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Faiella M, Botti G, Dalpiaz A, Gnudi L, Goyenvalle A, Pavan B, Perrone D, Bovolenta M, Marchesi E. In Vitro Studies to Evaluate the Intestinal Permeation of an Ursodeoxycholic Acid-Conjugated Oligonucleotide for Duchenne Muscular Dystrophy Treatment. Pharmaceutics 2024; 16:1023. [PMID: 39204368 PMCID: PMC11360444 DOI: 10.3390/pharmaceutics16081023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2024] [Revised: 07/24/2024] [Accepted: 07/29/2024] [Indexed: 09/04/2024] Open
Abstract
Delivery represents a major hurdle to the clinical advancement of oligonucleotide therapeutics for the treatment of disorders such as Duchenne muscular dystrophy (DMD). In this preliminary study, we explored the ability of 2'-O-methyl-phosphorothioate antisense oligonucleotides (ASOs) conjugated with lipophilic ursodeoxycholic acid (UDCA) to permeate across intestinal barriers in vitro by a co-culture system of non-contacting IEC-6 cells and DMD myotubes, either alone or encapsulated in exosomes. UDCA was used to enhance the lipophilicity and membrane permeability of ASOs, potentially improving oral bioavailability. Exosomes were employed due to their biocompatibility and ability to deliver therapeutic cargo across biological barriers. Exon skipping was evaluated in the DMD myotubes to reveal the targeting efficiency. Exosomes extracted from milk and wild-type myotubes loaded with 5'-UDC-3'Cy3-ASO and seeded directly on DMD myotubes appear able to fuse to myotubes and induce exon skipping, up to ~20%. Permeation studies using the co-culture system were performed with 5'-UDC-3'Cy3-ASO 51 alone or loaded in milk-derived exosomes. In this setting, only gymnotic delivery induced significant levels of exon skipping (almost 30%) implying a possible role of the intestinal cells in enhancing delivery of ASOs. These results warrant further investigations to elucidate the delivery of ASOs by gymnosis or exosomes.
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Affiliation(s)
- Marika Faiella
- Department of Translational Medicine, University of Ferrara, 44121 Ferrara, Italy; (M.F.); (M.B.)
| | - Giada Botti
- Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, 44121 Ferrara, Italy; (G.B.); (A.D.); (E.M.)
- Center for Translational Neurophysiology of Speech and Communication (CTNSC@UniFe), Italian Institute of Technology (IIT), 44121 Ferrara, Italy
| | - Alessandro Dalpiaz
- Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, 44121 Ferrara, Italy; (G.B.); (A.D.); (E.M.)
| | - Lorenzo Gnudi
- Department of Environmental and Prevention Sciences, University of Ferrara, 44121 Ferrara, Italy;
| | - Aurélie Goyenvalle
- University Paris-Saclay, UVSQ, Inserm, END-ICAP, 78000 Versailles, France;
| | - Barbara Pavan
- Center for Translational Neurophysiology of Speech and Communication (CTNSC@UniFe), Italian Institute of Technology (IIT), 44121 Ferrara, Italy
- Department of Neuroscience and Rehabilitation—Section of Physiology, University of Ferrara, 44121 Ferrara, Italy
| | - Daniela Perrone
- Department of Environmental and Prevention Sciences, University of Ferrara, 44121 Ferrara, Italy;
| | - Matteo Bovolenta
- Department of Translational Medicine, University of Ferrara, 44121 Ferrara, Italy; (M.F.); (M.B.)
| | - Elena Marchesi
- Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, 44121 Ferrara, Italy; (G.B.); (A.D.); (E.M.)
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Windhaber C, Heckl A, Csukovich G, Pratscher B, Burgener IA, Biermann N, Dengler F. A matter of differentiation: equine enteroids as a model for the in vivo intestinal epithelium. Vet Res 2024; 55:30. [PMID: 38493107 PMCID: PMC10943904 DOI: 10.1186/s13567-024-01283-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2023] [Accepted: 02/08/2024] [Indexed: 03/18/2024] Open
Abstract
Epithelial damage due to gastrointestinal disorders frequently causes severe disease in horses. To study the underlying pathophysiological processes, we aimed to establish equine jejunum and colon enteroids (eqJE, eqCE) mimicking the in vivo epithelium. Therefore, enteroids were cultivated in four different media for differentiation and subsequently characterized histomorphologically, on mRNA and on protein level in comparison to the native epithelium of the same donor horses to identify ideal culture conditions for an in vitro model system. With increasing enterocyte differentiation, the enteroids showed a reduced growth rate as well as a predominantly spherical morphology and less budding compared to enteroids in proliferation medium. Combined or individual withdrawal of stem cell niche pathway components resulted in lower mRNA expression levels of stem cell markers and concomitant differentiation of enterocytes, goblet cells and enteroendocrine cells. For eqCE, withdrawal of Wnt alone was sufficient for the generation of differentiated enterocytes with a close resemblance to the in vivo epithelium. Combined removal of Wnt, R-spondin and Noggin and the addition of DAPT stimulated differentiation of eqJE at a similar level as the in vivo epithelium, particularly with regard to enterocytes. In summary, we successfully defined a medium composition that promotes the formation of eqJE and eqCE consisting of multiple cell types and resembling the in vivo epithelium. Our findings emphasize the importance of adapting culture conditions to the respective species and the intestinal segment. This in vitro model will be used to investigate the pathological mechanisms underlying equine gastrointestinal disorders in future studies.
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Affiliation(s)
- Christina Windhaber
- Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine, Vienna, Austria
| | - Anna Heckl
- Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine, Vienna, Austria
| | - Georg Csukovich
- Division of Small Animal Internal Medicine, University of Veterinary Medicine, Vienna, Austria
| | - Barbara Pratscher
- Division of Small Animal Internal Medicine, University of Veterinary Medicine, Vienna, Austria
| | - Iwan Anton Burgener
- Division of Small Animal Internal Medicine, University of Veterinary Medicine, Vienna, Austria
| | - Nora Biermann
- Clinical Unit of Equine Surgery, University of Veterinary Medicine, Vienna, Austria
| | - Franziska Dengler
- Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine, Vienna, Austria.
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Moschandrea C, Kondylis V, Evangelakos I, Herholz M, Schneider F, Schmidt C, Yang M, Ehret S, Heine M, Jaeckstein MY, Szczepanowska K, Schwarzer R, Baumann L, Bock T, Nikitopoulou E, Brodesser S, Krüger M, Frezza C, Heeren J, Trifunovic A, Pasparakis M. Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes. Nature 2024; 625:385-392. [PMID: 38123683 PMCID: PMC10781618 DOI: 10.1038/s41586-023-06857-0] [Citation(s) in RCA: 10] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Accepted: 11/10/2023] [Indexed: 12/23/2023]
Abstract
Digested dietary fats are taken up by enterocytes where they are assembled into pre-chylomicrons in the endoplasmic reticulum followed by transport to the Golgi for maturation and subsequent secretion to the circulation1. The role of mitochondria in dietary lipid processing is unclear. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with specific ablation of the mitochondrial aspartyl-tRNA synthetase DARS2 (ref. 2), the respiratory chain subunit SDHA3 or the assembly factor COX10 (ref. 4) in intestinal epithelial cells showed accumulation of large lipid droplets (LDs) in enterocytes of the proximal small intestine and failed to thrive. Feeding a fat-free diet suppressed the build-up of LDs in DARS2-deficient enterocytes, which shows that the accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed an impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in intestinal epithelial cells. DARS2 deficiency caused a distinct lack of mature chylomicrons concomitant with a progressive dispersal of the Golgi apparatus in proximal enterocytes. This finding suggests that mitochondrial dysfunction results in impaired trafficking of chylomicrons from the endoplasmic reticulum to the Golgi, which in turn leads to storage of dietary lipids in large cytoplasmic LDs. Taken together, these results reveal a role for mitochondria in dietary lipid transport in enterocytes, which might be relevant for understanding the intestinal defects observed in patients with mitochondrial disorders5.
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Affiliation(s)
- Chrysanthi Moschandrea
- Institute for Genetics, University of Cologne, Cologne, Germany
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
| | - Vangelis Kondylis
- Institute for Pathology, Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany
- Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
| | - Ioannis Evangelakos
- Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Marija Herholz
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
- Institute for Mitochondrial Diseases and Aging, Medical Faculty, University of Cologne, Cologne, Germany
| | - Farina Schneider
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
- Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany
| | - Christina Schmidt
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
- Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany
- MRC Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge, UK
| | - Ming Yang
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
- Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany
- MRC Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge, UK
| | - Sandra Ehret
- Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Markus Heine
- Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Michelle Y Jaeckstein
- Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Karolina Szczepanowska
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
- Institute for Mitochondrial Diseases and Aging, Medical Faculty, University of Cologne, Cologne, Germany
| | - Robin Schwarzer
- Institute for Genetics, University of Cologne, Cologne, Germany
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
| | - Linda Baumann
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
- Institute for Mitochondrial Diseases and Aging, Medical Faculty, University of Cologne, Cologne, Germany
| | - Theresa Bock
- Institute for Genetics, University of Cologne, Cologne, Germany
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
| | - Efterpi Nikitopoulou
- MRC Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge, UK
| | - Susanne Brodesser
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
| | - Marcus Krüger
- Institute for Genetics, University of Cologne, Cologne, Germany
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
| | - Christian Frezza
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
- Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany
- MRC Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge, UK
| | - Joerg Heeren
- Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Aleksandra Trifunovic
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany.
- Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany.
- Institute for Mitochondrial Diseases and Aging, Medical Faculty, University of Cologne, Cologne, Germany.
| | - Manolis Pasparakis
- Institute for Genetics, University of Cologne, Cologne, Germany.
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany.
- Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany.
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Chaussé AM, Roche SM, Moroldo M, Hennequet-Antier C, Holbert S, Kempf F, Barilleau E, Trotereau J, Velge P. Epithelial cell invasion by salmonella typhimurium induces modulation of genes controlled by aryl hydrocarbon receptor signaling and involved in extracellular matrix biogenesis. Virulence 2023; 14:2158663. [PMID: 36600181 PMCID: PMC9828750 DOI: 10.1080/21505594.2022.2158663] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Salmonella is the only bacterium able to enter a host cell by the two known mechanisms: trigger and zipper. The trigger mechanism relies on the injection of bacterial effectors into the host cell through the Salmonella type III secretion system 1. In the zipper mechanism, mediated by the invasins Rck and PagN, the bacterium takes advantage of a cellular receptor for invasion. This study describes the transcriptomic reprogramming of the IEC-6 intestinal epithelial cell line to Salmonella Typhimurium strains that invaded cells by a trigger, a zipper, or both mechanisms. Using S. Typhimurium strains invalidated for one or other entry mechanism, we have shown that IEC-6 cells could support both entries. Comparison of the gene expression profiles of exposed cells showed that irrespective of the mechanism used for entry, the transcriptomic reprogramming of the cell was nearly the same. On the other hand, when gene expression was compared between cells unexposed or exposed to the bacterium, the transcriptomic reprogramming of exposed cells was significantly different. It is particularly interesting to note the modulation of expression of numerous target genes of the aryl hydrocarbon receptor showing that this transcription factor was activated by S. Typhimurium infection. Numerous genes associated with the extracellular matrix were also modified. This was confirmed at the protein level by western-blotting showing a dramatic modification in some extracellular matrix proteins. Analysis of a selected set of modulated genes showed that the expression of the majority of these genes was modulated during the intracellular life of S. Typhimurium.
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Affiliation(s)
| | | | - Marco Moroldo
- INRAE, AgroParisTech, Université Paris Saclay, Jouy-en-Josas, France
| | | | | | | | | | | | - Philippe Velge
- INRAE, ISP, Université de Tours, Nouzilly, France,CONTACT Philippe Velge
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5
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Sinnett-Smith J, Torres-Marquez ME, Chang JK, Shimizu Y, Hao F, Martin MG, Rozengurt E. Statins inhibit protein kinase D (PKD) activation in intestinal cells and prevent PKD1-induced growth of murine enteroids. Am J Physiol Cell Physiol 2023; 324:C807-C820. [PMID: 36779664 PMCID: PMC10042602 DOI: 10.1152/ajpcell.00286.2022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2022] [Revised: 01/10/2023] [Accepted: 01/10/2023] [Indexed: 02/14/2023]
Abstract
We examined the impact of statins on protein kinase D (PKD) activation by G protein-coupled receptor (GPCR) agonists. Treatment of intestinal IEC-18 cells with cerivastatin inhibited PKD autophosphorylation at Ser916 induced by angiotensin II (ANG II) or vasopressin in a dose-dependent manner with half-maximal inhibition at 0.2 µM. Cerivastatin treatment inhibited PKD activation stimulated by these agonists for different times (5-60 min) and blunted HDAC5 phosphorylation, a substrate of PKD. Other lipophilic statins, including simvastatin, atorvastatin, and fluvastatin also prevented PKD activation in a dose-dependent manner. Using IEC-18 cell lines expressing PKD1 tagged with EGFP (enhanced green fluorescent protein), cerivastatin or simvastatin blocked GPCR-mediated PKD1-EGFP translocation to the plasma membrane and its subsequent nuclear accumulation. Similar results were obtained in IEC-18 cells expressing PKD3-EGFP. Mechanistically, statins inhibited agonist-dependent PKD activation rather than acting directly on PKD catalytic activity since exposure to cerivastatin or simvastatin did not impair PKD autophosphorylation or PKD1-EGFP membrane translocation in response to phorbol dibutyrate, which bypasses GPCRs and directly stimulates PKC and PKD. Furthermore, cerivastatin did not inhibit recombinant PKD activity determined via an in vitro kinase assay. Using enteroids generated from intestinal crypt-derived epithelial cells from PKD1 transgenic mice as a model of intestinal regeneration, we show that statins oppose PKD1-mediated increase in enteroid area, complexity (number of crypt-like buds), and DNA synthesis. Our results revealed a previously unappreciated inhibitory effect of statins on receptor-mediated PKD activation and in opposing the growth-promoting effects of PKD1 on intestinal epithelial cells.
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Affiliation(s)
- James Sinnett-Smith
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, United States
- VA Greater Los Angeles Health Care System, Los Angeles, California, United States
| | - M Eugenia Torres-Marquez
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, United States
| | - Jen-Kuan Chang
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, United States
| | - Yuki Shimizu
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, United States
| | - Fang Hao
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, United States
| | - Martin G Martin
- Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, California, United States
| | - Enrique Rozengurt
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, United States
- VA Greater Los Angeles Health Care System, Los Angeles, California, United States
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6
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Complexification of In Vitro Models of Intestinal Barriers, A True Challenge for a More Accurate Alternative Approach. Int J Mol Sci 2023; 24:ijms24043595. [PMID: 36835003 PMCID: PMC9958734 DOI: 10.3390/ijms24043595] [Citation(s) in RCA: 20] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2022] [Revised: 01/27/2023] [Accepted: 02/03/2023] [Indexed: 02/15/2023] Open
Abstract
The use of cell models is common to mimic cellular and molecular events in interaction with their environment. In the case of the gut, the existing models are of particular interest to evaluate food, toxicants, or drug effects on the mucosa. To have the most accurate model, cell diversity and the complexity of the interactions must be considered. Existing models range from single-cell cultures of absorptive cells to more complex combinations of two or more cell types. This work describes the existing solutions and the challenges that remain to be solved.
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7
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Li R, Rao JN, Smith AD, Chung HK, Xiao L, Wang JY, Turner DJ. miR-542-5p targets c-myc and negates the cell proliferation effect of SphK1 in intestinal epithelial cells. Am J Physiol Cell Physiol 2023; 324:C565-C572. [PMID: 36622069 PMCID: PMC9942902 DOI: 10.1152/ajpcell.00145.2022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2022] [Revised: 12/22/2022] [Accepted: 12/28/2022] [Indexed: 01/10/2023]
Abstract
Intestinal epithelial barrier defects occur commonly during a variety of pathological conditions, though their underlying mechanisms are not completely understood. Sphingosine-1-phosphate (S1P) has been shown to be a critical regulator of proliferation and of maintenance of an intact intestinal epithelial barrier, as is also sphingosine kinase 1 (SphK1), the rate-limiting enzyme for S1P synthesis. SphK1 has been shown to modulate its effect on intestinal epithelial proliferation through increased levels of c-myc. We conducted genome-wide profile analysis to search for differential microRNA expression related to overexpressed SphK1 demonstrating adjusted expression of microRNA 542-5p (miR-542-5p). Here, we show that miR-542-5p is regulated by SphK1 activity and is an effector of c-myc translation that ultimately serves as a critical regulator of the intestinal epithelial barrier. miR-542-5p directly regulates c-myc translation through direct binding to the c-myc mRNA. Exogenous S1P analogs administered in vivo protect murine intestinal barrier from damage due to mesenteric ischemia reperfusion, and damaged intestinal tissue had increased levels of miR-542-5p. These results indicate that miR-542-5p plays a critical role in the regulation of S1P-mediated intestinal barrier function, and may highlight a novel role in potential therapies.
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Affiliation(s)
- Ruiyun Li
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland
- Baltimore VA Medical Center, Baltimore, Maryland
| | - Jaladanki N Rao
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland
- Baltimore VA Medical Center, Baltimore, Maryland
| | - Alexis D Smith
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland
| | - Hee Kyoung Chung
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland
- Baltimore VA Medical Center, Baltimore, Maryland
| | - Lan Xiao
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland
- Baltimore VA Medical Center, Baltimore, Maryland
| | - Jian-Ying Wang
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland
- Baltimore VA Medical Center, Baltimore, Maryland
- Cell Biology Group, Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland
| | - Douglas J Turner
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland
- Baltimore VA Medical Center, Baltimore, Maryland
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8
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Boger KD, Sheridan AE, Ziegler AL, Blikslager AT. Mechanisms and modeling of wound repair in the intestinal epithelium. Tissue Barriers 2022; 11:2087454. [PMID: 35695206 PMCID: PMC10161961 DOI: 10.1080/21688370.2022.2087454] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022] Open
Abstract
The intestinal epithelial barrier is susceptible to injury from insults, such as ischemia or infectious disease. The epithelium's ability to repair wounded regions is critical to maintaining barrier integrity. Mechanisms of intestinal epithelial repair can be studied with models that recapitulate the in vivo environment. This review focuses on in vitro injury models and intestinal cell lines utilized in such systems. The formation of artificial wounds in a controlled environment allows for the exploration of reparative physiology in cell lines modeling diverse aspects of intestinal physiology. Specifically, the use of intestinal cell lines, IPEC-J2, Caco-2, T-84, HT-29, and IEC-6, to model intestinal epithelium is discussed. Understanding the unique systems available for creating intestinal injury and the differences in monolayers used for in vitro work is essential for designing studies that properly capture relevant physiology for the study of intestinal wound repair.
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Affiliation(s)
- Kasey D Boger
- Comparative Medicine Institute, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
| | - Ana E Sheridan
- Comparative Medicine Institute, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
| | - Amanda L Ziegler
- Comparative Medicine Institute, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
| | - Anthony T Blikslager
- Comparative Medicine Institute, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
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9
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Kaur N, Lum M, Lewis RE, Black AR, Black JD. A novel anti-proliferative PKCα-Ras-ERK signaling axis in intestinal epithelial cells. J Biol Chem 2022; 298:102121. [PMID: 35697074 PMCID: PMC9270260 DOI: 10.1016/j.jbc.2022.102121] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2021] [Revised: 05/05/2022] [Accepted: 05/31/2022] [Indexed: 01/02/2023] Open
Abstract
We have previously shown that the serine/threonine kinase PKCα triggers MAPK/ERK kinase (MEK)-dependent G1→S cell cycle arrest in intestinal epithelial cells, characterized by downregulation of cyclin D1 and inhibitor of DNA-binding protein 1 (Id1) and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. Here, we use pharmacological inhibitors, genetic approaches, siRNA-mediated knockdown, and immunoprecipitation to further characterize anti-proliferative ERK signaling in intestinal cells. We show that PKCα signaling intersects the Ras-Raf-MEK-ERK kinase cascade at the level of Ras small GTPases, and that anti-proliferative effects of PKCα require active Ras, Raf, MEK and ERK, core ERK pathway components that are also essential for pro-proliferative ERK signaling induced by epidermal growth factor (EGF). However, PKCα-induced anti-proliferative signaling differs from EGF signaling in that it is independent of the Ras guanine nucleotide exchange factors (Ras-GEFs), SOS1/2, and involves prolonged rather than transient ERK activation. PKCα forms complexes with A-Raf, B-Raf and C-Raf that dissociate upon pathway activation, and all three Raf isoforms can mediate PKCα-induced anti-proliferative effects. At least two PKCα-ERK pathways that collaborate to promote growth arrest were identified: one pathway requiring the Ras-GEF, RasGRP3, and H-Ras, leads to p21Cip1 upregulation, while additional pathway(s) mediate PKCα-induced cyclin D1 and Id1 downregulation. PKCα also induces ERK-dependent SOS1 phosphorylation, indicating possible negative crosstalk between anti-proliferative and growth-promoting ERK signaling. Importantly, the spatio-temporal activation of PKCα and ERK in the intestinal epithelium in vivo supports the physiological relevance of these pathways and highlights the importance of anti-proliferative ERK signaling to tissue homeostasis in the intestine.
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Affiliation(s)
- Navneet Kaur
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Michelle Lum
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Robert E Lewis
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Adrian R Black
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Jennifer D Black
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.
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10
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Yang J, Chen W, Sun Y, Xia P, Liu J, Zhang W. The role of microRNAs in regulating cadmium-induced apoptosis by targeting Bcl-2 in IEC-6 cells. Toxicol Appl Pharmacol 2021; 432:115737. [PMID: 34662668 DOI: 10.1016/j.taap.2021.115737] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2021] [Revised: 08/28/2021] [Accepted: 09/12/2021] [Indexed: 02/07/2023]
Abstract
Cadmium (Cd) is one of the most harmful environmental pollutants and has been found to have adverse effects on the gut. However, the toxic effects and potential mechanism of Cd on intestinal epithelial cells (IECs) are poorly understood. This study evaluated the effects of Cd exposure (0, 0.25, 0.5, 1, 2, and 4 μM) on IEC-6 cells in terms of cell viability and apoptosis, as well as apoptosis-associated gene expression. The results indicated that low doses (0.25- 1 μM) of Cd exhibited hormetic effects, while high doses of Cd (2 and 4 μM) reduced cell viability. The apoptotic effect increased in a dose-dependent pattern. Moreover, the mRNA levels of the Bcl-2, Bax and Caspase 3 genes were altered, which was in agreement with their protein expression. Based on sequencing analysis, the expression pattern of the microRNAs (miRNAs) changed significantly in the 2 μM Cd-treated group. QRT-PCR verified that 7 miRNAs, including miR-124-3p and miR-370-3p, were all upregulated with dose-effect relationship. Besides, transfection of miR-124-3p and miR-370-3p mimics /inhibitor and Bcl-2 siRNA into IEC-6 cells verified that these two miRNAs could regulate Cd-induced apoptosis by targeting Bcl-2. Finally, the direct targeting relationship between miR-370-3p and Bcl-2 gene was confirmed by luciferase reporter assay. Overall, the results demonstrated that Cd exposure could induce apoptosis in IEC-6 cells. The potential mechanism may be interference with the regulation of Bcl-2 gene expression by miR-370-3p and miR-124-3p.
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Affiliation(s)
- Jinsong Yang
- Department of Infectious Disease Prevention and Control, Fujian Center for Disease Control and Prevention, Fujian Provincial Key Laboratory of Zoonosis Research, Jintai Road No.76, 350001, China.
| | - Wei Chen
- Department of Infectious Disease Prevention and Control, Fujian Center for Disease Control and Prevention, Fujian Provincial Key Laboratory of Zoonosis Research, Jintai Road No.76, 350001, China
| | - Yi Sun
- Department of Preventive Medicine, Fujian Provincial Key Laboratory of Environmental Factors and Cancer, Key Laboratory of Environment and Health, School of Public Health, Fujian Medical University, Xueyuan Road No. 1, Minhou Coudslanty, Fuzhou 350108, China
| | - Pincang Xia
- Department of Infectious Disease Prevention and Control, Fujian Center for Disease Control and Prevention, Fujian Provincial Key Laboratory of Zoonosis Research, Jintai Road No.76, 350001, China
| | - Jin Liu
- Department of Preventive Medicine, Fujian Provincial Key Laboratory of Environmental Factors and Cancer, Key Laboratory of Environment and Health, School of Public Health, Fujian Medical University, Xueyuan Road No. 1, Minhou Coudslanty, Fuzhou 350108, China
| | - Wenchang Zhang
- Department of Preventive Medicine, Fujian Provincial Key Laboratory of Environmental Factors and Cancer, Key Laboratory of Environment and Health, School of Public Health, Fujian Medical University, Xueyuan Road No. 1, Minhou Coudslanty, Fuzhou 350108, China.
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11
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Zhao W, Wang Q. Knockdown of TRIM9 attenuates irinotecan‑induced intestinal mucositis in IEC‑6 cells by regulating DUSP6 expression via the P38 pathway. Mol Med Rep 2021; 24:867. [PMID: 34676875 PMCID: PMC8554382 DOI: 10.3892/mmr.2021.12507] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Accepted: 09/01/2021] [Indexed: 12/14/2022] Open
Abstract
Intestinal mucositis is a common side effect of cancer chemotherapy and it limits the dose of chemotherapy given to a patient. Tripartite motif family (TRIM) proteins have been reported to be implicated in the regulation of cancer chemotherapy. The present study aimed to investigate the effect of TRIM9 on irinotecan‑induced intestinal mucositis in the rat intestinal epithelial cell line IEC‑6. The expression of several TRIMs, such as TRIM1, TRIM9, TRIM18, TRIM36, TRIM46 and TRIM67, was examined. After TRIM9 knockdown or overexpression by lentivirus infection, cell proliferation and apoptosis, epithelial barrier tight‑junction proteins, inflammatory cytokines, transepithelial electrical resistance (TEER) and FITC dextran were measured. Treatment with irinotecan significantly inhibited cell proliferation and induced cell apoptosis, TRIM9 expression, intestinal mucosal barrier impairment, the levels of inflammatory cytokines and P38 phosphorylation in IEC‑6 cells, while the expression levels of epithelial barrier tight‑junction protein ZO‑1 and Claudin‑4 were decreased. Knockdown of TRIM9 partly counteracted the effect of irinotecan treatment, and inhibition of P38 potently reversed the effect of TRIM9 overexpression in IEC‑6 cells. Moreover, co‑immunoprecipitation showed an interaction between TRIM9 and DUSP6 in IEC‑6 cells, and overexpression of DUSP6 notably counteracted the effect of TRIM9 overexpression. The results demonstrated that TRIM9 knockdown may benefit patients with intestinal mucositis by inhibiting inflammatory cytokine expression and repairing intestinal barrier functions, which was probably due to inhibition of the activation of the P38 pathway via targeting DUSP6.
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Affiliation(s)
- Wenjun Zhao
- Department of Anorectal Section, Shanghai Baoshan District Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai 201999, P.R. China
| | - Qingming Wang
- Department of Anorectal Section, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, P.R. China
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12
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Araújo RS, Cristina Oliveira M, Cardoso VN, Keefe DMK, Stringer AM. The effect of free and encapsulated cisplatin into long-circulating and pH-sensitive liposomes on IEC-6 cells during wound healing in the presence of host-microbiota. J Pharm Pharmacol 2021; 74:711-717. [PMID: 34791381 DOI: 10.1093/jpp/rgab156] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2021] [Accepted: 10/18/2021] [Indexed: 11/12/2022]
Abstract
OBJECTIVES To circumvent cisplatin (CDDP) toxic effects and improve the antitumoural effect, our research group developed long-circulating and pH-sensitive liposomes containing CDDP (SpHL-CDDP). This study aimed to evaluate whether SpHL-CDDP is associated with intestinal protection under in-vitro conditions in the presence of host-microbiota, compared with free CDDP. METHODS The cytotoxicity of CDDP and SpHL-CDDP were evaluated by colorimetric MTT and sulforhodamine B (SRB) assays. Epithelial proliferation was assessed by using an in-vitro wounding model in the presence of host-microbiota with intestinal epithelial cell line 6 (IEC-6) monolayers. Cytokines were determined by ELISA. KEY FINDINGS Reduced cytotoxicity of SpHL-CDDP in IEC-6 cells (minimum of 1.3-fold according to the IC50 values) was observed when compared with CDDP. The presence of microbiota or CDDP reduced the wound healing. The association of microbiota and SpHL-CDDP improved the wound healing and cell number in IEC-6 cells when compared with control. These beneficial results can be associated with increased IL-6 and IL-10 levels induced by SpHL-CDDP which were affected by the presence of microbiota. CONCLUSIONS These results indicate that the presence of microbiota associated with SpHL-CDDP provided less intestinal cellular damages compared with CDDP and constitutes a promising candidate for clinical use.
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Affiliation(s)
- Raquel Silva Araújo
- Department of Clinical and Toxicological Analysis, Faculty of Pharmacy, Federal Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
| | - Mônica Cristina Oliveira
- Department of Pharmaceutical Products, Faculty of Pharmacy, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
| | - Valbert Nascimento Cardoso
- Department of Clinical and Toxicological Analysis, Faculty of Pharmacy, Federal Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
| | - Dorothy M K Keefe
- Department of Medicine, Mucositis Research Group, The University of Adelaide, Adelaide, SA, Australia
| | - Andrea M Stringer
- Clinical and Health Sciences, University of South Australia, Adelaide, SA, Australia
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13
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Kacar S, Sahinturk V. The Protective Agents Used against Acrylamide Toxicity: An In Vitro Cell Culture Study-Based Review. CELL JOURNAL 2021; 23:367-381. [PMID: 34455711 PMCID: PMC8405082 DOI: 10.22074/cellj.2021.7286] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Subscribe] [Scholar Register] [Received: 11/14/2019] [Accepted: 01/19/2020] [Indexed: 01/23/2023]
Abstract
Acrylamide is a dangerous electrophile with the potency to react with many biological moieties including proteins, and nucleic acids as well as other macromolecules. Acrylamide was first only known a chemical exposed in working areas as a neurotoxicant, it was later discovered that beyond just being a neurotoxicant exposed in industrial areas, acrylamide is exposed via daily foods as well. As such, several strategies have been sought to be developed to relieve the toxic spectrum of this chemical. The utilization of a protective agent against acrylamide toxicity was one of those strategies. To date, many agents with protective potency have been investigated. Herein, we compiled these agents and their effects shown in in vitro studies. We used the search engines of Web of Knowledge and searched the keywords "acrylamide" and "protect" in the titles along with the keyword "cell" in the topics. Twenty-one directly related articles out of 35 articles were examined. Briefly, all agents used against acrylamide were reported to exhibit protective activity. In most of these reports, 5 mM concentration of acrylamide and 24-hour treatment were the employed dose and duration. Usually, the beneficial agents were pre-treated to the cells. PC12 cells were the most utilized cell line, and the mitogen-activated protein kinase (MAPK) and nuclear factor erythroid 2-related factor 2 (NRF2) pathways were the most studied pathways. This study, beside other importance, can be utilized as a guide for how the protective studies against acrylamide were done and which parameters were investigated in in vitro acrylamide studies. In conclusion, taking measures is of utmost importance to prevent or alleviate the toxicity of acrylamide, to which we are daily exposed even in our homes. Therefore, future studies should persist in focusing on mitigating acrylamide toxicity.
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Affiliation(s)
- Sedat Kacar
- Department of Histology and Embryology, Faculty of Medicine, Eskisehir Osmangazi University, Eskisehir, Turkey.
| | - Varol Sahinturk
- Department of Histology and Embryology, Faculty of Medicine, Eskisehir Osmangazi University, Eskisehir, Turkey
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14
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Vitamin D Receptor Protects against Radiation-Induced Intestinal Injury in Mice via Inhibition of Intestinal Crypt Stem/Progenitor Cell Apoptosis. Nutrients 2021; 13:nu13092910. [PMID: 34578802 PMCID: PMC8466099 DOI: 10.3390/nu13092910] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2021] [Revised: 08/13/2021] [Accepted: 08/20/2021] [Indexed: 12/13/2022] Open
Abstract
It is urgent to seek new potential targets for the prevention or relief of gastrointestinal syndrome in clinical radiation therapy for cancers. Vitamin D, mediated through the vitamin D receptor (VDR), has been identified as a protective nutrient against ionizing radiation (IR)-induced damage. This study investigated whether VDR could inhibit IR-induced intestinal injury and explored underlying mechanism. We first found that vitamin D induced VDR expression and inhibited IR-induced DNA damage and apoptosis in vitro. VDR was highly expressed in intestinal crypts and was critical for crypt stem/progenitor cell proliferation under physiological conditions. Next, VDR-deficient mice exposed to IR significantly increased DNA damage and crypt stem/progenitor cell apoptosis, leading to impaired intestinal regeneration as well as shorter survival time. Furthermore, VDR deficiency activated the Pmaip1-mediated apoptotic pathway of intestinal crypt stem/progenitor cells in IR-treated mice, whereas inhibition of Pmaip1 expression by siRNA transfection protected against IR-induced cell apoptosis. Therefore, VDR protects against IR-induced intestinal injury through inhibition of crypt stem/progenitor cell apoptosis via the Pmaip1-mediated pathway. Our results reveal the importance of VDR level in clinical radiation therapy, and targeting VDR may be a useful strategy for treatment of gastrointestinal syndrome.
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15
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Rathor N, Chung HK, Song JL, Wang SR, Wang JY, Rao JN. TRPC1-mediated Ca 2+ signaling enhances intestinal epithelial restitution by increasing α4 association with PP2Ac after wounding. Physiol Rep 2021; 9:e14864. [PMID: 33991460 PMCID: PMC8123541 DOI: 10.14814/phy2.14864] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2020] [Revised: 01/02/2021] [Accepted: 01/13/2021] [Indexed: 11/24/2022] Open
Abstract
Gut epithelial restitution after superficial wounding is an important repair modality regulated by numerous factors including Ca2+ signaling and cellular polyamines. Transient receptor potential canonical-1 (TRPC1) functions as a store-operated Ca2+ channel in intestinal epithelial cells (IECs) and its activation increases epithelial restitution by inducing Ca2+ influx after acute injury. α4 is a multiple functional protein and implicated in many aspects of cell functions by modulating protein phosphatase 2A (PP2A) stability and activity. Here we show that the clonal populations of IECs stably expressing TRPC1 (IEC-TRPC1) exhibited increased levels of α4 and PP2A catalytic subunit (PP2Ac) and that TRPC1 promoted intestinal epithelial restitution by increasing α4/PP2Ac association. The levels of α4 and PP2Ac proteins increased significantly in stable IEC-TRPC1 cells and this induction in α4/PP2Ac complexes was accompanied by an increase in IEC migration after wounding. α4 silencing by transfection with siRNA targeting α4 (siα4) or PP2Ac silencing destabilized α4/PP2Ac complexes in stable IEC-TRPC1 cells and repressed cell migration over the wounded area. Increasing the levels of cellular polyamines by stable transfection with the Odc gene stimulated α4 and PP2Ac expression and enhanced their association, thus also promoting epithelial restitution after wounding. In contrast, depletion of cellular polyamines by treatment with α-difluoromethylornithine reduced α4/PP2Ac complexes and repressed cell migration. Ectopic overexpression of α4 partially rescued rapid epithelial repair in polyamine-deficient cells. These results indicate that activation of TRPC1-mediated Ca2+ signaling enhances cell migration primarily by increasing α4/PP2Ac associations after wounding and this pathway is tightly regulated by cellular polyamines.
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Affiliation(s)
- Navneeta Rathor
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA.,Baltimore Veterans Affairs Medical Center, Baltimore, MD, USA
| | - Hee Kyoung Chung
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA.,Baltimore Veterans Affairs Medical Center, Baltimore, MD, USA
| | - Jia-Le Song
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA
| | - Shelley R Wang
- Baltimore Veterans Affairs Medical Center, Baltimore, MD, USA
| | - Jian-Ying Wang
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA.,Baltimore Veterans Affairs Medical Center, Baltimore, MD, USA.,Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, USA
| | - Jaladanki N Rao
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA.,Baltimore Veterans Affairs Medical Center, Baltimore, MD, USA
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16
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Wang L, Wang X, Shi Z, Shen L, Zhang J, Zhang J. Bovine milk exosomes attenuate the alteration of purine metabolism and energy status in IEC-6 cells induced by hydrogen peroxide. Food Chem 2021; 350:129142. [PMID: 33610842 DOI: 10.1016/j.foodchem.2021.129142] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2020] [Revised: 01/13/2021] [Accepted: 01/17/2021] [Indexed: 12/13/2022]
Abstract
Evidence suggests that dietary depletion of bovine milk exosomes and their cargos causes a loss of circulating microRNAs and a series of health problems. The aim of the current study was to determine whether bovine milk exosomes affect purine nucleotide metabolism and energy metabolism in oxidatively stressed intestinal crypt epithelial cells (IEC-6). Cells were pretreated with exosomes, followed by H2O2 to induce oxidative stress. Reactive oxidative species (ROS) levels, purine nucleotides, purine metabolic key enzyme activities, cell energy status, and AMPK protein expression were analysed. Exosome pretreatment reduced ROS level and the activities of adenosine deaminase and xanthine oxidase induced by H2O2 in cells. Total adenine nucleotides and energy charge were increased with exosome pretreatment, while the AMPK phosphorylation level was downregulated. The results indicated that bovine milk exosomes could attenuate purine nucleotide catabolism and improve energy status in oxidatively stressed IEC-6 cells and exerted protective effects against oxidative stress.
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Affiliation(s)
- Lanfang Wang
- Research Center for Translational Medicine at Shanghai East Hospital, Tongji University School of Medicine, Tongji University, Shanghai, PR China; Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai, PR China
| | - Xinyan Wang
- The People's Hospital of Zhaoyuan City, Shandong Province, PR China
| | - Zhexi Shi
- Research Center for Translational Medicine at Shanghai East Hospital, Tongji University School of Medicine, Tongji University, Shanghai, PR China
| | - Li Shen
- Research Center for Translational Medicine at Shanghai East Hospital, Tongji University School of Medicine, Tongji University, Shanghai, PR China
| | - Jing Zhang
- Research Center for Translational Medicine at Shanghai East Hospital, School of Life Science and Technology, Tongji University, Shanghai, PR China; Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai, PR China.
| | - Jun Zhang
- Research Center for Translational Medicine at Shanghai East Hospital, Tongji University School of Medicine, Tongji University, Shanghai, PR China; Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai, PR China.
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17
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Rapa SF, Di Paola R, Cordaro M, Siracusa R, D’Amico R, Fusco R, Autore G, Cuzzocrea S, Stuppner H, Marzocco S. Plumericin Protects against Experimental Inflammatory Bowel Disease by Restoring Intestinal Barrier Function and Reducing Apoptosis. Biomedicines 2021; 9:biomedicines9010067. [PMID: 33445622 PMCID: PMC7826791 DOI: 10.3390/biomedicines9010067] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2020] [Revised: 01/08/2021] [Accepted: 01/09/2021] [Indexed: 02/07/2023] Open
Abstract
Intestinal epithelial barrier impairment plays a key pathogenic role in inflammatory bowel diseases (IBDs). In particular, together with oxidative stress, intestinal epithelial barrier alteration is considered as upstream event in ulcerative colitis (UC). In order to identify new products of natural origin with a potential activity for UC treatment, this study evaluated the effects of plumericin, a spirolactone iridoid, present as one of the main bioactive components in the bark of Himatanthus sucuuba (Woodson). Plumericin was evaluated for its ability to improve barrier function and to reduce apoptotic parameters during inflammation, both in intestinal epithelial cells (IEC-6), and in an animal experimental model of 2, 4, 6-dinitrobenzene sulfonic acid (DNBS)-induced colitis. Our results indicated that plumericin increased the expression of adhesion molecules, enhanced IEC-6 cells actin cytoskeleton rearrangement, and promoted their motility. Moreover, plumericin reduced apoptotic parameters in IEC-6. These results were confirmed in vivo. Plumericin reduced the activity of myeloperoxidase, inhibited the expression of ICAM-1, P-selectin, and the formation of PAR, and reduced apoptosis parameters in mice colitis induced by DNBS. These results support a pharmacological potential of plumericin in the treatment of UC, due to its ability to improve the structural integrity of the intestinal epithelium and its barrier function.
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Affiliation(s)
- Shara Francesca Rapa
- Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Italy; (S.F.R.); (G.A.)
| | - Rosanna Di Paola
- Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, 98168 Messina, Italy; (R.D.P.); (R.S.); (R.D.); (R.F.); (S.C.)
| | - Marika Cordaro
- Department of Biomedical, Dental, Morphological and Functional Imaging Sciences, University of Messina, Via Consolare Valeria, 98125 Messina, Italy;
| | - Rosalba Siracusa
- Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, 98168 Messina, Italy; (R.D.P.); (R.S.); (R.D.); (R.F.); (S.C.)
| | - Ramona D’Amico
- Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, 98168 Messina, Italy; (R.D.P.); (R.S.); (R.D.); (R.F.); (S.C.)
| | - Roberta Fusco
- Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, 98168 Messina, Italy; (R.D.P.); (R.S.); (R.D.); (R.F.); (S.C.)
| | - Giuseppina Autore
- Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Italy; (S.F.R.); (G.A.)
| | - Salvatore Cuzzocrea
- Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, 98168 Messina, Italy; (R.D.P.); (R.S.); (R.D.); (R.F.); (S.C.)
| | - Hermann Stuppner
- Institute of Pharmacy/Pharmacognosy, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria;
| | - Stefania Marzocco
- Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Italy; (S.F.R.); (G.A.)
- Correspondence: ; Tel.: +89-969159
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18
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Fan J, Li BR, Zhang Q, Zhao XH, Wang L. Pretreatment of IEC-6 cells with quercetin and myricetin resists the indomethacin-induced barrier dysfunction via attenuating the calcium-mediated JNK/Src activation. Food Chem Toxicol 2021; 147:111896. [PMID: 33276066 DOI: 10.1016/j.fct.2020.111896] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2020] [Revised: 11/22/2020] [Accepted: 11/28/2020] [Indexed: 12/11/2022]
Abstract
This study investigated the protective effect of two flavonols quercetin and myricetin on barrier function of rat intestinal epithelial (IEC-6) cells with indomethacin injury. When the cells were pretreated with the heated or unheated flavonols of 2.5-10 μmol/L for 24-48 h and then injured by 300 μmol/L indomethacin for 24 h, they showed reduced lactate dehydrogenase release (LDH) but increased cell viability; however, the flavonols of 20 μmol/L exerted a little effect to increase cell viability or decrease LDH release. Cell pretreatment with 5 μmol/L flavonols also resisted cell barrier dysfunction by increasing transepithelial resistance, reducing paracellular permeability, and promoting mRNA and protein expression of three tight junction proteins zonula occluden-1, occludin, and claudin-1. Although indomethacin injury increased intracellular Ca2+ concentration ([Ca2+]i) and consequently caused JNK/Src activation, the flavonols could decrease [Ca2+]i and attenuate the calcium-mediated JNK/Src activation. Quercetin with less hydroxyl groups was more efficient than myricetin to resist barrier dysfunction, while the unheated flavonols were more active than the heated counterparts to perform this effect. It is thus proposed that quercetin and myricetin could resist barrier dysfunction of the intestine once injured by indomethacin, but heat treatment of flavonols had a negative impact on barrier-protective function of flavonols.
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Affiliation(s)
- Jing Fan
- School of Biology and Food Engineering, Guangdong University of Petrochemical Technology, 525000, Maoming, PR China; Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, 150030, Harbin, PR China
| | - Bai-Ru Li
- School of Mechanical and Electrical Engineering Guangdong University of Petrochemical Technology, 525000, Maoming, PR China
| | - Qiang Zhang
- School of Biology and Food Engineering, Guangdong University of Petrochemical Technology, 525000, Maoming, PR China
| | - Xin-Huai Zhao
- School of Biology and Food Engineering, Guangdong University of Petrochemical Technology, 525000, Maoming, PR China; Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, 150030, Harbin, PR China; Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong University of Petrochemical Technology, 525000, Maoming, PR China.
| | - Li Wang
- School of Biology and Food Engineering, Guangdong University of Petrochemical Technology, 525000, Maoming, PR China.
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Fan J, Zhao XH, Li TJ. Heat treatment of galangin and kaempferol inhibits their benefits to improve barrier function in rat intestinal epithelial cells. J Nutr Biochem 2020; 87:108517. [PMID: 33011286 DOI: 10.1016/j.jnutbio.2020.108517] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2020] [Revised: 09/07/2020] [Accepted: 09/24/2020] [Indexed: 12/14/2022]
Abstract
Flavonols are bioactive substances in plant foods. In this study, two flavonols galangin and kaempferol were heated at 100°C for 30 min prior to assessing their effects on barrier function of rat intestinal epithelial (IEC-6) cells. Both heated and unheated flavonols (2.5-20 µmol/L dosages) were nontoxic to the cells up to 48 h post-treatment, and could promote cell viability values to 102.2-141.2% of control. By treatment with 5 µmol/L flavonols for 24 and 48 h, the treated cells time-dependently showed better improved physical and biological barrier functions than the control cells without any flavonol treatment, including higher transepithelial electrical resistance and antibacterial effect but reduced paracellular permeability and bacterial translocation. The results from real-time PCR and western-blot assays indicated that the cells treated with heated and unheated flavonols of 5 µmol/L dosage had up-regulated mRNA (1.13-1.81 folds) and protein (1.15-5.11 folds) expression for zonula occluden-1, occludin, and claudin-1 that are vital to the tight junctions of the cells. Moreover, protein expression of RhoA and ROCK were down-regulated into 0.41-0.98 and 0.40-0.92 folds, respectively, demonstrating a Rho inactivation that led to enhanced cell barrier integrity via the RhoA/ROCK pathway. Overall, galangin was more active than kaempferol to perform three biofunctions like improving cell barrier function, up-regulating tight junctions protein expression, and down-regulating RhoA/ROCK expression. Moreover, the heated flavonols were less effective than the unheated counterparts to perform these biofunctions. It is concluded that this heat treatment of galangin and kaempferol could inhibit their benefits to improve barrier function of IEC-6 cells.
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Affiliation(s)
- Jing Fan
- Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin, PR China
| | - Xin-Huai Zhao
- Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin, PR China; School of Biology and Food Engineering, Guangdong University of Petrochemical Technology, Maoming, PR China.
| | - Tie-Jing Li
- Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin, PR China; College of Light Industry, Liaoning University, Shenyang, PR China.
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Jung ES, Jang HJ, Hong EM, Lim HL, Lee SP, Kae SH, Lee J. The Protective Effect of 5-Aminosalicylic Acid Against Non-Steroidal Anti-Inflammatory Drug-Induced Injury Through Free Radical Scavenging in Small Intestinal Epithelial Cells. ACTA ACUST UNITED AC 2020; 56:medicina56100515. [PMID: 33019698 PMCID: PMC7600857 DOI: 10.3390/medicina56100515] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Revised: 09/23/2020] [Accepted: 09/28/2020] [Indexed: 12/22/2022]
Abstract
Background and objectives: Non-steroidal anti-inflammatory drugs (NSAIDs) have been among the major causes of small intestinal injury in clinical practice. As such, the current study investigated the protective effect of 5-aminosalicylic acid (5-ASA) against an NSAID-induced small intestinal injury. Materials and Methods: IEC-6 cells were treated with various concentrations of indomethacin with or without 5-ASA in a serum-free medium, after which an 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Dromide (MTT) assay, a cell apoptosis assay, a caspase-3 activity assay, a reactive oxygen species (ROS) content and Superoxide dismutase 2 (SOD2) activity measurement, a Western blotting for occludin and zonula occludens-1 (ZO-1) and a wound healing assay were conducted. Results: 5-ASA ameliorated indomethacin-induced cell apoptosis and an increase in the intracellular ROS content while augmenting the indomethacin-induced suppression of SOD2 activity in IEC-6 cells. Moreover, 5-ASA reversed the indomethacin-induced attenuation of occludin and ZO-1 expression and promoted faster wound healing effects in IEC-6 cells following an indomethacin-induced injury. Conclusions: Our results suggested that 5-ASA protects small intestinal cells against an NSAID-induced small intestinal injury by scavenging free radicals. Therefore, 5-ASA could be a potential treatment for an NSAID-induced small intestinal injury.
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Polysaccharide from spore of Ganoderma lucidum ameliorates paclitaxel-induced intestinal barrier injury: Apoptosis inhibition by reversing microtubule polymerization. Biomed Pharmacother 2020; 130:110539. [DOI: 10.1016/j.biopha.2020.110539] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2020] [Revised: 06/28/2020] [Accepted: 07/20/2020] [Indexed: 12/20/2022] Open
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Chen J, Cui Y, Wang Z, Liu G. Identification and characterization of PEDV infection in rat crypt epithelial cells. Vet Microbiol 2020; 249:108848. [PMID: 32979749 PMCID: PMC7497550 DOI: 10.1016/j.vetmic.2020.108848] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2020] [Accepted: 09/08/2020] [Indexed: 01/07/2023]
Abstract
The rat crypt epithelial cells (IEC-6) were highly susceptible to different subtypes of PEDV. The PEDV replication capacity in IEC-6 cells was similar to Vero cells and superior to that in IPEC-J2 cells. PEDV infection activated a robust immune response in IEC-6 cells. Porcine epidemic diarrhea (PED) is a devastating enteric disease to the world's swine production. Porcine epidemic diarrhea virus (PEDV), as the PED causative agent, has been commonly propagated and investigated in Vero cells, as well as in IPEC-J2, a porcine epithelial cell-jejunum 2. However, Vero cells, which are defective in interferon production, cannot represent the host response in enteric cells while PEDV replicates poorly in IPEC-J2 cells. In this study, we observed that rat crypt epithelial cells (IEC-6) were highly susceptible to different subtypes of PEDV. The replication kinetics of PEDV in IEC-6 cells is similar to that in Vero cells, but it is much higher than in IPEC-J2 cells. Besides that, PEDV infection in IEC-6 cells can induce the production of inflammatory cytokines and interferon, especially the type III IFNs. Collectively, our findings suggest that IEC-6 is an ideal cell line for PEDV replication and immune response studies.
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Affiliation(s)
- Jianing Chen
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu 730046, China
| | - Yaru Cui
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu 730046, China
| | - Zemei Wang
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu 730046, China
| | - Guangliang Liu
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu 730046, China.
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Lu S, Sung T, Amaro M, Hirakawa B, Jessen B, Hu W. Phenotypic Characterization of Targeted Knockdown of Cyclin-Dependent Kinases in the Intestinal Epithelial Cells. Toxicol Sci 2020; 177:226-234. [PMID: 32556214 DOI: 10.1093/toxsci/kfaa092] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Cyclin-dependent kinases (CDKs) are serine/threonine kinases that regulate cell cycle and have been vigorously pursued as druggable targets for cancer. There are over 20 members of the CDK family. Given their structural similarity, selective inhibition by small molecules has been elusive. In addition, collateral damage to highly proliferative normal cells by CDK inhibitors remains a safety concern. Intestinal epithelial cells are highly proliferative and the impact of individual CDK inhibition on intestinal cell proliferation has not been well studied. Using the rat intestinal epithelial (IEC6) cells as an in vitro model, we found that the selective CDK4/6 inhibitor palbociclib lacked potent anti-proliferative activity in IEC6 relative to the breast cancer cell line MCF7, indicating the absence of intestinal cell reliance on CDK4/6 for cell cycle progression. To further illustrate the role of CDKs in intestinal cells, we chose common targets of CDK inhibitors (CDK 1, 2, 4, 6, and 9) for targeted gene knockdown to evaluate phenotypes. Surprisingly, only CDK1 and CDK9 knockdown demonstrated profound cell death or had moderate growth effects, respectively. CDK2, 4, or 6 knockdowns, whether single, double, or triple combinations, did not have substantial impact. Studies evaluating CDK1 knockdown under various cell seeding densities indicate direct effects on viability independent of proliferation state and imply a potential noncanonical role for CDK1 in intestinal epithelial biology. This research supports the concept that CDK1 and CDK9, but not CDKs 2, 4, or 6, are essential for intestinal cell cycle progression and provides safety confidence for interphase CDK inhibition.
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Affiliation(s)
- Shuyan Lu
- Drug Safety Research and Development, Pfizer Inc., San Diego, California 92121
| | - Tae Sung
- Drug Safety Research and Development, Pfizer Inc., San Diego, California 92121
| | - Marina Amaro
- Drug Safety Research and Development, Pfizer Inc., San Diego, California 92121
| | - Brad Hirakawa
- Drug Safety Research and Development, Pfizer Inc., San Diego, California 92121
| | - Bart Jessen
- Drug Safety Research and Development, Pfizer Inc., San Diego, California 92121
| | - Wenyue Hu
- Drug Safety Research and Development, Pfizer Inc., San Diego, California 92121
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Zhu M, Li Y, Niu Y, Li J, Qin Z. Effects of bisphenol A and its alternative bisphenol F on Notch signaling and intestinal development: A novel signaling by which bisphenols disrupt vertebrate development. ENVIRONMENTAL POLLUTION (BARKING, ESSEX : 1987) 2020; 263:114443. [PMID: 32311622 DOI: 10.1016/j.envpol.2020.114443] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/16/2019] [Revised: 03/20/2020] [Accepted: 03/22/2020] [Indexed: 06/11/2023]
Abstract
We previously found bisphenol A (BPA) alternative, bisphenol F (BPF) upregulated Notch-related gene expression in intestines of the African clawed frog Xenopus laevis, suggesting an agonistic action on Notch signaling, a crucial signaling in multiple biological processes during development. Here, we aimed to confirm the actions of BPA and BPF on Notch signaling and to reveal their effects on intestinal development. Using X. laevis, an excellent model for developmental biology, we found that 10-1000 nM BPA and BPF significantly elevated Notch-related gene expression in a concentration-dependent manner. Subsequently, exceptional cell proliferation as well as intestinal histological changes were observed in treated intestines. Importantly, Notch inhibitor markedly suppressed the effects of BPA and BPF described above. Furthermore, we employed rat intestinal epithelium cells (IEC-6), an ideal in vitro model of intestinal epithelial cell differentiation, to confirm the effects of bisphenols. As expected, BPA and BPF upregulated Notch-related gene expression and induced the translocation of the Notch intracellular domain to the nucleus, followed by exceptional cell proliferation and differentiation, whereas Notch inhibitor antagonized the effects caused by BPA and BPF. All results strongly demonstrate that both BPA and BPF activate Notch signaling and subsequently disrupt intestinal development in vertebrates. Given its fundamental roles in multiple developmental processes, we propose that Notch signaling is an important and general target signaling of bisphenols in many developing tissues of vertebrates including humans.
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Affiliation(s)
- Min Zhu
- State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China; University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Yuanyuan Li
- State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China; University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Yue Niu
- State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China; College of Chemistry and Environmental Science, Hebei University, Baoding, 071002, China
| | - Jinbo Li
- State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China; University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Zhanfen Qin
- State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
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Fan J, Li TJ, Zhao XH. Barrier-promoting efficiency of two bioactive flavonols quercetin and myricetin on rat intestinal epithelial (IEC-6) cells via suppressing Rho activation. RSC Adv 2020; 10:27249-27258. [PMID: 35516969 PMCID: PMC9055572 DOI: 10.1039/d0ra04162a] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2020] [Accepted: 07/15/2020] [Indexed: 01/13/2023] Open
Abstract
Polyphenols are beneficial to human health because of their bio-activities. In this study, two flavonols quercetin and myricetin with or without heat treatment at 100 °C for 30 min were assessed for their barrier-promoting efficiency in rat intestinal epithelial (IEC-6) cells. The results indicated that the heated and unheated flavonols at dose levels of 2.5-20 μmol L-1 had a nontoxic effect on the cells treated for 24 and 48 h but enhanced the values of cell viability larger than 100% (especially at a dose level of 5 μmol L-1). Moreover, the cells exposed to these flavonols of 5 μmol L-1 for 24 and 48 h had improved barrier integrity compared to the control cells without any flavonol treatment, reflected by enhanced transepithelial electrical resistance and anti-bacterial effect but decreased paracellular permeability and bacterial translocation. Moreover, the results from both mRNA and protein expression verified 1.1-3.4 fold up-regulation of zonula occludens-1, occludin, and claudin-1 that are critical to tight junctions and barrier function of cells. Furthermore, the expression of other two proteins RhoA and ROCK in the treated cells was also down-regulated, demonstrating suppressed Rho activation and consequently barrier promotion via the RhoA/ROCK signaling pathway. Overall quercetin, due to its lower molecular polarity, mostly gave higher barrier-promoting efficiency than myricetin, while the heated flavonols were always less efficient than the unheated counterparts to promote barrier integrity of IEC-6 cells. It is thus highlighted that flavonols can provide barrier-promoting effects on intestinal epithelial cells with a promoting efficiency dependent on flavonol polarity; however, heat treatment especially excessive heat treatment of plant foods might lead to damaged flavonol activity.
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Affiliation(s)
- Jing Fan
- Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University 150030 Harbin PR China
| | - Tie-Jing Li
- College of Light Industry, Liaoning University 110136 Shenyang PR China
| | - Xin-Huai Zhao
- Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University 150030 Harbin PR China
- School of Biology and Food Engineering, Guangdong University of Petrochemical Technology 525000 Maoming PR China
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Arachidonic Acid Promotes Intestinal Regeneration by Activating WNT Signaling. Stem Cell Reports 2020; 15:374-388. [PMID: 32649903 PMCID: PMC7419670 DOI: 10.1016/j.stemcr.2020.06.009] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2019] [Revised: 06/09/2020] [Accepted: 06/09/2020] [Indexed: 12/17/2022] Open
Abstract
Intestinal regeneration is crucial for functional restoration after injury, and nutritional molecules can play an important role in this process. Here, we found that arachidonic acid (AA) serves as a direct proliferation promoter of intestinal epithelial cells that facilitates small intestinal regeneration in both three-dimensional cultured organoids and mouse models. As shown in the study, during post-irradiation regeneration, AA positively regulates intestinal epithelial cell proliferation by upregulating the expression of Ascl2 and activating WNT signaling, but negatively regulates intestinal epithelial cell differentiation. AA acts as a delicate regulator that efficiently facilitates epithelial tissue repair by activating radiation-resistant Msi1+ cells rather than Lgr5+ cells, which are extensively considered WNT-activated crypt base stem cells. Additionally, short-term AA treatment maintains optimal intestinal epithelial homeostasis under physiological conditions. As a result, AA treatment can be considered a potential therapy for irradiation injury repair and tissue regeneration.
AA promotes regeneration of intestinal epithelium after irradiation injury AA triggers Ascl2 expression and activates WNT signaling in intestinal epithelium AA facilitates intestinal repair by activating Msi1+ populations
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Molecular pharmacokinetic mechanism of the drug-drug interaction between genistein and repaglinide mediated by P-gp. Biomed Pharmacother 2020; 125:110032. [PMID: 32187961 DOI: 10.1016/j.biopha.2020.110032] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2019] [Revised: 02/15/2020] [Accepted: 02/17/2020] [Indexed: 01/12/2023] Open
Abstract
This study was devised to investigate if P-glycoprotein (P-gp) mediated the drug-drug interaction (DDI) between genistein and repaglinide. When genistein was added, the plasma concentrations of repaglinide in rats were increased. The maximum plasma concentration (Cmax) of repaglinide increased from 70.80 ± 7.98 ng/mL to 124.71 ± 9.02 ng/mL and the area under the plasma concentration-time curve (AUC) increased from 134.89 ± 13.65 μg·h/L to 245.95 ± 7.24 μg·h/L. Intestinal absorption of repaglinide was markedly enhanced by genistein or P-gp inhibitor verapamil (Ver), both in situ rat jejunal perfusion studies and in vitro transport assays using everted rat intestinal sac preparations. Furthermore, the accumulation of repaglinide in both Caco-2 cells and IEC-6 cells also increased significantly in the presence of genistein and Ver. The transepithelial transport rate of repaglinide from basolateral-to-apical in MDR1-MDCK cells was 3.6-fold higher than the apical-to-basolateral rate with a net efflux ratio of 1.92 compared with mock-MDCK cells, which was significantly decreased following co-administration with genistein or Ver. In an intracellular accumulation experiment using Rhodamine 123 as a P-gp substrate, genistein significantly increased the intracellular fluorescence of Rhodamine 123. These results indicated that genistein had an inhibitory effect on the efflux function of P-gp. Through molecular docking assays we further found that genistein could bind to the nucleotide-binding domains (NBD) in the cytoplasm of P-gp, thus affecting the functions of P-gp. In conclusion, genistein inhibited the efflux of repaglinide mediated by P-gp in rats and in vitro. The findings suggested that the DDI between genistein and repaglinide is mediated by P-gp, and a dosage adjustment may be needed when they are co-administered in a clinical setting.
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Al-Wabli RI, Almomen AA, Almutairi MS, Keeton AB, Piazza GA, Attia MI. New Isatin-Indole Conjugates: Synthesis, Characterization, and a Plausible Mechanism of Their in vitro Antiproliferative Activity. DRUG DESIGN DEVELOPMENT AND THERAPY 2020; 14:483-495. [PMID: 32099332 PMCID: PMC7006853 DOI: 10.2147/dddt.s227862] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/19/2019] [Accepted: 01/09/2020] [Indexed: 01/24/2023]
Abstract
Background Cancer remains the leading cause of human morbidity universally. Hence, we sought to assess the in vitro antiproliferative activity of new isatin-based conjugates (5a–s) against three human cancer cell lines. Methods The antiproliferative activities of compounds 5a–s were evaluated in vitro and their ADME (absorption, distribution, metabolism and excretion) was carried out using standard protocols. Subsequently, Western blot analysis was conducted to elucidate the potential antiproliferative mechanism of compounds 5a–s. Results The in vitro antiproliferative activities of compounds 5a–s against the tested cancer cell lines ranged from 20.3 to 95.9%. Compound 5m had an IC50 value of 1.17 µM; thus, its antiproliferative potency was approximately seven-fold greater than that of sunitinib (IC50 = 8.11 µM). In-depth pharmacological testing was conducted with compound 5m to gain insight into the potential antiproliferative mechanism of this class of compounds. Compound 5m caused an increase in the number of cells in the G1 phase, with a concomitant reduction of those in the G2/M and S phases. Additionally, compound 5m significantly and dose-dependently reduced the amount of phosphorylated retinoblastoma protein detected. Compound 5m enhanced expression of B cell translocation gene 1, cell cycle-associated proteins (cyclin B1, cyclin D1, and phosphorylated cyclin-dependent kinase 1), and a pro-apoptotic protein (Bcl-2-associated X protein gene), and activated caspase-3. ADME predictions exposed the oral liability of compounds 5a-s. Conclusion Herein, we revealed the antiproliferative activity and ADME predictions of the newly-synthesized compounds 5a–s and provided a detailed insight into the pharmacological profile of compound 5m. Thus, compounds 5a–s can potentially be exploited as new antiproliferative lead compounds for cancer chemotherapeutic.
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Affiliation(s)
- Reem I Al-Wabli
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
| | - Aliyah A Almomen
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
| | - Maha S Almutairi
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
| | - Adam B Keeton
- Department of Oncologic Sciences and Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604-1405, USA
| | - Gary A Piazza
- Department of Oncologic Sciences and Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604-1405, USA
| | - Mohamed I Attia
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia.,Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre (ID: 60014618), Giza 12622, Egypt
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Li J, Luo J, Zhang Y, Tang C, Wang J, Chen C. Silencing of soluble epoxide hydrolase 2 gene reduces H 2O 2-induced oxidative damage in rat intestinal epithelial IEC-6 cells via activating PI3K/Akt/GSK3β signaling pathway. Cytotechnology 2020; 72:23-36. [PMID: 31907700 PMCID: PMC7002799 DOI: 10.1007/s10616-019-00354-x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2019] [Revised: 11/03/2019] [Accepted: 11/07/2019] [Indexed: 12/18/2022] Open
Abstract
Oxidative stress plays a vital role in the occurrence and development of intestinal injury. Soluble epoxide hydrolase 2 gene (EPHX2) is a class of hydrolytic enzymes. We aim to explore the effects and molecular mechanism of siEPHX2 on H2O2-induced oxidative damage in rat intestinal epithelial IEC-6 cells. IEC-6 cells were transfected with EPHX2-siRNA and control si RNA plasmids by lipofectamine™ 2000 transfection reagent. The transfected samples were treated with H2O2 (50, 100, 200, 300, 400, and 500 µmol/L) for 12, 24, and 48 h, respectively. Cell viability was determined by cell counting kit-8 (CCK-8). Lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were assessed by respective detection kits. Mitochondrial membrane potential (MMP), cell apoptosis and reactive oxygen species (ROS) and the levels of factors were determined by flow cytometer, quantitative real-time PCR (qRT-PCR) and western blot assays, respectively. We found that the IC50 of H2O2 was 200 µmol/L at 24 h, and the transfection of siEHPX2 in H2O2-induced IEC-6 cells significantly promoted the cell viability, SOD activity and MMP rate, and reduced the rates of ROS and apoptosis as well as LDH and MDA contents. siEHPX2 up-regulated the B-cell lymphoma-2 (Bcl-2) level and down-regulated the levels of fibroblast-associated (Fas), Fas ligand (Fasl), Bcl-2 associated X protein (Bax), and Caspase-3. Moreover, the phosphorylation levels of phosphoinositide 3 kinase (PI3K), protein kinase B (Akt), and glycogen synthase kinase3β (GSK3β) were up-regulated. We proved that siEPHX2 had a protective effect on H2O2-induced oxidative damage in IEC-6 cells through activating PI3K/Akt/GSK3β signaling pathway.
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Affiliation(s)
- Jun Li
- Department of Gastrointestinal Surgery, Hunan Provincial People's Hospital, No. 61, Jiefang West Road, Furong District, Changsha, 410000, Hunan, China
| | - Jihui Luo
- Department of Surgical Oncology, Chenzhou No.1 People's Hospital, Chenzhou, China
| | - Yang Zhang
- Department of Burn Plastic Surgery, Hunan Provincial People's Hospital, Changsha, Hunan, China
| | - Chunming Tang
- Department of Hepatobiliary Surgery, Hunan Provincial People's Hospital, Changsha, Hunan, China
| | - Jiang Wang
- Department of Gastrointestinal Surgery, Hunan Provincial People's Hospital, No. 61, Jiefang West Road, Furong District, Changsha, 410000, Hunan, China
| | - Chaowu Chen
- Department of Gastrointestinal Surgery, Hunan Provincial People's Hospital, No. 61, Jiefang West Road, Furong District, Changsha, 410000, Hunan, China.
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Almutairi MS, Hassan ES, Keeton AB, Piazza GA, Abdelhameed AS, Attia MI. Antiproliferative activity and possible mechanism of action of certain 5-methoxyindole tethered C-5 functionalized isatins. DRUG DESIGN DEVELOPMENT AND THERAPY 2019; 13:3069-3078. [PMID: 31695325 PMCID: PMC6718129 DOI: 10.2147/dddt.s208241] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/10/2019] [Accepted: 08/19/2019] [Indexed: 01/09/2023]
Abstract
Background Cancer is one of the most dreaded human diseases, that has become an ever-increasing health problem and is a prime cause of death globally. The potential antiproliferative activity of certain indole-isatin molecular hybrids 5a-w was evaluated in vitro against three human cancer cell lines. Methods Standard protocols were adopted to examine the antiproliferative potential and mechanisms of compounds 5a-w. Western blot analysis was carried out on compound 5o. Results Compounds 5a-w demonstrated in vitro antiproliferative activity in the range of 22.6-97.8%, with compounds 5o and 5w being the most active antiproliferative compounds with IC50 values of 1.69 and 1.91 µM, which is fivefold and fourfold more potent than sunitinib (IC50=8.11 µM), respectively. Compound 5o was selected for in-depth pharmacological testing to understand its possible mechanism of antiproliferative activity. It caused a lengthening of the G1 phase and a reduction in the S and G2/M phases of the cell cycle and had an IC50 value of 10.4 μM with the resistant NCI-H69AR cancer cell line. Moreover, compound 5o significantly decreased the amount of phosphorylated Rb protein in a dose-dependent fashion, which was confirmed via Western blot analysis. Conclusion The current investigation highlighted the potential antiproliferative activity of compounds 5a-w as well as the antiproliferative profile of compound 5o. These compounds can be harnessed as new lead antiproliferatives in the preclinical studies of cancer chemotherapy.
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Affiliation(s)
- Maha S Almutairi
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
| | - Eman S Hassan
- Department of Medical Laboratory Sciences, Al-Ghad International Medical Sciences College, Female Section, Riyadh 13315, Saudi Arabia
| | - Adam B Keeton
- Department of Oncologic Sciences and Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604-1405, USA
| | - Gary A Piazza
- Department of Oncologic Sciences and Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604-1405, USA
| | - Ali S Abdelhameed
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
| | - Mohamed I Attia
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia.,Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre (ID: 60014618), Giza 12622, Egypt
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31
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Regal JF, Lund JM, Wing CR, Root KM, McCutcheon L, Bemis LT, Gilbert JS, Fleming SD. Interactions between the complement and endothelin systems in normal pregnancy and following placental ischemia. Mol Immunol 2019; 114:10-18. [PMID: 31326653 DOI: 10.1016/j.molimm.2019.06.015] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2019] [Revised: 06/22/2019] [Accepted: 06/23/2019] [Indexed: 01/01/2023]
Abstract
Preeclampsia is characterized by new onset hypertension and fetal growth restriction and is associated with aberrant activation of the innate immune complement system and stressed or ischemic placenta. Previous studies have suggested a role for both endothelin and complement system activation products in new onset hypertension in pregnancy, but inter-relationships of the pathways are unclear. We hypothesized that complement activation following placental ischemia stimulates the endothelin pathway to cause hypertension and impair fetal growth. The Reduced Uterine Perfusion Pressure (RUPP) model results in hypertension and fetal growth restriction in a pregnant rat due to placental ischemia caused by mechanical obstruction of blood flow to uterus and placenta. The effect of inhibitor of complement activation soluble Complement Receptor 1 (sCR1) and endothelin A receptor (ETA) antagonist atrasentan on hypertension, fetal weight, complement activation (systemic circulating C3a and local C3 placental deposition) and endothelin [circulating endothelin and message for preproendothelin (PPE), ETA and endothelin B receptor (ETB) in placenta] in the RUPP rat model were determined. Following placental ischemia, sCR1 attenuated hypertension but increased message for PPE and ETA in placenta, suggesting complement activation causes hypertension via an endothelin independent pathway. With ETA antagonism the placental ischemia-induced increase in circulating C3a was unaffected despite inhibition of hypertension, indicating systemic C3a alone is not sufficient. In normal pregnancy, inhibiting complement activation increased plasma endothelin but not placental PPE message. Atrasentan treatment increased fetal weight, circulating endothelin and placental ETA message, and unexpectedly increased local complement activation in placenta (C3 deposition) but not C3a in circulation, suggesting endothelin controls local placental complement activation in normal pregnancy. Atrasentan also significantly decreased message for endogenous complement regulators Crry and CD55 in placenta and kidney in normal pregnancy. Results of our study indicate that complement/endothelin interactions differ in pregnancies complicated with placental ischemia vs normal pregnancy, as well as locally vs systemically. These data clearly illustrate the complex interplay between complement and endothelin indicating that perturbations of either pathway may affect pregnancy outcomes.
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Affiliation(s)
- Jean F Regal
- Department of Biomedical Sciences, 1035 University Dr., University of Minnesota Medical School, Duluth Campus, Duluth, Minnesota, 55812, USA.
| | - Jenna M Lund
- Department of Biomedical Sciences, 1035 University Dr., University of Minnesota Medical School, Duluth Campus, Duluth, Minnesota, 55812, USA.
| | - Cameron R Wing
- Department of Biomedical Sciences, 1035 University Dr., University of Minnesota Medical School, Duluth Campus, Duluth, Minnesota, 55812, USA.
| | - Kate M Root
- Department of Biomedical Sciences, 1035 University Dr., University of Minnesota Medical School, Duluth Campus, Duluth, Minnesota, 55812, USA.
| | - Luke McCutcheon
- Department of Biomedical Sciences, 1035 University Dr., University of Minnesota Medical School, Duluth Campus, Duluth, Minnesota, 55812, USA.
| | - Lynne T Bemis
- Department of Biomedical Sciences, 1035 University Dr., University of Minnesota Medical School, Duluth Campus, Duluth, Minnesota, 55812, USA.
| | - Jeffrey S Gilbert
- Department of Biomedical Sciences, 1035 University Dr., University of Minnesota Medical School, Duluth Campus, Duluth, Minnesota, 55812, USA.
| | - Sherry D Fleming
- Division of Biology, 18 Ackert, Kansas State University, 1717 Claflin Rd, Manhattan, Kansas, 66506, USA.
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32
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Adesso S, Ruocco M, Rapa SF, Piaz FD, Raffaele Di Iorio B, Popolo A, Autore G, Nishijima F, Pinto A, Marzocco S. Effect of Indoxyl Sulfate on the Repair and Intactness of Intestinal Epithelial Cells: Role of Reactive Oxygen Species' Release. Int J Mol Sci 2019; 20:ijms20092280. [PMID: 31072046 PMCID: PMC6539031 DOI: 10.3390/ijms20092280] [Citation(s) in RCA: 32] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2019] [Revised: 05/05/2019] [Accepted: 05/06/2019] [Indexed: 12/17/2022] Open
Abstract
Chronic kidney disease (CKD) is characterized by an oxidative stress status, driving some CKD-associated complications, even at the gastrointestinal level. Indoxyl Sulfate (IS) is a protein-bound uremic toxin, poorly eliminated by dialysis. This toxin is able to affect the intestinal system, but its molecular mechanism/s in intestinal epithelial cells (IECs) remain poorly understood. This study's aim was to evaluate the effect of IS (31.2-250 µM) on oxidative stress in IEC-6 cells and on the intactness of IECs monolayers. Our results indicated that IS enhanced oxidative cell damage by inducing reactive oxygen species (ROS) release, reducing the antioxidant response and affecting Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) nuclear translocation as well its related antioxidant enzymes. In the wound healing assay model, IS reduced IEC-6 migration, slightly impaired actin cytoskeleton rearrangement; this effect was associated with connexin 43 alteration. Moreover, we reported the effect of CKD patients' sera in IEC-6 cells. Our results indicated that patient sera induced ROS release in IEC-6 cells directly related to IS sera content and this effect was reduced by AST-120 serum treatment. Results highlighted the effect of IS in inducing oxidative stress in IECs and in impairing the intactness of the IECs cell monolayer, thus significantly contributing to CKD-associated intestinal alterations.
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Affiliation(s)
- Simona Adesso
- Department of Pharmacy, University of Salerno, I-84084, Fisciano, Salerno, Italy.
| | - Marco Ruocco
- Department of Pharmacy, University of Salerno, I-84084, Fisciano, Salerno, Italy.
| | - Shara Francesca Rapa
- Department of Pharmacy, University of Salerno, I-84084, Fisciano, Salerno, Italy.
| | - Fabrizio Dal Piaz
- Department of Medicine, University of Salerno, I-84084 Fisciano, Salerno, Italy.
| | | | - Ada Popolo
- Department of Pharmacy, University of Salerno, I-84084, Fisciano, Salerno, Italy.
| | - Giuseppina Autore
- Department of Pharmacy, University of Salerno, I-84084, Fisciano, Salerno, Italy.
| | - Fuyu Nishijima
- Pharmaceuticals Division, Kureha Corporation, 169-8503 Tokyo, Japan.
| | - Aldo Pinto
- Department of Pharmacy, University of Salerno, I-84084, Fisciano, Salerno, Italy.
| | - Stefania Marzocco
- Department of Pharmacy, University of Salerno, I-84084, Fisciano, Salerno, Italy.
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33
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Gronke K, Hernández PP, Zimmermann J, Klose CSN, Kofoed-Branzk M, Guendel F, Witkowski M, Tizian C, Amann L, Schumacher F, Glatt H, Triantafyllopoulou A, Diefenbach A. Interleukin-22 protects intestinal stem cells against genotoxic stress. Nature 2019; 566:249-253. [PMID: 30700914 PMCID: PMC6420091 DOI: 10.1038/s41586-019-0899-7] [Citation(s) in RCA: 270] [Impact Index Per Article: 45.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2018] [Accepted: 01/02/2019] [Indexed: 02/07/2023]
Abstract
Environmental genotoxic factors pose a challenge to the genomic integrity of epithelial cells at barrier surfaces that separate host organisms from the environment. They can induce mutations that, if they occur in epithelial stem cells, contribute to malignant transformation and cancer development1-3. Genome integrity in epithelial stem cells is maintained by an evolutionarily conserved cellular response pathway, the DNA damage response (DDR). The DDR culminates in either transient cell-cycle arrest and DNA repair or elimination of damaged cells by apoptosis4,5. Here we show that the cytokine interleukin-22 (IL-22), produced by group 3 innate lymphoid cells (ILC3) and γδ T cells, is an important regulator of the DDR machinery in intestinal epithelial stem cells. Using a new mouse model that enables sporadic inactivation of the IL-22 receptor in colon epithelial stem cells, we demonstrate that IL-22 is required for effective initiation of the DDR following DNA damage. Stem cells deprived of IL-22 signals and exposed to carcinogens escaped DDR-controlled apoptosis, contained more mutations and were more likely to give rise to colon cancer. We identified metabolites of glucosinolates, a group of phytochemicals contained in cruciferous vegetables, to be a widespread source of genotoxic stress in intestinal epithelial cells. These metabolites are ligands of the aryl hydrocarbon receptor (AhR)6, and AhR-mediated signalling in ILC3 and γδ T cells controlled their production of IL-22. Mice fed with diets depleted of glucosinolates produced only very low levels of IL-22 and, consequently, the DDR in epithelial cells of mice on a glucosinolate-free diet was impaired. This work identifies a homeostatic network protecting stem cells against challenge to their genome integrity by AhR-mediated 'sensing' of genotoxic compounds from the diet. AhR signalling, in turn, ensures on-demand production of IL-22 by innate lymphocytes directly regulating components of the DDR in epithelial stem cells.
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Affiliation(s)
- Konrad Gronke
- Laboratory of Innate Immunity, Department of Microbiology, Infectious Diseases and Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany
- Berlin Institute of Health (BIH), Berlin, Germany
- Mucosal and Developmental Immunology, Deutsches Rheuma-Forschungszentrum, Berlin, Germany
- Department of Medical Microbiology and Hygiene, University Medical Centre, University of Mainz, Mainz, Germany
- Department of Medical Microbiology, University of Freiburg, Freiburg, Germany
- Max Planck Institute for Immunobiology and Epigenetics, Freiburg, Germany
| | - Pedro P Hernández
- Department of Medical Microbiology, University of Freiburg, Freiburg, Germany
- Max Planck Institute for Immunobiology and Epigenetics, Freiburg, Germany
- Macrophages and Development of Immunity, Institute Pasteur, Paris, France
| | - Jakob Zimmermann
- Department of Medical Microbiology, University of Freiburg, Freiburg, Germany
| | - Christoph S N Klose
- Laboratory of Innate Immunity, Department of Microbiology, Infectious Diseases and Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany
- Department of Medical Microbiology, University of Freiburg, Freiburg, Germany
- Jill Roberts Institute for Research in Inflammatory Bowel Disease, Joan and Sanford I. Weill Department of Medicine, Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY, USA
| | - Michael Kofoed-Branzk
- Laboratory of Innate Immunity, Department of Microbiology, Infectious Diseases and Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany
- Berlin Institute of Health (BIH), Berlin, Germany
- Mucosal and Developmental Immunology, Deutsches Rheuma-Forschungszentrum, Berlin, Germany
- Department of Medical Microbiology and Hygiene, University Medical Centre, University of Mainz, Mainz, Germany
- Department of Medical Microbiology, University of Freiburg, Freiburg, Germany
- Max Planck Institute for Immunobiology and Epigenetics, Freiburg, Germany
| | - Fabian Guendel
- Laboratory of Innate Immunity, Department of Microbiology, Infectious Diseases and Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany
- Berlin Institute of Health (BIH), Berlin, Germany
- Mucosal and Developmental Immunology, Deutsches Rheuma-Forschungszentrum, Berlin, Germany
- Department of Medical Microbiology and Hygiene, University Medical Centre, University of Mainz, Mainz, Germany
- Department of Medical Microbiology, University of Freiburg, Freiburg, Germany
| | - Mario Witkowski
- Laboratory of Innate Immunity, Department of Microbiology, Infectious Diseases and Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany
- Berlin Institute of Health (BIH), Berlin, Germany
- Mucosal and Developmental Immunology, Deutsches Rheuma-Forschungszentrum, Berlin, Germany
- Department of Medical Microbiology and Hygiene, University Medical Centre, University of Mainz, Mainz, Germany
| | - Caroline Tizian
- Laboratory of Innate Immunity, Department of Microbiology, Infectious Diseases and Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany
- Berlin Institute of Health (BIH), Berlin, Germany
- Mucosal and Developmental Immunology, Deutsches Rheuma-Forschungszentrum, Berlin, Germany
- Department of Medical Microbiology and Hygiene, University Medical Centre, University of Mainz, Mainz, Germany
| | - Lukas Amann
- Department of Medical Microbiology, University of Freiburg, Freiburg, Germany
- Institute of Neuropathology, Medical Faculty, University of Freiburg, Freiburg, Germany
| | - Fabian Schumacher
- Department of Nutritional Toxicology, Institute of Nutritional Science, University of Potsdam, Nuthetal, Germany
- Department of Molecular Biology, University of Duisburg-Essen, Essen, Germany
| | - Hansruedi Glatt
- German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE), Potsdam, Germany
- Department Food Safety, Federal Institute for Risk Assessment, Berlin, Germany
| | - Antigoni Triantafyllopoulou
- Department of Rheumatology and Clinical Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany
- Innate Immunity in Rheumatic Diseases, Deutsches Rheuma-Forschungszentrum, Berlin, Germany
| | - Andreas Diefenbach
- Laboratory of Innate Immunity, Department of Microbiology, Infectious Diseases and Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany.
- Berlin Institute of Health (BIH), Berlin, Germany.
- Mucosal and Developmental Immunology, Deutsches Rheuma-Forschungszentrum, Berlin, Germany.
- Department of Medical Microbiology and Hygiene, University Medical Centre, University of Mainz, Mainz, Germany.
- Department of Medical Microbiology, University of Freiburg, Freiburg, Germany.
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34
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Shi J, Zhao XH. Effect of caseinate glycation with oligochitosan and transglutaminase on the intestinal barrier function of the tryptic caseinate digest in IEC-6 cells. Food Funct 2019; 10:652-664. [DOI: 10.1039/c8fo01785a] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
The oligochitosan-glycated caseinate digest has higher activity than the caseinate digest to strengthen the intestinal barrier function of IEC-6 cells.
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Affiliation(s)
- Jia Shi
- Key Laboratory of Dairy Science
- Ministry of Education
- Northeast Agricultural University
- Harbin 150030
- PR China
| | - Xin-Huai Zhao
- Key Laboratory of Dairy Science
- Ministry of Education
- Northeast Agricultural University
- Harbin 150030
- PR China
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35
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Shi J, Zhao XH. Influence of the Maillard-type caseinate glycation with lactose on the intestinal barrier activity of the caseinate digest in IEC-6 cells. Food Funct 2019; 10:2010-2021. [DOI: 10.1039/c8fo02607f] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The glycated caseinate digest of the Maillard-type shows lower capability than the caseinate digest to enhance the intestinal barrier function of IEC-6 cells.
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Affiliation(s)
- Jia Shi
- Key Laboratory of Dairy Science
- Ministry of Education
- Northeast Agricultural University
- Harbin 150030
- PR China
| | - Xin-Huai Zhao
- Key Laboratory of Dairy Science
- Ministry of Education
- Northeast Agricultural University
- Harbin 150030
- PR China
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36
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Liang Q, Lv X, Cai Q, Cai Y, Zhao B, Li G. Novobiocin, a Newly Found TRPV1 Inhibitor, Attenuates the Expression of TRPV1 in Rat Intestine and Intestinal Epithelial Cell Line IEC-6. Front Pharmacol 2018; 9:1171. [PMID: 30374305 PMCID: PMC6196238 DOI: 10.3389/fphar.2018.01171] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2018] [Accepted: 09/26/2018] [Indexed: 01/26/2023] Open
Abstract
Background and Purpose: Novobiocin (NOVO), an ABC transporter inhibitor, decreases intestinal wall permeability of capsaicin (CAP), an ABC transporter substrate. However, the mechanism of this effect is not consistent with the action of NOVO as an ABC transporter inhibitor. We previously found that CAP can also be transported via TRPV1, which was site-specific in the permeability of CAP across the intestine. We explored the regulation by NOVO of TRPV1 in the present study. Methods: Rats and transfected IEC-6 cells were used as the models to assess intestinal permeability and expression of TRPV1. Ussing chamber and intracellular accumulation were used to evaluate the influence of NOVO on the transport of CAP in vitro. The expression of TRPV1 was detected after administration of NOVO by qRT-PCR, western blot and immunofluorescent imaging. In addition, MTT and lactate dehydrogenase (LDH) were used to evaluate the cytotoxicity of NOVO in both rat and cell models. Finally, the effect of NOVO on the absorption of CAP in vivo was studied by LC-MS/MS. Results: In vitro data showed that there existed a dose-dependent relationship in the range of concentration between 5 and 50 μM, and even 5 μM NOVO could decrease intestinal permeability of CAP across the intestine. Meanwhile, cytosolic accumulation of CAP decreased when NOVO was used simultaneously or 24 h in advance. NOVO exhibited an inhibition level similar to that of ruthenium red (RR) or SB-705498, a TRPV1-specific inhibitor. NOVO down-regulated TRPV1 expression in the intestine and in transfected cells in a concentration-dependent fashion, hinting that its inhibition of the permeability of CAP is due to its inhibition of TRPV1 expression. Immunofluorescent imaging data showed that the fluorescence intensity of TRPV1 was reduced after pre-treatment with NOVO and SB-705498. In vivo data further demonstrated that oral co-administration of NOVO decreased Cmax and AUC of CAP in dosage-dependent ways, consistent with its role as a TRPV1 inhibitor. Conclusion: NOVO could be a potential TRPV1 inhibitor by attenuating the expression of TRPV1 and may be used to attenuate permeability of TRPV1 substrates.
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Affiliation(s)
- Qianying Liang
- Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou, China.,Rational Medication Evaluation and Drug Delivery Technology Lab, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Xueli Lv
- Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou, China.,Rational Medication Evaluation and Drug Delivery Technology Lab, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Qing Cai
- General Hospital of Guangzhou Military Command of PLA, Guangzhou, China
| | - Yun Cai
- Department of Pharmacy, the Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Boxin Zhao
- Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou, China.,Rational Medication Evaluation and Drug Delivery Technology Lab, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Guofeng Li
- Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou, China.,Rational Medication Evaluation and Drug Delivery Technology Lab, Nanfang Hospital, Southern Medical University, Guangzhou, China.,Guangdong Provincial Key Laboratory of New Drug Screening, Southern Medical University, Guangzhou, China
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37
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Okumura M, Ichihara H, Matsumoto Y. Hybrid liposomes showing enhanced accumulation in tumors as theranostic agents in the orthotopic graft model mouse of colorectal cancer. Drug Deliv 2018; 25:1192-1199. [PMID: 29790374 PMCID: PMC6058724 DOI: 10.1080/10717544.2018.1475517] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Hybrid liposomes (HLs) can be prepared by simply sonicating a mixture of vesicular and micellar molecules in a buffer solution. This study aimed to elucidate the therapeutic effects and ability of HLs to detect (diagnosis) cancer in an orthotopic graft mouse model of colorectal cancer with HCT116 cells for the use of HLs as theranostic agents. In the absence of a chemotherapeutic drug, HLs exhibited therapeutic effects by inhibiting the growth of HCT116 colorectal cancer cells in vitro, possibly through an increase in apoptosis. Intravenously administered HLs also caused a remarkable reduction in the relative cecum weight in an orthotopic graft mouse model of colorectal cancer. A decrease in tumor size in the cecal sections was confirmed by histological analysis using HE staining. TUNEL staining indicated an induction of apoptosis in HCT116 cells in the orthotopic graft mouse model of colorectal cancer. For the detection (diagnosis) of colorectal cancer by HLs, the accumulation of HLs encapsulating a fluorescent probe (ICG) was observed in HCT116 cells in the in vivo colorectal cancer model following intravenous administration. These data indicate that HLs can accumulate in tumor cells in the cecum of the orthotopic graft mouse model of colorectal cancer for a prolonged period of time, and inhibit the growth of HCT116 cells.
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Affiliation(s)
- Masaki Okumura
- a Division of Applied Life Science, Graduate School of Engineering , Sojo University , Nishi-ku, Kumamoto , Japan
| | - Hideaki Ichihara
- a Division of Applied Life Science, Graduate School of Engineering , Sojo University , Nishi-ku, Kumamoto , Japan
| | - Yoko Matsumoto
- a Division of Applied Life Science, Graduate School of Engineering , Sojo University , Nishi-ku, Kumamoto , Japan
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38
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Polysaccharide extracts of Astragalus membranaceus and Atractylodes macrocephala promote intestinal epithelial cell migration by activating the polyamine-mediated K+ channel. Chin J Nat Med 2018; 16:674-682. [DOI: 10.1016/s1875-5364(18)30107-9] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2017] [Indexed: 12/22/2022]
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39
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Boyle KA, Van Wickle J, Hill RB, Marchese A, Kalyanaraman B, Dwinell MB. Mitochondria-targeted drugs stimulate mitophagy and abrogate colon cancer cell proliferation. J Biol Chem 2018; 293:14891-14904. [PMID: 30087121 DOI: 10.1074/jbc.ra117.001469] [Citation(s) in RCA: 97] [Impact Index Per Article: 13.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2017] [Revised: 07/20/2018] [Indexed: 12/13/2022] Open
Abstract
Mutations in the KRAS proto-oncogene are present in 50% of all colorectal cancers and are increasingly associated with chemotherapeutic resistance to frontline biologic drugs. Accumulating evidence indicates key roles for overactive KRAS mutations in the metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis in cancer cells. Here, we sought to exploit the more negative membrane potential of cancer cell mitochondria as an untapped avenue for interfering with energy metabolism in KRAS variant-containing and KRAS WT colorectal cancer cells. Mitochondrial function, intracellular ATP levels, cellular uptake, energy sensor signaling, and functional effects on cancer cell proliferation were assayed. 3-Carboxyl proxyl nitroxide (Mito-CP) and Mito-Metformin, two mitochondria-targeted compounds, depleted intracellular ATP levels and persistently inhibited ATP-linked oxygen consumption in both KRAS WT and KRAS variant-containing colon cancer cells and had only limited effects on nontransformed intestinal epithelial cells. These anti-proliferative effects reflected the activation of AMP-activated protein kinase (AMPK) and the phosphorylation-mediated suppression of the mTOR target ribosomal protein S6 kinase B1 (RPS6KB1 or p70S6K). Moreover, Mito-CP and Mito-Metformin released Unc-51-like autophagy-activating kinase 1 (ULK1) from mTOR-mediated inhibition, affected mitochondrial morphology, and decreased mitochondrial membrane potential, all indicators of mitophagy. Pharmacological inhibition of the AMPK signaling cascade mitigated the anti-proliferative effects of Mito-CP and Mito-Metformin. This is the first demonstration that drugs selectively targeting mitochondria induce mitophagy in cancer cells. Targeting bioenergetic metabolism with mitochondria-targeted drugs to stimulate mitophagy provides an attractive approach for therapeutic intervention in KRAS WT and overactive mutant-expressing colon cancer.
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Affiliation(s)
- Kathleen A Boyle
- From the Department of Microbiology & Immunology.,MCW Cancer Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
| | | | - R Blake Hill
- MCW Cancer Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.,Department of Biochemistry
| | - Adriano Marchese
- MCW Cancer Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.,Department of Biochemistry
| | - Balaraman Kalyanaraman
- MCW Cancer Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.,Department of Biophysics
| | - Michael B Dwinell
- From the Department of Microbiology & Immunology, .,MCW Cancer Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.,Department of Surgery, and
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Austin K, Tsang D, Chalmers JA, Maalouf MF, Brubaker PL. Insulin-like growth factor-binding protein-4 inhibits epithelial growth and proliferation in the rodent intestine. Am J Physiol Gastrointest Liver Physiol 2018; 315:G206-G219. [PMID: 29631376 DOI: 10.1152/ajpgi.00349.2017] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Insulin-like growth factor-binding protein-4 (IGFBP-4) is a binding protein that modulates the action of insulin-like growth factor-1 (IGF-1), a growth factor whose presence is required for the intestinotrophic effects of glucagon-like peptide-2 (GLP-2). GLP-2 is a gut hormone that uses both IGF-1 and epidermal growth factor (EGF) as intermediary factors to promote intestinal growth. Therefore, to elucidate the mechanism through which IGFBP-4 regulates IGF-1 activity in the intestine, proliferation assays were conducted using rat intestinal epithelial cells (IEC-6). IGF-1 and EGF synergistically enhanced proliferation, an effect that was dose-dependently decreased by IGFBP-4 ( P < 0.05-0.001) in an IGF-1 receptor (R)- and MEK1/2- but not a phosphatidylinositol 3-kinase-dependent manner ( P > 0.05 for IGFBP-4 effects with IGF-1R and MEK1/2 inhibitors). Intestinal organoids derived from IGFBP-4 knockout mice demonstrated significantly greater Ki-67 expression and an enhanced surface area increase in response to IGF-1 treatment, compared with organoids from control mice ( P < 0.05-0.01). GLP-2 is also known to increase the mucosal expression of IGFBP-4 mRNA. To investigate whether this occurs through the actions of its intermediaries, IGF-1 and EGF, inducible intestinal epithelial-IGF-1R knockout and control mice were treated for 10 days with and without the pan-ErbB inhibitor, CI-1033. However, no differences in mucosal IGFBP-4 mRNA expression were found for any of the treatment groups ( P > 0.05). Consistently, IEC-6 cells treated with IGF-1 and/or EGF displayed no alteration in IGFBP-4 mRNA or in cellular and secreted IGFBP-4 protein ( P > 0.05). Overall, this study establishes that endogenous IGFBP-4 plays an important role in inhibiting IGF-1-induced intestinal epithelial proliferation and that mucosal IGFBP-4 expression is independent of IGF-1 and EGF. NEW & NOTEWORTHY This study demonstrates, for the first time, the inhibitory role of locally expressed insulin-like growth factor-binding protein-4 (IGFBP-4) on the intestinal proliferative actions of IGF-1 and supports the notion of the synergistic roles of IGF-1 and EGF in promoting intestinal epithelial growth. In turn, intestinal IGFBP-4 expression was not found to be regulated by IGF-1 and/or EGF.
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Affiliation(s)
- Kaori Austin
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
| | - Derek Tsang
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
| | | | - Michael F Maalouf
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
| | - Patricia L Brubaker
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada.,Department of Medicine, University of Toronto, Toronto, Ontario, Canada
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41
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Giardipain-1, a protease secreted by Giardia duodenalis trophozoites, causes junctional, barrier and apoptotic damage in epithelial cell monolayers. Int J Parasitol 2018; 48:621-639. [DOI: 10.1016/j.ijpara.2018.01.006] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2017] [Revised: 12/23/2017] [Accepted: 01/01/2018] [Indexed: 12/11/2022]
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42
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Eldehna WM, Al-Wabli RI, Almutairi MS, Keeton AB, Piazza GA, Abdel-Aziz HA, Attia MI. Synthesis and biological evaluation of certain hydrazonoindolin-2-one derivatives as new potent anti-proliferative agents. J Enzyme Inhib Med Chem 2018; 33:867-878. [PMID: 29707975 PMCID: PMC7011955 DOI: 10.1080/14756366.2018.1462802] [Citation(s) in RCA: 46] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
In connection with our research program on the development of novel indolin-2-one-based anticancer candidates, herein we report the design and synthesis of different series of hydrazonoindolin-2-ones 3a-e, 5a-e, 7a-c, and 10a-l. The synthesised derivatives were in vitro evaluated for their anti-proliferative activity towards lung A-549, colon HT-29, and breast ZR-75 human cancer cell lines. Compounds 5b, 5c, 7b, and 10e emerged as the most potent derivatives with average IC50 values of 4.37, 2.53, 2.14, and 4.66 µM, respectively, which are superior to Sunitinib (average IC50 = 8.11 µM). Furthermore, compounds 7b and 10e were evaluated for their effects on cell cycle progression and levels of phosphorylated retinoblastoma (Rb) protein in the A-549 cancer cell line. Moreover, 7b and 10e inhibited the cell growth of the multidrug-resistant lung cancer NCI-H69AR cell line with IC50 = 16 µM. In addition, the cytotoxic activities of 7b and 10e were assessed towards three non-tumorigenic cell lines (Intestine IEC-6, Breast MCF-10A, and Fibroblast Swiss-3t3) where both compounds displayed mean tumor selectivity index (1.6 and 1.8) higher than that of Sunitinib (1.4).
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Affiliation(s)
- Wagdy M Eldehna
- a Department of Pharmaceutical Chemistry, Faculty of Pharmacy , Kafrelsheikh University , Kafrelsheikh , Egypt
| | - Reem I Al-Wabli
- b Department of Pharmaceutical Chemistry, College of Pharmacy , King Saud University , Riyadh , Saudi Arabia
| | - Maha S Almutairi
- b Department of Pharmaceutical Chemistry, College of Pharmacy , King Saud University , Riyadh , Saudi Arabia
| | - Adam B Keeton
- c Department of Oncologic Sciences and Pharmacology , Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama , Mobile , AL , USA
| | - Gary A Piazza
- c Department of Oncologic Sciences and Pharmacology , Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama , Mobile , AL , USA
| | - Hatem A Abdel-Aziz
- d Department of Applied Organic Chemistry , National Research Centre , Giza , Egypt
| | - Mohamed I Attia
- b Department of Pharmaceutical Chemistry, College of Pharmacy , King Saud University , Riyadh , Saudi Arabia.,e Medicinal and Pharmaceutical Chemistry Department , Pharmaceutical and Drug Industries Research Division, National Research Centre (ID: 60014618) , Giza , Egypt
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43
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Rathor N, Chung HK, Wang SR, Qian M, Turner DJ, Wang JY, Rao JN. β-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding. Am J Physiol Gastrointest Liver Physiol 2018; 314:G399-G407. [PMID: 29191942 PMCID: PMC5899242 DOI: 10.1152/ajpgi.00296.2017] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Early gut mucosal restitution is a process by which intestinal epithelial cells (IECs) migrate over the wounded area, and its defective regulation occurs commonly in various critical pathological conditions. This rapid reepithelialization is mediated by different activating small GTP-binding proteins, but the exact mechanism underlying this process remains largely unknown. Recently, it has been reported that interaction between p21-activated kinase-interacting exchange factor (β-PIX) and G protein-coupled receptor kinase-interacting protein 1 (GIT1) activates small GTPases and plays an important role in the regulation of cell motility. Here, we show that induced association of β-PIX with GIT1 is essential for the stimulation of IEC migration after wounding by activating Rac1. Levels of β-PIX and GIT1 proteins and their association in differentiated IECs (line of IEC-Cdx2L1) were much higher than those observed in undifferentiated IECs (line of IEC-6), which was associated with an increase in IEC migration after wounding. Decreased levels of endogenous β-PIX by its gene-silencing destabilized β-PIX/GIT1 complexes, repressed Rac1 activity and inhibited cell migration over the wounded area. In contrast, ectopic overexpression of β-PIX increased the levels of β-PIX/GIT1 complexes, stimulated Rac1 activity, and enhanced intestinal epithelial restitution. Increased levels of cellular polyamines also stimulated β-PIX/GIT1 association, increased Rac1 activity, and promoted the epithelial restitution. Moreover, polyamine depletion decreased cellular abundances of β-PIX/GIT1 complex and repressed IEC migration after wounding, which was rescued by ectopic overexpression of β-PIX or GIT1. These results indicate that β-PIX/GIT1/Rac1 association is necessary for stimulation of IEC migration after wounding and that this signaling pathway is tightly regulated by cellular polyamines. NEW & NOTEWORTHY Our current study demonstrates that induced association of β-PIX with GIT1 is essential for the stimulation of intestinal epithelial restitution by activating Rac1, and this signaling pathway is tightly regulated by cellular polyamines.
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Affiliation(s)
- Navneeta Rathor
- 1Department of Surgery, Cell Biology Group, University of Maryland School of Medicine, Baltimore, Maryland,2Baltimore Veterans Affairs Medical Center, Baltimore, Maryland
| | - Hee Kyoung Chung
- 1Department of Surgery, Cell Biology Group, University of Maryland School of Medicine, Baltimore, Maryland,2Baltimore Veterans Affairs Medical Center, Baltimore, Maryland
| | - Shelley R. Wang
- 1Department of Surgery, Cell Biology Group, University of Maryland School of Medicine, Baltimore, Maryland,2Baltimore Veterans Affairs Medical Center, Baltimore, Maryland
| | - Michael Qian
- 1Department of Surgery, Cell Biology Group, University of Maryland School of Medicine, Baltimore, Maryland
| | - Douglas J. Turner
- 1Department of Surgery, Cell Biology Group, University of Maryland School of Medicine, Baltimore, Maryland,2Baltimore Veterans Affairs Medical Center, Baltimore, Maryland
| | - Jian-Ying Wang
- 1Department of Surgery, Cell Biology Group, University of Maryland School of Medicine, Baltimore, Maryland,2Baltimore Veterans Affairs Medical Center, Baltimore, Maryland,3Department of Pathology, Cell Biology Group, University of Maryland School of Medicine, Baltimore, Maryland
| | - Jaladanki N. Rao
- 1Department of Surgery, Cell Biology Group, University of Maryland School of Medicine, Baltimore, Maryland,2Baltimore Veterans Affairs Medical Center, Baltimore, Maryland
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Maresca M, Pinton P, Ajandouz EH, Menard S, Ferrier L, Oswald IP. Overview and Comparison of Intestinal Organotypic Models, Intestinal Cells, and Intestinal Explants Used for Toxicity Studies. Curr Top Microbiol Immunol 2018; 430:247-264. [PMID: 30259111 DOI: 10.1007/82_2018_142] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
The intestine is a complex organ formed of different types of cell distributed in different layers of tissue. To minimize animal experiments, for decades, researchers have been trying to develop in vitro/ex vivo systems able to mimic the cellular diversity naturally found in the gut. Such models not only help our understanding of the gut physiology but also of intestinal toxicity. This review describes the different systems used to evaluate the effects of drugs/contaminants on intestinal functions and compares their advantages and limitations. The comparison showed that the organotypic model is the best available model to perform intestinal toxicity studies, including on human tissues.
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Affiliation(s)
- Marc Maresca
- Aix Marseille Univ, CNRS, Centrale Marseille, iSm2, Marseille, France
| | - Philippe Pinton
- Toxalim (Research Centre in Food Toxicology), Université de Toulouse, INRA, ENVT, INP-Purpan, UPS, Toulouse, France
| | | | - Sandrine Menard
- Toxalim (Research Centre in Food Toxicology), Université de Toulouse, INRA, ENVT, INP-Purpan, UPS, Toulouse, France
| | - Laurent Ferrier
- Toxalim (Research Centre in Food Toxicology), Université de Toulouse, INRA, ENVT, INP-Purpan, UPS, Toulouse, France
| | - Isabelle P Oswald
- Toxalim (Research Centre in Food Toxicology), Université de Toulouse, INRA, ENVT, INP-Purpan, UPS, Toulouse, France.
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McManus S, Chababi W, Arsenault D, Dubois CM, Saucier C. Dissecting Oncogenic RTK Pathways in Colorectal Cancer Initiation and Progression. Methods Mol Biol 2018; 1765:27-42. [PMID: 29589299 DOI: 10.1007/978-1-4939-7765-9_2] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/08/2023]
Abstract
Colorectal cancer (CRC) is a progressive disorder associated with an accumulation of multiple heterogeneous genetic alterations in intestinal epithelial cells (IEC). However, when these cells undergo neoplastic transformation and become cancerous and metastatic, they invariably acquire hallmarks conferring them the ability to hyperproliferate, escape growth-inhibitory and death-inducing cues, and promote angiogenesis as well as epithelial-to-mesenchymal transformation (EMT), fostering their invasive dissemination from primary tumor into distant tissues. Compelling clinical and experimental evidence suggest that aberrant engagement of cell surface growth factor receptor tyrosine kinase (RTK) signaling, like that of the hepatocyte growth factor (HGF)/MET receptor, underlies CRC metastatic progression by promoting these cancer hallmarks. To date, though, the use of RTK-targeting agents has been viewed as a promising approach for the treatment of metastatic CRC, clinical success has been modest.Our vision is that the prospect of designing RTK-based, improved and innovative CRC therapies and prognostic markers likely rests on a comprehensive understanding of the biological processes and underlying regulatory molecular mechanisms by which deregulation of RTK signaling governs IEC's neoplastic transformation and their transition from noninvasive to metastatic and malignant cells. Herein, we describe our scheme for defining the full scope of oncogenic MET-driven cancer biological processes, in cellulo and in vivo, as well as the individual contribution of MET-binding effectors in a nontransformed IEC model, the IEC-6 cell line.
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Affiliation(s)
- Stephen McManus
- Department of Anatomy and Cell Biology, Faculty of Medicine and Health Science, Université de Sherbrooke, Sherbrooke, QC, Canada
| | - Walid Chababi
- Department of Anatomy and Cell Biology, Faculty of Medicine and Health Science, Université de Sherbrooke, Sherbrooke, QC, Canada
| | - Dominique Arsenault
- Department of Pediatrics, Faculty of Medicine and Health Science, Université de Sherbrooke, Sherbrooke, QC, Canada
- Department of Immunology Division, Faculty of Medicine and Health Science, Université de Sherbrooke, Sherbrooke, QC, Canada
| | - Claire M Dubois
- Department of Pediatrics, Faculty of Medicine and Health Science, Université de Sherbrooke, Sherbrooke, QC, Canada
- Department of Immunology Division, Faculty of Medicine and Health Science, Université de Sherbrooke, Sherbrooke, QC, Canada
| | - Caroline Saucier
- Department of Anatomy and Cell Biology, Faculty of Medicine and Health Science, Université de Sherbrooke, Sherbrooke, QC, Canada.
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Birch D, Christensen MV, Staerk D, Franzyk H, Nielsen HM. Fluorophore labeling of a cell-penetrating peptide induces differential effects on its cellular distribution and affects cell viability. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2017; 1859:2483-2494. [DOI: 10.1016/j.bbamem.2017.09.015] [Citation(s) in RCA: 43] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/06/2017] [Revised: 09/12/2017] [Accepted: 09/13/2017] [Indexed: 01/28/2023]
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Abdel-Aziz HA, Eldehna WM, Keeton AB, Piazza GA, Kadi AA, Attwa MW, Abdelhameed AS, Attia MI. Isatin-benzoazine molecular hybrids as potential antiproliferative agents: synthesis and in vitro pharmacological profiling. DRUG DESIGN DEVELOPMENT AND THERAPY 2017; 11:2333-2346. [PMID: 28848327 PMCID: PMC5557401 DOI: 10.2147/dddt.s140164] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
In continuation of our endeavor with respect to the development of potent and effective isatin-based anticancer agents, we adopted the molecular hybridization approach to design and synthesize four different sets of isatin-quinazoline (6a-f and 7a-e)/phthalazine (8a-f)/quinoxaline (9a-f) hybrids. The antiproliferative activity of the target hybrids was assessed towards HT-29 (colon), ZR-75 (breast) and A-549 (lung) human cancer cell lines. Hybrids 8b-d emerged as the most active antiproliferative congener in this study. Compound 8c induced apoptosis via increasing caspase 3/7 activity by about 5-fold in the A-549 human cancer cell line. In addition, it exhibited an increase in the G1 phase and a decrease in the S and G2/M phases in the cell cycle effect assay. Furthermore, it displayed an inhibitory concentration 50% value of 9.5 µM against multidrug-resistant NCI-H69AR lung cancer cell line. The hybrid 8c was also subjected to in vitro metabolic investigations through its incubation with rat liver microsomes and analysis of the resulting metabolites with the aid of liquid chromatography-mass spectrometry.
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Affiliation(s)
| | - Wagdy M Eldehna
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt
| | - Adam B Keeton
- Department of Oncologic Sciences and Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA
| | - Gary A Piazza
- Department of Oncologic Sciences and Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA
| | - Adnan A Kadi
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
| | - Mohamed W Attwa
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
| | - Ali S Abdelhameed
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
| | - Mohamed I Attia
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.,Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Giza, Egypt
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Nyegaard S, Andreasen T, Rasmussen JT. Lactadherin orthologs inhibit migration of human, porcine and murine intestinal epithelial cells. Food Sci Nutr 2017; 5:934-942. [PMID: 28748083 PMCID: PMC5520951 DOI: 10.1002/fsn3.479] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2016] [Revised: 10/24/2016] [Accepted: 02/21/2017] [Indexed: 01/25/2023] Open
Abstract
Lactadherin was originally described due to its appearance in milk, but is abundantly expressed especially by professional and nonprofessional phagocytes. The proteins has been shown to have a multitude of bioactive effects, including inhibition of inflammatory phospholipases, induction of effero‐ and phagocytosis, prevent rotavirus induced gastroenteritis, and modulate intestinal homeostasis by regulating epithelial cell migration. The level of expression seems to be important in a row of serious pathologies linked to the intestinal epithelial barrier function, vascular‐ and autoimmune disease. This study examines the ability of lactadherin to modulate migration of intestinal epithelium. A cell exclusion assay is used to quantify the ability of human, bovine and murine lactadherin orthologs to affect migration of primary small intestine epithelium cells. Previous reports show that recombinant murine lactadherin stimulate rat small intestine cell migration. The present study could not confirm this. Conversely, 10 μg/ml lactadherin inhibits migration. Therefore, as lactadherins enteroprotective properties is well established using in vivo models we conclude that the protective effects are linked to lactadherins ability operate as an opsonin, or other modulating effects, and not a direct lactadherin‐cell induction of migration. Thus, the molecular mechanism behind the enteroprotective role of lactadherin remains to be established.
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Affiliation(s)
- Steffen Nyegaard
- Department of Molecular Biology University of Aarhus Aarhus C Denmark
| | - Trine Andreasen
- Department of Molecular Biology University of Aarhus Aarhus C Denmark
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Attia MI, Eldehna WM, Afifi SA, Keeton AB, Piazza GA, Abdel-Aziz HA. New hydrazonoindolin-2-ones: Synthesis, exploration of the possible anti-proliferative mechanism of action and encapsulation into PLGA microspheres. PLoS One 2017; 12:e0181241. [PMID: 28742842 PMCID: PMC5526551 DOI: 10.1371/journal.pone.0181241] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2017] [Accepted: 06/28/2017] [Indexed: 01/31/2023] Open
Abstract
The synthesis and molecular characterization of new isatin-based hydrazonoindolin-2-ones 4a-o and 7a-e are reported. The in vitro anti-proliferative potential of the synthesized compounds 4a-o and 7a-e was examined against HT-29 (colon), ZR-75 (breast) and A549 (lung) human cancer cell lines. Compounds 7b, 7d and 7e were the most active congeners against the tested human cancer cell lines with average IC50 values of 4.77, 3.39 and 2.37 μM, respectively, as compared with the reference isatin-based drug, sunitinib, which exhibited an average IC50 value of 8.11 μM. Compound 7e was selected for further pharmacological evaluation in order to gain insight into its possible mechanism of action. It increased caspase 3/7 activity by 2.4- and 1.85-fold between 4 and 8 h of treatment, respectively, at 10 μM and it caused a decrease in the percentage of cells in the G1 phase of the cell cycle with a corresponding increase in the S-phase. In addition, compound 7e increased phosphorylated tyrosine (p-Tyr) levels nearly two-fold with an apparent IC50 value of 3.8 μM. The 7e-loaded PLGA microspheres were prepared using a modified emulsion-solvent diffusion method. The average encapsulation efficiency of the 7e-loaded PLGA microspheres was 85% ± 1.3. While, the in vitro release profile of the 7e-loaded microspheres was characterized by slow and continuous release of compound 7e during 21 days and the release curve was fitted to zero order kinetics. Incorporation of 7e into PLGA microspheres improved its in vitro anti-proliferative activity toward the human cancer cell line A549 after 120 h incubation period with an IC50 value less than 0.8 μM.
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Affiliation(s)
- Mohamed I. Attia
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
- Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre (ID: 60014618), Dokki, Giza, Egypt
| | - Wagdy M. Eldehna
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt
| | - Samar A. Afifi
- Department of Pharmaceutics, National Organization for Drug Control and Research, Giza, Egypt
- Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
| | - Adam B. Keeton
- Department of Oncologic Sciences and Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, United States of America
| | - Gary A. Piazza
- Department of Oncologic Sciences and Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, United States of America
| | - Hatem A. Abdel-Aziz
- Department of Applied Organic Chemistry, National Res earch Centre, (ID: 60014618), Dokki, Giza, Egypt
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Matsui T, Ichikawa H, Fujita T, Takagi T, Osada-Oka M, Minamiyama Y. Histidine deficiency attenuates cell viability in rat intestinal epithelial cells by apoptosis via mitochondrial dysfunction. JOURNAL OF NUTRITION & INTERMEDIARY METABOLISM 2017. [DOI: 10.1016/j.jnim.2017.05.002] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
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