Skin Fibroblast Cell Line Research Articles

Abstract 4321: Epigenetic regulation of TP73 expression in HCC and GI cancer

Abstract Tumorigenesis results from an accumulation of mutational and epigenetic changes that alter normal cell growth and survival pathways. TP73 is the homologue of the master tumor suppressor TP53 involved in cellular responses to stress and development. The TP73 gene encodes two different protein isoforms, TAp73 and ΔNp73, and maps to a region on chromosome 1p36 that is frequently deleted in neuroblastoma and other tumors, and is thought to contain multiple tumor suppressor genes. However, the analysis of p73-knockout mice yielded conflicting results with respect to tumor suppression. World-wide efforts in sequencing the TP73 gene in patient tumor samples have not provided evidence for genetic alterations as a common cause of TP73 inactivation in human cancer. The role of TP73 in tumorigenesis has remained elusive to date. In this study, we isolated two stem cell lines from normal young and old human liver tissues to determine TP73 expression in normal human liver stem cell and hepatocellular cancer (HCC) cell lines (HepG2, SNU398, SNU449 & SNU475), gastrointestinal cancer (GI) cell lines (Caco2 & HCT116) and a normal skin fibroblast cell line (HS27). The results show that TP73 only expresses in cancer cell lines. Further immunohistochemistry studies on human normal liver tissues as well as adjacent normal and cancerous liver tissues from HCC patients confirm that TP73 only expresses in the cancerous tissues. Moreover, methylation-specific PCR and bisulfite sequencing studies revealed that TP73 promoter is activated only in cancer cell lines by DNA methylation. Furthermore, ChIP assay results demonstrated that CTCF (a chromosomal networking protein CCCTC binding factor) binds to TP73 promoter and regulates TP73 expression. Our observation may prove significant for the development of future therapeutic and diagnostic applications. Citation Format: Zhixing Yao, Cristina Di Poto, Habtom W. Ressom, Zaki A. Sherif. Epigenetic regulation of TP73 expression in HCC and GI cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4321.

Safety Assessment of Natural Color Foundation for Atopy

Purpose: As a therapeutic makeup product, foundation is used to reduce the anxiety and unease associated with appearance in people with atopic skin problems. The aim of the present study was to prepare and evaluate the safety of natural color foundation for atopy. Methods: Color foundations containing organic kaolin and zinc oxide were prepared. Safety of color foundation was evaluated by MTT cell viability assay (CCD-986sk human skin fibroblast cell line), and human repeat insult patch test for atopic dermatits. Results: Results of the MTT cell viability assay showed that the growth activity of human skin fibroblast cells was over 88% in all concentrations of the natural color foundations (sample C and D). Patch test revealed that the natural color foundations were safe. While two cases showed weak positive reactions (+) to sample A and B (general foundation on the market). Conclusion: Natural color foundation with kaolin and zinc oxide was verified safe for atopic dermatitis and it appears to be beneficial for use as a foundation for atopy.

Design, synthesis, and docking study of new quinoline derivatives as antitumor agents.

New quinolines substituted with various heterocycles and chalcone moieties were synthesized and evaluated as antitumor agents. All the synthesized compounds were in vitro screened against 60 human cancer cell lines. Compound 13 showed the highest cytotoxicity toward 58 cell lines, exhibiting distinct growth inhibition values (GI50 ) against the majority of them, including SR, HL-60 (TB) strains (leukemia), and MDA-MB-435 strains (melanoma), with GI50 values of 0.232, 0.260, and 0.300 µM, respectively. It exhibited great selectivity toward cancer cell lines, with less toxic effect against normal cells represented by skin fibroblast (BJ) and breast epithelial cell lines (MCF-10F). The enzyme inhibitory activity of compound 13 was evaluated against topoisomerase 1 (Topo 1), epidermal growth factor receptor and vascular endothelial growth factor receptor 2, where it displayed worthy Topo 1 inhibition activity with an IC50 value of 0.278 µM compared with camptothecin as a reference drug (IC50 0.224 µM). Docking studies were performed to investigate the recognition profile of compound 13 with the Topo 1 enzyme binding site.

Biocomposites conductive scaffold based on PEDOT:PSS/nHA/chitosan/PCL: Fabrication and characterization

Biomaterial-based scaffolds with suitable characteristics are highly desired in tissue engineering (TE) application. Biocomposites based on polymer and ceramics increase the chance for modulating the properties of scaffold. In recent years, researchers have considered conductive polymers to be used in TE application, due to their conductivity. This property has a good impact on tissue regeneration. A suitable design for bone substitute that consists of considerations such as material component, fabrication technique and mechanical properties. The previous studies on PEDOT:PSS/nHA/CS showed high wettability rate but low mechanical properties. Polycaprolactone (PCL) is a biodegradable and biocompatible polymer with a low wettability. The incorporation of PCL inside biocomposite can lead to the decrement in wettability and increment in mechanical property. In addition, this paper would examine the feasibility of blending of PCL and chitosan to fabricate PEDOT:PSS/nHA/CS composite scaffold. The fabrication technique of freezing/ lyophilization was used in this study. The scaffolds were characterized morphologically using scanning electron microscopy (SEM). Wettability was studied using a contact angle instrument. The attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) spectra interpreted the presence of polymeric ingredients within composite scaffold. Conductivity of the scaffolds was measured using a Digital Multimeter. In-vitro biological evaluation of the scaffolds was studied using human skin Fibroblast (HSF) cell line. The morphological study of biocomposite PEDOT:PSS/nHA/CS/PCL scaffold revealed random pore sizes and 66% porosity. Contact angle of the scaffold was increased and the swelling property and pore sizes were decreased after blending of PCL polymer. The viability of HSF cells on biocomposite PEDOT:PSS/nHA/CS/PCL scaffold was 85%. After 7 days, SEM analysis revealed the presence of cells on the surface of scaffold. In conclusion, the results suggested that PEDOT:PSS/nHA/CS/PCL biocomposite scaffold was non-toxic to cells and has suitable properties.

Nanoparticles for Bioapplications: Study of the Cytotoxicity of Water Dispersible CdSe(S) and CdSe(S)/ZnO Quantum Dots.

Semiconductor nanocrystals or quantum dots (QDs) have unique optical and physical properties that make them potential imaging tools in biological and medical applications. However, concerns over the aqueous dispersivity, toxicity to cells, and stability in biological environments may limit the use of QDs in such applications. Here, we report an investigation into the cytotoxicity of aqueously dispersed CdSe(S) and CdSe(S)/ZnO core/shell QDs in the presence of human colorectal carcinoma cells (HCT-116) and a human skin fibroblast cell line (WS1). The cytotoxicity of the precursor solutions used in the synthesis of the CdSe(S) QDs was also determined in the presence of HCT-116 cells. CdSe(S) QDs were found to have a low toxicity at concentrations up to 100 µg/mL, with a decreased cell viability at higher concentrations, indicating a highly dose-dependent response. Meanwhile, CdSe(S)/ZnO core/shell QDs exhibited lower toxicity than uncoated QDs at higher concentrations. Confocal microscopy images of HCT-116 cells after incubation with CdSe(S) and CdSe(S)/ZnO QDs showed that the cells were stable in aqueous concentrations of 100 µg of QDs per mL, with no sign of cell necrosis, confirming the cytotoxicity data.

In vitro biological evaluations of Fe3O4 compared with core–shell structures of chitosan-coated Fe3O4 and polyacrylic acid-coated Fe3O4 nanoparticles

Today the use of magnetic nanoparticles (MNPs) is widely investigated because of their biocompatibility and nontoxicity. The objective of this study was to synthesis a uniform superparamagnetic Fe3O4, the core–shell structures of chitosan-coated Fe3O4 (cc/Fe3O4) and polyacrylic acid-coated Fe3O4 (pc/Fe3O4) nanoparticles using an in situ co-precipitation process for evaluation of biomedical applications. To structurally characterize the synthesized nanoparticles, morphological and magnetic properties, Fourier transform infrared, X-ray diffraction, transmission electron microscopy, dynamic light scattering and vibrating sample magnetometry studies were employed. Our results showed that Fe3O4, cc/Fe3O4 and pc/Fe3O4 nanoparticles possess an average mean diameter of 10, 15 and 11 nm (dry samples) per Scherrer's equation in a dry state with saturation magnetization (MS) values of 70.64, 38.65 and 23.53 emu/g, respectively. The cytotoxicity of nanoparticles was evaluated by MTT assay using breast cancer (MCF7) and human normal skin (fibroblast) cell lines. Both of the two core–shell structures did not show toxicity on the fibroblast cell line even after 24 and 48 h. The viability rate of the bare Fe3O4 MNPs on the MCF7 cell line was significantly less than two the coated nanoparticles. MTT assays demonstrated that core–shell nanoparticles have less cytotoxicity than bare Fe3O4 MNPs (in 100 μg/ml during 24 h, the viability of bare Fe3O4 MNPs, cc/Fe3O4 and pc/Fe3O4 nanoparticles was 68.01%, 85.91%, and 88.13%, respectively). Moreover, hemolysis assay was performed to measure cytotoxicity of the nanoparticles on red blood cells (RBCs). The cc/Fe3O4 structure at all concentrations had less hemolysis percentage than the pc/Fe3O4 structure. The chitosan-coated Fe3O4 nanoparticle showed the highest biocompatibility.

3D printed chitosan dressing crosslinked with genipin for potential healing of chronic wounds

Recently, various additive manufacturing (3D printing) approaches have been employed to fabricate dressings such as film scaffolds that possess well defined architecture and orientation at the micro level. In this study, crosslinked chitosan (CH) based film matrices were prepared using 3D printing with genipin (GE) as a crosslinker, with glycerol (GLY) and poly ethylene glycol (PEG) as plasticizer. The 3D printed films were functionally characterized using (tensile, fluid handling, mucoadhesion, drug dissolution, morphological properties and cell viability as well physico-chemical characterization using scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction. CH-GE-PEG600 3D printed films having the ratio of 1:1 polymer: plasticizer was selected due to their appropriate flexibility. Fourier transform infrared results showed intermolecular interaction between CH, GE and PEG which was confirmed by X-ray diffraction showing amorphous matrix structure. In vitro mucoadhesion studies of CH-GE-PEG600 films showed the capability of the 3D printed film to adhere to the epithelial surface. Scanning electron microscopy images showed that the surface of the plasticised films were smooth indicating content uniformity of CH, GE and PEG whilst micro cracks in unplasticised films confirmed their brittle nature. Plasticised films also showed high swelling capacity which enhanced water absorption. Cytotoxicity (MTT) assay using human skin fibroblast cell lines demonstrated that more than 90% of cells were viable after 48 h confirming non-toxic nature of the 3D printed CH-GE-PEG600 films and therefore promising dressing for chronic wound healing applications.

Mono- or di-substituted imidazole derivatives for inhibition of acetylcholine and butyrylcholine esterases

Mono- or di-substituted imidazole derivatives were synthesized using a one-pot, two-step strategy. All imidazole derivatives were tested for AChE and BChE inhibition and showed nanomolar activity similar to that of the test compound donepezil and higher than that of tacrine. Structure activity relationship studies, docking studies to on X-ray crystal structure of AChE with PDB code 1B41, and adsorption, distribution, metabolism, and excretion (ADME) predictions were performed. The synthesized core skeleton was bound to important regions of the active site of AChE such as the peripheral anionic site (PAS), oxyanion hole (OH), and anionic subsite (AS). Selectivity of the reported test compounds was calculated and enzyme kinetic studies revealed that they behave as competitive inhibitors, while two of the test compounds showed noncompetitive inhibitory behavior. ADME predictions revealed that the synthesized molecules might pass through the blood brain barrier and intestinal epithelial barrier and circulate freely in the blood stream without binding to human serum albumin. While the toxicity of one compound on the WS1 (skin fibroblast) cell line was 1790 µM, its toxicity on the SH-SY5Y (neuroblastoma) cell line was 950 µM.

Design and synthesis of 2-phenyl benzimidazole derivatives as VEGFR-2 inhibitors with anti-breast cancer activity.

Three new series of 2-phenyl benzimidazole-based derivatives were designed, synthesized, and evaluated for their in vitro cytotoxic activity against breast cancer (MCF-7) cell lines. Three compounds 8, 9, and 15 showed high cytotoxic activities, with IC50 values of 3.37, 6.30, and 5.84μM, respectively, while they showed comparable cytotoxicity to the standard drug doxorubicin against human normal cells, including nontumorigenic breast epithelial cell line (MCF-10F), skin fibroblast cell line (BJ), and lung fibroblast cell line (MRC-5). Six of the synthesized compounds were screened against vascular endothelial growth factor receptor 2 (VEGFR-2) where compounds 8, 9, 12, and 15 exhibited an outstanding potency in comparison with sorafenib, with IC50 values of 6.7-8.9nM. Molecular docking study assessed the good binding patterns of the most potent compounds with the reported conserved amino acids of VEGFR-2 active site.

Synthesis and Cytotoxicity of 1,4-Naphthoquinone Oxime Derivatives

A series of hydroxylated 1,4-naphthoquinone oximes were designed and synthesized. The in vitro cytotoxicity of these compounds was evaluated against five human cancer cell lines and human skin fibroblast cell line. Among them, compounds (1E,4E)-6-{1-[(5-Hydroxypentyl)oxy]-2,2-dimethylbut-3-en-1-yl}-5,8- dimethoxynaphthalene-1,4-dione dioxime and (1E,4E)-6-{1-[(6-Hydroxyhexyl)oxy]-2,2-dimethylbut-3-en-1-yl}-5,8-dimethoxynaphthalene-1,4-dione dioxime displayed higher cytotoxicity in three cancer cell lines than the positive drug 5-fluorouracil

Concentration- and Time-Dependent Cytotoxicity of Silver Nanoparticles on Normal Human Skin Fibroblast Cell Line

Background: A wide use of Silver Nanoparticles (AgNPs) in consumer products, industry, pharmaceuticals, and medical sciences, including dermatology, has always caused concerns related to its adverse effects on the environment and human safety. Nano-sized particles can be uptaken by the body through various routes, especially the skin, which is the widest organ and the first contact surface for different nanomaterials and other foreign agents. Objectives: The aim of this investigation was to assess the viability of a vulnerable normal skin cell line to cytotoxicity of AgNPs with the same effective size and concentrations used for various microbial agents, such as Candida species. Methods: Synthesized AgNPs were characterized using UV-visible absorption spectroscopy, X-ray diffraction, Dynamic Light Scattering (DLS), and Transmission Electron Microscopy (TEM). Different concentrations of the aqueous colloidal AgNPs (2.17 to 69.5 µg/mL) were tested on normal Human Skin Fibroblast (HSF) cell line using MTT assay after 24 and 48 hours of exposure. Results: The analysis demonstrated that the synthesized AgNPs were mainly spherical with an average size of 6.03 nm. The MTT findings showed a significant concentration- and time-dependent manner in the reduction of cell viability. The calculated IC50 values were 30.64 and 14.98 µg/mL for 24 and 48 hours of incubation, respectively. Conclusions: The cytotoxic effect of AgNPs was higher for Human Skin Fibroblast cells after 48 hours of exposure compared with 24 hours. According to the observed results, small nano-sized silver particles could potentially have cytotoxic effects on normal cells, especially at high concentrations and prolonged exposure times.

Biological and anticancer properties of Inonotus obliquus extracts

Abstract Inonotus obliquus (IO) has traditionally been used in folk medicine in the treatment of gastrointestinal cancer, cardiovascular disease and diabetes. The aim of our study was to evaluate the biological and metabolic properties of IO extracts. Free radical scavenging properties, inhibition of the activity of xanthine oxidase (XOi), induction of reactive oxygen species (ROS) production, cell viability and expression of superoxide dismutase (SOD1), catalase (CAT) and KI67 mRNA in the human colorectal adenocarcinoma (Caco-2) and normal human skin fibroblast (BJ) cell lines were measured. It was found that 80% ethanol extract of IO exhibited the highest properties inhibiting the activity of XO and radical scavenging activities. PBS, ethanol and methanol extracts of IO increased ROS production in the Caco-2 cell line. Moreover, the ethanol extract of IO increased SOD1, CAT and KI67 mRNA expression and decreased ROS production in the BJ cell line. Alternately, PBS, ethanol and methanol extracts of IO decreased ROS production and SOD1, CAT and KI67 mRNA expression in Caco-2. The present study revealed, for the first time that IO extract acts in different way in normal and cancerous cell culture. Moreover cytotoxic mechanism of action can be partially ROS-dependent.

PO-420 Mechanisms of action of anti-proliferative lichen compound protolichesterinic acid

Introduction Protolichesterinic acid (PA), an aliphatic α-methylene γ-lactone isolated from the lichen Iceland moss ( Cetraria islandica ) has an anti-proliferative effect against a variety human cancer cells. In multiple myeloma cells PA induces apoptosis. The anti-proliferative effect of PA is not mediated by the known inhibitory action of PA on 5- and 12-lipoxygenases and effects on growth factor signalling are secondary. PA inhibits DNA replication and earlier studies indicated direct effects of PA on HIV reverse transcriptase (RT) and DNA polymerase. RT inhibitors are known to affect mitochondria. Material and methods Molecular modelling software (Glide, Schrodinger, LLC) was used to test for the ability of PA to bind onto DNA polymerases α, β, γ and θ. The crystal structures of polymerases were acquired from Protein Data Bank (PDB ID: 4QCL, 5TB8, 4ZTZ and 5A9F, respectively) and PA was docked into each structure. The effects of PA on mitochondria were assessed morphologically by electron microscopy and metabolically by measuring extracellular glucose and lactate concentrations in cell medium after 6 and 24 hour (ABL90 FLEX blood gas analyzer) in the pancreatic cancer cell line Aspc-1, the breast cancer cell line T47D, normal skin fibroblast and MCF-10 non-tumorigenic breast epithelial cell line. Results and discussions PA docked to all four types of DNA polymerases, creating multiple hydrogen bonds directly with the enzymes/polymerases and with the metal ion (Mg 2+ ) cofactor. Hydrogen bonds to the metal ions bridging the interaction to the enzyme appeared important and might be necessary/important for the activity of protolichesterinic acid and explain its mechanism of action. Morphological changes in mitochondria were observed following exposure to PA in both cancer cell lines, but these were difficult to interpret. Aspc-1 pancreatic cancer showed a significant increase in lactate production after 24 hour exposure to PA. This was not observed in the breast cell lines, T47D and MCF-10. Aspc-1 is less sensitive to the anti-proliferative effects of PA than the breast cell lines. Further metabolomics studies are in progress. Conclusion Docking study of crystal structures of DNA polymerases indicates that PA could affect activity of several types of DNA polymerase, but this remains to be tested experimentally. PA may affect mitochondrial structure but increased lactate production is associated with relative resistance to its anti-proliferative effects.

Fracture resistant, antibiofilm adherent, self-assembled PMMA/ZnO nanoformulations for biomedical applications: physico-chemical and biological perspectives of nano reinforcement

Antimicrobial, antibiofilm adherent, fracture resistant nano zinc oxide (ZnO NP) formulations based on poly methyl methacrylate (PMMA) matrix were developed using a facile ex situ compression moulding technique. These formulations demonstrated potent, long-term biofilm-resisting effects against Candida albicans (9000 CFU to 1000 CFU) and Streptococcus mutans. Proposed mechanism of biofilm resistance was the release of metallic ions/metal oxide by ‘particle-corrosion’. MTT and cellular proliferation assays confirmed both qualitatively and quantitatively equal human skin fibroblast cell line proliferations (approximately 75%) on both PMMA/ZnO formulation and neat PMMA. Mechanical performance was evaluated over a range of filler loading, and theoretical models derived from Einstein, Guth, Thomas and Quemade were chosen to predict the modulus of the nanoformulations. All the models gave better fitting at lower filler content, which could be due to restricted mobility of the polymer chains by the constrained zone/interfacial rigid amorphous zone and also due to stress absorption by the highly energized NPs. Fracture mechanics were clearly described based on substantial experimental evidence surrounding crack prevention in the initial zones of fracture. Filler−polymer interactions at the morphological and structural levels were elucidated through FTIR, XRD, SEM, TEM and AFM analyses. Major clinical challenges in cancer patient rehabilitation and routine denture therapy are frequent breakage of the prostheses and microbial colonization on the prostheses/tissues. In the present study, we succeeded in developing an antimicrobial, mechanically improved fracture resistant, biocompatible nanoformulation in a facile manner without the bio-toxic effects of surface modifiers/functionalization. This PMMA/ZnO nanoformulation could serve as a cost effective breakthrough biomaterial in the field of prosthetic rehabilitation and local drug delivery scaffolds for abused tissues.

Blackcurrant Anthocyanins Increase the Levels of Collagen, Elastin, and Hyaluronic Acid in Human Skin Fibroblasts and Ovariectomized Rats.

Blackcurrants (Ribes nigrum L.) contain high levels of anthocyanin polyphenols, which have beneficial effects on health, owing to their antioxidant and anticarcinogenic properties. Phytoestrogens are plant-derived substances with estrogenic activity, which could have beneficial effects on the skin. Estradiol secretion decreases during menopause, reducing extracellular matrix (ECM) component production by skin fibroblasts. Using a normal human female skin fibroblast cell line (TIG113) and ovariectomized rats, the present study investigated whether an anthocyanin-rich blackcurrant extract (BCE) and four blackcurrant anthocyanins have novel phytoestrogenic activities that could benefit the skin in menopausal women. In TIG113 cells, a microarray and the Ingenuity® Pathway Analysis showed that 1.0 μg/mL of BCE upregulated the expression of many estrogen signaling-related genes. A quantitative RT-PCR analysis confirmed that BCE (1.0 or 10.0 μg/mL) and four types of anthocyanins (10 μM) altered the mRNA expression of ECM proteins and enzymes involved in ECM turnover. Immunofluorescence staining indicated that the anthocyanins stimulated the expression of ECM proteins, such as collagen (types I and III) and elastin. Dietary administration of 3% BCE to ovariectomized rats for 3 months increased skin levels of collagen, elastin, and hyaluronic acid. This is the first study to show that blackcurrant phytoestrogens have beneficial effects on skin experimental models.

Sterically modulated silver(I) complexes of coumarin substituted benzimidazol–2–ylidenes: Synthesis, crystal structures and evaluation of their antimicrobial and antilung cancer potentials

In this contribution, a series of sterically–encumbered coumarin substituted benzimidazole–based N–heterocyclic carbene (NHC) precursors (1−12) and their silver(I)–NHC complexes (13–24) are reported. Molecular structure of NHC precursors 8 and 12 and cationic complexes 15 and 16 was established by single crystal X–ray diffraction method. The silver(I) complexes demonstrated various significant intramolecular agostic–like interactions operating between the metal center and the hydrogen atoms of the substituents alongside a variety of feeble π–π stacking interactions. A distorted linear coordination geometry is documented at the silver(I) center with the anti–arrangement of the ligands. Further, the complexes demonstrated promising antibacterial properties against Gram positive and Gram negative bacterial strains, especially complex 18 displayed a minimum inhibitory concentration (MIC) of 2 and 4 μg/mL against S. aureus and E. coli, and P. aeruginosa, respectively. Furthermore, complexes 14, 15, 16 and 18 were found cytotoxic against the human lung cancer cell lines A549 and H1975 with the IC50 (concentration of the test sample required to kill 50% of the cell population) value under 10 μM, while mono–NHC complex 20 displayed a potential drug window with the IC50 of 13.7 ± 2.70 and 14.5 ± 1.20 μM against the cancer cell lines H1975 and A549, respectively. Notably, these complexes displayed relatively lesser cytotoxic behaviour against the normal skin fibroblast cell line, Hs68. All the NHC precursors displayed significantly lower biological activities compared with their respective complexes, indicating the utility of silver(I) ions in antimicrobial and antilung cancer applications.

Integrin Molecular Tensions in Live Cells are Altered by Substrate Rigidity

Mounting evidence suggests that mammalian cells are capable of sensing the rigidity of their local environment and responding to it. However, it remains unclear whether cells sense cellular force alteration caused by matrix compliance (stress sensing) or sense cytoskeleton remodeling caused by matrix deformation (strain sensing). Because integrins are the major mechano-sensitive receptors that transmit cellular force at the cell-matrix interface, the tension transmitted by integrin molecules (integrin tension) may serve as fundamental mechanical signal in cell mechanotransduction and rigidity sensing. Therefore, measuring and mapping integrin molecular tension in live cells on elastic substrates will shed light to the mechanism of cell rigidity sensing. In this work, we applied integrative tension sensor (ITS), a sensor converting molecular tension above a threshold to fluorescent signal, to calibrate and map integrin tension directly by fluorescence imaging. In experiments, ITS with 54 pN tension threshold was grafted on PDMS (Polydimethylsiloxane) substrates with Young's modulus ranging from 1kPa to 1.8 MPa. Integrin tension above 54 pN in live cells was mapped with high resolution (0.4 µm) and sensitivity on these substrates. The results show that cell spreading area and integrin tension activity both increased with increasing PDMS gel stiffness for CHO-K1, NIH-3T3 and skin fibroblast cell lines. Also, keratocytes migrated faster on softer PDMS substrates and integrin tension activity was the highest on the stiffest PDMS sample. These experiments for the first time monitored integrin tension in live cells on elastic substrates, and clearly demonstrated that the substrate rigidity affects integrin tension activity, providing evidence in favor of stress sensing hypothesis.

Localization Microscopy Analyses of MRE11 Clusters in 3D-Conserved Cell Nuclei of Different Cell Lines.

In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell’s decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair.

Signatures of protein expression revealed by secretome analyses of cancer associated fibroblasts and melanoma cell lines

The imbalance of cellular homeostasis during oncogenesis together with the high heterogeneity of tumor-associated stromal cells have a marked effect on the repertoire of the proteins secreted by malignant cells (the secretome). Hence, the study of tumoral secretomes provides insights for understanding the cross-talk between cells within the tumor microenvironment as well as the key effectors for the establishment of the pre-metastatic niche in distant tumor sites. In this context, we performed a proteomic analysis of the secretomes derived from four cell lines: a paired set of fibroblasts - Hs 895. T, a cell line obtained from a lung node metastatic site from a patient who had melanoma and Hs 895.Sk, a skin fibroblast cell line (derived from the same patient); two malignant metastatic melanoma cell lines - A375, a malignant melanoma cell line from primary source and SH-4, a cell line derived from pleural effusion of a patient with metastatic melanoma. Clustering of expression profiles together with functional enrichment analysis resulted in patterns that mirrored each cell type. In addition, these patterns might be the result of cell-specific protein expression programs and reveal the emergence of trends in the co-expression of functionally related proteins in cellular melanoma models. SignificanceMelanoma is an aggressive skin cancer and a lethal melanocytic neoplasm with increasing annual number of cases, faster than any other solid tumor. In this context, the imbalance of cellular homeostasis during oncogenesis together with the high heterogeneity of tumor-associated stromal cells have a marked effect on the repertoire of the proteins secreted by malignant cells (the secretome). Therefore, the identification of protein expression patterns in malignant cells together with functional enrichment analysis provide insights into cell-specific protein expression programs and may reveal the emergence of trends in the co-expression of functionally related proteins regardless of cell type. Moreover, the identification of networks of protein interactions together with their expression profiles can be used for the targeted analysis of co-expressed proteins, allowing the identification of regulatory motifs in melanoma protein-protein interaction networks.

A Schiff Base Macrocycle Ligand and Its Mg(II) and Cd(II) Complexes: Spectral Properties with Theoretical Understanding and Biological Activity

AbstractSimple Schiff base condensation is employed to prepare a macrocyclic ligand (L1H4) with a support of intramolecular hydrogen templating effect. Reaction with Mg(II) and Cd(II) a total number of three complexes namely, [L14–(Mg2+)2] (1), [L14–(Cd2+)2(H2O)2] (2) and [H2L12– Cd2+] (3) is obtained under same reaction condition and same solvent. Ligand and the complexes are identified and characterized by different spectroscopic techniques. Further the structural geometry and absorption spectra were analyzed by DFT (for ligand)/ semi‐empherical method (for complexes). Considering the involvement of metal ions in the regulation of cell proliferation we further explored the effect of these macrocycle confined metal complexes on human skin fibroblast cell line (GM00637) with or without the presence of fibroblasts stimulant (extracts from Calendula officinalis and Morinda citrifolia). Also, same study has been performed with ligand and metal ion without mixing them. Among these Cd(II) complexes are found most promising. Luminescent property for the ligand and all the complexes are also examined and analyzed theoretically.