Ex vivo Study of Partial Bladder Resection in a Porcine ModelEx vivo Study of Partial Bladder Resection in a Porcine Model.

INTRODUCTION: Partial cystectomy aims to preserve bladder function, yet its urodynamic impacts remain unclear. We investigate these effects using an ex-vivo porcine model, evaluating bladder volume, compliance, and wall thickness, alongside with thermal damage after bi- and monopolar resection. METHODS: Within an artificial human pelvis, we conducted partial bladder wall resections (5 cm2, 10 cm2). Urodynamic tests and sonography assessed volume, compliance, and thickness changes. Traction force for catheter retrieval and thermal collagen destruction were measured. RESULTS: Bladder compliance decreased by 1.12 and 1.5 after 5 cm2 and 10 cm2 resections respectively, with volume reductions of 3-6% and 10-18%. Wall thickness decreased by 20% and 30% post-resection. Comparable thermal damage was observed with mono- and bipolar resection methods. CONCLUSION: Our study outlines urodynamic impacts and technical considerations of partial cystectomy, affirming its endoscopic feasibility while highlighting potential bladder dysfunction risks.

Urine protects urothelial cells against killing with nanosecond pulsed electric fields.

The quest for safe and effective ablation resulted in the development of nanosecond pulsed electric fields (nsPEF) technology for tumor treatment. For future applications of nsPEF in urothelial cancer treatment, we evaluated the effect of urine presence at the ablation site. We prepared artificial urine (AU) with compounds commonly present in the healthy human urine at physiological concentrations. We compared nsPEF cytotoxicity for cancerous (T24) and non-cancerous (SV-HUC-1) human urothelial cell lines pulsed either in the AU or in a physiological solution (PS). Cell monolayers were exposed to trains of 300-ns, 10-Hz pulses using a two-needle electrode assembly placed orthogonal to the monolayer. The assembly produced the electric field gradually weakening with the distance from the electrodes. The electric field which killed 50 % of cells (LD50) was measured by staining with propidium iodide and matching the stained area with the simulated electric field strength. nsPEF exposure in PS was more cytotoxic to cancer cells. The AU protected both healthy and cancer urothelial cells, increasing their LD50 1.4 and 1.6 times, respectively. Omitting urea from the AU reduced the LD50 for healthy and cancer urothelial cells. Testing the role of other AU components, we found that it was the high concentration of phosphates what also rendered the protective effect of the AU. Our findings suggest that the nsPEF ablation of bladder cancer will be less efficient if the bladder is filled with urine.

Boosting the Immune Response-Combining Local and Immune Therapy for Prostate Cancer Treatment.

Due to its slow progression and susceptibility to radical forms of treatment, low-grade PC is associated with high overall survival (OS). With the clinical progression of PC, the therapy is becoming more complex. The immunosuppressive tumor microenvironment (TME) makes PC a difficult target for most immunotherapeutics. Its general immune resistance is established by e.g., immune evasion through Treg cells, synthesis of immunosuppressive mediators, and the defective expression of surface neoantigens. The success of sipuleucel-T in clinical trials initiated several other clinical studies that specifically target the immune escape of tumors and eliminate the immunosuppressive properties of the TME. In the settings of PC treatment, this can be commonly achieved with radiation therapy (RT). In addition, focal therapies usually applied for localized PC, such as high-intensity focused ultrasound (HIFU) therapy, cryotherapy, photodynamic therapy (PDT), and irreversible electroporation (IRE) were shown to boost the anti-cancer response. Nevertheless, the present guidelines restrict their application to the context of a clinical trial or a prospective cohort study. This review explains how RT and focal therapies enhance the immune response. We also provide data supporting the combination of RT and focal treatments with immune therapies.

How to alleviate cardiac injury from electric shocks at the cellular level.

Electric shocks, the only effective therapy for ventricular fibrillation, also electroporate cardiac cells and contribute to the high-mortality post-cardiac arrest syndrome. Copolymers such as Poloxamer 188 (P188) are known to preserve the membrane integrity and viability of electroporated cells, but their utility against cardiac injury from cardiopulmonary resuscitation (CPR) remains to be established. We studied the time course of cell killing, mechanisms of cell death, and protection with P188 in AC16 human cardiomyocytes exposed to micro- or nanosecond pulsed electric field (µsPEF and nsPEF) shocks. A 3D printer was customized with an electrode holder to precisely position electrodes orthogonal to a cell monolayer in a nanofiber multiwell plate. Trains of nsPEF shocks (200, 300-ns pulses at 1.74 kV) or µsPEF shocks (20, 100-µs pulses at 300 V) produced a non-uniform electric field enabling efficient measurements of the lethal effect in a wide range of the electric field strength. Cell viability and caspase 3/7 expression were measured by fluorescent microscopy 2-24 h after the treatment. nsPEF shocks caused little or no caspase 3/7 activation; most of the lethally injured cells were permeable to propidium dye already at 2 h after the exposure. In contrast, µsPEF shocks caused strong activation of caspase 3/7 at 2 h and the number of dead cells grew up to 24 h, indicating the prevalence of the apoptotic death pathway. P188 at 0.2-1% reduced cell death, suggesting its potential utility in vivo to alleviate electric injury from defibrillation.

Atorvastatin Modulates the Efficacy of Electroporation and Calcium Electrochemotherapy.

Electroporation is influenced by the features of the targeted cell membranes, e.g., the cholesterol content and the surface tension of the membrane. The latter is eventually affected by the organization of actin fibers. Atorvastatin is a statin known to influence both the cholesterol content and the organization of actin. This work analyzes the effects of the latter on the efficacy of electroporation of cancer cells. In addition, herein, electroporation was combined with calcium chloride (CaEP) to assess as well the effects of the statin on the efficacy of electrochemotherapy. Cholesterol-rich cell lines MDA-MB231, DU 145, and A375 underwent (1) 48 h preincubation or (2) direct treatment with 50 nM atorvastatin. We studied the impact of the statin on cholesterol and actin fiber organization and analyzed the cells' membrane permeability. The viability of cells subjected to PEF (pulsed electric field) treatments and CaEP with 5 mM CaCl2 was examined. Finally, to assess the safety of the therapy, we analyzed the N-and E-cadherin localization using confocal laser microscopy. The results of our investigation revealed that depending on the cell line, atorvastatin preincubation decreases the total cholesterol in the steroidogenic cells and induces reorganization of actin nearby the cell membrane. Under low voltage PEFs, actin reorganization is responsible for the increase in the electroporation threshold. However, when subject to high voltage PEF, the lipid composition of the cell membrane becomes the regulatory factor. Namely, preincubation with atorvastatin reduces the cytotoxic effect of low voltage pulses and enhances the cytotoxicity and cellular changes induced by high voltage pulses. The study confirms that the surface tension regulates of membrane permeability under low voltage PEF treatment. Accordingly, to reduce the unfavorable effects of preincubation with atorvastatin, electroporation of steroidogenic cells should be performed at high voltage and combined with a calcium supply.

Effects of high-frequency nanosecond pulses on prostate cancer cells.

Electroporation with pulsed electric fields show a potential to be applied as an experimental focal therapy of tumors. Sub-microsecond regime of electric pulses displays unique electrophysical features operative in cells and membranes. Recently, MHz compression of nanosecond pulses electric fields (nsPEFs) bursts proved to enhance the effectiveness of the therapy. High morbidity of prostate cancer (PCa) and risk of overtreatment associated with this malignancy call for new minimal-invasive treatment alternative. Herein we present the in vitro study for developing applications based on this new technology. In this study, we used flow cytometric analysis, cell viability assay, caspase activity analysis, wound healing assay, confocal microscopy study, and immunofluorescence to investigate the biological effect of high-frequency nsPEFs on PCa cells. Our results show that high-frequency nsPEFs induces the permeabilization and cell death of PCa cells. The cytotoxicity is significantly enhanced in MHz compression of pulses and with the presence of extracellular Ca2+. High-frequency nsPEFs trigger changes in PCa cells' cytoskeleton and their mobility. The presented data show a therapeutic potential of high-frequency nsPEFs in a PCa setting. The sub-microsecond regime of pulses can potentially be applied in nanosecond electroporation protocols for PCa treatment.

Mechanisms of curcumin-based photodynamic therapy and its effects in combination with electroporation: An in vitro and molecular dynamics study.

Photodynamic therapy (PDT) and electrochemotherapy (ECT) are two methods designed to enhance the anticancer potential of various drugs. Various clinical trials proved the efficacy of both ECT and PDT in melanoma treatment. Curcumin is a natural polyphenolic compound with high anticancer potential against melanoma due to its light absorption properties and toxicity towards cancer cells; however, high reactivity and amphipathic structure of curcumin are limiting its utility. This study aimed to propose the most effective protocol for antimelanoma combination of both therapies (PDT and ECT) in the context of curcumin. The in vitro studies were carried on melanotic melanoma (A375), amelanotic melanoma (C32) and fibroblast (HGF) cell lines. In molecular dynamics studies curcumin presented the single-layer localization in the water-membrane interphase. Further, the mass spectrometry studies exposed that during the PDT treatment curcumin is degraded to vanillin, feruloylmethane, and ferulic acid. Instant ECT with curcumin followed by PDT is the most efficient approach due to its selective genotoxicity towards malignant cells. The metabolic activity of fibroblasts decreased, however, at the same time the fragmentation of DNA did not occur. Additionally, instant PDT with curcumin followed by ECT after 3 h of incubation was a therapy selective towards melanotic melanoma.

In Vitro Study of Calcium Microsecond Electroporation of Prostate Adenocarcinoma Cells.

Electroporation, applied as a non-thermal ablation method has proven to be effective for focal prostate treatment. In this study, we performed pre-clinical research, which aims at exploring the specific impact of this so-called calcium electroporation on prostate cancer. First, in an in-vitro study of DU 145 cell lines, microsecond electroporation (µsEP) parameters were optimized. We determined hence the voltage that provides both high permeability and viability of these prostate cancer cells. Subsequently, we compared the effect of µsEP on cells' viability with and without calcium administration. For high-voltage pulses, the cell death's mechanism was evaluated using flow-cytometry and confocal laser microscopy. For lower-voltage pulses, the influence of electroporation on prostate cancer cell mobility was studied using scratch assays. Additionally, we applied calcium-binding fluorescence dye (Fluo-8) to observe the calcium uptake dynamic with the fluorescence microscopy. Moreover, the molecular dynamics simulation visualized the process of calcium ions inflow during µsEP. According to our results calcium electroporation significantly decreases the cells viability by promoting apoptosis. Furthermore, our data shows that the application of pulsed electric fields disassembles the actin cytoskeleton and influences the prostate cancer cells' mobility.

Electroporation-Based Treatments in Urology.

The observation that an application of a pulsed electric field (PEF) resulted in an increased permeability of the cell membrane has led to the discovery of the phenomenon called electroporation (EP). Depending on the parameters of the electric current and cell features, electroporation can be either reversible or irreversible. The irreversible electroporation (IRE) found its use in urology as a non-thermal ablative method of prostate and renal cancer. As its mechanism is based on the permeabilization of cell membrane phospholipids, IRE (as well as other treatments based on EP) provides selectivity sparing extracellular proteins and matrix. Reversible EP enables the transfer of genes, drugs, and small exogenous proteins. In clinical practice, reversible EP can locally increase the uptake of cytotoxic drugs such as cisplatin and bleomycin. This approach is known as electrochemotherapy (ECT). Few in vivo and in vitro trials of ECT have been performed on urological cancers. EP provides the possibility of transmission of genes across the cell membrane. As the protocols of gene electrotransfer (GET) over the last few years have improved, EP has become a well-known technique for non-viral cell transfection. GET involves DNA transfection directly to the cancer or the host skin and muscle tissue. Among urological cancers, the GET of several plasmids encoding prostate cancer antigens has been investigated in clinical trials. This review brings into discussion the underlying mechanism of EP and an overview of the latest progress and development perspectives of EP-based treatments in urology.

Oxidative Effects during Irreversible Electroporation of Melanoma Cells-In Vitro Study.

Irreversible electroporation (IRE) is today used as an alternative to surgery for the excision of cancer lesions. This study aimed to investigate the oxidative and cytotoxic effects the cells undergo during irreversible electroporation using IRE protocols. To do so, we used IRE-inducing pulsed electric fields (PEFs) (eight pulses of 0.1 ms duration and 2-4 kV/cm intensity) and compared their effects to those of PEFs of intensities below the electroporation threshold (eight pulses, 0.1 ms, 0.2-0.4 kV/cm) and the PEFs involving elongated pulses (eight pulses, 10 ms, 0.2-0.4 kV/cm). Next, to follow the morphology of the melanoma cell membranes after treatment with the PEFs, we analyzed the permeability and integrity of their membranes and analyzed the radical oxygen species (ROS) bursts and the membrane lipids' oxidation. Our data showed that IRE-induced high cytotoxic effect is associated both with irreversible cell membrane disruption and ROS-associated oxidation, which is occurrent also in the low electric field range. It was shown that the viability of melanoma cells characterized by similar ROS content and lipid membrane oxidation after PEF treatment depends on the integrity of the membrane system. Namely, when the effects of the PEF on the membrane are reversible, aside from the high level of ROS and membrane oxidation, the cell does not undergo cell death.

Effects of Photosensitization of Curcumin in Human Glioblastoma Multiforme Cells.

BACKGROUND/AIM: There is no satisfactory treatment of glioblastoma multiforme, a highly invasive brain tumor. The aim of this study was to analyze the cytotoxic effects of curcumin (CUR) alone and as a photosensitizer on glioblastoma cells. MATERIALS AND METHODS: The SNB-19 cells where incubated for 2 and 24 h with 5-200 mM of CUR. The cells were radiated with blue light (6 J/cm2) and compared to non-irradiated ones. The effects of treatment were assessed by measuring mitochondrial activity with the MTT method and apoptosis progression by flow cytometry. To investigate CUR uptake, fluorescence imaging of cells was performed. RESULTS: Photosensitization of CUR decreased the EC50 6.3 times when the incubation time was 2 h and over 90% of cells underwent apoptosis. The study of the uptake of CUR showed that during the 2 h, CUR was placed in the entire cytoplasm, and over time, its amount decreased and localized in the subcellular compartments. CONCLUSION: CUR is a promising medicament that can be used as a photosensitizer in photodynamic therapy for glioma treatment.

In Search of Panacea-Review of Recent Studies Concerning Nature-Derived Anticancer Agents.

Cancers are one of the leading causes of deaths affecting millions of people around the world, therefore they are currently a major public health problem. The treatment of cancer is based on surgical resection, radiotherapy, chemotherapy or immunotherapy, much of which is often insufficient and cause serious, burdensome and undesirable side effects. For many years, assorted secondary metabolites derived from plants have been used as antitumor agents. Recently, researchers have discovered a large number of new natural substances which can effectively interfere with cancer cells' metabolism. The most famous groups of these compounds are topoisomerase and mitotic inhibitors. The aim of the latest research is to characterize natural compounds found in many common foods, especially by means of their abilities to regulate cell cycle, growth and differentiation, as well as epigenetic modulation. In this paper, we focus on a review of recent discoveries regarding nature-derived anticancer agents.