Human Fetal Lung Research Articles

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.

Isolation and Total Synthesis of Hoshinolactam, an Antitrypanosomal Lactam from a Marine Cyanobacterium.

In the search for new antiprotozoal substances, hoshinolactam, an antitrypanosomal lactam, was isolated from a marine cyanobacterium. The gross structure was elucidated by spectroscopic analyses, and the absolute configuration was determined by the first total synthesis. Hoshinolactam showed potent antitrypanosomal activity with an IC50 value of 3.9 nM without cytotoxicity against human fetal lung fibroblast MRC-5 cells (IC50 > 25 μM).

The Role of Serotonin Transporter in Human Lung Development and in Neonatal Lung Disorders.

Introduction. Failure of the vascular pulmonary remodeling at birth often manifests as pulmonary hypertension (PHT) and is associated with a variety of neonatal lung disorders including a uniformly fatal developmental disorder known as alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV). Serum serotonin regulation has been linked to pulmonary vascular function and disease, and serotonin transporter (SERT) is thought to be one of the key regulators in these processes. We sought to find evidence of a role that SERT plays in the neonatal respiratory adaptation process and in the pathomechanism of ACD/MPV. Methods. We used histology and immunohistochemistry to determine the timetable of SERT protein expression in normal human fetal and postnatal lungs and in cases of newborn and childhood PHT of varied etiology. In addition, we tested for a SERT gene promoter defect in ACD/MPV patients. Results. We found that SERT protein expression begins at 30 weeks of gestation, increases to term, and stays high postnatally. ACD/MPV patients had diminished SERT expression without SERT promoter alteration. Conclusion. We concluded that SERT/serotonin pathway is crucial in the process of pulmonary vascular remodeling/adaptation at birth and plays a key role in the pathobiology of ACD/MPV.

P2.01-076 Drebrin: A New Targetable Molecular Marker of Lung Adenocarcinoma

Embryonic antigens, such as carcinoembryonic antigen (CEA) and alfa-fetoprotein (AFP), are routinely employed as serum and immunohistochemical tumor markers in clinical medicine. Since they are not expressed in adult human tissues, it is reasonable to conclude that embryonic antigens are extremely specific tumor markers. However, no systematic studies have yet identified clinically useful embryonic antigens for tumor diagnosis. In the present study, we developed a strategy for systematic identification of lung adenocarcinoma markers using monoclonal antibodies generated from embryonic swine tissue. Swine mRNA shows more than 80% homology with human mRNA, and embryonic swine tissue is thought to be a useful material for detection of human embryonic markers. In order to produce mouse monoclonal antibodies, we immunized BALB/c mice by injection of fetal swine lung nuclear fraction into the hock, and used a human lung adenocarcinoma nuclear fraction for the second immunization. We recovered lymph nodes from the mice, and fused mouse B lymphocytes with the murine myeloma cell line SP2/0 using polyethylene glycol. The resulting hybridomas were then selectively cultured. For selection of interesting hybridoma clones, we performed immunohistochemical staining using the supernatant from each one, with tissue microarray loading swine fetal and adult lung, human lung cancer and normal lung tissue. Immunohistochemical screening of 284-clones showed that the antibodies derived from four of them were strongly reactive with the cytoplasm and cytomembrane of fetal swine lung and human lung cancer. We then focused on one clone (B246) whose antibody reacted most clearly with human lung adenocarcinoma cells. Protein microarray analysis confirmed that B246 reacted specifically with “drebrin”, one of the actin binding proteins, originally identified in neuronal cells. There are two drebrin isoforms in human tissue: drebrin E (embryonic) is abundant in the developing neurons, and drebrin A (adult) is expressed in adult brain. We then examined the association of the drebrin expression pattern with the pathological features and prognosis of lung adenocarcinoma using 200 selected cases for which formalin-fixed and paraffin-embedded samples were available. Drebrin immunohistochemistry delineated the samples into those with strong (n=85) and weak (n=115) drebrin expression. Kaplan-Meier analysis demonstrated a significant difference in disease-free survival (DFS) between the groups with strong and weak drebrin expression (p=0.033). Drebrin is expressed specifically in lung adenocarcinoma and is associated with outcome. The present findings indicate that drebrin is a new marker of lung adenocarcinoma and indicative of prognosis.

MA02.03 Expression of Oncofetal miRNAs Inactivates NFIB, a Developmental Transcription Factor Linked to Tumor Aggressiveness in Lung Adenocarcinoma

Fetal and tumor development share striking similarities, such as intense cell proliferation, angiogenesis, increased cell motility, and immune evasion. Molecular regulators, including microRNAs (miRNAs), play important roles in both fetal lung development and in the malignant transformation of adult lung cells. Consequently, investigation of lung tumor biology in the context of lung development may reveal key regulatory mechanisms that tumors hijack from normal development, which potentially play critical roles in the pathology of lung cancer. 131 pairs of non-small cell lung cancer (NSCLC) tumor and non-malignant lung tissues and 15 human fetal lung tissue samples were profiled by miRNA-sequencing. Genes controlled by the oncofetal miRNAs identified were first investigated by miRNA-Data-Integration-Portal (mirDIP) prediction, followed by luciferase-reporter assays. Associations between patient survival and mRNA expression of oncofetal miRNA-gene targets were evaluated in independent samples (>1,400 cases) across multiple NSCLC cohorts. Immunohistochemical analysis of oncofetal miRNA targets was performed on 96 lung adenocarcinoma (LUAD) specimens. We describe for the first time a comprehensive characterization of miRNA expression in human fetal lung tissue, and identified numerous miRNAs that recapitulate their fetal expression patterns in NSCLC. Nuclear Factor I/B (NFIB), a transcription factor essential for lung development, was identified as being frequently targeted by these oncofetal miRNAs. Overexpression of the oncofetal miRNA miR-92b-3p, significantly reduced NFIB levels in vitro. Concordantly, analysis of NFIB expression in multiple NSCLC cohorts revealed its frequent underexpression in tumors (∼40-70%). This is in contrast with its recurrent oncogenic overexpression recently reported in SCLC. Low expression of NFIB was significantly associated with poorer survival in LUAD patients but not in squamous cell carcinoma patients, consistent with the functional role of NFIB in distal lung cell differentiation (i.e., precursor cells of LUAD). Furthermore, an NFIB-related gene signature was identified in LUAD tumors, comprising several well-known lung differentiation markers (e.g., TTF-1, SFTPB, ABCA3). The underexpression of NFIB protein was ultimately validated in LUAD specimens, which also revealed that tumors presenting lower levels of this transcription factor are associated with higher grade, biologically more aggressive LUAD (invasive mucinous, micropapillary and solid subtypes). This work has revealed a prominent mechanism for the downregulation of NFIB, a transcription factor essential for lung differentiation, which we found to be associated with aggressive phenotypes of LUAD and consequently, poor patient survival. Restoration of NFIB expression, specifically in LUAD, has the potential to induce lung cell differentiation and thereby reduce tumor aggressiveness.

Resveratrol raises in vitro anticancer effects of paclitaxel in NSCLC cell line A549 through COX-2 expression.

The aim of this study was to determine the raising anticancer effects of resveratrol (Res) on paclitaxel (PA) in non-small cell lung cancer (NSCLC) cell line A549. The 10 µg/ml of Res had no effect on human fetal lung fibroblast MRC-5 cells or on A549 cancer cells and the 5 or 10 µg/ml of PA also had no effect on MRC-5 normal cells. PA-L (5 µg/ml) and PA-H (10 µg/ml) had the growth inhibitory effects in NSCLC cell line A549, and Res increased these growth inhibitory effects. By flow cytometry experiment, after Res (5 µg/ml)+PA-H (10 µg/ml) treatment, the A549 cells showed the most apoptosic cells compared to other group treatments, and after additional treatment with Res, the apoptosic cells of both two PA concentrations were raised. Res+PA could reduce the mRNA and protein expressions of COX-2, and Res+PA could reduce the COX-2 related genes of VEGF, MMP-1, MMP-2, MMP-9, NF-κB, Bcl-2, Bcl-xL, procollagen I, collagen I, collagen III and CTGF, TNF-α, IL-1β, iNOS and raise the TIMP-1, TIMP-2, TIMP-3, IκB-α, p53, p21, caspase-3, caspase-8, caspase-9, Bax genes compared to the control cells and the PA treated cells. From these results, it can be suggested that Res could raise the anticancer effects of PA in A549 cells, thus Res might be used as a good sensitizing agent for PA.

Probed adhesion force of living lung cells with a tip-modified atomic force microscope.

The mechanical properties of the extracellular matrix play an important role in bio-microenvironment activities. Herein, atomic force microscope (AFM) was used to measure the interaction between Au and Ag nanoparticle (NP) clusters on the surface of human fetal lung cells. Using (3-mercapto-propyl) triethoxysilane (MPTMS), NP clusters were grafted onto the apex of AFM tip, and then, the adhesion force between the tip and the cell was analyzed. The measured adhesion force increased from 92 pN for AFM tip to 332 pN for that modified with MPTMS. The increase is most probably contributed by the nonspecific interactions between the apex of the modified AFM tip and the surface of the cells. The adhesion forces between the surface of NPs clusters grafted AFM tip and that of lung cells were dramatically reduced as NPs clusters were replaced by MPTMS. For the former, as the Au NPs cluster was applied, the adhesion force reached to 122 pN, whereas it significantly augmented with the addition of the cluster's size and dimension on the AFM tip. For the case of Ag cluster grafted on AFM tip, its adhesion force with the surface of the cells significantly lowered and reduced to 56 pN. Presumably, the electrostatic or van der Waals force between the two surfaces results in the variation of measurements. It is also very likely that the cell-surface interactions are probably varied by the nature of the contact surfaces, like the force-distance of attraction. The result is significant for understanding the the nature of the interactions between the surface of NPs and the membrane of lung cells.

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.

Antiinflammatory Effects of Budesonide in Human Fetal Lung.

Lung inflammation in premature infants contributes to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease with long-term sequelae. Pilot studies administering budesonide suspended in surfactant have found reduced BPD without the apparent adverse effects that occur with systemic dexamethasone therapy. Our objective was to determine budesonide potency, stability, and antiinflammatory effects in human fetal lung. We cultured explants of second-trimester fetal lung with budesonide or dexamethasone and used microscopy, immunoassays, RNA sequencing, liquid chromatography/tandem mass spectrometry, and pulsating bubble surfactometry. Budesonide suppressed secreted chemokines IL-8 and CCL2 (MCP-1) within 4 hours, reaching a 90% decrease at 12 hours, which was fully reversed 72 hours after removal of the steroid. Half-maximal effects occurred at 0.04-0.05 nM, representing a fivefold greater potency than for dexamethasone. Budesonide significantly induced 3.6% and repressed 2.8% of 14,500 sequenced mRNAs by 1.6- to 95-fold, including 119 genes that contribute to the glucocorticoid inflammatory transcriptome; some are known targets of nuclear factor-κB. By global proteomics, 22 secreted inflammatory proteins were hormonally regulated. Two glucocorticoid-regulated genes of interest because of their association with lung disease are CHI3L1 and IL1RL1. Budesonide retained activity in the presence of surfactant and did not alter its surface properties. There was some formation of palmitate-budesonide in lung tissue but no detectable metabolism to inactive 16α-hydroxy prednisolone. We concluded that budesonide is a potent and stable antiinflammatory glucocorticoid in human fetal lung in vitro, supporting a beneficial antiinflammatory response to lung-targeted budesonide:surfactant treatment of infants for the prevention of BPD.

161 - Redox-Dependent Calcium-Mediated Signaling Networks That Control the Senescence-Associated Secretory Phenotype

Cellular senescence has evolved as one of the protective mechanisms to arrest the growth of cells with oncogenic potential. While senescent cells lose the ability to divide, they remain metabolically active and adapt a senescence-associated secretory phenotype (SASP). The SASP is central to the progression of several age-associated disease pathologies perpetuated by inflammation. A key modulator of SASP is the pro-inflammatory cytokine interleukin-1 alpha (IL-1α) whose expression and activity is responsive to the senescence-associated (SA) oxidant production and the accompanying disruption of calcium (Ca2+) homeostasis. Members of the Transient Receptor Potential (TRP) channel family control cellular Ca2+ and are sensitive to activation by steady state hydrogen peroxide (ss-H2O2). This study aims at determining the molecular identity of the Ca2+ channel(s) that control the activity of the SASP regulator IL-1α. Using primary IMR-90 human fetal lung fibroblasts as a cellular senescence model, we have begun to explore the likely channel responsible for increased Ca2+ entry in senescent cells. We establish the idea that the redox-responsive TRPM2/TRPC5 channel activity is compromised during senescence due to desensitization by increased ss-H2O2 and TRPC6 channel activity is elevated to compensate for this loss of H2O2-responsive channel activity, thereby modulating SA dysregulation of Ca2+. We hypothesize that modification of this Ca2+ channel in the senescent cells can limit the downstream processing and regulation of IL-1α thereby reversing the SASP. This can establish a new paradigm of communication between the mitochondrial reactive oxygen species (ROS), cytosolic Ca2+ and nuclear IL-1α in senescence. Also, IL-1 α and TRPC6 have been shown to be independently regulated by mechanistic target of rapamycin (mTOR) signaling, a nutrient sensing pathway involved in regulation of aging processes. Overall, these studies identifying the TRPC6-mediated IL1α regulatory loop in senescence have the potential to identify new therapeutic approaches that limit the disease promoting properties of senescent cells without impairing their ability to defend against oncogenic insult.

64 - Low Dose Cadmium Stimulates Myofibroblast Differentiation and Lung Fibrosis by Activation of SMAD and Nuclear Thioredoxin-1

Background Toxic effects of cadmium (Cd) from cigarette smoke and occupational sources are well studied, yet low-level Cd also occurs in human diet. Cd accumulates in vivo due to a twenty-year biological half-life. Increasing evidence suggests that exposure to dietary Cd can impact human health by potentiating inflammatory mechanism. Submicromolar Cd can alter cellular redox control mechanism, contributing to stimulation of pulmonary diseases. The pathogenesis of idiopathic pulmonary fibrosis involves excessive myofibroblast activation which leads to increased contractures in lung. Substantial evidence supports that myofibroblast differentiation is associated with disruption of redox homeostasis, yet the mechanistic effects of exogenous exposure remains unknown. Objectives To test our hypothesis that low dietary levels of Cd stimulate pulmonary fibrosis via activation of SMAD transcription factor and stimulation of myofibroblast differentiation. Results Using human fetal lung fibroblasts (HFLF), we found that two differentiation markers, α-Smooth-Muscle-actin (α-SM-actin) and Extra-Domain-A-containing fibronectin (ED-A FN), were significantly elevated in transcriptional and translational levels by low-dose Cd (0.5, 1.0 and 2.0 μM). Under the same Cd treatment, an increased formation of stress fibers was observed in HFLF. To examine the effects of low-dose Cd on SMAD activation and redox regulatory mechanism, HFLF was transfected with nuclear exporting or nuclear localized signal conjugated thioredoxin (Trx)-1 to express in cytoplasm or nucleus, respectively. The results show that Cd increased SMAD activity by 3 fold and stimulated translocation of Trx1 from cytoplasm to nucleus. Overexpression of Trx1 in cytoplasm diminished Cd induced SMAD activation, while overexpression of Trx1 in nuclei mimicked the Cd effect. This suggests that low-dose Cd stimulates differentiation of HFLF to myofibroblast by mechanism involving nuclear translocation of Trx1 followed by activation of SMAD and subsequent gene expression of α-SM-actin and ED-A FN for differentiation and fibrosis. Conclusion Low dietary exposure to Cd presents a potential risk for pulmonary fibrosis by affecting redox signaling mechanism. The improved understanding of redox regulation mechanism in differentiation and fibrosis can provide therapeutic targets to maintain pulmonary health.

Haploinsufficiency of the folliculin gene leads to impaired functions of lung fibroblasts in patients with Birt-Hogg-Dubé syndrome.

Birt–Hogg–Dubé syndrome (BHDS) is an autosomal dominant inherited disorder caused by germline mutations in the FLCN gene, and characterized by skin fibrofolliculomas, multiple lung cysts, spontaneous pneumothorax, and renal neoplasms. Pulmonary manifestations frequently develop earlier than other organ involvements, prompting a diagnosis of BHDS. However, the mechanism of lung cyst formation and pathogenesis of pneumothorax have not yet been clarified. Fibroblasts were isolated from lung tissues obtained from patients with BHDS (n = 12) and lung cancer (n = 10) as controls. The functional abilities of these lung fibroblasts were evaluated by the tests for chemotaxis to fibronectin and three‐dimensional (3‐D) gel contraction. Fibroblasts from BHDS patients showed diminished chemotaxis as compared with fibroblasts from controls. Expression of fibronectin and TGF‐β1 was significantly reduced in BHDS fibroblasts when assessed by qPCR. Addition of TGF‐β1 in culture medium of BHDS lung fibroblasts significantly restored these cells' abilities of chemotaxis and gel contraction. Human fetal lung fibroblasts (HFL‐1) exhibited reduced chemotaxis and 3‐D gel contraction when FLCN expression was knocked down. To the contrary, a significant increase in chemotactic activity toward to fibronectin was demonstrated when wild‐type FLCN was overexpressed, whereas transduction of mutant FLCN showed no effect on chemotaxis. Our results suggest that FLCN is associated with chemotaxis in lung fibroblasts. Together with reduced TGF‐β1 expression by BHDS lung fibroblasts, a state of FLCN haploinsufficiency may cause lung fibroblast dysfunction, thereby impairing tissue repair. These may reveal one mechanism of lung cyst formation and pneumothorax in BHDS patients.

Drebrin: A new oncofetal biomarker associated with prognosis of lung adenocarcinoma

ObjectivesWith the aim of searching for novel oncofetal tumor biomarkers of lung adenocarcinoma other than carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), we developed a strategy involving monoclonal antibodies generated from embryonic tissue of miniature swine. Materials and methodsUsing immunohistochemistry, we selected suitable hybridoma clones that were reactive against swine fetal lung but not adult lung using tissue microarray loading of human normal lung, lung cancer, and fetal and adult swine tissues. ResultsThe selected clones included several that were uniquely reactive against both swine fetal lung and human lung adenocarcinoma, and protein microarray revealed that the antigen they recognized was “drebrin” (DBN1). We then examined the association between the pattern of drebrin expression and the clinicopathological characteristics of lung adenocarcinoma using surgically resected samples of human lung adenocarcinoma. Two hundred formalin-fixed and paraffin-embedded tumor samples were immunostained for drebrin using clone B246, one of the clones that were reactive against drebrin. The cases were divided into those with strong (n=85) and weak (n=115) drebrin expression. In terms of disease-free survival, cases showing strong drebrin expression had a significantly poorer prognosis than those with weak drebrin expression (p=0.033). ConclusionThe present findings indicate that “drebrin” is a unique oncofetal protein that can be applied as a new biomarker of lung adenocarcinoma.

The Ser82 RAGE Variant Affects Lung Function and Serum RAGE in Smokers and sRAGE Production In Vitro.

IntroductionGenome-Wide Association Studies have identified associations between lung function measures and Chronic Obstructive Pulmonary Disease (COPD) and chromosome region 6p21 containing the gene for the Advanced Glycation End Product Receptor (AGER, encoding RAGE). We aimed to (i) characterise RAGE expression in the lung, (ii) identify AGER transcripts, (iii) ascertain if SNP rs2070600 (Gly82Ser C/T) is associated with lung function and serum sRAGE levels and (iv) identify whether the Gly82Ser variant is functionally important in altering sRAGE levels in an airway epithelial cell model.MethodsImmunohistochemistry was used to identify RAGE protein expression in 26 human tissues and qPCR was used to quantify AGER mRNA in lung cells. Gene expression array data was used to identify AGER expression during lung development in 38 fetal lung samples. RNA-Seq was used to identify AGER transcripts in lung cells. sRAGE levels were assessed in cells and patient serum by ELISA. BEAS2B-R1 cells were transfected to overexpress RAGE protein with either the Gly82 or Ser82 variant and sRAGE levels identified.ResultsImmunohistochemical assessment of 6 adult lung samples identified high RAGE expression in the alveoli of healthy adults and individuals with COPD. AGER/RAGE expression increased across developmental stages in human fetal lung at both the mRNA (38 samples) and protein levels (20 samples). Extensive AGER splicing was identified. The rs2070600T (Ser82) allele is associated with higher FEV1, FEV1/FVC and lower serum sRAGE levels in UK smokers. Using an airway epithelium model overexpressing the Gly82 or Ser82 variants we found that HMGB1 activation of the RAGE-Ser82 receptor results in lower sRAGE production.ConclusionsThis study provides new information regarding the expression profile and potential role of RAGE in the human lung and shows a functional role of the Gly82Ser variant. These findings advance our understanding of the potential mechanisms underlying COPD particularly for carriers of this AGER polymorphism.

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.

A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids.

Human pluripotent stem cell (hPSC) derived tissues often remain developmentally immature in vitro, and become more adult-like in their structure, cellular diversity and function following transplantation into immunocompromised mice. Previously we have demonstrated that hPSC-derived human lung organoids (HLOs) resembled human fetal lung tissue in vitro (Dye et al., 2015). Here we show that HLOs required a bioartificial microporous poly(lactide-co-glycolide) (PLG) scaffold niche for successful engraftment, long-term survival, and maturation of lung epithelium in vivo. Analysis of scaffold-grown transplanted tissue showed airway-like tissue with enhanced epithelial structure and organization compared to HLOs grown in vitro. By further comparing in vitro and in vivo grown HLOs with fetal and adult human lung tissue, we found that in vivo transplanted HLOs had improved cellular differentiation of secretory lineages that is reflective of differences between fetal and adult tissue, resulting in airway-like structures that were remarkably similar to the native adult human lung.

Study of the effects of bisphenol A using human fetal lung fibroblasts

Study of the effects of bisphenol A using human fetal lung fibroblasts