Dynamics of cell membrane lesions and adaptive conductance under the electrical stress.

Exceeding physiological limits of the cell membrane potential compromises structural integrity, enabling the passage of normally impermeant solutes and disrupting cell function. Electropermeabilization has been studied extensively at the cellular scale, but not at the individual membrane lesion level. We employed fast total internal reflection fluorescence (TIRF) imaging of Ca entry transients to discern individual lesions in a hyperpolarized cell membrane and characterize their focality, thresholds, electrical conductance, and the lifecycle.

Real-time imaging of individual electropores proves their longevity in cells.

With over 50 years of electroporation research, the nature of cell membrane permeabilization remains elusive. The lifetime of electropores in molecular models is limited to nano- or microseconds, whereas the permeabilization of electroporated cells can last minutes. This study aimed at resolving a longstanding debate on whether the prolonged permeabilization is due to the formation of long-lived pores in cells.

Silencing of ATP1A1 attenuates cell membrane disruption by nanosecond electric pulses.

This study explored the role of the Na/K-ATPase (NKA) in membrane permeabilization induced by nanosecond electric pulses. Using CRISPR/Cas9 and shRNA, we silenced the ATP1A1 gene, which encodes α1 NKA subunit in U937 human monocytes. Silencing reduced the rate and the cumulative uptake of YoPro-1 dye after electroporation by 300-ns, 7-10 kV/cm pulses, while ouabain, a specific NKA inhibitor, enhanced YoPro-1 entry.

Identification of Proteins Involved in Cell Membrane Permeabilization by Nanosecond Electric Pulses (nsEP).

The study was aimed at identifying endogenous proteins which assist or impede the permeabilized state in the cell membrane disrupted by nsEP (20 or 40 pulses, 300 ns width, 7 kV/cm). We employed a LentiArray CRISPR library to generate knockouts (KOs) of 316 genes encoding for membrane proteins in U937 human monocytes stably expressing Cas9 nuclease. The extent of membrane permeabilization by nsEP was measured by the uptake of Yo-Pro-1 (YP) dye and compared to sham-exposed KOs and control cells transduced with a non-targeting (scrambled) gRNA.

The role of ESCRT-III and Annexin V in the repair of cell membrane permeabilization by the nanosecond pulsed electric field.

Exposure of living cells to intense nanosecond pulsed electric field (nsPEF) increases membrane permeability to small solutes, presumably by the formation of nanometer-size membrane lesions. Mechanisms responsible for the restoration of membrane integrity over the course of minutes after nsPEF have not been identified. This study explored if ESCRT-III and Annexin V calcium-dependent repair mechanisms, which play critical role in resealing large membrane lesions, are also activated by electroporation and contribute to the membrane resealing.

Epigenetic Regulation of APAF-1 Through DNA Methylation in Pancreatic Cancer.

Background/aim: Apoptotic peptidase activating factor 1 (APAF-1) is essential regulator of apoptosis and inactivation by DNA methylation is common event in numerous cancer types. We investigated the regulation of APAF-1 through DNA methylation in pancreatic cancer.

Materials And Methods: Datasets from 44 patients after pancreatoduodenectomy and the pancreatic adenocarcinoma (PDAC) cell lines Capan-2 and MIA PaCa-2 treated with decitabine were analyzed by RT-PCR, immunoblotting, methylation-specific PCR analysis, apoptosis and viability assays to identify effects of APAF-1 regulation.

Stimulated upregulation of is associated with inadequate response of gastric and ovarian cancer cell lines to hyperthermia and cisplatin treatment.

Heme oxygenase (HO)-1 is a heat shock protein induced by hyperthermia, responsible for cellular resistance to temperature. The aim of this study was to clarify the response of gastric and ovarian cancer cells to hyperthermic intraperitoneal chemotherapy, following the modulation of expression. AGS and OVCAR-3 cells were treated with different temperature regimens, either alone or in combination with an IC dose of cisplatin for 1 h.

Hyperthermia potentiates cisplatin cytotoxicity and negative effects on mitochondrial functions in OVCAR-3 cells.

The aim of this study was to determine the effects of hyperthermia, cisplatin and their combination on mitochondrial functions such as glutamate dehydrogenase (GDH) activity and mitochondrial respiration rates, as well as survival of cultured ovarian adenocarcinoma OVCAR-3 cells. Cells treated for 1 h with hyperthermia (40 and 43 °C) or cisplatin (IC50) or a combination of both treatments were left for recovery at 37 °C temperature for 24 h or 48 h. The obtained results revealed that 43 °C hyperthermia potentiated effects of cisplatin treatment: combinatory treatment more strongly suppressed GDH activity and expression, mitochondrial functions, and decreased survival of OVCAR-3 cells in comparison to separate single treatments.

Human antigen R mediated post-transcriptional regulation of inhibitors of apoptosis proteins in pancreatic cancer.

Aim: To determine the association of human antigen R (HuR) and inhibitors of apoptosis proteins (IAP1, IAP2) and prognosis in pancreatic cancer.

Methods: Protein and mRNA expression levels of IAP1, IAP2 and HuR in pancreatic ductal adenocarcinoma (PDAC) were compared with normal pancreatic tissue. The correlations among IAP1/IAP2 and HuR as well as their respective correlations with clinicopathological parameters were analyzed.

Impact of Gender and Age on Hyperthermia-Induced Changes in Respiration of Liver Mitochondria.

: This study aimed to compare hyperthermia-induced changes in respiration and generation of reactive oxygen species (ROS) in liver mitochondria derived from animals of different gender and age. : The effects of hyperthermia (40⁻47 °C) on oxidation of different substrates and ROS production were estimated in mitochondria isolated from the liver of male and female rats of the 1⁻1.5, 3⁻4, or 6⁻7 months age.

Contribution of mitochondria to injury of hepatocytes and liver tissue by hyperthermia.

Objective: The aim of this study was to investigate functional changes of liver mitochondria within the experimentally modeled transition zone of radiofrequency ablation and to estimate possible contribution of these changes to the energy status of liver cells and the whole tissue.

Materials And Methods: Experiments were carried out on mitochondria isolated from the perfused liver and isolated hepatocytes of male Wistar rats. Hyperthermia was induced by changing the temperature of perfusion medium in the range characteristic for the transition zone (38-52°C).

Upregulation of cugbp2 increases response of pancreatic cancer cells to chemotherapy.

Purpose: Altered expression and/or function of ribosomal RNA (rRNA)-binding proteins CUGBP2/CELF2 might influence post-transcriptional regulation of the HO-1- and COX-2-mediated cytoprotective pathways and represents an important therapeutic target. The aim of this study was to assess the effects of CUGBP2-mediated post-transcriptional regulation of COX-2 and HO-1 in pancreatic cancer cells in regard of response to gemcitabine (GEM) treatment.

Methods: Expression of CUGBP2, COX-2, and HO-1 was evaluated using qRT-PCR and Western blot methods.

HuR mediated post-transcriptional regulation as a new potential adjuvant therapeutic target in chemotherapy for pancreatic cancer.

Aim: To investigate the expression of HuR in pancreatic ductal adenocarcinoma (PDA) and to assess the effects of HuR silencing on the expression of cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1) and the in vitro response to gemcitabine (GEM) treatment in pancreatic cell lines.

Methods: We compared the expression of HuR, COX-2, and HO-1 in PDA and normal pancreatic tissue using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. In addition, the HuR, COX-2 and HO-1 were analyzed in four types of cancer cell lines (MiaPaca2, Su.