NIH3T3 Mouse Fibroblasts Research Articles

Biocompatible hydrogels of chitosan-alkali lignin for potential wound healing applications

Biocompatible hydrogels were prepared by mixing aqueous-acidic solution of chitosan with alkali lignin, a major by-product of the paper producing industries, for the first time, by sustainable means. Electrostatic interactions between the phenoxide groups in lignin and the ammonium groups on the chitosan backbone were found to be responsible for the ionotropic cross-linking. These gels were non-toxic to Mesenchymal stem cells, in vitro, and to zebrafish up to 100 μg/ml, in vivo. In addition, these gels provided a conducive surface for cell attachment and proliferation, making it suitable for application as scaffolds in tissue engineering. In presence of the hydrogel, NIH 3T3 mouse fibroblast cells showed good cell migration characteristics suggesting that the gel might be suitable for wound healing application. The chitosan-alkali lignin gelation system was further capable of removing ferric ions from contaminated water by way of complexation and coagulation. Cross-linked films of chitosan and alkali lignin could also be prepared by simply immersing chitosan films into a solution of alkali lignin. Alkali lignin was observed to diffuse into the chitosan “crystal”, forming electrostatic cross-links between the chitosan chains. The choice of lignin, in comparison to the other ionotropic cross-linkers for chitosan, makes the cross-linking system, inexpensive and sustainable.

Crosstalk between p38 and Erk 1/2 in Downregulation of FGF1-Induced Signaling.

Mitogen-activated protein kinases (MAPK): Erk1 and Erk2 are key players in negative-feedback regulation of fibroblast growth factor (FGF) signaling. Upon activation, Erk1 and Erk2 directly phosphorylate FGF receptor 1 (FGFR1) at a specific serine residue in the C-terminal part of the receptor, substantially reducing the tyrosine phosphorylation in the receptor kinase domain and its signaling. Similarly, active Erks can also phosphorylate multiple threonine residues in the docking protein FGF receptor substrate 2 (FRS2), a major mediator of FGFR signaling. Here, we demonstrate that in NIH3T3 mouse fibroblasts and human osteosarcoma U2OS cells stably expressing FGFR1, in addition to Erk1 and Erk2, p38 kinase is able to phosphorylate FRS2. Simultaneous inhibition of Erk1/2 and p38 kinase led to a significant change in the phosphorylation pattern of FRS2 that in turn resulted in prolonged tyrosine phosphorylation of FGFR1 and FRS2 and in sustained signaling, as compared to the selective inhibition of Erks. Furthermore, excessive activation of p38 with anisomycin partially compensated the lack of Erks activity. These experiments reveal a novel crosstalk between p38 and Erk1/2 in downregulation of FGF-induced signaling.

Chlorambucil Conjugated Ugi Dendrimers with PAMAM-NH₂ Core and Evaluation of Their Anticancer Activity.

Herein, a new Ugi multicomponent reaction strategy is described to enhance activity and solubility of the chemotherapeutic drug chlorambucil through its conjugation to poly(amidoamine) (PAMAM-NH2) dendrimers with the simultaneous introduction of lipidic (i-Pr) and cationic (–NH2) or anionic (–COOH) groups. Standard viability assays were used to evaluate the anticancer potential of the water-soluble dendrimers against PC-3 prostate and HT-29 colon cancer cell lines, as well as non-cancerous mouse NIH3T3 fibroblasts. It could be demonstrated that the anticancer activity against PC-3 cells was considerably improved when both chlorambucil and –NH2 (cationic) groups were present on the dendrimer surface (1b). Additionally, this dendrimer showed activity only against the prostate cancer cells (PC-3), while it did not affect colon cancer cells and fibroblasts significantly. The cationic chlorambucil-dendrimer 1b blocks PC-3 cells in the G2/M phase and induces caspase independent apoptosis.

ER stress activation impairs the expression of circadian clock and clock-controlled genes in NIH3T3 cells via an ATF4-dependent mechanism

Endoplasmic reticulum (ER) stress and circadian clockwork signaling pathways mutually regulate various cellular functions, but the details regarding the cross-talk between these pathways in mammalian cells are unclear. In this study, whether perturbation of ER stress signaling affects the cellular circadian clockwork and transcription of clock-controlled genes was investigated in NIH3T3 mouse fibroblasts. An NIH3T3 cell model stably expressing luciferase (Luc) under the control of the Bmal1 clock gene promoter was established using a lentiviral system. Then, Luc activity was monitored in real-time to detect Bmal1-Luc oscillations. The ER stress activators thapsigargin (Tg) and tunicamycin (Tm) markedly reduced Bmal1-Luc oscillation amplitudes and induced phase delay shifts in NIH3T3 cells. Treatment with Tg/Tm activated ER stress signaling by upregulating GRP78, CHOP, ATF6, and ATF4 and simultaneously significantly decreased BMAL1 protein levels and inhibited the transcription of circadian clock (Bmal1, Per2, Nr1d1, and Dbp) and clock-controlled (Scad1, Fgf7, and Arnt) genes. 4-Phenylbutyric acid, an ER stress inhibitor, alleviated the transcriptional repression of the circadian clock genes and partially restored Bmal1-Luc oscillation amplitudes in Tg- or Tm-treated NIH3T3 cells. More importantly, knock-down of ATF4, but not ATF6, in Tg-treated NIH3T3 cells partially rescued Bmal1-Luc oscillation amplitudes and mRNA expression of the four circadian clock genes. Taken together, our study demonstrates that ER stress activation inhibits the transcription of circadian clock and clock-controlled genes via an ATF4-dependent mechanism.

Smad3 Suppresses Epithelial Cell Migration and Proliferation via the Clock Gene Dec1, Which Negatively Regulates the Expression of Clock Genes Dec2 and Per1

Smad3 has circadian expression; however, whether Smad3 affects the expression of clock genes is poorly understood. Here, we investigated the regulatory mechanisms between Smad3 and the clock genes Dec1, Dec2, and Per1. In Smad3 knockout mice, the amplitude of locomotor activity was decreased, and Dec1 expression was decreased in the suprachiasmatic nucleus, liver, kidney, and tongue compared with control mice. Conversely, Dec2 and Per1 expression was increased compared with that of control mice. In Smad3 knockout mice, immunohistochemical staining revealed that Dec1 expression decreased, whereas Dec2 and Per1 expression increased in the endothelial cells of the kidney and liver. In NIH3T3 cells, Smad3 overexpression increased Dec1 expression, but decreased Dec2 and Per1 expression. In a wound-healing experiment that used Smad3 knockout mice, Dec1 expression decreased in the basal cells of squamous epithelium, promoting wound healing of the mucosa. Finally, the migration and proliferation of Smad3 knockdown squamous carcinoma cells was suppressed by Dec1 overexpression but was promoted by Dec2 overexpression. Dec1 overexpression decreased E-cadherin and proliferating cell nuclear antigen expression, whereas these expression levels were increased by Dec2 overexpression. These results suggest Smad3 is relevant to circadian rhythm and regulates cell migration and proliferation through Dec1, Dec2, and Per1 expression.

Apelin receptor (Aplnr) signaling promotes fibroblast migration.

Cell migration is one of the major cellular processes in development, tissue regeneration, wound healing, cancer and immune function. Underlying molecular mechanism behind the fibroblast cell migration and matrix interactions generates the basis of tissue homeostasis. The role of Apelin receptor (Aplnr) signaling in gastrulation movements has emerged in recent years but how Aplnr regulates cell movement remains unclear. In the current study, the migratory activity of Aplnr signaling has been shown in cultured fibroblast cells in vitro by scratch assay, gene and protein expression analyses. Aplnr signaling was activated and knocked down in MEFs and NIH-3T3 mouse fibroblast cells to analyze whether cell migration is affected by Aplnr signalling. Activation of Aplnr signaling by Apelin peptide and small molecule ML-233 increased cell movement and expression of migration related gene and proteins including Actin and Vimentin. Similarly, reducing the expression of Aplnr and Apelin (Apln) by siRNA exposure inhibited cell migration of mouse fibroblast cells. Application of Apelin peptide and small molecule ML-233 to human fibroblast cells enhanced scratch closure rate significantly compared to control. This study demonstrated that activation of Aplnr signaling promotes cell migration of fibroblast cells in vitro. Aplnr signaling could be a potential therapeutic candidate as a migration related regulatory mechanism in cancer and wound healing for further research.

Hippo pathway controls cell adhesion and context-dependent cell competition to influence skin engraftment efficiency.

Cell competition is involved in mammalian embryogenesis and tumor elimination and progression. It was previously shown that, whereas NIH3T3 mouse fibroblasts expressing high levels of the yes-associated protein 1(YAP1) target TEA domain family (TEAD) transcription factors become "winners" in cell competitions, Madin-Darby canine kidney cells expressing activated YAP1 become "losers" and are eliminated from culture monolayers. Thus, YAP1's role in cell competitions is clearly context dependent. Here, we show that keratinocytes overexpressing a constitutively activated YAP1 mutant lose in in vitro competitions with control cells conducted in standard tissue culture dishes and undergo apical extrusion. Similarly, cells in which endogenous YAP1 is activated by NF2 knockdown become losers. The YAP1-overexpressing cells exhibit a decrease in cell-matrix adhesion because of defective expression of adhesion molecules such as fibronectin-1. Cell adhesion-mediated proliferation is also impaired. However, because of intrinsic factors, YAP1-expressing cells proliferate faster than control cells when cocultured in dishes impeding cell adhesion. In vivo, Mob1a/b-deficient (YAP1-activated) epidermis, which shows decreased expression of type XVII collagen, cannot be engrafted successfully onto donor mice. YAP1-activated skin grafts shrink away from surrounding control skin, and the epidermis peels off the basement membrane. Our data show that YAP1 activation controls cell competition in part by decreasing cell adhesion.-Nishio, M., Miyachi, Y., Otani, J., Tane, S., Omori, H., Ueda, F., Togashi, H., Sasaki, T., Mak, T. W., Nakao, K., Fujita, Y., Nishina, H., Maehama, T., Suzuki, A. Hippo pathway controls cell adhesion and context-dependent cell competition to influence skin engraftment efficiency.

Low-temperature argon and ammonia plasma treatment of poly-3-hydroxybutyrate films: Surface topography and chemistry changes affect fibroblast cells in vitro

Poly-3-hydroxybutyrate (PHB) films were plasma-treated using pure NH3, pure Ar or mixtures of the two different proportions (20%, 30%, 40%, 50%, 70% NH3 in Ar). Surface chemistry and surface topography changes of PHB films were observed after plasma processing in all plasma regimes. The XPS results confirmed the absence of chemical modification in the case of pure Ar plasma treatment. Nitrogen-containing groups (e.g., NCO) were detected on the surfaces of P3HB films treated with NH3-containing plasma. The surfaces of the untreated P3HB films were hydrophobic, and plasma treatment turned the surfaces hydrophilic, irrespective of the treatment. A significant decrease in the contact angle and an increase in the free surface energy were observed. An insignificant surface ageing effect was observed when P3HB samples were exposed to air for 10 days. In NIH 3T3 mice fibroblast cells, cell adhesion was significantly improved after plasma treatment in an Ar atmosphere, which is likely related to the fact that there was a surface ξ potential of 88.6 mV at neutral pH, causing a cleavage of the polymer chains and an increase in surface roughness.

Transcriptional Activity of FOXO Transcription Factors Measured by Luciferase Assays.

The Forkhead box O (FOXO) family of transcription factors translates environmental cues into gene expression. FOXO factors are crucial for the maintenance of cell homeostasis, with important roles in cell fate decisions and differentiation. Identification of FOXO target genes requires strict validation by several methods. Luciferase-based reporters are a valuable starting point for determining the transcription-promoting capacity of potential FOXO-binding sites in candidate genes. Luciferase, an enzyme found in bioluminescent organisms catalyzes oxidation of luciferin to produce oxyluciferin together with light, which can be easily detected and measured with a luminometer. Due to their many advantages, transcriptional assays based on luciferase activity are widely used; they are easy, highly reproducible, and very sensitive. Continued improvements in luciferase-based vectors and measurement reagents confer considerable versatility. Luciferase-based reporters are also a reliable approach in the search for unknown components in the signaling pathways that control FOXO factor activity.We previously reported that FOXO transcription factors control expression of the enzyme diacylglycerol kinase α (DGKα) in T cells. DGKα consumes diacylglycerol, a lipid that activates several mitogenic pathways. Here, we describe the use of a luciferase-based promoter bearing the FOXO-binding sites of the DGKα gene to explore the relationship between the expression of this enzyme and stress conditions in NIH3T3 mouse fibroblasts. Our data support a role for FOXO factors in promoting high DGKα levels in conditions of growth factor deprivation. DGKα regulation by FOXO factors correlates with the reported alterations in DGKα expression during cell transformation and cancer progression.

Modification of chitosan grafted with collagen peptide by enzyme crosslinking

Free radicals are closely related to the occurrence and development of aging, cancer and inflammation. In this paper, the microbial transglutaminase (MTGase) was used as a catalyst to graft the collagen peptide (COP) molecules on the amino group of carboxymethyl chitosan sulfate (CMCS) to improve the antioxidant effects. FT-IR and NMR spectroscopy were used to confirm the successful grafting of COP to CMCS. Degree of substitution (DS) of CMCS-COP could be controlled by adjusting the reaction conditions. With the increase of concentration, the ability of each sample on scavenging capacity and reducibility tends to increase obviously. The results of anticoagulant experiments showed that the ability of CMCS and CMCS-COP with three different degrees of substitution on activated partial thrombin time (APTT) and prothrombin time (PT) values were all increased to compare with the control group. No relevant cytotoxicity against NIH-3T3 mouse fibroblasts was found for the copolymers. These results suggested that CMCS-COP would appear to be a promising candidate for wound dressing application.

Surface modification of poly (ethylene terephthalate) fabric by soy protein isolate hydrogel for wound dressing application

In this study, a composite of poly (ethylene terephthalate) (PET) fabric and soy protein isolate (SPI) hydrogel loaded with gabapentin was developed. For covalent attachment of SPI on the surface of PET fabric, graft polymerization of acrylic acid (AA) on the surface of PET fabric was performed and then carboxyl groups available in the structure of AA were activated using EDAC and then SPI was coated on the surface of PET fabric. The results revealed appropriate connection between hydrogel and modified fabric. The hydrogel was characterized by swelling test and the drug release behavior was investigated. It was found that the casting temperature affects the swelling ratio of the hydrogel and an appropriate release profile of the drug was observed. The surface of fabric was characterized by contact angle measurement, electron microscopy, and infrared spectroscopy. In vitro cell culture study was performed using NIH 3T3 mouse fibroblasts to investigate the biocompatibility of final composite and MTS results along with morphology of cells on the surface of PET fabric coated with SPI revealed the biocompatibility of final product and no cell cytotoxicity was observed in modified PET fabric.

Hydrothermal deposition of high strength calcium phosphate coatings on magnesium alloy for biomedical applications

Magnesium and Mg-alloys are suitable replacement to metallic implants (Titanium, stainless steel, Co-Cr alloys) in terms of stress shielding effect and repeated surgery requirement. However, the only hindrance in their successful use as biodegradable orthopedic implants is their high corrosion rates in physiological environment. Thus, in present study, we developed the single step hydrothermal process to deposit bioactive coatings that may lower the corrosion rate. Highly crystalline, cytocompatible, bioresorbable and high strength monetite (CaHPO4) coating successfully produced on Ca-containing Mg-alloy via hydrothermal process. Deposition parameters have significant influence on coatings morphology and degree of crystallinity. XRD pattern indicates sharp, intense and well-defined peaks of monetite along with minor peaks of brucite and spinel phases. SEM study reveals that compact and defects free coatings were deposited at 100 °C. FTIR analysis showed all characteristic peaks that confirms the presence of monetite. Corrosion behavior of coated and uncoated specimen were analyzed using potentiodynamic polarization scan in simulated body fluid (SBF) at 37 °C. Calcium phosphate (CaP) coating significantly improve the degradation rate of Mg-alloy. Corrosion rate was 80% lower in CaP coated Mg-alloy specimens than bare Mg-alloy. Adhesion strength of coatings was determined using lap shear test demonstrating a cohesive failure at 21.89 MPa stress. Furthermore, cytotoxicity of coated and uncoated alloy was assessed by using Alamar Blue (AB) assay on NIH3T3 mouse fibroblast cell line for 9 days indicate no significant difference in proliferation of cells for CaP coated Mg-alloy and positive control. These results suggest that hydrothermal method reported here can potentially be used to deposit compact, highly crystalline, bioresorbable, cytocompatible and protective coatings with high adhesion strength on intricate geometries for degradable implant applications.

Novel spiroindoline HDAC inhibitors: Synthesis, molecular modelling and biological studies

This paper describes the rational development of a series of novel spiroindoline derivatives endowed with selective inhibitory activity on the HDAC6 isoform. A convenient multicomponent one-pot protocol was applied for the assembly of the desired N1-substituted spiroindoline core which allowed a straightforward analoging. Computational studies and in vitro determination of inhibitory potency for the developed compounds against HDAC6 and HDAC1 isoforms were flanked by cell-based studies on histone H3 and α-tubulin acetylation. The effects on cancer cell cycle and apoptosis of the best performing derivatives were assessed on cancer cell lines highlighting a promising antitumor potential. In view of cell-based data and calculated drug-like properties, the selective HDAC6 inhibitor 5b, with a spiroindoline-based hydroxamate bearing a tert-butyl carbamate functionality, was selected to be further investigated for its potential in inhibiting tumor cells migration. It was able to potently inhibit cell migration in SH-SY5Y neuroblastoma cells and did not display toxicity in NIH3T3 mouse fibroblasts. Taken together, these data foster further investigation and optimization for this class of compounds as novel anticancer agents.

Production and properties of bacterial cellulose by the strain Komagataeibacter xylinus B-12068.

A strain of acetic acid bacteria, Komagataeibacter xylinus B-12068, was studied as a source for bacterial cellulose (BC) production. The effects of cultivation conditions (carbon sources, temperature, and pH) on BC production and properties were studied in surface and submerged cultures. Glucose was found to be the best substrate for BC production among the sugars tested; ethanol concentration of 3% (w/v) enhanced the productivity of BC. Optimization of medium and cultivation conditions ensures a high production of BC on glucose and glycerol, up to 2.4 and 3.3g/L/day, respectively. C/N elemental analysis, emission spectrometry, SEM, DTA, and X-ray were used to investigate the structure and physical and mechanical properties of the BC produced under different conditions. MTT assay and SEM showed that native cellulose membrane did not cause cytotoxicity upon direct contact with NIH 3T3 mouse fibroblast cells and was highly biocompatible.

In vitro cytotoxicity of superheated steam hydrolyzed oligo((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) and characteristics of its blend with poly(L-lactic acid) for biomaterial applications.

In order to clarify the in vitro cytotoxicity effect of superheated steam (SHS) treated poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) (PHBHHx) for biomaterial applications, SHS-treated PHBHHx oligoester samples: P(HB-co-6%-HHx) and P(HB-co-11%-HHx) with low and high percentages of unsaturated chain ends were evaluated for their cytotoxicity effects toward the growth of mouse fibroblast cell line NIH 3T3. From the results obtained after 24 and 48 h of the growth test, the SHS-treated PHBHHx oligoesters were found to be nontoxic to the growth of mouse fibroblast NIH 3T3 cell line with cell viability percentages of more than 95%. In order to serve as a potential resorbable medical suture, PHBHHx oligoesters were blended with poly(L-lactic acid) (PLLA) with a weight ratio of PHBHHx oligoester/PLLA = 20:80 (wt/wt) to improve mechanical properties of PHBHHx oligoesters. The PHBHHx oligoesters/PLLA blend films were evaluated for their thermal, mechanical, and surface wetting properties. Thermal properties of the blend films suggested a good compatibility between PHBHHx oligoesters and PLLA components. Mechanical properties of the blend films were determined to be close enough to a desirable strength range of medical sutures. Moreover, contact angle range of 65 < θ < 70° for the blend samples could provide desirable cell adhesion when used as biomaterials. Therefore, the blend of SHS-treated PHBHHx oligoesters and PLLA would be an ideal choice to be used as biomedical materials.

Immobilization of silk fibroin on the surface of PCL nanofibrous scaffolds for tissue engineering applications

ABSTRACTPoly(ɛ‐caprolactone) (PCL) is explored in tissue engineering (TE) applications due to its biocompatibility, processability, and appropriate mechanical properties. However, its hydrophobic nature and lack of functional groups in its structure are major drawbacks of PCL‐based scaffolds limiting appropriate cell adhesion and proliferation. In this study, silk fibroin (SF) was immobilized on the surface of electrospun PCL nanofibers via covalent bonds in order to improve their hydrophilicity. To this end, the surface of PCL nanofibers was activated by ultraviolet (UV)–ozone irradiation followed by carboxylic functional groups immobilization on their surface by their immersion in acrylic acid under UV radiation and final immersion in SF solution. Furthermore, morphological, mechanical, contact angle, and Attenuated total reflection‐ Fourier transform infrared (ATR‐FTIR) were measured to assess the properties of the surface‐modified PCL nanofibers grafted with SF. ATR‐FTIR results confirmed the presence of SF on the surface of PCL nanofibers. Moreover, contact angle measurements of the PCL nanofibers grafted with SF showed the contact angle of zero indicating high hydrophilicity of modified nanofibers. In vitro cell culture studies using NIH 3T3 mouse fibroblasts confirmed enhanced cytocompatibility, cell adhesion, and proliferation of the SF‐treated PCL nanofibers. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 46684.

Effect of halloysite nanotube structure on physical, chemical, structural and biological properties of elastic polycaprolactone/gelatin nanofibers for wound healing applications

Nanofibrous elastic material based on the blend of hydrophobic poly(ε-caprolactone) (PCL) and hydrophilic gelatin (Gel) reinforced with halloysite nanotubes (HNTs) was prepared by electrospinning process by respecting principles of “green chemistry” required for tissue engineering and drug delivery carriers. Three different kinds of HNTs with similar aspect ratio, but different length and inner diameter were examined to explain the effect of HNT concentration and geometry on a structure, morphology, chemical composition, mechanical properties and biocompatibility of nanostructured materials. Reinforcing effect of each type of HNTs has been confirmed up to 6 wt%. However, the highest improvement of mechanical properties was exhibited by addition just 0.5 wt% of HNTs. All HNT modified nanofibers have been confirmed as non-cytotoxic based on the interaction with mouse fibroblasts NIH-3T3 cells and therefore suitable for biomedical applications, e.g. as wound healing coverings with controlled drug delivery.

Effect of Ginger Powder Extracts on Actin Assembly In Vitro

ObjectiveThe components of ginger root (Zingiber officinale Roscoe) have been widely used for various medicinal purposes in Asia. Several bioactive compounds have been identified in ginger, including 6‐ and 8‐gingerols and 6‐shogaol, which are agonists of the capsaicin receptor TRPV1[1, 2]. Recently, we demonstrated that ginger powder might affect human metabolism in vivo[3]. However, the effects of the bioactive compounds of ginger on live cells have not been completely elucidated. In this study, we investigated whether ginger powder extracts (GPE) induced signal transduction, especially actin assembly, in vitro.MethodThe active components of GPE were characterized by high‐performance liquid chromatography. NIH3T3 mouse fibroblast cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. These cells were treated with GPE (10 ng/mL) for 10 min, followed by analysis of the activation of mitogen‐activated protein kinases (MAPKs) ERK and p38 MAPK, and actin assembly.ResultsThe levels of phosphorylated ERK and p38 MAPK increased significantly after a 10‐ min stimulation with GPE. In addition, lamellipodia formation, which is known to facilitate cell migration[4], occurred at cell edges after GPE stimulation.ConclusionsGPE activated MAPKs and increased lamellipodia formation in mouse fibroblasts. These results suggest that GPE modifies signal transduction, including actin assembly, in vitro.Support or Funding InformationKAKENHI, JapanThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiotensin-converting enzyme inhibitor reduces scar formation by inhibiting both canonical and noncanonical TGF-\u03b21 pathways

Angiotensin-converting enzyme inhibitors (ACEIs) can improve the fibrotic processes in many internal organs. Recent studies have shown a relationship between ACEI with cutaneous scar formation, although it has not been confirmed, and the underlying mechanism is unclear. In this study, we cultured mouse NIH 3T3 fibroblasts with different concentrations of ACEI. We measured cell proliferation with a Cell Counting Kit-8 and collagen expression with a Sirius Red Collagen Detection Kit. Flow cytometry and western blotting were used to detect transforming growth factor β1 (TGF-β1) signaling. We also confirmed the potential antifibrotic activity of ACEI in a rat scar model. ACEI reduced fibroblast proliferation, suppressed collagen and TGF-β1 expression, and downregulated the phosphorylation of SMAD2/3 and TAK1, both in vitro and in vivo. A microscopic examination showed that rat scars treated with ramipril or losartan were not only narrower than in the controls, but also displayed enhanced re-epithelialization and neovascularization, and the formation of organized granulation tissue. These data indicate that ACEI inhibits scar formation by suppressing both TGF-β1/SMAD2/3 and TGF-β1/TAK1 pathways, and may have clinical utility in the future.

New histone demethylase LSD1 inhibitor selectively targets teratocarcinoma and embryonic carcinoma cells

LSD1/KDM1 is a histone demethylase that preferentially removes methyl groups from the mono- and di-methylated lysine 4 in histone H3 (H3K4), key marks for active chromatin for transcriptional activation. LSD1 is essential for pluripotent embryonic stem cells and embryonic teratocarcinoma/carcinoma cells and its expression is often elevated in various cancers. We developed a new LSD1 inhibitor, CBB3001, which potently inhibited LSD1 activity both in vitro and in vivo. CBB3001 also selectively inhibited the growth of human ovarian teratocarcinoma PA-1 and mouse embryonic carcinoma F9 cells, caused the downregulation of pluripotent stem cell proteins SOX2 and OCT4. However, CBB3001 does not have significant inhibition on the growth of human colorectal carcinoma HCT116 cells or mouse fibroblast NIH3T3 cells that do not express these stem cell proteins. Our studies strongly indicate that CBB3001 is a specific LSD1 inhibitor that selectively inhibits teratocarcinoma and embryonic carcinoma cells that express SOX2 and OCT4.