Human Fetal Lung Fibroblast Cells Research Articles

New insights regarding the selectivity and the uptake potential of nanoceria by human cells

Lately, cerium oxide nanoparticles (CeO2-NPs, nanoceria) have gained a great interest for applications in diagnose and treatment of many diseases, including cancer. Herein, we have synthesized CeO2-NPs and performed in vitro studies on HeLa (human cervical cancer) and HFL-1 (normal human fetal lung fibroblast) cell lines. The nanosized CeO2-NPs, 6–16nm in diameter, were obtained by a simple, precipitation method. In order to identify the crystalline phases and to estimate the average particle sizes, an X-ray diffraction investigation was performed. The structural and morphological characterization of the nanoceria was assessed by Raman Spectroscopy, Transmission Electron Microscopy (TEM), thermogravimetric analysis, and Zeta potential analysis. The in vitro tests were aimed at studying the CeO2-NPs cellular cytotoxicity, uptake, and ability to induce generation of reactive oxygen species on the HeLa and HFL-1 cells. The results obtained demonstrate a selective behaviour of the CeO2-NP towards both cell lines. The nanoparticles are, to a large extent, internalized in the HeLa cells, while very little nanoceria internalization is observed on the HFL-1 cells. Also, the CeO2-NPs are more cytotoxic to HeLa cells compared to HFL-1. Moreover, the level of reactive oxygen species upon exposure to CeO2-NPs is significantly higher on the HeLa cells. Such findings might have implications in targeted cancer therapies. The more nanoceria exhibit selective properties toward tumor cells and avoid interaction with non-tumor cells, the more potential they have to become an efficient system for targeted drug delivery.

Sox9 Protects against Human Lung Fibroblast Cell Apoptosis Induced by LPS through Activation of the AKT/GSK3β Pathway.

Sex-determining region Y-box 9 (Sox9) is an important transcription factor that has been identified as a key regulator of several types of diseases. In this study, we explored the correlation of Sox9 with cell proliferation, apoptosis, inflammatory factor expression, and the possible signaling pathway in human lung fibroblast cell line to investigate the possible mechanism of neonatal pneumonia. Therefore, in the present study, pc-Sox9 and si-Sox9 were transfected into MRC-5 (human fetal lung fibroblast cell line) to promote or inhibit expression of Sox-9. Quantitative reverse-transcription polymerase chain reaction and Western blot were used to determine the expression level of Sox-9 at mRNA and protein level. Then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and flow cytometry were used to explore, respectively, proliferation and apoptosis in vitro. We found that Sox9 could significantly upregulate the proliferation rate and inhibit apoptosis rate and inflammatory factor expression of MRC-5 cells compared with a control group. Moreover, the signaling pathway study confirmed that Sox9 protected MRC-5 from lipopolysaccharide injury through the AKT/GSK3β pathway. All these findings suggest that Sox9 acts as a novel marker for neonatal pneumonia and could be a new therapeutic target for this disease.

Upregulation of RGS2: A New Mechanism for Pirfenidone Amelioration of Pulmonary Fibrosis

BackgroundPirfenidone was recently approved for treatment of idiopathic pulmonary fibrosis. However, the therapeutic dose of pirfenidone is very high, causing side effects that limit its doses and therapeutic effectiveness. Understanding the molecular mechanisms of action of pirfenidone could improve its safety and efficacy. Because activated fibroblasts are critical effector cells associated with the progression of fibrosis, this study investigated the genes that change expression rapidly in response to pirfenidone treatment of pulmonary fibroblasts and explored their contributions to the anti‐fibrotic effects of pirfenidone.MethodsWe used the GeneChip microarray to screen for genes that were rapidly up‐regulated upon exposure of human lung fibroblast cells to pirfenidone, with confirmation for specific genes by real‐time PCR and western blots. Biochemical and functional analyses were used to establish their anti‐fibrotic effects in cellular and animal models of pulmonary fibrosis.ResultsWe identified Regulator of G‐protein Signaling 2 (RGS2) as an early pirfenidone‐induced gene. Treatment with pirfenidone significantly increased RGS2 mRNA and protein expression in both a human fetal lung fibroblast cell line and primary pulmonary fibroblasts isolated from patients without or with idiopathic pulmonary fibrosis. Pirfenidone treatment or direct overexpression of recombinant RGS2 in human lung fibroblasts inhibited the profibrotic effects of thrombin, whereas loss of RGS2 exacerbated bleomycin‐induced pulmonary fibrosis and mortality in mice. Pirfenidone treatment reduced bleomycin‐induced pulmonary fibrosis in wild‐type but not RGS2 knockout mice.ConclusionsEndogenous RGS2 exhibits anti‐fibrotic functions. Upregulated RGS2 contributes significantly to the anti‐fibrotic effects of pirfenidone.Support or Funding InformationThis work was supported by NIH grant R01HL116849 to Y. Tu and T.B. Casale; R01HL097796, P01 HL114471 and P30 ES013508 to R.A. Panettieri; Nebraska State LB595 grant to Y. Tu and P.W. Abel; and the NIH National Center for Research Resources G20‐RR024001.

Antibacterial mode of action of violacein from Chromobacterium violaceum UTM5 against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA)

Violacein, violet pigment produced by Chromobacterium violaceum, has attracted much attention recently due to its pharmacological properties including antibacterial activity. The present study investigated possible antibacterial mode of action of violacein from C. violaceum UTM5 against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) strains. Violet fraction was obtained by cultivating C. violaceum UTM5 in liquid pineapple waste medium, extracted, and fractionated using ethyl acetate and vacuum liquid chromatography technique. Violacein was quantified as major compound in violet fraction using HPLC analysis. Violet fraction displayed bacteriostatic activity against S. aureus ATCC 29213 and methicillin-resistant S. aureus ATCC 43300 with minimum inhibitory concentration (MIC) of 3.9μg/mL. Fluorescence dyes for membrane damage and scanning electron microscopic analysis confirmed the inhibitory effect by disruption on membrane integrity, morphological alternations, and rupture of the cell membranes of both strains. Transmission electron microscopic analysis showed membrane damage, mesosome formation, and leakage of intracellular constituents of both bacterial strains. Mode of action of violet fraction on the cell membrane integrity of both strains was shown by release of protein, K+, and extracellular adenosine 5'-triphosphate (ATP) with 110.5μg/mL, 2.34μg/mL, and 87.24ng/μL, respectively, at 48h of incubation. Violet fraction was toxic to human embryonic kidney (HEK293) and human fetal lung fibroblast (IMR90) cell lines with LC50 value of 0.998±0.058 and 0.387±0.002μg/mL, respectively. Thus, violet fraction showed a strong antibacterial property by disrupting the membrane integrity of S. aureus and MRSA strains. This is the first report on the possible mode of antibacterial action of violet fraction from C. violaceum UTM5 on S. aureus and MRSA strains.

Melatonin regulates PARP1 to control the senescence-associated secretory phenotype (SASP) in human fetal lung fibroblast cells.

Cellular senescence is an important tumor-suppressive mechanism. However, acquisition of a senescence-associated secretory phenotype (SASP) in senescent cells has deleterious effects on the tissue microenvironment and, paradoxically, promotes tumor progression. In a drug screen, we identified melatonin as a novel SASP suppressor in human cells. Strikingly, melatonin blunts global SASP gene expression upon oncogene-induced senescence (OIS). Moreover, poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage, was identified as a new melatonin-dependent regulator of SASP gene induction upon OIS. Here, we report two different but potentially coherent epigenetic strategies for melatonin regulation of SASP. The interaction between the telomeric repeat-containing RNA (TERRA) and PARP-1 stimulates the SASP, which was attenuated by 67.9% (illustrated by the case of IL8) by treatment with melatonin. Through binding to macroH2A1.1, PARP-1 recruits CREB-binding protein (CBP) to mediate acetylation of H2BK120, which positively regulates the expression of target SASP genes, and this process is interrupted by melatonin. Consequently, the findings provide novel insight into melatonin's epigenetic role via modulating PARP-1 in suppression of SASP gene expression in OIS-induced senescent cells. Our studies identify melatonin as a novel anti-SASP molecule, define PARP-1 as a new target by which melatonin regulates SASP, and establish a new epigenetic paradigm for a pharmacological mechanism by which melatonin interrupts PARP-1 interaction with the telomeric long noncoding RNA(lncRNA) or chromatin.

Synthesis characterization and biological activity of mixed ligand silver(I) complex of 2-benzimidazolylurea and triphenylphosphine

The treatment with triphenylphosphine (TPP) in DMF solution of the white precipitation derived from the reaction of silver(I) nitrate with 2-benzimidazolylurea (BZIMU) results in the [Ag(TPP)2(BZIMU)NO3] (1) complex. The product was characterized by elemental analysis and spectroscopic techniques (FT-IR and 1H, 13C NMR). Single crystal X-ray diffraction analysis of 1 have been carried out at 293K. Mixed ligand complexes of silver(I) nitrate with urea derivatives and phosphines which exhibits significant biological effects are rare in the literature.Complex 1 was evaluated for its in vitro cytotoxic activity (cell viability) against: (i) human breast adenocarcinoma cancer cell line (MCF-7) and (ii) normal human fetal lung fibroblast cells (MRC-5) with SRB assay. The results showed that 1 mediates a strong cytotoxic response to the tested normal (MRC-5) and cancer cell lines (MCF-7). It is also exhibits four time stronger activity against cancer cells (MCF-7) than cisplatin while it is less toxic against normal cells (MRC-5). The therapeutic potency index (TPI) which is define as the IC50 value of 1 against normal cells divided by its IC50 value against cancerous cells is 0.9 in contrast to the 0.2 of cisplatin. The influence of the complex 1, upon the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically and theoretically studied.

The Expression and Degradation of SM22-Alpha/Transgelin are Regulated by Mechanical Tension in the Cytoskeleton

SM22-alpha, also named transgelin, encoded by the gene TAGLN is a calponin-related protein found in smooth muscle, fibroblast and cancer cells. SM22-alpha was discovered three decades ago but its biological function remains unclear. In addition to an application as a differentiation marker for smooth muscle cells, SM22-alpha has been reported to regulate the structure and dynamics of actin cytoskeleton and cell motility in fibroblasts and cancer cells. Here we report a novel finding that the expression and degradation of SM22-alpha/transgelin are both regulated by mechanical tension. Mass spectrometry detected that SM22-alpha was significantly decreased in mouse aortic rings after incubation under low mechanical tension. Using specific monoclonal antibodies developed against chicken gizzard SM22-alpha, we found high levels of SM22-alpha in human fetal lung fibroblast cells line MRC-5 and primary neonatal mouse skin fibroblasts. Similar to that of calponin 2, the level of SM22-alpha is positively dependent on the mechanical tension in the cytoskeleton as determined by the stiffness of culture substrate. Quantitative RT-PCR demonstrated a transcriptional regulation of TAGLN gene expression by mechanical tension in the cytoskeleton. The cellular localization of SM22-alpha overlaps with that of myosin IIA and blebbistatin inhibition of myosin motor decreased the expression of SM22-alpha. The level of SM22-alpha is decreased in skin fibroblasts isolated from calponin 2 knockout mice compared to that in calponin 2-positive wild type cells, suggesting their correlated functions. With the close phylogenetic relationship between TAGLN and the calponin genes, SM22-alpha is identified as a calponin-like cytoskeleton regulatory protein. These findings laid a groundwork for understanding the physiological function of SM22-alpha in mechanoregulation of cytoskeleton and cell motility and its relationship with calponins.

QSAR studies on antimony(III) halide complexes with N-substituted thiourea derivatives

The synthesis, characterization and biological studies of three novel antimony(III) complexes (SbX3, X=Cl and Br) with N-substituted thioureas; N,N-dimethylthiourea (DMTU) and N,N-diethylthiourea (DETU) of formulae [fac-SbCl3(DMTU)3] (1), [mer-SbBr3(DMTU)3] (2) and b,c,d-Cl-[SbCl3(DETU)2] (3) are reported. The compounds were characterized by melting point (m.p.), elemental analysis (e.a.), molar conductivity, FT-IR, FT-Raman, 1H, 13C NMR, UV–Vis spectroscopic techniques and Thermogravimetric–Differential Thermal Analysis (TG–DTA). The crystal structures of complexes were also determined by X-ray diffraction. Complexes 1 and 2 are monomers with octahedral (Oh) geometry around the metal ion, which is formed by three sulfur and three halide atoms. However the coordination mode of the ligands varied around the metal centers. Thus, 1 possesses the facial isomeric form, while 2 the meridional form. In case of 3 two sulfur atoms from thiourea ligands and three chlorides form a trigonal pyramidal geometry b,c,d-Cl-[SbCl3(DETU)2] which finally turns to be a polymeric one through μ–Cl bonds and Oh geometry around each Sb ion. A chloride counter anion neutralizes the whole complex. Complexes 1–3 were evaluated for their in vitro cytotoxic activity against human breast (MCF-7) and cervix (HeLa) adenocarcinoma cells. The toxicity of the complexes was studied against human fetal lung fibroblast cells (MRC-5). The apoptotic type of the cells death was confirmed by cell cycle arrest. The influence of 1–3 upon the catalytic peroxidation of linoleic acid to hyperoxolinoleic acid by the enzyme lipoxygenase (LOX) was kinetically studied. QSAR study reveals a linear correlation between experimental and calculated IC50 values. The model predict the activity of Sb complexes successfully, if log(IC50)>0.

TGF-beta and glutathione promote tissue repair in cigarette smoke induced injury.

We studied the effect of cigarette smoke extract (CSE) on a three-dimensional (3-D) co-culture model with epithelial cells and mesenchymal cells to clarify how epithelial cells protect lung tissue from cigarette smoke in-vivo. Two types of gels were prepared. The one was the co-culture of human fetal lung fibroblasts (HFL-I) embedded in type-I collagen gel, with alveolar epithelial cells (A549) cultured covering the top of the gel. The other was HFL-I cells alone. After 48 hours from CSE exposure, gel contraction, levels of fibronectin, transforming growth factor (TGF)-beta and GSH were assessed. CSE inhibited fibroblast-mediated gel contraction and this inhibition was lessened in co-culture associated with higher GSH concentration and TGF-beta1 level as compared to the level in HFL-I cells alone. CSE lowered fibronectin level to a lesser extent in co-culture as compared to the level in HFL-I cells alone. Exogenous TGF-beta1 restored the inhibition of gel contraction by CSE independent of GSH level. Cigarette smoke may interfere with 3-D co-culture gel contraction by diminishing GSH, fibronectin and TGF-beta1 action in the epithelial-mesenchymal interaction.

MicroRNA-7 inhibits cell proliferation, migration and invasion in human non-small cell lung cancer cells by targeting FAK through ERK/MAPK signaling pathway.

ObjectiveTo investigate the effects of microRNA-7 (miR-7) on the proliferation, migration and invasion of non-small cell lung cancer NSCLC) cells by targeting FAK through ERK/MAPK signaling pathway.MethodsNSCLC tissues and adjacent normal tissues were obtained from 160 NSCLC patients after operation. NSCLC cell lines (A549, H1299 and H1355) and a normal human fetal lung fibroblast cell line (MRC-5) were obtained. NSCLC cells were assigned into miR-7 inhibitors, miR-7 mimics, blank, miR-7 mimics control, miR-7 inhibitors control, FAK siRNA and miR-7 inhibitors + FAK siRNA groups. The expressions of miR-7 and FAK mRNA in tissues and cell lines were detected by qRT-PCR and Western-Blotting. Cell proliferation, migration and invasion were detected by MTT assay, wound scratch assay and Transwell assay.ResultsCompared with adjacent normal tissues, miR-7 expression was down-regulated, but the mRNA and protein expressions of FAK, ERK and MAPK were up-regulated. Compared with the blank and mimics control groups, miR-7 significantly increased but FAK, ERK and MAPK expressions decreased in miR-7 mimics and FAK siRNA groups. Cell proliferation, migration and invasion were inhibited in the miR-7 mimics and FAK siRNA groups, while opposite regarding miR-7 inhibitors group.ConclusionThe miR-7 can inhibit the activation of ERK/MAPK signaling pathway by down-regulating FAK expression, thereby suppressing the proliferation, migration and invasion of NSCLC cells. The miR-7 and its target gene FAK may be novel targets for the diagnosis and treatment of NSCLC.

Platinum(II)-thiosemicarbazone drugs override the cell resistance due to glutathione; assessment of their activity against human adenocarcinoma cells

New platinum(II) compounds of the thiosemicarbazone 1-(1H-Benzimidazol-2-yl)ethan-1-one thiosemicarbazone (BzimetTSCH), [Pt(BzimetTSCH)Cl]·2H2O (1) and [Pt(BzimetTSCH)(tpp)]Cl·H2O·MeCN (2) were synthesized. The complexes were characterized by FT-IR spectroscopy and 1H NMR spectroscopy. The crystal structures of 1 and 2 were determined with single-crystal X-ray diffraction analysis. The coordination around platinum is square planar in both complexes. Compounds 1 and 2 were evaluated for their in vitro cytotoxic activity against human adenocarcinoma breast (MCF-7) and cervix (HeLa) cells. The apoptotic pathway of cell death was confirmed by cell cycle arrest test. Since deactivation of cisplatin caused by glutathione (GSH) seems to be an important determinant of its cytotoxic effects, the reactions of 1 and 2 with GSH were investigated by UV-absorption spectroscopy. The genotoxicities on normal human fetal lung fibroblast cells (MRC-5) caused by 1 and 2 were evaluated by fluorescence microscopy. The absence of micronucleus in MRC-5 cells confirms the in vitro non toxic behavior of the compounds. Moreover, the in vivo genotoxicities of 1 and 2 were evaluated by the Allium cepa test. Due to negligible genototoxic effect and high antitumor activity which is similar to that of cisplatin, 2 could be a candidate for further study as potential drug since the mitotic index is unchanged.

Silver(I) complexes of methyl xanthate against human adenocarcinoma breast cancer cells

The sodium salt of methyl xanthate derives from the reaction of carbon disulfide and sodium hydroxide (1:1) in methanol. Addition of an acetonitrile solution of silver nitrate and tri-aryl-phosphine in 1:2 molar ratio, form the complexes with general formula [Ag(CH3OCS2)(Ar3P)2] (Ar3P=triphenylphosphine (tpp, 1), tri(p-tolyl)phosphine (tptp, 2) and tri(m-tolyl)phosphine (tmtp, 3)). The complexes have been characterized by melting point, FT-IR, UV–Vis, 1H, 31P NMR spectroscopic data and X-ray crystallography. The stability of the Ag–P bond in solution was determined by 31P NMR resonances signals. The photosensitivity of 1–3 against UVB radiation is studied by means of UV–Vis spectroscopy. The stronger photosensitivity is exhibited by 1. The LogP was calculated in order to correlate the influence of lipophilicity on biological activity. The steric demands of arylphosphine were assessed through determination of the cone angles, volumes and H-all atom contacts. Complex of tri(m-tolyl)phosphine exert the greatest steric repulsion (higher cone angle). Hirshfeld surface volumes (2 occupied the greatest space) were analyzed to clarify the nature of the intermolecular interactions. Complexes 1–3 were tested for their in vitro cytotoxic activity against human adenocarcinoma cancer cell lines: MCF-7 (breast, estrogen receptor (ER) positive), MDA-MB-231 (breast, estrogen receptor (ER) negative) and MRC-5 (normal human fetal lung fibroblast cells) with sulforhodamine B (SRB) colorimetric assay. Molecule 3 with the lower H-all atoms inter-molecular interactions exhibits the higher activity against MCF-7 cells. The results are correlated with those obtained from heteroleptic silver complexes of tri-aryl-phosphines and carboxylates analogs to 1–3 where the xanthate is replaced by carboxylates. The structure activity relationships (SAR) study underlines the significance of the ligand type on the bioactivity of these compounds.

CRISPLD2 (LGL1) inhibits proinflammatory mediators in human fetal, adult, and COPD lung fibroblasts and epithelial cells.

Chronic lung disease of prematurity/bronchopulmonary dysplasia (BPD) is the leading cause of perinatal morbidity in developed countries. Inflammation is a prominent finding. Currently available interventions have associated toxicities and limited efficacy. While BPD often resolves in childhood, survivors of preterm birth are at risk for acquired respiratory disease in early life and are more likely to develop chronic obstructive pulmonary disease (COPD) in adulthood. We previously cloned Crispld2 (Lgl1), a glucocorticoid‐regulated mesenchymal secretory protein that modulates lung branching and alveogenesis through mesenchymal–epithelial interactions. Absence of Crispld2 is embryonic lethal. Heterozygous Crispld2+/− mice display features of BPD, including distal airspace enlargement, disruption of elastin, and neonatal lung inflammation. CRISPLD2 also plays a role in human fetal lung fibroblast cell expansion, migration, and mesenchymal–epithelial signaling. This study assessed the effects of endogenous and exogenous CRISPLD2 on expression of proinflammatory mediators in human fetal and adult (normal and COPD) lung fibroblasts and epithelial cells. CRISPLD2 expression was upregulated in a lipopolysaccharide (LPS)‐induced human fetal lung fibroblast line (MRC5). LPS‐induced upregulation of the proinflammatory cytokines IL‐8 and CCL2 was exacerbated in MRC5‐CRISPLD2 knockdown cells. siRNA suppression of endogenous CRISPLD2 in adult lung fibroblasts (HLFs) led to augmented expression of IL‐8, IL‐6, CCL2. LPS‐stimulated expression of proinflammatory mediators by human lung epithelial HAEo‐ cells was attenuated by purified secretory CRISPLD2. RNA sequencing results from HLF‐CRISPLD2 knockdown suggest roles for CRISPLD2 in extracellular matrix and in inflammation. Our data suggest that suppression of CRISPLD2 increases the risk of lung inflammation in early life and adulthood.

Structural, photolysis and biological studies of novel mixed metal Cu(I)-Sb(III) mixed ligand complexes

Direct reaction of copper(I) halides with triphenylstibine (tpSb) and 2-mercapto-thiazolidine (tzdtH) in 1:1:1 molar ratio, results in the formation of the [CuX(μ2-S)-tzdtH)(tpSb)]2 (X=Cl (1), Br (2) and I (3)) complexes. The complexes have been characterized by melting point, FT-IR, UV–vis, 1H NMR spectroscopic data and X-ray crystallography. Complexes 1–3 are di-nuclear and they are the first examples of mixed metals (CuSb), mixed ligand (thioamide, stibine and halogen) containing complexes. Two μ2-S (1–3) atoms bridge the two copper(I) ions with tetrahedral geometry. The coordination sphere around copper atoms is completed by one Sb from tpSb and one halogen (chlorine, bromine or iodine) atom. Intermolecular via N-H⋯X (Cl (1) and Br (2)) interactions stabilized the assembly. The short coppercopper bond distances of 3.103 (1), 3.061 (2) and 3.110, 3.108 (3) Ǻ found in 1–3 indicates d10-d10 interaction between metal centers. The complexes exhibit high photo-sensitivity to UVB light.The complexes 1–3 and the already known [Cu(μ2-I)(tpSb)2]2 (4) were tested for their in vitro cytotoxic activity against human cancer cell lines: MCF-7 (breast, estrogen receptor (ER) positive), MDA-MB-231 (breast, estrogen receptor (ER) negative) and MRC-5 (normal human fetal lung fibroblast cells) with sulforhodamine B (SRB) colorimetric assay. Since estrogen receptors (ERs) are located in MCF-7, in contrast to MDA-MB-231 cells, the estrogenic effect of 1–4 on MCF-7 cells was studied by the mean of methylene blue assay. Compound 4 exhibits the highest estrogenic effect. None of 1–4 exceeds the activity of cisplatin against MCF-7 cells, but they are more active than cisplatin towards MDA-MB-231 cells. UVB light increases the effectiveness of complexes on MCF-7 cells which in the case of 4 is up to 28% higher than the corresponding initial complex (without irradiation).

Upregulation of RGS2: a new mechanism for pirfenidone amelioration of pulmonary fibrosis.

BackgroundPirfenidone was recently approved for treatment of idiopathic pulmonary fibrosis. However, the therapeutic dose of pirfenidone is very high, causing side effects that limit its doses and therapeutic effectiveness. Understanding the molecular mechanisms of action of pirfenidone could improve its safety and efficacy. Because activated fibroblasts are critical effector cells associated with the progression of fibrosis, this study investigated the genes that change expression rapidly in response to pirfenidone treatment of pulmonary fibroblasts and explored their contributions to the anti-fibrotic effects of pirfenidone.MethodsWe used the GeneChip microarray to screen for genes that were rapidly up-regulated upon exposure of human lung fibroblast cells to pirfenidone, with confirmation for specific genes by real-time PCR and western blots. Biochemical and functional analyses were used to establish their anti-fibrotic effects in cellular and animal models of pulmonary fibrosis.ResultsWe identified Regulator of G-protein Signaling 2 (RGS2) as an early pirfenidone-induced gene. Treatment with pirfenidone significantly increased RGS2 mRNA and protein expression in both a human fetal lung fibroblast cell line and primary pulmonary fibroblasts isolated from patients without or with idiopathic pulmonary fibrosis. Pirfenidone treatment or direct overexpression of recombinant RGS2 in human lung fibroblasts inhibited the profibrotic effects of thrombin, whereas loss of RGS2 exacerbated bleomycin-induced pulmonary fibrosis and mortality in mice. Pirfenidone treatment reduced bleomycin-induced pulmonary fibrosis in wild-type but not RGS2 knockout mice.ConclusionsEndogenous RGS2 exhibits anti-fibrotic functions. Upregulated RGS2 contributes significantly to the anti-fibrotic effects of pirfenidone.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0418-4) contains supplementary material, which is available to authorized users.

Nimesulide Silver Metallodrugs, Containing the Mitochondriotropic, Triaryl Derivatives of Pnictogen; Anticancer Activity against Human Breast Cancer Cells.

Novel silver(I) metallo-drugs of the nonsteroidal anti-inflammatory drug nimesulide (nim) and the mitochondriotropic triaryl derivatives of pnictogen ligands (tpE, E = P (tpp, tptp, or totp), As (tpAs), Sb (tpSb)) with the formulas {[Ag(nim) (tpp)2]DMF} (1), [Ag(nim) (tptp)2] (2), [Ag(nim) (totp)] (3), [Ag(nim) (tpAs)2] (4), and [Ag(nim) (tpSb)3] (5) ((tpp = triphenyphosphine, tptp = tri(p-tolyl)phosphine, totp = tri(o-tolyl)phosphine, tpAs = triphenylarsine, tpSb = triphenylantimony, and DMF = dimethylformamide) were synthesized and characterized by melting point, vibrational spectroscopy (mid-Fourier transform IR), (1)H NMR, UV-visible spectroscopic techniques, and X-ray crystallography. The in vitro cytotoxic activity of 1-5 against human breast adenocarcinoma cancer cell lines: MCF-7 (estrogen receptor (ER) positive) and MDA-MB-231 (ER negative) was determined. The genotoxicity on normal human fetal lung fibroblast cells (MRC-5) caused by 1-5 was evaluated by fluorescence microscopy. The absence of micronucleus in MRC-5 cells confirms the in vitro non toxicity behavior of the compounds. Because of the morphology of the cells, an apoptotic pathway was concluded for the cell death. The apoptotic pathway, especially though the mitochondrion damage, was confirmed by DNA fragmentation, cell cycle arrest, and permeabilization of the mitochondrial membrane tests. The molecular mechanism of action of 1-5 was further studied by (i) the binding affinity of 1-5 toward the calf thymus (CT) DNA, (ii) the inhibitory activity of 1-5 against lipoxygenase (an enzyme that oxidizes polyunsaturated fatty acids to leukotrienes or prostaglandins), and (iii) the catalytic activity of 1-5 on the oxidation of linoleic acid (an acid that partakes in membrane fluidity, membrane enzyme activities, etc.) to hyperoxolinoleic acid by oxygen.

Synthesis and biological evaluation of a series of non-hemiacetal ester derivatives of artemisinin

In an attempt to improve the efficacy and stability of current, clinically used artemisinins, a series non-hemiacetal ester derivatives of artemisinin were synthesized and evaluated for their in vitro antiplasmodial and anticancer activities as well as cytotoxicities. These esters were synthesized through the reaction of acid anhydrides, or acid chlorides with artemisinin derived alcohol. In vitro antiplasmodial activity assessments were conducted against intraerythrocytic NF54 and Dd2 Plasmodium falciparum strains. Cytotoxicities were assessed, using normal human fetal lung fibroblast (WI-38) and Chinese hamster ovarian (CHO) mammalian cell lines, while anticancer activities were tested by using panels with three cell lines, consisting of renal (TK10), melanoma (UACC62) and breast (MCF7) cancer cells. Most compounds were found active against the breast cancer cell line. Since antiplasmodial activities for most compounds were found comparable only to that of artesunate, this study did not yield any esters with significantly improved antimalarial efficacies, nor did it deliver any promising antitumor hits. However, from the outcomes of this study, compounds with good safety profiles and increased thermal stabilities, compared to the clinically used artemisinins, were identified. The benzoate derivative 11 was found to have antimalarial activity, comparable to that of dihydroartemisinin and was it subsequently identified as a candidate for further investigation in the urgent search for new, safe and effective antimalarial drugs.

Synthesis, characterisation and cytotoxic activity of organoruthenium(II)-halido complexes with 1H-benzimidazole-2-carboxylic acid

Three new ruthenium(II)–arene halido complexes of general formula [(η6-p-cymene)RuX(L)] (C1C3) were synthesised by reaction of [(η6-p-cymene)RuX2]2 (X− = Cl−, Br−, I−) with 1H-benzimidazole-2-carboxylic acid (HL) in ethanol. The complexes were characterised by elemental analysis, mass spectrometry, IR, 1H and 13C NMR spectroscopy. The 1H-benzimidazole-2-carboxylate was found to act as a bidentate N,O–chelating ligand. Single-crystal X-ray diffraction analysis confirmed the “piano-stool” geometry of C3. The cytotoxic activity of the ligand precursor and ruthenium complexes was tested in human cancer cell lines: cervical carcinoma (HeLa), breast carcinoma (MDA-MB-231), myelogenous leukemia (K562) as well as in one normal human fetal lung fibroblast cell line (MRC-5), by MTT assay. The results show that ruthenium(II)-arene complexes possess enhanced cytotoxicity when compared to that of HL. The latter was devoid of activity in the range of concentrations up to 300 μM. Complex C3, carrying an iodido leaving ligand, exhibited moderate, but selective cytotoxicity toward HeLa, MDA-MB-231 and K562 cell lines, with IC50 values: 73.7, 60.9 and 53.9 μM, respectively, being less toxic against MRC-5 cells (IC50 = 175.9 μM). Complexes C1 and C2 showed moderate to low cytotoxicity in HeLa and K562 cells.

Varicella zoster virus infection of human fetal lung cells alters mitochondrial morphology.

Varicella zoster virus (VZV) is a ubiquitous alphaherpesvirus that establishes latency in ganglionic neurons throughout the neuraxis after primary infection. Here, we show that VZV infection induces a time-dependent significant change in mitochondrial morphology, an important indicator of cellular health, since mitochondria are involved in essential cellular functions. VZV immediate-early protein 63 (IE63) was detected in mitochondria-rich cellular fractions extracted from infected human fetal lung fibroblasts (HFL) by Western blotting. IE63 interacted with cytochrome c oxidase in bacterial 2-hybrid analyses. Confocal microscopy of VZV-infected HFL cells at multiple times after infection revealed the presence of IE63 in the nucleus, mitochondria, and cytoplasm. Our data provide the first evidence that VZV infection induces alterations in mitochondrial morphology, including fragmentation, which may be involved in cellular damage and/or death during virus infection.

Ochratoxin A and T-2 Toxin Induce Clonogenicity and Cell Migration in Human Colon Carcinoma and Fetal Lung Fibroblast Cell Lines.

T-2 toxin and Ochratoxin A (OTA) are toxic secondary metabolites produced by various fungi, and together they contaminate feedstuffs worldwide. T-2 toxin and OTA may exert carcinogenic action in rodent. Despite the various in vivo experiments, carcinogenicity of these two mycotoxins has not yet been proven for human. In this current study, we proposed to investigate, in Human colon carcinoma cells and fetal lung fibroblast-like cells transfected with MYC, the effect of T-2 toxin and OTA on cell clonogenicity and cell migration. Results of the present investigation showed that T2-toxin as well as OTA has an important clonogenic effect in all cell lines, suggesting that these mycotoxins could promote the transcription of c-myc gene. Furthermore, T-2 toxin and OTA enhanced the migration effect of HCT116 cells at very low concentrations, proposing that these mycotoxins may exhibit carcinogenesis-like properties in the studied cells.