Human BJ Fibroblasts Research Articles

Synthesis, Aromatase Inhibition, Cytotoxicity and Molecular Docking Studies of New Fluorinated and Non-fluorinated Thiourea Derivatives of Desloratadine.

Aromatase inhibitors are among the most effective treatment of the breast cancer. Aromatase catalyzes estrogen biosynthesis, which is a long-term cause of breast cancer. Current study describes the synthesis, purification of 26 new fluorinated and non-fluorinated thiourea derivatives of desloratadine (5), and their aromatase inhibition activity, cytotoxicity against cancer cell line (MDA-MB-231). Compounds 7v and 7l exhibited a significant anti-aromatase activity, while compounds 7a, 7g-h, 7m and 7u were also significant active against MDA-MB-231 cell line. Furthermore, the molecular docking studies revealed that active compounds form key interactions with the crucial amino acid of aromatase active site including TRP224, LEU477, CYS437, ALA438, MET374, ARG115, ILE305, and PHE221, which are responsible for the binding interaction of aromatase. All analogues were new, except 7b and 7k and also lacked cytotoxicity BJ human fibroblasts, with the exception of 5 and 7x. This selectivity makes this series particularly interesting for further studies.

A recombinant fragment antigen-binding (Fab) of trastuzumab displays low cytotoxic profile in adult human cardiomyocytes: first evidence and the key implication of FcγRIIA receptor.

Fragment crystallizable gamma receptors (FcγRs) mediate various cellular responses with significant cardiovascular implications. They contribute to the anticancer activity of trastuzumab (TRZ), a recombinant humanized monoclonal antibody that interferes with human epidermal growth factor receptor 2 (HER2), thereby blocking its physiological function in cardiac cells. This is responsible for cardiac complications that hamper TRZ clinical application. In this study we investigated the involvement of FcγRs in the TRZ cardiotoxicity. We used a recombinant antigen-binding fragment (Fab) of TRZ (rFab-HER2) to examine whether the absence of the Fc region resulted in fewer cardiomyocyte toxicity while preserving TRZ's ability to inhibit HER2. When exposed to rFab-HER2, AC16 human adult ventricular cardiomyocytes were less vulnerable to damage and death, than to TRZ. Specifically, TRZ exhibited cytotoxicity at a lower concentration (150 µg/mL, corresponding to ~1 µM) compared to rFab-HER2 (250 µg/mL, corresponding to ~5 µM). Like TRZ, rFab-HER2 negatively modulated HER2 levels in cardiomyocyte (without inducing cytotoxic activity in BJ human fibroblast cells that either did not express or express very low levels of HER2) and inhibited the downstream ERK/AKT cascades. But rFab-HER2 did not alter cardiomyocyte mitochondrial dynamic balance, and affect apoptosis and inflammation, while it limited cytosolic and mitochondrial ROS indicators. On contrary, the Fc region (50-250 μg/mL) exerted direct cytotoxic action on cardiomyocytes (but not on human fibroblasts that lacked Fc receptors). TRZ (150 μg/mL) markedly upregulated the expression level of FcγRIIA (a FcγRs strongly involved in TRZ-induced antibody-dependent cellular toxicity) in cardiomyocytes, whereas the Fab fragment (150 μg/mL) had no effect. Our results demonstrate that Fc region plays an important pathogenic role in TRZ-induced cardiomyocyte toxicity. In addition, targeting FcγRIIA might contribute to the off-target effects of TRZ therapy.

Phytochemical investigation of Chrysanthellum americanum Vatke and its constituents- a targeted approach for the treatment of leishmaniasis

The present study is focused on the isolation and identification of new therapeutic candidates from Chrysanthellum americanum Vatke., and their efficacy against pteridine reductase-1 (PTR1), a valid chemotherapeutic target in the Leishmania parasite. Henceforth, a new compound, chrysanamerine (1), along with 7 known compounds, polyacetylene 2, and flavonoids 3–8, were isolated from C. americanum. Their structures were determined by chemical and spectroscopic analyses and compared with the reported spectroscopic data. All compounds were evaluated for their anti-leishmanial activity against PTR1 via biochemical mechanism-based assay. The in vitro results showed five potential hits including a new compound, chrysanamerine (1), and four known compounds against the PTR1 enzyme. Among them, compound 1 showed a potent enzyme inhibition with an IC50 of 31.02 ± 2.36 μM, whereas a moderate inhibition was observed in cases of compounds 5 and 6 (IC50 = 59.86 ± 3.32, and 45.32 ± 3.5 μM, respectively). Whereas, compounds 3 and 8 showed mild inhibition (IC50 = 72.12 ± 1.12, and 97.18 ± 1.23 μM, respectively) against PTR1, compared with trimethoprim (positive control) (IC50 = 21.07 ± 1.6 μM). Moreover, the results were further validated via molecular docking and molecular dynamics (MD) simulations. Compound 1 showed a strong affinity to the binding site with a docking score of −11.83, along with the formation of a stable protein-ligand complex over the trajectory of 100 ns. Besides, compounds 1–8 were found to be non-cytotoxic on BJ (human fibroblast) cells.

Clioquinol derivatives as potent inhibitors of Methicillin-Resistant Staphylococcus aureus and their mechanistic study

AimsMethicillin-Resistant Staphylococcus aureus cause many nosocomial, and community-acquired infections, burdening healthcare systems globally. The aim of the current study was to identify the potent inhibitors of MRSA strains as first step towards drug discovery against MRSA related infections. Methods and ResultsClioquinol derivatives 1–10 were evaluated against MRSA strains, using microplate alamar blue assay (MABA). Among them compounds 1–3, and 6–8 were found to be active with MIC values within the range of 61.08–213.58 µM. Compounds 1, and 7 were found highly potent with MIC values of 64.04 and 61.08 µM, respectively. Both these compounds also showed non-covalent interactions with active site key residues of topoisomerase IV enzyme in molecular docking studies. Mechanisms of action of the most active compounds 1, and 7 were further studied through AFM, and fluorescence microscopy. It was observed that compounds 1, and 7 caused a massive destruction in shape, and greatly reduced the size and number of viable cells. Flow cytometry analysis showed a significant rise in DiBAC4(3) fluorescence, and a non-significant rise in PI fluorescence indicating increased bacterial membrane depolarization of treated cells. Most importantly the clioquinol derivatives 1, and 7 did not cause any hemolytic effect. Moreover, all the compounds were found to be non-cytotoxic to BJ (Human fibroblast) normal cell line. ConclusionOur findings suggest that clioquinol derivatives 1, and 7 possess highly potent antibacterial activity against three MDR, and one sensitive strains of S. aureus. Significance and impact of the studyClioquinol derivatives 1, and 7 could be further studied as potential antibacterial agents.

Nanoarchitectonics and Biological Properties of Nanocomposite Thermosensitive Chitosan Hydrogels Obtained with the Use of Uridine 5'-Monophosphate Disodium Salt.

Currently, an important group of biomaterials used in the research in the field of tissue engineering is thermosensitive chitosan hydrogels. Their main advantage is the possibility of introducing their precursors (sols) into the implantation site using a minimally invasive method-by injection. In this publication, the results of studies on the new chitosan structures in the form of thermosensitive hydrogels containing graphene oxide as a nanofiller are presented. These systems were prepared from chitosan lactate and chitosan chloride solutions with the use of a salt of pyrimidine nucleotide-uridine 5'-monophosphate disodium salt-as the cross-linking agent. In order to perform the characterization of the developed hydrogels, the sol-gel transition temperature of the colloidal systems was first determined based on rheological measurements. The hydrogels were also analyzed using FTIR spectroscopy and SEM. Biological studies assessed the cytotoxicity (resazurin assay) and genotoxicity (alkaline version of the comet assay) of the nanocomposite chitosan hydrogels against normal human BJ fibroblasts. The conducted research allowed us to conclude that the developed hydrogels containing graphene oxide are an attractive material for potential use as scaffolds for the regeneration of damaged tissues.

Biochemical and In Silico Studies on Triazole Derivatives as Tyrosinase Inhibitors: Potential Treatment of Hyperpigmentation Related Skin Disorders.

Tyrosinase is a versatile, glycosylated copper-containing oxidase enzyme that mainly catalyzes the biosynthesis of melanin in mammals. Its overexpression leads to the formation of excess melanin, resulting in hyperpigmentary skin disorders, such as dark spots, melasma, freckles, etc. Therefore, inhibition of tyrosinase is a therapeutic approach for the treatment of hyperpigmentation. The current study focused on evaluating tyrosinase inhibitory activities of triazole derivatives 1-20, bearing different substituents on the phenyl ring. 17 derivatives have shown a potent tyrosinase inhibition with IC50 values between 1.6 to 13 μM, as compared to the standard drug, i.e., kojic acid (IC50 = 24.1 ± 0.5 μM). Particularly, compounds 11 and 15 displayed 12 times more potent inhibitory effects than the kojic acid. The structure-activity relationship revealed that substituting halogens at the C-4 position of the benzene ring renders remarkable anti-tyrosinase activities. Compounds 1-3 and 8 showed a competitive type of inhibition, while compounds 5, 11, and 15 showed a non-competitive mode of inhibition. Next, we performed molecular docking analyses to study the binding modes and interactions between the ligands (inhibitors) and the active site of the tyrosinase enzyme (receptor). Besides this, we have assessed the toxicity profile of inhibitors on the BJ human fibroblast cell line. The majority of the newly identified tyrosinase inhibitors were found to be noncytotoxic. The results presented herein form the basis of further studies on triazole derivatives as potential drug leads against tyrosinase-related diseases.

In vivo assessment of dual-function submicron textured nitric oxide releasing catheters in a 7-day rabbit model

Catheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. Herein, a dualfunction submicron textured nitric oxide (NO)-releasing catheter was developed. The hemocompatibility and antithrombotic activity of vascular catheters were evaluated in both 20 h in vitro blood loop and 7 d in vivo rabbit model. Surface characterization assessments via atomic force microscopy show the durability of the submicron pattern after incorporation of NO donor S-nitroso-N-acetylpenicillamine (SNAP). The SNAP-doped catheters exhibited prolonged and controlled NO release mimicking the levels released by endothelium. Fabricated catheters showed cytocompatibility when evaluated against BJ human fibroblast cell lines. After 20h in vitro evaluation of catheters in a blood loop, textured-NO catheters exhibited a 13-times reduction in surface thrombus formation compared to the control catheters, which had 83% of the total area covered by clots. After the 7 d in vivo rabbit model, analysis on the catheter surface was examined via scanning electron microscopy, where significant reduction of platelet adhesion, fibrin mesh, and thrombi can be observed on the NO-releasing textured surfaces. Moreover, compared to relative controls, a 63% reduction in the degree of thrombus formation within the jugular vein was observed. Decreased levels of fibrotic tissue decomposition on the jugular vein and reduced platelet adhesion and thrombus formation on the texture of the NO-releasing catheter surface are indications of mitigated foreign body response. This study demonstrated a biocompatible and robust dual-functioning textured NO PU catheter in limiting fouling-induced complications for longer-term blood-contacting device applications. Statement of significanceCatheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. This study demonstrated a robust, biocompatible, dual-functioning textured nitric oxide (NO) polyurethane catheter in limiting fouling-induced complications for longer-term blood-contacting device applications.The fabricated catheters exhibited prolonged and controlled NO release that mimics endothelium levels. After the 7 d in vivo model, a significant reduction in platelet adhesion, fibrin mesh, and thrombi was observed on the NO-releasing textured catheters, along with decreased levels of fibrotic tissue decomposition on the jugular vein. Results illustrate that NO-textured catheter surface mitigates foreign body response.

Identification, crystallization, and first X-ray structure analyses of phenyl boronic acid-based inhibitors of human carbonic anhydrase-II

Human carbonic anhydrases (hCAs) play a central role in various physiological processes in the human body. HCAs catalyze the reversible hydration of CO2 into HCO3−, and hence maintains the fluid and pH balance. Overexpression of CA II is associated with diseases, such as glaucoma, and epilepsy. Therefore, CAs are important clinical targets and inhibition of different isoforms, especially hCA II is used in treatment of glaucoma, altitude sickness, and epilepsy. Therapeutically used CA inhibitors (CAI) are sulfonamide-based, such as acetazolamide, dichlorphenamide, methazolamide, ethoxzolamide, etc. However, they exhibit several undesirable effects such as numbness, tingling of extremities, malaise, metallic taste, fatigue, renal calculi, and metabolic acidosis. Therefore, there is an urgent need to identify safe and effective inhibitors of the hCAs. In this study, different phenyl boronic acids 1–5 were evaluated against bovine (bCA II) and hCA II. Among all, compound 1 (4-acetylphenyl boronic acid) was found to be active against bCAII and hCA II with IC50 values of 246 ± 0.48 and 281.40 ± 2.8 μM, respectively, while the remaining compounds were found in-active. Compound 1 was identified as competitive inhibitor of hCA II enzyme (Ki = 283.7 ± 0.002 μM). Additionally, compound 1 was found to be non-toxic against BJ Human fibroblast cell line. The X-ray crystal structure for hCA II in-complex with compound 1 was evaluated to a resolution of 2.6 Å. In fact, this the first structural analysis of a phenyl boron-based inhibitor bound to hCA II, allowing an additional structure-activity analysis of the compounds. Compound 1 was found to be directly bound in the active site of hCA II by interacting with His94, His119, and Thr199 residues. In addition, a bond of 3.11 Å between the zinc ion and coordinated boron atom of the boronic acid moiety of compound 1 was also observed, contributing to binding affinity of compound 1 for hCA II. PDB ID8IGF.

Electrospun Nanofibers Encapsulated with Natural Products: A Novel Strategy to Counteract Skin Aging.

The skin is the primary tissue affected by wounds and aging, significantly impacting its protective function. Natural products are widely used in cosmetics, representing a new approach to preventing age-related damage. Nanomedicine combines nanotechnology and traditional treatments to create innovative drugs. The main targets of nanotechnological approaches are wound healing, regeneration, and rejuvenation of skin tissue. The skin barrier is not easily permeable, and the creation of modern nanodevices is a way to improve the passive penetration of substances. In this study, Helichrysum italicum oil (HO) was combined with different types of electrospun nanofibers to study their protective activity on the skin and to evaluate their future application for topical treatments. In the present research, we used biodegradable polymers, including polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP), which were characterized by a scanning electron microscope (SEM). All results show a positive trend in cell proliferation and viability of human skin stem cells (SSCs) and BJ fibroblasts pre-treated with combined nanofibers and then exposed to UV stress. Gene expression analysis revealed the activation of a molecular rejuvenation program in SSCs treated with functionalized nanofibers before UV exposure. Understanding the mechanisms involved in skin changes during aging allows for the future application of nanomaterials combined with HO directly to the patients.

New pyranopyrazole based derivatives: Design, synthesis, and biological evaluation as potential topoisomerase II inhibitors, apoptotic inducers, and antiproliferative agents

A new series of pyranopyrazole-based derivatives were designed and synthesized. The synthesized compounds were assessed for their cytotoxic efficacy against A549 human lung carcinoma and MCF-7 human breast carcinoma cell lines. Three compounds (1b, 4b, and 7b) exhibited 1.3- to 2.3-fold more antiproliferative activity than that of doxorubicin against the A549 cell line. In comparison to doxorubicin, compounds 1d and 3b were 4.1- and 1.04-fold, respectively more powerful against MCF-7 cancer cells. All the synthesized compounds were found to be more selective toward A549 cancer cells than the normal human fibroblast BJ cells. Of interest, compounds 1b and 7b exhibited promising cytotoxicity and SIs of 27.72 and 25.30, respectively, towards A549 cancer cells, higher than that of doxorubicin (SI 4.81). The most potent compounds 1b, 1d, 3b, 4b, and 7b were then subjected to in vitro Topo II inhibition assay. They showed IC50 values in the range of 2.07 to 8.86 µM. Of particular interest, compound 7b (IC50 = 2.07 µM), exhibited higher Topo II inhibitory activity than that of doxorubicin (IC50 = 2.56 µM). The significant Topo II inhibition of compound 7b was explained by molecular docking simulations into the Topo II active site. Compound 7b halted the cell cycle in the S phase in A549 cancer cells. It induced total apoptosis and necrosis of 20.73- and 4-fold, respectively, greater than the control. This evidence was supported by a 3.59-fold increase in the level of apoptotic caspase-9 and a remarkable elevation of the Bax/BCL-2 ratio. The physiochemical parameters of compound 7b were aligned with Lipinski’s rule of five.

A novel modular opto-biomechatronics bioreactor for simultaneous isotropic mechanical stretch application and fluorescence microscopy under cell and tissue culture conditions

Mechanical stresses are an environmental challenge virtually all tissues in the body are exposed to and thus, are of fundamental interest to study cell reactions in mechanobiology. Yet, unlike acute short-term mechanical cell stimulations, long-term or cyclic mechano-stimulation as experienced in the body is difficult to reproduce. Bioreactors are designed to control cell culture conditions, but still, there are yet no technical solutions available to merge bioreactor and opto-biomechatronics technologies for cyclic stretch-applications and simultaneous live cell imaging. To close this gap, we have engineered an opto-biomechatronics module, consisting of our in-house developed IsoStretcher technology and customised epifluorescence optics, into an automated bioreactor platform. For this, redesigned polydimethylsiloxane (PDMS) chambers with closed geometry (∽700μL internal volume) to warrant sterile operation were developed. Those chambers could be flushed with cell solution for cell seeding in a sterile manner. The epifluorescence imaging module was engineered into the reactor underneath the IsoStretcher to allow for continuous image acquisition during long-term stretch cycles (hours to days). The system was validated on human fibroblast BJ foreskin cells, and Cal-520 Ca2＋ fluorescence was stably imaged using our in-built autofocus functionality. Cultures for 24h within the IsoStretcher-bioreactor preserved a normal cell morphology as compared to external incubator control cultures. Isotropic stretch was reliably transferred to the cell membranes. Our system with in-built bioreactor and opto-biomechatronics functionality provides a holistic technology platform for the growing field of mechanobiology to allow long-term observations of cultured single cells and confluent cell layers that are subjected to cyclic long-term isotropic stretch protocols.

Radioprotective effect of the anti-diabetic drug metformin.

Metformin is a biguanide currently used in the treatment of diabetes mellitus type 2. Besides its anti-glycemic effects, metformin has been reported to induce different cellular pleiotropic effects, depending on concentration and time of treatment. Here we report one administration of metformin (0.5 mM) has radioprotective effects in vitro on BJ human fibroblasts, increasing DNA damage repair and increasing SOD1 expression in the nucleus. Importantly, metformin (200 mg/kg) pre-administration for only 3 days in wild type 129/sv mice, decreases the formation of micronuclei in bone marrow cells and DNA damage in colon and lung tissues compared to control irradiated mice at sub-lethal and lethal doses, increasing the overall survival fraction by 37% after 10Gy total body irradiation. We next pre-treated with metformin and then exposed 129/sv mice, to a galactic cosmic rays simulation (GCRsim), at the NASA Space Radiation Laboratory (NSRL). We found metformin pre-treatment decreases the presence of bone marrow micronuclei and DNA damage in colon and lung tissues and an increase of 8-oxoguanine DNA glycosylase-1 (OGG1) expression. Our data highlight a radioprotective effect of metformin through an indirect modulation of the gene expression involved in the cellular detoxification rather than its effects on mitochondria.

Decavanadate-Bearing Guanidine Derivatives Developed as Antimicrobial and Antitumor Species.

To obtain biologically active species, a series of decavanadates (Hpbg)4[H2V10O28]·6H2O (1) (Htbg)4[H2V10O28]·6H2O; (2) (Hgnd)2(Hgnu)4[V10O28]; (3) (Hgnu)6[V10O28]·2H2O; and (4) (pbg = 1-phenyl biguanide, tbg = 1-(o-tolyl)biguanide, gnd = guanidine, and gnu = guanylurea) were synthesized and characterized by several spectroscopic techniques (IR, UV-Vis, and EPR) as well as by single crystal X-ray diffraction. Compound (1) crystallizes in space group P-1 while (3) and (4) adopt the same centrosymmetric space group P21/n. The unusual signal identified by EPR spectroscopy was assigned to a charge-transfer π(O)→d(V) process. Both stability in solution and reactivity towards reactive oxygen species (O2- and OH·) were screened through EPR signal modification. All compounds inhibited the development of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis bacterial strains in a planktonic state at a micromolar level, the most active being compound (3). However, the experiments conducted at a minimal inhibitory concentration (MIC) indicated that the compounds do not disrupt the biofilm produced by these bacterial strains. The cytotoxicity assayed against A375 human melanoma cells and BJ human fibroblasts by testing the viability, lactate dehydrogenase, and nitric oxide levels indicated compound (1) as the most active in tumor cells.

Abstract 17130: Metabolic and Epigenetic Coupled Regulation in Angiogenic Transdifferentiation and Vascular Recovery

Recent studies from our group and others have characterized the phenomenon of "angiogenic transdifferentiation” from fibroblasts to endothelial cells as a novel endogenous mechanism for microvasculature expansion and perfusion restoration in murine models of hindlimb ischemia and myocardial infarction. Metabolic regulation of epigenetic reprogramming has emerged as a key regulatory process controlling cell fate transition. To study the comprehensive role of metabolism in this healing process, we conducted liquid chromatography-mass spectrometry (LC-MS) based untargeted and targeted metabolomics during transdifferentiation. Our analysis revealed the upregulation of key metabolites, including UDP-GlcNAc (substrate for post-translational modification O-GlcNAcylation) and acetyl-CoA (substrate for histone acetylation). With our in vitro transdifferentiation protocol, we found that pharmacologic or genetic inhibition of OGT (O-GlcNAc transferase, the enzyme responsible for protein O-GlcNAcylation) or ACL (ATP citrate lyase, the enzyme converting citrate to acetyl-CoA) in human BJ fibroblasts impairs transdifferentiation. Employing a preclinical peripheral arterial disease (PAD) hindlimb ischemia model with fibroblast lineage tracing mice, we discovered that O-GlcNAcylation and ACL were accumulated in fibroblast lineage cells in ischemic limbs, and pharmacologic inhibition of OGT or ACL diminished the transdifferentiation cell population in the ischemic tissue. Furthermore, we identified fibroblast-specific OGT or ACL KO impaired vascular recovery post femoral artery ligation in the mice. Finally, we showed that metabolism-dependent post-translational modifications on histones (acetylation of H3K9, H3K27, H3K14) or histone chaperons (O-GlcNAcylation of HIRA, a histone H3.3 chaperone) mechanistically enhanced chromatin openness, thereby facilitating cell fate plasticity and vascular regeneration. In conclusion, our studies demonstrate the significant regulatory potential of metabolic factors in angiogenic transdifferentiation which presents an innovative avenue to enhance vascular recovery in ischemic vascular diseases such as PAD.

High-Level Secretion of Pregnancy Zone Protein Is a Novel Biomarker of DNA Damage-Induced Senescence and Promotes Spontaneous Senescence.

Identification of unique and specific biomarkers to better detect and quantify senescent cells remains challenging. By a global proteomic profiling of senescent human skin BJ fibroblasts induced by ionizing radiation (IR), the cellular level of pregnancy zone protein (PZP), a presumable pan-protease inhibitor never been linked to cellular senescence before, was found to be decreased by more than 10-fold, while the level of PZP in the conditioned medium was increased concomitantly. This observation was confirmed in a variety of senescent cells induced by IR or DNA-damaging drugs, indicating that high-level secretion of PZP is a novel senescence-associated secretory phenotype. RT-PCR examination verified that the transcription of the PZP gene is enhanced in various cells at senescence or upregulated following DNA damage treatment in a p53-independent manner. Moreover, pretreatment with late pregnancy serum containing a high level of PZP led to inhibition of doxorubicin-induced senescence in A549 cells, and depletion of PZP in the pregnancy serum could enhance such inhibition. Finally, the addition of immuno-precipitated PZP complexes into tissue culture attenuated the growth of A549 cells and promoted the spontaneous senescence. Therefore, we revealed that high-level secretion of PZP is a novel and unique feature associated with DNA damage-induced senescence, and secreted PZP is a positive regulator of cellular senescence, particularly during the late stage of gestation.

Influence of chlorpyrifos exposure on UVB irradiation induced toxicity in human skin cells

BackgroundAlthough chlorpyrifos (CPS) has been banned in many developed countries, it still remains one of the best-selling pesticides in the world. Widespread environmental and occupational exposure to CPS pose a serious risk to human health. Another environmental factor that can adversely affect human health is ultraviolet radiation B (UVB, 280–315 nm wave length). Here we attempt determine if exposure to CPS can modify toxic effects of UVB. Such situation might be a common phenomenon in agriculture workers, where exposure to both factors takes place.MethodsTwo skin cell lines; namely human immortalized keratinocytes HaCaT and BJ human fibroblasts were used in this study. Cytotoxicity was investigated using a cell membrane damage detection assay (LDH Cytotoxicity Assay), a DNA damage detection assay (Comet Assay), an apoptosis induction detection assay (Apo-ONE Homogeneous Caspase-3/7 Assay) and a cell reactive oxygen species detection assay (ROS-Glo H2O2 assay). Cytokine IL-6 production was also measured in cells using an ELISA IL-6 Assay.ResultsPre-incubation of skin cells with CPS significantly increased UVB-induced toxicity at the highest UVB doses (15 and 20 mJ/cm2). Also pre-exposure of BJ cells to CPS significantly increased the level of DNA damage, except for 20 mJ/cm2 UVB. In contrast, pre-exposure of HaCaT cells, to CPS prior to UVB radiation did not cause any significant changes. A decrease in caspase 3/7 activity was observed in HaCaT cells pre-exposed to 250 µM CPS and 5 mJ/cm2 UVB. Meanwhile, no statistically significant changes were observed in fibroblasts. In HaCaT cells, pre-exposure to CPS resulted in a statistically significant increase in ROS production. Also, in BJ cells, similar results were obtained except for 20 mJ/cm2. Interestingly, CPS seems to inhibited IL-6 production in HaCaT and BJ cells exposed to UVB (in the case of HaCaT cells for all UVB doses, while for BJ cells only at 15 and 20 mJ/cm2).ConclusionsIn conclusion, the present study indicates that CPS may contribute to the increased UVB-induced toxicity in skin cells, which was likely due to the induction of ROS formation along with the generation of DNA damage. However, further studies are required to gain better understanding of the mechanisms involved.

An amino acid mixture, enriched with Krebs cycle intermediates, enhances extracellular matrix gene expression in cultured human fibroblasts

In the human body, the skin is one of the organs most affected by the aging process. Nutritional approaches aimed to counteract the age-induced decline of extracellular matrix (ECM) deposition could be a valuable tool to decrease the degenerative processes underlying skin aging. Here, we investigated the ability of a six-amino acid plus hyaluronic acid (6AAH) formulation enriched with tricarboxylic acid (TCA) intermediates to stimulate ECM gene expression. To this aim, human BJ fibroblasts were treated with 6AAH alone or plus succinate or malate alone or succinate plus malate (6AAHSM), and mRNA levels of several ECM markers were evaluated. 6AAHSM increased the expression of all the ECM markers significantly above 6AAH alone or plus only succinate or malate. Furthermore, in an in vitro oxidative damage model, 6AAHSM blunted the hydrogen peroxide-induced decline in ECM gene expression. Our data suggest that feeding cells with 6AAH enriched with TCAs could efficiently be employed as a non-pharmacological approach for counteracting skin aging.

Cocrystallization of methyltestosterone with salicylic acid and 2,2′-disulfanediyldibenzoic acid - Effect on solubility of methyltestosterone and growth of leishmania parasite (amastigotes and promastigotes)

Methyltestosterone is a class II anabolic androgenic steroidal drug, structurally resembled to natural testosterone, produced by body. Two cocrystals of methyltestosterone with thiosalicylic acid and salicylic acid were successfully furnished by using solid state grinding method with the aim to study changes in biological activities and solubility. During cocrystallization the coformer thiosalicylic acid dimerize into 2, 2′-disulfanediyldibenzoic acid and furnished co-crystal I. Characterization of cocrystals 1 and II was carried out on the basis of single-crystal X-ray diffraction technique, IR spectroscopy, DSC/TGA analysis. The solubility of prepared cocrystals I and II were found to be significantly improved in different dissolution media i.e. phosphate buffer (pH 6.8, 7.4), 0.1 N HCl and distill water. Methyltestosterone and both cocrystals were found to be non-cytotoxic while tested against BJ human normal fibroblast cell line. Non-cytotoxic cocrystals I and II were further evaluated for anti-leishmanial activity (amastigotes and promastigotes). Both cocrystal I (IC50 = 11.9 µM) and II (IC50 = 27.2 µM) shows potent anti-leishmanial activity against L. major promastigote (extracellular / in blood parasite stage) and amastigote (intracellular / inside macrophage cell) stage in comparison to the standard drugs miltefosine (IC50 = 25.5 ± 0.42 µM) and methyltestosterone (IC50 = 37.42±0.57 µM). Infected macrophage cellular assay together with AFM analysis are carried out to study the effect of synthesized cocrystals on growth of amastigotes stage of L. major. The promising result demonstrates the effect of cocrystallization on solubility and anti-leishmanial potential of anabolic androgenic steroidal drug methyltestosterone for the first time.

Ketorolac Tromethamine Loaded Nano-Spray Dried Nanoparticles: Preparation, Characterization, Cell Viability, COL1A1 Gene Simulation and Determination of Anti-inflammatory Activity by In vivo HET-CAM Assay.

Ketorolac tromethamine (KT) is a non-steroidal anti-inflammatory drug from the heteroaryl acetic acid derivatives family. The most widely used new nanotechnological approaches for topical drug delivery are polymeric nanoparticles (NPs). Successful results have been obtained with low doses in many treatments, such as cancer, antimicrobial, pain, made with nanoparticle formulations of drug active ingredients. NPs were prepared using Nano Spray-Dryer. The cytotoxicity of the optimum formulation in BJ (ATCC® CRL-2522™) human fibroblast cells was determined by the WST- 1 method and the gene activity was elucidated by mRNA isolation and real-time polymerase chain reaction (RT-PCR). The in vivo HET- CAM assay was performed for anti-inflammatory activity. NPs presented PDI values lower than 0.5, and therefore particle size distribution was decided to be monodisperse. Positive zeta potential values of NPs highlighted the presence of the cationic ammonium group of Eudragit® RS 100. The release rates observed from KT-NP coded formulations after 24 hours were 78.4% ± 2.9, demonstrating extended release from all formulations, relative to pure KT. The lowest concentration of KT-NP increased fibroblast cell proliferation higher than the highest concentration of KT. The 5-fold increased effect of KT-NP formulation on collagen gene expression compared to KT is also related to the enhanced anti-inflammatory effect in line with the in vivo HET-CAM assay results. With the obtained cell viability, gene expression, and HET-CAM results, it has the hope of a successful nano-topical formulation, especially in both wound healing and anti-inflammatory treatment.

Modifications in cellular viability, DNA damage and stress responses inflicted in cancer cells by copper-64 ions.

Due to combined therapeutical emissions, a high linear energy transfer Auger-electrons with the longer ranged β- particles, 64Cu-based radiopharmaceuticals raise particular theragnostic interest in cancer, by joined therapeutic and real-time PET imaging properties. The in vitro study aimed to investigate the biological and molecular background of 64CuCl2 therapy by analyzing the damages and stress responses inflicted in various human normal and tumor cell lines. Colon (HT29 and HCT116) and prostate carcinoma (DU145) cell lines, as well as human normal BJ fibroblasts, were treated up to 72 h with 2-40 MBq/mL 64CuCl2. Radioisotope uptake and retention were assessed, and cell viability/death, DNA damage, oxidative stress, and the expression of 84 stress genes were investigated at various time points after [64Cu]CuCl2 addition. All the investigated cells incorporated 64Cu ions similarly, independent of their tumoral or normal status, but their fate after exposure to [64Cu]CuCl2 was cell-dependent. The most striking cytotoxic effects of the radioisotope were registered in colon carcinoma HCT116 cells, for which a substantial decrease in the number of metabolically active cells, and an increased DNA damage and oxidative stress were registered. The stress gene expression study highlighted the activation of both death and repair mechanisms in these cells, related to extrinsic apoptosis, necrosis/necroptosis or autophagy, and cell cycle arrest, nucleotide excision repair, antioxidant, and hypoxic responses, respectively. The in vitro study indicated that 40 MBq/mL [64Cu]CuCl2 delivers a therapeutic effect in human colon carcinoma, but its use is limited by harmful, yet lower effects on normal fibroblasts. The exposure of tumor cells to 20 MBq/mL [64Cu]CuCl2, might be used for a softer approach aiming for a lower radiotoxicity in normal fibroblasts as compared to tumor cells. This radioactive concentration was able to induce a persistent decrease in the number of metabolically active cells, accompanied by DNA damage and oxidative stress, associated with significant changes in stress gene expression in HCT116 colon cancer cells.