Cold Atmospheric Plasma: A Promising Complementary Therapy for Squamous Head and Neck Cancer

<div><p>Head and neck squamous cell cancer (HNSCC) is the 7^th most common cancer worldwide. Despite the development of new therapeutic agents such as monoclonal antibodies, prognosis did not change for the last decades. Cold atmospheric plasma (CAP) presents the most promising new technology in cancer treatment. In this study the efficacy of a surface micro discharging (SMD) plasma device against two head and neck cancer cell lines was proved. Effects on the cell viability, DNA fragmentation and apoptosis induction were evaluated with the MTT assay, alkaline microgel electrophoresis (comet assay) and Annexin-V/PI staining. MTT assay revealed that the CAP treatment markedly decreases the cell viability for all tested treatment times (30, 60, 90, 120 and 180 s). IC 50 was reached within maximal 120 seconds of CAP treatment. Comet assay analysis showed a dose dependent high DNA fragmentation being one of the key players in anti-cancer activity of CAP. Annexin-V/PI staining revealed induction of apoptosis in CAP treated HNSCC cell lines but no significant dose dependency was seen. Thus, we confirmed that SMD Plasma technology is definitely a promising new approach on cancer treatment.</p></div

Annexin V/PI Staining of HNSCC cells.

<p>Results obtained by fluorescence microscopy show that early apoptotic cells have bound Annexin-FITC to the phosphatidylserin on the membrane surface (green cell). As apoptosis progressed, the plasma membrane integrity gets lost, and the propidium iodide is able to bind to nucleotid DNA, so that late apoptotic or necrotic cells appear in red.</p

Apoptotic OSC-19 cells after different CAP treatment times.

<p>Apoptotic OSC-19 cells after different CAP treatment times.</p

Standard box-plot of apoptotic cells for FaDu cell line after different CAP treatment times.

<p>Standard box-plot of apoptotic cells for FaDu cell line after different CAP treatment times.</p

Measured cell viability reduction for different CAP treatment times.

<p>For both HNSCC cell lines the viability decreases for increasing CAP treatment times. Standard errors of the mean, depicted by whisker blots.</p

Image of the CAP device–MiniFlatPlaSter®- used in this study.

<p>Image of the CAP device–MiniFlatPlaSter®- used in this study.</p

Comet assay images after DNA-staining with ethidium bromide.

<p><b>A</b> Undamaged OSC-19 cell with intact DNA and no migration. DNA fragmentation leads to a faster and further migration into the electric field, which results in a figure shaped like a comet with undamaged DNA in the head and damaged DNA in the tail (<b>B+C</b>). The brighter and longer the tail, the higher the level of DNA fragmentation. <b>B</b> OSC-19 cell with a moderate CAP induced DNA-damage. <b>C</b> Representative image of high DNA-fragmentation.</p

Experimental setup for CAP treatment of the HNSCC cell lines OSC-19 and FaDu.

<p>Experimental setup for CAP treatment of the HNSCC cell lines OSC-19 and FaDu.</p

Detection of DNA damage (using the comet assay) in FaDu cells after different CAP treatment times.

<p>Standard box-plots (lower quartile, median, upper quartile) were used to illustrate the results. Dots denote mild statistical outliers (between 1.5 and 3 times interquartile range (IQR)); asterisks denote extreme statistical outliers (more than 3 times IQR).</p

Detection of DNA damage (using the comet assay) in OSC-19 cells after different CAP treatment times.

<p>Standard box-plots (lower quartile, median, upper quartile) were used to illustrate the results. Dots denote mild statistical outliers (between 1.5 and 3 times interquartile range (IQR)).</p