Acidified Nitrite Contributes to the Antitumor Effect of Cold Atmospheric Plasma on Melanoma Cells.

Cold atmospheric plasma (CAP) is partially ionized gas near room temperature with previously reported antitumor effects. Despite extensive research and growing interest in this technology, active components and molecular mechanisms of CAP are not fully understood to date. We used Raman spectroscopy and colorimetric assays to determine elevated nitrite and nitrate levels after treatment with a MiniFlatPlaster CAP device. Previously, we demonstrated CAP-induced acidification. Cellular effects of nitrite and strong extracellular acidification were assessed using live-cell imaging of intracellular Ca2+ levels, cell viability analysis as well as quantification of p21 and DNA damage. We further characterized these observations by analyzing established molecular effects of CAP treatment. A synergistic effect of nitrite and acidification was found, leading to strong cytotoxicity in melanoma cells. Interestingly, protein nitration and membrane damage were absent after treatment with acidified nitrite, thereby challenging their contribution to CAP-induced cytotoxicity. Further, phosphorylation of ERK1/2 was increased after treatment with both acidified nitrite and indirect CAP. This study characterizes the impact of acidified nitrite on melanoma cells and supports the importance of RNS during CAP treatment. Further, it defines and evaluates important molecular mechanisms that are involved in the cancer cell response to CAP.

Thermotoga maritima NusG: domain interaction mediates autoinhibition and thermostability.

NusG, the only universally conserved transcription factor, comprises an N- and a C-terminal domain (NTD, CTD) that are flexibly connected and move independently in Escherichia coli and other organisms. In NusG from the hyperthermophilic bacterium Thermotoga maritima (tmNusG), however, NTD and CTD interact tightly. This closed state stabilizes the CTD, but masks the binding sites for the interaction partners Rho, NusE and RNA polymerase (RNAP), suggesting that tmNusG is autoinhibited. Furthermore, tmNusG and some other bacterial NusGs have an additional domain, DII, of unknown function. Here we demonstrate that tmNusG is indeed autoinhibited and that binding to RNAP may stabilize the open conformation. We identified two interdomain salt bridges as well as Phe336 as major determinants of the domain interaction. By successive weakening of this interaction we show that after domain dissociation tmNusG-CTD can bind to Rho and NusE, similar to the Escherichia coli NusG-CTD, indicating that these interactions are conserved in bacteria. Furthermore, we show that tmNusG-DII interacts with RNAP as well as nucleic acids with a clear preference for double stranded DNA. We suggest that tmNusG-DII supports tmNusG recruitment to the transcription elongation complex and stabilizes the tmNusG:RNAP complex, a necessary adaptation to high temperatures.

Cold atmospheric plasma treatment inhibits growth in colorectal cancer cells.

Plasma oncology is a relatively new field of research. Recent developments have indicated that cold atmospheric plasma (CAP) technology is an interesting new therapeutic approach to cancer treatment. In this study, p53 wildtype (LoVo) and human p53 mutated (HT29 and SW480) colorectal cancer cells were treated with the miniFlatPlaSter - a device particularly developed for the treatment of tumor cells - that uses the Surface Micro Discharge (SMD) technology for plasma production in air. The present study analyzed the effects of plasma on colorectal cancer cells in vitro and on normal colon tissue ex vivo. Plasma treatment had strong effects on colon cancer cells, such as inhibition of cell proliferation, induction of cell death and modulation of p21 expression. In contrast, CAP treatment of murine colon tissue ex vivo for up to 2 min did not show any toxic effect on normal colon cells compared to H2O2 positive control. In summary, these results suggest that the miniFlatPlaSter plasma device is able to kill colorectal cancer cells independent of their p53 mutation status. Thus, this device presents a promising new approach in colon cancer therapy.

Bile canaliculi formation and biliary transport in 3D sandwich-cultured hepatocytes in dependence of the extracellular matrix composition.

Primary human hepatocytes (PHH) are still considered as gold standard for investigation of in vitro metabolism and hepatotoxicity in pharmaceutical research. It has been shown that the three-dimensional (3D) cultivation of PHH in a sandwich configuration between two layers of extracellular matrix (ECM) enables the hepatocytes to adhere three dimensionally leading to formation of in vivo like cell-cell contacts and cell-matrix interactions. The aim of the present study was to investigate the influence of different ECM compositions on morphology, cellular arrangement and bile canaliculi formation as well as bile excretion processes in PHH sandwich cultures systematically. Freshly isolated PHH were cultured for 6 days between two ECM layers made of collagen and/or Matrigel in four different combinations. The cultures were investigated by phase contrast microscopy and immunofluorescence analysis with respect to cell-cell connections, repolarization as well as bile canaliculi formation. The influence of the ECM composition on cell activity and viability was measured using the XTT assay and a fluorescent dead or alive assay. Finally, the bile canalicular transport was analyzed by live cell imaging to monitor the secretion and accumulation of the fluorescent substance CDF in bile canaliculi. Using collagen and Matrigel in different compositions in sandwich cultures of hepatocytes, we observed differences in morphology, cellular arrangement and cell activity of PHH in dependence of the ECM composition. Sandwich-cultured hepatocytes with an underlay of collagen seem to represent the best in vivo tissue architecture in terms of formation of trabecular cell arrangement. Cultures overlaid with collagen were characterized by the formation of abundant bile canaliculi, while the bile canaliculi network in hepatocytes cultured on a layer of Matrigel and overlaid with collagen showed the most branched and stable canalicular network. All cultures showed a time-dependent leakage of CDF from the bile canaliculi into the culture supernatant with variations in dependence on the used matrix combination. In conclusion, the results of this study show that the choice of ECM has an impact on the morphology, cell assembly and bile canaliculi formation in PHH sandwich cultures. The morphology and the multicellular arrangement were essentially influenced by the underlaying matrix, while bile excretion and leakage of sandwich-cultured hepatocytes were mainly influenced by the overlay matrix. Leaking and damaged bile canaliculi could be a limitation of the investigated sandwich culture models in long-term excretion studies.

Acidification is an Essential Process of Cold Atmospheric Plasma and Promotes the Anti-Cancer Effect on Malignant Melanoma Cells.

(1) Background: Cold atmospheric plasma (CAP) is ionized gas near room temperature. The anti-cancer effects of CAP were confirmed for several cancer types and were attributed to CAP-induced reactive species. However, the mode of action of CAP is still not well understood. (2) Methods: Changes in cytoplasmic Ca2+ level after CAP treatment of malignant melanoma cells were analyzed via the intracellular Ca2+ indicator fura-2 AM. CAP-produced reactive species were determined by fluorescence spectroscopic and protein nitration by Western Blot analysis. (3) Results: CAP caused a strong acidification of water and solutions that were buffered with the so-called Good buffers, while phosphate-buffered solutions with higher buffer capacity showed minor pH reductions. The CAP-induced Ca2+ influx in melanoma cells was stronger in acidic pH than in physiological conditions. NO formation that is induced by CAP was dose- and pH-dependent and CAP-treated solutions only caused protein nitration in cells under acidic conditions. (4) Conclusions: We describe the impact of CAP-induced acidification on the anti-cancer effects of CAP. A synergistic effect of CAP-induced ROS, RNS, and acidic conditions affected the intracellular Ca2+ level of melanoma cells. As the microenvironment of tumors is often acidic, further acidification might be one reason for the specific anti-cancer effects of CAP.

Metabolism of remimazolam in primary human hepatocytes during continuous long-term infusion in a 3-D bioreactor system.

BACKGROUND: Remimazolam is an ultra-short acting benzodiazepine under development for procedural sedation and general anesthesia. It is hydrolyzed by CES1 to an inactive metabolite (CNS7054). PURPOSE: In this study, the effect of continuous remimazolam exposure on its metabolism and on CES1 expression was investigated in a dynamic 3-D bioreactor culture model inoculated with primary human hepatocytes. METHODS: Remimazolam was continuously infused into bioreactors for 5 days at a final concentration of 3,000 ng/ml (6.8 µM). In parallel, 2-D cultures were run with cells from the same donors, but with discontinuous exposure to remimazolam. RESULTS: Daily measurement of clinical chemistry parameters (glucose, lactate, urea, ammonia, and liver enzymes) in culture supernatants indicated no noxious effect of remimazolam on hepatocyte integrity as compared to untreated controls. Concentrations of remimazolam reached steady-state values of around 250 ng/ml within 8 hours in 3-D bioreactors whereas in 2-D cultures remimazolam concentrations declined to almost zero within the same time frame. Levels of CNS7054 showed an inverse time-course reaching average values of 1,350 ng/ml in perfused 3-D bioreactors resp. 2,800 ng/ml in static 2-D cultures. Analysis of mRNA expression levels of CES1 indicated no changes in gene expression over the culture period. CONCLUSION: The results indicated a stable metabolism of remimazolam during 5 days of continuous exposure to clinically relevant concentrations of the drug. Moreover, there was no evidence for a harmful effect of remimazolam exposure on the integrity and metabolic activity of in vitro cultivated primary human hepatocytes.

Cold atmospheric plasma causes a calcium influx in melanoma cells triggering CAP-induced senescence.

Cold atmospheric plasma (CAP) is a promising approach in anti-cancer therapy, eliminating cancer cells with high selectivity. However, the molecular mechanisms of CAP action are poorly understood. In this study, we investigated CAP effects on calcium homeostasis in melanoma cells. We observed increased cytoplasmic calcium after CAP treatment, which also occurred in the absence of extracellular calcium, indicating the majority of the calcium increase originates from intracellular stores. Application of previously CAP-exposed extracellular solutions also induced cytoplasmic calcium elevations. A substantial fraction of this effect remained when the application was delayed for one hour, indicating the chemical stability of the activating agent(s). Addition of ryanodine and cyclosporin A indicate the involvement of the endoplasmatic reticulum and the mitochondria. Inhibition of the cytoplasmic calcium elevation by the intracellular chelator BAPTA blocked CAP-induced senescence. This finding helps to understand the molecular influence and the mode of action of CAP on tumor cells.

Characterization of in vitro antifungal activities of small and American cranberry (Vaccinium oxycoccos L. and V. macrocarpon Aiton) and lingonberry (Vaccinium vitis-idaea L.) concentrates in sugar reduced fruit spreads.

In this study, cranberry and lingonberry concentrates were added to commercial sugar-reduced fruit spreads (raspberry-Aloe vera, strawberry-guava, and strawberry-lime), and tested for their antifungal activities. Selected strains of the species Absidia glauca, Penicillium brevicompactum, Saccharomyces cerevisiae and Zygosaccharomyces bailii, as well as xerophilic environmental isolates of the genera Penicillium and Eurotium were used for challenge testing. Initially, varying concentrations of synthetic antifungal agents, such as sodium benzoate, potassium sorbate and butyl 4-hydroxybenzoate were tested against these fungi on wort agar containing 31% fructose at different pH values. Subsequently, the experiments were conducted in fruit spreads containing different concentrations of cranberry and lingonberry concentrates. The results of this study demonstrate that these concentrates were able to inhibit growth of visible colonies of xerophilic and non-xerophilic fungi. Cranberry and lingonberry concentrates are interesting candidates for natural preservation against fungal growth in sugar reduced fruit spreads.