GABA-B receptor activation inhibits the in vitro migration of malignant hepatocytes

2011 ◽  
Vol 89 (6) ◽  
pp. 393-400 ◽  
Author(s):  
Carly Lodewyks ◽  
Jose Rodriguez ◽  
Jing Yan ◽  
Betty Lerner ◽  
Jeremy Lipschitz ◽  
...  
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Cell Lines ◽  
Adenosine Monophosphate ◽  
Receptor Activation ◽  
Receptor Expression ◽  
Camp Signaling ◽  
Hepatocyte Proliferation ◽  
Camp Levels

There are conflicting data regarding whether activation of γ-aminobutyric acid-B (GABA-B) receptors results in inhibition of tumor growth and invasion. The objectives of this study were to document the effects of the GABA-B receptor agonist baclofen on malignant hepatocyte proliferation and migration. We also sought to determine whether any effects on cell migration were mediated by changes in cyclic adenosine monophosphate (cAMP) signaling or matrix metalloproteinase (MMP) expression. Finally, GABA-B1 and -B2 receptor expression was documented in 2 malignant hepatocyte cell lines (PLC/PRF/5 and Huh-7) and 12 sets of human hepatocellular carcinoma and adjacent nontumor tissues. Cell proliferative activity was documented by WST-1 absorbance, migration by wound healing assays, cAMP levels by enzyme-linked immunoassay (ELISA), MMP by immunohistochemistry and ELISA, and GABA-B receptor expression by flow cytometry and reverse transcriptase – polymerase chain reaction. Although baclofen had no effect on cell proliferation, wound healing was delayed, an effect that was reversed by the GABA-B receptor antagonist CGP. cAMP levels were decreased in Huh-7 but not PLC cells exposed to baclofen. MMP expression remained unaltered in both cell lines. Finally, GABA-B1 receptor expression was present and consistently expressed, but GABA-B2 expression was limited and varied with the number of cell passages and (or) duration of culture. In conclusion, activation of GABA-B receptors has no effect on malignant hepatocyte proliferation but does decrease cell migration. This inhibitory effect may involve cAMP signaling but not MMP expression. GABA-B2 receptor expression is limited and variable, which may help to explain discrepancies with previously published results.

scholarly journals The role of the integrated stress response in keratinocyte migration

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Jennifer Hartman ◽  
Miguel Barriera Diaz ◽  
Ronald C. Wek ◽  
Dan F. Spandau
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Stress Response ◽  
Protein Expression ◽  
Cell Lines ◽  
Actin Binding ◽  
Collective Cell Migration ◽  
Integrated Stress Response ◽  
Keratinocyte Migration

Background and Hypothesis: Cutaneous wound healing involves: hemostatic, inflammatory, proliferative, and tissue remodeling phases. Re-epithelialization can be modeled in vitro using human keratinocytes and artificial wounds. Previous work showed undifferentiated keratinocytes closing wounds in vitro using individual cell migration (ICM), whilst differentiated keratinocytes utilize collective cell migration (KCCM). Therefore, we hypothesize that ICM in vitro is equivalent to keratinocyte migration during squamous cell carcinoma metastasis in vivo and KCCM is a model for wound re-epithelialization. Furthermore, we hypothesize that the integrated stress response (ISR) is important in ICM and KCCM. The ISR is activated by environmental stresses that protein kinases (GCN2 and PERK) can detect and phosphorylate translation factor, eIF2a. Our goal is to define how the ISR, specifically GCN2 and PERK, influence keratinocyte migration. Methods: We will evaluate in vitro wound healing and kinetic variation in protein expression and cytoskeleton remodeling. We will utilize four keratinocyte cell lines, control human keratinocyte NTERTs, and CRISPR-derived gene knockouts of GCN2, PERK, and ISR effector gene ATF4. Quantitative analysis of wound healing is accomplished using an IncuCyte ZOOM instrument. Protein expression is measured via immunoblots following high density wounding. Cytoskeletal analyses was done by immunofluorescence. Results: Preliminary results show PERK-KO and GCN2-KO cells have reduced expression of F-actin. Immunoblots showed actin-binding protein, phospho-cofilin, at lower levels in PERK-KO and GCN2-KO cells than in NTERT cells. Wound healing assays showed differentiated keratinocytes healing faster than undifferentiated in all cells, except GCN2-KO. GCN2-KO cells healed significantly slower than other differentiated cells and undifferentiated GCN2-KO cells. Wound healing assays showed undifferentiated PERK-KO cells healing slower than other undifferentiated cell lines. Conclusion/Potential Impact: The results indicate PERK and GCN2 could be key components in ICM and CCM respectfully. GCN2 and PERK could thus be potential therapeutic targets to provide cost-effective therapeutics to enhance/inhibit keratinocyte migration.

scholarly journals The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

2011 ◽  
Vol 193 (6) ◽  
pp. 1009-1020 ◽  
Author(s):  
Martijn Gloerich ◽  
Marjolein J. Vliem ◽  
Esther Prummel ◽  
Lars A.T. Meijer ◽  
Marije G.A. Rensen ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Negative Regulator ◽  
Camp Signaling ◽  
Nuclear Pore ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Wide Range

Cyclic adenosine monophosphate (cAMP) is a second messenger that relays a wide range of hormone responses. In this paper, we demonstrate that the nuclear pore component RanBP2 acts as a negative regulator of cAMP signaling through Epac1, a cAMP-regulated guanine nucleotide exchange factor for Rap. We show that Epac1 directly interacts with the zinc fingers (ZNFs) of RanBP2, tethering Epac1 to the nuclear pore complex (NPC). RanBP2 inhibits the catalytic activity of Epac1 in vitro by binding to its catalytic CDC25 homology domain. Accordingly, cellular depletion of RanBP2 releases Epac1 from the NPC and enhances cAMP-induced Rap activation and cell adhesion. Epac1 also is released upon phosphorylation of the ZNFs of RanBP2, demonstrating that the interaction can be regulated by posttranslational modification. These results reveal a novel mechanism of Epac1 regulation and elucidate an unexpected link between the NPC and cAMP signaling.

A Method for Quantitative Analysis of the Kinematics of Fibroblast Migration in a Monolayer Wound Model

2011 ◽  
Author(s):  
Gil Topman ◽  
Orna Sharabani-Yosef ◽  
Amit Gefen
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Wound Healing Assay ◽  
Complete Coverage ◽  
Time Intervals ◽  
Fibroblast Migration ◽  
Wound Model ◽  
Regular Time

A wound healing assay is simple but effective method to study cell migration in vitro. Cell migration in vitro was found to mimic migration in vivo to some extent [1,2]. In wound healing assays, a “wound” is created by either scraping or mechanically crushing cells in a monolayer, thereby forming a denuded area. Cells migrate into the denuded area to complete coverage, and thereby “heal” the wound. Micrographs at regular time intervals are captured during such experiments for analysis of the process of migration.

Abstract 470: Recording Cell Migration Dynamics in Mouse Tissues With Cells Secreting Fluorescence Protein-tagged Collagen

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Zhongming Chen
Keyword(s):  
Cell Migration ◽  
Cell Lines ◽  
Wild Type Mouse ◽  
Cardiac Progenitor Cells ◽  
Wild Type ◽  
Mouse Tissues ◽  
Migration Dynamics ◽  
Fluorescence Protein

Background: Cell migration is an important step involved in heart regeneration and many cardiovascular diseases. However, cell migration dynamics in vivo is poorly understood due to the challenges from mammal hearts, which are opaque and fast beating, and thus individual cardiac cells cannot be imaged or tracked. Aims: In this study, cell migration dynamics in the heart is recorded with a novel strategy, in which fluorescence protein-tagged collagen is secreted from cells and deposited into extracellular matrix, forming visible trails when cells are moving in tissues. As a proof-of-concept, transplanted migration dynamics of cardiac progenitor cells in mouse hearts were investaged. Methods: Stable cell lines expressing mCherry-tagged type I collagen were generated from isolated cardiac progenitor cells, ABCG2 + CD45 - CD31 - cells (side populations), or c-kit + CD45 - CD31 - cells (c-kit + CPCs). The cell migration dynamics were monitored and measured based on the cell trails after cell transplantation into mouse tissues. Results: The stable cell lines form red cell trails both in vitro and in vivo (Fig. 1A & 1B, Green: GFP; Red: mCherry-collagen I, Blue: DAPI, bar: 50 microns). In culture dishes, the cells form visible cell trails of fluorescence protein. The cell moving directions are random, with a speed of 288 +/- 79 microns/day (side populations, n=3) or 143 +/-37 microns/day (c-kit + CPCs, n=3). After transplantation into wild-type mouse hearts, the cells form highly tortuous trails along the gaps between the heart muscle fibers. Angle between a cell trail and a muscle fiber is 16+/-16 degree (n=3). Side populations migrate twice as fast as c-kit+ CPCs in the heart (16.0 +/-8.7 microns/day vs. 8.1+/-0.0 microns/day, n=3, respectively), 18 time slower than the respective speeds in vitro . Additionally, side populations migrate significantly faster in the heart than in the skeletal muscles (26.4+/-5.8 microns/day, n=3). The side populations move significantly faster in immunodeficient mouse hearts (36.7+/-13.3 microns/day, n=3, typically used for studying cell therapies) than in wild-type mouse hearts. Conclusion: For the first time, cell migration dynamics in living hearts is monitored and examined with genetically modified cell lines. This study may greatly advance the fields of cardiovascular biology.

Dioscorea villosa Leaf Extract Enhances in vitro Wound Healing and Expression of Extra Cellular Matrix Factors Transforming Growth Factor-Beta 1 and Collagen-1 in L929 Cell Lines

2019 ◽  
Vol 15 (66) ◽  
pp. 483
Author(s):  
SurapaneniKrishna Mohan ◽  
Murad Alsawalha ◽  
AbeerMohammed Al-Subaie ◽  
ReemYousuf Al-Jindan ◽  
SrinivasaRao Bolla ◽  
...  
Keyword(s):  
Wound Healing ◽  
Growth Factor ◽  
Cell Lines ◽  
Transforming Growth Factor Beta ◽  
Leaf Extract ◽  
Transforming Growth Factor ◽  
L929 Cell ◽  
Growth Factor Beta ◽  
Matrix Factors

scholarly journals Interleukin-3, GM-CSF, and G-CSF receptor expression on cell lines and primary leukemia cells: receptor heterogeneity and relationship to growth factor responsiveness

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 56-65 ◽  
Author(s):  
LS Park ◽  
PE Waldron ◽  
D Friend ◽  
HM Sassenfeld ◽  
V Price ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Lines ◽  
Specific Binding ◽  
Absolute Number ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Interleukin 3 ◽  
Gm Csf ◽  
In Vitro Cell Lines

Abstract Recombinant human granulocyte-macrophage (GM) colony-stimulating factor (GM-CSF), G-CSF, and interleukin-3 (IL-3) labeled with 125I were used to study the characteristics and distribution of receptors for these factors on in vitro cell lines and on cells from patients with acute nonlymphocytic leukemia (ANL) and acute lymphocytic leukemia (ALL). Receptors for GM-CSF and G-CSF were restricted to a subset of myelomonocytic cell lines whereas IL-3 receptors were also found on pre- B- or early B-cell lines. Receptors for all three CSFs were broadly distributed on ANL cells, with considerable variability in levels of expression. Measurement of the colony-forming ability of ANL cells in response to the CSFs showed that there was no direct correlation between the ability of the cells to respond to a growth factor and the absolute number of receptors expressed for that growth factor. Binding of radiolabeled IL-3 and GM-CSF to ANL cells produced complex biphasic curves. Further analysis showed that both IL-3 and GM-CSF were able to partially compete for specific binding of the heterologous radiolabeled ligand to cells from several ANL patients, suggesting that heterogeneity may exist in human CSF receptors. These results provide new insights into the complex role that CSFs may play in ANL.

scholarly journals Kava constituents exert selective anticancer effects in oral squamous cell carcinoma cells in vitro

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Antonio Celentano ◽  
Callisthenis Yiannis ◽  
Rita Paolini ◽  
Pangzhen Zhang ◽  
Camile S. Farah ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Squamous Cell Carcinoma ◽  
Cell Migration ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Cell Lines ◽  
Squamous Cell ◽  
Vitro Assay ◽  
Anticancer Effects

Abstract Kava is a beverage made from the ground roots of the plant Piper Methysticum. Active compounds of Kava have previously been demonstrated to exert an antiproliferative effect through cell cycle arrest and promotion of apoptosis. Our aim was to investigate the in vitro effects of the main constituents derived from Kava on oral squamous cell carcinoma (OSCC) activity. Gas chromatography mass spectrometry (GCMS) was used to characterise the main constituents of two Kava preparations. Cell proliferation was assessed in two human OSCC cell lines (H400 and BICR56) and in normal oral keratinocytes (OKF6) treated with the identified Kava constituents, namely Flavokawain A (FKA), Flavokawain B (FKB), yangonin, kavain and methysticin using an MTS in vitro assay. Cell migration at 16 h was assessed using a Transwell migration assay. Cell invasion was measured at 22 h using a Matrigel assay. Cell adhesion was assessed at 90 min with a Cytoselect Adhesion assay. The two Kava preparations contained substantially different concentrations of the main chemical constituents. Treatment of malignant and normal oral keratinocyte cell lines with three of the identified constituents, 10 μg/ml FKA, 2.5 μg/ml FKB and 10 μg/ml yangonin, showed a significant reduction in cell proliferation in both H400 and BICR56 cancer cell lines but not in normal OKF6 cells. Remarkably, the same Kava constituents induced a significant reduction of OSCC cell migration and invasion. We have demonstrated, for the first time, that Kava constituents, FKA, FKB and yangonin have potential anticancer effects on OSCC. This highlights an avenue for further research of Kava constituents in the development of future cancer therapies to prevent and treat OSCC.

scholarly journals Role of Berry Anthocyanins and Phenolic Acids on Cell Migration and Angiogenesis: An Updated Overview

Nutrients ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 1075 ◽  
Author(s):  
Panagiotis Tsakiroglou ◽  
Natalie E. VandenAkker ◽  
Cristian Del Bo’ ◽  
Patrizia Riso ◽  
Dorothy Klimis-Zacas
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Phenolic Acids ◽  
Rho Gtpase ◽  
Small Gtpases ◽  
Endothelial Cell Migration ◽  
Berry Extracts

Cell migration is a critical process that is highly involved with normal and pathological conditions such as angiogenesis and wound healing. Important members of the RHO GTPase family are capable of controlling cytoskeleton conformation and altering motility characteristics of cells. There is a well-known relationship between small GTPases and the PI3K/AKT pathway. Endothelial cell migration can lead to angiogenesis, which is highly linked to wound healing processes. Phenolics, flavonoids, and anthocyanins are major groups of phytochemicals and are abundant in many natural products. Their antioxidant, antimicrobial, anti-inflammatory, antidiabetic, angiogenenic, neuroprotective, hepatoprotective, and cardioprotective properties have been extensively documented. This comprehensive review focuses on the in vitro and in vivo role of berry extracts and single anthocyanin and phenolic acid compounds on cell migration and angiogenesis. We aim to summarize the most recent published studies focusing on the experimental model, type of berry extract, source, dose/concentration and overall effect(s) of berry extracts, anthocyanins, and phenolic acids on the above processes.

scholarly journals Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132

2018 ◽  
Vol 115 (37) ◽  
pp. 9252-9257 ◽  
Author(s):  
Jamie L. Lahvic ◽  
Michelle Ammerman ◽  
Pulin Li ◽  
Megan C. Blair ◽  
Emma R. Stillman ◽  
...  
Keyword(s):  
Fatty Acids ◽  
G Protein ◽  
Cell Lines ◽  
Saturated Fatty Acids ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Hydroxy Fatty Acids ◽  
High Affinity ◽  
G Protein Coupled

Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based β-arrestin recruitment assay. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure–activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis.

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