The Regulation of Plasmid DNA Expression by Using The Cytototix Effect of Bleomycin After Simultaneous Electrotransfer of Both Molecules

Elektroporacija yra fizikinis metodas, kurio metu taikomi trumpi ir intensyvūs elektriniai impulsai, sukeliantys laikiną ląstelės plazminės membranos pralaidumą natūraliai pro ją nepraeinančioms molekulėms. Šis klinikinis metodas yra taikomas priešvėžinio gydymo metu, siekiant palengvinti priešvėžinių vaistų (bleomicino) patekimą į naviko ląsteles. Toks elektroporacijos ir priešvėžinio vaisto derinio taikymas pavadintas elektrochemoterapija (ECT). Šis lokalus ECT metodas iki šiol yra optimizuojamas, pavyzdžiui, naudojant vienalaikę priešvėžinio vaisto ir genų elektropernašą. Tokiu būdu galima gauti terapinį (priešvėžinį) gydymo poveikį kartu sukeliant pernešto geno ekspresiją. Dėl šios priežasties, darbe buvo siekta ištirti plazmidinės DNR ekspresijos reguliavimą naudojant bleomicino sukeltą citotoksiškumo efektą po vienalaikės abiejų molekulių elektropernašos. Remiantis gautais rezultatais, matoma, kad terpėje esant plazmidinei DNR ir priešvėžiniam vaistui bleomicinui, galima vienalaikė abiejų molekulių elektropernaša. Nustatyta, kad plazmidinės DNR buvimas terpėje gali padidinti bleomicino elektropernašą, taip padidinant priešvėžinio vaisto citotoksiškumą. Taip pat, buvo nustatyta, kad bleomicino buvimas terpėje kartu su skirtingo dydžio plazmidinėmis DNR, gali pakeisti genų ekspresijos laiką ir intensyvumą. Gauti rezultatai gali būti pritaikyti keičiant elektrochemoterapijos efektyvumą (vienalaikė bleomicino ir genų pernaša), iš lokalaus į sisteminį atsaką.Electroporation is a physical method where short and intense electrical pulses are applied thus causing the temporal permeability of the cellular membrane to exogenus molecules. This clinical method is used in the treatment of cancer to facilitate the entry of anticancer drugs (bleomycin) into tumor cells. This application of a combination of electroporation and an anticancer drug is called electrochemotherapy (ECT). Such local, ECT approach so far has been optimized by using simultaneous anti-cancer drug and gene electrotransfer. In this way, a therapeutic (anti-cancer) effect of the treatment can be obtained by simultaneously inducing the expression of the transferred gene. For this reason, the aim of this work was to determine the regulation of plasmid DNA expression using bleomycin-induced cytotoxic effect after simultaneous electrotransfer of both molecules. Based on the results obtained, it can be seen that the presence of plasmid DNA and the anticancer drug bleomycin in the medium allows simultaneous electransfer of both molecules. It has been found that the presence of plasmid DNA in the medium can increase the electrotransfer permeability of bleomycin, thereby increasing the cytotoxicity of the anticancer drug. Also, it has been found that the presence of bleomycin in the medium together with plasmid DNA of different sizes can alter the time and intensity of gene expression. The obtained results can be applied by changing the efficiency of electrochemotherapy (simultaneous bleomycin and gene transfer), from local to systemic response.Gamtos mokslų fakultetasBiologijos katedr

In vitro analysis of the possibility to simultaneously transfer bleomycin and plasmid DNA in cells via electroporation

Electroporation is a process when as a result of electric fields effect induced transmembrane potential create transient pores in the plasma membrane, leading to a large increase in drug transport, delivery of macromolecules, a quick drug effect onset. Hence, anticancer drugs can be easily transported into targeted tumour cells by using this technique. Nowadays, electrochemotherapy is used in clinics for the cancer treatment. Electrochemotherapy is a process, when electroporation is combined with the injection of cytotoxic drugs (e.g. bleomycin (BLM). A single molecule of bleomycin can cause around 8–10 DNA breaks causing the cell death, which explains its high cytotoxicity when present inside the cell. However, it is only a local response. In order to initiate a systemic response rate, one has to induce immune response. One way to do it is to transfect a gene that express a compound that enhance immune response. In this study, we show the possibility to simultaneously transfer bleomycin and plasmid DNA in cells via electroporation. Chinese hamster ovary (CHO) cell line was used for experiments. The final concentration of BLM in experiments ranged from 0.1 ng/ml to 20 ng/ml. For that different concentrations of anticancer drugs were used together with pDNA transfection. pMAX FGP coding plasmid in concentraton of 200 μg/ml was used. Electroporation was performed by using combination of 1 electric pulse that induced electric fields at the amplitude of 1400 V/cm for the duration of 100 μs. Cells were electroporated in laboratory made EP medium (pH 7.1, conductivity 0.1 S/m, osmolarity 270 mOsm). Afterwards, comet assay was performed to evaluate DNA damage. In addition, clonogenic assay was done to evaluate cell viability. Transfection efficiency was measured using flow cytometry (BD accuri C6).[...]Biologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Bleomycin electrotransfer enhancement by using Plasmid Dna

Over the last decade a new cancer treatment method termed electrochemotherapy has emerged. Such anticancer therapy is based on the process of electroporation. This process initiates when applied electric field is increasing transmembrane voltage of the affected cells, hence increasing cell membrane permeability that leads to increased uptake of exogenous molecules such as anticancer drugs. Currently, the combination of anticancer drug (mainly bleomycin) and the method of electroporation is used in clinics. Once inside the cell, the anticancer drug bleomycin induces the cellular DNA cleavage, that in turn leads to cell death. Previously it has been shown that big molecules (dextrans) with high net charge increase electrotransfer of bleomycin into the cells. Here we show the enhanced bleomycin electrotransfer when plasmid DNA (as a big molecule with charge) was in the media during the electroporation We analysed bleomycin electrotransfer in Chinese Hamster Ovary (CHO) cells. For that different concentrations of anticancer drugs were used together with pDNA transfection. pMAX FGP coding plasmid in concentraton of 200 µg/ml was used. The anticancer drug bleomycin was used for electrotransfer experiments in the concentrations ranging from 0.1 to 20 ng/ml. Electroporation was performed by using combination of 1 electric pulse that induced electric fields at the amplitude of 1400 V/cm for the duration of 100 µs. Afterwards, comet assay was performed to evaluate DNA damage. In addition, clonogenic assay was done to evaluate cell viability. Obtained results showed bigger bleomycin electrotransfer after usage of combination of bleomycin, pDNA and electroporation. In conditions of 1HV electroporation intensity, 200 µg/ml pDNA and 20 ng/ml BLM concentrations, DNA cleavage reaches up to 10% [...]Aplinkos tyrimų centrasGamtos mokslų fakultetasVytauto Didžiojo universiteta

Elektrochemoterapijos kaip sisteminio priešvėžinio gydymo koncepto pagrindimas in vitro

Be dažniausia naudojamų, konvencinių, priešvėžinių gydymo būdų (chirurginis naviko išpjovimas (rezekcija), radioterapija, chemoterapija, imunoterapija), taip pat pradėti vystyti pažangūs, gydymo nuo vėžio būdai, turintys tik lokalų poveikį. Vienas tokių, jau klinikinėje praktikoje taikomų, lokalių priešvėžinės terapijos būdų, yra elektrochemoterapija (ECT ). ECT metu lokalų priešvėžinių vaistų poveikį sukelia elektroporacijos procesas, kurio metu dėl atitinkamo stiprumo elektrinių laukų poveikio ląstelių plazminė membrana yra laikinai pralaidinama įvairioms hidrofilinėms ar amfifilinėms molekulėms, tokioms kaip priešvėžinis vaistas bleomicinas (BLM) [1]. Įrodyta, kad naudojant elektroporacijos pernašos metodą galima sumažinti priešvėžinių vaistų koncentraciją iki 1 000 kartų, išlaikant analogišką terapinį poveikį [2]. ECT klinikinės terapijos metu net iki 85 proc. pacientų gydymas baigiasi remisija. Žinoma, kad BLM patekęs į ląstelę sukarpo vėžinių ląstelių DNR, todėl ląstelė žūsta [3]. Tačiau toks gydymo būdas yra lokalus, t. y. veikiamos ląstelės, kurios yra paveiktos elektriniais laukais. Tokiu atveju elektrochemoterapija nepaveikia metastazinių audinių, paplitusių toliau nuo naviko. Šio tyrimo idėja ta, kad apoptotiškai žūdama ląstelė dar gali ekspresuoti sėkmingai transfekuotą geną. Tokiu būdu, pasirinkus atitinkamą ekspresuojamą baltymą (PVZ IFN-1 beta, arba TNF-alfa), žūdamos ląstelės dar gali sukelti imuninį sisteminį atsaką [4]. Atitinkamai panaudojus šiuos mechanizmus, lokalų elektrochemoterapijos procesą galima paversti sisteminį atsaką sukeliančių gydymu. Tai galima pasiekti navikines ląsteles transfekuojant atitinkamais signalinius baltymus koduojančiais genais. Šiame tyrime parodyta kombinuota priešvėžinio vaisto bleomicino elektropernaša bei reporterinio geno transfekcija.[...]Biologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Plazmidinės DNR ekspresijos reguliavimas naudojant bleomicino sukeltą citotoksiškumo efektą po vienalaikės abiejų molekulių elektropernašos

Leidinys: https://www.lmt.lt/lt/doclib/2aa2wt4rkd9g4zep1nuxhw9e9a197v7sPasaulio sveikatos organizacijos 2018 metų duomenimis [1], vėžys yra 9,6 milijonų žmonių mirties priežastimi, tad intensyviai tiriama kontroliuojama vaistų ir genų pernaša į ląsteles bei audinius. Egzogeninių molekulių pernašai į ląstelę gali būti naudojami įvairūs fizikiniai metodai, kurie pasižymi efektyvesne didelių molekulių pernaša bei nesukelia tokio intensyvaus imuninio atsako, kaip virusiniai [2]. Vienas iš galimų lokalių priešvėžinės terapijos būdų, naudojant fizikinius vaistų pernašos metodus, yra elektrochemoterapija (ECT) [3]. ECT metu, vykstant elektroporacijai, ląstelių plazminė membrana tampa laikinai laidi įvairioms hidrofilinėms medžiagoms, tokioms, kaip priešvėžiniam vaistui bleomicinui (BLM ), taip padidinant lokalų priešvėžinių vaistų poveikį. Membranos pralaidumo padidėjimą lemia elektrinių laukų sukeltas porų atsiradimas, kai indukuotas transmembraninis potencialas viršija kritinę ribą [4]. Į ląstelę patekus BLM , susiformuoja aktyviosios deguonies formos (RO S). Patekęs į ląstelę BLM sukelia užprogramuotą (apoptotinę) ląstelės žūtį, taip sukeliama daug viengrandžių ir dvigrandžių genominės DNR trūkių, lemiančių nekrotinę arba apoptotinę ląstelės žūtį [5]. Vėžio gydyme naudojant jonizuojančiąją spinduliuotę, ląstelėse-taikiniuose taip pat susidaro RO S, kurios šiose ląstelėse suformuoja daug viengrandžių ir dvigrandžių genominės DNR trūkių, sukeliančių ląstelės žūtį. Yra žinoma, kad radiacija paveiktos ląstelės žūdamos į aplinką išskiria signalines molekules, kurios paveikia šalia esančias, tačiau tiesiogiai jonizuojančia spinduliuote nepaveiktas, ląsteles. Toks reiškinys pavadintas „Bystander“ efektu [6]. Nors „Bystander“ efektas yra žinomas radioterapijoje, tačiau elektrochemoterapijoje jis yra beveik netyrinėtas – nėra atlikta duomenų,[...]Biologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

The assessment of cellular DNA damage after bleomycin electrotransfer using comet assay in vitro

Online ISSN: 2335-8718When 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. Presently one of electroporation applications is local anticancer drug delivery to tumour, termed electrochemotherapy. Bleomycin, a well know anticancer drug, is used, for electrochemotherapy. Once inside the cell, this anticancer drug induces single and double strand breaks to genomic DNA, thus ultimately causing cell death. Nevertheless, to our knowledge, there is no studies published, that indicate the quantification of DNA damage induced by BLM electrotransfer. Here we performed in vitro analysis of DNA damage of BLM electrotransfered cells using Comet assay. Chinese hamster ovary (CHO) cell line was used for experiments. The final concentration of BLM in experiments ranged from 0.2 ng/ml to 20 μg/ml. Electroporation was performed by using electric field at 1400 V/cm voltage for the duration of 100 μs. Cells were electroporated in laboratory made EP medium (pH 7.1, conductivity 0.1 S/m, osmolarity 270 mOsm).[...]Biologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

In vitro analysis of DNA damage after cell electroporation in the presence of bleomycin

Electroporation is based on the formation of pores in cell membrane when exposed to electric fields. Creation of pores leads to ion leakage, escape of metabolites, and increased uptake of various small hydrophilic molecules, including anticancer drugs. Currently electroporation is applied for local anticancer drug delivery in clinics for antitumor electrochemotherapy. During electrochemotherapy usually anticancer drug bleomycin (BLM) is used. This drug act as endonuclease inducing DNA breaks, and ultimately causing cell death. However, to our knowledge there is no studies published, that indicate the quantification of DNA damage induced by BLM electrotransfer. Here we performed in vitro analysis of DNA damage of BLM electrotransfered cells using Comet assay. Chinese Hamster Ovary (CHO) cell culture was used for bleomycin electrotransfer. Used BLM concentration was from 20 to 2000 μg/ml. Cell were suspended in electroporation medium (conductivity 0.1 S/m, osmolarity 270 mOsm, pH 7.1,) for the delivery of electric pulses. Electroporation was performed with 1 pulse at 1400 V/cm for the duration of 100 μs. Comet assay was performed to evaluate DNA damage. Clonogenic assay was performed to evaluate cell viability. Results indicate a significant cells DNA damage increase when higher BLM concentrations of were used. After bleomycin electrotransfer DNA breaks varied from 10 % at 20 ng/ml to 80 % at 2 mg/ml. No DNA damage was observed when cells were incubated with the same concentrations of BLM without performance of electroporation. Cell viability decrease was observed only when electroporation was performed in the presence of BLM and started to be significant at concentrations ranging between 0.2 to 2 ng/ml. At higher concentrations cell viability decreased to was about 5 %Biologijos katedraFizikos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

In vitro analysis of DNA damage after cell electroporation in the presence of bleomycin

Electroporation is based on the formation of pores in cell membrane when exposed to electric fields. Creation of pores leads to ion leakage, escape of metabolites, and increased uptake of various small hydrophilic molecules, including anticancer drugs. Currently electroporation is applied for local anticancer drug delivery in clinics for antitumor electrochemotherapy. During electrochemotherapy usually anticancer drug bleomycin (BLM) is used. This drug act as endonuclease inducing DNA breaks, and ultimately causing cell death. However, to our knowledge there is no studies published, that indicate the quantification of DNA damage induced by BLM electrotransfer. Here we performed in vitro analysis of DNA damage of BLM electrotransfered cells using Comet assay. Chinese Hamster Ovary (CHO) cell culture was used for bleomycin electrotransfer. Used BLM concentration was from 20 to 2000 μg/ml. Cell were suspended in electroporation medium (conductivity 0.1 S/m, osmolarity 270 mOsm, pH 7.1,) for the delivery of electric pulses. Electroporation was performed with 1 pulse at 1400 V/cm for the duration of 100 μs. Comet assay was performed to evaluate DNA damage. Clonogenic assay was performed to evaluate cell viability. Results indicate a significant cells DNA damage increase when higher BLM concentrations of were used. After bleomycin electrotransfer DNA breaks varied from 10 % at 20 ng/ml to 80 % at 2 mg/ml. No DNA damage was observed when cells were incubated with the same concentrations of BLM without performance of electroporation. Cell viability decrease was observed only when electroporation was performed in the presence of BLM and started to be significant at concentrations ranging between 0.2 to 2 ng/ml. At higher concentrations cell viability decreased to was about 5 %Biologijos katedraFizikos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Enhancement of bleomycin cytotoxic effect and regulation of gene expression using simultaneous plasmid DNA and bleomycin electotransfer

Electrochemotherapy (ETC) is an effective physical technique that enables cytotoxic drugs to have direct access to the cytosol. This budding mode of therapy uses electroporation and anticancer drugs (mainly bleomycin) to trigger cell death in cancerous cells. At this moment, various methods are being explored to increase the impact of ETC, such as gene transfer. Our preliminary studies have shown that plasmid DNA and bleomycin in the medium allows simultaneous transport of these molecules. In addition, we have shown that the presence of DNA in the medium can increase bleomycin transport and thereby cytotoxicity, whereas the presence of bleomycin may alter the strength and timing of gene expression. These effects can be used to increase the efficiency of ECT (due to more efficient BLM electrotransfer) and to regulate the immune response, by delivering genes, encoding specific cytokines. In this work, the cytotoxicity effect of the electrotransfer of different concentrations of bleomycin and different size of plasmid DNA by using the same parameters of the electric pulses on Chinese Hamster Ovary cells was determined. Electroporation was performed by using combination of 1 electric pulse of 1400 V/cm pulse strength and 100 μs pulse duration. pMAX GFP (3.5 kb), pEGFP (4.7 kb) and piggyBac (7.1 kb) coding plasmids in concentrations of 200 μg/ml were used. The obtained results showed that a combination of plasmid DNA, bleomycin, and electroporation increases the cytotoxic effect of the anticancer drug but achieves lower transfection efficiency. Further studies have shown that the cytotoxic effects of anticancer drug (BLM) and transfection efficiency are dependent on plasmid DNA sizeBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

A Novel method for controlled gene expression via combined bleomycin and plasmid DNA electrotransfer

Article no. 4047Electrochemotherapy is an e cient method for the local treatment of cutaneous and subcutaneous metastases, but its e cacy as a systemic treatment remains low. The application of gene electrotransfer (GET) to transfer DNA coding for immune system modulating molecules could allow for a systemic e ect, but its applications are limited because of possible side e ects, e.g., immune system overactivation and autoimmune response. In this paper, we present the simultaneous electrotransfer of bleomycin and plasmid DNA as a method to increase the systemic e ect of bleomycin-based electrochemotherapy. With appropriately selected concentrations of bleomycin and plasmid DNA, it is possible to achieve e cient cell transfection while killing cells via the cytotoxic e ect of bleomycin at later time points. We also show the dynamics of both cell electrotransfection and cell death after the simultaneous electrotransfer of bleomycin and plasmid DNA. Therefore, this method could have applications in achieving the transient, cell death-controlled expression of immune system activating genes while retaining e cient bleomycin mediated cell killingBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta