[Paper Review] The Strong Cell-based Hydrogen Peroxide Generation Triggered by Cold Atmospheric Plasma

Dayun Yan, Haitao Cui+5 more|arXiv (Cornell University)|May 28, 2017

Plasma Applications and Diagnostics49 references19 citations

TL;DR

This study demonstrates that cold atmospheric plasma (CAP) induces rapid, micromolar-level hydrogen peroxide (H2O2) generation in breast cancer and pancreatic adenocarcinoma cells within just 1 minute of direct treatment. The cell-based H2O2 production is modulated by medium volume, cell confluence, and discharge voltage, revealing a previously unknown cellular response to CAP with implications for cancer therapy mechanisms.

ABSTRACT

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.

Motivation & Objective

• To investigate whether cold atmospheric plasma (CAP) induces endogenous hydrogen peroxide (H2O2) generation in cancer cells.
• To determine the conditions under which cell-based H2O2 production is maximized, including medium volume and cell confluence.
• To explore the role of CAP parameters such as discharge voltage in modulating H2O2 output.
• To identify a novel cellular response to CAP that may contribute to its anti-cancer effects.
• To provide mechanistic insight into CAP-induced oxidative stress in cancer cells through endogenous H2O2 production.

Proposed method

• Direct application of cold atmospheric plasma (CAP) to monolayer cultures of MDA-MB-231 breast cancer and BxPC-3 pancreatic adenocarcinoma cells.
• Measurement of extracellular H2O2 levels using a fluorometric assay with Amplex Red reagent.
• Systematic variation of medium volume (100–500 µL) and cell confluence (20–100%) to assess their effects on H2O2 generation.
• Adjustment of discharge voltage (10–20 kV) to evaluate its influence on H2O2 production kinetics.
• Use of a helium-based CAP device operating at near-ambient temperature to minimize thermal damage.
• Quantitative analysis of H2O2 production over time, with peak levels recorded after 1 minute of exposure.

Experimental results

Research questions

• RQ1 Does cold atmospheric plasma induce endogenous hydrogen peroxide (H2O2) production in cancer cells?
• RQ2 How does the volume of culture medium affect CAP-induced H2O2 generation in adherent cancer cells?
• RQ3 What is the impact of cell confluence on the magnitude of cell-based H2O2 production following CAP treatment?
• RQ4 How does the discharge voltage of the CAP device influence the rate and level of H2O2 generation?
• RQ5 Is there a previously unreported cellular response to CAP involving endogenous H2O2 production?

Key findings

• Breast cancer cells (MDA-MB-231) produced up to 1.2 µM of H2O2 within 1 minute of direct CAP treatment.
• Pancreatic adenocarcinoma cells (BxPC-3) generated up to 1.5 µM of H2O2 in the same time frame, indicating strong cell-based H2O2 production.
• H2O2 generation was significantly reduced when medium volume increased from 100 µL to 500 µL, suggesting diffusion or quenching effects.
• Higher cell confluence (100%) led to greater H2O2 output compared to lower confluence (20%), indicating cell density dependence.
• Increased discharge voltage (from 10 to 20 kV) enhanced H2O2 production, confirming a dose-dependent response.
• The observed H2O2 levels were attributed to endogenous cellular production rather than plasma-derived species alone, indicating a novel cellular response.