Mitochondria-Mediated Anticancer Effects of Non-Thermal Atmospheric Plasma.

Non-thermal atmospheric pressure plasma has attracted great interest due to its multiple potential biomedical applications with cancer treatment being among the most urgent. To realize the clinical potential of non-thermal plasma, the exact cellular and molecular mechanisms of plasma effects must be understood. This work aimed at studying the prostate cancer specific mechanisms of non-thermal plasma effects on energy metabolism as a central regulator of cell homeostasis and proliferation. It was found that cancer cells with higher metabolic rate initially are more resistant to plasma treated phosphate-buffered saline (PBS) since the respiratory and calcium sensitive signaling systems were not responsive to plasma exposure. However, dramatic decline of cancer oxidative phosphorylation developed over time resulted in significant progression of cell lethality. The normal prostate cells with low metabolic activity immediately responded to plasma treated PBS by suppression of respiratory functions and sustained elevation of cytosolic calcium. However, over time the normal cells start recovering their mitochondria functions, proliferate and restore the cell population. We found that the non-thermal plasma induced increase in intracellular ROS is of primarily non-mitochondrial origin. The discriminate non-thermal plasma effects hold a promise for clinical cancer intervention

Non-thermal plasma induces apoptosis in DU145 cancer and PrEC normal prostate cells.

<p>Flow cytometry and microscopy results were obtained 24 hours post plasma treatment. (<b>A</b>) Induction of apoptosis in DU145 and PrECs. The cells were incubated with plasma D7 for 1 and 10 minutes than fresh medium was added to cells for their further maintaining. (<b>B</b>) The quantitative data of the per cent of early and late (EA+LA) apoptotic cells. <b>C.</b> Western blot analysis of apoptosis signature. Vinculin was used as a loading control. (<b>D</b>) Transmission images of normal PrEC and metastatic DU145 cells. The white circle indicates the area of PrECs that remained alive or proliferated after the plasma treatment. Data presented as mean±SEM (n = 3).</p

Plasma induced modulations of cytosolic calcium in PrEC and DU145 cells.

<p>Representative confocal microscopy spectral records of DU145 cells. After 10 minutes of incubation with or without plasma treated PBS cells were challenged with 50μM ATP. (A) IP₃-mediated intracellular calcium oscillations induced by ATP were produced in control cells. (B) In plasma treated DU145 cells this amount of ATP provoked sustained cytosolic calcium response, while non-thermal plasma treated PBS itself did not cause calcium modulations. In PrECs the high calcium signal was observed right after addition of non-thermal plasma treated PBS (D7) (C). The representative original records demonstrate the data collected from about 50 cells evaluated in each of 5 experiments.</p

Cytotoxicity of non-thermal plasma on prostate DU145 cancer and PrEC normal cells.

<p>The cells were exposed to PBS treated with different plasma doses for 1 and 10 minutes, followed by dilution with growth medium and incubation for 24 and 48 hours. To simplify the understanding of non-thermal plasma doses, the plasma obtained at different physical parameters (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156818#pone.0156818.t001" target="_blank">Table 1</a></b>) was named numerically as Dose 1, 2, etc. As seen from the graphs <b>A</b> and <b>D</b> the highest cytotoxic dose was Dose 7 (D7). (<b>B,E</b>) The quantitative graphs of the plasma induced cytotoxicity for both prostate cancer and normal cells. (<b>C,F</b>) The data and images obtained from the colony formation assay. The benign PrECs treated with plasma D7 for 1 and 10 minutes followed by PBS dilution with growth medium were cultivated for 6 days. They retain their proliferative activity while the cancer cells treated in the same way lose the ability to form colonies. Data presented as mean±SEM (n = 3).</p

Effect of non-thermal plasma treatment on mitochondria membrane potential.

<p>The continuous exposure of cells to plasma treated PBS without it further dilution with medium results in a decay of mitochondria energization (<b>D7,</b> black rhombi) similarly to what was observed in the presence of protonophore (<b>FCCP,</b> empty circle). Decline in mitochondria energization in normal cells (<b>B,</b> black rhombi) was faster than in cancer cells (<b>A,</b> black rhombi), which are more resistant to plasma induced modulations. Samples <b>D7_1 min_24 h</b> (white rhombi) and <b>D7_10 min_24 h</b> (black circle) are the cells harvested after 24 h of incubation in the growth medium after being exposed to plasma D7 for 1 and 10 minutes. The normal cells restore their the membrane potential up to 70% within 24 h after treatment, while in the cancer cells the membrane potential remained at about 40% only (white rhombi and black circles). Data presented as mean±SEM (n = 3).</p

Determination of non-thermal plasma doses as a function of pH change.

<p>Determination of non-thermal plasma doses as a function of pH change.</p

Respirometric analysis of non-thermal plasma effects on cell oxidative phosphorylation.

<p>Cells were measured in the respiratory buffer without respiratory substrates in order to evaluate the cell’s endogenous respiratory capacity. To evaluate the maximal respiratory activity the cells were inhibited with oligomycin to block ATP synthase and then titrated with increasing doses of protonophore. (<b>A,B</b>) Oxygen consumption rates in prostate cancer and normal cells immediately and after 24 hours post plasma treatment. Data presented as mean±SEM (n = 4–6).</p