Non-thermal plasma induces a stress response in mesothelioma cells resulting in increased endocytosis, lysosome biogenesis and autophagy

Our plain-English summary of Non-thermal plasma induces a stress response in mesothelioma cells resulting in increased endocytosis, lysosome biogenesis and autophagy, by L. Shi, F. Ito, Y. Wang, et al (Free Radical Biology & Medicine, 2017).

The study

• Authors: L. Shi, F. Ito, Y. Wang, Y. Okazaki, H. Tanaka, M. Mizuno, M. Hori, T. Hirayama, H. Nagasawa, D.R. Richardson, S. Toyokuni
• Journal: Free Radical Biology & Medicine
• Published: 2017;108:904-917
• Cited by: 68 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Non-thermal plasma (NTP) is a potential new therapeutic modality for cancer. However, its mechanism of action remains unclear. Herein, we studied the effect of NTP on mesothelioma cells and fibroblasts to understand its anti-proliferative efficacy. Interestingly, NTP demonstrated greater selective anti-proliferative activity against mesothelioma cells relative to fibroblasts than cisplatin, which is used for mesothelioma treatment. The anti-proliferative effect of NTP was enhanced by pre-incubation with the cellular iron donor, ferric ammonium citrate (FAC), and inhibited by iron chelation using desferrioxamine (DFO). Three oxidative stress probes (CM-H2DCFDA, MitoSOX and C11-BODIPY) demonstrated reactive oxygen species (ROS) generation by NTP, which was inhibited by DFO. Moreover, NTP decreased transferrin receptor-1 and increased ferritin-H and -L chain expression that was correlated with decreased iron-regulatory protein expression and RNA-binding activity. This regulation was potentially due to increased intracellular iron in lysosomes, which was demonstrated via the Fe(II)-selective probe, HMRhoNox-M, and was consistent with autophagic-induction. Immunofluorescence using LysoTracker and Pepstatin A probes demonstrated increased cellular lysosome content, which was confirmed by elevated LAMP1 expression. The enhanced lysosomal biogenesis after NTP could be due to the observed increase in fluid-phase endocytosis and early endosome formation. These results suggest NTP acts as a stressor, which results in increased endocytosis, lysosome content and autophagy. In fact, NTP rapidly increased autophagosome formation, as judged by increased LC3B-II expression, which co-localized with LAMP1, indicating autophagolysosome formation. Autophagic-induction by NTP was confirmed using electron microscopy. In summary, NTP acts as a cellular stressor to rapidly induce fluid-phase endocytosis, lysosome biogenesis and autophagy.

Copyright © 2017 Elsevier Inc. All rights reserved.

In plain English

This laboratory study used cells in dishes, not people. The authors tested non-thermal plasma (NTP), an activated argon gas proposed as a cancer therapy, on fibroblasts and on two malignant mesothelioma cell lines, SM2 and EM2, established from rats injected with asbestos. The cancer drug cisplatin showed little selectivity: its IC50 was 3.4 ± 1.5 µM in EM2 cells and 10.0 ± 1.2 µM in SM2 cells, against 9.9 ± 1.2 µM in IMR 90SV fibroblasts. NTP for 60 to 120 seconds slowed mesothelioma cell growth more than fibroblast growth. Argon gas alone did not significantly affect SM2 growth. In separate SM2 experiments, iron boosted a 30-second NTP treatment, and the iron-binding drug DFO at 12.5 µM totally blocked a 90-second treatment's effect. In SM2 cells, NTP increased oxidation of two reactive-oxygen-species probes 8- to 16-fold over untreated cells; in fibroblasts, probe signals rose 1.3- to 2-fold. NTP also increased markers of lysosomes and autophagy, cells digesting their own parts. The authors concluded that NTP acts as a stress stimulus increasing endocytosis, lysosome formation and autophagy.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) cell-culture experiments
• Who was studied: Rat malignant mesothelioma cell lines SM2 (sarcomatoid sub-type) and EM2 (epithelioid sub-type), established from rats injected with asbestos. Comparison cells were the human fibroblast cell line IMR 90SV and the rat fibroblast cell line Rat-1. No human subjects were studied.
• Exposure: Non-thermal plasma generated from argon gas, applied for 30 to 120 seconds. Comparisons were cisplatin, argon gas alone, the iron donor ferric ammonium citrate (FAC) and the iron chelator desferrioxamine (DFO). Asbestos was used only to create the rat mesothelioma cell lines and was not itself studied.
• Main results:
  • Cisplatin showed little selectivity. IC50 was 3.4 ± 1.5 µM in EM2 mesothelioma cells and 10.0 ± 1.2 µM in SM2 mesothelioma cells, compared with 9.9 ± 1.2 µM in IMR 90SV fibroblasts.
  • NTP treatments of 60, 90 and 120 seconds had greater anti-proliferative activity against both mesothelioma cell lines than against IMR 90SV fibroblasts (p less than 0.001).
  • In SM2 cells, argon gas alone (without electrical activation) had no significant effect on cell proliferation.
  • In SM2 cells, pre-loading with iron (FAC) significantly increased the anti-proliferative effect of a 30-second NTP treatment.
  • In a separate experiment in SM2 cells, the iron chelator DFO at 12.5 µM totally inhibited the anti-proliferative activity of a 90-second NTP treatment.
  • In SM2 cells, NTP produced an 8-16-fold increase in oxidation of two reactive oxygen species probes (CM-H2DCFDA and C11-BODIPY) compared with untreated control cells, and a 2-fold increase with the MitoSOX probe.
  • Measured with the three probes, IMR 90SV fibroblasts showed the lowest fold-change after NTP (1.3-2-fold relative to control) of the three cell types.
  • In SM2 cells, NTP significantly decreased the iron-regulatory proteins IRP1 (from 4 to 24 hours) and IRP2 (from 0.5 to 24 hours).
  • In SM2 cells, co-localization of a ferrous iron probe with a lysosome marker increased after NTP, with the coefficient rising from 0.75 to 0.83.
  • In SM2 cells, the autophagy marker LC3B-II rose significantly from 0.5 hours after NTP, peaked at 2 hours, then decreased.
  • The LC3B-II autophagy marker increased more in the mesothelioma cells than in the fibroblasts after NTP.
  • Electron microscopy of NTP-treated SM2 cells showed a pronounced increase in autophagic vacuoles.
• The authors' conclusion: The authors concluded that NTP acts as a stress stimulus that increases endocytosis, early endosome and lysosome formation, and autophagy. They linked this to reactive oxygen species generation that depends on iron inside the cells.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelioma, Fibroblast, Cisplatin, Oxidative Stress, Reactive Oxygen Species (ROS), Ferritin.

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