EXTH-74. INCREASING CANCER CELL MEMBRANE PERMEABILITY THROUGH APPLICATION OF TUMOR TREATING FIELDS (TTFIELDS)

Abstract INTRODUCTION Tumor Treating Fields (TTFields) are intermediate frequency, alternating electric fields with anti-mitotic effects on cancerous cells. TTFields are delivered non-invasively through arrays placed on the skin at the tumor region. TTFields therapy is approved in several territories for treatment of glioblastoma (GBM) and mesothelioma. Recently, TTFields have been shown to increase GBM cell membrane permeability. The current study aimed to explore this effect in multiple cell lines and examine the potential benefits of combining TTFields with other anticancer agents. METHODS TTFields were delivered to GBM (U-87 MG), uterine sarcoma (MES-SA), and breast adenocarcinoma (MCF-7) cell lines for 72hr across a range of frequencies (50-500kHz). Cytotoxicity of TTFields was examined by cell counts, and intracellular accumulation of 7-aminoactinomycin D (7-AAD) was measured by flow cytometry. Exposing the cells to 7-AAD at different time points relative to TTFields application cessation was used to determine the kinetics of cell membrane permeability. The potential of TTFields to facilitate intracellular accumulation of anthracycline chemotherapeutics was tested in chemotherapy-sensitive and chemotherapy-resistant cells. RESULTS Elevated intracellular accumulation of 7-AAD was observed in all examined cell lines treated with TTFields, at an optimal frequency that differed from that for maximal TTFields-induced cytotoxicity. No intracellular accumulation of 7-AAD was seen for measurements performed after termination of TTFields application, indicating that increased cell membrane permeability by TTFields was temporary and reversible. Lastly, the accumulation of chemotherapeutic agents in chemotherapy-resistant cancer cells was elevated to the same extent as in matched chemotherapy-sensitive cells when TTFields were delivered concomitant with chemotherapy. CONCLUSIONS TTFields increased cancer cell permeability in a transient and reversible manner across multiple cancer cell types. The increased permeability enhanced intracellular accumulation of chemotherapeutics, even within chemotherapy-resistant cells.

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EXTH-69. INCREASED CANCER CELL PERMEABILITY FOLLOWING TUMOR TREATING FIELDS (TTFIELDS) APPLICATION IN VITRO

Neuro-Oncology ◽

10.1093/neuonc/noaa215.423 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii102-ii102

Author(s):

Tali Voloshin ◽

Yaara Porat ◽

Noa Kaynan ◽

Anat Klein-Goldberg ◽

Rom Paz ◽

...

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

Cell Permeability ◽

Intracellular Accumulation ◽

Optimal Frequency ◽

Tumor Treating Fields ◽

Cell Counts ◽

Cellular Permeability ◽

Resistant Cells

Abstract INTRODUCTION Tumor Treating Fields (TTFields), encompassing alternating electric fields within the intermediate frequency range, is an anticancer treatment delivered to the tumor region through transducer arrays placed non-invasively on the skin. This novel loco-regional treatment has demonstrated efficacy and safety and is FDA-approved in patients with glioblastoma (GBM) and malignant pleural mesothelioma. TTFields are currently being investigated in other solid tumors in ongoing trials. Recently, TTFields were reported to alter the cellular membrane structure of GBM cells, rendering them more permeable. The objective of this study was to characterize TTFields-induced cellular permeability in cancerous cell lines. METHODS TTFields were applied to uterine sarcoma, glioblastoma, and breast adenocarcinoma cell lines across a range of frequencies (50–500 kHz). Cellular permeability was assessed by quantifying the percentages of cells with accumulated 7-aminoactinomycin D (7-AAD) using flow cytometry and cytotoxicity was assessed based on cell counts. Kinetics were determined using different 7-AAD exposure times relative to TTFields treatment end. Changes in intracellular accumulation of anthracycline chemotherapeutics were evaluated in chemotherapy-sensitive and chemotherapy-resistant cells. RESULTS TTFields induced cellular permeability to 7-AAD in all 4 cancer cell lines tested. The optimal frequency for TTFields-induced cellular permeability was different from the optimal cytotoxic frequency. Kinetics measurements demonstrated that TTFields-induced permeability is transient and is effective only during application of TTFields. In combination experiments, TTFields improved intracellular accumulation of chemotherapeutic agents. Furthermore, combining chemotherapy with TTFields treatment facilitated accumulation of chemotherapeutics in chemotherapy-resistant cells to levels comparable with accumulation in chemotherapy-sensitive cancer cells. CONCLUSIONS This study demonstrates that TTFields can transiently increase cancer cell permeability in vitro with an optimal frequency that is variable from the frequency that is used to induce cancer cell cytotoxicity. Moreover, this effect is reversible and cellular permeability is restored to a normal state upon TTFields treatment cessation.

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Cold Atmospheric Plasma Treatment of Chondrosarcoma Cells Affects Proliferation and Cell Membrane Permeability

International Journal of Molecular Sciences ◽

10.3390/ijms21072291 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2291

Author(s):

Lyubomir Haralambiev ◽

Andreas Nitsch ◽

Josephine M. Jacoby ◽

Silas Strakeljahn ◽

Sander Bekeschus ◽

...

Keyword(s):

Cell Membrane ◽

Membrane Permeability ◽

Surgical Resection ◽

Cell Lines ◽

Atp Release ◽

Cell Types ◽

Atmospheric Plasma ◽

Cell Membrane Permeability ◽

Membrane Function ◽

Cold Atmospheric Plasma

Chondrosarcoma is the second most common malign bone tumor in adults. Surgical resection of the tumor is recommended because of its resistance to clinical treatment such as chemotherapy and radiation therapy. Thus, the prognosis for patients mainly depends on sufficient surgical resection. Due to this, research on alternative therapies is needed. Cold atmospheric plasma (CAP) is an ionized gas that contains various reactive species. Previous studies have shown an anti-oncogenic potential of CAP on different cancer cell types. The current study examined the effects of treatment with CAP on two chondrosarcoma cell lines (CAL-78, SW1353). Through proliferation assay, the cell growth after CAP-treatment was determined. A strong antiproliferative effect for both cell lines was detected. By fluorescein diacetate (FDA) assay and ATP release assay, alterations in the cell membrane and associated translocation of low molecular weight particles through the cytoplasmic membrane were observed. In supernatant, the non-membrane-permeable FDA and endogenously synthesized ATP detected suggest an increased membrane permeability after CAP treatment. Similar results were shown by the dextran-uptake assay. Furthermore, fluorescence microscopic G-/F-actin assay was performed. G- and F-actin were selectively dyed, and the ratio was measured. The presented results indicate CAP-induced changes in cell membrane function and possible alterations in actin-cytoskeleton, which may contribute to the antiproliferative effects of CAP.

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EFFECTS OF HERBICIDES OF DIFFERENT MODES OF ACTION ON LEAF-CELL MEMBRANE PERMEABILITY IN PHASEOLUS VULGARIS

Canadian Journal of Plant Science ◽

10.4141/cjps80-088 ◽

1980 ◽

Vol 60 (2) ◽

pp. 613-620 ◽

Cited By ~ 4

Author(s):

JACINTA CROWLEY ◽

G. N. PRENDEVILLE

Keyword(s):

Phaseolus Vulgaris ◽

Cell Membrane ◽

Membrane Permeability ◽

Leaf Damage ◽

Cell Permeability ◽

Cell Membrane Permeability ◽

Leaf Cell ◽

Visible Injury ◽

Modes Of Action ◽

2,4 Dichlorophenoxyacetic Acid

Leakage of electrolytes from leaf discs of treated Phaseolus vulgaris L. plants was used to study the effects of several herbicides of different modes of action on leaf-cell membrane permeability. Linuron (N-(3,4-dichlorophenyl)-N-methoxy-N-methylurea), prometryne (4,6-bisisopropylamino-2-methylthio-1,3,5-triazine), bromacil (5,bromo-6-methyl-3-(1-methyl-n-propyl) uracil), sodium azide and dalapon (2,2-dichloropropionic acid) increased leaf-cell permeability at 24 h after treatment and this occurred without appearance of leaf necrosis. Glyphosate (N-(phosphonomethyl) glycine) increased leaf-cell permeability at 96 h and this was always associated with visible injury, including wilting. Paraquat (1,1-dimethyl-4,4-bipyridylium) at 10−5M increased leaf-cell permeability 48 h after treatment, without apparent leaf damage, but at higher concentrations, increased permeability was always associated with visible effects. Chlorpropham (isopropyl N-(d)3-chlorophenyl) carbamate), picloram (4-amino-3,5,6-trichloropicolinic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) did not alter cell permeability even though epinastic symptoms in leaves became evident 24 h after treatment with picloram and 2,4-D.

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R-2HG/KDM2B/RIPK1 Signaling Mediates Necroptosis in Myelodysplastic Syndromes

Blood ◽

10.1182/blood-2019-125482 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5049-5049

Author(s):

Shuanghong Zhu ◽

Chen Mei ◽

Hongyan Tong ◽

Jie Jin

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Cell Membrane ◽

Membrane Permeability ◽

Cell Lines ◽

Myelodysplastic Syndromes ◽

Poor Prognosis ◽

Cell Membrane Permeability ◽

Expression Levels ◽

Low Expression

Introduction: Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic stem cell disorders and manifested as ineffective hematopoiesis, refractory cytopenia and a propensity to evolve into acute myeloid leukemia (AML). Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in both MDS and AML. Mutated IDH produces R-2-hydroxyglutarate (R-2HG) which inhibits multiple α-ketoglutarate/α-KG-dependent dioxygenases by competing against α-KG binding. Recent studies have demonstrated that R-2-HG can abrogates leukemic growth and induce leukemia cell death. Several nonapoptotic cell death have been identified, including phosphoribosyl pyrophosphate (PPRP)-1 mediated necrosis, pyroptosis, ferroptosis and necroptosis. By now, the specific type of cell death by which R-2-HG exerts anti-tumor effects is still unknown. Results: (1) (2R)-Octyl-2-HG exhibited anti-tumor effect on SKM-1, THP-1, Molm-13, HL-60 cell lines and bone marrow mononuclear cells from MDS and AML patients in a dose- and time-dependent manner. The overexpression of IDH2 mutation induced by doxycycline significantly decreased the viability of AML cell lines, and the inhibition rate was related to the dose of doxycycline. (2R)-Octyl-2-HG promotes apoptosis and causes G0/G1 phase arrest. (2) R-2-HG leads to increased expression of RIPK1 in high-risk MDS cells. The results of gene enrichment analysis indicated that the apoptotic pathway was enriched in (2R)-Octyl-2-HG groups, and the expression of RIPK1 gene was increased in all three (SKM-1, NOMO-1 and MA9.3ITD) (2R)-Octyl-2-HG groups. After treatment with (2R)-Octyl-2-HG, the mRNA and protein expression levels of RIPK1 gene were increased. (3) R-2-HG triggers RIPK1-dependent necroptosis and occurs earlier than apoptosis. Within 10 hours after treatment with (2R)-Octyl-2-HG, cell membrane permeability was disrupted, interactions between RIPK1 and caspase 8 increased, as well as phosphorylated MLKL level. However, caspase activity did not increase significantly, suggesting that necroptosis occurs earlier than apoptosis. With RIPK1 inhibitor Necrostatin-1 and (2R)-Octyl-2-HG co-treating cells, proliferation inhibition was reduced, cell membrane permeability was more stable and RIPK1-caspase8 complex was difficult to form. The same phenomenon occurs in SKM-1 cells stably transfected with RIPK1 shRNA virus, suggesting that RIPK1-dependent necroptosis is involved in cell death caused by (2R)-Octyl-2-HG. (4) In vivo experiments demonstrated that necroptosis by R-2-HG is dependent on the expression of RIPK. In RIPK1 shRNA MDS mice, the tumor burden showed a decreasing trend, but did not show a significant change in the R-2-HG treatment group. Treatment of scramble shRNA MDS mice with R-2-HG resulted in significantly smaller tumors in the spleen and less engrafment of CD45+ cells in bone marrow. (5) Low RIPK1 expression predicts poor prognosis in MDS patients. Data from the TCGA and GEO public databases indicate that RIPK1 expression is reduced in MDS and AML patients compared to healthy controls. Survival analysis showed that patients with lower RIPK1 expression levels had significantly shorter overall survival (OS) than patients with higher RIPK1 expression levels. MDS patients with lower RIPK1 expression levels progress to leukemia more frequently. (6) Inhibition of KDM2B induces necroptosis independently. Western blot assay shows the knockdown of KDM2B, upregulation of RIPK1 and increased levels of p-MLKL. Analysis of cell numbers showed that proliferation ceased from 4 days after doxycycline treatment onwards in shKDM2B cells. Co-IP assay shows the formation of RIPK1-caspase8 complex. Conclusion: This study confirmed that R-2-HG inhibited the viability of MDS and AML cells. R-2-HG increased the expression of RIPK1 in MDS cells, inducing necroptosis. Necroptosis occurred earlier than apoptosis. Inhibition of RIPK1 can alleviate the inhibitory effect of R-2-HG on MDS and AML cells. Clinical studies have shown that low expression of the RIPK1 gene is associated with poor prognosis in patients with MDS and AML. MDS patients with low expression of RIPK1 was more likely to progress to leukemia. Inhibition of KDM2B can induce necroptosis independently. Disclosures No relevant conflicts of interest to declare.

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Sarcolemmal permeability changes during early myocardial anoxia

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084089 ◽

1978 ◽

Vol 36 (2) ◽

pp. 642-643

Author(s):

M. Ashraf ◽

L. Landa ◽

L. Nimmo ◽

C. M. Bloor

Keyword(s):

Cell Membrane ◽

Membrane Permeability ◽

Coronary Artery Occlusion ◽

Artery Occlusion ◽

Cell Membrane Permeability ◽

Enzyme Release ◽

Sprague Dawley ◽

Sprague Dawley Rats ◽

Sarcolemmal Permeability ◽

Membrane Changes

Following coronary artery occlusion, the myocardial cells lose intracellular enzymes that appear in the serum 3 hrs later. By this time the cells in the ischemic zone have already undergone irreversible changes, and the cell membrane permeability is variably altered in the ischemic cells. At certain stages or intervals the cell membrane changes, allowing release of cytoplasmic enzymes. To correlate the changes in cell membrane permeability with the enzyme release, we used colloidal lanthanum (La+++) as a histological permeability marker in the isolated perfused hearts. The hearts removed from sprague-Dawley rats were perfused with standard Krebs-Henseleit medium gassed with 95% O2 + 5% CO2. The hypoxic medium contained mannitol instead of dextrose and was bubbled with 95% N2 + 5% CO2. The final osmolarity of the medium was 295 M osmol, pH 7. 4.

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Membrane-Interacting DNA Nanotubes Induce Cancer Cell Death

Nanomaterials ◽

10.3390/nano11082003 ◽

2021 ◽

Vol 11 (8) ◽

pp. 2003

Author(s):

Samet Kocabey ◽

Aslihan Ekim Kocabey ◽

Roger Schneiter ◽

Curzio Rüegg

Keyword(s):

Drug Delivery ◽

Cell Death ◽

Cytochrome C ◽

Membrane Permeability ◽

Cancer Cell ◽

Cell Membrane Permeability ◽

Caspase Activity ◽

Cancer Cell Death ◽

Modified Dna ◽

Dna Nanotubes

DNA nanotechnology offers to build nanoscale structures with defined chemistries to precisely position biomolecules or drugs for selective cell targeting and drug delivery. Owing to the negatively charged nature of DNA, for delivery purposes, DNA is frequently conjugated with hydrophobic moieties, positively charged polymers/peptides and cell surface receptor-recognizing molecules or antibodies. Here, we designed and assembled cholesterol-modified DNA nanotubes to interact with cancer cells and conjugated them with cytochrome c to induce cancer cell apoptosis. By flow cytometry and confocal microscopy, we observed that DNA nanotubes efficiently bound to the plasma membrane as a function of the number of conjugated cholesterol moieties. The complex was taken up by the cells and localized to the endosomal compartment. Cholesterol-modified DNA nanotubes, but not unmodified ones, increased membrane permeability, caspase activation and cell death. Irreversible inhibition of caspase activity with a caspase inhibitor, however, only partially prevented cell death. Cytochrome c-conjugated DNA nanotubes were also efficiently taken up but did not increase the rate of cell death. These results demonstrate that cholesterol-modified DNA nanotubes induce cancer cell death associated with increased cell membrane permeability and are only partially dependent on caspase activity, consistent with a combined form of apoptotic and necrotic cell death. DNA nanotubes may be further developed as primary cytotoxic agents, or drug delivery vehicles, through cholesterol-mediated cellular membrane interactions and uptake.

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