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

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

doi:10.1038/s41598-019-49013-3

Abstract

Room temperature Cold Atmospheric Plasma (CAP) has shown promising efficacy for the treatment of cancer but the exact mechanisms of action remain unclear. Both apoptosis and necrosis have been implicated as the mode of cell death in various cancer cells. We have previously demonstrated a caspase-independent mechanism of cell death in p53-mutated glioblastoma multiforme (GBM) cells exposed to plasma. The purpose of this study was to elucidate the molecular mechanisms involved in caspase-independent cell death induced by plasma treatment. We demonstrate that plasma induces rapid cell death in GBM cells, independent of caspases. Accumulation of vesicles was observed in plasma treated cells that stained positive with acridine orange. Western immunoblotting confirmed that autophagy is not activated following plasma treatment. Acridine orange intensity correlates closely with the lysosomal marker Lyso TrackerTM Deep Red. Further investigation using isosurface visualisation of confocal imaging confirmed that lysosomal accumulation occurs in plasma treated cells. The accumulation of lysosomes was associated with concomitant cell death following plasma treatment. In conclusion, we observed rapid accumulation of acidic vesicles and cell death following CAP treatment in GBM cells. We found no evidence that either apoptosis or autophagy, however, determined that a rapid accumulation of late stage endosomes/lysosomes precedes membrane permeabilisation, mitochondrial membrane depolarisation and caspase independent cell death.

Highlights

Glioblastoma multiforme (GBM), a grade IV malignant astrocytoma, is highly proliferative, invasive and resistant to most therapies
The current study aims to investigate further the triggers involved in activation of CICD and the biochemical alterations evident when reactive oxygen species (ROS)-resistant glioblastoma multiforme (GBM) cells are exposed to toxic doses of plasma
Our results indicate that ROS-dependent activation of apoptosis is not necessary for cell death when U373MG GBM cells are exposed to Cold Atmospheric Plasma (CAP)

Summary

Introduction

Glioblastoma multiforme (GBM), a grade IV malignant astrocytoma, is highly proliferative, invasive and resistant to most therapies. Many studies have shown that plasma can induce a cytotoxic response in vitro in a variety of cell lines, for example glioblastoma, cervical, breast, colorectal, and lung[11,12,13,14,15], of which the cell death mechanisms have been reported as apoptosis[16,17], cell cycle arrest[18,19], autophagy[20] and necrosis[20] depending on the tumour model studied and the plasma device/system used This is not unexpected given the wide array of chemical and physical alterations that plasma can induce in cells and the interconnectivity of initiation and signal transduction between different subtypes of cell death. A form of necrosis that is energy-dependent and regulated in cells has been discovered and is known as necroptosis, suggesting that most forms of physiological cell death are energy dependent involving intracellular and sometimes extracellular signal transduction

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