scholarly journals Targeting a Lipid Desaturation Enzyme, SCD1, Selectively Eliminates Colon Cancer Stem Cells through the Suppression of Wnt and NOTCH Signaling

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 106
Author(s):  
Yeongji Yu ◽  
Hyejin Kim ◽  
SeokGyeong Choi ◽  
JinSuh Yu ◽  
Joo Yeon Lee ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Notch Signaling ◽  
Cultured Cells ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Dependent Manner ◽  
Lipid Desaturation ◽  
Novel Target

The elimination of the cancer stem cell (CSC) population may be required to achieve better outcomes of cancer therapy. We evaluated stearoyl-CoA desaturase 1 (SCD1) as a novel target for CSC-selective elimination in colon cancer. CSCs expressed more SCD1 than bulk cultured cells (BCCs), and blocking SCD1 expression or function revealed an essential role for SCD1 in the survival of CSCs, but not BCCs. The CSC potential selectively decreased after treatment with the SCD1 inhibitor in vitro and in vivo. The CSC-selective suppression was mediated through the induction of apoptosis. The mechanism leading to selective CSC death was investigated by performing a quantitative RT-PCR analysis of 14 CSC-specific signaling and marker genes after 24 and 48 h of treatment with two concentrations of an inhibitor. The decrease in the expression of Notch1 and AXIN2 preceded changes in the expression of all other genes, at 24 h of treatment in a dose-dependent manner, followed by the downregulation of most Wnt- and NOTCH-signaling genes. Collectively, we showed that not only Wnt but also NOTCH signaling is a primary target of suppression by SCD1 inhibition in CSCs, suggesting the possibility of targeting SCD1 against colon cancer in clinical settings.

scholarly journals Recombinant Water Stress Protein 1 (Re-WSP1) Suppresses Colon Cancer Cell Growth Through Mir-539/Β-Catenin Signaling Pathway

2021 ◽  
Author(s):  
Songjia Guo ◽  
Shuhua Shan ◽  
Haili Wu ◽  
huiqiang hao ◽  
Zhuoyu Li
Keyword(s):  
Colon Cancer ◽  
Water Stress ◽  
Cell Growth ◽  
Molecular Mechanisms ◽  
Stress Protein ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Nostoc Commune

Abstract Nostoc commune Vauch is a nitrogen-fixing blue-green algae, contains a large number of active molecules with medicinal functions. Our previous study found that a water stress protein (WSP1) from Nostoc commune Vauch and its the recombinant protein (Re-WSP1) exhibited significant anti-colon cancer (CRC) activity both in vitro and in vivo. However, the underlying mechanism remains unknown. In this study, the CCK8 and clonogenic assays showed that Re-WSP1 restrained the colon cancer growth in a dose-dependent manner. Mechanistically, Re-WSP1 inhibited the expression of β-catenin, which was partly reversed by LiCl treatment, demonstrating a key role in Re-WSP1-induced inhibition of cell growth. Quantitative PCR analysis showed that the expression of microRNA-539 (miR-539) was significantly up-regulated upon Re-WSP1 treatment. Moreover, miR-539 negatively regulateed the expression of β-catenin through directly binds to the 3’UTR of β-catenin mRNA. Taken together, our data demonstrate that Re-WSP1 suppresses the CRC growth via miR-539/β-catenin axis, which provides new insights into the molecular mechanisms underlying Re-WSP1 against CRC.

scholarly journals Effect of rs67085638 in long non-coding RNA (CCAT1) on colon cancer chemoresistance to paclitaxel through modulating the microRNA-24-3p and FSCN1 signaling

2020 ◽  
Author(s):  
Caihong Zhang ◽  
Yonglin Wang
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Luciferase Assay ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Colon Cancer Cells ◽  
Protein Levels ◽  
The Difference

Abstract BackgroundIt has been reported that rs67085638 in lncRNA-CCAT1 was associated with the risk of tumorigenesis. Also, CCAT1 could affect chemoresistance of cancer cells to PTX via regulating miR-24-3p and FSCN1 expression. In this study, we aimed to investigate the effect of rs67085638 on the expression of CCAT1/miR-24-3p/FSCN1 and the response of colon cancer to the treatment of PTX.Method48 colon cancer patients were recruited and grouped by their genotypes of rs67085638 polymorphism as a CC group (N=28) and a CT group (N=20). Colon cancer cells were collected from the patients and cancer cell xenografts were transplanted into mice. PCR analysis, IHC assay and Western blot were performed to observe the expression of lncRNA-CCAT1, miR-24-3p and FSCN1 in vivo and in vitro, and the relationships among the expression of lncRNA-CCAT1, miR-24-3p and FSCN1 were validated by computational analysis and luciferase assay. TUNEL assay and flow cytometry were conducted to observe tumor cell apoptosis and survival.ResultLncRNA-CCAT1 and FSCN1 mRNA/protein were over-expressed while miR-24-3p was down-regulated in the CT-genotyped patients and cells compared with those in the CC-genotyped patients and cells. The survival of colon cancer cells was decreased while the apoptosis of colon cancer cells was increased by PTX treatment in a dose-dependent manner. However, the survival rate of CT-genotyped cells was higher while the apoptosis rate of CT-genotyped cells was lower than that of the CC-genotyped cells, and the difference was partly eliminated by the knockdown of lncRNA-CCAT1. MiR-24-3p was validated to target lncRNA-CCAT1 and FSCN1 mRNA, and the over-expression of CCAT1 could reduce the expression of miR-24-3p while elevating the expression of FSCN1. The growth of CT-genotyped tumors in mice was more suppressed in compared with the growth of CC-genotyped tumors, while the knockdown of lncRNA-CCAT1 partly reversed the effect of the CT genotype. Furthermore, compared with the rs67085638-CC mice, the lncRNA-CCAT1 and FSCN1 mRNA/protein levels in the rs67085638-CT+NC shRNA mice were increased while their miR-24-3p level was decreased, and the knockdown of lncRNA-CCAT1 partly reversed the dysregulation of these genes.ConclusionThe findings of this study demonstrated that the presence of the minor allele of rs67085638 increased the expression of CCAT1 and accordingly enhanced the resistance to PTX. Downregulation of CCAT1 partially, but significantly, re-stored the sensitivity to PTX of colon cancer cells.

scholarly journals Neurofascin as a novel target for autoantibody-mediated axonal injury

2007 ◽  
Vol 204 (10) ◽  
pp. 2363-2372 ◽  
Author(s):  
Emily K. Mathey ◽  
Tobias Derfuss ◽  
Maria K. Storch ◽  
Kieran R. Williams ◽  
Kimberly Hales ◽  
...  
Keyword(s):  
Axonal Injury ◽  
Dependent Manner ◽  
Nodes Of Ranvier ◽  
Native Form ◽  
Neuronal Protein ◽  
Axonal Pathology ◽  
Immune Mediated ◽  
Novel Target

Axonal injury is considered the major cause of disability in patients with multiple sclerosis (MS), but the underlying effector mechanisms are poorly understood. Starting with a proteomics-based approach, we identified neurofascin-specific autoantibodies in patients with MS. These autoantibodies recognize the native form of the extracellular domains of both neurofascin 186 (NF186), a neuronal protein concentrated in myelinated fibers at nodes of Ranvier, and NF155, the oligodendrocyte-specific isoform of neurofascin. Our in vitro studies with hippocampal slice cultures indicate that neurofascin antibodies inhibit axonal conduction in a complement-dependent manner. To evaluate whether circulating antineurofascin antibodies mediate a pathogenic effect in vivo, we cotransferred these antibodies with myelin oligodendrocyte glycoprotein–specific encephalitogenic T cells to mimic the inflammatory pathology of MS and breach the blood–brain barrier. In this animal model, antibodies to neurofascin selectively targeted nodes of Ranvier, resulting in deposition of complement, axonal injury, and disease exacerbation. Collectively, these results identify a novel mechanism of immune-mediated axonal injury that can contribute to axonal pathology in MS.

CNP gene expression is activated by Wnt signaling and correlates with Wnt4 expression during renal injury

2003 ◽  
Vol 284 (4) ◽  
pp. F653-F662 ◽  
Author(s):  
Kameswaran Surendran ◽  
Theodore C. Simon
Keyword(s):  
Wnt Signaling ◽  
Renal Injury ◽  
Cultured Cells ◽  
Proximal Promoter ◽  
Dependent Manner ◽  
Binding Factor ◽  
Identical Cell ◽  
Renal Tubular

C-type natriuretic peptide (CNP) regulates salt excretion, vascular tone, and fibroblast proliferation and activation. CNP inhibits fibroblast activation in vitro and fibrosis in vivo, but endogenous CNP gene ( Nppc) expression during tissue fibrosis has not been reported. We determined that Nppc is induced in renal tubular epithelia and then in interstitial myofibroblasts after unilateral ureteral obstruction (UUO). Induction of Nppcoccurred in identical cell populations to those in which Wnt4 is induced after renal injury. In addition, Nppc was activated in Wnt4-expressing cells during nephrogenesis. Wnt signaling components β-catenin and T cell factor/lymphoid enhancer binding factor (TCF/LEF) specifically bound to cognate elements in the Nppc proximal promoter. Wnt-4, β-catenin, and LEF-1 activated an Nppc transgene in cultured cells, and transgene activation by Wnt-4 and LEF-1 was dependent on the presence of intact cognate elements. These findings suggest that Wnt-4 stimulates Nppc in a TCF/LEF-dependent manner after renal injury and thus may contribute to limiting renal fibrosis.

scholarly journals JAK Inhibitors Suppress Colon Cancer Cachexia-Associated Anorexia and Adipose Wasting in Mice

2020 ◽  
Author(s):  
Gurpreet Arora ◽  
Arun Gupta ◽  
Tong Guo ◽  
Aakash Gandhi ◽  
Aaron Laine ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Colon Adenocarcinoma ◽  
Serum Levels ◽  
Immunoblot Analysis ◽  
Dependent Manner ◽  
Jak Inhibitors ◽  
Rt Pcr ◽  
Stat3 Phosphorylation

ABSTRACTBackgroundCachexia (CX), a syndrome of muscle atrophy, adipose loss, and anorexia, is associated with reduced survival in cancer patients. The colon adenocarcinoma C26c20 cell line secretes the cytokine leukemia inhibitor factor (LIF) which induces CX. We characterized how LIF promotes CX-associated weight loss and anorexia in mice through JAK-dependent changes in adipose and hypothalamic tissues.MethodsCX was induced in vivo with C26c20 colon adenocarcinoma cells or recombinant LIF administration in the absence or presence of JAK inhibitors. Blood, adipose, and hypothalamic tissues were collected and processed for cyto/adipokine ELISAs, immunoblot analysis, and quantitative RT-PCR. CX was induced in vitro by stimulating differentiated adipocytes with recombinant LIF or IL-6 in the absence or presence of lipase or JAK inhibitors. These activated adipocytes were processed for lipolysis, immunoblot analysis, and RT-PCR.ResultsTumor-secreted LIF induced changes in adipose tissue expression and serum levels of IL-6 and leptin in a JAK-dependent manner influencing CX-associated adipose wasting and anorexia. We identified two JAK inhibitors that block cytokine-mediated adipocyte lipolysis and IL-6 induction using an in vitro CX lipolysis assay. JAK inhibitors administered to in vivo colon cancer CX mouse models led to 1) a decrease in STAT3 phosphorylation in hypothalamic and adipose tissues, 2) a reverse in the CX serum cyto/adipokine signature, 3) a delay in colon cancer CX-associated anorexia and adipose loss, and 4) an improvement in overall survival.ConclusionsJAK inhibitors suppress cytokine-associated adipose loss and anorexia in multiple in vitro and in vivo models of cancer CX.

scholarly journals Insertion of EGFP into the replicase gene of Semliki Forest virus results in a novel, genetically stable marker virus

2007 ◽  
Vol 88 (4) ◽  
pp. 1225-1230 ◽  
Author(s):  
Nele Tamberg ◽  
Valeria Lulla ◽  
Rennos Fragkoudis ◽  
Aleksei Lulla ◽  
John K. Fazakerley ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Semliki Forest Virus ◽  
Cultured Cells ◽  
Marker Gene ◽  
Virus Production ◽  
Marker Genes ◽  
Replicase Gene ◽  
Marker Virus

Alphavirus-based vector and replicon systems have been extensively used experimentally and are likely to be used in human and animal medicine. Whilst marker genes can be inserted easily under the control of a duplicated subgenomic promoter, these constructs are often genetically unstable. Here, a novel alphavirus construct is described in which an enhanced green fluorescent protein (EGFP) marker gene is inserted into the virus replicase open reading frame between nsP3 and nsP4, flanked by nsP2 protease-recognition sites. This construct has correct processing of the replicase polyprotein, produces viable virus and expresses detectable EGFP fluorescence upon infection of cultured cells and cells of the mouse brain. In comparison to parental virus, the marker virus has an approximately 1 h delay in virus RNA and infectious virus production. Passage of the marker virus in vitro and in vivo demonstrates good genetic stability. Insertion of different markers into this novel construct has potential for various applications.

scholarly journals qPCR analysis of mesenchymal stem cell marker expression during the long-term culture of canine adipocyte derived stem cells

2020 ◽  
Vol 8 (4) ◽  
pp. 139-145
Author(s):  
Rut Bryl ◽  
Claudia Dompe ◽  
Maurycy Jankowski ◽  
Katarzyna Stefańska ◽  
Afsaneh Golkar Narenji ◽  
...  
Keyword(s):  
Stem Cells ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Stem Cell Marker ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Long Term Culture ◽  
Marker Expression

AbstractDue to its availability and accessibility, adipose tissue has been the subject of various studies in many different medical fields and is believed to be a useful source of stem cells. The ability of ASCs to differentiate towards different cell lineages, with possibility of directing this differentiation, increases their possible clinical applications, and they have been widely employed in multiple therapies and treatment of different pathologies. However, a deeper understanding of the molecular mechanisms underlying the ASCs osteoblastic and chondrocyte differentiation may lead to novel applications treating a multitude of different bone-related diseases through techniques more likely meeting worldwide consensus. In this study, the RT-qPCR method was used to determine the changes in expression of ASC specific markers (CD105, CD73, CD14, CD34, CD90 and CD45) before and after long-term (14-day) in vitro cultures. To confirm the identity of the investigated cells, flow cytometry was used to evaluate the presence of positive (CD44, CD90) and negative (CD45, CD34) ASC markers. Overall, the results of the PCR analysis showed a significant change in expression of most of the marker genes, indicating significant changes in the cultured cells caused by their long-term culture, potentially altering their original stem-like characteristics.Running title: ASC marker expression during long-term in vitro culture

scholarly journals AMPK directly activates mTORC2 to promote cell survival during acute energetic stress

2019 ◽  
Vol 12 (585) ◽  
pp. eaav3249 ◽  
Author(s):  
Dubek Kazyken ◽  
Brian Magnuson ◽  
Cagri Bodur ◽  
Hugo A. Acosta-Jaquez ◽  
Deqiang Zhang ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cultured Cells ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Downstream Signaling ◽  
Energetic Stress ◽  
Diabetes Drug ◽  
Tumor Suppressive Function

AMP-activated protein kinase (AMPK) senses energetic stress and, in turn, promotes catabolic and suppresses anabolic metabolism coordinately to restore energy balance. We found that a diverse array of AMPK activators increased mTOR complex 2 (mTORC2) signaling in an AMPK-dependent manner in cultured cells. Activation of AMPK with the type 2 diabetes drug metformin (GlucoPhage) also increased mTORC2 signaling in liver in vivo and in primary hepatocytes in an AMPK-dependent manner. AMPK-mediated activation of mTORC2 did not result from AMPK-mediated suppression of mTORC1 and thus reduced negative feedback on PI3K flux. Rather, AMPK associated with and directly phosphorylated mTORC2 (mTOR in complex with rictor). As determined by two-stage in vitro kinase assay, phosphorylation of mTORC2 by recombinant AMPK was sufficient to increase mTORC2 catalytic activity toward Akt. Hence, AMPK phosphorylated mTORC2 components directly to increase mTORC2 activity and downstream signaling. Functionally, inactivation of AMPK, mTORC2, and Akt increased apoptosis during acute energetic stress. By showing that AMPK activates mTORC2 to increase cell survival, these data provide a potential mechanism for how AMPK paradoxically promotes tumorigenesis in certain contexts despite its tumor-suppressive function through inhibition of growth-promoting mTORC1. Collectively, these data unveil mTORC2 as a target of AMPK and the AMPK-mTORC2 axis as a promoter of cell survival during energetic stress.

scholarly journals Brefeldin A-dependent Membrane Tubule Formation Reconstituted In Vitro Is Driven by a Cell Cycle–regulated Microtubule Motor

2000 ◽  
Vol 11 (3) ◽  
pp. 941-955 ◽  
Author(s):  
Alasdair M. Robertson ◽  
Victoria J. Allan
Keyword(s):  
Cell Cycle ◽  
Cultured Cells ◽  
Brefeldin A ◽  
Binding Activity ◽  
Membrane Dynamics ◽  
Dependent Manner ◽  
Tubule Formation ◽  
Early Endosomes

Treatment of cultured cells with brefeldin A (BFA) induces the formation of extensive membrane tubules from the Golgi apparatus,trans-Golgi network, and early endosomes in a microtubule-dependent manner. We have reconstituted this transport process in vitro using Xenopus egg cytosol and a rat liver Golgi-enriched membrane fraction. The presence of BFA results in the formation of an intricate, interconnected tubular membrane network, a process that, as in vivo, is inhibited by nocodazole, the H1 anti-kinesin monoclonal antibody, and by membrane pretreatment with guanosine 5′-O-(3-thiotriphosphate). Surprisingly, membrane tubule formation is not due to the action of conventional kinesin or any of the other motors implicated in Golgi membrane dynamics. Two candidate motors of ∼100 and ∼130 kDa have been identified using the H1 antibody, both of which exhibit motor properties in a biochemical assay. Finally, BFA-induced membrane tubule formation does not occur in metaphase cytosol, and because membrane binding of both candidate motors is not altered after incubation in metaphase compared with interphase cytosol, these results suggest that either the ATPase or microtubule-binding activity of the relevant motor is cell cycle regulated.

scholarly journals Androgen Receptor Acetylation Governs trans Activation and MEKK1-Induced Apoptosis without Affecting In Vitro Sumoylation and trans-Repression Function

2002 ◽  
Vol 22 (10) ◽  
pp. 3373-3388 ◽  
Author(s):  
Maofu Fu ◽  
Chenguang Wang ◽  
Jian Wang ◽  
Xueping Zhang ◽  
Toshiyuki Sakamaki ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Cultured Cells ◽  
Nuclear Hormone Receptor ◽  
Dependent Manner ◽  
Wild Type ◽  
Acetylation Site ◽  
Induced Apoptosis

ABSTRACT The androgen receptor (AR) is a nuclear hormone receptor superfamily member that conveys both trans repression and ligand-dependent trans-activation function. Activation of the AR by dihydrotestosterone (DHT) regulates diverse physiological functions including secondary sexual differentiation in the male and the induction of apoptosis by the JNK kinase, MEKK1. The AR is posttranslationally modified on lysine residues by acetylation and sumoylation. The histone acetylases p300 and P/CAF directly acetylate the AR in vitro at a conserved KLKK motif. To determine the functional properties governed by AR acetylation, point mutations of the KLKK motif that abrogated acetylation were engineered and examined in vitro and in vivo. The AR acetylation site point mutants showed wild-type trans repression of NF-κB, AP-1, and Sp1 activity; wild-type sumoylation in vitro; wild-type ligand binding; and ligand-induced conformational changes. However, acetylation-deficient AR mutants were selectively defective in DHT-induced trans activation of androgen-responsive reporter genes and coactivation by SRC1, Ubc9, TIP60, and p300. The AR acetylation site mutant showed 10-fold increased binding of the N-CoR corepressor compared with the AR wild type in the presence of ligand. Furthermore, histone deacetylase 1 (HDAC1) bound the AR both in vivo and in cultured cells and HDAC1 binding to the AR was disengaged in a DHT-dependent manner. MEKK1 induced AR-dependent apoptosis in prostate cancer cells. The AR acetylation mutant was defective in MEKK1-induced apoptosis, suggesting that the conserved AR acetylation site contributes to a pathway governing prostate cancer cellular survival. As AR lysine residue mutations that abrogate acetylation correlate with enhanced binding of the N-CoR repressor in cultured cells, the conserved AR motif may directly or indirectly regulate ligand-dependent corepressor disengagement and, thereby, ligand-dependent trans activation.

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