Ketoprofen Combined with UVA Irradiation Exerts Higher Selectivity in the Mode of Action against Melanotic Melanoma Cells than against Normal Human Melanocytes

Malignant melanoma is responsible for the majority of skin cancer-related deaths. The methods of cancer treatment include surgical removal, chemotherapy, immunotherapy, and targeted therapy. However, neither of these methods gives satisfactory results. Therefore, the development of new anticancer therapeutic strategies is very important and may extend the life span of people suffering from melanoma. The aim of this study was to examine the effect of ketoprofen (KTP) and UVA radiation (UVAR) therapy on cell proliferation, apoptosis, and cell cycle distribution in both melanotic melanoma cells (COLO829) and human melanocytes (HEMn-DP) in relation to its supportive effect in the treatment of melanoma. The therapy combining the use of pre-incubation with KTP and UVAR causes a significant increase in the anti-proliferative properties of ketoprofen towards melanoma cells and the co-exposure of melanotic melanoma cells induced apoptosis shown as the mitochondrial membrane breakdown, cell-cycle deregulation, and DNA fragmentation. Moreover, co-treatment led to GSH depletion showing its pro-apoptotic effect dependent on ROS overproduction. The treatment did not show a significant effect on normal cells—melanocytes—which indicates its high selectivity. The results suggest a possible benefit from the use of the ketoprofen and ultraviolet A irradiation as a new concept of melanotic melanoma therapy.

Mitotic inactivation of the cGAS‒MITA/STING pathways

The cyclic GMP‒AMP synthase (cGAS)‒mediator of interferon response factor 3 activation/stimulator of interferon genes (MITA/STING) axis has emerged as a major pathway, which senses microbial or mis-located cellular DNA in the cytosol to trigger innate immune responses. cGAS senses cytosolic DNA without a preference of self or non-self DNA. How the cGAS‒MITA/STING axis is inactivated upon nuclear membrane breakdown (NEBD) at mitotic entry in vertebrate cells to avoid self DNA sensing remains unclear until very recently. In this review, we summarize the recent advances on how cGAS responds to chromosomes upon NEBD and the mechanisms involved in the inactivation of the cGAS‒MITA/STING pathways in mitosis.

Small Extracellular Vesicles and Metastasis—Blame the Messenger

Cancer is a complex disease, driven by genetic defects and environmental cues. Systemic dissemination of cancer cells by metastasis is generally associated with poor prognosis and is responsible for more than 90% of cancer deaths. Metastasis is thought to follow a sequence of events, starting with loss of epithelial features, detachment of tumor cells, basement membrane breakdown, migration, intravasation and survival in the circulation. At suitable distant niches, tumor cells reattach, extravasate and establish themselves by proliferating and attracting vascularization to fuel metastatic growth. These processes are facilitated by extensive cross-communication of tumor cells with cells in the primary tumor microenvironment (TME) as well as at distant pre-metastatic niches. A vital part of this communication network are small extracellular vesicles (sEVs, exosomes) with a size of 30–150 nm. Tumor-derived sEVs educate recipient cells with bioactive cargos, such as proteins, and in particular, major nucleic acid classes, to drive tumor growth, cell motility, angiogenesis, immune evasion and formation of pre-metastatic niches. Circulating sEVs are also utilized as biomarker platforms for diagnosis and prognosis. This review discusses how tumor cells facilitate progression through the metastatic cascade by employing sEV-based communication and evaluates their role as biomarkers and vehicles for drug delivery.

Bacterial survival at solid-air interfaces: from individual response to population ecology

Acclimation and flexible response mechanisms are ancient survival modes allowing prokaryotic cells to conquer diverse habitats and maintain viability in nature. Evidently, lack of water significantly impacts cellular response making it vital for individual cells to benefit from population support and access survival means available beyond the cell boundaries. Microscopy of dried bacterial cells at solid-air interfaces suggested that cell membrane breakdown didn’t occur for months. Viable counts for Gram-positive Arthrobacter spp. and Gram-negative Pseudomonas stutzeri were significantly higher for larger population density during prolonged periods of desiccation and nutrient starvation. Presence of homogenized cells extended short-term survival of Arthrobacter spp. cells, while heat-killed cells significantly extended viability of all populations. It appeared that huddled zombielike cells allow preservation and recycling of essential survival components within a population. Understanding population-mediated survival at solid-air interfaces is key for monitoring and controlling bacteria including in deep geological repositories for nuclear waste management.

Bacterial survival at solid-air interfaces: from individual response to population ecology

Acclimation and flexible response mechanisms are ancient survival modes allowing prokaryotic cells to conquer diverse habitats and maintain viability in nature. Evidently, lack of water significantly impacts cellular response making it vital for individual cells to benefit from population support and access survival means available beyond the cell boundaries. Microscopy of dried bacterial cells at solid-air interfaces suggested that cell membrane breakdown didn’t occur for months. Viable counts for Gram-positive Arthrobacter spp. and Gram-negative Pseudomonas stutzeri were significantly higher for larger population density during prolonged periods of desiccation and nutrient starvation. Presence of homogenized cells extended short-term survival of Arthrobacter spp. cells, while heat-killed cells significantly extended viability of all populations. It appeared that huddled zombielike cells allow preservation and recycling of essential survival components within a population. Understanding population-mediated survival at solid-air interfaces is key for monitoring and controlling bacteria including in deep geological repositories for nuclear waste management.

Neutrophil Extracellular Traps in the Autoimmunity Context

The formation of neutrophil extracellular traps (NETs) is a strategy utilized by neutrophils for capturing infective agents. Extracellular traps consist in a physical net made of DNA and intracellular proteins externalized from neutrophils, where bacteria and viruses are entrapped and killed by proteolysis. A complex series of events contributes to achieving NET formation: signaling from infectious triggers comes first, followed by decondensation of chromatin and extrusion of the nucleosome components (DNA, histones) from the nucleus and, after cell membrane breakdown, outside the cell. NETs are composed of either DNA or nucleosome proteins and hundreds of cytoplasm proteins, a part of which undergo post-translational modification during the steps leading to NETs. There is a thin balance between the production and the removal of circulating NETs from blood where digestion of DNA by circulating DNases 1 and IL3 has a critical role. A delay in NET removal may have consequences for autoimmunity. Recent studies have shown that circulating NET levels are increased in systemic lupus erythematosus (SLE) for a functional block of NET removal mediated by anti-DNase antibodies or, in rare cases, by DNase IL3 mutations. In SLE, the persistence in circulation of NETs signifies elevated concentrations of either free DNA/nucleosome components and oxidized proteins that, in some cases, are recognized as non-self and presented to B-cells by Toll-like receptor 9 (TLR9). In this way, it is activated as an immunologic response, leading to the formation of IgG2 auto-antibody. Monitoring serum NET levels represents a potential new way to herald the development of renal lesions and has clinical implications. Modulating the balance between NET formation and removal is one of the objectives of basic research that are aimed to design new drugs for SLE.Clinical Trial Registration Number: The Zeus study was registered at https://clinicaltrials.gov (study number: NCT02403115).

Dual role of ER stress in response to metabolic co-targeting and radiosensitivity in head and neck cancer cells

Arginine deprivation therapy (ADT) is a new metabolic targeting approach with high therapeutic potential for various solid cancers. Combination of ADT with low doses of the natural arginine analog canavanine effectively sensitizes malignant cells to irradiation. However, the molecular mechanisms determining the sensitivity of intrinsically non-auxotrophic cancers to arginine deficiency are still poorly understood. We here show for the first time that arginine deficiency is accompanied by global metabolic changes and protein/membrane breakdown, and results in the induction of specific, more or less pronounced (severe vs. mild) ER stress responses in head and neck squamous cell carcinoma (HNSCC) cells that differ in their intrinsic ADT sensitivity. Combination of ADT with canavanine triggered catastrophic ER stress via the eIF2α-ATF4(GADD34)-CHOP pathway, thereby inducing apoptosis; the same signaling arm was irrelevant in ADT-related radiosensitization. The particular strong supra-additive effect of ADT, canavanine and irradiation in both intrinsically more and less sensitive cancer cells supports the rational of ER stress pathways as novel target for improving multi-modal metabolic anti-cancer therapy.

The Fission Yeast RNA-Binding Protein Meu5 Is Involved in Outer Forespore Membrane Breakdown during Spore Formation

In Schizosaccharomyces pombe, the spore wall confers strong resistance against external stress. During meiosis II, the double-layered intracellular forespore membrane (FSM) forms de novo and encapsulates the nucleus. Eventually, the inner FSM layer becomes the plasma membrane of the spore, while the outer layer breaks down. However, the molecular mechanism and biological significance of this membrane breakdown remain unknown. Here, by genetic investigation of an S. pombe mutant (E22) with normal prespore formation but abnormal spores, we showed that Meu5, an RNA-binding protein known to bind to and stabilize more than 80 transcripts, is involved in this process. We confirmed that the E22 mutant does not produce Meu5 protein, while overexpression of meu5+ in E22 restores the sporulation defect. Furthermore, electron microscopy revealed that the outer membrane of the FSM persisted in meu5∆ spores. Investigation of the target genes of meu5+ showed that a mutant of cyc1+ encoding cytochrome c also showed a severe defect in outer FSM breakdown. Lastly, we determined that outer FSM breakdown occurs coincident with or after formation of the outermost Isp3 layer of the spore wall. Collectively, our data provide novel insights into the molecular mechanism of spore formation.

CTNI-70. LONG-TERM SURVIVAL IN GLIOBLASTOMA PATIENTS AFTER TUMOR TREATING FIELDS (TTFIELDS) THERAPY

PURPOSE Glioblastoma multiforme (GBM) is the most common and malignant primary intracranial tumor and traditionally has a median survival of only 10 to 14 months, with only 3 to 5% of patients surviving more than three years. Recurrence (RGBM) is nearly universal, and further decreases the median survival to only 5 to 7 months with optimal therapy. Thus, advances in treatment that may improve survival in these patients are highly desirable. METHODS Tumor treating fields (TTFields) therapy (Novocure Ltd., Haifa, Israel) is an approved antimitotic treatment for patients with newly diagnosed and recurrent GBM. TTFields are low-intensity (1–3 V/cm), intermediate-frequency (100–300 kHz) alternating electric fields that selectively kill or arrest the growth of rapidly dividing cells by inhibiting the proper formation of the mitotic spindle and by causing rapid membrane breakdown during cytokinesis. RESULTS Our center was the first in the world to apply TTFields treatment to histologically proven GBM in a pilot study of 20 individuals (10 GBM and 10 RGBM) in 2004 and 2005, and 4 of the original 20 patients are still alive today (2 GBM, 2 RGBM), in good health and no longer receiving any treatment roughly 15 years (range 14.2–15.9 years) after initiating TTFields therapy, with no clinical or radiological evidence of recurrence. The diagnosis of GBM was confirmed in all patients in two independent laboratories. Two of the 4 surviving patients exhibited radiological signs of tumor growth initially, before the tumor regressed in size after a median of 4 months of continuous treatment. CONCLUSIONS Our results indicate that TTFields treatment may be remarkably successful for both newly diagnosed and recurrent GBM patients. We recommend that TTFields treatment should be applied for a sufficient amount of time, and that initial radiologic progression following treatment initiation should not be considered a reason to discontinue treatment.

CpG preconditioning reduces accumulation of lysophosphatidylcholine in ischemic brain tissue after middle cerebral artery occlusion

Ischemic stroke is one of the major causes of death and permanent disability in the world. However, the molecular mechanisms surrounding tissue damage are complex and further studies are needed to gain insights necessary for development of treatment. Prophylactic treatment by administration of cytosine-guanine (CpG) oligodeoxynucleotides has been shown to provide neuroprotection against anticipated ischemic injury. CpG binds to Toll-like receptor 9 (TLR9) causing initialization of an inflammatory response that limits visible ischemic damages upon subsequent stroke. Here, we use nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging (MSI) to characterize molecular effects of CpG preconditioning prior to middle cerebral artery occlusion (MCAO) and reperfusion. By doping the nano-DESI solvent with appropriate internal standards, we can study and compare distributions of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) in the ischemic hemisphere of the brain despite the large changes in alkali metal abundances. Our results show that CpG preconditioning not only reduces the infarct size but it also decreases the degradation of PC and accumulation of LPC species, which indicates reduced cell membrane breakdown and overall ischemic damage. Our findings show that molecular mechanisms of PC degradation are intact despite CpG preconditioning but that these are limited due to the initialized inflammatory response.