Confluent Cells Research Articles

De Novo Design and Development of a Nutrient-Rich Perfusate for Ex Vivo Lung Perfusion with Cell Culture Models.

Ex vivo lung perfusion (EVLP) has increased donor lung utilization through assessment of "marginal" lungs prior to transplantation. To develop it as a donor lung reconditioning platform, prolonged EVLP is necessary, and new perfusates are required to provide sufficient nutritional support. Human pulmonary microvascular endothelial cells and epithelial cells were used to test different formulas for basic cellular function. A selected formula was further tested on an EVLP cell culture model, and cell confluence, apoptosis, and GSH and HSP70 levels were measured. When a cell culture medium (DMEM) was mixed with a current EVLP perfusate-Steen solution, DMEM enhanced cell confluence and migration and reduced apoptosis in a dose-dependent manner. A new EVLP perfusate was designed and tested based on DMEM. The final formula contains 5 g/L Dextran-40 and 7% albumin and is named as D05D7A solution. It inhibited cold static storage and warm reperfusion-induced cell apoptosis, improved cell confluence, and enhanced GSH and HSP70 levels in human lung cells compared to Steen solution. DMEM-based nutrient-rich EVLP perfusate could be a promising formula to prolong EVLP and support donor lung repair, reconditioning and further improve donor lung quality and quantity for transplantation with better clinical outcome.

Transformed cells maintain survival by downregulating autophagy at a high cell confluency.

Autophagy plays a dual role in tumorigenesis by functioning as both a tumor suppressor and promoter, depending on the stage of tumorigenesis. However, it is still unclear at what stage the role of autophagy changes during tumorigenesis. Herein, we investigated the differences in the basal levels and roles of autophagy in five cell lines at different stages of cell transformation. We found that cell lines at higher transformation stages were more sensitive to the autophagy inhibitors, suggesting that autophagy plays a more important role as the transformation progresses. Our ptfLC3 imaging analysis to measure Atg5/LC3-dependent autophagy showed increased autophagic flux in transformed cells compared to untransformed cells. However, the Cyto-ID analysis, which measures Atg5-dependent and -independent autophagic flux, showed high levels of autophagosome formation not only in the transformed cells but also in the initiated cell and Atg5 KO cell line. These results indicate that Atg5-independent autophagy may be more critical in initiated and transformed cell lines than in untransformed cells. Specially, we observed that transformed cells maintained relatively high basal autophagy levels under rapidly proliferating conditions but exhibited much lower basal autophagy levels at high confluency; however, autophagic flux was not significantly reduced in untransformed cells, even at high confluency. In addition, when continuously cultured for 3 weeks without passage, senescent cells were significantly less sensitive to autophagy inhibition than their actively proliferating counterparts. These results imply that once a cell has switched from a proliferative state to a senescent state, the inhibition of autophagy has only a minimal effect. Taken together, our results suggest that autophagy can be differentially regulated in cells at different stages of tumorigenesis under stressful conditions.

Towards the Standardization of Mesenchymal Stem Cell Secretome-Derived Product Manufacturing for Tissue Regeneration.

Mesenchymal stem cell secretome or conditioned medium (MSC-CM) is a combination of biomolecules and growth factors in cell culture growth medium, secreted by mesenchymal stem cells (MSCs), and the starting point of several derived products. MSC-CM and its derivatives could be applied after injuries and could mediate most of the beneficial regenerative effects of MSCs without the possible side effects of using MSCs themselves. However, before the clinical application of these promising biopharmaceuticals, several issues such as manufacturing protocols and quality control must be addressed. This review aims to underline the influence of the procedure for conditioned medium production on the quality of the secretome and its derivatives and highlights the questions considering cell sources and donors, cell expansion, cell passage number and confluency, conditioning period, cell culture medium, microenvironment cues, and secretome-derived product purification. A high degree of variability in MSC secretomes is revealed based on these parameters, confirming the need to standardize and optimize protocols. Understanding how bioprocessing and manufacturing conditions interact to determine the quantity, quality, and profile of MSC-CM is essential to the development of good manufacturing practice (GMP)-compliant procedures suitable for replacing mesenchymal stem cells in regenerative medicine.

An automated approach for fibroblast cell confluency characterisation and sample handling using AIoT for bio-research and bio-manufacturing

An automated approach for fibroblast cell confluency characterisation and sample handling using AIoT for bio-research and bio-manufacturing

Cobalt/Bioglass Nanoparticles Enhanced Dermal Regeneration in a 3-Layered Electrospun Scaffold.

Due to the multilayered structure of the skin tissue, the architecture of its engineered scaffolds needs to be improved. In the present study, 45s5 bioglass nanoparticles were selected to induce fibroblast proliferation and their protein secretion, although cobalt ions were added to increase their potency. A 3-layer scaffold was designed as polyurethane (PU) - polycaprolactone (PCL)/ collagen/nanoparticles-PCL/collagen. The scaffolds examined by scanning electron microscopy (SEM), Fourier transform infrared (FTIR), tensile, surface hydrophilicity and weight loss. Biological tests were performed to assess cell survival, adhesion and the pattern of gene expression. The mechanical assay showed the highest young modulus for the scaffold with the doped nanoparticles and the water contact angle of this scaffold after chemical crosslinking of collagen was reduced to 52.34±7.7°. In both assessments, the values were statistically compared to other groups. The weight loss of the corresponding scaffold was the highest value of 82.35±4.3 % due to the alkaline effect of metal ions and indicated significant relations in contrast to the scaffold with non-doped particles and bare one (P value<0.05). Moreover, better cell expansion, greater cell confluence and a lower degree of toxicity were confirmed. The up-regulation of TGF β1 and VEGF genes introduced this scaffold as a better model for the fibroblasts commitment to a new skin tissue among bare and nondoped scaffold (P value<0.05). The 3-layered scaffold which is loaded with cobalt ions-bonded bioglass nanoparticles, is a better substrate for the culture of the fibroblasts.

Isolation of porcine reproductive and respiratory syndrome virus from feed ingredients and complete feed, with subsequent RT-qPCR analysis.

We used virus isolation (VI) to determine tissue culture infectivity and reverse-transcription quantitative PCR (RT-qPCR) to determine the stability of porcine reproductive and respiratory syndrome virus 2 (PRRSV) strain P129 in solvent-extracted soybean meal (SBM), dried distillers grains with solubles (DDGS), complete swine feed (FEED), or medium (DMEM) at 4°C, 23°C, or 37°C for up to 3 d. Samples of each treatment were taken at regular intervals and processed. Supernatant was titrated and used to inoculate confluent MARC-145 cells to determine infectivity. RNA was extracted from each supernatant sample and tested by RT-qPCR to determine any change in detectable virus RNA across matrix type, temperature, and time. An interaction (p = 0.028) was observed for matrix × temperature × hour for live virus detected by VI. At 4°C, the concentration of infectious virus was greatest in DMEM, intermediate in SBM, and lowest in DDGS and FEED. DMEM also had the greatest concentration of infectious PRRSV at 23°C over time; a higher infectious virus concentration was maintained in SBM for longer than in DDGS or FEED. At 37°C, a greater concentration of infectious virus was sustained in DMEM than in the feedstuffs, with concentrations decreasing until 48 h post-inoculation. Only matrix type influenced the quantity of viral RNA detected by RT-qPCR (p = 0.032). More viral RNA was detected in the virus control than in DDGS; SBM and FEED were intermediate. By VI, we found that infectious virus could be harbored in SBM, DDGS, and FEED for a short time.

Investigating ionizing radiation-induced changes in breast cancer cells using stimulated Raman scattering microscopy.

Altered lipid metabolism of cancer cells has been implicated in increased radiation resistance. A better understanding of this phenomenon may lead to improved radiation treatment planning. Stimulated Raman scattering (SRS) microscopy enables label-free and quantitative imaging of cellular lipids but has never been applied in this domain. We sought to investigate the radiobiological response in human breast cancer MCF7 cells using SRS microscopy, focusing on how radiation affects lipid droplet (LD) distribution and cellular morphology. MCF7 breast cancer cells were exposed to either 0 or 30Gy (X-ray) ionizing radiation and imaged using a spectrally focused SRS microscope every 24hrs over a 72-hr time period. Images were analyzed to quantify changes in LD area per cell, lipid and protein content per cell, and cellular morphology. Cell viability and confluency were measured using a live cell imaging system while radiation-induced lipid peroxidation was assessed using BODIPY C11 staining and flow cytometry. The LD area per cell and total lipid and protein intensities per cell were found to increase significantly for irradiated cells compared to control cells from 48 to 72hrs post irradiation. Increased cell size, vacuole formation, and multinucleation were observed as well. No significant cell death was observed due to irradiation, but lipid peroxidation was found to be greater in the irradiated cells than control cells at 72hrs. This pilot study demonstrates the potential of SRS imaging for investigating ionizing radiation-induced changes in cancer cells without the use of fluorescent labels.

Development of buffalo ear skin fibroblast cell line for somatic cell nuclear transfer

Fibroblast is the principal active cell of connective tissue. It can be used as a useful tool for reproductive and therapeutic studies for its ability to easily grow in culture, create a preferable environment to growth and can be used as both for primary and permanent cell lines. Somatic cell nuclear transfer (SCNT) is commonly used for complete genetic reprogramming of a fully differentiated cell (e.g., fibroblast). SCNT is influential means for studying genomic imprinting, nuclear-cytoplasmic interaction, totipotency, and the contribution of paternal and maternal genomes to developing embryos. For reproductive cloning, SCNT is a fundamental step. So, the objective of this study was to adapt buffalo fibroblast cell culture protocol in order to develop fibroblast cell line for SCNT. River type buffalo ear skin tissues were used for developing primary cell culture to establish fibroblast cell line. Cells were grown in vitro. The general morphology and growth of cell population and presence of any microbial contaminants were checked regularly under an inverted microscope in phase contrast. Cells were counted by using hemocytometers. Sliced tissue was sown in 15 culture dishes to create primary and subcultures, and cells were cultivated in 6 of those dishes. Cell confluence varied from 70 to 80%. There were performed subcultures. Cell confluence in passages 1 through 10 varied from 73 to 90%. Cell concentration was 2.72 x 105, 2.68 x 105, and 2.65 x 105 per ml in passages 1, 5, and 10, respectively. Viable cells ranged from 84-93% of the total cells in passages 1 through 10. Different passages of cultivated fibroblast cells showed significantly varying viability (p&lt; 0.05). Cultured fibroblast cell lines could now be preserved using cryotechnology.

Dead or alive: Distinguishing active from passive particles using supervised learning (a)

A longstanding open question in the field of dense disordered matter is how precisely structure and dynamics are related to each other. With the advent of machine learning, it has become possible to agnostically predict the dynamic propensity of a particle in a dense liquid based on its local structural environment. Thus far, however, these machine-learning studies have focused almost exclusively on simple liquids composed of passive particles. Here we consider a mixture of both passive and active (i.e., self-propelled) Brownian particles, with the aim to identify the active particles from minimal local structural information. We compare a state-of-the-art machine learning approach for passive systems with a new method we develop based on Voronoi tessellation. Both methods accurately identify the active particles based on their structural properties at high activity and low concentrations of active particles. Our Voronoi method is, however, substantially faster to train and deploy because it requires fewer, and easy to compute, input features. Notably, both become ineffective when the activity is low, suggesting a fundamentally different structural signature for dynamic propensity and non-equilibrium activity. Ultimately, these efforts might also find relevance in the context of biological active glasses such as confluent cell layers, where subtle changes in the microstructure can hint at pathological changes in cell dynamics.

The Tumor Suppressor DAB2IP Is Regulated by Cell Contact and Contributes to YAP/TAZ Inhibition in Confluent Cells.

External and internal mechanical forces modulate cell morphology, movement, proliferation and metabolism, and represent crucial inputs for tissue homeostasis. The transcriptional regulators YAP and TAZ are important effectors of mechanical signaling and are frequently activated in solid tumors, correlating with metastasis, chemoresistance, and shorter patient survival. YAP/TAZ activity is controlled by various pathways that sense cell shape, polarity, contacts, and mechanical tension. In tumors, aberrant YAP/TAZ activation may result from cancer-related alterations of such regulatory networks. The tumor suppressor DAB2IP is a Ras-GAP and scaffold protein that negatively modulates multiple oncogenic pathways and is frequently downregulated or inactivated in solid tumors. Here, we provide evidence that DAB2IP expression is sustained by cell confluency. We also find that DAB2IP depletion in confluent cells alters their morphology, reducing cell packing while increasing cell stiffness. Finally, we find that DAB2IP depletion in confluent cells favors YAP/TAZ nuclear localization and transcriptional activity, while its ectopic expression in subconfluent cells increases YAP/TAZ retention in the cytoplasm. Together, these data suggest that DAB2IP may function as a sensor of cell interactions, contributing to dampening cellular responses to oncogenic inputs in confluent cells and that DAB2IP loss-of-function would facilitate YAP/TAZ activation in intact epithelia, accelerating oncogenic transformation.

#4451 MONOCYTE MIGRATION AND ACTIVATION IN ANCA-ASSOCIATED GLOMERULONEPHRITIS

Abstract Background and Aims Glomerular injury in Anti-Neutrophil Cytoplasmic Antibodies (ANCA)-associated glomerulonephritis (AGN) is associated with macrophage infiltration. To date, their role and origin remain to be fully elucidated. Activated blood monocytes likely transmigrate and differentiate into kidney macrophages depending on the local microenvironment. Even in remission, some studies indicate sustained monocyte activation and upregulation of adhesion markers. The aim of this study was to research the intrinsic migration capacity of monocytes in AGN. Targeting monocyte migration might open new therapeutic avenues in the treatment of AGN. Method Transendothelial migration of monocytes was tested in AGN patients with active disease (n = 2), in remission (n = 8) and healthy controls (n = 6). To assess their adhesive and migratory capacity, freshly isolated CD14 positive monocytes were added to confluent TNF-α or IL1β overnight-stimulated Human Aortic Endothelial Cell (HAEC) for 30 minutes and subsequently fixed. Monocyte adhesion and transmigration was visualized by phase-contrast microscopy and quantified using ImageJ. To unravel the potential mechanisms driving these differences in monocyte migration, we performed bulk RNA-sequencing of monocytes from AGN patients with active (n = 4) and stable (n = 10) disease, and healthy controls (HC) (n = 6). Results Monocyte adhesion, but not migration, was significantly increased during active disease, independently of the stimuli used to mimic the pro-inflammatory phenotype of endothelial cells (EC) (Figure 1). During remission, decreased adhesion was found on the IL1β-stimulated ECs. While CD11b mRNA expression was upregulated, CD11a expression was downregulated in monocytes from active AGN patients compared to HC. Two genes involved in paracellular monocyte transmigration (JAML, PECAM-1) were significantly decreased. Most AGN patients were treated with corticosteroids at time of experiments. Conclusion These results suggest a remarkable increase in monocyte adhesion, but lower intrinsic migration capacity, in a 30 minute time-frame during active AGN. Our findings on migration are surprising in the light of theorized enhanced monocyte extravasation towards diseased AGN kidneys. Therefore, it could be speculated that after a longer period of time, a subset of the adherent monocytes would ultimately migrate and differentiate into kidney macrophages. In remission, long-term immunosuppressive treatment or chronic inflammation might decrease monocyte adhesion. This was the first ex-vivo study to research monocyte migration in AGN, further research is required to validate findings and to develop new therapies targeting monocyte migration.

Extracellular and intracellular productions of lysophosphatidic acids and cyclic phosphatidic acids by lysophospholipase D from exogenously added lysophosphatidylcholines to cultured NRK52E cells

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that is a notable biomarker of kidney injury. However, it is not clear how LPA is produced in renal cells. In this study, we explored LPA generation and its enzymatic pathway in a rat kidney-derived cell, NRK52E cells. Culturing of NRK52E cells with acyl lysophosphatidylcholine (acyl LPC), or lyso-platelet activating factor (lysoPAF, alkyl LPC) was resulted in increased extracellular level of choline, co-product with LPA by lysophospholipase D (lysoPLD). Their activities were enhanced by addition of calcium ions to the cell culture medium, but failed to be inhibited by S32826, an autotaxin (ATX)-specific inhibitor. Liquid chromatography-tandem mass spectrometric analysis revealed the small, but significant extracellular production of acyl LPA/cyclic phosphatidic acid (cPA) and alkyl LPA/cPA. The mRNA expression of glycerophosphodiesterase (GDE) 7 with lysoPLD activity was elevated in confluent NRK52E cells cultured over 3 days. GDE7 plasmid-transfection of NRK52E cells augmented both extracellular and intracellular productions of LPAs (acyl and alkyl) as well as extracellular productions of cPAs (acyl and alkyl) from exogenous LPCs (acyl and alkyl). These results suggest that intact NRK52E cells are able to produce choline and LPA/cPA from exogenous LPCs through the enzymatic action of GDE7 that is located on the plasma membranes and intracellular membranes.

Effects of Bacterial Lysates and Metabolites on Collagen Homeostasis in TNF-α-Challenged Human Dermal Fibroblasts.

During skin aging, the production of extracellular matrix (ECM) proteins, such as type I collagen, decreases and the synthesis of ECM-degrading matrix metalloproteinases (MMPs) rises, leading to an imbalance in homeostasis and to wrinkle formation. In this study, we examined the effects of bacterial lysates and metabolites from three bifidobacteria and five lactobacilli on collagen homeostasis in human dermal fibroblasts during challenge with tumor necrosis factor alpha (TNF-α), modeling an inflammatory condition that damages the skin's structure. Antiaging properties were measured, based on fibroblast cell viability and confluence, amount of type I pro-collagen, ratio of MMP-1 to type I pro-collagen, cytokines, and growth factors. The TNF-α challenge increased the MMP-1/type I pro-collagen ratio and levels of proinflammatory cytokines, as expected. With the probiotics, differences were clearly dependent on bacterial species, strain, and form. In general, the lysates elicited less pronounced responses in the biomarkers. Of all strains, the Bifidobacterium animalis ssp. lactis strains Bl-04 and B420 best maintained type I pro-collagen production and the MMP-1/collagen type I ratio under no-challenge and challenge conditions. Metabolites that were produced by bifidobacteria, but not their lysates, reduced several proinflammatory cytokines (IL-6, IL-8, and TNF-α) during the challenge, whereas those from lactobacilli did not. These results indicate that B. animalis ssp. lactis-produced metabolites, especially those of strains Bl-04 and B420, could support collagen homeostasis in the skin.

Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells.

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds toGM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.

Phenotypic and Genotypic Antimicrobial Susceptibility Testing of Chlamydia trachomatis Isolates from Patients with Persistent or Clinical Treatment Failure in Spain

The aim of this multicentre project (seven hospitals across the Spanish National Health Service) was to study the phenotypic and genotypic susceptibility of C. trachomatis to the main antimicrobials used (macrolides, doxycycline, and quinolones) in isolates from patients with clinical treatment failure in whom reinfection had been ruled out. During 2018–2019, 73 clinical isolates were selected. Sixty-nine clinical specimens were inoculated onto confluent McCoy cell monolayers for phenotypic susceptibility testing. The minimum inhibitory concentration for azithromycin and doxycycline was defined as the lowest concentration associated with an at least 95% reduction in inclusion-forming units after one passage in the presence of the antibiotic compared to the initial inoculum for each strain (control). Sequencing analysis was performed for the genotypic detection of resistance to macrolides, analysing mutations in the 23S rRNA gene (at positions 2057, 2058, 2059, and 2611), and quinolones, analysing a fragment of the gyrA gene, and searching for the G248T mutation (Ser83-&gt;Ile). For tetracyclines, in-house RT-PCR was used to test for the tet(C) gene. The phenotypic susceptibility testing was successful for 10 isolates. All the isolates had minimum inhibitory concentrations for azithromycin ≤ 0.125 mg/L and for doxycycline ≤ 0.064 mg/L and were considered sensitive. Of the 73 strains studied, no mutations were found at positions T2611C or G248T of the gyrA gene. We successfully sequenced 66 isolates. No macrolide resistance-associated mutations were found at positions 2057, 2058, 2059, or T2611C. None of the isolates carried the tet(C) gene. We found no evidence for genomic resistance in this large, clinically relevant dataset.

Metformin induces autophagy of cisplatin-resistant human gastric cancer cells in addition to apoptosis.

Metformin has been used to treat cases of type 2 diabetes mellitus, and mounting studies have shown that metformin can act alone or in synergy with other anticancer agents to achieve anti-cancer efficacies on various types of tumors. However, the role of metformin in either inducing autophagy and cisplatin-resistance of human gastric cancer (GC) cells has never been examined. The study has established a cisplatin-resistant GC cell line and investigated the effects of metformin on inducing autophagy on it. The results demonstrated that treatment with metformin can concentration-dependently suppress the cell viability and cell confluence of cisplatin-resistant GC cells, while having no effects on human primary stomach epithelial cells (HPSEC). For the first time, we found that metformin can significantly increase the acidic vesicular organelles (AVO) level and decrease the acridine orange (AO) level spontaneously in the cisplatin-resistant GC cells. Thus, we further checked the other markers, Atg5, Atg12 and LC3-II, which showed that metformin indeed induced autophagy in the cisplatin-resistant GC cells. In addition, treatment of 3-Methyladenine (3-MA) can significantly rescue the metformin-induced autophagy. At the same time, metformin can induce the alterations of apoptosis-associated signal molecules, such as caspase-3 and caspase-7 activities. Overall, the pilot study provided evidence for metformin induced autophagy in addition to apoptosis, making it as an effective anticancer drug for the therapy of cisplatin-resistant GC. Killing the cisplatin-resistant GC cells with non-toxic metformin via both autophagy and apoptosis might extend its usefulness in our fighting with chemo-resistance of gastric cancer cells.

A Cartilaginous Construct with Bone Collar Exerts Bone-Regenerative Property Via Rapid Endochondral Ossification.

Three-dimensional clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes (C-MSCs) can be implanted into tissue defects with no artificial scaffolds. In addition, the cellular properties and characteristics of the ECM in C-MSCs can be regulated in vitro. Most bone formation in the developmental and healing process is due to endochondral ossification, which occurs after bone collar formation surrounding cartilage derived from MSCs. Thus, to develop a rapid and reliable bone-regenerative cell therapy, the present study aimed to generate cartilaginous tissue covered with a mineralized bone collar-like structure from human C-MSCs by combining chondrogenic and osteogenic induction. Human bone marrow-derived MSCs were cultured in xeno-free/serum-free (XF) growth medium. Confluent cells that formed cellular sheets were detached from the culture plate using a micropipette tip. The floating cellular sheet contracted to round clumps of cells (C-MSCs). C-MSCs were maintained in XF-chondro-inductive medium (CIM) and XF-osteo-inductive medium (OIM). The biological and bone-regenerative properties of the generated cellular constructs were assessed in vitro and in vivo. C-MSCs cultured in CIM/OIM formed cartilaginous tissue covered with a mineralized matrix layer, whereas CIM treatment alone induced cartilage with no mineralization. Transplantation of the cartilaginous tissue covered with a mineralized matrix induced more rapid bone reconstruction via endochondral ossification in the severe combined immunodeficiency mouse calvarial defect model than that of cartilage generated using only CIM. These results highlight the potential of C-MSC culture in combination with CIM/OIM to generate cartilage covered with a bone collar-like structure, which can be applied for novel bone-regenerative cell therapy.

Identification of 2',4',6'-trihydroxyacetophenone as promising cysteine conjugate beta-lyase inhibitor for preventing cisplatin-induced nephrotoxicity.

Cisplatin is a chemotherapeutic agent used to treat many types of malignant tumors. However, irrespective of its potent anticancer properties and efficacy, nephrotoxicity is the dose-limiting factor of cisplatin treatment. Cisplatin infiltrates renal tubular cells in the kidneys and is metabolized by cysteine conjugate-beta lyase 1 (CCBL1) to form highly reactive thiol-cisplatin; this may mediate cisplatin's nephrotoxicity. Therefore, CCBL1 inhibition may prevent cisplatin-induced nephrotoxicity. Using a high-throughput screening assay, we identified 2',4',6'-trihydroxyacetophenone (THA) as an inhibitor of CCBL1. THA inhibited human CCBL1 beta-elimination activity in a concentration-dependent manner. We further investigated the preventive effect of THA on cisplatin-induced nephrotoxicity. THA attenuated the effect of cisplatin on the viability of confluent renal tubular cells (LLC-PK1 cells) but had no effect on cisplatin-induced reduction of proliferation in the tumor cell lines (LLC and MDA-MB-231). THA pre-treatment significantly attenuated cisplatin-induced increases in blood urea nitrogen, creatinine, cell damage score, and apoptosis of renal tubular cells in mice in a dose-dependent manner. Furthermore, THA pre-treatment attenuated cisplatin-induced nephrotoxicity without compromising its anti-tumor activities in mice bearing subcutaneous syngeneic LLC tumors. THA could help prevent cisplatin-induced nephrotoxicity and may provide a new strategy for cisplatin-inclusive cancer treatments.

UVA/UVB Irradiation Exerts a Distinct Phototoxic Effect on Human Keratinocytes Compared to Human Malignant Melanoma Cells.

Solar ultraviolet radiation (UVR) is responsible for the development of many skin diseases, including malignant melanoma (MM). This study assessed the phototoxic effects of UVA, and UVB radiations on healthy and pathologic skin cells by evaluating the behavior of human keratinocytes (HaCaT) and MM cells (A375) at 24 h post-irradiation. The main results showed that UVA 10 J/cm2 exerted no cytotoxicity on HaCaT and A375 cells, while UVB 0.5 J/cm2 significantly reduced cell viability and confluence, induced cell shrinkage and rounding, generated nuclear and F-actin condensation, and induced apoptosis by modulating the expressions of Bax and Bcl-2. The association of UVA 10 J/cm2 with UVB 0.5 J/cm2 (UVA/UVB) induced the highest cytotoxicity in both cell lines (viability < 40%). However, the morphological changes were different-HaCaT cells showed signs of necrosis, while in A375 nuclear polarization and expulsion from the cells were observed, features that indicate enucleation. By unraveling the impact of different UVR treatments on the behavior of normal and cancer skin cells and describing enucleation as a novel process involved in the cytotoxicity of UVA/UVB irradiation, these findings bridge the gap between the current and the future status of research in the field.

Combination of PARP and WEE1 inhibitors invitro: Potential for use in the treatment of SHH medulloblastoma.

Medulloblastoma (MB), grouped as either WNT‑activated, Sonic hedgehog (SHH)‑activated, or non-WNT/non-SHH group 3, accounts for almost 20% of all childhood brain cancers. In spite of current intensive treatments, not all patients are cured and survivors suffer from severe side‑effects. The present study therefore examined the effects of the poly‑ADP‑ribose polymerase (PARP) and WEE1‑like protein kinase (WEE1) inhibitors, BMN673 and MK‑1775, respectively, alone or in combination on four MB cell lines. More specifically, the MB cell lines, DAOY, UW228‑3, MED8A and D425, were tested for their sensitivity to BMN673 and MK‑1775 alone or in combination, using cell viability, cell confluency and cytotoxicity assays. The effects on the cell cycle phases were also examined using FACS analysis. Monotherapy with BMN673 and MK‑1775 exerted dose‑dependent inhibitory effects on the viability of almost all MB cell lines. Notably, when BMN673 and MK‑1775 were used in combination, synergistic effects were noted in the SHH group cell lines (DAOY and UW228‑3), but not in the already WEE1‑sensitive group 3 (MED8A and D425) lines. Moreover, the combination treatment decreased the percentage of cells in the G1 phase and induced the novel distribution of both DAOY and UW228‑3 cells in the S and G2/M phases, with the UW228‑3 cells exhibiting a greater delay. To conclude, MK‑1775 was efficient in all and BMN673 in most cell lines, and their combined use exerted synergistic effects on the SHH, but not the group 3 cell lines. These data suggest that MK‑1775 alone may be of interest for all MB cell lines, and that the combination of PARP/WEE1 inhibitors may provide possible therapeutic opportunities for the therapy of SHH MBs. Their use warrants further investigations in the future.