P2.14-50 Low Temperature Plasma Needle Induces Cell Cycle Arrest of Epithelial Lung Cancer Cells in Vitro via a p21-Dependent Pathway

Abstract

Low temperature plasma sources which operate at atmospheric pressure produce reactive oxygen species (ROS) and reactive nitrogen species which can cause cancer cell death. In this study we investigated the effect of our low temperature plasma needle system on a non-small cell lung cancer cell line A549 and studied its mechanism of action. The housing of the pen lookalike atmospheric plasma source (plasma needle) was made of Teflon. A Pyrex glass tube (o.d. 6 mm and i.d. 4 mm) through which 1 slm of helium was released as the feeding gas was positioned within the housing. The central electrode, a tungsten wire, was powered with a 13.56 MHz sine wave and placed within a ceramic tube inside the glass tube. The central electrode was sticking 1 mm outside of the ceramic and glass tubes, so the discharge occurred on its tip as a weak glow. The cytotoxic activity was determined using MTT assay. The potential of inducing cell cycle perturbations and apoptosis, and changes in the level of ROS was investigated by flow cytometry. The influence of plasma treatment on growth inhibition of multicellular tumor spheroids (MCTS) was also investigated. Evaluation of gene expression was performed by qPCR. All experiments were performed in triplicate, with statistical significance set at p<0.05. Our plasma needle exerted a cytotoxic effect with lower sensitivity towards BEAS-2B normal cells than towards A549 cells. A decrease in the number of cells in the G1 phase (up to 45%), increase in the G2 phase (up to 27%), and an increase in the sub-G1 phase (up to 12%) with fragmented DNA was detected upon treatment. The plasma treatment exerted a mild apoptotic effect (around 15% of apoptotic cells), and an increase in the level of ROS in a power dependent manner. There was no significant reduction in growth of MCTS after plasma treatment under investigated experimental conditions. A statistically non-significant proapoptotic effect (increase in Bax, decrease in Bcl2 and decrease in SKP2) was observed at the genetic level. A significant overexpression of the cyclin-dependent kinase inhibitor 1 (p21) was also observed at the genetic level in a power dependent manner. Our low temperature plasma needle induced cell cycle arrest of epithelial lung cancer cells through overexpression of p21. The effect of combined plasma treatment with existing treatment modalities (cisplatin, PARP inhibitors) is currently under in vitro investigation.