Selective adhesion inhibition and hyaluronan envelope reduction of dermal tumor cells by cold plasma-activated medium

The sensitivity to cold plasma is specific to tumor cells while leaving normal tissue cells unaffected. This is the desired challenge in cancer therapy. Therefore, the focus of this work was a comparative study concerning the plasma sensitivity of dermal tumor cells (A-431) versus non-tumorigenic dermal cells (HaCaT) regarding their adhesion capacity. We found a selective inhibiting effect of plasma-activated medium on the adhesion of tumor cells while hardly affecting normal cells. We attributed this to a lower basal gene expression for the adhesion-relevant components CD44, hyaluronan synthase 2 (HAS2), HAS3, and the hyaluronidases in A431. Noteworthy, after plasma exposure, we revealed a significantly higher expression and synthesis of the hyaluronan envelope, the HAS3 gene, and the transmembrane adhesion receptors in non-tumorigenic HaCaTs.</p

Argon plasma treatment and medium temperature.

<p>The average temperature in DMEM during plasma treatment up to 120 s is 25°C and remains constant up to a plasma treatment time of 180 s. Thus, negative thermal effects in the plasma-treated cell culture medium DMEM are excluded.</p

Online-monitoring of cell adhesion of living mHepR1 cells.

<p>Note that complete DMEM plasma-treated for 60 s, which was stored for 4 days at 37°C, induced the cells to detach from the sensor chip indicating the loss of adhesion capacity. The arrow indicates the time point of medium addition. (Bionas 1500 analysing system, n = 3, student's <i>t</i>-test * p<0.05 for all values from the time point of 4 hours).</p

Workflow.

<p>Cell biological investigations after cultivation with argon plasma-treated, long-time stored cell medium DMEM.</p

Tight junction architecture in the mHepR1 epithelial cell monolayer.

<p><i>a)</i> Note the continuous ZO-1 bands between mHepR1 cells in normal, untreated cell medium DMEM (left column). In contrast, 60 s plasma-treated DMEM stored for 1 d induces large openings between cell margins (arrow, right above), indicating a break of tight junctions. Plasma-treated DMEM (60 s) stored for 7 d induces an irregular cytoplasmic distribution of the ZO-1 proteins (arrow, right below). (AxioObserver.Z1, Carl Zeiss, bars = 5, 10 µm). <i>b)</i> The plasma treatment time of 120 s of the DMEM and storage of 7 d induces not only a retraction of the ZO-1 protein into the cytoplasm but also a nearly complete loss of the cell-cell contacts (arrow), while normal cells show a strong bonding (arrowhead). Note the long-lasting argon plasma effect of cell medium on structural organisation of cells. (AxioObserver.Z1, bar = 50 µm, the windows indicate the areas of the magnified view below).</p

Concentration of hydrogen peroxide in 60 s plasma-treated DMEM.

<p>Note the continuous decrease of hydrogen peroxide over various time periods. (n = 7, Mann-Whitney <i>U</i>-test, ***p<0.001).</p

Cell membrane morphology of mHepR1 cells.

<p><i>a)</i> The surface structure of cells after 24 h in normal, untreated medium (left column) presents elongated microvilli with high densities, which extend over the entire cell surface. In contrast, the mHepR1 cells in 60 s plasma-treated medium (right column) show extremely shortened microvilli accompanied by a reduced density (bar = 4 µm, 5,000× magnification, SEM DSM 960 A, Carl Zeiss). <i>b)</i> The significantly shortened microvilli are impressively visible using a higher magnification (bar = 2 µm, 15,000× magnification, FE-SEM Zeiss Merlin VP compact, Carl Zeiss) (p<0.001, student's <i>t-</i>test, n = 410–500 microvilli). <i>c)</i> Scheme: microvilli at the cell surface in normal epithelial cells (left) and cells exposed to plasma-treated DMEM (right).</p

Oxygen content of 60 s plasma-treated DMEM.

<p>Note that in the plasma-treated complete DMEM the molecular oxygen concentration is decreased by half, which is equalised after only one day. (PreSens oxygen sensor, n = 3, student's <i>t</i>-test, *p<0.05, ** p<0.01, ***p<0.001).</p

Representative gel filtration chromatogram resulting from: (a) application of untreated complete medium, (b) complete argon-gas treated medium, (c) complete medium with 60 s or (d) 120 s of plasma treatment.

<p>The arrowheads mark the one peak which alters considerably after plasma treatment. (n = 6 independent experiments).</p

Oxygen content of cell culture medium immediately after plasma treatment for 60 and 120 s. (PreSens oxygen sensor, mean ± SD, n = 3; student's <i>t</i>-test).

<p>*p<0.05 <i>vs.</i> untreated,</p><p>***p<0.001 <i>vs.</i> untreated.</p><p>Oxygen content of cell culture medium immediately after plasma treatment for 60 and 120 s. (PreSens oxygen sensor, mean ± SD, n = 3; student's <i>t</i>-test).</p