Osteosarcoma and Ewing’s sarcoma are the most common malignant bone tumors. Conventional therapies such as polychemotherapy, local surgery, and radiotherapy improve the clinical outcome for patients. However, they are accompanied by acute and chronic side effects that affect the quality of life of patients, motivating novel research lines on therapeutic options for the treatment of sarcomas. Previous experimental work with physical plasma operated at body temperature (cold atmospheric plasma, CAP) demonstrated anti-oncogenic effects on different cancer cell types. This study investigated the anti-cancer effect of CAP on two bone sarcoma entities, osteosarcoma and Ewing’s sarcoma, which were represented by four cell lines (U2-OS, MNNG/HOS, A673, and RD-ES). A time-dependent anti-proliferative effect of CAP on all cell lines was observed. CAP-induced alterations in cell membrane functionality were detected by performing a fluorescein diacetate (FDA) release assay and an ATP release assay. Additionally, modifications of the cell membrane and modifications in the actin cytoskeleton composition were examined using fluorescence microscopy monitoring dextran-uptake assay and G-/F-actin distribution. Furthermore, the CAP-induced induction of apoptosis was determined by TUNEL and active caspases assays. The observations suggest that a single CAP treatment of bone sarcoma cells may have significant anti-oncogenic effects and thus may be a promising extension to existing applications.

Chondrosarcoma is the second most common malign bone tumor in adults. Surgical resection of the tumor is recommended because of its resistance to clinical treatment such as chemotherapy and radiation therapy. Thus, the prognosis for patients mainly depends on sufficient surgical resection. Due to this, research on alternative therapies is needed. Cold atmospheric plasma (CAP) is an ionized gas that contains various reactive species. Previous studies have shown an anti-oncogenic potential of CAP on different cancer cell types. The current study examined the effects of treatment with CAP on two chondrosarcoma cell lines (CAL-78, SW1353). Through proliferation assay, the cell growth after CAP-treatment was determined. A strong antiproliferative effect for both cell lines was detected. By fluorescein diacetate (FDA) assay and ATP release assay, alterations in the cell membrane and associated translocation of low molecular weight particles through the cytoplasmic membrane were observed. In supernatant, the non-membrane-permeable FDA and endogenously synthesized ATP detected suggest an increased membrane permeability after CAP treatment. Similar results were shown by the dextran-uptake assay. Furthermore, fluorescence microscopic G-/F-actin assay was performed. G- and F-actin were selectively dyed, and the ratio was measured. The presented results indicate CAP-induced changes in cell membrane function and possible alterations in actin-cytoskeleton, which may contribute to the antiproliferative effects of CAP.

Background: Cold atmospheric plasma (CAP) has a variety of anticancer effects on different cancer cell types. In osteosarcoma (OS) cells, CAP reduces growth and motility, induces apoptosis, and alters secretion of cellular factors. The influence of CAP on membrane integrity of OS cells is unknown. Materials and Methods: Two different OS cell lines (U-2 OS and MNNG-HOS) were treated with CAP.Proliferation assays for cell growth after treatment was performed. Alterations in membrane permeability and the associated translocation of low molecular weight particles through the cytoplasmic membrane of OS cells after CAP treatment were shown in fluorescein diacetate (FDA) assays. Results: FDA increasingly passed the membrane after CAP treatment and this effect depended on the duration of treatment. It was also shown that after CAP treatment, FDA was able to diffuse into the cells from the outside as well as out of the cell interior. These effects were observed when CAP-treated buffer was used and therefore no direct contact between cells and CAP occurred. Conclusion: The observations suggest that changes in membrane permeability and function may contribute to the antiproliferative effects of CAP.

Interaction of Staphylococcus aureus alpha-toxin (hemolysin A, Hla) with eukaryotic cell membranes is mediated by proteinaceous receptors and certain lipid domains in host cell plasma membranes. Hla is secreted as a 33 kDa monomer that forms heptameric transmembrane pores whose action compromises maintenance of cell shape and epithelial tightness. It is not exactly known whether certain membrane lipid domains of host cells facilitate adhesion of Ha monomers, oligomerization, or pore formation. We used sphingomyelinase (hemolysin B, Hlb) expressed by some strains of staphylococci to pre-treat airway epithelial model cells in order to specifically decrease the sphingomyelin (SM) abundance in their plasma membranes. Such a pre-incubation exclusively removed SM from the plasma membrane lipid fraction. It abrogated the formation of heptamers and prevented the formation of functional transmembrane pores. Hla exposure of rHlb pre-treated cells did not result in increases in [Ca2+]i, did not induce any microscopically visible changes in cell shape or formation of paracellular gaps, and did not induce hypo-phosphorylation of the actin depolymerizing factor cofilin as usual. Removal of sphingomyelin from the plasma membranes of human airway epithelial cells completely abrogates the deleterious actions of Staphylococcus aureus alpha-toxin.

Background/Aim: The structural integrity of the eukaryotic cytoplasmic membrane is of crucial importance for its cell biological function and thus for the survival of the cell. Physical and chemical noxae can interact in various ways with components of the cytoplasmic membrane, influence its permeability and thus mediate toxic effects. In the study presented, changes in membrane permeability were quantified by intracellular accumulation of a fluorescent dye and by the release of the fluorescent dye from dye-loaded cells. Materials and Methods: Non-malignant (RC-124) and malignant (786-O, Caki-1) renal cells were permeabilized with different concentrations of Triton X-100. The permeability of the membrane was determined at the single-cell level by the uptake of the dye into the cell inner by flow cytometry. In addition, a fluorescence plate reader was used to detect and quantify the release of the dye into the cell culture supernatant. Results: Both malignant and non-malignant cells showed a dosedependent alteration of membrane permeability after treatment with Triton X-100. In the presence of the fluorescent dye, significantly more dye was introduced into the permeabilized cells compared to control incubations. Vice versa, Triton X-100-treated and dye-loaded cells released significantly more dye into the cell culture supernatant. Conclusion: The combination of measurement of intracellular accumulated and extracellular released dye can quantifiably detect changes in membrane permeability due to cell-membrane damage. The combination of two different measurement methods offers additional value in reliable detection of membrane-damaging, potentially toxic influences.

(1) Background: Chondrosarcoma (CS) is a malignant primary bone tumor with a cartilaginous origin. Its slow cell division and severely restricted vascularization are responsible for its poor responsiveness to chemotherapy and radiotherapy. The decisive factor for the prognosis of CS patients is the only adequate therapy—surgical resection. Cold atmospheric pressure plasma (CAP) is emerging as a new option in anti-cancer therapy. Its effect on chondrosarcomas has been poorly investigated. (2) Methods: Two CS cell lines—SW 1353 and CAL 78—were used. Various assays, such as cell growth kinetics, glucose uptake, and metabolic activity assay, along with two different apoptosis assays were performed after CAP treatment. A radius cell migration assay was used to examine cell motility. (3) Results: Both cell lines showed different growth behavior, which was taken into account when using the assays. After CAP treatment, a reduction in metabolic activity was observed in both cell lines. The immediate effect of CAP showed a reduction in cell numbers and in influence on this cell line’s growth rate. The measurement of the glucose concentration in the cell culture medium showed an increase after CAP treatment. Live-dead cell imaging shows an increase in the proportion of dead cells over the incubation time for both cell lines. There was a significant increase in apoptotic signals after 48 h and 72 h for both cell lines in both assays. The migration assay showed that CAP treatment inhibited the motility of chondrosarcoma cells. The effects in all experiments were related to the duration of CAP exposure. (4) Conclusions: The CAP treatment of CS cells inhibits their growth, motility, and metabolism by initiating apoptotic processes.

Background: Cold atmospheric plasma (CAP) is increasingly used in the field of oncology. Many of the mechanisms of action of CAP, such as inhibiting proliferation, DNA breakage, or the destruction of cell membrane integrity, have been investigated in many different types of tumors. In this regard, data are available from both in vivo and in vitro studies. Not only the direct treatment of a tumor but also the influence on its blood supply play a decisive role in the success of the therapy and the patient’s further prognosis. Whether the CAP influences this process is unknown, and the first indications in this regard are addressed in this study. Methods: Two different devices, kINPen and MiniJet, were used as CAP sources. Human endothelial cell line HDMEC were treated directly and indirectly with CAP, and growth kinetics were performed. To indicate apoptotic processes, caspase-3/7 assay and TUNEL assay were used. The influence of CAP on cellular metabolism was examined using the MTT and glucose assay. After CAP exposure, tube formation assay was performed to examine the capillary tube formation abilities of HDMEC and their migration was messured in separate assays. To investigate in a possible mutagenic effect of CAP treatment, a hypoxanthine-guanine-phosphoribosyl-transferase assay with non malignant cell (CCL-93) line was performed. Results: The direct CAP treatment of the HDMEC showed a robust growth-inhibiting effect, but the indirect one did not. The MMT assay showed an apparent reduction in cell metabolism in the first 24 h after CAP treatment, which appeared to normalize 48 h and 72 h after CAP application. These results were also confirmed by the glucose assay. The caspase 3/7 assay and TUNEL assay showed a significant increase in apoptotic processes in the HDMEC after CAP treatment. These results were independent of the CAP device. Both the migration and tube formation of HDMEC were significant inhibited after CAP-treatment. No malignant effects could be demonstrated by the CAP treatment on a non-malignant cell line.

Background/Aim: Mammary carcinoma (MC) remains one of the leading causes of morbidity and mortality in the female population worldwide. Cold physical plasma at atmospheric pressure (CAP) has an antioncogenic effect on tumor cells, and its anticancer properties may complement or even extend existing treatment options. In the present study, the efficacy of CAP was characterized on an MC in vitro cell culture system. Materials and Methods: MC cells (MCF-7, MDA-MB-231) were directly treated with CAP or incubated with CAP-treated cell culture medium. Cell growth, cell mobility and apoptosis were subsequently analyzed. Results: A single treatment of MC cells with CAP and CAP treated medium led to a treatment-time dependent reduction of cell growth. Furthermore, CAP exposure led to a loss of cellular motility and induced apoptosis. Conclusion: Due to its anticancer properties, CAP treatment is an innovative and promising physical approach to expand and complement the treatment options for MC. In particular, a combination of CAP application with surgical and/or chemotherapeutic interventions might significantly improve the therapeutic outcomes.Physical procedures, such as radiotherapy or electrosurgery entered medicine as early as the 1990s. The technical advancement of physical plasma led to the development of another physical therapy field, 'plasma oncology' (1). Cold physical plasma under atmospheric pressure (cold atmospheric plasma: CAP) is generated by applying high electrical voltage to two electrodes through which a carrier gas flows. In contact with the surrounding atmosphere, numerous excited and charged particles, free radicals, and electromagnetic radiation with high biological reactivity are generated (2, 3). In particular, reactive oxygen and nitrogen species (ROS, RNS) are formed, which play a major role in the biological effects of CAP treatment (4). It has been shown that after treatment of tumor cells with CAP, produced antiproliferative effects in varying tumor entities including carcinomas of the lung, ovaries, pancreas, and prostate, as well as osteosarcoma (5-9). In addition, CAP possesses antimicrobial, antiseptic, immunomodulating, antiinflammatory and wound healing promoting properties (10,11), which is why CAP has also been used in the treatment of body surfaces (skin diseases including skin tumours, tumours of the oral cavity) (12). Due to the short half-life of the reactive particles, CAP effects are temporally and locally limited. The intraoperative use of CAP in surgical oncology would be a further therapeutic alternative. Furthermore, the antimicrobial and wound healing promoting properties of CAP treatment may reduce postoperative complications.The mammary carcinoma (MC) is one of the most common cancers in the female population and is the main cause of morbidity and mortality in women. Worldwide, MC is responsible for about 25% of all cancers and about 15% of all cancer-related deaths, although the incidence can vary considerably from country to country (13, 14). Five options are avail...