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Regulatory changes occurring early in colorectal cancer development remain poorly investigated. Since the majority of cases develop from polyps in the adenoma-carcinoma transition, a search of early molecular features, such as aberrations in miRNA expression occurring prior to cancer development, would enable identification of potentially causal, rather than consequential, candidates in the progression of polyp to cancer. In the current study, by employing small RNA-seq profiling of colon biopsy samples, we described differentially expressed miRNAs and their isoforms in the adenoma-carcinoma transition. Analysis of healthy-adenoma-carcinoma sequence in an independent validation group enabled us to identify early deregulated miRNAs including hsa-miR-1246 and hsa-miR-215-5p, the expressions of which are, respectively, gradually increasing and decreasing. Loss-of-function experiments revealed that inhibition of hsa-miR-1246 lead to reduced cell viability, colony formation, and migration rate, thereby indicating an oncogenic effect of this miRNA in vitro. Subsequent western blot and luciferase reporter assay provided evidence of hsa-miR-1246 being involved in the regulation of target AXIN2 and CFTR genes' expression. To conclude, the present study revealed possible involvement of hsa-miR-1246 in early colorectal cancer development and regulation of tumor suppressors AXIN2 and CFTR.

Control of membrane permeability to exogenous compounds by membrane electroporation can lead to cell death, which is related to permanent membrane damage, oxidation stress, leakage of intracellular molecules. In this study, we show that the predominant cell death modality after the application of high voltage electric pulses is related with inability to reseal of initial pores (first stage irreversible electroporation, FirEP). After moderately strong electric pulses, initial pores reseal, however, some cell still die later on due to electric field induced cell stress which leads to delayed cell death (late-stage irreversible electroporation, LirEP). According to our data, the period in which the majority of cells commit to either pore resealing or complete loss of barrier function depends on the intensity of electric field treatment but did not exceed 35 min. Additionally, we show that after electroporation using electric pulse parameters that induce LirEP, some cells can be rescued by supplementing medium with compounds obtained from irreversibly electroporated cells. We determined that the intracellular molecules that contribute to the increase of cell viability are larger than 30 kDa. This serves to prove that the loss of intracellular compounds plays a significant role in the decrease of cell viability after electroporation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Deregulated microRNA (miRNA) expression profiles and their contribution to carcinogenesis have been observed in virtually all types of human cancer. However, their role in the pathogenesis of rare mesenchymal gastrointestinal stromal tumors (GISTs) is not well defined, yet. In this study, we aimed to investigate the role of two miRNAs strongly downregulated in GIST-miR-375-3p and miR-200b-3p-in the pathogenesis of GIST. To achieve this, miRNA mimics were transfected into GIST-T1 cells and changes in the potential target gene mRNA and protein expression, as well as alterations in cell viability, migration, apoptotic cell counts and direct miRNA-target interaction, were evaluated. Results revealed that overexpression of miR-375-3p downregulated the expression of KIT mRNA and protein by direct binding to KIT 3'UTR, reduced GIST cell viability and migration rates. MiR-200b-3p lowered expression of ETV1 protein, directly targeted and lowered expression of EGFR mRNA and protein, and negatively affected cell migration rates. To conclude, the present study identified that miR-375-3p and miR-200b-3p have a tumor-suppressive role in GIST.

Electroporation-a transient electric-field-induced increase in cell membrane permeability-can be used to facilitate the delivery of anticancer drugs for antitumour electrochemotherapy. In recent years, Ca 2+ electroporation has emerged as an alternative modality to electrochemotherapy. The antitumor effect of calcium electroporation is achieved as a result of the introduction of supraphysiological calcium doses. However, calcium is also known to play a key role in membrane resealing, potentially altering the pore dynamics and molecular delivery during electroporation. To elucidate the role of calcium for the electrotransfer of small charged molecule into cell we have performed experiments using nano- and micro-second electric pulses. The results demonstrate that extracellular calcium ions inhibit the electrotransfer of small charged molecules. Experiments revealed that this effect is related to an increased rate of membrane resealing. We also employed mathematical modelling methods in order to explain the differences between the CaCl 2 effects after the application of nano- and micro-second duration electric pulses. Simulation showed that these differences occur due to the changes in transmembrane voltage generation in response to the increase in specific conductivity when CaCl 2 concentration is increased., Competing Interests: The authors declare no conflict of interest.

Gastric cancer (GC) is one of the most common and lethal gastrointestinal malignancies worldwide. Many studies have shown that development of GC and other malignancies is mainly driven by alterations of cellular signaling pathways. MicroRNAs (miRNAs) are small noncoding molecules that function as tumor-suppressors or oncogenes, playing an essential role in a variety of fundamental biological processes. In order to understand the functional relevance of miRNA dysregulation, studies analyzing their target genes are of major importance. Here, we chose to analyze two miRNAs, miR-20b and miR-451a, shown to be deregulated in many different malignancies, including GC. Deregulated expression of miR-20b and miR-451a was determined in GC cell lines and the INS-GAS mouse model. Using Western Blot and luciferase reporter assay we determined that miR-20b directly regulates expression of PTEN and TXNIP , and miR-451a: CAV1 and TSC1 . Loss-of-function experiments revealed that down-regulation of miR-20b and up-regulation of miR-451a expression exhibits an anti-tumor effect in vitro (miR-20b: reduced viability, colony formation, increased apoptosis rate, and miR-451a: reduced colony forming ability). To summarize, the present study identified that expression of miR-20b and miR-451a are deregulated in vitro and in vivo and have a tumor suppressive role in GC through regulation of the PI3K/AKT/mTOR signaling pathway., Competing Interests: The authors declare no conflict of interest.

Electrochemotherapy is an efficient method for the local treatment of cutaneous and subcutaneous metastases, but its efficacy 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 effect, but its applications are limited because of possible side effects, 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 effect of bleomycin-based electrochemotherapy. With appropriately selected concentrations of bleomycin and plasmid DNA, it is possible to achieve efficient cell transfection while killing cells via the cytotoxic effect 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 efficient bleomycin mediated cell killing.

Electroporation is a widely established method for molecular delivery across electric field perturbed plasma membrane. It can be used as a non-viral DNA transfection method, or as a way to achieve small molecule delivery to or extraction from cells. We examined the possibility of combining the DNA delivery to the cells with small molecule transport across electroporated plasma membrane. The results show that the presence of DNA in electroporation medium increases the extraction of fluorescent dye calcein from calcein-AM loaded cells as well as the delivery of small-molecule drug bleomycin to the cells. We propose that these results may have implications in enhanced drug delivery using electroporation both in vivo and in clinics., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recent discoveries have shown that self-replicating RNA viruses can produce small RNAs (sRNAs) in host cells. Given their potential to be modified to generate short-term transgene expression without integrating viral sequences into the host genome, these viruses could be used as safe delivery vehicles for sRNAs to induce long-term transcriptional gene silencing (TGS). This might open new avenues for therapeutic approaches, where a single administration would safely induce long-term therapeutic effects for various diseases. Here, we introduce and discuss the possible use of cytoplasmic alphaviruses, flaviviruses, Sendai virus (SeV), and nucleoplasmic Influenza A (IAV) and Borna disease (BoDV) viruses to induce long-term TGS., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Electrospray is a process based on creation and acceleration of small sized droplets based on electrostatic repulsion. Spraying plasmid containing liquids this process may be used to transfer genes into cells. Within this paper we report on a method for accessing and evaluating the spray modalities using high speed imaging system with a post processing of image data to obtain estimated volume and velocity of emerging droplets first. Second we investigate on the impact of different media on the spray modalities. Third we evaluate the impact of the spray on cell viability and on transfection efficiency of an eGFP plasmid as reporter gene obtained in an in vitro setup on alveolar epithelial like cells (A549).

Long-term transcriptional gene silencing has been hampered by delivery issues. A potential solution is the application of RNA viruses that generate small RNAs without any DNA intermediate. Long-term therapy for various diseases is expected after a single administration., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The axonal growth cone is a specialized structure enabling axon extension and proper guidance to its target by sensing the extracellular environment. A growth cone collapse assay is a popular approach designed to characterize the inhibitory effect of secreted guidance cues in vitro. However, the actin cytoskeleton of the growth cone is very sensitive to various factors like physical impact, temperature, and acidity of environment that may also induce responses resembling those of guidance signals. Herein, we provide an easy and reproducible method to analyze growth cone sensitivity to the prototypic guidance molecule family class 3 semaphorin. This protocol is intended to present a systematic approach that is easy to apply to any soluble factors with a potential to impact axon elongation.

Commercially available chitins and the chitin isolated from mushrooms, insect cuticles, shells of shrimp, crab and crayfish reported in the literature are in forms of powder, flake or granule. Three-dimensional chitins have been only known from the sponges but still three-dimensional chitosan has not been reported yet. In this study, we produced three-dimensional chitin and chitosan rings from the body segments of a diplopod species (Julus terrestris). Obtained chitin and chitosan rings were characterized (by FT-IR, SEM, TGA, XRD, dilute solution viscometry and EA) and compared with commercial chitin and chitosan. The interactions with plasmid DNA was studied at varying concentrations of chitosan (0.04, 0.4 and 4mg/mL). Antitumor activity tests were conducted (L929 and HeLa), low cytotoxicity and high antiproliferative activity was observed. Antimicrobial activities of J. terrestris chitosan were investigated on twelve microorganisms and maximum inhibition (15.6±1.154mm) was recorded for common human pathogen Staphylococcus aureus., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Ultrasound induced microbubble (MB) cavitation is used to significantly enhance cell membrane permeabilization, thereby allowing delivery of various therapeutic agents into cells. In order to monitor and quantitatively control the extent of cavitation the uniform dosimetry model is needed. In present study we have simultaneously performed quantitative evaluation of three main sonoporation factors: (1) MB concentration, (2) MB cavitation extent, and (3) doxorubicin (DOX) sonotransfer into Chinese hamster ovary cells. MB concentration measurement results and passively recorded MB cavitation signals were used for MB sonodestruction rate and spectral root-mean-square (RMS) calculations, respectively. Subsequently, time to maximum value of RMS and inertial cavitation dose (ICD) quantifications were performed for every acoustic pressure value. This comprehensive research has led not only to explanation of relation of ICD and MB sonodestruction rate but also to the development of a new sonoporation metric: the inverse of time to maximum value of RMS (1/time to maximum value of RMS). ICD and MB sonodestruction rate intercorrelation and correlation with DOX sonotransfer suggest inertial cavitation to be the key mechanism for cell sonoporation. All these metrics were successfully used for doxorubicin sonotransfer prediction (R(2) > 0.9, p < 0.01) and therefore shows feasibility to be applied for future dosimetric applications for ultrasound-mediated drug and gene delivery.

Background: Cell exposure to ultrasound (US) in the presence of contrast agent microbubbles (MBs) can result in cell sonoporation that can be exploited for drug or gene delivery. Anticancer drug bleomycin (BLM), used in sonoporation, can effectively eliminate tumor cells in vitro and in vivo. Nevertheless, sonoporation mechanism is not known, thus different US parameters and MB types are used. Recently, we proposed that efficiency of cell sonoporation can be related to the efficiency of MB sonodestruction., Purpose: We analyzed human tumor cells viability in response to BLM, US and MB treatment., Methods: Human glioblastoma astrocytoma (U-87 MG) or colon cancer (HCT-116) cells were exposed to US in the presence of BLM and either SonoVue® or BR14® MBs. MB sonodestruction was evaluated according to US signal attenuation., Results: Both HCT-116 and U-87 MG cell viability following US exposure decreased up to 30%. Decrease in cell viability followed similar tendency as MB sonodestruction, which suggests direct relationship between MB sonodestruction and BLM intracellular delivery., Conclusion: Sonoporation is a feasible method to deliver BLM in to several types of human cancer cell lines. Efficiency of cell sonoporation correlated well with MB sonodestruction, providing a possibility to optimize US parameters by measuring MB sonodestruction.

Objectives: The efficiency of sonoporation is directly related to microbubble cavitation and can be dependent on the microbubble sonodestruction rate. The objective of this study was to investigate whether the rate of microbubble sonodestruction can be used as a parameter to develop an implicit dosimetric method for sonoporation efficiency evaluation., Methods: To evaluate the rate of microbubble sonodestruction as a function of the ultrasound (US) peak negative ultrasound pressure, 12-MHz diagnostic US was used in the B-scan mode. Chinese hamster ovary cells were exposed to therapeutic US at 880 kHz in the absence or presence of microbubbles. The sonoporation efficiency was evaluated by the sonotransfer of bleomycin, a cytotoxic, membrane-impermeable anticancer drug., Results: At a low microbubble sonodestruction rate of 1/τ < 0.5 second(-1) (τ providing the time necessary to decrease the microbubble concentration to 37% of its initial value), cell viability remained basically unaffected, but the percentage of sonoporated cells did not reach 10%. At higher microbubble sonodestruction rates, the efficiencies of irreversible and reversible sonoporation started to increase linearly and reached the plateau at 5 seconds(-1)., Conclusions: These results show that the microbubble sonodestruction rate can be used to predict the percentage of reversible and irreversible sonoporation.

Cell sonoporation enables the delivery of various exogenous molecules into the cells. To maximize the percentage of reversibly sonoporated cells and to increase cell viability we propose a model for implicit dosimetry for adjustment of ultrasound (US) exposure duration. The Chinese hamster ovary cell suspension was supplemented with microbubbles (MB) and exposed to US, operating at the frequency of 880kHz, with a 100% duty cycle and with an output peak negative pressure (PNP) of 500kPa for durations ranging from 0.5 to 30s. Using diagnostic B-scan imaging we showed that the majority of the MB at 500kPa US peak negative pressure undergo sonodestruction in less than a second. During this time maximal number of reversibly sonoporated cells was achieved. Increase of US exposure duration did not increase sonoporated cell number, however it induced additional cell viability decrease. Therefore aiming to achieve the highest level of reversibly sonoporated cells and also to preserve the highest level of cell viability, the duration of US exposure should not exceed the duration needed for complete MB sonodestruction.

Introduction: Intracellular gene electrotransfer by means of electroporation has been on the increase during the past decade. Significant progress has been achieved both in characterizing mechanisms of gene electrotransfer and in optimizing the protocol in many preclinical trials. Recently this has led to initiation of clinical trials of gene electrotransfer to treat metastatic melanomas. Further progress with the method in various clinical trials requires better understanding of mechanisms of gene electrotransfer., Areas Covered: A summary of recent progress in understanding mechanisms of gene electrotransfer, imparting general knowledge of cell electroporation and intracellular molecule electrotransfer., Expert Opinion: Gene electrotransfer into cells and tissues is a complex process involving multiple steps that lead to plasmid DNA passage from the extracellular region to the cell nucleus crossing the barriers of the plasma membrane, cytoplasm and nucleus membrane. Electrical parameters of pulses used for gene electrotransfer affect the initial steps of DNA translocation through the plasma membrane and play a crucial role in determining the transfection efficiency. When considering gene electrotransfer into tissues it becomes clear that other nonelectrical conditions are also of primary importance.

DNA electrotransfer in vivo for gene therapy is a promising method. For further clinical developments, the efficiency of the method should be increased. It has been shown previously that high efficiency of gene electrotransfer in vivo can be achieved using high-voltage (HV) and low-voltage (LV) pulses. In this study we evaluated whether HV and LV pulses could be optimized in vitro for efficient DNA electrotransfer. Experiments were performed using Chinese hamster ovary (CHO) cells. To evaluate the efficiency of DNA electrotransfer, two different plasmids coding for GFP and luciferase were used. For DNA electrotransfer experiments 50 microl of CHO cell suspension containing 100, 10 or 1 microg/ml of the plasmid were placed between plate electrodes and subjected to various combinations of HV and LV pulses. The results showed that at 100 microg/ml plasmid concentration LV pulse delivered after HV pulse increased neither the percentage of transfected cells nor the total transfection efficiency (luciferase activity). The contribution of the LV pulse was evident only at reduced concentration (10 and 1 microg/ml) of the plasmid. In comparison to HV (1,200 V/cm, 100 micros) pulse, addition of LV (100 V/cm, 100 ms) pulse increased transfection efficiency severalfold at 10 microg/ml and fivefold at 1 microg/ml. At 10 microg/ml concentration of plasmid, application of four LV pulses after HV pulse increased transfection efficiency by almost 10-fold. Thus, these results show that contribution of electrophoretic forces to DNA electrotransfer can be investigated in vitro using HV and LV pulses.

Background: Electric pulses delivered to cells that are in close contact may induce cell fusion, by a process termed electrofusion. Recently it has been shown that electrofusion in tumours in vivo depends on tumour type. The aim of this work was to examine the differences in electrofusion in various cell lines, both in vivo and in vitro., Materials and Methods: LPB, B16F10 and DC-3F cells in vitro and LLC tumours in vivo were exposed to various electric pulses. The number of fused cells was then evaluated., Results: Cell electropermeabilization was confirmed to be a necessary but non-exclusive condition to obtain a high level of cell electrofusion. The extent of electrofusion depends both on the degree of permeabilization and cell type., Conclusion: It was observed that metastatic tumour cells easily electrofuse, suggesting that cell type-specific membrane properties and/or secretion of proteases determine the extent of electrofusion.

There is increasing evidence for a crucial role of proteases and metalloproteinases during axon growth and guidance. In this context, we recently described a functional link between the chemoattractive Sema3C and Matrix metalloproteinase 3 (MMP3). Here, we provide data demonstrating the involvement of MMP-2 to trigger the growth-promoting effect of Sema3A in cortical dendrites. The in situ analysis of MMP-2 expression and activity is consistent with a functional growth assay demonstrating in vitro that the pharmacological inhibition of MMP-2 reduces the growth of cortical dendrites in response to Sema3A. Hence, our results suggest that the selective recruitment and activation of MMP-2 in response to Sema3A requires a PKC alpha dependent mechanism. Altogether, we provide a second set of data supporting MMPs as effectors of the growth-promoting effects of semaphorins, and we identify the potential signalling pathway involved.

The aim of our study was to determine if electroporation could improve the efficacy of photodynamic tumor therapy. A disadvantage of photodynamic therapy is a slow and in some cases insufficient accumulation of photosensitizer in tumor tissue, which could restrict the achievement of an efficient dose. Under the action of electric pulses, cells undergo membrane electroporation, which results in an increased permeability to various exogenous molecules. In this study, murine hepatoma MH22A cells were exposed to light in vitro in the presence of a photosensitizer, either chlorin e6 or aluminum phthalocyanine tetrasulfonate, following electroporation. Accumulation of the photosensitizers was registered by fluorescence microscopy. Cell viability was determined by the MTT assay. Our results demonstrate that electroporation improves an access of chlorin e6 and aluminum phthalocyanine tetrasulfonate to MH22A cells. Electroporation in combination with photosensitization significantly reduces viability of the treated cells even at low doses of photosensitizers.

While initially thought to be essentially a developmental characteristic, observed in artificial in vitro models, local protein synthesis in growth cones has been described in the adult, and more interestingly, during nerve regeneration. This emerging field is under intense investigation, revealing new functions of localized protein synthesis that include axon guidance, growth cone adaptation and sensitivity modulation at intermediate targets or axon regeneration. Here, we will review these functions and provide a short survey of the current knowledge on mechanisms of mRNA transport and regulation of localized protein synthesis. In addition, we will consider what lessons can be learned from localized protein synthesis in dendrites, and what developments can be expected next in the field. This latter question relates to the crucial point of which technical strategy to adopt for an ideal and pertinent analysis of the phenomenon.

When cells are exposed to a strong enough external electric field, transient aqueous pores are formed in the membrane. The fraction of electroporated cells can be determined by measuring the release of intracellular potassium ions. The current work is the first study where such a method was employed successfully not only with cells suspended in the medium with a rather high concentration of potassium (4-5 mM) but also with cells that release some part of intracellular potassium responding, in this way, to the stress caused by manipulation procedures during the preparation of the cell suspension. Experiments were carried out on mouse hepatoma MH-A22 cells exposed to a square-wave electric pulse. The curves showing the dependence of the fraction of the cells that have become permeable to bleomycin, a membrane-impermeable cytotoxic drug, are close to the ones showing the release of intracellular potassium ions.

Efficient DNA electrotransfer can be achieved with combinations of short high-voltage (HV) and long low voltage (LV) pulses that cover two effects of the pulses, namely, target cell electropermeabilization and DNA electrophoresis within the tissue. Because HV and LV can be delivered with a lag up to 3000 sec between them, we considered that it was possible to analyze separately the respective importance of the two types of effects of the electric fields on DNA electrotransfer efficiency. The tibialis cranialis muscles of C57BL/6 mice were injected with plasmid DNA encoding luciferase or green fluorescent protein and then exposed to various combinations of HV and LV pulses. DNA electrotransfer efficacy was determined by measuring luciferase activity in the treated muscles. We found that for effective DNA electrotransfer into skeletal muscles the HV pulse is prerequisite; however, its number and duration do not significantly affect electrotransfer efficacy. DNA electrotransfer efficacy is dependent mainly on the parameters of the LV pulse(s). We report that different LV number, LV individual duration, and LV strength can be used, provided the total duration and field strength result in convenient electrophoretic transport of DNA toward and/or across a permeabilized membrane.

Efficient cell electrotransfection can be achieved using combinations of high-voltage (HV; 800 V/cm, 100 micros) and low-voltage (LV; 80 V/cm, 100 ms) pulses. We have developed equipment allowing the generation of various HV and LV combinations with precise control of the lag between the HV and LV pulses. We injected luciferase-encoding DNA in skeletal muscle, before or after pulse delivery, and measured luciferase expression after various pulse combinations. In parallel, we determined permeabilization levels using uptake of (51)Cr-labeled EDTA. High voltage alone resulted in a high level of muscle permeabilization for 300 seconds, but very low DNA transfer. Combinations of one HV pulse followed by one or four LV pulses did not prolong the high permeabilization level, but resulted in a large increase in DNA transfer for lags up to 100 seconds in the case of one HV + one LV and up to 3000 seconds in the case of one HV + four LV. DNA expression also reached similar levels when we injected the DNA between the HV and LV pulses. We conclude that the role of the HV pulse is limited to muscle cell permeabilization and that the LV pulses have a direct effect on DNA. In vivo DNA electrotransfer is thus a multistep process that includes DNA distribution, muscle permeabilization, and DNA electrophoresis.

Intramuscular plasmid DNA injection results in long-term but low and variable expression of the injected genes. Optimization is difficult because the mechanism of naked DNA uptake by the cells in vivo is not yet determined. Here we used injections of plasmid DNA encoding luciferase to further characterize this mechanism. We analyzed the kinetics of naked DNA uptake by means of DNase I or heparin injections, using the level of luciferase expression as the indicator of DNA uptake. We demonstrated that in vivo heparin inhibits DNA uptake without affecting the expression of DNA internalized by means of electric pulses. Inhibition by heparin is dose dependent and compatible with the competition for the binding to a receptor. As shown also with DNase I, DNA uptake by muscle cells is slow: a progressive accumulation of the DNA in the myofibers can be found for at least 4 hours after naked DNA injection. Physical presence of DNA molecules during the uptake period, but not later, was confirmed by the facilitation of DNA uptake with appropriate electric pulses. Therefore, uptake proceeds for the entire time during which intact DNA is present in the extracellular compartment. Our results support evidence for a DNA uptake mechanism based on receptor-mediated endocytosis.