Nanosecond PEF Induces Oxidative Stress and Apoptosis via Proteasomal Activity Inhibition in Gastric Adenocarcinoma Cells with Drug Resistance
Nanosecond (ns) pulsed electric field (PEF) is a technology in which the application of ultra-short electrical pulses can be used to disrupt the barrier function of cell plasma and internal membranes. Disruptions of the membrane integrity cause a substantial imbalance in cell homeostasis in which ox...
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MDPI AG
2022-10-01
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| Series: | International Journal of Molecular Sciences |
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| author | Julita Kulbacka Nina Rembiałkowska Anna Szewczyk Joanna Rossowska Małgorzata Drąg-Zalesińska Marek Kulbacki Anna Choromańska |
| author_facet | Julita Kulbacka Nina Rembiałkowska Anna Szewczyk Joanna Rossowska Małgorzata Drąg-Zalesińska Marek Kulbacki Anna Choromańska |
| author_sort | Julita Kulbacka |
| collection | DOAJ |
| description | Nanosecond (ns) pulsed electric field (PEF) is a technology in which the application of ultra-short electrical pulses can be used to disrupt the barrier function of cell plasma and internal membranes. Disruptions of the membrane integrity cause a substantial imbalance in cell homeostasis in which oxidative stress is a principal component. In the present study, nsPEF-induced oxidative stress was investigated in two gastric adenocarcinoma cell lines (EPG85-257P and EPG85-257RDB) which differ by their sensitivity to daunorubicin. Cells were exposed to 200 pulses of 10 ns duration, with the amplitude and pulse repetition frequency at 1 kHz, with electric field intensity varying from 12.5 to 50 kV/cm. The electroporation buffer contained either 1 mM or 2 mM calcium chloride. CellMask DeepRed visualized cell plasma permeabilization, Fluo-4 was used to visualize internal calcium ions content, and F-actin was labeled with AlexaFluor<sup>®</sup>488 for the cytoskeleton. The cellular viability was determined by MTT assay. An alkaline and neutral comet assay was employed to detect apoptotic and necrotic cell death. The luminescent method estimated the modifications in GSSG/GSH redox potential and the imbalance of proteasomal activity (chymotrypsin-, trypsin- and caspase-like). The reactive oxygen species (ROS) level was measured by flow cytometry using dihydroethidium (DHE) dye. Morphological visualization indicated cell shrinkage, affected cell membranes (characteristic bubbles and changed cell shape), and the reorganization of actin fibers with sites of its dense concentration; the effect was more intense with the increasing electric field strength. The most significant decrease in cell viability and GSSG/GSH redox potential was noted at the highest amplitude of 50 kV/cm, and calcium ions amplified this effect. nsPEF, particularly with calcium ions, inhibited proteasomal activities, resulting in increased protein degradation. nsPEF increased the percentage of apoptotic cells and ROS levels. The EPG85-257 RDB cell line, which is resistant to standard chemotherapy, was more sensitive to applied nsPEF protocols. The applied nsPEF method disrupted the metabolism of cancer cells and induced apoptotic cell death. The nsPEF ability to cause apoptosis, oxidative stress, and protein degradation make the nsPEF methodology a suitable alternative to current anticancer pharmacological methods. |
| first_indexed | 2024-03-09T19:01:58Z |
| format | Article |
| id | doaj.art-d0a106bd854e433496a40898d965c617 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T19:01:58Z |
| publishDate | 2022-10-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-d0a106bd854e433496a40898d965c6172023-11-24T04:59:38ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-10-0123211294310.3390/ijms232112943Nanosecond PEF Induces Oxidative Stress and Apoptosis via Proteasomal Activity Inhibition in Gastric Adenocarcinoma Cells with Drug ResistanceJulita Kulbacka0Nina Rembiałkowska1Anna Szewczyk2Joanna Rossowska3Małgorzata Drąg-Zalesińska4Marek Kulbacki5Anna Choromańska6Department of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211 A, 50-556 Wroclaw, PolandDepartment of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211 A, 50-556 Wroclaw, PolandDepartment of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211 A, 50-556 Wroclaw, PolandHirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 50-422 Wroclaw, PolandDivision of Histology and Embryology, Division of Human Morpholog and Embryology, Faculty of Medicine, Wroclaw Medical University, 50-368 Wroclaw, PolandPolish-Japanese Academy of Information Technology, 02-008 Warsaw, PolandDepartment of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211 A, 50-556 Wroclaw, PolandNanosecond (ns) pulsed electric field (PEF) is a technology in which the application of ultra-short electrical pulses can be used to disrupt the barrier function of cell plasma and internal membranes. Disruptions of the membrane integrity cause a substantial imbalance in cell homeostasis in which oxidative stress is a principal component. In the present study, nsPEF-induced oxidative stress was investigated in two gastric adenocarcinoma cell lines (EPG85-257P and EPG85-257RDB) which differ by their sensitivity to daunorubicin. Cells were exposed to 200 pulses of 10 ns duration, with the amplitude and pulse repetition frequency at 1 kHz, with electric field intensity varying from 12.5 to 50 kV/cm. The electroporation buffer contained either 1 mM or 2 mM calcium chloride. CellMask DeepRed visualized cell plasma permeabilization, Fluo-4 was used to visualize internal calcium ions content, and F-actin was labeled with AlexaFluor<sup>®</sup>488 for the cytoskeleton. The cellular viability was determined by MTT assay. An alkaline and neutral comet assay was employed to detect apoptotic and necrotic cell death. The luminescent method estimated the modifications in GSSG/GSH redox potential and the imbalance of proteasomal activity (chymotrypsin-, trypsin- and caspase-like). The reactive oxygen species (ROS) level was measured by flow cytometry using dihydroethidium (DHE) dye. Morphological visualization indicated cell shrinkage, affected cell membranes (characteristic bubbles and changed cell shape), and the reorganization of actin fibers with sites of its dense concentration; the effect was more intense with the increasing electric field strength. The most significant decrease in cell viability and GSSG/GSH redox potential was noted at the highest amplitude of 50 kV/cm, and calcium ions amplified this effect. nsPEF, particularly with calcium ions, inhibited proteasomal activities, resulting in increased protein degradation. nsPEF increased the percentage of apoptotic cells and ROS levels. The EPG85-257 RDB cell line, which is resistant to standard chemotherapy, was more sensitive to applied nsPEF protocols. The applied nsPEF method disrupted the metabolism of cancer cells and induced apoptotic cell death. The nsPEF ability to cause apoptosis, oxidative stress, and protein degradation make the nsPEF methodology a suitable alternative to current anticancer pharmacological methods.https://www.mdpi.com/1422-0067/23/21/12943gastric adenocarcinomadrug resistancensPEFcalcium ionsapoptosisoxidative stress |
| spellingShingle | Julita Kulbacka Nina Rembiałkowska Anna Szewczyk Joanna Rossowska Małgorzata Drąg-Zalesińska Marek Kulbacki Anna Choromańska Nanosecond PEF Induces Oxidative Stress and Apoptosis via Proteasomal Activity Inhibition in Gastric Adenocarcinoma Cells with Drug Resistance International Journal of Molecular Sciences gastric adenocarcinoma drug resistance nsPEF calcium ions apoptosis oxidative stress |
| title | Nanosecond PEF Induces Oxidative Stress and Apoptosis via Proteasomal Activity Inhibition in Gastric Adenocarcinoma Cells with Drug Resistance |
| title_full | Nanosecond PEF Induces Oxidative Stress and Apoptosis via Proteasomal Activity Inhibition in Gastric Adenocarcinoma Cells with Drug Resistance |
| title_fullStr | Nanosecond PEF Induces Oxidative Stress and Apoptosis via Proteasomal Activity Inhibition in Gastric Adenocarcinoma Cells with Drug Resistance |
| title_full_unstemmed | Nanosecond PEF Induces Oxidative Stress and Apoptosis via Proteasomal Activity Inhibition in Gastric Adenocarcinoma Cells with Drug Resistance |
| title_short | Nanosecond PEF Induces Oxidative Stress and Apoptosis via Proteasomal Activity Inhibition in Gastric Adenocarcinoma Cells with Drug Resistance |
| title_sort | nanosecond pef induces oxidative stress and apoptosis via proteasomal activity inhibition in gastric adenocarcinoma cells with drug resistance |
| topic | gastric adenocarcinoma drug resistance nsPEF calcium ions apoptosis oxidative stress |
| url | https://www.mdpi.com/1422-0067/23/21/12943 |
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