, a known phenomenon in radiation biology, where irradiated cells release signals which cause damage to nearby, unirradiated cells, has not been explored in electroporated cells yet. Therefore, our aim was to determine whether bystander effect is present in electroporated melanoma cells in vitro, by determining viability of non-electroporated cells exposed to medium from electroporated cells and by the release of microvesicles as potential indicators of the bystander effect. Here, we demonstrated that electroporation of cells induces bystander effect: Cells exposed to electric pulses mediated their damage to the non-electroporated cells, thus decreasing cell viability. We have shown that shedding microvesicles may be one of the ways used by the cells to mediate the death signals to the neighboring cells. The murine melanoma B16F1 cell line was found to be more electrosensitive and thus more prone to bystander effect than the canine melanoma CMeC-1 cell line. In B16F1 cell line, bystander effect was present above the level of electropermeabilization of the cells, with the threshold at 800 V/cm. Furthermore, with increasing electric field intensities and the number of pulses, the bystander effect also increased. In conclusion, electroporation can induce bystander effect which may be mediated by microvesicles, and depends on pulse amplitude, repetition frequency and cell type.

Electrotransfer Parameters As a Tool for Controlled and Targeted Gene Expression in Skin

Molecular Therapy. Nucleic Acids. Aug, 2016  |  Pubmed ID: 27574782

Skin is an attractive target for gene electrotransfer. It consists of different cell types that can be transfected, leading to various responses to gene electrotransfer. We demonstrate that these responses could be controlled by selecting the appropriate electrotransfer parameters. Specifically, the application of low or high electric pulses, applied by multi-electrode array, provided the possibility to control the depth of the transfection in the skin, the duration and the level of gene expression, as well as the local or systemic distribution of the transgene. The influence of electric pulse type was first studied using a plasmid encoding a reporter gene (DsRed). Then, plasmids encoding therapeutic genes (IL-12, shRNA against endoglin, shRNA against melanoma cell adhesion molecule) were used, and their effects on wound healing and cutaneous B16F10 melanoma tumors were investigated. The high-voltage pulses resulted in gene expression that was restricted to superficial skin layers and induced a local response. In contrast, the low-voltage electric pulses promoted transfection into the deeper skin layers, resulting in prolonged gene expression and higher transgene production, possibly with systemic distribution. Therefore, in the translation into the clinics, it will be of the utmost importance to adjust the electrotransfer parameters for different therapeutic approaches and specific mode of action of the therapeutic gene.

Identification and Quantification of Bleomycin in Serum and Tumor Tissue by Liquid Chromatography Coupled to High Resolution Mass Spectrometry

Talanta. Nov, 2016  |  Pubmed ID: 27591601

Bleomycin is a cytotoxic antibiotic available as a compost of structurally strongly related glycopeptides, which is in vivo found chelated with several metals. Its pharmacotherapy has merely been based on experimental dose - response data, whereas its biodistribution and pharmacokinetics remain fundamentally unknown. This is reasoned by an absence of a specific and sensitive mass spectrometry-based analytical method for its determination in biological tissues. We herein reveal the results of our study on the mass spectrometric behavior of two main bleomycin fractions A2 and B2, including their metal complexes, particularly the predominant copper chelates. In the electrospray ion source bleomycin forms double charged species, where for the metal-free fraction A2 and its copper complex m/z 707.76 and m/z 707.21 are seen, respectively. Hence, the second isotopic ion of the chelate (m/z 707.71) nearly coincides with the first isotopic ion of the metal-free fraction. This phenomenon can only be followed by high-resolution mass spectrometry, and is considered the plausible reason, why the attempts to determine bleomycin with mass spectrometry have been so scarce. The presented paper further describes a sensitive and selective liquid chromatography - mass spectrometry analytical method for determination of bleomycin in serum and tumor tissues. This newly developed method was employed for bleomycin pharmacokinetic studies in serum and tumors of laboratory animals. Additionally, the method was employed for determination of bleomycin pharmacokinetic parameters in elderly patients in order to determine the effective therapeutic window of electrochemotherapy with bleomycin.

Electrochemotherapy with Bleomycin is Effective in BRAF Mutated Melanoma Cells and Interacts with BRAF Inhibitors

Radiology and Oncology. Sep, 2016  |  Pubmed ID: 27679543

The aim of the study was to explore the effectiveness of electrochemotherapy (ECT) during the treatment of melanoma patients with BRAF inhibitors. Its effectiveness was tested on BRAF mutated and non-mutated melanoma cells in vitro and in combination with BRAF inhibitors.

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