Cold Atmospheric Plasma induces accumulation of lysosomes and caspase-independent cell death in U373MG glioblastoma multiforme cells

Conway, Gillian E; He, Zhonglei; Hutanu, Ana Lacramioara; Cribaro, George P.; Manaloto, Eline; Casey, Alan; Traynor, Damien; Milosavljević, Vladimir; Howe, Orla; Barcia, Carlos; Murray, James T.; Cullen, Patrick J.; Curtin, James F.

doi:10.1038/s41598-019-49013-3

Cited by 39 publications

(34 citation statements)

References 60 publications

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“…2e), indicating a high level of lipid peroxidation is induced by CAP treatment. In our previous study, the IC 50 of CAP treatment (75 kV) was determined to be 74.26 s (95% confidence range of 47.24-116.8 s) for U373MG cells 36 and we demonstrated that 60 s CAP treatment induces rapid permeabilisation of U373MG cell membranes 37 . Here, we confirm that no significant increase of PI uptake is evident following 30 s CAP treatment compared to the control (Fig.…”

Section: Cap Treatment Induces Lipid Peroxidation Ros Induces Lipid mentioning

confidence: 62%

Cold Atmospheric Plasma Stimulates Clathrin-Dependent Endocytosis to Repair Oxidised Membrane and Enhance Uptake of Nanomaterial in Glioblastoma Multiforme Cells

Liu

Scally

et al. 2020

Sci Rep

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cold atmospheric plasma (cAp) enhances uptake and accumulation of nanoparticles and promotes synergistic cytotoxicity against cancer cells. However, the mechanisms are not well understood. In this study, we investigate the enhanced uptake of theranostic nanomaterials by CAP. Numerical modelling of the uptake of gold nanoparticle into U373MG Glioblastoma multiforme (GBM) cells predicts that CAP may introduce a new uptake route. We demonstrate that cell membrane repair pathways play the main role in this stimulated new uptake route, following non-toxic doses of dielectric barrier discharge CAP. CAP treatment induces cellular membrane damage, mainly via lipid peroxidation as a result of reactive oxygen species (ROS) generation. Membranes rich in peroxidised lipids are then trafficked into cells via membrane repairing endocytosis. We confirm that the enhanced uptake of nanomaterials is clathrindependent using chemical inhibitors and silencing of gene expression. Therefore, CAP-stimulated membrane repair increases endocytosis and accelerates the uptake of gold nanoparticles into U373MG cells after CAP treatment. We demonstrate the utility of CAP to model membrane oxidative damage in cells and characterise a previously unreported mechanism of membrane repair to trigger nanomaterial uptake. This knowledge will underpin the development of new delivery strategies for theranostic nanoparticles into cancer cells. Cold atmospheric plasma (CAP) is increasingly studied in a growing number of clinical trials for cancer treatment 1,2 and research is ongoing to explore the combination of CAP with other therapies, including nanoparticles, radiotherapy and chemotherapy 3-5. Gold nanoparticles (AuNPs) are known to be weakly-toxic to human cells and be readily manufactured and designed for targeting delivery of various therapeutic compounds into cells. Citrate-capped cationic AuNPs may adsorb serum proteins onto their surface and thereby stimulate receptor-mediated endocytosis 6. Without special surface functionalisation, AuNPs enter cells and become trapped in vesicles 6-8 or enter the nucleus, depending on their size/shape 9,10. Meanwhile, AuNPs with functionalised surface chemistries/ligands can directly penetrate the membrane and enter the cytoplasm 11 .

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Section: Cap Treatment Induces Lipid Peroxidation Ros Induces Lipid mentioning

confidence: 62%

Cold Atmospheric Plasma Stimulates Clathrin-Dependent Endocytosis to Repair Oxidised Membrane and Enhance Uptake of Nanomaterial in Glioblastoma Multiforme Cells

Liu

Scally

et al. 2020

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“…The generated RONS in the CAP-treated culture medium, including hydrogen peroxide, nitrite and nitrate, were also quantitatively measured (As seen in Figure 2c). By comparison with our previous study 38,42 , after the same low does CAP treatment (30 s, 75 kV), the levels of generated H2O2, NO -2, and NO -3 in culture medium are relatively low cytotoxic to U373MG cells, but still are able to induce lipid peroxidation.…”

Section: Discussionmentioning

confidence: 46%

“…It has been shown that CAP treatment can alter membrane structures, which may be partly due to the reactive species-caused lipid peroxidation 42,51,53,54 . However, the cell membrane remains PI impermeable after exposing to 75 kV CAP for 30 s, which demonstrates that the oxidised membrane may remain mechanically intact and the U373MG cells retain viability after a low dose of CAP treatment (30 s, 75 kV) (Figure 2f), which aligns with the results indicating low concentration of RONS in the CAP-treated medium (Figure 2c).…”

Section: Discussionmentioning

confidence: 99%

“…The IC50 of CAP treatment (75 kV) was determined to be 74.26 s (95% confidence range of 47.24-116.8 s) for U373MG cells 33 . We have previously determined that 60 s CAP treatment induces rapid permeabilisation of U373MG cell membranes 42 . Figure 2f demonstrated no significant increase of PI uptake following 30 s CAP treatment compared to the control, which indicated that the permeability of the cell membrane to PI, an indicator of Figure S2).…”

Section: Cap Treatment Induces Lipid Peroxidationmentioning

confidence: 99%

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Low dose Cold Atmospheric Plasma induces membrane oxidation, stimulates endocytosis and enhances uptake of nanomaterials in Glioblastoma multiforme cells

Liu

Scally

et al. 2019

Preprint

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Cold atmospheric plasma (CAP) has demonstrated synergistic cytotoxic effects with nanoparticles, especially promoting the uptake and accumulation of nanoparticles inside cells.However, the mechanisms driving the effects need to be explored. In this study, we investigate the enhanced uptake of theranostic nanomaterials by CAP. Numerical modelling of the uptake of gold nanoparticle into U373MG Glioblastoma multiforme (GBM) cells predicts that CAP may introduce a new uptake route. We demonstrate that cell membrane repair pathways play the main role in this stimulated new uptake route, following non-toxic doses of dielectric barrier discharge CAP (30 s, 75 kV). CAP treatment induces cellular membrane damage, mainly via lipid peroxidation as a result of reactive oxygen species (ROS) generation. Membranes rich in peroxidated lipids are then trafficked into cells via membrane repairing endocytosis. We confirm that the enhanced uptake of nanomaterials is clathrin-dependent using chemical inhibitors and silencing of gene expression. Therefore, CAP-stimulated membrane repair increases endocytosis and accelerates the uptake of gold nanoparticles into U373MG cells after CAP treatment. Our data demonstrate the utility of CAP to model membrane oxidative damage in cells and characterise a previously unreported mechanism of membrane repair to trigger nanomaterial uptake which will be useful for developing more efficient deliveries of nanoparticles and pharmaceuticals into cancer cells for tumour therapy and diagnosis. This mechanism of RONS-induced endocytosis will also be of relevance to other cancer therapies that induce an increase in extracellular RONS.

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“…Figure 6c demonstrates co-localisation, as depicted in blue in the merged panel. As previously described, AO also binds with single-stranded nucleic acids and emits orange fluorescents which can be seen in both the merged panel of both the untreated control and UA treated cells (Conway et al, 2019).…”

Section: Ua Triggers the Formation Of Lysosomesmentioning

confidence: 57%

Ursolic acid inhibits cell migration and promotes JNK-dependent lysosomal associated cell death in Glioblastoma multiforme cells

Conway

Zizyte

Mondala

et al. 2020

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Running title: Ursolic Acid is Pro-Apoptotic and Anti-Invasive in GBM Abbreviations: UA, Ursolic acid; GBM, Glioblastoma Multiforme; TMZ, temozolomide; 3-MA, 3methyladenine. AbstractUrsolic acid (UA) is a bioactive compound which has demonstrated therapeutic efficacy in a variety of cancer cell lines. UA activates various signalling pathways in Glioblastoma multiforme (GBM), however, the relationship between cell death and migration has yet to be elucidated. UA induces a dose dependent cytotoxic response demonstrated by flow cytometry and biochemical cytotoxicity assays. Inhibitor and fluorescent probe studies demonstrated that UA induces a caspase independent, JNK dependent, mechanism of cell death. Migration studies established that UA inhibits GBM cell migration in a time dependent manner that is independent of the JNK signalling pathway. The cytotoxic insult induced by UA resulted in the formation of acidic vesicle organelles (AVOs), speculating activation of autophagy. However, inhibitor and spectrophotometric analysis demonstrated that autophagy was not responsible for the formation of the AVOs and confocal microscopy identified the AVO's as lysosomes. Further investigation using isosurface visualisation of confocal imaging determined co-localisation of lysosomes with the previously identified acidic vesicles, thus providing evidence that lysosomes are likely to be playing a role in UA induced cell death.Collectively, our data identifies that UA rapidly induces a lysosomal associated mechanism of cell death in addition to UA acting as an inhibitor of GBM cell migration.

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Cold Atmospheric Plasma induces accumulation of lysosomes and caspase-independent cell death in U373MG glioblastoma multiforme cells

Cited by 39 publications

References 60 publications

Cold Atmospheric Plasma Stimulates Clathrin-Dependent Endocytosis to Repair Oxidised Membrane and Enhance Uptake of Nanomaterial in Glioblastoma Multiforme Cells

Cold Atmospheric Plasma Stimulates Clathrin-Dependent Endocytosis to Repair Oxidised Membrane and Enhance Uptake of Nanomaterial in Glioblastoma Multiforme Cells

Low dose Cold Atmospheric Plasma induces membrane oxidation, stimulates endocytosis and enhances uptake of nanomaterials in Glioblastoma multiforme cells

Ursolic acid inhibits cell migration and promotes JNK-dependent lysosomal associated cell death in Glioblastoma multiforme cells

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