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Peters DE, Norris LD, Tenora L, Šnajdr I, Ponti AK, Zhu X, Sakamoto S, Veeravalli V, Pradhan M, Alt J, Thomas AG, Majer P, Rais R, McDonald C, Slusher BS. A gut-restricted glutamate carboxypeptidase II inhibitor reduces monocytic inflammation and improves preclinical colitis. Sci Transl Med 2023; 15:eabn7491. [PMID: 37556558 PMCID: PMC10661206 DOI: 10.1126/scitranslmed.abn7491] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2021] [Accepted: 07/21/2023] [Indexed: 08/11/2023]
Abstract
There is an urgent need to develop therapeutics for inflammatory bowel disease (IBD) because up to 40% of patients with moderate-to-severe IBD are not adequately controlled with existing drugs. Glutamate carboxypeptidase II (GCPII) has emerged as a promising therapeutic target. This enzyme is minimally expressed in normal ileum and colon, but it is markedly up-regulated in biopsies from patients with IBD and preclinical colitis models. Here, we generated a class of GCPII inhibitors designed to be gut-restricted for oral administration, and we interrogated efficacy and mechanism using in vitro and in vivo models. The lead inhibitor, (S)-IBD3540, was potent (half maximal inhibitory concentration = 4 nanomolar), selective, gut-restricted (AUCcolon/plasma > 50 in mice with colitis), and efficacious in acute and chronic rodent colitis models. In dextran sulfate sodium-induced colitis, oral (S)-IBD3540 inhibited >75% of colon GCPII activity, dose-dependently improved gross and histologic disease, and markedly attenuated monocytic inflammation. In spontaneous colitis in interleukin-10 (IL-10) knockout mice, once-daily oral (S)-IBD3540 initiated after disease onset improved disease, normalized colon histology, and attenuated inflammation as evidenced by reduced fecal lipocalin 2 and colon pro-inflammatory cytokines/chemokines, including tumor necrosis factor-α and IL-17. Using primary human colon epithelial air-liquid interface monolayers to interrogate the mechanism, we further found that (S)-IBD3540 protected against submersion-induced oxidative stress injury by decreasing barrier permeability, normalizing tight junction protein expression, and reducing procaspase-3 activation. Together, this work demonstrated that local inhibition of dysregulated gastrointestinal GCPII using the gut-restricted, orally active, small-molecule (S)-IBD3540 is a promising approach for IBD treatment.
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Affiliation(s)
- Diane E. Peters
- Johns Hopkins Drug Discovery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Lauren D. Norris
- Johns Hopkins Drug Discovery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Lukáš Tenora
- Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 160 00 Prague, Czechia
| | - Ivan Šnajdr
- Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 160 00 Prague, Czechia
| | - András K. Ponti
- Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
| | - Xiaolei Zhu
- Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Shinji Sakamoto
- Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Vijayabhaskar Veeravalli
- Johns Hopkins Drug Discovery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Manisha Pradhan
- Johns Hopkins Drug Discovery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Jesse Alt
- Johns Hopkins Drug Discovery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Ajit G. Thomas
- Johns Hopkins Drug Discovery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Pavel Majer
- Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 160 00 Prague, Czechia
| | - Rana Rais
- Johns Hopkins Drug Discovery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Christine McDonald
- Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
| | - Barbara S. Slusher
- Johns Hopkins Drug Discovery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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Histopathological and ultra-structural investigation of the damaging effects of hypoinsulinemia, hyper glycaemia and oxidative stress caused by parenteral nutrition combined with fasting on the small intestine of rabbits. Arab J Gastroenterol 2023:S1687-1979(23)00012-6. [PMID: 36890026 DOI: 10.1016/j.ajg.2023.02.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/20/2021] [Revised: 09/27/2022] [Accepted: 02/19/2023] [Indexed: 03/08/2023]
Abstract
BACKGROUND AND STUDY AIMS Parenteral nutrition (PN) is a life-saving practice when the use of the gastrointestinal tract is not appropriate. Despite its great benefits, however, PN may cause several complications. In this study, we conducted histopathological and ultra-structural examinations of the effect of PN combined with starvation on the small intestines of rabbits. MATERIALS AND METHODS Rabbits were divided into four groups. A fasting + PN group was left completely unfed and received all its daily required energy by PN through an intravenous central catheter. An oral feeding + PN group received half the necessary daily calories by oral feeding and the other half through PN. A semi-starvation group received only half the necessary daily calories by oral feeding and no PN. The fourth group, serving as a control, was supplied with its entire daily energy requirements through oral feeding. After 10 days, the rabbits were euthanized. Blood and small intestine tissue samples were collected from all groups. Blood samples were biochemically analysed, and tissue samples were examined by light and transmission electron microscopy. RESULTS The fasting + PN group exhibited lower insulin levels, higher glucose levels, and increased systemic oxidative stress than the other groups. Ultra-structural and histopathological examinations revealed a significant increase in apoptotic activity in this group's small intestines and a significant decrease in villus length and crypt depth. Severe damage to the intracellular organelles and nuclei of enterocytes was also observed. CONCLUSION PN combined with starvation appears to cause apoptosis in the small intestine due to oxidative stress and hyperglycaemia with hypoinsulinemia, with destructive effects on small intestine tissue. Adding enteral nutrition to PN may reduce these destructive effects.
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Adipose-Derived Stem Cell Extracellular Vesicles Improve Wound Closure and Angiogenesis in Diabetic Mice. Plast Reconstr Surg 2023; 151:331-342. [PMID: 36696316 DOI: 10.1097/prs.0000000000009840] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
BACKGROUND Currently, there is a lack in therapy that promotes the reepithelialization of diabetic wounds as an alternative to skin grafting. Here, the authors hypothesized that extracellular vesicles from adipose-derived stem cells (ADSC-EVs) could accelerate wound closure through rescuing the function of keratinocytes in diabetic mice. METHODS The effect of ADSC-EVs on the biological function of human keratinocyte cells was assayed in vitro. In vivo, 81 male severe combined immune deficiency mice aged 8 weeks were divided randomly into the extracellular vesicle-treated diabetes group (n = 27), the phosphate-buffered saline-treated diabetes group (n = 27), and the phosphate-buffered saline-treated normal group (n = 27). A round, 8-mm-diameter, full-skin defect was performed on the back skin of each mouse. The wound closure kinetics, average healing time, reepithelialization rate, and neovascularization were evaluated by histological staining. RESULTS In vitro, ADSC-EVs improved proliferation, migration, and proangiogenic potential, and inhibited the apoptosis of human keratinocyte cells by suppressing Fasl expression with the optimal dose of 40 μg/mL. In vivo, postoperative dripping of ADSC-EVs at the dose of 40 μg/mL accelerated diabetic wound healing, with a 15.8% increase in closure rate and a 3.3-day decrease in average healing time. ADSC-EVs improved reepithelialization (18.2%) with enhanced epithelial proliferation and filaggrin expression, and suppressed epithelial apoptosis and Fasl expression. A 2.7-fold increase in the number of CD31-positive cells was also observed. CONCLUSION ADSC-EVs improve diabetic wound closure and angiogenesis by enhancing keratinocyte-mediated reepithelialization and vascularization. CLINICAL RELEVANCE STATEMENT ADSC-EVs could be developed as a regenerative medicine for diabetic wound care.
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Livanova AA, Fedorova AA, Zavirsky AV, Krivoi II, Markov AG. Dose- and Segment-Dependent Disturbance of Rat Gut by Ionizing Radiation: Impact of Tight Junction Proteins. Int J Mol Sci 2023; 24:ijms24021753. [PMID: 36675266 PMCID: PMC9863103 DOI: 10.3390/ijms24021753] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2022] [Revised: 12/30/2022] [Accepted: 01/12/2023] [Indexed: 01/18/2023] Open
Abstract
The damaging effect of ionizing radiation (IR) exposure results in the disturbance of the gut natural barrier, followed by the development of severe gastrointestinal injury. However, the dose and application segment are known to determine the effects of IR. In this study, we demonstrated the dose- and segment-specificity of tight junction (TJ) alteration in IR-induced gastrointestinal injury in rats. Male Wistar rats were subjected to a total-body X-ray irradiation at doses of 2 or 10 Gy. Isolated jejunum and colon segments were tested in an Ussing chamber 72 h after exposure. In the jejunum, 10-Gy IR dramatically altered transepithelial resistance, short-circuit current and permeability for sodium fluorescein. These changes were accompanied by severe disturbance of histological structure and total rearrangement of TJ content (increased content of claudin-1, -2, -3 and -4; multidirectional changes in tricellulin and occludin). In the colon of 10-Gy irradiated rats, lesions of barrier and transport functions were less pronounced, with only claudin-2 and -4 altered among TJ proteins. The 2-Gy IR did not change electrophysiological characteristics or permeability in the colon or jejunum, although slight alterations in jejunum histology were noted, emphasized with claudin-3 increase. Considering that TJ proteins are critical for maintaining epithelial barrier integrity, these findings may have implications for countermeasures in gastrointestinal acute radiation injury.
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Affiliation(s)
- Alexandra A. Livanova
- Department of General Physiology, St. Petersburg State University, 199034 St. Petersburg, Russia
| | - Arina A. Fedorova
- Department of General Physiology, St. Petersburg State University, 199034 St. Petersburg, Russia
| | - Alexander V. Zavirsky
- Department of Military Toxicology and Radiation Defense, S. M. Kirov Military Medical Academy, 194044 St. Petersburg, Russia
| | - Igor I. Krivoi
- Department of General Physiology, St. Petersburg State University, 199034 St. Petersburg, Russia
| | - Alexander G. Markov
- Department of General Physiology, St. Petersburg State University, 199034 St. Petersburg, Russia
- Correspondence:
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Hageb A, Thalheim T, Nattamai KJ, Möhrle B, Saçma M, Sakk V, Thielecke L, Cornils K, Grandy C, Port F, Gottschalk KE, Mallm JP, Glauche I, Galle J, Mulaw MA, Geiger H. Reduced adhesion of aged intestinal stem cells contributes to an accelerated clonal drift. Life Sci Alliance 2022; 5:5/8/e202201408. [PMID: 35487692 PMCID: PMC9057243 DOI: 10.26508/lsa.202201408] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Revised: 04/11/2022] [Accepted: 04/12/2022] [Indexed: 11/30/2022] Open
Abstract
Analysis of clonal dynamics of intestinal stem cells supports an accelerated clonal drift upon aging, likely because of reduced adhesion of aged ISCs because of reduced canonical Wnt signaling. Upon aging, the function of the intestinal epithelium declines with a concomitant increase in aging-related diseases. ISCs play an important role in this process. It is known that ISC clonal dynamics follow a neutral drift model. However, it is not clear whether the drift model is still valid in aged ISCs. Tracking of clonal dynamics by clonal tracing revealed that aged crypts drift into monoclonality substantially faster than young ones. However, ISC tracing experiments, in vivo and ex vivo, implied a similar clonal expansion ability of both young and aged ISCs. Single-cell RNA sequencing for 1,920 high Lgr5 ISCs from young and aged mice revealed increased heterogeneity among subgroups of aged ISCs. Genes associated with cell adhesion were down-regulated in aged ISCs. ISCs of aged mice indeed show weaker adhesion to the matrix. Simulations applying a single cell–based model of the small intestinal crypt demonstrated an accelerated clonal drift at reduced adhesion strength, implying a central role for reduced adhesion for affecting clonal dynamics upon aging.
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Affiliation(s)
- Ali Hageb
- Institute of Molecular Medicine, Ulm University, Ulm, Germany
| | - Torsten Thalheim
- Interdisciplinary Centre for Bioinformatics, University Leipzig, Leipzig, Germany
| | - Kalpana J Nattamai
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, OH, USA
| | - Bettina Möhrle
- Institute of Molecular Medicine, Ulm University, Ulm, Germany
| | - Mehmet Saçma
- Institute of Molecular Medicine, Ulm University, Ulm, Germany
| | - Vadim Sakk
- Institute of Molecular Medicine, Ulm University, Ulm, Germany
| | - Lars Thielecke
- Institute for Medical Informatics and Biometry, Technische Universität Dresden, Dresden, Germany
| | - Kerstin Cornils
- Clinic of Pediatric Hematology and Oncology, Division of Pediatric Stem Cell Transplantation and Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.,Research Institute Children's Cancer Center Hamburg, Hamburg, Germany
| | - Carolin Grandy
- Institute for Experimental Physics, Ulm University, Ulm, Germany
| | - Fabian Port
- Institute for Experimental Physics, Ulm University, Ulm, Germany
| | - Kay-E Gottschalk
- Institute for Experimental Physics, Ulm University, Ulm, Germany
| | - Jan-Philipp Mallm
- Division of Chromatin Networks, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Ingmar Glauche
- Institute for Medical Informatics and Biometry, Technische Universität Dresden, Dresden, Germany
| | - Jörg Galle
- Interdisciplinary Centre for Bioinformatics, University Leipzig, Leipzig, Germany
| | - Medhanie A Mulaw
- Central Unit Single Cell Sequencing, Medical Faculty, Ulm University, Ulm, Germany
| | - Hartmut Geiger
- Institute of Molecular Medicine, Ulm University, Ulm, Germany
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Deets KA, Nichols Doyle R, Rauch I, Vance RE. Inflammasome activation leads to cDC1-independent cross-priming of CD8 T cells by epithelial cell-derived antigen. eLife 2021; 10:e72082. [PMID: 34939932 PMCID: PMC8719880 DOI: 10.7554/elife.72082] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2021] [Accepted: 12/21/2021] [Indexed: 12/17/2022] Open
Abstract
The innate immune system detects pathogens and initiates adaptive immune responses. Inflammasomes are central components of the innate immune system, but whether inflammasomes provide sufficient signals to activate adaptive immunity is unclear. In intestinal epithelial cells (IECs), inflammasomes activate a lytic form of cell death called pyroptosis, leading to epithelial cell expulsion and the release of cytokines. Here, we employed a genetic system to show that simultaneous antigen expression and inflammasome activation specifically in IECs is sufficient to activate CD8+ T cells. By genetic elimination of direct T cell priming by IECs, we found that IEC-derived antigens were cross-presented to CD8+ T cells. However, cross-presentation of IEC-derived antigen to CD8+ T cells only partially depended on IEC pyroptosis. In the absence of inflammasome activation, cross-priming of CD8+ T cells required Batf3+ dendritic cells (conventional type one dendritic cells [cDC1]), whereas cross-priming in the presence of inflammasome activation required a Zbtb46+ but Batf3-independent cDC population. These data suggest the existence of parallel inflammasome-dependent and inflammasome-independent pathways for cross-presentation of IEC-derived antigens.
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Affiliation(s)
- Katherine A Deets
- Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, BerkeleyBerkeleyUnited States
| | - Randilea Nichols Doyle
- Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, BerkeleyBerkeleyUnited States
| | - Isabella Rauch
- Department of Molecular Microbiology and Immunology, Oregon Health and Science UniversityPortlandUnited States
| | - Russell E Vance
- Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, BerkeleyBerkeleyUnited States
- Cancer Research Laboratory, University of California, BerkeleyBerkeleyUnited States
- Howard Hughes Medical Institute, University of California, BerkeleyBerkeleyUnited States
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Rowles JL, Wallig MA, Selting KA, Fan TM, Miller RJ, O'Brien WD, Erdman JW. A 10% Tomato Diet Selectively Reduces Radiation-Induced Damage in TRAMP Mice. J Nutr 2021; 151:3421-3430. [PMID: 34386819 DOI: 10.1093/jn/nxab257] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2021] [Revised: 03/30/2021] [Accepted: 07/09/2021] [Indexed: 11/12/2022] Open
Abstract
BACKGROUND Tomatoes contain carotenoids that have the potential to alter the effects of external beam radiation therapy (EBRT). OBJECTIVES We hypothesized that dietary lyophilized tomato paste (TP) would reduce apoptosis within carotenoid-containing nonneoplastic tissues in EBRT-treated TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice. METHODS Male TRAMP mice (n = 73) were provided an AIN-93G diet or a modified AIN-93G diet containing 10% TP (wt:wt) at 4 wk of age. Prostate tumor growth was monitored by ultrasound. The caudal half of the mouse was irradiated with 7.5 Gy (Rad) or 0 Gy (sham) at 24 wk of age or after the tumor volume exceeded 1000 mm3 with a Cobalt-60 source. Mice were euthanized 24 h postradiation. Carotenoids and α-tocopherol were measured by HPLC and compared by a t test. Tissues were assessed for radiation-induced changes (hematoxylin and eosin) and apoptosis [cleaved caspase-3 (CC3)] and compared by Kruskal-Wallis test or Freedman-Lane's permutation test. RESULTS Serum concentrations of lycopene (52% lower), phytoene (26% lower), and α-tocopherol (22% lower) were decreased in TP-fed irradiated mice (TP-Rad) compared with TP-fed sham mice (P < 0.05). CC3 scores increased within the prostate tumor with radiation treatments (P < 0.05), but were not affected by tomato consumption. In nonneoplastic tissues, TP-Rad had a lower percentage of CC3-positive cells within the cranial (67% lower) and caudal (75% lower) duodenum than irradiated mice on the control diet (Rad) (P < 0.005). Likewise, CC3 scores within the dorsolateral prostate of TP-Rad trended toward lower scores than for Rad (P = 0.07). CONCLUSIONS TP selectively reduces radiation-induced apoptosis in extratumoral tissues without decreasing radiation-induced apoptosis within the prostate tumor in TRAMP mice. Additional studies are needed to confirm and expand upon these findings.
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Affiliation(s)
- Joe L Rowles
- Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Matthew A Wallig
- Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA.,Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Kimberly A Selting
- Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA.,Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Timothy M Fan
- Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA.,Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Rita J Miller
- Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - William D O'Brien
- Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA.,Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - John W Erdman
- Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA.,Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, IL, USA.,Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, USA
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Matsuu-Matsuyama M, Shichijo K, Tsuchiya T, Kondo H, Miura S, Matsuda K, Sekine I, Nakashima M. Protective effects of a cystine and theanine mixture against acute radiation injury in rats. ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 2020; 78:103395. [PMID: 32325407 DOI: 10.1016/j.etap.2020.103395] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/25/2019] [Revised: 04/07/2020] [Accepted: 04/09/2020] [Indexed: 06/11/2023]
Abstract
This study aims to examine the effects of cystine and theanine (CT), which increases glutathione biosynthesis, on the survival rate and acute radiation injury of the small intestine and bone marrow using a rat model. CT pre-treatment (280 mg/kg for 5 days) significantly improved weight loss and survival rate of rats as compared with the control group after 5 Gy. CT pre-treatment significantly increased the rate of mucosa and crypt length, and decreased the number of apoptotic cells, TUNEL and cleaved caspase-3 positive cells, while increasing the number of mitotic cells and Ki-67 positive cells in jejunal crypts and villi compared to control rats post-irradiation. CT also suppressed bone marrow cell loss and reduced the number of apoptotic cells in bone marrow. These results suggest a protective effect of CT pre-treatment for acute injury after irradiation through apoptosis inhibition and increased proliferative activity in jejunal crypt cells and bone marrow cells.
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Affiliation(s)
- Mutsumi Matsuu-Matsuyama
- Tissue and Histopathology Section, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
| | - Kazuko Shichijo
- Department of Tumor and Diagnostic Pathology, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
| | - Takashi Tsuchiya
- Sendai City Medical Center, 5-22-1 Tsurugaya, Miyagino, Miyagi 983-0824, Japan.
| | - Hisayoshi Kondo
- Biostatistics Section, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
| | - Shiro Miura
- Department of Pathology, National Hospital Organization Nagasaki Medical Center, 2-1001-1 Kubara, Omura, Nagasaki 856-8562, Japan.
| | - Katsuya Matsuda
- Department of Tumor and Diagnostic Pathology, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
| | - Ichiro Sekine
- Department of Tumor and Diagnostic Pathology, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
| | - Masahiro Nakashima
- Tissue and Histopathology Section, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; Department of Tumor and Diagnostic Pathology, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
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9
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Akpolat M, Oz ZS, Gulle K, Hamamcioglu AC, Bakkal BH, Kececi M. X irradiation induced colonic mucosal injury and the detection of apoptosis through PARP-1/p53 regulatory pathway. Biomed Pharmacother 2020; 127:110134. [PMID: 32361637 DOI: 10.1016/j.biopha.2020.110134] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2019] [Revised: 03/25/2020] [Accepted: 03/27/2020] [Indexed: 12/13/2022] Open
Abstract
This study aimed to explore whether PARP-1 regulatory pathway mediated X irradiation induced cell cycle arrest and apoptosis or not. In this regard, colonic mucosal injury caused by whole-body X-irradiation induced apoptosis through PARP-1, caspase 3 and p53 regulatory pathway were evaluated in experimental rat models. Eighteen Wistar albino rats were divided into three groups. Two radiation groups received 8.3 Gy dose of whole-body X-irradiation as a single dose and the control group received physiological saline intraperitoneally. Radiation groups were sacrificed after 6 h and 4 days of irradiation. PARP-1 and caspase 3 expression in the nuclei of colonic crypt cells significantly increased 6 h after irradiation, and declined 4 days after irradiation. In conflict with other studies that reported p53 as not being expressed widely in colonic mucosa, in our study the expressions of p53 were elevated both in the cytoplasm and in the nucleus of the crypt cells, especially 6 h after irradiation. In the radiation groups, colonic mucosal injury score was significantly elevated compared with that of the control group. Our data demonstrated that PARP-1, caspase-3 and p53 expression increased in colonic mucosa 6 h after irradiation.
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Affiliation(s)
- Meryem Akpolat
- Zonguldak Bulent Ecevit University, Faculty of Medicine, Department of Histology and Embryology, Zonguldak, Turkey.
| | - Zehra Safi Oz
- Zonguldak Bulent Ecevit University Faculty of Medicine, Department of Medical Biology, Zonguldak, Turkey.
| | - Kanat Gulle
- Suleyman Demirel University, Faculty of Medicine, Department of Histology and Embryology, Isparta, Turkey.
| | - Ayse C Hamamcioglu
- Zonguldak Bulent Ecevit University, Faculty of Pharmacy, Department of Biochemistry, Zonguldak, Turkey.
| | - Bekir H Bakkal
- Zonguldak Bulent Ecevit University, Faculty of Medicine, Department of Radiation Oncology, Zonguldak, Turkey.
| | - Mete Kececi
- Zonguldak Bulent Ecevit University, Faculty of Medicine, Department of Histology and Embryology, Zonguldak, Turkey
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10
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Lizárraga-Verdugo E, Ruiz-García E, López-Camarillo C, Bermúdez M, Avendaño-Félix M, Ramos-Payán R, Romero-Quintana G, Ayala-Ham A, Villegas-Mercado C, Pérez-Plasencia C, Aguilar-Medina M. Cell Survival Is Regulated via SOX9/BCL2L1 Axis in HCT-116 Colorectal Cancer Cell Line. JOURNAL OF ONCOLOGY 2020; 2020:5701527. [PMID: 32411238 PMCID: PMC7206885 DOI: 10.1155/2020/5701527] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/18/2020] [Revised: 03/20/2020] [Accepted: 04/10/2020] [Indexed: 12/13/2022]
Abstract
Colorectal cancer (CRC) is one of the most frequent types of malignancies and one of the major causes of cancer-related death worldwide. Sex-determining region Y (SRY)-box 9 protein (SOX9) is a member of the SOX family of transcription factors which are involved in the regulation of differentiation and development. Recently, several reports suggest an important role of SOX9 in tumorigenesis since its overexpression correlates with tumor progression and poor outcome in several types of cancer; however, its role in CRC is not clear until now. Therefore, in this work, we searched for novel SOX9-regulated genes involved in cell survival of CRC. We silenced SOX9 in the poorly differentiated HCT-116 cell line, using a specific siRNA, to identify differential expressed genes by DNA microarrays and analyzed the role or candidate genes in apoptosis and autophagy. Transcriptome analysis showed that diverse cellular pathways, associated with CRC carcinogenesis such as Wnt/β-catenin, MAPK, TGF-β, and mTOR, were modulated after SOX9 silencing. Interestingly, we found that SOX9 silencing promotes downregulation of BCL2L1 and overexpression of CASP3, proteins related to apoptosis, which was further confirmed in SW-480, a moderated-differentiated cell line, but not in HT-29, well-differentiated cell line. Moreover, inhibition of BCL2L1 by ABT-737 (BH3 mimetic) in SOX9-silenced HCT-116 cells resulted in an increased apoptosis percentage. However, downregulation of BCL2L1 was not enough to induce autophagy. This is the first report, suggesting that cell survival in poorly and moderated-differentiated CRC cells lines is regulated by SOX9/BCL2L1 axis, but not in well-differentiated cell lines.
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Affiliation(s)
- Erik Lizárraga-Verdugo
- Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, Mexico
| | | | - César López-Camarillo
- Posgrado en Ciencias Genómicas, Universidad Autónoma de La Ciudad de México, CDMX, Mexico
| | - Mercedes Bermúdez
- Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, Mexico
| | - Mariana Avendaño-Félix
- Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, Mexico
| | - Rosalío Ramos-Payán
- Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, Mexico
| | - Geovanni Romero-Quintana
- Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, Mexico
| | - Alfredo Ayala-Ham
- Facultad de Odontología, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, Mexico
| | | | | | - Maribel Aguilar-Medina
- Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, Mexico
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11
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Qiu X, Dong K, Guan J, He J. Hydrogen attenuates radiation-induced intestinal damage by reducing oxidative stress and inflammatory response. Int Immunopharmacol 2020; 84:106517. [PMID: 32361189 DOI: 10.1016/j.intimp.2020.106517] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2020] [Revised: 04/13/2020] [Accepted: 04/13/2020] [Indexed: 01/23/2023]
Abstract
The small intestine is known to be particularly sensitive to radiation, and the major limiting factor of radiotherapy is the gastrointestinal syndrome that subsequently develops after its administration. The detrimental effects of radiation are mostly mediated via the overproduction of reactive oxygen species (ROS), especially the hydroxyl radical (·OH). Because hydrogen is a selective ·OH scavenger, we hypothesized that hydrogen might exert a protective effect against radiation-induced intestinal damage. Herein, radiation models were built both in mice and in an intestinal crypt epithelial cell (IEC-6) line. In the animal experiment, we demonstrated that hydrogen-rich saline significantly reduced radiation-induced intestinal mucosal damage, improved intestinal function, and increased the survival rate. In addition, radiation-induced oxidative stress damage and systemic inflammatory response were also mitigated by hydrogen treatment. Moreover, hydrogen treatment decreased cell apoptosis and maintained intestinal epithelial cell proliferation in mice. In vitro experiments using the IEC-6 cell line showed that hydrogen-rich medium significantly inhibited ROS formation, maintained cell viability, and inhibited cell apoptosis. Importantly, hydrogen treatment prevented mitochondrial depolarization, cytochrome c release, and activity of caspase-3, caspase-9, and PARP. Moreover, the decreased expression of Bcl-xl and Bcl-2 and the increased expression of Bax protein were also blocked by hydrogen treatment. In conclusion, our study concurrently demonstrated that hydrogen provides an obviously protective effect on radiation-induced intestinal and cell injuries. Our work demonstrated that this protective effect might be due to the blockage of the mitochondrial apoptotic pathway.
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Affiliation(s)
- Xiaochen Qiu
- Department of General Surgery, The Eighth Medical Center, Chinese PLA(People's Liberation Army) General Hospital, Beijing 100091, China
| | - Kaisheng Dong
- Department of Oncology, The Eighth Medical Center, Chinese PLA (People's Liberation Army) General Hospital, Beijing 100091, China; Postgraduate Department of Hebei North University, Zhangjiakou 075000, China
| | - Jingzhi Guan
- Department of Oncology, The Eighth Medical Center, Chinese PLA (People's Liberation Army) General Hospital, Beijing 100091, China
| | - JianMiao He
- Department of General Surgery, The Eighth Medical Center, Chinese PLA(People's Liberation Army) General Hospital, Beijing 100091, China.
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12
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Chen Q, Wang C, Zhao FQ, Liu J, Liu H. Effects of methionine partially replaced by methionyl-methionine dipeptide on intestinal function in methionine-deficient pregnant mice. J Anim Physiol Anim Nutr (Berl) 2019; 103:1610-1618. [PMID: 31106911 DOI: 10.1111/jpn.13126] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2018] [Revised: 04/24/2019] [Accepted: 05/03/2019] [Indexed: 12/17/2022]
Abstract
This study was to compare the effects of parenteral supplementation of methionyl-methionine (Met-Met) or Met on intestinal barrier function in Met-deficient pregnant mice. Pregnant mice were randomly divided into three groups. The Control group was provided a diet containing Met and received i.p. injection of saline. The Met group was fed the same diet but without Met and received daily i.p. injection of 35% of the Met contained in the control diet. The Met-Met group was treated the same as the Met group, except that 25% of the Met injected was replaced with Met-Met. Met-Met promoted villus surface area in ileum compared with Met alone. In addition, the mRNA abundance of amino acid and glucose transporters in the small intestine was altered with Met-Met. Moreover, Met-Met increased tight junction protein and decreased apoptosis-related proteins expression in the jejunum and ileum. These results suggest that Met-Met can promote intestinal function over Met alone in Met-deficient mice.
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Affiliation(s)
- Qiong Chen
- Institute of Dairy Science, College of Animal Sciences, Zhejiang University, Hangzhou, China
| | - Caihong Wang
- Institute of Dairy Science, College of Animal Sciences, Zhejiang University, Hangzhou, China
| | - Feng-Qi Zhao
- Institute of Dairy Science, College of Animal Sciences, Zhejiang University, Hangzhou, China.,Department of Animal and Veterinary Sciences, University of Vermont, Burlington, Vermont
| | - Jianxin Liu
- Institute of Dairy Science, College of Animal Sciences, Zhejiang University, Hangzhou, China
| | - Hongyun Liu
- Institute of Dairy Science, College of Animal Sciences, Zhejiang University, Hangzhou, China
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13
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Davis HW, Vallabhapurapu SD, Chu Z, Vallabhapurapu SL, Franco RS, Mierzwa M, Kassing W, Barrett WL, Qi X. Enhanced phosphatidylserine-selective cancer therapy with irradiation and SapC-DOPS nanovesicles. Oncotarget 2019; 10:856-868. [PMID: 30783515 PMCID: PMC6368238 DOI: 10.18632/oncotarget.26615] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2018] [Accepted: 12/29/2018] [Indexed: 02/06/2023] Open
Abstract
Normal living cells exhibit phosphatidylserine (PS) primarily within the intracellular leaflet of the plasma membrane. In contrast, viable cancer cells have high levels of PS on the external surface, and exhibit a broad range of surface PS, even within specific types of cancer. Agents that target surface PS have recently been developed to treat tumors and are expected to be more effective with higher surface PS levels. In this context, we examined whether surface PS is increased with irradiation. In vitro irradiation of cancer cell lines selected surviving cells that had higher surface PS in a dose- and time-dependent manner. This was more pronounced if surface PS was initially in the lower range for cancer cells. Radiation also increased the surface PS of tumor cells in subcutaneous xenografts in nude mice. We found an inverse relationship between steady state surface PS level of cancer cell lines and their sensitivity to radiation-induced cell death. In addition, serial irradiation, which selected surviving cells with higher surface PS, also increased resistance to radiation and to some chemotherapeutic drugs, suggesting a PS-dependent mechanism for development of resistance to therapy. On the other hand, fractionated radiation enhanced the effect of a novel anti-cancer, PS-targeting drug, SapC-DOPS, in some cancer cell lines. Our data suggest that we can group cancer cells into cells with low surface PS, which are sensitive to radiation, and high surface PS, which are sensitive to SapC-DOPS. Combination of these interventions may provide a potential new combination therapy.
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Affiliation(s)
- Harold W Davis
- Division of Hematology/Oncology, Translational Research Laboratory, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, USA
| | - Subrahmanya D Vallabhapurapu
- Division of Hematology/Oncology, Translational Research Laboratory, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, USA
| | - Zhengtao Chu
- Division of Hematology/Oncology, Translational Research Laboratory, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, USA
| | - Swarajya L Vallabhapurapu
- Division of Hematology/Oncology, Translational Research Laboratory, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, USA
| | - Robert S Franco
- Division of Hematology/Oncology, Translational Research Laboratory, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, USA
| | - Michelle Mierzwa
- Department of Radiation Oncology, University of Cincinnati College of Medicine, Cincinnati, OH, USA
| | - William Kassing
- Department of Radiation Oncology, University of Cincinnati College of Medicine, Cincinnati, OH, USA
| | - William L Barrett
- Department of Radiation Oncology, University of Cincinnati College of Medicine, Cincinnati, OH, USA
| | - Xiaoyang Qi
- Division of Hematology/Oncology, Translational Research Laboratory, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, USA.,Division of Human Genetics, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
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Yu J, Wang W, Wang J, Wang C, Li C. Short-term toxicity of dibutyl phthalate to mice intestinal tissue. Toxicol Ind Health 2018; 35:20-31. [PMID: 30453839 DOI: 10.1177/0748233718807303] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The objective of this study was to investigate changes in intestinal histopathology and expression of heat-shock proteins (HSPs) in the small intestinal tissue of mouse after acute exposure to dibutyl phthalate (DBP). Forty-eight 60-day-old Institute of Cancer Research (ICR) mice were administered DBP by gavage once a day for 10 days. The mice were divided into three groups of 16 mice each: the high-dose group was administered 500 mg/kg body weight (BW) DBP; the low-dose group was administered 50 mg/kg BW; and the control group was not administered DBP. Significant increases in the uterine index, ovary index, and testicular index were observed in the DBP-exposed groups compared to those in the control group. Villus height and V/ C ratio significantly increased ( p < 0.05) in the duodenum and decreased ( p < 0.05) in the jejunum after the administration of DBP. The goblet cell number decreased in both the duodenum and the jejunum of mice exposed to DBP ( p < 0.05) compared to the number in the control group mice. Damage to the structure of the small intestine was accompanied by a marked increase in HSP27 expression and a decrease in the expression of HSP70 and HSP90 in both high-dose and low-dose groups. These results indicate that elevated HSP27 levels in the duodenum and jejunum may be important markers for acute DBP exposure and that HSP27 may act as a protective protein involved in intestinal mucosa repair.
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Affiliation(s)
- Jimian Yu
- 1 Ningbo College of Health Sciences, Ningbo, China
| | - Wei Wang
- 2 College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo, China
| | - Jianfeng Wang
- 3 Ningbo Academy of Inspection and Quarantine, Ningbo, China
| | - Chun Wang
- 3 Ningbo Academy of Inspection and Quarantine, Ningbo, China
| | - Caiyan Li
- 2 College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo, China
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15
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Pandurangan AK, Divya T, Kumar K, Dineshbabu V, Velavan B, Sudhandiran G. Colorectal carcinogenesis: Insights into the cell death and signal transduction pathways: A review. World J Gastrointest Oncol 2018; 10:244-259. [PMID: 30254720 PMCID: PMC6147765 DOI: 10.4251/wjgo.v10.i9.244] [Citation(s) in RCA: 56] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/14/2018] [Revised: 06/05/2018] [Accepted: 06/28/2018] [Indexed: 02/05/2023] Open
Abstract
Colorectal carcinogenesis (CRC) imposes a major health burden in developing countries. It is the third major cause of cancer deaths. Despite several treatment strategies, novel drugs are warranted to reduce the severity of this disease. Adenomatous polyps in the colon are the major culprits in CRC and found in 45% of cancers, especially in patients 60 years of age. Inflammatory polyps are currently gaining attention in CRC, and a growing body of evidence denotes the role of inflammation in CRC. Several experimental models are being employed to investigate CRC in animals, which include the APCmin/+ mouse model, Azoxymethane, Dimethyl hydrazine, and a combination of Dextran sodium sulphate and dimethyl hydrazine. During CRC progression, several signal transduction pathways are activated. Among the major signal transduction pathways are p53, Transforming growth factor beta, Wnt/β-catenin, Delta Notch, Hippo signalling, nuclear factor erythroid 2-related factor 2 and Kelch-like ECH-associated protein 1 pathways. These signalling pathways collaborate with cell death mechanisms, which include apoptosis, necroptosis and autophagy, to determine cell fate. Extensive research has been carried out in our laboratory to investigate these signal transduction and cell death mechanistic pathways in CRC. This review summarizes CRC pathogenesis and the related cell death and signal transduction pathways.
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Affiliation(s)
- Ashok kumar Pandurangan
- Cell Biology Laboratory, Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, India
- School of Life sciences, B.S. Abdur Rahman Crescent Institute of Science and Technology, Chennai 600048, India
| | - Thomas Divya
- Cell Biology Laboratory, Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, India
| | - Kalaivani Kumar
- School of Life sciences, B.S. Abdur Rahman Crescent Institute of Science and Technology, Chennai 600048, India
| | - Vadivel Dineshbabu
- Cell Biology Laboratory, Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, India
| | - Bakthavatchalam Velavan
- Cell Biology Laboratory, Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, India
| | - Ganapasam Sudhandiran
- Cell Biology Laboratory, Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, India
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Abstract
The intestinal tract is a site of intense immune cell activity that is poised to mount an effective response against a pathogen and yet maintain tolerance toward commensal bacteria and innocuous dietary antigens. The role of cell death in gut pathologies is particularly important as the intestinal epithelium undergoes self-renewal every 4-7 days through a continuous process of cell death and cell division. Cell death is also required for removal of infected, damaged, and cancerous cells. Certain forms of cell death trigger inflammation through release of damage-associated molecular patterns. Further, molecules involved in cell death decisions also moonlight as critical nodes in immune signaling. The manner of cell death is, therefore, highly instructive of the immunological consequences that ensue. Perturbations in cell death pathways can impact the regulation of the immune system with deleterious consequences. In this review, we discuss the various forms of cell death with a special emphasis on lytic cell death pathways of pyroptosis and necroptosis and their implications in inflammation and cancer in the gut. Understanding the implications of distinct cell death pathways will help in the development of therapeutic interventions in intestinal pathologies.
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Affiliation(s)
- Deepika Sharma
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA
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17
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Tang S, Yang R, Zhou X, Pan H, Liu J. Expression of GOLPH3 in patients with non-small cell lung cancer and xenografts models. Oncol Lett 2018; 15:7555-7562. [PMID: 29849795 PMCID: PMC5962867 DOI: 10.3892/ol.2018.8340] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2017] [Accepted: 10/26/2017] [Indexed: 01/06/2023] Open
Abstract
Increased expression of Golgi phosphoprotein 3 (GOLPH3) has been reported to be associated with several types of human cancer. Patient-derived cancer xenograft models have demonstrated great potential in preclinical studies. In the present study, the link between GOLPH3 expression and survival was examined in patients with non-small cell lung cancer (NSCLC). Patient-derived lung cancer xenograft models were established with two different methods. Lastly, the association between GOLPH3 expression and establishment of the xenograft models was explored. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry analysis were used to examine GOLPH3 expression in 60 NSCLC tissues and matched adjacent non-cancerous tissues (ANT). In addition, tumor pieces from the 60 NSCLC tissues were implanted in the subcutaneous layer and in the subrenal kidney capsule of nude mice. RT-qPCR, histopathology and immunohistochemistry were used to confirm the human origin of the xenograft tumors. RT-qPCR was also used to research the mutation status of GOLPH3 in the xenograft tumors. The results demonstrated that NSCLC tissues had higher expression of GOLPH3, at the mRNA and protein level, compared with ANT. High expression of GOLPH3 correlated with poor survival in patients with NSCLC. Successful engraftment was established for 27 tissues in the subrenal kidney capsule and for 16 in the subcutaneous layer of nude mice. The subrenal kidney capsule group demonstrated significantly higher engraftment rates than the subcutaneous layer group. In addition, higher GOLPH3 expression in the tumor tissues was significantly correlated with higher engraftment rates in mice. In both groups, few xenografts lost the GOLPH3 mutation. In summary, GOLPH3 may be an important diagnosis and prognosis indicator in patients with NSCLC. The genotype and phenotype of the xenograft tumors derived from patient lung cancer tissues exhibited significant similarities to the originating primary tumors. High GOLPH3 expression may promote the successful establishment of xenograft models for NSCLC.
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Affiliation(s)
- Shicong Tang
- Department of Breast Surgery, The Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
| | - Rirong Yang
- Department of Immunology, School of Preclinical Medicine, Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
| | - Xin Zhou
- Department of Thoracic Surgery, The Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
| | - Hong Pan
- Department of Thoracic Surgery, The Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
| | - Jianlun Liu
- Department of Breast Surgery, The Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
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Abstract
Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease.IMPORTANCE Acute viral infections are thought to be cleared by the host with few lasting consequences. However, there may be much broader and long-lasting effects of viruses on immune homeostasis. Infection with reovirus, a common, nonpathogenic virus, triggers inflammation against innocuous food antigens, implicating this virus in the development of celiac disease, an autoimmune intestinal disorder triggered by exposure to dietary gluten. Using two reovirus strains that differ in the capacity to abrogate oral tolerance, we found that strain-specific differences in the capacity to replicate in the intestine inversely correlate with the capacity to induce apoptotic death of intestinal epithelial cells, providing a host-mediated process to restrict intestinal infection. This work contributes new knowledge about virus-host interactions in the intestine and establishes a foundation for future studies to define mechanisms by which viruses break oral tolerance in celiac disease.
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19
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Holen I, Lefley DV, Francis SE, Rennicks S, Bradbury S, Coleman RE, Ottewell P. IL-1 drives breast cancer growth and bone metastasis in vivo. Oncotarget 2018; 7:75571-75584. [PMID: 27765923 PMCID: PMC5342762 DOI: 10.18632/oncotarget.12289] [Citation(s) in RCA: 160] [Impact Index Per Article: 22.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2016] [Accepted: 09/15/2016] [Indexed: 01/19/2023] Open
Abstract
Background We have recently identified interleukin 1B (IL-1B) as a potential biomarker for predicting breast cancer patients at increased risk for developing bone metastasis. In mouse models, IL-1B and its receptor (IL-1R1) are upregulated in breast cancer cells that metastasise to bone compared with cells that do not. We have now investigated the functional role of IL-1 by blocking IL-1R signalling with the clinically licensed antagonist, anakinra. Methodology 6-week old female BALB/c mice received a subcutaneous or intra-venous injection of MDA-MB-231-IV or MCF7 cells. Anakinra (1mg/kg/day) or placebo was administered 3 days before (preventative) or 7 days later (treatment). Tumour volume, apoptosis (TUNEL, Caspase 3), proliferation (Ki67) and angiogenesis (CD34, VEGF and endothelin) were analysed. Effects on bone were measured by uCT, and TRAP, P1NP, IL-1B, TNF alpha and IL-6 ELISA. Results Anakinra significantly reduced growth of MDA-MB-231-IV tumours in bone from 6.50+/3.00mm2 (placebo) to 2.56+/−1.07mm2 (treatment) and 0.63+/−0.18mm2 (preventative). Anakinra also reduced the number of mice that developed bone metastasis from 90% (placebo) to 40% (treatment) and 10% (preventative). Anti-tumour effects were not confined to bone, subcutaneous tumour volumes reduced from 656.68mm3 (placebo) to 160.47mm3 (treatment) and 31.08mm3 (preventative). Anakinra did not increase tumour cell apoptosis but reduced proliferation and angiogenesis in addition to exerting significant effects on the tumour environment reducing bone turnover markers, IL-1B and TNF alpha. Conclusions Our novel data demonstrate a functional role of IL-1 signalling in breast tumour progression and metastasis, supporting that anakinra could be repurposed for the treatment of breast cancer bone metastasis.
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Affiliation(s)
- Ingunn Holen
- Academic Unit of Clinical Oncology, Department of Oncology and Metabolism, Mellanby Centre for Bone Research, University of Sheffield, Sheffield S10 2RX, UK
| | - Diane V Lefley
- Academic Unit of Clinical Oncology, Department of Oncology and Metabolism, Mellanby Centre for Bone Research, University of Sheffield, Sheffield S10 2RX, UK
| | - Sheila E Francis
- Department of Infection Immunity and Cardiovascular Disease, University of Sheffield, Sheffield S10 2RX, UK
| | - Sarah Rennicks
- Academic Unit of Clinical Oncology, Department of Oncology and Metabolism, Mellanby Centre for Bone Research, University of Sheffield, Sheffield S10 2RX, UK
| | - Steven Bradbury
- Academic Unit of Clinical Oncology, Department of Oncology and Metabolism, Mellanby Centre for Bone Research, University of Sheffield, Sheffield S10 2RX, UK
| | - Robert E Coleman
- Academic Unit of Clinical Oncology, Department of Oncology and Metabolism, Mellanby Centre for Bone Research, University of Sheffield, Sheffield S10 2RX, UK
| | - Penelope Ottewell
- Academic Unit of Clinical Oncology, Department of Oncology and Metabolism, Mellanby Centre for Bone Research, University of Sheffield, Sheffield S10 2RX, UK
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Linking DNA Damage and Age-Related Promoter DNA Hyper-Methylation in the Intestine. Genes (Basel) 2018; 9:genes9010017. [PMID: 29303998 PMCID: PMC5793170 DOI: 10.3390/genes9010017] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2017] [Revised: 12/21/2017] [Accepted: 12/21/2017] [Indexed: 12/22/2022] Open
Abstract
Aberrant DNA methylation in stem cells is a hallmark of aging and tumor development. Here, we explore whether and how DNA damage repair might impact on these time-dependent changes, in particular in proliferative intestinal stem cells. We introduce a 3D multiscale computer model of intestinal crypts enabling simulation of aberrant DNA and histone methylation of gene promoters during aging. We assume histone state-dependent activity of de novo DNA methyltransferases (DNMTs) and methylation-dependent binding of maintenance DNMTs to CpGs. We simulate aging with and without repeated DNA repair. Motivated by recent findings on the histone demethylase KDM2b, we consider that DNA repair is associated with chromatin opening and improved recruitment of de novo DNMTs. Our results suggest that methylation-dependent binding of maintenance DNMTs to CpGs, establishing bistable DNA methylation states, is a prerequisite to promoter hyper-methylation following DNA repair. With this, the transient increase in de novo DNMT activity during repair can induce switches from low to high methylation states. These states remain stable after repair, leading to an epigenetic drift. The switches are most frequent in genes with H3K27me3 modified promoters. Our model provides a mechanistic explanation on how even successful DNA repair might confer long term changes of the epigenome.
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Stansborough RL, Bateman EH, Al-Dasooqi N, Bowen JM, Keefe DMK, Yeoh ASJ, Logan RM, Yeoh EEK, Stringer AM, Gibson RJ. Fractionated abdominal irradiation induces intestinal microvascular changes in an in vivo model of radiotherapy-induced gut toxicity. Support Care Cancer 2017; 25:1973-1983. [PMID: 28175996 DOI: 10.1007/s00520-017-3601-3] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2016] [Accepted: 01/23/2017] [Indexed: 11/28/2022]
Abstract
PURPOSE Radiotherapy-induced gut toxicity (RIGT) is associated with diarrhoea, pain and rectal bleeding and can occur as an acute or chronic toxicity. The microvasculature has been shown to be altered in the development of RIGT; however, the features are not yet characterized. We hypothesized that apoptosis of microvascular cells would occur early in the gastrointestinal tract following fractionated irradiation, followed by late microvascular changes, including sclerosis and telangiectasis. METHODS Female Dark Agouti rats were treated with a 6-week fractionated radiation schedule of 3 × 2.5 Gy doses per week localized to the abdomen. At 3, 6 and 15 weeks, the intestines were assessed for markers of acute and chronic injury including morphological changes, collagen deposition, apoptosis and proliferation. RESULTS Apoptosis of microvascular cells significantly increased at 6 and 15 weeks in the jejunum (p = 0.0026 and p = 0.0062, respectively) and at 6 and 15 weeks in the colon (p < 0.0001 and p = 0.0005, respectively) in rats receiving fractionated radiation to the abdomen. Histopathological changes of the colon microvasculature were also seen from week 3, including thickening of the lamina propria and dilated, thickened, telangiectatic vessels. CONCLUSIONS Findings of this study provide evidence of regional and timing-specific changes in the intestinal microvasculature in response to fractionated radiotherapy which may play a role in development of both acute and chronic RIGT.
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Affiliation(s)
- Romany L Stansborough
- School of Medicine, University of Adelaide, North Terrace, Adelaide, 5005, Australia.
| | - Emma H Bateman
- School of Medicine, University of Adelaide, North Terrace, Adelaide, 5005, Australia
| | - Noor Al-Dasooqi
- School of Medicine, University of Adelaide, North Terrace, Adelaide, 5005, Australia
- Division of Health Sciences, University of South Australia, Adelaide, Australia
| | - Joanne M Bowen
- School of Medicine, University of Adelaide, North Terrace, Adelaide, 5005, Australia
| | - Dorothy M K Keefe
- School of Medicine, University of Adelaide, North Terrace, Adelaide, 5005, Australia
| | - Ann S J Yeoh
- School of Medicine, University of Adelaide, North Terrace, Adelaide, 5005, Australia
| | - Richard M Logan
- School of Medicine, University of Adelaide, North Terrace, Adelaide, 5005, Australia
| | - Eric E K Yeoh
- School of Medicine, University of Adelaide, North Terrace, Adelaide, 5005, Australia
| | - Andrea M Stringer
- School of Medicine, University of Adelaide, North Terrace, Adelaide, 5005, Australia
- Division of Health Sciences, University of South Australia, Adelaide, Australia
| | - Rachel J Gibson
- School of Medicine, University of Adelaide, North Terrace, Adelaide, 5005, Australia
- Division of Health Sciences, University of South Australia, Adelaide, Australia
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22
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Carr JS, King S, Dekaney CM. Depletion of enteric bacteria diminishes leukocyte infiltration following doxorubicin-induced small intestinal damage in mice. PLoS One 2017; 12:e0173429. [PMID: 28257503 PMCID: PMC5336284 DOI: 10.1371/journal.pone.0173429] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2016] [Accepted: 02/19/2017] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND & AIMS While enteric bacteria have been shown to play a critical role in other forms of intestinal damage, their role in mediating the response to the chemotherapeutic drug Doxorubicin (Doxo) is unclear. In this study, we used a mouse model of intestinal bacterial depletion to evaluate the role enteric bacteria play in mediating Doxo-induced small intestinal damage and, more specifically, in mediating chemokine expression and leukocyte infiltration following Doxo treatment. An understanding of this pathway may allow for development of intervention strategies to reduce chemotherapy-induced small intestinal damage. METHODS Mice were treated with (Abx) or without (NoAbx) oral antibiotics in drinking water for four weeks and then with Doxo. Jejunal tissues were collected at various time points following Doxo treatment and stained and analyzed for apoptosis, crypt damage and restitution, and macrophage and neutrophil number. In addition, RNA expression of inflammatory markers (TNFα, IL1-β, IL-10) and cytokines (CCL2, CC7, KC) was assessed by qRT-PCR. RESULTS In NoAbx mice Doxo-induced damage was associated with rapid induction of apoptosis in jejunal crypt epithelium and an increase weight loss and crypt loss. In addition, we observed an increase in immune-modulating chemokines CCL2, CCL7 and KC and infiltration of macrophages and neutrophils. In contrast, while still positive for induction of apoptosis following Doxo treatment, Abx mice showed neither the overall weight loss nor crypt loss seen in NoAbx mice nor the increased chemokine expression and leukocyte infiltration. CONCLUSION Enteric bacteria play a critical role in Doxo-induced small intestinal damage and are associated with an increase in immune-modulating chemokines and cells. Manipulation of enteric bacteria or the damage pathway may allow for prevention or treatment of chemotherapy-induced small intestinal damage.
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Affiliation(s)
- Jacquelyn S. Carr
- Department of Surgery, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Stephanie King
- Department of Molecular Biomedical Sciences, NC State University, Raleigh, North Carolina, United States of America
| | - Christopher M. Dekaney
- Department of Surgery, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Department of Molecular Biomedical Sciences, NC State University, Raleigh, North Carolina, United States of America
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23
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Wang Y, George SP, Roy S, Pham E, Esmaeilniakooshkghazi A, Khurana S. Both the anti- and pro-apoptotic functions of villin regulate cell turnover and intestinal homeostasis. Sci Rep 2016; 6:35491. [PMID: 27765954 PMCID: PMC5073230 DOI: 10.1038/srep35491] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2016] [Accepted: 09/15/2016] [Indexed: 12/11/2022] Open
Abstract
In the small intestine, epithelial cells are derived from stem cells in the crypts, migrate up the villus as they differentiate and are ultimately shed from the villus tips. This process of proliferation and shedding is tightly regulated to maintain the intestinal architecture and tissue homeostasis. Apoptosis regulates both the number of stem cells in the crypts as well as the sloughing of cells from the villus tips. Previously, we have shown that villin, an epithelial cell-specific actin-binding protein functions as an anti-apoptotic protein in the gastrointestinal epithelium. The expression of villin is highest in the apoptosis-resistant villus cells and lowest in the apoptosis-sensitive crypts. In this study we report that villin is cleaved in the intestinal mucosa to generate a pro-apoptotic fragment that is spatially restricted to the villus tips. This cleaved villin fragment severs actin in an unregulated fashion to initiate the extrusion and subsequent apoptosis of effete cells from the villus tips. Using villin knockout mice, we validate the physiological role of villin in apoptosis and cell extrusion from the gastrointestinal epithelium. Our study also highlights the potential role of villin’s pro-apoptotic function in the pathogenesis of inflammatory bowel disease, ischemia-reperfusion injury, enteroinvasive bacterial and parasitic infections.
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Affiliation(s)
- Yaohong Wang
- Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA
| | - Sudeep P George
- Department of Biology and Biochemistry, University of Houston, Houston TX 77204, USA
| | - Swati Roy
- Department of Biology and Biochemistry, University of Houston, Houston TX 77204, USA
| | - Eric Pham
- Department of Biology and Biochemistry, University of Houston, Houston TX 77204, USA
| | | | - Seema Khurana
- Department of Biology and Biochemistry, University of Houston, Houston TX 77204, USA.,Baylor College of Medicine, Houston TX 77030, USA
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24
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Rigby RJ, Carr J, Orgel K, King SL, Lund PK, Dekaney CM. Intestinal bacteria are necessary for doxorubicin-induced intestinal damage but not for doxorubicin-induced apoptosis. Gut Microbes 2016; 7:414-23. [PMID: 27459363 PMCID: PMC5046166 DOI: 10.1080/19490976.2016.1215806] [Citation(s) in RCA: 72] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Doxorubicin (DOXO) induces significant, but transient, increases in apoptosis in the stem cell zone of the jejunum, followed by mucosal damage involving a decrease in crypt proliferation, crypt number, and villus height. The gastrointestinal tract is home to a vast population of commensal bacteria and numerous studies have demonstrated a symbiotic relationship between intestinal bacteria and intestinal epithelial cells (IEC) in maintaining homeostatic functions of the intestine. However, whether enteric bacteria play a role in DOXO-induced damage is not well understood. We hypothesized that enteric bacteria are necessary for induction of apoptosis and damage associated with DOXO treatment. Conventionally raised (CONV) and germ free (GF) mice were given a single injection of DOXO, and intestinal tissue was collected at 6, 72, and 120 h after treatment and from no treatment (0 h) controls. Histology and morphometric analyses quantified apoptosis, mitosis, crypt depth, villus height, and crypt density. Immunostaining for muc2 and lysozyme evaluated Paneth cells, goblet cells or dual stained intermediate cells. DOXO administration induced significant increases in apoptosis in jejunal epithelium regardless of the presence of enteric bacteria; however, the resulting injury, as demonstrated by statistically significant changes in crypt depth, crypt number, and proliferative cell number, was dependent upon the presence of enteric bacteria. Furthermore, we observed expansion of Paneth and goblet cells and presence of intermediate cells only in CONV and not GF mice. These findings provide evidence that manipulation and/or depletion of the enteric microbiota may have clinical significance in limiting chemotherapy-induced mucositis.
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Affiliation(s)
- Rachael J. Rigby
- Faculty of Health and Medicine, Lancaster University, Lancaster, United Kingdom
| | - Jacquelyn Carr
- Department of Surgery, University of North Carolina, Chapel Hill, North Carolina, USA
| | - Kelly Orgel
- Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina, USA,Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, North Carolina, USA
| | - Stephanie L. King
- Department of Molecular Biomedical Sciences, NC State University, Raleigh, North Carolina, USA
| | - P. Kay Lund
- Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, North Carolina, USA
| | - Christopher M. Dekaney
- Department of Surgery, University of North Carolina, Chapel Hill, North Carolina, USA,Department of Molecular Biomedical Sciences, NC State University, Raleigh, North Carolina, USA
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25
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Fukuda K, Uehara Y, Nakata E, Inoue M, Shimazu K, Yoshida T, Kanda H, Nanjo H, Hosoi Y, Yamakoshi H, Iwabuchi Y, Shibata H. A diarylpentanoid curcumin analog exhibits improved radioprotective potential in the intestinal mucosa. Int J Radiat Biol 2016; 92:388-94. [PMID: 27043482 DOI: 10.3109/09553002.2016.1164910] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
PURPOSE To best enhance the effects of radiotherapy, it is important to minimize adverse events, including free radical-induced intestinal cell damage. Given the threat of nuclear power plant accidents or nuclear terrorism, there is an urgent need for radioprotectants to counteract the radiation-induced toxicity and/or injuries. Curcumin exhibits protective effects against gamma irradiation; however, its in vivo efficacy is decreased due to the low bioavailability. We examined the radioprotective effect of a newly synthesized curcumin analog, GO-Y031, on 11-Gy X-ray-induced intestinal mucosal damage in mice. MATERIALS AND METHODS The radioprotection experiments were conducted by using C57BL/6J or Jcl:ICR mice. Molecules related to radiation damage, including p53, Bax, Bcl-2, cleaved caspase-3, and reactive carbonyl species (RCS), were investigated immunohistochemically. RESULTS GO-Y031 protected against crypt hypoplasia relative to a mock treatment at 0.5% (weight/weight); the number of crypts were 11.00 ± 2.00/circumference (mm) in treated versus 6.86 ± 0.99/mm in mock-treated C57BL/6 mice (p = 0.0079). GO-Y031 also reduced the levels of RCS, p53, and cleaved caspase-3 accumulation in the irradiated intestinal cells. CONCLUSIONS GO-Y031 suppresses the accumulation of RCS and apoptosis-related molecules in irradiated cells. This compound may be a good primary radioprotective compound.
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Affiliation(s)
- Koji Fukuda
- a Department of Clinical Oncology , Graduate School of Medicine, Akita University , Akita , Japan
| | - Yoshihiko Uehara
- b Laboratory for Radiation Biology , Tohoku University , Sendai , Japan
| | - Eiko Nakata
- c Department of Radiation Oncology , Graduate School of Medicine, Tohoku University , Sendai , Japan
| | - Masahiro Inoue
- a Department of Clinical Oncology , Graduate School of Medicine, Akita University , Akita , Japan
| | - Kazuhiro Shimazu
- a Department of Clinical Oncology , Graduate School of Medicine, Akita University , Akita , Japan
| | - Taichi Yoshida
- a Department of Clinical Oncology , Graduate School of Medicine, Akita University , Akita , Japan
| | - Hiroaki Kanda
- d Division of Pathology , Cancer Institute, Japanese Foundation for Cancer Research , Tokyo , Japan
| | - Hiroshi Nanjo
- e Department of Clinical Pathology , Graduate School of Medicine, Akita University , Akita , Japan
| | - Yoshio Hosoi
- b Laboratory for Radiation Biology , Tohoku University , Sendai , Japan
| | - Hiroyuki Yamakoshi
- f Department of Organic Chemistry , Graduate School of Pharmaceutical, Tohoku University , Sendai , Japan
| | - Yoshiharu Iwabuchi
- f Department of Organic Chemistry , Graduate School of Pharmaceutical, Tohoku University , Sendai , Japan
| | - Hiroyuki Shibata
- a Department of Clinical Oncology , Graduate School of Medicine, Akita University , Akita , Japan
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26
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Trumpi K, Steller EJA, de Leng WW, Raats DA, Nijman IJ, Morsink FHM, Borel Rinkes IHM, Kranenburg O. Mice lacking functional CD95-ligand display reduced proliferation of the intestinal epithelium without gross homeostatic alterations. Med Mol Morphol 2015; 49:110-8. [PMID: 26700225 DOI: 10.1007/s00795-015-0129-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2015] [Accepted: 12/08/2015] [Indexed: 01/06/2023]
Abstract
Homeostasis of the continuously self-renewing intestinal tract involves cell proliferation, migration, differentiation along the crypt-villus-axis and shedding of cells into the gut lumen. CD95-ligand (FAS-ligand, CD95L) is a cytokine that is known for its capacity to induce apoptosis by binding its cognate receptor, CD95 (Fas). More recently, it was discovered that CD95L can also induce other cellular responses, such as proliferation, differentiation and cell migration. CD95L is highly expressed in Paneth cells of the small intestine which are in close contact with intestinal stem cells. This suggests a potential role for CD95L in controlling stem cell function and, possibly, intestinal homeostasis. We analyzed the intestines of mice deficient for functional CD95L (gld) for potential alterations in the diversity of stem-cell-lineages and parameters of intestinal homeostasis. Stem cell diversity was assessed by analyzing methylation patterns of the non-transcribed mMYOD gene. Proliferation was analyzed by BrdU labeling and differentiation was assessed by immunohistochemistry. Of all parameters analyzed, only epithelial cell proliferation was significantly reduced in the small intestines of gld-mice, but not in their colons which lack CD95L expression. We conclude that CD95L has a proliferation-stimulating role during normal turnover of the small intestine, but has a marginal effect on overall intestinal homeostasis.
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Affiliation(s)
- Kari Trumpi
- Department of Surgery, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands
| | - Ernst J A Steller
- Department of Surgery, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands
| | - Wendy W de Leng
- Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584CX, Utrecht, The Netherlands
| | - Daniëlle A Raats
- Department of Surgery, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands
| | - Isaäc J Nijman
- Hubrecht Institute, The Royal Dutch Academy of Arts and Sciences, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Folkert H M Morsink
- Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584CX, Utrecht, The Netherlands
| | - Inne H M Borel Rinkes
- Department of Surgery, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands
| | - Onno Kranenburg
- Department of Surgery, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
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27
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Richmond CA, Shah MS, Deary LT, Trotier DC, Thomas H, Ambruzs DM, Jiang L, Whiles BB, Rickner HD, Montgomery RK, Tovaglieri A, Carlone DL, Breault DT. Dormant Intestinal Stem Cells Are Regulated by PTEN and Nutritional Status. Cell Rep 2015; 13:2403-2411. [PMID: 26686631 DOI: 10.1016/j.celrep.2015.11.035] [Citation(s) in RCA: 75] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2015] [Revised: 10/13/2015] [Accepted: 11/10/2015] [Indexed: 11/25/2022] Open
Abstract
The cellular and molecular mechanisms underlying adaptive changes to physiological stress within the intestinal epithelium remain poorly understood. Here, we show that PTEN, a negative regulator of the PI3K→AKT→mTORC1-signaling pathway, is an important regulator of dormant intestinal stem cells (d-ISCs). Acute nutrient deprivation leads to transient PTEN phosphorylation within d-ISCs and a corresponding increase in their number. This release of PTEN inhibition renders d-ISCs functionally poised to contribute to the regenerative response during re-feeding via cell-autonomous activation of the PI3K→AKT→mTORC1 pathway. Consistent with its role in mediating cell survival, PTEN is required for d-ISC maintenance at baseline, and intestines lacking PTEN have diminished regenerative capacity after irradiation. Our results highlight a PTEN-dependent mechanism for d-ISC maintenance and further demonstrate the role of d-ISCs in the intestinal response to stress.
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Affiliation(s)
- Camilla A Richmond
- Division of Gastroenterology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
| | - Manasvi S Shah
- Division of Endocrinology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
| | - Luke T Deary
- Division of Endocrinology, Boston Children's Hospital, Boston, MA 02115, USA
| | - Danny C Trotier
- Division of Endocrinology, Boston Children's Hospital, Boston, MA 02115, USA
| | - Horatio Thomas
- Division of Endocrinology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
| | - Dana M Ambruzs
- Division of Endocrinology, Boston Children's Hospital, Boston, MA 02115, USA
| | - Lijie Jiang
- Division of Endocrinology, Boston Children's Hospital, Boston, MA 02115, USA
| | - Bristol B Whiles
- Harvard Stem Cell Institute, 7 Divinity Avenue, Cambridge, MA 02138, USA
| | - Hannah D Rickner
- Division of Endocrinology, Boston Children's Hospital, Boston, MA 02115, USA
| | - Robert K Montgomery
- Division of Gastroenterology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
| | - Alessio Tovaglieri
- Division of Endocrinology, Boston Children's Hospital, Boston, MA 02115, USA
| | - Diana L Carlone
- Division of Endocrinology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, 7 Divinity Avenue, Cambridge, MA 02138, USA
| | - David T Breault
- Division of Endocrinology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, 7 Divinity Avenue, Cambridge, MA 02138, USA.
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28
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Parry L, Young M, El Marjou F, Clarke AR. Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models. J Vis Exp 2015. [PMID: 26649885 PMCID: PMC4755722 DOI: 10.3791/53429] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022] Open
Abstract
The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the ‘‘stemness’’ of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.
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Affiliation(s)
- Lee Parry
- European Cancer Stem Cell Research Institute, Cardiff University;
| | - Madeleine Young
- European Cancer Stem Cell Research Institute, Cardiff University
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29
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Harmse L, Dahan-Farkas N, Panayides JL, van Otterlo W, Penny C. Aberrant Apoptotic Response of Colorectal Cancer Cells to Novel Nucleoside Analogues. PLoS One 2015; 10:e0138607. [PMID: 26390405 PMCID: PMC4577089 DOI: 10.1371/journal.pone.0138607] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2015] [Accepted: 09/01/2015] [Indexed: 12/19/2022] Open
Abstract
Despite the increased understanding of colorectal cancer and the introduction of targeted drug therapy, the metastatic phase of the disease remains refractory to treatment. Since the deregulation of normal apoptosis contributes to the pathogenesis of colorectal cancer, novel nucleoside analogues were synthesized here and evaluated for their ability to induce apoptosis and cause cell death in two colorectal adeno-carcinoma cell lines, Caco-2 and HT-29. Three novel nucleoside analogues assessed here showed cytotoxic activity, as measured by the MTT assay against both cell lines: the IC50 values ranged between 3 and 37 μM, with Caco-2 cells being more sensitive than HT-29 cells. Compared to camptothecin, the positive control, the nucleoside analogues were significantly less toxic to normal unstimulated leukocytes (p>0.05). Moreover, the nucleosides were able to induce apoptosis as measured by an increase in caspase 8 and caspase 3 activity above that of the control. This was additionally supported by data derived from Annexin V-FITC assays. Despite marginal changes to the mitochondrial membrane potential, all three nucleosides caused a significant increase in cytosolic cytochrome c (p>0.05), with a corresponding decrease in mitochondrial cytochrome c. Morphological analysis of both cell lines showed the rapid appearance of vacuoles following exposure to two of the nucleosides, while a third caused cellular detachment, delayed cytoplasmic vacuolisation and nuclear abnormalities. Preliminary investigations, using the autophagic indicator monodansylcadaverine and chloroquine as positive control, showed that two of the nucleosides induced the formation of autophagic vacuoles. In summary, the novel nucleoside analogues showed selective cytotoxicity towards both cancer cell lines and are effective initiators of an unusual apoptotic response, demonstrating their potential to serve as structural scaffolds for more potent analogues.
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Affiliation(s)
- Leonie Harmse
- Division of Pharmacology, Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193, South Africa
- * E-mail:
| | - Nurit Dahan-Farkas
- Division of Pharmacology, Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193, South Africa
| | - Jenny-Lee Panayides
- Molecular Sciences Institute, School of Chemistry, Faculty of Science, University of the Witwatersrand, Private Bag 3, Johannesburg 2050, South Africa
| | - Willem van Otterlo
- Molecular Sciences Institute, School of Chemistry, Faculty of Science, University of the Witwatersrand, Private Bag 3, Johannesburg 2050, South Africa
- Department of Chemistry and Polymer Sciences, Stellenbosch University, Private Bag XI, Matieland 7602, South Africa
| | - Clement Penny
- Department of Internal Medicine, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, South Africa
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30
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Duckworth CA, Abuderman AA, Burkitt MD, Williams JM, O'Reilly LA, Pritchard DM. bak deletion stimulates gastric epithelial proliferation and enhances Helicobacter felis-induced gastric atrophy and dysplasia in mice. Am J Physiol Gastrointest Liver Physiol 2015; 309:G420-30. [PMID: 26159699 PMCID: PMC4572407 DOI: 10.1152/ajpgi.00404.2014] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/11/2014] [Accepted: 07/01/2015] [Indexed: 01/31/2023]
Abstract
Helicobacter infection causes a chronic superficial gastritis that in some cases progresses via atrophic gastritis to adenocarcinoma. Proapoptotic bak has been shown to regulate radiation-induced apoptosis in the stomach and colon and also susceptibility to colorectal carcinogenesis in vivo. Therefore we investigated the gastric mucosal pathology following H. felis infection in bak-null mice at 6 or 48 wk postinfection. Primary gastric gland culture from bak-null mice was also used to assess the effects of bak deletion on IFN-γ-, TNF-α-, or IL-1β-induced apoptosis. bak-null gastric corpus glands were longer, had increased epithelial Ki-67 expression, and contained fewer parietal and enteroendocrine cells compared with the wild type (wt). In wt mice, bak was expressed at the luminal surface of gastric corpus glands, and this increased 2 wk post-H. felis infection. Apoptotic cell numbers were decreased in bak-null corpus 6 and 48 wk following infection and in primary gland cultures following cytokine administration. Increased gastric epithelial Ki-67 labeling index was observed in C57BL/6 mice after H. felis infection, whereas no such increase was detected in bak-null mice. More severe gastric atrophy was observed in bak-null compared with C57BL/6 mice 6 and 48 wk postinfection, and 76% of bak-null compared with 25% of C57BL/6 mice showed evidence of gastric dysplasia following long-term infection. Collectively, bak therefore regulates gastric epithelial cell apoptosis, proliferation, differentiation, mucosal thickness, and susceptibility to gastric atrophy and dysplasia following H. felis infection.
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Affiliation(s)
- C. A. Duckworth
- 1Department of Gastroenterology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom;
| | - A. A. Abuderman
- 1Department of Gastroenterology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom;
| | - M. D. Burkitt
- 1Department of Gastroenterology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom;
| | - J. M. Williams
- 1Department of Gastroenterology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom;
| | - L. A. O'Reilly
- 2The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia; and ,3Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia
| | - D. M. Pritchard
- 1Department of Gastroenterology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom;
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31
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Burkitt MD, Hanedi AF, Duckworth CA, Williams JM, Tang JM, O'Reilly LA, Putoczki TL, Gerondakis S, Dimaline R, Caamano JH, Pritchard DM. NF-κB1, NF-κB2 and c-Rel differentially regulate susceptibility to colitis-associated adenoma development in C57BL/6 mice. J Pathol 2015; 236:326-36. [PMID: 25727407 PMCID: PMC4737252 DOI: 10.1002/path.4527] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2014] [Revised: 02/11/2015] [Accepted: 02/23/2015] [Indexed: 12/24/2022]
Abstract
NF-κB signalling is an important factor in the development of inflammation-associated cancers. Mouse models of Helicobacter-induced gastric cancer and colitis-associated colorectal cancer have demonstrated that classical NF-κB signalling is an important regulator of these processes. In the stomach, it has also been demonstrated that signalling involving specific NF-κB proteins, including NF-κB1/p50, NF-κB2/p52, and c-Rel, differentially regulate the development of gastric pre-neoplasia. To investigate the effect of NF-κB subunit loss on colitis-associated carcinogenesis, we administered azoxymethane followed by pulsed dextran sodium sulphate to C57BL/6, Nfkb1(-/-), Nfkb2(-/-), and c-Rel(-/-) mice. Animals lacking the c-Rel subunit were more susceptible to colitis-associated cancer than wild-type mice, developing 3.5 times more colonic polyps per animal than wild-type mice. Nfkb2(-/-) mice were resistant to colitis-associated cancer, developing fewer polyps per colon than wild-type mice (median 1 compared to 4). To investigate the mechanisms underlying these trends, azoxymethane and dextran sodium sulphate were administered separately to mice of each genotype. Nfkb2(-/-) mice developed fewer clinical signs of colitis and exhibited less severe colitis and an attenuated cytokine response compared with all other groups following DSS administration. Azoxymethane administration did not fully suppress colonic epithelial mitosis in c-Rel(-/-) mice and less colonic epithelial apoptosis was also observed in this genotype compared to wild-type counterparts. These observations demonstrate different functions of specific NF-κB subunits in this model of colitis-associated carcinogenesis. NF-κB2/p52 is necessary for the development of colitis, whilst c-Rel-mediated signalling regulates colonic epithelial cell turnover following DNA damage.
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Affiliation(s)
- Michael D Burkitt
- Department of Gastroenterology, Institute of Translational Medicine, University of Liverpool, UK
| | | | - Carrie A Duckworth
- Department of Gastroenterology, Institute of Translational Medicine, University of Liverpool, UK
| | - Jonathan M Williams
- Department of Gastroenterology, Institute of Translational Medicine, University of Liverpool, UK
| | - Joseph M Tang
- Department of Gastroenterology, Institute of Translational Medicine, University of Liverpool, UK
| | - Lorraine A O'Reilly
- The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia.,Department of Medical Biology, The University of Melbourne, Australia
| | - Tracy L Putoczki
- The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia.,Department of Medical Biology, The University of Melbourne, Australia
| | - Steve Gerondakis
- Australian Centre for Blood Diseases, Monash University Central Clinical School, Melbourne, Australia
| | - Rod Dimaline
- Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, UK
| | - Jorge H Caamano
- IBR-MRC Centre for Immune Regulation, College of Medicine and Dental Sciences, University of Birmingham, UK
| | - D Mark Pritchard
- Department of Gastroenterology, Institute of Translational Medicine, University of Liverpool, UK
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Yang C, Dai W, Chen H, Wu B. Application of human bone marrow-derived mesenchymal stem cells in the treatment of radiation-induced Gastrointestinal syndrome. SCIENCE CHINA-LIFE SCIENCES 2014; 57:1177-82. [PMID: 25205377 DOI: 10.1007/s11427-014-4721-3] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/26/2013] [Accepted: 10/22/2013] [Indexed: 01/14/2023]
Abstract
Nuclear accidents and terrorism present a serious threat for mass casualty. Accidental or intended radiation exposure leads to radiation-induced gastrointestinal (GI) syndrome. However, currently there are no approved medical countermeasures for GI syndrome. Thus, developing novel treatments for GI syndrome is urgent. Mesenchymal stem cells (MSCs) derived from bone marrow are a subset of multipotent adult somatic stem cells that have the ability to undergo self-renewal, proliferation and pluripotent differentiation. MSCs have advantages over other stem cells; they can be easily isolated from patients or donors, readily expanded ex vivo, and they possess reparative and immunomodulatory properties. Moreover, MSCs have been shown to be powerful tools in gene therapy and can be effectively transduced with vectors containing therapeutic genes. Therefore, the therapeutic potential of MSCs has been brought into the spotlight for the clinical treatment of GI syndrome. In this review, we discuss the possible role of MSCs in radiation-induced GI syndrome.
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Affiliation(s)
- Chao Yang
- Gastrointestinal Department of Southern Building, General Hospital of Chinese PLA, Beijing, 100853, China
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Phesse TJ, Myant KB, Cole AM, Ridgway RA, Pearson H, Muncan V, van den Brink GR, Vousden KH, Sears R, Vassilev LT, Clarke AR, Sansom OJ. Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo. Cell Death Differ 2014; 21:956-66. [PMID: 24583641 PMCID: PMC4013513 DOI: 10.1038/cdd.2014.15] [Citation(s) in RCA: 80] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2013] [Revised: 12/13/2013] [Accepted: 01/08/2014] [Indexed: 12/20/2022] Open
Abstract
Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.
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Affiliation(s)
- T J Phesse
- School of Biosciences, University of Cardiff.CF10 3US, Cardiff, UK
- Ludwig Institute for Cancer Research, Melbourne, Australia
- The Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia
- Department of Medical Biology, University of Melbourne, Melbourne, Australia
| | - K B Myant
- Beatson Institute for Cancer Research, Glasgow, UK
| | - A M Cole
- Beatson Institute for Cancer Research, Glasgow, UK
| | - R A Ridgway
- Beatson Institute for Cancer Research, Glasgow, UK
| | - H Pearson
- School of Biosciences, University of Cardiff.CF10 3US, Cardiff, UK
| | - V Muncan
- Department of Gastroenterology & Hepatology, Leiden University Medical Center, Leiden, The Netherlands
| | - G R van den Brink
- Department of Gastroenterology & Hepatology, Leiden University Medical Center, Leiden, The Netherlands
| | - K H Vousden
- Beatson Institute for Cancer Research, Glasgow, UK
| | - R Sears
- Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA
| | - L T Vassilev
- Discovery Oncology, Roche Research Center, Nutley, NJ, USA
| | - A R Clarke
- School of Biosciences, University of Cardiff.CF10 3US, Cardiff, UK
| | - O J Sansom
- Beatson Institute for Cancer Research, Glasgow, UK
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Murano T, Kagawa Y, Tsuneda S. Mathematical modelling of spatio-temporal cell dynamics in colonic crypts following irradiation. Cell Prolif 2014; 47:347-55. [PMID: 24828339 DOI: 10.1111/cpr.12110] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2014] [Accepted: 03/17/2014] [Indexed: 02/06/2023] Open
Abstract
OBJECTIVES Modelling the apoptotic process is essential for simulating and understanding tumour growth, as most tumour tissues carry mutations in apoptotic signalling pathways. Thus here, we have aimed to construct a mathematical model of colonic crypts that explicitly incorporates the apoptotic mechanism. METHODS A murine colonic crypt was described as being a two-dimensional rectangular surface model. In this system, three types of cells with different proliferating and differentiating potentials migrate. Apoptosis was described as a process activated by irradiation that progresses in a stepwise manner. Parameter values in the model were determined to be consistent with experimental data for changes in the apoptotic cell ratio within murine transverse colonic crypts following irradiation. RESULTS First, we constructed a model reproducing cell proliferation dynamics in normal murine colonic crypts; next, we applied the apoptotic mechanism to this model. As a result, we succeeded in simultaneous reproduction of both spatial and temporal changes in distribution of apoptotic cells in murine colonic crypts by determining parameter values in numerical simulations. Through this adjustment process, we were able to predict that stem cells and transit amplifying (TA) cells in each generation must react distinctly from each other, to apoptosis-inducing stimuli. CONCLUSIONS We constructed a mathematical model with which we could quantitatively describe cell proliferative and apoptotic dynamics in a murine colonic crypt. Using this model, we were able to make novel predictions that sensitivity to apoptosis-inducing stimuli is dependent on cell type.
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Affiliation(s)
- T Murano
- Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan
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Bing SJ, Kim MJ, Ahn G, Im J, Kim DS, Ha D, Cho J, Kim A, Jee Y. Acidic polysaccharide of Panax ginseng regulates the mitochondria/caspase-dependent apoptotic pathway in radiation-induced damage to the jejunum in mice. Acta Histochem 2014; 116:514-21. [PMID: 24380494 DOI: 10.1016/j.acthis.2013.11.012] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2013] [Revised: 11/01/2013] [Accepted: 11/06/2013] [Indexed: 12/17/2022]
Abstract
Owing to its susceptibility to radiation, the small intestine of mice is valuable for studying radioprotective effects. When exposed to radiation, intestinal crypt cells immediately go through apoptosis, which impairs swift differentiation necessary for the regeneration of intestinal villi. Our previous studies have elucidated that acidic polysaccharide of Panax ginseng (APG) protects the mouse small intestine from radiation-induced damage by lengthening villi with proliferation and repopulation of crypt cells. In the present study, we identified the molecular mechanism involved. C57BL/6 mice were irradiated with gamma-rays with or without APG and the expression levels of apoptosis-related molecules in the jejunum were investigated using immunohistochemistry. APG pretreatment strongly decreased the radiation-induced apoptosis in the jejunum. It increased the expression levels of anti-apoptotic proteins (Bcl-2 and Bcl-XS/L) and dramatically reduced the expression levels of pro-apoptotic proteins (p53, BAX, cytochrome c and caspase-3). Therefore, APG attenuated the apoptosis through the intrinsic pathway, which is controlled by p53 and Bcl-2 family members. Results presented in this study suggest that APG protects the mouse small intestine from irradiation-induced apoptosis through inhibition of the p53-dependent pathway and the mitochondria/caspase pathway. Thus, APG may be a potential agent for preventing radiation induced injuries in intestinal cells during radio-therapy such as in cancer treatment.
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Systemic overexpression of antizyme 1 in mouse reduces ornithine decarboxylase activity without major changes in tissue polyamine homeostasis. Transgenic Res 2013; 23:153-63. [DOI: 10.1007/s11248-013-9763-y] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2013] [Accepted: 10/21/2013] [Indexed: 11/27/2022]
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Parry L, Young M, El Marjou F, Clarke AR. Evidence for a crucial role of paneth cells in mediating the intestinal response to injury. Stem Cells 2013; 31:776-85. [PMID: 23335179 PMCID: PMC3744757 DOI: 10.1002/stem.1326] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2012] [Accepted: 12/17/2012] [Indexed: 01/29/2023]
Abstract
The identification of the intestinal stem cell (ISC) markers Lgr5 and Bmi-1 has furthered our understanding of how they accomplish homeostasis in this rapidly self-renewing tissue. Recent work indicates that these markers identify a cycling Lgr5(+) ISC which can be replaced by a quiescent Bmi-1(+) ISC. Currently, there is little data on how these cells interact to control intestinal crypt homeostasis and regeneration. This interaction likely involves other differentiated cells within the niche as it has previously been demonstrated that the "stemness" of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. To investigate this, we used two conditional mouse models to delete the transcription factor β-catenin within the intestinal crypt. Critically these differ in their ability to drive recombination within Paneth cells and therefore allow us to compare the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. After gene deletion, the intestines in the model in which Paneth cells were retained showed a rapid recovery and repopulation of the crypt-villus axis presumably from either a spared ISC or the hypothetical quiescent ISCs. However, in the absence of Paneth cells the recovery ability was compromised resulting in complete loss of intestinal epithelial integrity. This data indicates that the Paneth cells play a crucial role within the in vivo ISC niche in aiding recovery following substantial insult.
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Affiliation(s)
- Lee Parry
- School of Bioscience Biosciences, Cardiff University, Cardiff, United Kingdom
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38
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Dutta P, Bhansali A, Vaiphei K, Dutta U, Ravi Kumar P, Masoodi S, Mukherjee KK, Varma A, Kochhar R. Colonic neoplasia in acromegaly: increased proliferation or deceased apoptosis? Pituitary 2012; 15:166-73. [PMID: 21451936 DOI: 10.1007/s11102-011-0300-9] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Patients with acromegaly have higher prevalence of colorectal neoplasms. The pathogenetic mechanism is still unclear and may be related to sustained increase in serum GH-IGF1. We aimed to evaluate the proliferative and apoptotic markers in samples of colonic mucosa obtained during screening colonoscopic biopsy from patients with acromegaly and study their relationship to serum IGF-1 and GH levels. The study subjects included 32 patients with acromegaly (4 female), 10 healthy controls (irritable bowel syndrome) and 10 positive controls (non-acromegalic colonic adenocarcinoma). Patients with acromegaly were divided into two groups, active disease (AD) and disease in remission (AR). Two biopsies each were obtained during colonoscopy from the right colon, transverse colon and rectosigmoid region. All the polyps were biopsied and subjected to histopathological examination. Immunohistochemistry for proliferation marker (Ki-67) and apoptotic markers (caspase-3 and TdT-Mediated dUTP Nick-End Labeling (TUNEL) was carried out in the histopathological samples. Indices of proliferation were significantly different in patients with acromegaly as compared to healthy controls. The mean Ki-67 positivity was 45.1 ± 17.7% in AD and 45.6 ± 23.1% in AR, as compared to 10 ± 5% in healthy controls. While none of the healthy controls had Ki-67 positivity beyond the lower third of crypts, among patients with acromegaly 12/32 (37.5%) had mid-third positivity (P = 0.000) and 15/32 (46.8%) had full length of crypt positively (P = 0.00). Immunostaining for caspase-3 was negative in patients with acromegaly and healthy controls. TUNEL was strongly positive in patients with colonic adenocarcinoma but not in healthy controls and patients with acromegaly. IGF-1 levels were higher in those with Ki-67 positivity in the superficial mucosa. Patients with acromegaly have increased proliferation of colonic epithelial cells. Elevated levels of serum IGF1 are associated with increase proliferation in the superficial crypt cells.
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Affiliation(s)
- Pinaki Dutta
- Departments of Endocinology, Postgraduate Institute of Medical Education and Research, Chandigarh, 160012, India.
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De Matteis G, Graudenzi A, Antoniotti M. A review of spatial computational models for multi-cellular systems, with regard to intestinal crypts and colorectal cancer development. J Math Biol 2012. [PMID: 22565629 DOI: 10.1007/s00285‐012‐0539‐4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Colon rectal cancers (CRC) are the result of sequences of mutations which lead the intestinal tissue to develop in a carcinoma following a "progression" of observable phenotypes. The actual modeling and simulation of the key biological structures involved in this process is of interest to biologists and physicians and, at the same time, it poses significant challenges from the mathematics and computer science viewpoints. In this report we give an overview of some mathematical models for cell sorting (a basic phenomenon that underlies several dynamical processes in an organism), intestinal crypt dynamics and related problems and open questions. In particular, major attention is devoted to the survey of so-called in-lattice (or grid) models and off-lattice (off-grid) models. The current work is the groundwork for future research on semi-automated hypotheses formation and testing about the behavior of the various actors taking part in the adenoma-carcinoma progression, from regulatory processes to cell-cell signaling pathways.
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Affiliation(s)
- Giovanni De Matteis
- Department of Mathematics "F. Enriques", University of Milan, Via Saldini 50, 20133 Milan, Italy
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40
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A review of spatial computational models for multi-cellular systems, with regard to intestinal crypts and colorectal cancer development. J Math Biol 2012; 66:1409-62. [PMID: 22565629 DOI: 10.1007/s00285-012-0539-4] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2011] [Revised: 04/11/2012] [Indexed: 02/06/2023]
Abstract
Colon rectal cancers (CRC) are the result of sequences of mutations which lead the intestinal tissue to develop in a carcinoma following a "progression" of observable phenotypes. The actual modeling and simulation of the key biological structures involved in this process is of interest to biologists and physicians and, at the same time, it poses significant challenges from the mathematics and computer science viewpoints. In this report we give an overview of some mathematical models for cell sorting (a basic phenomenon that underlies several dynamical processes in an organism), intestinal crypt dynamics and related problems and open questions. In particular, major attention is devoted to the survey of so-called in-lattice (or grid) models and off-lattice (off-grid) models. The current work is the groundwork for future research on semi-automated hypotheses formation and testing about the behavior of the various actors taking part in the adenoma-carcinoma progression, from regulatory processes to cell-cell signaling pathways.
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Clarke JM, Young GP, Topping DL, Bird AR, Cobiac L, Scherer BL, Winkler JG, Lockett TJ. Butyrate delivered by butyrylated starch increases distal colonic epithelial apoptosis in carcinogen-treated rats. Carcinogenesis 2011; 33:197-202. [PMID: 22080572 PMCID: PMC3276328 DOI: 10.1093/carcin/bgr254] [Citation(s) in RCA: 76] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Animal studies show that increasing large bowel butyrate concentration through ingestion of butyrylated or resistant starches opposes carcinogen-induced tumorigenesis, which is consistent with population data linking greater fiber consumption with lowered colorectal cancer (CRC) risk. Butyrate has been shown to regulate the apoptotic response to DNA damage. This study examined the impact of increasing large bowel butyrate concentration by dietary butyrylated starch on the colonic epithelium of rats treated with the genotoxic carcinogen azoxymethane (AOM). Four groups of 10 male rats were fed AIN-93G based-diets containing either low amylose maize starch (LAMS), LAMS with 3% tributyrin, 10% high amylose maize starch (HAMS) or 10% butyrylated HAMS (HAMSB). HAMS and HAMSB starches were cooked by heating in water. After 4 weeks, rats were injected once with AOM and killed 6 h later. Rates of apoptosis and proliferation were measured in colonic epithelium. Short-chain fatty acid concentrations in large bowel digesta and hepatic portal venous plasma were higher in HAMSB than all other groups. Apoptotic rates in the distal colon were increased by HAMSB and correlated with luminal butyrate concentrations but cellular proliferation rates were unaffected by diet. The increase in apoptosis was most marked in the base and proliferative zone of the crypt. Regulation of luminal butyrate using HAMSB increases the rates of apoptotic deletion of DNA-damaged colonocytes. We propose this pro-apoptotic function of butyrate plays a major role reducing tumour formation in the AOM-treated rat and that these data support a potential protective role of butyrate in CRC.
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Affiliation(s)
- Julie M Clarke
- Preventative Health National Research Flagship, Commonwealth Scientific and Industrial Research Organisation (CSIRO) Food and Nutritional Sciences, PO Box 10041, Adelaide BC, Adelaide, South Australia 5000, Australia.
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Liu J, Chen D, Mao X, Yu B. Effects of maternal folic acid supplementation on morphology and apoptosis-related gene expression in jejunum of newborn intrauterine growth retarded piglets. Arch Anim Nutr 2011; 65:376-85. [DOI: 10.1080/1745039x.2011.594352] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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43
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Combination therapy inhibits development and progression of mammary tumours in immunocompetent mice. Breast Cancer Res Treat 2011; 133:523-36. [DOI: 10.1007/s10549-011-1782-x] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2011] [Accepted: 09/13/2011] [Indexed: 10/17/2022]
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44
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Dong L, Jiang K, Zhang Y, Zhang H, Zhuo H, Cui Z, Ye Y, Wang S. BAP31 is frequently overexpressed in patients with primary colorectal cancer and correlates with better prognosis. ACTA ACUST UNITED AC 2011. [DOI: 10.1007/s11434-011-4610-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
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45
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Zeilstra J, Joosten SP, Wensveen FM, Dessing MC, Schütze DM, Eldering E, Spaargaren M, Pals ST. WNT signaling controls expression of pro-apoptotic BOK and BAX in intestinal cancer. Biochem Biophys Res Commun 2011; 406:1-6. [DOI: 10.1016/j.bbrc.2010.12.070] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2010] [Accepted: 12/15/2010] [Indexed: 01/30/2023]
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46
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Buske P, Galle J, Barker N, Aust G, Clevers H, Loeffler M. A comprehensive model of the spatio-temporal stem cell and tissue organisation in the intestinal crypt. PLoS Comput Biol 2011; 7:e1001045. [PMID: 21253562 PMCID: PMC3017108 DOI: 10.1371/journal.pcbi.1001045] [Citation(s) in RCA: 127] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2010] [Accepted: 12/01/2010] [Indexed: 12/20/2022] Open
Abstract
We introduce a novel dynamic model of stem cell and tissue organisation in murine intestinal crypts. Integrating the molecular, cellular and tissue level of description, this model links a broad spectrum of experimental observations encompassing spatially confined cell proliferation, directed cell migration, multiple cell lineage decisions and clonal competition. Using computational simulations we demonstrate that the model is capable of quantitatively describing and predicting the dynamic behaviour of the intestinal tissue during steady state as well as after cell damage and following selective gain or loss of gene function manipulations affecting Wnt- and Notch-signalling. Our simulation results suggest that reversibility and flexibility of cellular decisions are key elements of robust tissue organisation of the intestine. We predict that the tissue should be able to fully recover after complete elimination of cellular subpopulations including subpopulations deemed to be functional stem cells. This challenges current views of tissue stem cell organisation. In the murine small intestine there are more than a million organized groups of proliferating cells, the crypts, each of which contains about 250–300 cells. About 60% of these cells are in rapid cycle. The functional stem cells of this tissue have been demonstrated to reside at defined positions at the lower third of the crypt and to give rise to four different cell types. Considering this simple structure the murine intestine is an ideal system to study general aspects of tissue organization. Here, we introduce a comprehensive and predictive computer model of the spatio-temporal organization of the murine intestine which describes how cell production and cell fate decisions could be organized in steady state as well as under perturbations. The model is based on single cells acting as individual agents, updating their status within a certain set of options governed by some active rules and on signals received from the environment. This kind of self-organization enables effective tissue regeneration without assuming an explicit stem cell population that maintains itself by asymmetric division. Thus, the model offers a novel systems biological view on crypt stem cell and tissue organisation.
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Affiliation(s)
- Peter Buske
- Interdisciplinary Centre for Bioinformatics, University Leipzig, Leipzig, Germany.
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47
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Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells. Proc Natl Acad Sci U S A 2010; 108:179-84. [PMID: 21173232 DOI: 10.1073/pnas.1013004108] [Citation(s) in RCA: 429] [Impact Index Per Article: 28.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
The intestinal epithelium is maintained by a population of rapidly cycling (Lgr5(+)) intestinal stem cells (ISCs). It has been postulated, however, that slowly cycling ISCs must also be present in the intestine to protect the genome from accumulating deleterious mutations and to allow for a response to tissue injury. Here, we identify a subpopulation of slowly cycling ISCs marked by mouse telomerase reverse transcriptase (mTert) expression that can give rise to Lgr5(+) cells. mTert-expressing cells distribute in a pattern along the crypt-villus axis similar to long-term label-retaining cells (LRCs) and are resistant to tissue injury. Lineage-tracing studies demonstrate that mTert(+) cells give rise to all differentiated intestinal cell types, persist long term, and contribute to the regenerative response following injury. Consistent with other highly regenerative tissues, our results demonstrate that a slowly cycling stem cell population exists within the intestine.
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48
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The role of glucagon-like peptide-2 on apoptosis, cell proliferation, and oxidant-antioxidant system at a mouse model of intestinal injury induced by tumor necrosis factor-alpha/actinomycin D. Mol Cell Biochem 2010; 350:13-27. [PMID: 21153865 DOI: 10.1007/s11010-010-0678-0] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2010] [Accepted: 12/02/2010] [Indexed: 12/18/2022]
Abstract
Tumor necrosis factor-alpha (TNF-α) is a multifunctional cytokine, which has the ability to produce cytotoxicity via induction of cell death and cell cycle arrest. Blocking the synthesis of protective proteins through a transcriptional inhibitor such as actinomycin D (Act D) sensitizes many cell types to TNF-α toxicity. Teduglutide, h[Gly(2)]GLP-2, is a protease-resistant synthetic analog of glucagon-like peptide-2 (GLP-2) which is an intestinotrophic peptide. In this study, we evaluated this potential of GLP-2 on apoptosis, cell proliferation, and oxidant-antioxidant system on a mouse model of intestinal injury induced by TNF-α/Act D. The intestinal injury was induced by intraperitoneal administration of 15 μg/kg TNF-α and 800 μg/kg Act D per mouse. Animals were injected subcutaneously 200 μg/kg h[Gly(2)]GLP-2 every 12 h for 10 consecutive days prior to the administration of TNF-α and Act D. The model of intestinal injury induced by TNF-α/Act D, which is the new animal model for the intestinal disorders, was characterized by the degeneration of intestinal mucosa, an increase in apoptotic index, expression of active caspase-3, lipid peroxidation and glutathione (GSH) levels, glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities; a decrease in cell proliferation and catalase (CAT) activity. h[Gly(2)]GLP-2 pretreatment prevented the TNF-α/Act D-induced oxidative injury by a significant reduction in the intestinal injury, apoptotic index, expression of active caspase-3, lipid peroxidation and GSH levels, GPx and SOD activities; a markedly increase in cell proliferation, and CAT activity. These results demonstrate that GLP-2 has a protective, antiapoptotic, proliferative, and antioxidant effects against to TNF-α/Act D-induced intestinal injury. It is suggested that GLP-2 may potentially be useful as a therapeutic agent in TNF-α-mediated intestinal disorders.
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Colon J, Hsieh N, Ferguson A, Kupelian P, Seal S, Jenkins DW, Baker CH. Cerium oxide nanoparticles protect gastrointestinal epithelium from radiation-induced damage by reduction of reactive oxygen species and upregulation of superoxide dismutase 2. NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE 2010; 6:698-705. [DOI: 10.1016/j.nano.2010.01.010] [Citation(s) in RCA: 241] [Impact Index Per Article: 16.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/20/2009] [Revised: 01/20/2010] [Accepted: 01/25/2010] [Indexed: 10/19/2022]
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Mirpuri J, Brazil JC, Berardinelli AJ, Nasr TR, Cooper K, Schnoor M, Lin PW, Parkos CA, Louis NA. Commensal Escherichia coli reduces epithelial apoptosis through IFN-alphaA-mediated induction of guanylate binding protein-1 in human and murine models of developing intestine. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2010; 184:7186-95. [PMID: 20483731 PMCID: PMC3145319 DOI: 10.4049/jimmunol.0903116] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Appropriate microbial colonization protects the developing intestine by promoting epithelial barrier function and fostering mucosal tolerance to luminal bacteria. Commensal flora mediate their protective effects through TLR9-dependent activation of cytokines, such as type I IFNs (alpha, beta) and IL-10. Although IFN-beta promotes apoptosis, IFN-alpha activates specific antiapoptotic target genes whose actions preserve epithelial barrier integrity. We have recently identified guanylate binding protein-1 (GBP-1) as an antiapoptotic protein, regulated by both type I and type II IFNs, that promotes intestinal epithelial barrier integrity in mature intestine. However, the mechanisms by which commensal bacteria regulate epithelial apoptosis during colonization of immature intestine and the contributions of GBP-1 are unknown. The healthy newborn intestine is initially colonized with bacterial species present in the maternal gastrointestinal tract, including nonpathogenic Escherichia coli. Therefore, we examined the influence of commensal E. coli on cytokine expression and candidate mediators of apoptosis in preweaned mice. Specifically, enteral exposure of 2 wk-old mice to commensal E. coli for 24 h selectively increased both IFN-alphaA and GBP-1 mRNA expression and prevented staurosporine-induced epithelial apoptosis. Exogenous IFN-alphaA treatment also induced GBP-1 expression and protected against staurosporine-induced apoptosis in a GBP-1 dependent manner, both in vitro and ex vivo. These findings identify a role for IFN-alphaA-mediated GBP-1 expression in the prevention of intestinal epithelial apoptosis by commensal bacteria. Thus IFN-alphaA mediates the beneficial effects of commensal bacteria and may be a promising therapeutic target to promote barrier integrity and prevent the inappropriate inflammatory responses seen in developing intestine as in necrotizing enterocolitis.
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Affiliation(s)
- Julie Mirpuri
- Division of Neonatal-Perinatal Medicine, Emory University, Atlanta, Georgia
| | - Jennifer C. Brazil
- Division of Neonatal-Perinatal Medicine, Emory University, Atlanta, Georgia
| | | | - Tala R. Nasr
- Division of Neonatal-Perinatal Medicine, Emory University, Atlanta, Georgia
| | | | - Michael Schnoor
- Department of Vascular Biology, Max Planck Institute for Molecular Biomedicine, Munster, Germany
| | - Patricia W. Lin
- Division of Neonatal-Perinatal Medicine, Emory University, Atlanta, Georgia
| | - Charles A. Parkos
- Division of Pathology and Laboratory Medicine, Emory University, Atlanta, GA
| | - Nancy A. Louis
- Division of Neonatal-Perinatal Medicine, Emory University, Atlanta, Georgia
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