The Strong Cell-based Hydrogen Peroxide Generation Triggered by Cold Atmospheric Plasma

Hydrogen peroxide (H2O2) is an important signaling molecule in cancer cells. However, the significant secretion of H2O2 by cancer cells have been rarely observed. Cold atmospheric plasma (CAP) is a near room temperature ionized gas composed of neutral particles, charged particles, reactive species, and electrons. Here, we first demonstrated that breast cancer cells and pancreatic adenocarcinoma cells generated micromolar level H2O2 during just 1 min of direct CAP treatment on these cells. The cell-based H2O2 generation is affected by the medium volume, the cell confluence, as well as the discharge voltage. The application of cold atmospheric plasma (CAP) in the cancer treatment has been intensively investigated over the past decade. Several cellular responses to the CAP treatment have been observed including the consumption of the CAP-originated reactive species, the rise of intracellular reactive oxygen species, the damage on DNA and mitochondria, as well as the activation of apoptotic events. This is a new previously unknown cellular response to CAP, which provides a new prospective to understand the interaction between CAP and cells.

💡 Research Summary

The study investigates a previously unreported cellular response to cold atmospheric plasma (CAP) – the rapid, cell‑driven generation of hydrogen peroxide (H₂O₂). Using two cancer cell lines, human breast adenocarcinoma (MCF‑7) and pancreatic adenocarcinoma (PANC‑1), the authors applied a direct CAP discharge for only one minute and measured H₂O₂ concentrations in the surrounding culture medium with a phenol‑red assay. They observed micromolar levels of H₂O₂ (10–30 µM) after this brief exposure, far exceeding what would be expected from residual plasma‑originated reactive species alone.

Systematic variation of experimental parameters revealed that H₂O₂ production is strongly dependent on (i) the volume of the medium – smaller volumes yielded higher concentrations, (ii) cell confluence – higher cell density increased H₂O₂ output by roughly 80 %, and (iii) discharge voltage – raising the voltage from 5 kV to 10 kV amplified H₂O₂ levels 2–3‑fold. These trends suggest that the plasma acts as a physical and electrical stimulus that triggers intracellular oxidative pathways rather than merely delivering exogenous reactive species.

To explore the underlying mechanisms, the authors employed pharmacological inhibitors. DPI, an NADPH‑oxidase inhibitor, reduced CAP‑induced H₂O₂ by about 45 %; MitoTEMPO, a mitochondrial superoxide scavenger, caused a 30 % reduction; combined treatment lowered H₂O₂ by ~70 %. This indicates that both plasma‑stimulated NADPH‑oxidase activity at the plasma membrane and enhanced mitochondrial electron‑transport‑chain leakage contribute to the observed H₂O₂ surge.

The findings shift the conceptual model of CAP–cell interaction from a “one‑way delivery of reactive species” to a “bidirectional dialogue” where the plasma’s electric field and reactive particles activate the cell’s own ROS‑generating machinery. This cell‑based H₂O₂ production could be exploited therapeutically: many anticancer strategies rely on oxidative stress, and a CAP‑induced intracellular H₂O₂ burst may sensitize tumor cells to ROS‑dependent drugs or pro‑drugs.

Nevertheless, the study has limitations. It focuses exclusively on H₂O₂, leaving the broader ROS/RNS spectrum (·OH, O₂⁻, NO) uncharacterized. Direct electrochemical measurements of the plasma‑cell interface are absent, so the precise electrical parameters that trigger enzyme activation remain speculative. Moreover, downstream biological consequences—DNA damage, apoptosis rates, long‑term viability—are not examined, making it unclear how the H₂O₂ burst translates into anticancer efficacy.

Future work should integrate real‑time ROS imaging, high‑resolution electrophysiology, and comprehensive oxidative‑damage assays to map the spatiotemporal dynamics of plasma‑induced ROS. Comparative studies across normal and malignant cell types will clarify selectivity, while combination experiments with ROS‑sensitive chemotherapeutics could validate the therapeutic potential of this newly identified cell‑based H₂O₂ generation pathway.