Restoration of Sensitivity in Chemo — Resistant Glioma Cells by Cold Atmospheric Plasma

et al. (2013) Restoration of Sensitivity in Chemo - Resistant Glioma Cells by Cold Atmospheric Plasma. PLoS ONE 8(5): e64498. doi:10.1371/journal.pone.0064498 Restoration of Sensitivity in Chemo - Resistant Glioma Cells by Cold Atmospheric Plasma Julia Ko ritzer 0 Veronika Boxhammer 0 Andrea Scha fer 0 Tetsuji Shimizu 0 Tobias G. Kla mpfl 0 Yang-Fang Li 0 Christian Welz 0 Sabina Schwenk-Zieger 0 Gregor E. Morfill 0 Julia L. Zimmermann 0 Ju rgen Schlegel 0 Michael Lim, Johns Hopkins Hospital, United States of America 0 1 Max Planck Institute for Extraterrestrial Physics , Garching, Germany , 2 Department of Neuropathology, Technical University Munich , Munich, Germany , 3 Department of Head and Neck Cancer, Ludwig Maximilians University , Munich , Germany 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. - Funding: The work was supported by the grant (M.TT.A.EXT00002) of the Max Planck Institute for Extraterrestrial Physics, Garching, Germany. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults and is classified according to the world health organization (WHO) as a grade IV tumor. These tumors are a highly invasive, rapidly spreading form of central nervous system cancer which are resistant to surgical and medical treatment. Particular challenges of treating GBM are its distinct tumor heterogeneity, the inability of treatments to reach all tumor cells, the delivery of drugs across the blood-brain barrier and the high likelihood of relapse, which is often rapid and aggressive. Although some advances have been made in recent years, treatment remains palliative for most patients as a cure remains elusive. Looking at the numbers the median survival has improved from 12.1 to 14.6 months, but still less than 16% of the patients survive three years postdiagnosis [1]. The first-line chemotherapeutic drug is the alkylating agent temozolomide (TMZ). Following oral absorption, TMZ is converted to an alkylating methyldiazonium cation that is known to damage DNA thereby leading to DNA double strand breaks [2],[3]. The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is capable of counteracting the cytotoxicity induced by TMZ [4],[5] - thus tumors expressing high levels of MGMT (MGMT positive, unfavorable) are more resistant to TMZ than those in which the enzyme has become silenced by promoter methylation (MGMT negative, favorable). MGMT promoter methylation is associated with a favorable outcome and predicts a benefit from alkylating agent chemotherapy in patients with newly diagnosed glioblastoma [6],[7],[8],[9]. In a large randomized multicenter trial an unmethylated MGMT promoter (protein is expressed - unfavorable MGMT status) was observed in more than half of the patients and those therefore did not benefit from the TMZ treatment [10]. Thus, there is a clinical need to establish additional novel therapy regimes to overcome TMZ resistance. Therefore in the present study the concomitant treatment of GBM with cold atmospheric plasma (CAP) and TMZ in overcoming TMZ resistance was investigated. In the past years CAP a partially ionized gas - proved its effectiveness for different applications in health care and medicine. In a combined effort of physicists, engineers, chemists, biologists and medical doctors several different CAP sources were developed, characterized and to some extent optimized for their respective application. All these plasma sources have in common that they generate CAP thereby initiating reactions in the surrounding air, which lead to the production of a reactive mix of electrons, ions, neutrals, reactive species and UV light. Nevertheless depending on the plasma source properties, composition and concentrations of the produced species can be varied and therefore initiate different reactions with the respective target. Several developed CAP sources have proven to successfully inactivate bacteria, fungi, virus and spores in a dose-dependent manner [11],[12],[13],[14],[15]. Healthcare applications such as the sterilization of surgical instruments [16],[17],[18], skin [19],[20] and wound disinfection [21],[22] therefore paved its way into medical care. Further generations of CAP sources however also showed anti-cancer properties. Main targets for CAP in cancer cell lines were growth inhibition [23], inhibition of cell migration and invasion (in colorectal cancer cells [24]) or induction of apoptosis (in melanoma cells [25], [26], mouse lung carcinoma [27]). The production of reactive oxygen and nitrogen species by CAP is thought to be a key player initiating the anticancer properties of CAP. It was shown that intracellular ROS levels increase after CAP treatment, thereby inducing DNA damage and apoptosis in the cells [28], [25], [29], [30]. In this study we show that TMZ resistant cells with an unfavorable MGMT status as well as TMZ sensitive cell lines are susceptible to CAP treatment. Furthermore we are able to demonstrate a synergistic effect of a combined treatment with TMZ and CAP in cells with unfavorable MGMT status. Materials and Methods Plasma Device The CAP device employed in this study uses the Surface Micro Discharge (SMD) technology for plasma production as shown in figure 1 and has been published and characterized in detail in Morfill et al. [11] and Maisch et al. [12]. In short, the electrode for plasma production is located at the top inside a closed box (figure 1 A). The electrode consists of a Teflon (insulator) plate sandwiched by a planar brass plate (sheet (...truncated)