The apoptotic effect of ozone therapy on mitochondrial activity of highly metastatic breast cancer cell line MDA-MB-231 using in vitro approaches

Background: looking for selective ways to slow aggressive breast cancer cells

MDA-MB-231 is a widely studied human breast cancer cell line. It is triple-negative and highly metastatic, meaning it lacks the receptors many standard breast cancer therapies target and spreads readily in the laboratory. Because these cells are difficult to treat, researchers often use them to screen agents that might slow their growth. Ozone, a reactive form of oxygen used for more than a century as a disinfectant because of its strong oxidising effect, has been examined for a range of effects on living tissue. This laboratory study set out to test, in a controlled dish environment, how measured doses of ozone behave against these breast cancer cells and against normal cells.

Study design: measured ozone doses applied to cultured cancer and normal cells

This was an in vitro study, meaning all work was carried out on cells grown in culture rather than in animals or people. The researchers used two cell types: MDA-MB-231 breast cancer cells and HUVEC cells (human umbilical vein endothelial cells), which are normal cells used as a healthy comparison. Ozone was applied across a range of concentrations, roughly 5 to 20 micrograms per millilitre. The team then assessed the cells using several standard laboratory methods: viability testing to measure how many cells survived, Annexin V and propidium iodide staining read by flow cytometry to detect apoptosis (programmed cell death), cell cycle analysis, a migration assay to gauge how readily the cells moved, and RT-PCR to measure the expression of apoptosis-related genes. Mitochondrial activity, a marker of the cells' energy state, was also examined.

Findings: dose-dependent cell death in the cultured cancer cells, little effect on the normal cells

The researchers reported that ozone reduced the growth of the MDA-MB-231 breast cancer cells in a dose-dependent way, with stronger effects at higher concentrations. At around 10 micrograms per millilitre, ozone increased the expression of apoptotic genes and raised the proportion of cells undergoing programmed cell death. The treated cancer cells also showed reduced migration potential in the dish. By contrast, the normal HUVEC cells showed little cytotoxicity at the doses tested, suggesting the effect on the cancer cells was more pronounced than on the healthy comparison cells under these specific laboratory conditions.

What the authors concluded

The authors concluded that, in this cell-culture setting, a specific dose of ozone could trigger apoptosis and enhance apoptotic gene expression in the breast cancer cells while causing little to no cytotoxicity in the normal cells. They described ozone as a promising avenue worth further investigation for cancer research. These conclusions relate strictly to cultured cells in the laboratory, not to treatment outcomes in patients.

Limitations of this study

This was an in vitro study using a single breast cancer cell line and one type of normal cell, tested over a narrow range of ozone doses and a short timeframe. Cells grown in a dish do not reproduce the complexity of a living body, including its blood supply, immune system and the way tissues interact, so results seen in culture do not necessarily carry over to animals or people. The specific mechanisms and the safety of any applied dose would need to be confirmed in further laboratory, animal and ultimately clinical research before any conclusions about people could be drawn. This study concerns ozone's effects on cultured breast cancer cells in the laboratory. It does not establish ozone as a treatment or cure for breast cancer itself. Ozone therapy is a complementary approach and should always be discussed with your oncology team.