NAC, tiron and trolox impair survival of cell cultures containing glioblastoma tumorigenic initiating cells by inhibition of cell cycle progression

Our plain-English summary of NAC, tiron and trolox impair survival of cell cultures containing glioblastoma tumorigenic initiating cells by inhibition of cell cycle progression, by M. Monticone, R. Taherian, S. Stigliani, et al (PloS One, 2014).

The study

• Authors: M. Monticone, R. Taherian, S. Stigliani, E. Carra, S. Monteghirfo, L. Longo, A. Daga, M. Dono, S. Zupo, W. Giaretti, P. Castagnola
• Journal: PloS One
• Published: 2014;9(2):e90085
• Cited by: 23 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Reactive oxygen species (ROS) are metabolism by-products that may act as signaling molecules to sustain tumor growth. Antioxidants have been used to impair cancer cell survival. Our goal was to determine the mechanisms involved in the response to antioxidants of a human cell culture (PT4) containing glioblastoma (GBM) tumorigenic initiating cells (TICs). ROS production in the absence or presence of N-acetyl-L-cysteine (NAC), tiron, and trolox was evaluated by flow cytometry (FCM). The effects of these antioxidants on cell survival and apoptosis were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) and FCM. The biological processes modulated by these drugs were determined by oligonucleotide microarray gene expression profiling. Our results showed that NAC, tiron and trolox impaired PT4 cell survival, had minor effects on ROS levels and caused wide deregulation of cell cycle genes. Furthermore, tiron and trolox caused inhibition of cell survival in two additional cell cultures containing TICs, FO-1 and MM1, established from a melanoma and a mesothelioma patient, respectively. NAC, instead, impaired survival of the MM1 cells but not of the FO-1 cells. However, when used in combination, NAC enhanced the inhibitory effect of PLX4032 (BRAF V600E inhibitor) and Gefitinib (EGFR inhibitor), on FO-1 and PT4 cell survival. Collectively, NAC, tiron and trolox modulated gene expression and impaired the growth of cultures containing TICs primarily by inhibiting cell cycle progression.

In plain English

This laboratory study of cultured human cancer cells in Genoa, Italy, did not study asbestos exposure or people. The authors tested the antioxidant drugs NAC, tiron and trolox mainly on PT4, a glioblastoma (brain tumor) culture containing tumor-initiating cells. They also tested cultures from a human malignant mesothelioma (MM1) and a melanoma (FO-1). Glioblastoma cultures produced more global reactive oxygen species than normal human astrocytes. Against a second astrocyte lot, PT2 and PT4 gave a lower mitochondrial superoxide signal. The authors said this may be explained by that lot's higher mitochondrial mass. All three drugs reduced PT4 survival as the dose rose. They changed oxygen-species levels only slightly and caused only a modest rise in apoptosis. Cell cycle genes changed widely; tiron first affected genes responding to oxygen levels. In six-day tests, all three drugs reduced mesothelioma-cell survival dose-dependently. In melanoma cells, tiron and trolox did but NAC did not. In longer tests at the lowest and intermediate doses, only tiron did. The authors concluded that the drugs impaired cell survival by inhibiting cell cycle progression, unrelated in their experiments to the cells' redox balance.

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) experiments on human tumor-derived cell cultures, using cell survival assays, flow cytometry, gene expression microarrays, qPCR and immunoblots
• Who was studied: Long-term cell cultures containing glioblastoma tumor-initiating cells, mainly PT4 (also PT2, PT1, PT5), from surgical samples at San Martino Hospital, Genoa. The authors also used normal human astrocytes, a malignant mesothelioma-derived culture (MM1) and a melanoma culture (FO-1).
• Exposure: The antioxidant drugs N-acetyl-L-cysteine (NAC), tiron and trolox. For PT4 cells the doses were 3 mM NAC, 0.7 mM tiron and 1.8 mM trolox for 6 days, with the drug-containing medium changed every other day. Some experiments also used PLX4032 (a BRAF V600E inhibitor) and gefitinib (an EGFR inhibitor). No asbestos exposure was studied.
• Main results:
  • All cultures containing glioblastoma tumor-initiating cells produced more global reactive oxygen species and more mitochondrial superoxide than the normal astrocyte culture NA1.
  • Compared with a second lot of normal astrocytes (NA2), PT2 and PT4 produced more global reactive oxygen species but showed lower mitochondrial superoxide. The authors noted that NA2 had a higher mitochondrial mass, which may explain its higher superoxide signal.
  • In PT4 cells, NAC, tiron and trolox each inhibited cell survival in a dose-dependent manner.
  • After 6 days with the drugs, PT4 cells showed only a modest increase in early and late apoptotic cells compared with controls.
  • After 48 hours, NAC increased the share of PT4 cells in the G0/G1 phase of the cell cycle. Tiron increased the share in the S and G2/M phases. Trolox did not change the cell cycle phases in PT4.
  • In PT4 cells the drugs caused only slight changes in oxygen-species levels. All three slightly reduced the mitochondrial superoxide signal. Only trolox reduced the global oxygen-species signal; NAC and tiron slightly increased it.
  • At 48 hours, NAC and trolox deregulated cell cycle genes in PT4 cells (171 genes for NAC and 244 for trolox in the 'cell cycle' category). Tiron instead affected genes for response to oxygen levels, glycolysis and angiogenesis. At 6 days, tiron deregulated cell cycle genes (178 genes).
  • In the six-day test, the mesothelioma-derived MM1 cells showed dose-dependent reduced survival after treatment with all three drugs.
  • In the six-day test on melanoma FO-1 cells, tiron and trolox reduced survival, but NAC did not cause major changes up to the 18 mM dose.
  • In longer incubations using the lowest and intermediate concentrations from the dose-response tests, FO-1 survival was reduced by tiron at 0.33 mM and 1 mM, but not by NAC or trolox. The authors suggested the FO-1 cells may develop resistance to trolox.
  • NAC further inhibited the FO-1 cell survival reduction caused by PLX4032. In PT4 cells, combining each antioxidant with gefitinib gave a statistically significant extra reduction in survival: very minor with tiron, moderate with trolox and strong with NAC.
• The authors' conclusion: The authors concluded that NAC, tiron and trolox acted as wide modulators of gene expression and impaired cell survival by inhibiting cell cycle progression. They also concluded that in their experimental context these effects were not related to modulation of cell redox balance.

Topics

Medical terms in this study, explained in our Lexicon: Reactive Oxygen Species (ROS), Apoptosis, Mesothelioma, BRAF V600E, EGFR.

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