One of the promising possibilities of the clinical application of cold plasma, so-called cold atmospheric plasma (CAP), is its application on malignant cells and cancer tissue using its anti-neoplastic effects, primarily through the delivery of reactive oxygen and nitrogen species (ROS, RNS). In this study, we investigated the impact of CAP on cellular proliferation and consecutive molecular response mechanisms in established prostate cancer (PC) cell lines. PC cells showed a significantly reduced cell growth following CAP treatment as a result of both an immediate increase of intracellular peroxide levels and through the induction of apoptosis indicated by annexin V assay, TUNEL assay, and the evaluation of changes in nuclear morphology. Notably, co-administration of N-acetylcysteine (NAC) completely neutralized CAP effects by NAC uptake and rapid conversion to glutathione (GSH). Vitamin C could not counteract the CAP induced effects on cell growth. In summary, relatively short treatments with CAP of 10 seconds were sufficient to induce a significant inhibition of cancer proliferation, as observed for the first time in urogenital cancer. Therefore, it is important to understand the mode of CAP related cell death and clarify and optimize CAP as cancer therapy. Increased levels of peroxides can alter redox-regulated signaling pathways and can lead to growth arrest and apoptosis. We assume that the general intracellular redox homeostasis, especially the levels of cellular GSH and peroxidases such as peroxiredoxins affect the outcome of the CAP treatment.

Glucose-6-phosphatase plays an important role in the regulation of hepatic glucose production, and insulin suppresses glucose-6-phosphatase gene expression. Recent studies indicate that protein kinase B and Forkhead proteins contribute to insulin-regulated gene expression in the liver. Here, we examined the role of protein kinase B and Forkhead proteins in mediating effects of insulin on glucose-6-phosphatase promoter activity. Transient transfection studies with reporter gene constructs demonstrate that insulin suppresses both basal and dexamethasone/cAMP-induced activity of the glucose-6-phosphatase promoter in H4IIE hepatoma cells. Both effects are partially mimicked by coexpression of protein kinase B␣. Coexpression of the Forkhead transcription factor FKHR stimulates the glucose-6-phosphatase promoter activity via interaction with an insulin response unit (IRU), and this activation is suppressed by protein kinase B. Coexpression of a mutated form of FKHR that cannot be phosphorylated by protein kinase B abolishes the regulation of the glucose-6-phosphatase promoter by protein kinase B and disrupts the ability of insulin to regulate the glucose-6-phosphatase promoter via the IRU. Mutation of the insulin response unit of the glucose-6-phosphatase promoter also prevents the regulation of promoter activity by FKHR and protein kinase B but only partially impairs the ability of insulin to suppress both basal and dexamethasone/ cAMP-stimulated promoter function. Taken together, these results indicate that signaling by protein kinase B to Forkhead proteins can account for the ability of insulin to regulate glucose-6-phosphatase promoter activity via the IRU and that other mechanisms that are independent of the IRU, protein kinase B, and Forkhead proteins also are important in mediating effects of in insulin on glucose-6-phosphatase gene expression.Glucose-6-phosphatase (Glc-6-Pase) 1 catalyzes the hydrolysis of glucose 6-phosphate to glucose, which is the terminal step of both hepatic gluconeogenesis and glycogen breakdown. Glc-6-Pase is induced in starved and diabetic animals (1, 2). In vitro models have shown that glucocorticoids and cAMP induce Glc-6-Pase gene expression. This effect is opposed by insulin, which also is able to reduce basal expression of the Glc-6-Pase gene (3-6). Identification of the signaling events that connect the insulin receptor to the Glc-6-Pase promoter, leading to the subsequent repression of gene transcription, is of particular interest because Glc-6-Pase plays a key role in the regulation of hepatic glucose production and blood glucose homeostasis.In H4IIE hepatoma cells, activation of class 1a phosphoinositide 3-kinase (PI 3-kinase), but not of the Ras/Raf/MAP kinase pathway, is necessary for the suppression of Glc-6-Pase promoter activity by insulin (6). The formation of PtdIns(3,4,5)P 3 catalyzed by PI 3-kinase has been shown to increase the activity of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and to result in a conformational change in PKB which renders it susceptible...

Despite recent advances in the treatment of metastatic castration-resistant prostate cancer (CRPC), outcome of patients remains poor due to the development of drug resistance. Thus, new drugs are urgently needed. We investigated efficacy, toxicity and mechanism of action of marine triterpene glycoside frondoside A (FrA) using CRPC cell lines in vitro and in vivo. FrA revealed high efficacy in human prostate cancer cells, while non-malignant cells were less sensitive. Remarkably, proliferation and colony formation of cells resistant to enzalutamide and abiraterone (due to the androgen receptor splice variant AR-V7) were also significantly inhibited by FrA. The marine compound caused cell type specific cell cycle arrest and induction of caspase-dependent or -independent apoptosis. Up-regulation or induction of several pro-apoptotic proteins (Bax, Bad, PTEN), cleavage of PARP and caspase-3 and down-regulation of anti-apoptotic proteins (survivin and Bcl-2) were detected in treated cells. Global proteome analysis revealed regulation of proteins involved in formation of metastases, tumor cell invasion, and apoptosis, like keratin 81, CrkII, IL-1b and cathepsin B. Inhibition of pro-survival autophagy was observed following FrA exposure. In vivo, FrA inhibited tumor growth of PC-3 and DU145 cells with a notable reduction of lung metastasis, as well as circulating tumor cells in the peripheral blood. Increased lymphocyte counts of treated animals might indicate an immune

The relationship between the apolipoprotein E (apoE) and beta-fibrinogen G/A-455 polymorphisms and cerebrovascular disease (CVD) was examined in the present study. We compared 227 patients with the subtypes of CVD (large-vessel disease, lacunar stroke, cardiac embolism, or undetermined pathomechanisms) with 225 control subjects. The occurrence of apoE isoforms (E2, E3, and E4) and the beta-fibrinogen G/A-455 genotype was determined in these individuals. No differences in apoE polymorphisms or allele frequencies between the CVD patients and control subjects were found. However, analysis of apoE genotypes as a function of stroke subtype revealed that the apoE4 allele was significantly more common in those patients with macroangiopathy-associated CVD. The only CVD risk factor that distinguished patients with the E4 allele from those with other apoE genotypes was elevated cholesterol. No association between the beta-fibrinogen G/A-455 polymorphism and CVD was found. However, homozygosity for the A allele was more common in patients with CVD resulting from large-vessel disease. These data demonstrate that the apoE4 allele and the AA genotype of the beta-fibrinogen G/A-455 polymorphism occur significantly more frequently in patients with CVD resulting from stenosis of large, brain-supplying vessels. Such genetic analyses may further our understanding of the etiology of cerebrovascular disease.

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