Cell confluence as crucial regulator of ROS-induced toxicity: Analysis of t-BuOOH-induced cell death in vitro
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Abstract
Oxidative stress due to excessive intracellular ROS formation can result in macromolecule damage and cell death. In vitro, ROS-induced apoptosis has been almost exclusively investigated in proliferating cells, although in vivo, cells establish cell-cell contacts and reside in the G1 cell cycle phase. Previous studies indicated that contact-inhibited compared to proliferating murine fibroblasts (NIH3T3) are resistant against t-BuOOH – a lipid ROS inducer, resembling oxidative stress in, e.g. neurodegenerative diseases and cancer. A role of cell-cell contacts in the resistance against ROS-induced cell death independent of a G1 arrest is so far not known.
Resistance of early confluent versus semiconfluent NIH3T3 cells and human epithelial cells (HaCaT, Caco-2) against t-BuOOH-induced necrosis was proved via setup I+II independent of a G1 arrest. Vitality assays, flow cytometry, Western Blot analysis and laser scanning microscopy demonstrated that (i) confluent cells proliferated at the time point of t-BuOOH exposure, and (ii) semiconfluent cultures (serum-depleted; U0126-treated) that exhibited a similar amount of cells in G1 as confluent cultures, were comparably sensitive to t-BuOOH as semiconfluent, proliferating cultures. Increased detoxification of t-BuOOH due to a higher cell number in confluent cultures was excluded via setup II. Further analyses and comet assays revealed a similar (i) cytosolic ROS increase, (ii) basal GSH level, (iii) replication block, as well as (iv) DNA SSB formation / repair in confluent and semiconfluent NIH3T3 cells. Confluent cultures were in contrast protected against (i) NADH oxidation, (ii) a decrease in ATP, (iii) collapse of the MMP (∆ψm), and (iv) an increase in DNA DSBs in the presence of t-BuOOH. Further experiments identified (i) t-BuOOH as a novel inducer of lipid ROS-induced ferroptosis, (ii) a prosurvival role of p38, JNK and ATR, as well as (iii) Ca2+ as crucial mediator of ferroptosis and cellular damage.
In summary, these findings present cell-cell contacts and Ca2+ as crucial regulators of t-BuOOH-induced ferroptosis in vitro and might help to develop therapies against ROS-induced diseases or multicellular resistance of solid tumors.