Morphological Transformation Of Cells Research Articles

The Role of Vimentin in Human Corneal Fibroblast Spreading and Myofibroblast Transformation.

Vimentin has been reported to play diverse roles in cell processes such as spreading, migration, cell-matrix adhesion, and fibrotic transformation. Here, we assess how vimentin impacts cell spreading, morphology, and myofibroblast transformation of human corneal fibroblasts. Overall, although knockout (KO) of vimentin did not dramatically impact corneal fibroblast spreading and mechanical activity (traction force), cell elongation in response to PDGF was reduced in vimentin KO cells as compared to controls. Blocking vimentin polymerization using Withaferin had even more pronounced effects on cell spreading and also inhibited cell-induced matrix contraction. Furthermore, although absence of vimentin did not completely block TGFβ-induced myofibroblast transformation, the degree of transformation and amount of αSMA protein expression was reduced. Proteomics showed that vimentin KO cells cultured in TGFβ had a similar pattern of protein expression as controls. One exception included periostin, an ECM protein associated with wound healing and fibrosis in other cell types, which was highly expressed only in Vim KO cells. We also demonstrate for the first time that LRRC15, a protein previously associated with myofibroblast transformation of cancer-associated fibroblasts, is also expressed by corneal myofibroblasts. Interestingly, proteins associated with LRRC15 in other cell types, such as collagen, fibronectin, β1 integrin and α11 integrin, were also upregulated. Overall, our data show that vimentin impacts both corneal fibroblast spreading and myofibroblast transformation. We also identified novel proteins that may regulate corneal myofibroblast transformation in the presence and/or absence of vimentin.

Heat Shock Proteins Are Differentially Expressed in the Domestic Cat (Felis catus) Testis, Epididymis, and Vas Deferens.

Heat shock proteins (HSPs) play key roles in controlling the morphological transformation of germ cells during spermatogenesis and posttesticular maturation of sperm. This study aims to reveal the presence and localization patterns of large adenosine triphosphate (ATP)-dependent HSPs family members in adult domestic cat testis and excurrent ducts utilizing Western blot, immunohistochemistry, and immunofluorescence techniques. The results indicated that the relative amounts of heat shock protein D1 (HSPD1)/HSP60, heat shock protein C (HSPC)/HSP90, and heat shock protein H (HSPH)/HSP105/110 were highest in the testis, while heat shock protein A (HSPA)/HSP70 was highest in the corpus epididymis. HSPs exhibited spermatogenic stage-dependent localization patterns in germ cells. Sertoli and Leydig cells were positive for other HSPs except for HSPC/HSP90. The tubules rectus and rete testis epithelia showed only HSPD1/HSP60 and HSPA/HSP70 immunoreactivity, while the ciliated cells of efferent ductules were positive for all HSPs. In the epididymis and vas deferens, HSPs localizations were cell and region specific. HSPD1/HSP60 was localized in the midpiece of the immature spermatozoa tail, while HSPA/HSP70 and HSP90 were found only in the proximal cytoplasmic droplet (CD). HSPH/HSP105 was observed in CD and the principal piece but not the midpiece. Overall, the different expression of HSPs throughout the domestic cat testis and excurrent ducts indicates their critical roles in maintaining reproductive functions under physiological conditions.

Growth Factor Cocktail to Facilitate Epithelial Differentiation of Exfoliated Deciduous Teeth Stem Cells

Epithelial cells are important in the regeneration of oral mucosal tissue. The cell source is commonly derived from tissue biopsy, which is obtained through surgery. Stem cells from human exfoliated deciduous teeth (SHED) were demonstrated to differentiate into multiple cell types. As it can be readily available from exfoliated deciduous teeth, it can be induced and become a potential source of epithelial cell for oral tissue study. This study aims to examine a mixture of growth factors in the differentiation of epithelial-like cells obtained from human exfoliated deciduous teeth (SHED) stem cells. This growth factor cocktail constitutes hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), insulin-like growth factor 2 (IGF-II), and epidermal growth factor (EGF). After introducing the cocktail, the treated SHED were assessed for epithelial characteristics and markers using cell proliferation test, morphological transformation, protein and gene expression by immunofluorescence staining and cytometry assessment, respectively. The proliferation rate was analysed statistically using Analysis of Variance (ANOVA) with Repeated Measures (p<0.05). SHED cells demonstrated morphological changes on the 7th day after using the cocktail. These transformations are in alignment with the identification of genes associated with epithelial cells and positive stain outcomes for pan-cytokeratin, E-cadherin, and p63. The cell proliferation test indicated that proliferation of cells and growth factor introduction were significantly correlated. The growth factor cocktail used for this research facilitated SHED differentiation for epithelial-like cells. The outcomes validate the production of epithelial cells using SHED; tissue production studies focus on these aspects immensely.

Quorum sensing molecules in yeast wastewater treatment and their regulation of yeast cell morphology

Yeast cells can transform from yeast form into mycelium form under some circumstances, resulting in poor cell settleability and seriously deteriorating the stability of system operation. The feasibility of mediating cell morphology by quorum sensing was carried out in this study. The results showed that the predominant species in the system, Candida tropicalis, could secrete quorum sensing molecules(QSMs) in some media. The supernatant of yeast extract-peptone-dextrose medium(YPD) or yeast filamentation-induced medium(YCBP) after cultivating Candida tropicalis could inhibit mycelial transformation in YCBP, implying that this strain could secrete QSMs. Further, QSMs-(E, E)-farnesol, 2-phenylethanol(2-PE) and a cell filamentation inhibitor 2,4-di-tert-butylphenol(2,4-di-TBP) were detected in YPD supernatant and a lab-scale SBR system in treating oil-containing wastewater. 2-PE and 2,4-di-TBP were detected in the supernatant of YCBP, indicating that the kinds of QSMs depended on substrate components. Finally, the detected compounds were added to a filamentation-induced media to investigate the optimal supplement time to antagonize cell morphological transformation. The results showed that the addition of (E, E)-farnesol, 2-PE, or 2,4-di-TBP could inhibit the morphological transformation from yeast form into mycelium form within the first 3 h of induction, with the maximum inhibition rates of mycelia number and length of 78.1 ± 3.5 % and 59.4 ± 12.7 % for 300 μM (E, E)-farnesol, respectively. For 100 μM 2,4-di-TBP, the highest mycelia number and length inhibition rates were achieved at 98.5 ± 2.3 % and 86.8 ± 5 %. Supplements of QSMs or filamentation inhibitors to the system are expected to inhibit yeast filamentation and establish an efficient and stable yeast wastewater treatment system.

RhoB Promotes Endometrial Stromal Cells Decidualization Via Semaphorin3A/PlexinA4 Signaling in Early Pregnancy.

Endometrial decidualization refers to a series of morphological changes and functional remodeling of the uterine endometrium to accept the embryo under the effect of estrogen and progesterone secreted by ovaries after ovulation. During decidualization, endometrial stromal cells (ESCs) proliferate and differentiate into decidual stromal cells, undergoing cytoskeletal rearrangement-mediated morphological changes and expressing decidualization markers, such as insulin-like growth factor-binding protein-1 and prolactin. Ras homology (Rho) proteins, a family of small G proteins, are well known as regulators of cellular morphology and involved in multiple other cellular processes. In this study, we found ras homolog family member B (RHOB) was the most significantly upregulated gene in the Rho protein family after the in vitro decidualization of human primary ESCs. RhoB expression was induced mainly by 3',5'-cyclic adenosine 5'-monophosphate (cAMP) / protein kinase A (PKA) / cyclic adenosine monophosphate-response element binding protein signaling and partly by progesterone signaling. Knockdown of RhoB in ESCs greatly inhibited actin cytoskeletal rearrangement, cell morphological transformation, and upregulation of insulin-like growth factor-binding protein-1, suggesting an indispensable role of RhoB in decidualization. Mechanistically, the downstream target of RhoB was semaphorin3A (Sema3A), which mediated its signaling via interacting with the receptor, plexinA4. More importantly, decreased expression of RhoB, Sema3A, and plexinA4 were detected in deciduas from patients with unexplained spontaneous miscarriage. Collectively, our results indicate that RhoB/Sema3A/plexinA4 signaling plays a positive role in endometrial decidualization and relates to unexplained spontaneous miscarriage, which is worthy of further exploration so as to provide new insights into therapeutic strategies for pregnancy diseases associated with poor decidualization.

Anti-inflammation of torachrysone-8-O-β-ᴅ-glucoside by hurdling over morphological changes of macrophages

Macrophages exhibit significant phenotypic plasticity to switch their functional phenotypes during inflammation and recovery. Pro-inflammatory (M1) macrophages transform their morphology from round in M0 phenotype to flat and rapidly adhere to lesion sites to recognize series of molecular patterns: damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). Macrophages could also reprogram their metabolism to influence their function.Torachrysone-8-O-β-ᴅ-glucoside (TG), a naphthalene glucoside from Polygonum multiflorum Thunb., exhibited remarkable anti-inflammatory effect. In this study, TG significantly inhibited the Tyr-phosphorylation of focal adhesion kinase (FAK), a key regulator of morphological transformation, and downregulated FAK-mediated transcription of cytoskeleton genes. Thus, TG greatly restrained LPS-induced morphological transformation of macrophage cells into M1 type and reduced their adhesion. The inhibition of TG on FAK phosphorylation also blocked the binding between phosphor-FAK and pyruvate kinase (PK), which contributed to the inhibition of PK activity and limited the high glycolysis rate of M1 metabolic phenotype.Moreover, TG ameliorated defective function of the TCA cycle by markedly increasing of succinate dehydrogenase activity and upregulating the transcription of three rate-limiting enzymes of TCA cycle in M1-polarized macrophage cells. TG enhanced the expression of M2 polarization makers, blunting the sensitivity of RAW 264.7 cells to DAMPs/PAMPs, and inhibited nuclear translocation of NF-κB p65, thus decreased the M1-associated the release of inflammatory factors. These results demonstrated that TG could be a potent anti-inflammatory agent that curbed both the morphological and metabolic phenotype changes of macrophages and warranted further investigations on anti-inflammation effects from angles of morphology, which were unfortunately mostly neglected.

Prohibitin Expression Level As a Companion Diagnostic Marker in Cytogenetically Normal Acute Myeloid Leukemia

Background: Cytogenetically normal acute myeloid leukemia (CN-AML) accounts for almost half of all AML and shows significantly heterogeneous clinical outcomes. Despite the advancements in the molecular pathophysiology of CN-AML, clinical outcome of this leukemia remains unsatisfactory. The recent studies of close relationship between mitochondrial alterations and pathologies of various tumors and degenerative diseases suggested that mitochondria have been an important target for the identification and development of novel companion diagnostic and prognostic marker. These findings prompted us to investigate whether mitochondrial markers could serve as a potential diagnostic, prognostic and therapeutic target in CN-AML patients.Patients and Methods: The proteome analysis of the mitochondria enriched cytoplasmic fractions in primary CN-AML and counterpart normal cells was performed using 2-DE and MALDI-TOF/TOF mass spectrometry. For the development of anti-leukemic drug candidates targeting PHB in CN-AML cells, we synthesized potent chemical substances that can alkylate identified biomarkers. Quantitative reverse transcriptase-polymerase chain reaction and immunohistochemical staining (IHCS) on paraffin-embedded bone marrow sections were performed in order to obtain clinicopathological implications of candidate biomarkers. To assess the functional significance of identified biomarkers, we investigated the effect of β-catenin accumulation on the expression of identified biomarkers because accumulation of β-catenin in the nucleus has been identified in leukemic cells.Results: Prohibitin (PHB /gi4505773) was highly expressed in primary CN-AML cells compared with normal cells. PHB2 protein expression using IHCS was assessed in 133 CN-AML patients. Patients with PHB2 overexpression showed inferior OS (23.7% vs 50.0%; P =0.026) and high hazard ratio (1.808; P = 0.028) compared to PHB2 expression negative patients in CN-AML. Quantitative expression of PHB1 g ene (ddCt) was higher in the control group; 0.31 to 28.32 (mean 4.40, median 2.16) in control group versus 0.22 to 6.52 (mean 1.83, median 1.56) in patient group. Meanwhile, PHB 2 gene expression was higher in the patient group; 0.0 to 0.46 (mean 0.16, median 0.15) in control group versus 0.15 to 3.49 (mean 0.80, median 3.49) in patient group. As a result of linear discrimination analysis, a linear equation (y = 0.5857 * x1 - 4.7252 * x2 - 0.18652; x1 was ddCt of PHB1 , x2 was ddCt of PHB 2) was derived. Estimating the prevalence of AML as 0.003%, the sensitivity of newly developed algorithm was 88.0%, specificity was 87.0%, negative predictive value (NPV) was 99.9% and positive predictive value (PPV) was 2.0%. The elevated level of PHB in CN-AML cells was the result of Wnt signals that were up-regulated in AML cells. PHB is a direct target of β-catenin, and TCF-4/LEF-1 binding motif, CATCTG, appears in the promoter region of PHB by site-directed mutagenesis and ChIP assay. This β-catenin-mediated activation of PHB expression is independent of c-MYC activation which is also a product of Wnt signaling. Potent chemical substances that can alkylate PHB in AML cells, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU). Proliferation suppression was observed in leukemic cell lines, THP-1, K-562 and Kasumi-1, which were treated with CCEU and ICEU in time and dose dependent manner. Moreover, notable morphological transformation of leukemic cells was observed when treated with 50 - 200 umol of CCEU and ICEU for 24 hours. Cell cycle analysis of CCEU-and-ICEU-treated- THP-1 and Kasumi-1 cell lines showed a remarkable increase of the sub-G1 phase.Conclusions: Overexpression of PHB2 protein was associated with poor prognosis in CN-AML. Newly developed algorithm using quantitative data of PHB1 and PHB2 mRNA expressions also showed the possibility as rapid diagnostic tools for leukemic cell identification, and novel alkylating chemotherapeutics, CCEU and ICEU, targeting PHB for selective killing of leukemic cells have been developed. PHB2 expression status can provide clinically valuable results for the diagnosis, prediction of the prognosis, detection of minimal residual disease and therapeutic targeting of AML with normal karyotype. High expression of PHB was served as a companion diagnostic marker in cytogenetically normal acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

A Novel Method for Effective Cell Segmentation and Tracking in Phase Contrast Microscopic Images.

Cell migration plays an important role in the identification of various diseases and physiological phenomena in living organisms, such as cancer metastasis, nerve development, immune function, wound healing, and embryo formulation and development. The study of cell migration with a real-time microscope generally takes several hours and involves analysis of the movement characteristics by tracking the positions of cells at each time interval in the images of the observed cells. Morphological analysis considers the shapes of the cells, and a phase contrast microscope is used to observe the shape clearly. Therefore, we developed a segmentation and tracking method to perform a kinetic analysis by considering the morphological transformation of cells. The main features of the algorithm are noise reduction using a block-matching 3D filtering method, k-means clustering to mitigate the halo signal that interferes with cell segmentation, and the detection of cell boundaries via active contours, which is an excellent way to detect boundaries. The reliability of the algorithm developed in this study was verified using a comparison with the manual tracking results. In addition, the segmentation results were compared to our method with unsupervised state-of-the-art methods to verify the proposed segmentation process. As a result of the study, the proposed method had a lower error of less than 40% compared to the conventional active contour method.

Quetiapine promotes oligodendroglial process outgrowth and membrane expansion by orchestrating the effects of Olig1.

Oligodendroglial lineage cells go through a series of morphological changes before myelination. Prior to myelination, cell processes and membrane structures enlarge by approximately 7,000 times, which is required to support axonal wrapping and myelin segment formation. Failure of these processes leads to maldevelopment and impaired myelination. Quetiapine, an atypical antipsychotic drug, was proved to promote oligodendroglial differentiation and (re)myelination, pending detailed effects and regulatory mechanism. In this study, we showed that quetiapine promotes morphological maturation of oligodendroglial lineage cells and myelin segment formation, and a short-term quetiapine treatment is sufficient to induce these changes. To uncover the underlying mechanism, we examined the effect of quetiapine on the Oligodendrocyte transcription factor 1 (Olig1). We found that quetiapine upregulates Olig1 expression level and promotes nuclear Olig1 translocation to the cytosol, where it functions not as a transcription modulator, but in a way that highly correlates with oligodendrocyte morphological transformation. In addition, quetiapine treatment reverses the negative regulatory effect of the Olig1-regulated G protein-coupled receptor 17 (GPR17) on oligodendroglial morphological maturation. Our results demonstrate that quetiapine enhances oligodendroglial differentiation and myelination by promoting cell morphological transformation. This would shed light on the orchestration of oligodendroglia developmental mechanisms, and provides new targets for further therapeutic research.

Interpreting the Mechanisms by which Integrins Promote the Differentiation of Mesenchymal Stem Cells and Integrin Application Prospects.

Transmembrane integrin receptors represent a major component of cell-extracellular matrix (ECM) communications that mediate cellular biological activities, including proliferation and differentiation. Stem cells, especially mesenchymal stem cells (MSC), have rapidly emerged as promising therapies for various diseases. Dynamic links exist between extracellular and intracellular environments that profoundly influence the cellular activities via integrin receptors, such as cell morphology transformation and differentiation. Interpreting the roles of integrin receptors in the regulation of MSC differentiation may potentially lead to an amplified therapeutic effect. In this review, we summarize, for the first time, the potential mechanisms by which integrins promote MSC multilineage differentiation, including integrin downstream signaling cascades and the interactions between integrin and ion channels, the cytoskeleton, and nuclear mechanoresponses. Furthermore, we focus on the current state and future prospects of the application of integrins to promote cell differentiation.

Hepatic Perisinusoidal Myofibroblast Proliferation and Systemic Inflammatory Response Precedes Sep/Tox Hepatitis in Broilers.

Septicemia-toxemia (sep/tox) falls under U.S. Department of Agriculture (USDA) food safety Category 1 and is the most common and economically significant cause of broiler carcass condemnations. Hepatic lesions are considered a possible consequence of septicemia and associated bacterial contamination of the carcass. Thus, these lesions are considered an indicator of sep/tox (sep/tox hepatitis). This study was undertaken to analyze the histologic lesions preceding grossly visible liver lesions leading to condemnation because of sep/tox at the processing plant. Livers from carcasses of broilers condemned by USDA inspectors for sep/tox were used to establish microscopic and gross criteria of end-stage sep/tox hepatitis. Following the characterization of sep/tox hepatitis, broilers from a farm with a history of sep/tox condemnations were submitted for postmortem examination and bacteriologic investigation at four intervals during the final 20 days of production. Five healthy and five clinically ill chickens were submitted from four houses at 18, 25, 32, and 38 days of production (160 total). Microscopic lesions representing hepatic perisinusoidal myofibroblast proliferation (HPMP), periportal extramedullary granulopoiesis (PEMG), splenic follicular histiocytosis, and bone marrow cellularity (BMC) were graded subjectively for each bird, and subjective grading was evaluated with digital quantitative techniques. Perisinusoidal hepatic stellate cell morphology and progressive transformation of these cells into myofibroblasts was confirmed by immunohistochemistry for smooth muscle actin and desmin. Aerobic cultures of livers and gall bladders from sep/tox birds yielded no growth of bacteria associated with septicemia. Mild to severe HPMP was observed in all age groups, representing 28% of examined birds. Increases in inflammatory cells observed by PEMG and BMC were positively correlated with progressive HPMP and end-stage sep/tox hepatitis in broiler chickens.

Antimicrobial and immunomodulatory responses of photodynamic therapy in Galleria mellonella model

BackgroundNew therapeutics are urgently needed for infectious diseases, especially for the fungal infection like Fonsecaea monophora. Photodynamic therapy has been showing antimicrobial activity on some pathogens. The combination of antimicrobial medicines and photodynamic therapy (PDT) might be a practical approach. However, whether the treatment of PDT could do benefits to the host immunity remains poorly documented.ResultsIn this study, Galleria mellonella larvae were employed as a model organism to evaluate the activity of PDT, and also to investigate the regulation of humoral immunity by PDT. Photosensitizer 5-aminolevulinic acid (ALA) was applied to the G. mellonella infection model. It was found that ALA-mediated PDT was non-toxic to G. mellonella, and could extend the median survival of infected larvae from 3 days to 5.5 days. We observed that larval hemocytes inhibited the growth of Candida albicans and Staphylococcus aureus, without any contribution by ALA-PDT. Furthermore, the application of ALA-PDT demonstrated the immunomodulation of larval innate immunity as increased hemocyte density counting, cell morphological transformation, and sensitivity to pathogens.ConclusionsG. mellonella could be considered as a useful model to study the immunoregulation of PDT. This model revealed that ALA-PDT positively defense against infections through inducing humoral immune responses of larvae.

Enhanced morphological transformation of human lung epithelial cells by continuous exposure to cellulose nanocrystals

Cellulose nanocrystals (CNC), also known as nanowhiskers, have recently gained much attention due to their biodegradable nature, advantageous chemical and mechanical properties, economic value and renewability thus making them attractive for a wide range of applications. However, before these materials can be considered for potential uses, investigation of their toxicity is prudent. Although CNC exposures are associated with pulmonary inflammation and damage as well as oxidative stress responses and genotoxicity in vivo, studies evaluating cell transformation or tumorigenic potential of CNC’s were not previously conducted. In this study, we aimed to assess the neoplastic-like transformation potential of two forms of CNC derived from wood (powder and gel) in human pulmonary epithelial cells (BEAS-2B) in comparison to fibrous tremolite (TF), known to induce lung cancer. Short-term exposure to CNC or TF induced intracellular ROS increase and DNA damage while long-term exposure resulted in neoplastic-like transformation demonstrated by increased cell proliferation, anchorage-independent growth, migration and invasion. The increased proliferative responses were also in-agreement with observed levels of pro-inflammatory cytokines. Based on the hierarchical clustering analysis (HCA) of the inflammatory cytokine responses, CNC powder was segregated from the control and CNC-gel samples. This suggests that CNC may have the ability to influence neoplastic-like transformation events in pulmonary epithelial cells and that such effects are dependent on the type/form of CNC. Further studies focusing on determining and understanding molecular mechanisms underlying potential CNC cell transformation events and their likelihood to induce tumorigenic effects in vivo are highly warranted.

Intrafamilial Infection of Helicobacter Pylori: Abnormal Gastric Epithelial Cells, Pedestal-Rich H. Pylori Adherence, and a Gene Mutation in a Child with Protein-Losing Gastroenteropathy

Abstract Helicobacter pylori, one of the most prevalent human pathogens, colonizes the gastric mucosa and is associated with gastric diseases, such as gastritis and peptic ulcers, and is also a bacterial risk factor for gastric cancer. Cytotoxin-associated gene A (CagA) protein, a major virulence factor of H. pylori, is phosphorylated in cells at its Glu-Pro-IIe-Tyr-Ala (EPIYA) motif and is considered to trigger gastric cancer. CagA is classified into two forms, Western CagA with EPIYA-ABC and East Asian CagA with EPIYA-ABD, with the latter associated with a high risk of developing gastric cancer. CagA causes morphological transformation of cells, yielding the “hummingbird” phenotype in AGS cells and possibly membranous pedestals in the gastric epithelium, albeit rarely. H. pylori adherence to the gastric mucosa is not yet fully understood. Here, we describe an intrafamilial infection case of H. pylori, focusing on the gastric epithelium, H. pylori adherence, and a gene mutation in a child with protein-losing gastroenteropathy (characterized by excessive loss of plasma proteins into the gastrointestinal tract). H. pylori, which also infected family members (mother and father), was genetically a single clone with the virulence genes of an East Asian type. The patient’ gastric mucosa exhibited some unique features. Endoscopy revealed the presence of protein plugs on the mucosal surface, which were immunoelectrophoretically similar to serum proteins. Electron microscopy revealed abnormal gastric epithelial cells, totally covered with the secretions or possessing small swollen structures and irregular microvilli. The patient’s H. pylori infection was characterized by frequently occurring thick pedestals, formed along adherent H. pylori. The serum protein level returned to normal and the protein plugs disappeared after the successful eradication of H. pylori, albeit with lag periods for healing. He had a mutation in the OCRL1 gene, associated with Dent disease (asymptomatic proteinuria). Thus, in the patient’s gastric mucosa, we found the abnormal gastric epithelial cells, which may be caused by an OCRL1 mutation or H. pylori, and pedestal-rich H. pylori infection, possibly caused by a higher level of action of CagA in the abnormal epithelial cells. The data suggests a novel H. pylori virulence factor associated with “excessive plasma protein release”.

Chemerin acts via CMKLR1 and GPR1 to stimulate migration and invasion of gastric cancer cells: putative role of decreased TIMP-1 and TIMP-2.

The chemokine-like peptide, chemerin, stimulates chemotaxis in several cell types. In this study we examined the expression of putative chemerin receptors in gastric cancer and the action of chemerin on cancer cell migration and invasion. Immunohistochemical studies of gastric tumors identified expression of two putative receptors, chemokine-like receptor-1 (CMKLR1) and G-protein coupled receptor 1(GPR1), in cancer cells; there was also some expression in stromal myofibroblasts although generally at a lower intensity. The expression of both receptors was detected in a gastric cancer cell line, AGS; chemerin itself was expressed in cultured gastric cancer myofibroblasts but not AGS cells. Chemerin stimulated (a) morphological transformation of AGS cells characterized by extension of processes and cell scattering, (b) migration in scratch wound assays and (c) both migration and invasion in Boyden chamber chemotaxis assays. These responses were inhibited by two putative receptor antagonists CCX832 and α-NETA. Inhibition of receptor expression by siRNA selectively reduced CMKLR1 or GPR1 and inhibited the action of chemerin indicating that both receptors contributed to the functional response. Using a proteomic approach employing stable isotope dynamic labeling of secretomes (SIDLS) to selectively label secreted proteins, we identified down regulation of tissue inhibitors of metalloproteinease (TIMP)1 and TIMP2 in media in response to chemerin. When cells were treated with chemerin and TIMP1 or TIMP2 the migration response to chemerin was reduced. The data suggest a role for chemerin in promoting the invasion of gastric cancer cells via CMKLR1 and GPR1at least partly by reducing TIMP1 and TIMP2 expression. Chemerin receptor antagonists have potential in inhibiting gastric cancer progression.

Ameliorating effects of Geranium Koreanum kom on esophageal damage in reflux esophagitis via nuclear factor-κB signaling-mediated anti-inflammatory activities

Background: Geranium koreanum, an herbaceous perennial plant, is widely used as a Chinese herbal medicine to treat several diseases. Objective: In the present study, we wanted to demonstrate the anti-inflammatory effects of G. koreanum extracts on esophagus damage in acute reflux esophagitis (RE)-induced rats. Materials and Methods: The anti-inflammatory effects of G. koreanum measured in nitric oxide (NO) production and inducible NO synthase (iNOS) protein expression on lipopolysaccharide (LPS)-induced Raw 264.7 macrophages. To evaluate the improvement in RE, rats were per orally treated with G. koreanum 100 and 200 mg/kg 90 min before RE induction surgery. After 5 h, the rats were sacrificed to confirm the degree of esophageal injury. The esophageal mucosal ulcer ratio and histological changes were examined using Image J program and hematoxylin and eosin (H and E) staining of the rat esophagus, respectively. In addition, expression levels of pro-inflammatory proteins on esophagus in RE rat were measured by western blotting. Results: G. koreanum exhibited anti-inflammatory effects against LPS-stimulated cells by significantly inhibiting NO production and iNOS protein expression. In addition, the LPS-induced morphological transformation of cells was recovered following pretreatment with G. koreanum. In vivo, the result show that G. koreanum 200 mg/kg extract-treated group was ameliorated esophageal damage on histological analysis. The inflammatory mechanism (nuclear factor-κB and mitogen-activated protein kinases signaling pathways) involved in the esophageal damage was investigated by using western blotting with esophageal tissue, and G. koreanum clearly inhibited the inflammatory response in esophagitis-induced rats. Conclusions: This study showed that G. koreanum has anti-inflammatory and ameliorating effects in acute RE. Abbreviations used: RE: Reflux esophagitis; G. koreanum: Geranium koreanum; NO: Nitric oxide; iNOS: inducible nitric oxide synthase; H and E: Hematoxyline and eosin

Lead facilitates foci formation in a Balb/c-3T3 two-step cell transformation model: role of Ape1 function

Several possible mechanisms have been examined to gain an understanding on the carcinogenic properties of lead, which include among others, mitogenesis, alteration of gene expression, oxidative damage, and inhibition of DNA repair. The aim of the present study was to explore if low concentrations of lead, relevant for human exposure, interfere with Ape1 function, a base excision repair enzyme, and its role in cell transformation in Balb/c-3T3. Lead acetate 5 and 30μM induced APE1 mRNA and upregulation of protein expression. This increase in mRNA expression is consistent throughout the chronic exposure. Additionally, we also found an impaired function of Ape1 through molecular beacon-based assay. To evaluate the impact of lead on foci formation, a Balb/c-3T3 two-step transformation model was used. Balb/c-3T3 cells were pretreated 1week with low concentrations of lead before induction of transformation with n-methyl-n-nitrosoguanidine (MNNG) (0.5μg/mL) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.1μg/mL) (a classical two-step protocol). Morphological cell transformation increased in response to lead pretreatment that was paralleled with an increase in Ape1 mRNA and protein overexpression and an impairment of Ape1 activity and correlating with foci number. In addition, we found that lead pretreatment and MNNG (transformation initiator) increased DNA damage, determined by comet assay. Our data suggest that low lead concentrations (5, 30μM) could play a facilitating role in cellular transformation, probably through the impaired function of housekeeping genes such as Ape1, leading to DNA damage accumulation and chromosomal instability, one of the most important hallmarks of cancer induced by chronic exposures.

TAGLN2 is a candidate prognostic biomarker promoting tumorigenesis in human gliomas

BackgroundTransgelin-2 (TAGLN2) is a member of the calponin family of actin-bundling proteins that is involved in the regulation of cell morphology, motility, and cell transformation. Here, the clinical significance and potential function of TAGLN2 in malignant gliomas were investigated.MethodsMolecular and clinical data was obtained from The Cancer Genome Atlas (TCGA) database. Gene ontology and pathway analysis was used to predict potential functions of TAGLN2. RNA knockdown was performed using siRNA or lentiviral contructs in U87MG and U251 glioma cell lines. Cells were characterized in vitro or implanted in vivo to generate orthotopic xenografts in order to assess molecular status, cell proliferation/survival, and invasion by Western blotting, flow cytometry, and 3D tumor spheroid invasion assay, respectively.ResultsIncreased TAGLN2 expression was associated with increasing tumor grade (P < 0.001), the mesenchymal molecular glioma subtype and worse prognosis in patients (P < 0.001). Immunohistochemistry performed with anti-TAGLN2 on an independent cohort of patients (n = 46) confirmed these results. Gene silencing of TAGLN2 in U87MG and U251 significantly inhibited invasion and tumor growth in vitro and in vivo. Western blot analysis revealed that epithelial-mesenchymal transition (EMT) molecular markers, such as N-cadherin, E-cadherin, and Snail, were regulated in a manner corresponding to suppression of the EMT phenotype in knockdown experiments. Finally, TAGLN2 was induced ~ 2 to 3-fold in U87MG and U251 cells by TGFβ2, which was also elevated in GBM and highly correlated with TAGLN2 mRNA levels (P < 0.001).ConclusionsOur findings indicate that TAGLN2 exerts a role in promoting the development of human glioma. The regulation and function of TAGLN2 therefore renders it as a candidate molecular target for the treatment of GBM.

SET/I2PP2A overexpression induces phenotypic, molecular, and metabolic alterations in an oral keratinocyte cell line.

The multifunctional SET/I2PP2A protein is known to be overexpressed in head and neck squamous cell carcinoma. However, SET has been reported to have apparently conflicting roles in promoting cancer cell survival under oxidative stress conditions and preventing invasion and metastasis, complicating efforts to understand the contribution of SET to carcinogenesis. In the present study, we overexpressed SETin a spontaneously immortalized oral keratinocyte cell line (NOK-SI SET) and demonstrated that SET upregulation alone was sufficient to transform cells. In comparison with NOK-SI cells, NOK-SI SET cells demonstrated increased levels of phosphorylated Akt, c-Myc and inactive/phosphorylated Rb, together with decreased total Rb protein levels. In addition, NOK-SI SET cells presented the following: (a) a spindle-cell shape morphology compared with the polygonal morphology of NOK-SI cells; (b) loss of mesenchymal stem cell markers CD44 and CD73, and epithelial cell markers CD71 and integrin α6/β4; (c) the ability to form xenograft tumors in nude mice; and (d) increased mitochondrial respiration accompanied by decreased ROSlevels. Overall, our results show that SEToverexpression promotes morphological and oncogenic cell transformation of an oral keratinocyte cell.

Activation of Glucagon-Like Peptide-1 Receptor Promotes Neuroprotection in Experimental Autoimmune Encephalomyelitis by Reducing Neuroinflammatory Responses.

The signaling axis of glucagon-like peptide-1 (GLP-1)/GLP-1 receptor (GLP-1R) has been an important component in overcoming diabetes, and recent reports have uncovered novel beneficial roles of this signaling axis in central nervous system (CNS) disorders, such as Alzheimer's disease, Parkinson's disease, and cerebral ischemia, accelerating processes for exendin-4 repositioning. Here, we studied whether multiple sclerosis (MS) could be a complement to the CNS disorders that are associated with the GLP-1/GLP-1R signaling axis. Both components of the signaling axis, GLP-1 and GLP-1R proteins, are expressed in neurons, astrocytes, and microglia in the spinal cord of normal mice. In particular, they are abundant in Iba1-positive microglia. Upon challenge by experimental autoimmune encephalomyelitis (EAE), an animal model of MS, the mRNA expression of both GLP-1 and GLP-1R was markedly downregulated in EAE-symptomatic spinal cords, indicating attenuated activity of GLP-1/GLP-1R signaling in EAE. Such a downregulation obviously occurred in LPS-stimulated rat primary microglia, a main cell type to express both GLP-1 and GLP-1R, further indicating attenuated activity of GLP-1/GLP-1R signaling in activated microglia. To investigate whether increased activity of GLP-1R has a therapeutic benefit, exendin-4 (5μg/kg, i.p.), a GLP-1R agonist, was administered daily to EAE-symptomatic mice. Exendin-4 administration to symptomatic EAE mice significantly improved the clinical signs of the disease, along with the reversal of histopathological sequelae such as cell accumulation, demyelination, astrogliosis, microglial activation, and morphological transformation of activated microglia in the injured spinal cord. Such an improvement by exendin-4 was comparable to that by FTY720 (3mg/kg, i.p.), a drug for MS. The neuroprotective effects of exendin-4 against EAE were also associated with decreased mRNA expression of proinflammatory cytokines, such as interleukin (IL)-17, IL-1β, IL-6, and tumor necrosis factor (TNF)-α, all of which are usually upregulated in injured sites of the EAE spinal cord. Interestingly, exendin-4 exposure similarly reduced mRNA levels of IL-1β and TNF-α in LPS-stimulated microglia. Furthermore, exendin-4 administration significantly attenuated activation of NF-κB signaling in EAE spinal cord and LPS-stimulated microglia. Collectively, the current study demonstrates the therapeutic potential of exendin-4 for MS by reducing immune responses in the CNS, highlighting the importance of the GLP-1/GLP-1R signaling axis in the development of a novel therapeutic strategy for MS.