Restoration of Sensitivity in Chemo

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. Despite multimodal treatments including surgery, chemotherapy and radiotherapy the prognosis remains poor and relapse occurs regularly. The alkylating agent temozolomide (TMZ) has been shown to improve the overall survival in patients with malignant gliomas, especially in tumors with methylated promoter of the O6-methylguanine-DNA-methyltransferase (MGMT) gene. However, intrinsic and acquired resistance towards TMZ makes it crucial to find new therapeutic strategies aimed at improving the prognosis of patients suffering from malignant gliomas. Cold atmospheric plasma is a new auspicious candidate in cancer treatment. In the present study we demonstrate the anti-cancer properties of different dosages of cold atmospheric plasma (CAP) both in TMZ-sensitive and TMZ-resistant cells by proliferation assay, immunoblotting, cell cycle analysis, and clonogenicity assay. Importantly, CAP treatment restored the responsiveness of resistant glioma cells towards TMZ therapy. Concomitant treatment with CAP and TMZ led to inhibition of cell growth and cell cycle arrest, thus CAP might be a promising candidate for combination therapy especially for patients suffering from GBMs showing an unfavorable MGMT status and TMZ resistance

Combined treatment of glioma cells with TMZ and CAP.

<p><b>A</b> LN18 cells were CAP treated once without medium; afterwards TMZ was applied consecutively for three days. <b>B</b> U87MG cells were CAP treated once and TMZ was applied consecutively afterwards for three days. <b>C</b> LN229 cells were CAP treated followed by TMZ treatment for three days. MTT assay for evaluating the cell viability was performed at day four. Controls were kept without medium and/or DMSO treated. P-value *** <0.001"</p

Clonogenic capacity of glioma cells treated with TMZ (B) or CAP (C).

<p>Glioma cells were either TMZ or CAP treated without medium and 24 h later 150 cells/well were seeded on a 6-well plate. Colonies formed after 12 days were stained and counted. P values *** <0.001. <b>A</b> Picture of the LN18 (MGMT positive) cells treated with CAP for 30 s, 60 s and 120 s. Afterwards the formed colonies were stained. <b>B</b> Treatment of glioma cells with TMZ with concentrations of up to 500 µM and colony formation assay was performed afterwards. <b>C</b> CAP treatment followed by the colony formation assay in either MGMT positive or MGMT negative cells.</p

Cell cycle arrest in G2/M-phase.

<p><b>A</b> Cell cycle analysis of U87MG cells was performed 24 h, 48 h and 72 h after CAP treatment (30 s, 60 s, 120 s and 180 s) by flow cytometry. Treatment was performed only with a thin film of liquid covering the cells. Similar results were observed for the LN229 (MGMT negative) and LN18 (MGMT positive) cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064498#pone.0064498.s003" target="_blank">Figure S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064498#pone.0064498.s004" target="_blank">Figure S4</a>). <b>B</b> Statistical significances of the observed arrest in the G2/M-phase in U87MG and <b>C</b> in LN18 cells. P-value *** <0.001.</p

Induction of DNA damage.

<p><b>A</b> Representative immunoblotting of LN229 cells (MGMT negative) and staining for cleaved PARP1, PARP1 and yH2AX as a marker for DNA damage was performed 48 h and 72 h after CAP treatment. GAPDH served as the loading control. <b>B</b> Similar results were observed 48 h and 72 h after the treatment for the MGMT positive cell line LN18.</p

Inhibition of proliferation by TMZ or CAP treatment.

<p><b>A</b> Immunoblotting of the investigated cell lines for expression of the MGMT protein. Lysates of U87MG, LN18 and LN229 cells were blotted against the anti-MGMT antibody, GAPDH served as a loading control. MGMT protein is expressed in the LN18 cell line, U87MG and LN229 cells do not express the MGMT protein. <b>B</b> U87MG, LN229 and LN18 glioma cells were treated with 50 µM, 100 µM and 200 µM TMZ for three days consecutively. The number of viable cells was measured using the MTT assay after the treatment with TMZ. Statistical significances are evaluated for 200 µM of TMZ in the LN229 and U87MG cell lines. P-value: 0.001 <b>C</b> The same cell lines were treated with CAP in a 96-well plate without medium. Fresh medium was added to the cells immediately after the CAP treatment. Cell growth was measured 48 h after CAP treatment. Statistical significances were observed after 60 seconds of treatment in all tested cell lines. P-value *** <0.001.</p

The CAP device based on Surface Micro Discharge technology.

<p><b>A</b> Photo of the FlatPlaSter 2.0 during plasma production. <b>B</b> Sketch of the plasma production on a Surface Micro-Discharge (SMD) electrode. The plasma discharge is ignited on the SMD electrode and the reactive species are produced in the plasma by electrons from air molecules. The produced reactive species reach the sample by diffusion and induce there biological reactions.</p

TMZ resistant cells respond with cell cycle arrest to combined treatment.

<p><b>A</b> LN18 glioma cells were TMZ treated for three days consecutively with 100 µM, 200 µM and 500 µM and cell cycle analysis was performed afterwards. In comparison, CAP treatment without medium for 60 seconds was applied once to LN18 glioma cells, followed by TMZ treatment with 50 µM, 100 µM and 200 µM for three days consecutively. Cell cycle distribution was determined afterwards. <b>B</b> Analysis of the percentage of cells in the G2/M-phase after treatment with TMZ and combined treatment with CAP. P-value *** <0.001.</p