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. We developed a method for dynamic monitoring and conductance measurements of individual electropores. This was accomplished by time-lapse total internal reflection fluorescence (TIRF) imaging in HEK cells loaded with CAL-520 dye and placed on an indium tin oxide (ITO) surface. Applying a 1-ms, 0 to -400 mV pulse between the patch pipette and ITO evoked focal Ca2+ transients that identified individual electropores. Some transients disappeared in milliseconds but others persisted for over a minute. Persistent transients ("Ca2+ plumes") faded over time to a stable or a randomly fluctuating level that could include periods of full quiescence. Single pore conductance, measured by 0 to -50 mV, 50 ms steps at 30 and 60 s after the electroporation, ranged from 80 to 200 pS. These experiments proved electropore longevity in cells, in stark contrast to molecular simulations and many findings in lipid bilayers.

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. We conclude that the α1 subunit supports the electropermeabilized membrane state, by forming or stabilizing electropores or by hindering repair mechanisms, and this role is independent of NKA's ion pump function.

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. Only two KOs, for SCNN1A and CLCA1 genes, showed a statistically significant reduction in YP uptake. The respective proteins could be part of electropermeabilization lesions or increase their lifespan. In contrast, as many as 39 genes were identified as likely hits for the increased YP uptake, meaning that the respective proteins contributed to membrane stability or repair after nsEP. The expression level of eight genes in different types of human cells showed strong correlation (R > 0.9, p < 0.02) with their LD50 for lethal nsEP treatments, and could potentially be used as a criterion for the selectivity and efficiency of hyperplasia ablations with nsEP.

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. The extent of membrane damage and the time course of resealing were monitored by the time-lapse imaging of propidium (Pr) uptake in human cervical carcinoma (HeLa) cells exposed to trains of 300-ns PEF. The removal of the extracellular Ca2+ slowed down the resealing, although did not prevent it. Recruitment of CHMP4B protein, a component of ESCRT-III complex, to the electroporated plasma membrane was not observed, thus providing no evidence for possible contribution of the macro-vesicle shedding mechanism. In contrast, silencing the AnxA5 gene impaired resealing and reduced the viability of nsPEF-treated cells. We conclude that Annexin V but not ESCRT-III was involved in the repair of HeLa cells permeabilized by 300-ns stimuli, but it was not the only and perhaps not the main repair mechanism.

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. RESULTS: APAF-1 mRNA and protein levels were significantly down-regulated, and APAF-1 methylation status was associated with perineural invasion in PDAC. Decitabine inhibited cell viability and increased apoptosis rates, however failed to restore APAF-1 mRNA and protein levels in cells. CONCLUSION: APAF-1 gene hypermethylation may contribute to the progression of PDAC through perineural invasion. Decitabine could sensitize pancreatic cancer cells to apoptosis and growth retardation, however, not directly through the APAF-1 demethylation process.

Stimulated upregulation of HO-1 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 in vitro study was to clarify the response of gastric and ovarian cancer cells to hyperthermic intraperitoneal chemotherapy, following the modulation of HO-1 expression. AGS and OVCAR-3 cells were treated with different temperature regimens, either alone or in combination with an IC50 dose of cisplatin for 1 h. Prior to treatment, HO-1 expression was silenced by short interfering RNA transfection. In OVCAR-3 cells, cisplatin increased HO-1 mRNA expression by 3.73-fold under normothermia and 2.4-fold under hyperthermia; furthermore, these factors similarly increased HO-1 protein expression levels. Exposure to cisplatin under hyperthermia reduced the viability of OVCAR-3 cells by 36% and HO-1-silencing enhanced this effect by 20%. HO-1-silencing under normothermia increased apoptotic rates in cisplatin-treated OVCAR-3 cells by 2.07-fold, and hyperthermia enhanced the effect by 3.09-fold. Semi-quantitative polymerase chain reaction (PCR) cell analysis indicated that exposure to cisplatin decreased the cell index under normothermia, and that hyperthermia boosted this effect in OVCAR-3. In AGS cells, only temperature increased cellular HO-1 levels. Silencing HO-1 in AGS cells at 37°C reduced viability by 16% and increased apoptotic rates 2.63-fold. Hyperthermia did not affect AGS viability; however, apoptosis was increased 6.84-fold. PCR analysis indicated no additional effects of hyperthermia on the AGS cell index. HO-1 is induced in cancer cells by different stressors in a variable manner. In tumors with highly inducible HO-1, prior silencing of this gene could improve the cellular response to hyperthermia and cisplatin.

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. We obtained evidence that in the OVCAR-3 cell line GDH was directly activated by hyperthermia (cisplatin eliminated this effect); however, this effect was followed by GDH inhibition after 48 h recovery. A combination of 43 °C hyperthermia with cisplatin induced stronger GDH inhibition in comparison to separate treatments, and negative effects exerted on GDH activity correlated with suppression of mitochondrial respiration with glutamate + malate. Cisplatin did not induce uncoupling of oxidative phosphorylation in OVCAR-3 cells but induced impairment of the outer mitochondrial membrane in combination with 43 °C hyperthermia. Hyperthermia (43 °C) potentiated cytotoxicity of cisplatin in an OVCAR-3 cell line.

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. The Kaplan-Meier method and log-rank tests were used for survival analysis. Immunoprecipitation assay was performed to demonstrate HuR binding to IAP1, IAP2 mRNA. PANC1 cells were transfected with either anti-HuR siRNA or control siRNA for 72 h and quantitative reverse transcription polymerase chain reaction (RT-PCR), western blot analysis was carried out. RESULTS: RT-PCR analysis revealed that HuR, IAP1, IAP2 mRNA expression were accordingly 3.3-fold, 5.5-fold and 8.4 higher in the PDAC when compared to normal pancreas (P < 0.05). Expression of IAP1 was positively strongly correlated with HuR expression (P < 0.05, r = 0.783). Western blot analysis confirmed RT-PCR results. High IAP1 expression, tumor resection status, T stage, lymph-node metastases, tumor differentiation grade, perineural and lymphatic invasion were identified as significant factors for shorter survival in PDAC patients (P < 0.05). Immunohistological analysis showed that HuR was mainly expressed in the ductal cancer cell's nucleus and less so in cytoplasm. RNA immunoprecipitation analysis confirmed IAP1 and IAP2 post-transcriptional regulation by HuR protein. Following siHuR transfection, IAP1 mRNA and protein levels were decreased, however IAP2 expression levels were increased. CONCLUSION: HuR mediated overexpression of IAP1 significantly correlates with poor outcomes and early progression of pancreatic cancer. Further studies are needed to assess the underlying mechanisms.

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

Aim: 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. Methods: 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. Results: Gender-dependent differences in response of respiration to hyperthermia were the highest at 3⁻4 months of age, less so at 6⁻7 months of age, and only minor at juvenile age. Mild hyperthermia (40⁻42 °C) stimulated pyruvate + malate oxidation in mitochondria of females, but inhibited in mitochondria of males in the 3⁻4 month age group. The resistance of mitochondrial membrane to hyperthermia was the highest at 3⁻4 month males, and the lowest in the 6⁻7 month age group. Inhibition of glutamate + malate oxidation by hyperthermia was caused by thermal inactivation of glutamate dehydrogenase. ROS generation at 37 °C was higher at 1⁻1.5 month of age, but the increase in ROS generation with rise in temperature in this age group was the smallest, and the strongest in 6⁻7 month old animals of both genders. Conclusions: The response to hyperthermia varies during the first 6⁻7 months of life of experimental animals: stronger gender dependence is characteristic at 3⁻4 months of age, while mitochondria from 6⁻7 months animals are less resistant to hyperthermia.

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). After 15-min perfusion, mitochondria were isolated to investigate changes in the respiration rates and the membrane potential. Adenine nucleotides extracted from isolated hepatocytes and perfused liver subjected to hyperthermic treatment were analyzed by HPLC. RESULTS: Hyperthermic liver perfusion at 42-52°C progressively impaired oxidative phosphorylation in isolated mitochondria. Significant inhibition of the respiratory chain components was observed after perfusion at 42°C, irreversible uncoupling became evident after liver perfusion at higher temperatures (46°C and above). After perfusion at 50-52°C energy supplying function of mitochondria was entirely compromised, and mitochondria turned to energy consumers. Hyperthermia-induced changes in mitochondrial function correlated well with changes in the energy status and viability of isolated hepatocytes, but not with the changes in the energy status of the whole liver tissue. CONCLUSIONS: In this study the pattern of the adverse changes in mitochondrial functions that are progressing with increase in liver perfusion temperature was established. Results of experiments on isolated mitochondria and isolated hepatocytes indicate that hyperthermic treatment significantly and irreversibly inhibits energy-supplying function of mitochondria under conditions similar to those existing in the radiofrequency ablation transition zone and these changes can lead to death of hepatocytes. However, it was not possible to estimate contribution of mitochondrial injury to liver tissue energy status by estimating only hyperthermia-induced changes in adenine nucleotide amounts on the whole tissue level.

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. Cell viability after treatment with GEM and/or curcumin and siCUGBP2 was evaluated using MTT and crystal violet tests. RNA immunoprecipitation analysis was used to confirm COX-2 and HO-1 post-transcriptional regulation by CUGBP2 protein. RESULTS: CUGBP2 expression at the messenger RNA (mRNA) level was 2.2-fold lower (p = 0.007), but HO-1 and COX-2 expression was increased 6.9- (p = 0.023) and 2.3- (p = 0.046) fold in pancreatic cancer tissues. The median survival of patients with low CUGBP2 expression from the lowest tercile was 13.8 months. The median survival of patients in terciles of middle and high CUGBP2 expression levels was 21.9 month (p = 0.123). Induction of CUGBP2 expression by curcumin resulted in the downregulation of HO-1 and COX-2 and strongly sensitized tumor cells to GEM treatment. However, CUGBP2 silencing upregulated HO-1 and COX-2 protein expression and had a high effect on cells viability. CONCLUSION: Decreased activity of CUGBP2 could be associated with high chemoresistance and early dissemination of pancreatic cancer through the HO-1- and COX-2-mediated cytoprotective and carcinogenesis pathways. Curcumin significantly increased the effectiveness of GEM treatment in vitro via the CUGBP2-mediated post-transcriptional regulation pathway.

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.86.86, Capan-1, and Capan-2) with and without GEM treatment. Immunocytofluorescence analysis was used to investigate HuR localization in cells. Cell viability and response to GEM after HuR silencing were determined with the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide test and the crystal violet clonogenic assay, respectively. To measure apoptosis, activation of caspases 3/7 was evaluated using immunofluorescence. RESULTS: In PDA tissue obtained from patients not treated with GEM, HuR mRNA expression was 3.2 times lower (P < 0.05) and COX-2 and HO-1 mRNA expression was 2.3-fold and 7.2-fold higher (P < 0.05), respectively, than normal pancreatic tissue (from organ donor). qRT-PCR analysis showed that HuR, COX-2, and HO-1 mRNA were overexpressed in all cancer cell lines treated with the half maximal inhibitory concentration (IC50) dose of GEM compared with control cells (P < 0.05). Western blot analysis revealed that COX-2 and HO-1 levels were significantly decreased in cancer cells after HuR silencing. Furthermore, HuR silencing increased the response to GEM treatment and decreased cell viability by 11.6%-53.7% compared to control cell lines. Caspases 3 and 7 were activated after HuR silencing and GEM treatment in all pancreatic cancer cell lines. In comparison, treatment with GEM alone did not activate caspases 3 and 7 in the same cell lines. CONCLUSION: HuR mediated post-transcriptional upregulation of COX-2 and HO-1 expression after GEM treatment in pancreatic cancer cells. HuR silencing significantly increased the effectiveness of GEM treatment in vitro.