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Jeong H, Lee B, Cho SY, Lee Y, Kim J, Hur S, Cho K, Kim KH, Kim SH, Nam KT. Microbiota-derived short-chain fatty acids determine stem cell characteristics of gastric chief cells. Dev Cell 2025; 60:599-612.e6. [PMID: 39642880 DOI: 10.1016/j.devcel.2024.11.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2023] [Revised: 08/07/2024] [Accepted: 11/08/2024] [Indexed: 12/09/2024]
Abstract
The gastric mucosa is a highly dynamic tissue that undergoes constant self-renewal through stem cell differentiation. Chief cells maintain a quiescent state in homeostasis but are responsible for regeneration after injury. Although the role of microbiome-host interactions in the intestine is well studied, less is known about these interactions in the stomach. Using the mouse organoid and germ-free mouse models, we show that microbiota-derived short-chain fatty acids (SCFAs) suppress the proliferation of chief cells in mice. This effect is mediated by activation of G-protein-coupled receptor 43. Most importantly, through metabolomics and transplantation studies, we show butyrate-producing Lactobacillus intestinalis modulates the proliferation of chief cells in mice. Our findings identify a mechanism by which the microbiota regulates the cell characteristics of chief cells, providing insight into the complex interplay between the host and its microbial environment and the mechanisms underlying gastric homeostasis, with potential therapeutic implications for gastric diseases.
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Affiliation(s)
- Haengdueng Jeong
- Department of Biomedical Science, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
| | - Buhyun Lee
- Department of Biomedical Science, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
| | - Soo Young Cho
- Department of Molecular and Life Science, Hanyang University, Ansan, Republic of Korea
| | - Yura Lee
- Department of Biomedical Science, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
| | - Jiseon Kim
- Department of Biomedical Science, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
| | - Sumin Hur
- Department of Biomedical Science, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
| | - Kyungrae Cho
- Department of Biomedical Science, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
| | - Kwang H Kim
- Department of Biomedical Science, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
| | - Sung-Hee Kim
- Department of Biomedical Science, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
| | - Ki Taek Nam
- Department of Biomedical Science, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, Republic of Korea.
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Cai X, Yao Y, Ren F, Zhang S. circTldc1 increases Tldc1 expression by targeting miR-485-5p to promote fibroblast-like synoviocytes proliferation in collagen-induced arthritis. Exp Cell Res 2024; 435:113928. [PMID: 38190869 DOI: 10.1016/j.yexcr.2024.113928] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2023] [Revised: 01/04/2024] [Accepted: 01/05/2024] [Indexed: 01/10/2024]
Abstract
Abnormalities in the function of fibroblast-like synoviocytes (FLSs) are crucial factors leading to joint damage of rheumatoid arthritis. In recent years, the role of circular RNA (circRNA) in RA has gradually been revealed. However, the functional regulation of FLSs mediated by circRNA and its potential mechanisms remain unclear. In this study, we elucidated the expression profile of circRNA in FLSs, as well as the role and molecular mechanisms of circTldc1. Through sequencing and validation experiments on primary FLSs derived from collagen-induced arthritis (CIA) rats, we found that circTldc1 can promote FLSs proliferation and exacerbate CIA-induced joint damage. The data revealed that circTldc1's parent gene, Tldc1, is homologous to human Tldc1, and circTldc1 is located in the cytoplasm of FLSs, belonging to the exonic circRNA category. The results from bioinformatics analysis, molecular experiments on FLSs (manipulating circTldc1 expression in vitro), and animal experiments (local regulation of circTldc1 expression in vivo) collectively confirmed that circTldc1 promotes Tldc1 expression by targeting miR-485-5p. High expression of Tldc1 further enhances FLSs proliferation and inflammatory responses, thereby worsening joint damage in CIA rats. High expression of circTldc1 and its parent gene Tldc1 may serve as biomarkers for RA. Local regulation of circTldc1 and Tldc1 gene levels in the joint cavity may represent a potential strategy to improve joint damage and inflammation in RA.
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Affiliation(s)
- Xiaoyu Cai
- Department of Pharmacy, Hangzhou First People's Hospital, Hangzhou, 310006, China.
| | - Yao Yao
- Department of Pharmacy, Women's Hospital, Zhejiang University School of Medicine, Hangzhou, 310006, China
| | - Fujia Ren
- Department of Pharmacy, Hangzhou Women's Hospital, Hangzhou, China
| | - Shiwei Zhang
- Department of Anesthesiology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China
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Kawahigashi T, Iwanami S, Takahashi M, Bhadury J, Iwami S, Yamazaki S. Age-related changes in the hematopoietic stem cell pool revealed via quantifying the balance of symmetric and asymmetric divisions. PLoS One 2024; 19:e0292575. [PMID: 38285676 PMCID: PMC10824414 DOI: 10.1371/journal.pone.0292575] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2023] [Accepted: 01/02/2024] [Indexed: 01/31/2024] Open
Abstract
Hematopoietic stem cells (HSCs) are somatic stem cells that continuously generate lifelong supply of blood cells through a balance of symmetric and asymmetric divisions. It is well established that the HSC pool increases with age. However, not much is known about the underlying cause for these observed changes. Here, using a novel method combining single-cell ex vivo HSC expansion with mathematical modeling, we quantify HSC division types (stem cell-stem cell (S-S) division, stem cell-progenitor cell (S-P) division, and progenitor cell-progenitor cell (P-P) division) as a function of the aging process. Our time-series experiments reveal how changes in these three modes of division can explain the increase in HSC numbers with age. Contrary to the popular notion that HSCs divide predominantly through S-P divisions, we show that S-S divisions are predominant throughout the lifespan of the animal, thereby expanding the HSC pool. We, therefore, provide a novel mathematical model-based experimental validation for reflecting HSC dynamics in vivo.
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Affiliation(s)
- Teiko Kawahigashi
- Division of Stem Cell Biology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, University of Tokyo, Tokyo, Japan
| | - Shoya Iwanami
- interdisciplinary Biology Laboratory (iBLab), Division of Natural Science, Graduate School of Science, Nagoya University, Nagoya, Japan
| | - Munetomo Takahashi
- Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
- Medical Research Council Toxicology Unit, Gleeson Building, Tennis Court Road, University of Cambridge, Cambridge, United Kingdom
| | - Joydeep Bhadury
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, United States of America
| | - Shingo Iwami
- interdisciplinary Biology Laboratory (iBLab), Division of Natural Science, Graduate School of Science, Nagoya University, Nagoya, Japan
- Institute of Mathematics for Industry, Kyushu University, Fukuoka, Japan
- Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan
- Interdisciplinary Theoretical and Mathematical Sciences Program (iTHEMS), RIKEN, Saitama, Japan
- NEXT-Ganken Program, Japanese Foundation for Cancer Research (JFCR), Tokyo, Japan
- Science Groove Inc., Fukuoka, Japan
| | - Satoshi Yamazaki
- Division of Stem Cell Biology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, University of Tokyo, Tokyo, Japan
- Laboratory of Stem Cell Therapy, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
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Yao Y, Cai X, Zhang M, Zheng Y, Fei W, Zhao M, Zheng C. Circ-Plod2 destabilizes Mpo mRNA by binding to IGF2BP2 to promote osteogenic differentiation of bone marrow mesenchymal stem cells. Eur J Pharmacol 2023; 961:176192. [PMID: 37981258 DOI: 10.1016/j.ejphar.2023.176192] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2023] [Revised: 10/11/2023] [Accepted: 11/07/2023] [Indexed: 11/21/2023]
Abstract
Osteogenic differentiation, proliferation, and/or apoptosis of bone marrow mesenchymal stem cells (BMSCs) are involved in the progression of postmenopausal osteoporosis (PMO). However, circular RNA (circRNA)-mediated changes in the cellular function of BMSCs in PMO are still unclear. This study revealed the excellent ability of circ-Plod2 to promote osteogenic differentiation of BMSCs and its molecular mechanisms. In this study, ovariectomized (OVX) rats and control (Sham) rats were used to simulate PMO. Initially, we found that the expression of circ-Plod2 in OVX BMSCs is down-regulated and the expression of the Mpo gene is up-regulated by sequencing and verification. Further, we confirmed that circ-Plod2 is located in the cytoplasm and belongs to exon-type circRNA. Interestingly, circ-Plod2 promotes Mpo-dependent osteogenic differentiation of BMSCs without affecting proliferation, apoptosis, adipogenic differentiation, or chondrogenic differentiation of BMSCs. Mechanistically, we demonstrated that circ-Plod2 specifically binds IGF2BP2 to form an RNA-protein complex that destabilizes Mpo mRNA. Overexpression of circ-Plod2 in the bone marrow cavity effectively alleviated osteoporosis in OVX rats and inhibited the expression of MPO in BMSCs. Together, this study reveals that circ-Plod2 destabilizes Mpo mRNA by binding to IGF2BP2 to promote osteogenic differentiation of BMSCs to alleviate osteoporosis. The findings of this study may provide biomarkers for the diagnosis of PMO, and may also provide potential strategies for the clinical treatment of PMO.
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Affiliation(s)
- Yao Yao
- Department of Pharmacy, Women's Hospital, Zhejiang University School of Medicine, Hangzhou, 310006, China
| | - Xiaoyu Cai
- Department of Clinical Pharmacology, Key Laboratory of Clinical Cancer Pharmacology and Toxicology Research of Zhejiang Province, Affiliated Hangzhou First People's Hospital, Cancer Center, Zhejiang University School of Medicine, Hangzhou, 310006, China
| | - Meng Zhang
- Department of Pharmacy, Women's Hospital, Zhejiang University School of Medicine, Hangzhou, 310006, China
| | - Yongquan Zheng
- Department of Pharmacy, Women's Hospital, Zhejiang University School of Medicine, Hangzhou, 310006, China
| | - Weidong Fei
- Department of Pharmacy, Women's Hospital, Zhejiang University School of Medicine, Hangzhou, 310006, China
| | - Mengdan Zhao
- Department of Pharmacy, Women's Hospital, Zhejiang University School of Medicine, Hangzhou, 310006, China.
| | - Caihong Zheng
- Department of Pharmacy, Women's Hospital, Zhejiang University School of Medicine, Hangzhou, 310006, China.
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5
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Tao X, Liu K, Li W, Zhao S, Liu C, Dai Q, Dong T, Wei P, Duan J, Wang J, Xi M. Saponin of Aralia taibaiensis promotes angiogenesis through VEGF/VEGFR2 signaling pathway in cerebral ischemic mice. JOURNAL OF ETHNOPHARMACOLOGY 2023; 317:116771. [PMID: 37308026 DOI: 10.1016/j.jep.2023.116771] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Revised: 05/28/2023] [Accepted: 06/09/2023] [Indexed: 06/14/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE Aralia taibaiensis is known for its ability to promote blood circulation and dispel blood stasis, activate meridians and remove arthralgia. The saponins of Aralia taibaiensis (sAT) are the main active components that are often used to treat cardiovascular and cerebrovascular diseases. However, it has not been reported whether sAT can improve ischemic stroke (IS) by promoting angiogenesis. AIM OF THE STUDY In this study, we investigated the potential of sAT to promote post-ischemic angiogenesis in mice and determined the underlying mechanism through in vitro experiments. METHODS To establish the middle cerebral artery occlusion (MCAO) mice model in vivo. First of all, we examined the neurological function, brain infarct volume, and degree of brain swelling in MCAO mice. We also observed pathological changes in brain tissue, ultrastructural changes in blood vessels and neurons, and the degree of vascular neovascularization. Additionally, we established the oxygen-glucose deprivation/reoxygenation (OGD/R) -human umbilical vein endothelial cells (HUVECs) model in vitro to detect the survival, proliferation, migration and tube formation of OGD/R HUVECs. Finally, we verified the regulatory mechanism of Src and PLCγ1 siRNA on sAT promoting angiogenesis by cell transfection technique. RESULTS In the cerebral ischemia-reperfusion mice, sAT distinctly improved the cerebral infarct volume, brain swelling degree, neurological dysfunction, and brain histopathological morphology due to cerebral ischemia/reperfusion injury. It also increased the double positive expression of BrdU and CD31 in brain tissue, promoted the release of VEGF and NO and decreased the release of NSE and LDH. In the OGD/R HUVECs, sAT significantly improved cell survival, proliferation, migration and tube formation, promoted the release of VEGF and NO, and increased the expression of VEGF, VEGFR2, PLCγ1, ERK1/2, Src and eNOS. Surprisingly, the effect of sAT on angiogenesis was inhibited by Src siRNA and PLCγ1 siRNA in OGD/R HUVECs. CONCLUSION The results proved that sAT promotes angiogenesis in cerebral ischemia-reperfusion mice and its mechanism is to regulate VEGF/VEGFR2 and then regulate Src/eNOS and PLCγ1/ERK1/2.
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Affiliation(s)
- Xingru Tao
- Department of Pharmacy, Xijing Hospital, Air Force Military Medical University, Xi'an City, Shaanxi Province, 710032, China; College of Pharmacy, Shaanxi University of TCM, Xianyang City, Shaanxi Province, 712046, China
| | - Kedi Liu
- TANK Medicinal Biology Institute of Xi'an, Xi'an City, Shaanxi Province, 710032, China
| | - Weihong Li
- College of Pharmacy, Shaanxi University of TCM, Xianyang City, Shaanxi Province, 712046, China
| | - Shi Zhao
- TANK Medicinal Biology Institute of Xi'an, Xi'an City, Shaanxi Province, 710032, China
| | - Chengzhao Liu
- College of Pharmacy, Shaanxi University of TCM, Xianyang City, Shaanxi Province, 712046, China
| | - Qi Dai
- College of Pharmacy, Shaanxi University of TCM, Xianyang City, Shaanxi Province, 712046, China
| | - Taiwei Dong
- College of Pharmacy, Shaanxi University of TCM, Xianyang City, Shaanxi Province, 712046, China
| | - Peifeng Wei
- National Drug Clinical Trial Institute, The Second Affiliated Hospital, Shaanxi University of TCM, Xianyang City, Shaanxi Province, 712000, China.
| | - Jialin Duan
- Institute of Medicine, Northwestern Polytechnical University, Xi'an City, Shaanxi Province, 710072, China.
| | - Jingwen Wang
- Department of Pharmacy, Xijing Hospital, Air Force Military Medical University, Xi'an City, Shaanxi Province, 710032, China.
| | - Miaomiao Xi
- TANK Medicinal Biology Institute of Xi'an, Xi'an City, Shaanxi Province, 710032, China; National Drug Clinical Trial Institute, The Second Affiliated Hospital, Shaanxi University of TCM, Xianyang City, Shaanxi Province, 712000, China.
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6
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Yang H, Zheng T, Ku CF, Ngai CK, Yue GGL, Lee HK, Lau CBS. Discovery of a heat-generated compound DHD derived from Patrinia villosa water extract with inhibitory effects on colon cancer cells viability and migration. Front Chem 2023; 11:1195883. [PMID: 37332894 PMCID: PMC10272523 DOI: 10.3389/fchem.2023.1195883] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2023] [Accepted: 05/18/2023] [Indexed: 06/20/2023] Open
Abstract
Introduction: The plant Patrinia villosa Juss. (PV) has long been used as a medicinal herb for treating intestinal disorders. Pharmacological activities such as anti-oxidation, anti-inflammation, and anti-cancer effects of compounds isolated from PV have been reported, but these bioactive compounds were not derived from PV water extract (PVW). Therefore, in the present study, we aimed to identify the active component(s) of PVW which exhibit inhibitory activities in colon cancer cells viability and migration. Methods: Human colon cancer HCT116 cells were treated with the isolated compounds of PVW and then subjected to MTT and transwell migration assays. Results: Our results showed that an active compound in PVW, 8,9-didehydro-7-hydroxydolichodial (DHD) inhibited cell viability of HCT116 cells, with IC50 value at 6.1 ± 2.2 μM. Interestingly, DHD was not detected in the herbal material of PV. Further investigation revealed that DHD is in fact a heat-generated compound derived from a natural compound present in PV, namely valerosidate. Valerosidate also reduced cell viability in HCT116 cells, with IC50 value at 22.2 ± 1.1 μM. Moreover, both DHD (2.75 μM) and valerosidate (10.81 μM) suppressed cell migration in HCT116 cells, with inhibitory rates at 74.8% and 74.6%, respectively. In addition, western blot results showed that DHD (5.5 μM) could significantly increase p53 expression by 34.8% and PTEN expression by 13.9%, while valerosidate (21.6 μM) could increase expressions of p53 and PTEN by 26.1% and 34.6%, respectively in HCT116 cells after 48 h treatment. Discussion: Taken together, this is the first report that a naturally-occurring valerosidate present in PV could actually transform to DHD by thermal hydrolysis, and both compounds exhibited inhibitory effects on cell viability and migration in HCT116 cells via increasing the expressions of tumor suppressors (p53 and PTEN). Our findings demonstrated that valerosidate is present in raw herb PV but not in PVW, while DHD is present in PVW rather than in raw herb PV. This difference in chemical profiles of raw herb and boiled water extract of PV may affect the anti-cancer activity, and hence further investigations are warranted.
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Affiliation(s)
- Huihai Yang
- Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
- State Key Laboratory of Research on Bioactivities and Clinical Applications of Medicinal Plants, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
| | - Tao Zheng
- Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
- State Key Laboratory of Research on Bioactivities and Clinical Applications of Medicinal Plants, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
| | - Chuen-Fai Ku
- Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
- State Key Laboratory of Research on Bioactivities and Clinical Applications of Medicinal Plants, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
| | - Cheuk Kit Ngai
- Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
| | - Grace Gar-Lee Yue
- Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
- State Key Laboratory of Research on Bioactivities and Clinical Applications of Medicinal Plants, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
| | - Hung Kay Lee
- Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
| | - Clara Bik-San Lau
- Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
- State Key Laboratory of Research on Bioactivities and Clinical Applications of Medicinal Plants, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
- Li Dak Sum Yip Yio Chin R&D Centre for Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
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7
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Ramezani M, Mirzaeian L, Ghezelayagh Z, Ghezelayagh Z, Ghorbanian MT. Comparing the mesenchymal stem cells proliferation rate with different labeling assessments. THE NUCLEUS 2023. [DOI: 10.1007/s13237-022-00415-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
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8
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Zhang T, Huang J, Zhang Z, Lv J, Zhang D, Qing R, Lan L. Flow cytometry and 5-ethynyl-2'-deoxyuridine (EdU) labeling to detect the cell cycle dynamics of Phaeodactylum tricornutum under light. JOURNAL OF PHYCOLOGY 2022; 58:555-567. [PMID: 35352350 DOI: 10.1111/jpy.13250] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/20/2021] [Revised: 02/28/2022] [Accepted: 02/28/2022] [Indexed: 06/14/2023]
Abstract
Cell cycle studies in plants and algae are highly dependent on reliable methods for detecting cellular DNA replication. With its short growth cycle and ease of genetic transformation, Phaeodactylum tricornutum is an important model organism for the study of pennate diatoms. Here we explored two different methods to detect the cell cycle of P. tricornutum, one using SYBR-green I to via flow cytometry, and the other using EdU labeling to observe cell cycle changes under fluorescence microscopy. Both EdU labeling fluorescence microscopy and SYBR-green I staining flow cytometry accurately indicated that the cells of P. tricornutum enter the G2/M phase after 12 h of light exposure. The results indicate that SYBR Green I was an adequate detection method for nuclear DNA quantitation in cells of P. tricornutum using a flow cytometer and without RNase A treatment. In addition, EdU can be applied to P. tricornutum to reliably detect cell proliferation. Besides, Mg-ProtoIX was able to reverse the cell cycle division inhibition of P. tricornutum and allow the nuclear DNA replication to proceed normally. Taken together, the photoperiodic division time point was clearly identified, which sheds light on the regulation of cell division mechanism in P. tricornutum.
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Affiliation(s)
- Ting Zhang
- Key Laboratory of Bio-Resource and Eco-Environment of the Ministry of Education, College of Life Science, Sichuan University, Chengdu, 610065, China
| | - Jingyi Huang
- Key Laboratory of Bio-Resource and Eco-Environment of the Ministry of Education, College of Life Science, Sichuan University, Chengdu, 610065, China
| | - Zhixia Zhang
- Key Laboratory of Bio-Resource and Eco-Environment of the Ministry of Education, College of Life Science, Sichuan University, Chengdu, 610065, China
| | - Jie Lv
- Key Laboratory of Bio-Resource and Eco-Environment of the Ministry of Education, College of Life Science, Sichuan University, Chengdu, 610065, China
| | - Dongqun Zhang
- Key Laboratory of Bio-Resource and Eco-Environment of the Ministry of Education, College of Life Science, Sichuan University, Chengdu, 610065, China
| | - Renwei Qing
- Key Laboratory of Bio-Resource and Eco-Environment of the Ministry of Education, College of Life Science, Sichuan University, Chengdu, 610065, China
| | - Liqiong Lan
- Key Laboratory of Bio-Resource and Eco-Environment of the Ministry of Education, College of Life Science, Sichuan University, Chengdu, 610065, China
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Islam S, Parker J, Dash BC, Hsia HC. Human iPSC-Vascular smooth muscle cell spheroids demonstrate size-dependent alterations in cellular viability and secretory function. J Biomed Mater Res A 2022; 110:1813-1823. [PMID: 35815599 DOI: 10.1002/jbm.a.37423] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Revised: 06/08/2022] [Accepted: 06/15/2022] [Indexed: 11/11/2022]
Abstract
Human-induced pluripotent stem cells (hiPSC) and their differentiated vascular cells have been revolutionizing the field of regenerative wound healing. These cells are shown to be rejuvenated with immense potentials in secreting paracrine factors. Recently, hiPSC-derived vascular smooth muscle cells (hiPSC-VSMC) have shown regenerative wound healing ability via their paracrine secretion. The quest to modulate the secretory function of these hiPSC-VSMC is an ongoing effort and involves the use of both biochemical and biophysical stimuli. This study explores the development and optimization of a reproducible, inexpensive protocol to form hiPSC-VSMC derived spheroids to investigate the implications of spheroid size on viability and paracrine secretion. Our data show the successful formation of different sizes of spheroids using various amount of hiPSC-VSMC. The hiPSC-VSMC spheroids formed with 10,000 cells strike an ideal balance between overall cell health and maximal paracrine secretion. The conditioned medium from these spheroids was found to be bioactive in enhancing human dermal fibroblast cell proliferation and migration. This research will inform future studies on the optimal spheroid size for regenerative wound healing applications.
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Affiliation(s)
- Sara Islam
- Section of Plastic Surgery, Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, USA
| | - Jackson Parker
- Section of Plastic Surgery, Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, USA
| | - Biraja C Dash
- Section of Plastic Surgery, Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, USA
| | - Henry C Hsia
- Section of Plastic Surgery, Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, USA.,Department of Biomedical Engineering, Yale University, New Haven, Connecticut, USA
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10
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Abstract
5-Bromo-2'-deoxyuridine (bromodeoxyuridine (BrdU)), a modified nucleotide and analog of thymidine, is commonly used for detecting proliferating cells. For detection, an anti-BrdU antibody (probe) with a fluorescent dye is applied to bind the BrdU label after DNA denaturation. In this protocol, we provide the BrdU labeling method for both in vitro and in vivo studies, along with immunocytochemistry (ICC)/immunofluorescence (IF) and immunohistochemistry (IHC) staining procedures, respectively. Multicolor staining is also presented as an option to detect the co-distribution of two or multiple antigens in the same sample, making it possible to visualize the location of different molecules at the same time.
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Affiliation(s)
- Jihang Yu
- Radiobiology and Health, Canadian Nuclear Laboratories, Chalk River, ON, Canada
| | - Zhixiang Wang
- Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada
| | - Yi Wang
- Radiobiology and Health, Canadian Nuclear Laboratories, Chalk River, ON, Canada.
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada.
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11
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Sherapura A, Malojirao VH, Thirusangu P, Sharath BS, Kandagalla S, Vigneshwaran V, Novak J, Ranganatha L, Ramachandra YL, Baliga SM, Khanum SA, Prabhakar BT. Anti-neoplastic pharmacophore benzophenone-1 coumarin (BP-1C) targets JAK2 to induce apoptosis in lung cancer. Apoptosis 2021; 27:49-69. [PMID: 34837562 DOI: 10.1007/s10495-021-01699-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/07/2021] [Indexed: 11/21/2022]
Abstract
Reigning of the abnormal gene activation associated with survival signalling in lung cancer leads to the anomalous growth and therapeutic failure. Targeting specific cell survival signalling like JAK2/STAT3 nexus has become a major focus of investigation to establish a target specific treatment. The 2-bromobenzoyl-4-methylphenoxy-acetyl hydra acetyl Coumarin (BP-1C), is new anti-neoplastic agent with apoptosis inducing capacity. The current study was aimed to develop antitumor phramacophore, BP-1C as JAK2 specific inhibitor against lung neoplastic progression. The study validates and identifies the molecular targets of BP-1C induced cell death. Cell based screening against multiple cancer cell lines identified, lung adenocarcinoma as its specific target through promotion of apoptosis. The BP-1C is able to induce, specific hall marks of apoptosis and there by conferring anti-neoplastic activity. Validation of its molecular mechanism, identified, BP-1C specifically targets JAK2Tyr1007/1008 phosphorylation, and inhibits its downstream STAT3Tyr705 signalling pathway to induce cell death. As a consequence, modulation in Akt/Src survival signal and altered expression of interwoven apoptotic genes were evident. The results were reproducible in an in-vivo LLC tumor model and in-ovo xenograft studies. The computational approaches viz, drug finger printing confers, BP-1C as novel class JAK2 inhibitor and molecular simulations studies assures its efficiency in binding with JAK2. Overall, BP-1C is a novel JAK2 inhibitor with experimental evidence and could be effectively developed into a promising drug for lung cancer treatment.
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Affiliation(s)
- Ankith Sherapura
- Molecular Biomedicine Laboratory, Postgraduate Department of Studies and Research in Biotechnology, Sahyadri Science College, Kuvempu University, Shivamogga, Karnataka, 577203, India
| | - Vikas H Malojirao
- Molecular Biomedicine Laboratory, Postgraduate Department of Studies and Research in Biotechnology, Sahyadri Science College, Kuvempu University, Shivamogga, Karnataka, 577203, India.,Division for DNA Repair Research, Department of Neurosurgery, Centre for Neuroregeneration, Houston Methodist, Fannin Street, Houston, TX, USA
| | - Prabhu Thirusangu
- Molecular Biomedicine Laboratory, Postgraduate Department of Studies and Research in Biotechnology, Sahyadri Science College, Kuvempu University, Shivamogga, Karnataka, 577203, India.,Department of Experimental Pathology and Laboratory Medicine, Mayo Clinic, Rochester, MN, USA
| | - B S Sharath
- School of System Biomedical Science and Department of Bioinformatics and Lifescience, Soongsil University, Seoul, South Korea
| | - Shivananda Kandagalla
- Laboratory of Computational Modelling of Drugs, Higher Medical and Biological School, South Ural State University, Chaikovskogo 20A, Chelyabinsk, Russia, 454008
| | - V Vigneshwaran
- Molecular Biomedicine Laboratory, Postgraduate Department of Studies and Research in Biotechnology, Sahyadri Science College, Kuvempu University, Shivamogga, Karnataka, 577203, India.,Department of Pharmacology and Centre for Lung and Vascular Biology, University of Illinois at Chicago, Chicago, 60612, USA
| | - Jurica Novak
- Laboratory of Computational Modelling of Drugs, Higher Medical and Biological School, South Ural State University, Chaikovskogo 20A, Chelyabinsk, Russia, 454008
| | - Lakshmi Ranganatha
- Department of Chemistry, The National Institute of Engineering, Mysuru, Karnataka, 570008, India
| | - Y L Ramachandra
- Department of Studies and Research in Biotechnology and Bioinformatics, Kuvempu University, Jnanasahyadri, Shankaraghatta, 577 451, India
| | - Shrinath M Baliga
- Department of Radiation Oncology, Mangalore Institute of Oncology, Mangalore, Karnataka, 575 002, India
| | - Shaukath Ara Khanum
- Department of Chemistry, Yuvaraja's College (Autonomous), University of Mysore, Mysuru, Karnataka, 570 005, India.
| | - B T Prabhakar
- Molecular Biomedicine Laboratory, Postgraduate Department of Studies and Research in Biotechnology, Sahyadri Science College, Kuvempu University, Shivamogga, Karnataka, 577203, India.
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12
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Recent advances in nucleotide analogue-based techniques for tracking dividing stem cells: An overview. J Biol Chem 2021; 297:101345. [PMID: 34717955 PMCID: PMC8592869 DOI: 10.1016/j.jbc.2021.101345] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2021] [Revised: 10/18/2021] [Accepted: 10/19/2021] [Indexed: 01/14/2023] Open
Abstract
Detection of thymidine analogues after their incorporation into replicating DNA represents a powerful tool for the study of cellular DNA synthesis, progression through the cell cycle, cell proliferation kinetics, chronology of cell division, and cell fate determination. Recent advances in the concurrent detection of multiple such analogues offer new avenues for the investigation of unknown features of these vital cellular processes. Combined with quantitative analysis, temporal discrimination of multiple labels enables elucidation of various aspects of stem cell life cycle in situ, such as division modes, differentiation, maintenance, and elimination. Data obtained from such experiments are critically important for creating descriptive models of tissue histogenesis and renewal in embryonic development and adult life. Despite the wide use of thymidine analogues in stem cell research, there are a number of caveats to consider for obtaining valid and reliable labeling results when marking replicating DNA with nucleotide analogues. Therefore, in this review, we describe critical points regarding dosage, delivery, and detection of nucleotide analogues in the context of single and multiple labeling, outline labeling schemes based on pulse-chase, cumulative and multilabel marking of replicating DNA for revealing stem cell proliferative behaviors, and determining cell cycle parameters, and discuss preconditions and pitfalls in conducting such experiments. The information presented in our review is important for rational design of experiments on tracking dividing stem cells by marking replicating DNA with thymidine analogues.
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13
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Abstract
Cell cycle involves a series of changes that lead to cell growth and division. Cell cycle analysis is crucial to understand cellular responses to changing environmental conditions. Since its inception, flow cytometry has been particularly useful for cell cycle analysis at single cell level due to its speed and precision. Previously, flow cytometric cell cycle analysis relied solely on the measurement of cellular DNA content. Later, methods were developed for multiparametric analysis. This review explains the journey of flow cytometry to understand different molecular and cellular events underlying cell cycle using various protocols. Recent advances in the field that overcome the shortcomings of traditional flow cytometry and expand its scope for cell cycle studies are also discussed.
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14
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Arhgef2 regulates mitotic spindle orientation in hematopoietic stem cells and is essential for productive hematopoiesis. Blood Adv 2021; 5:3120-3133. [PMID: 34406376 DOI: 10.1182/bloodadvances.2020002539] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2020] [Accepted: 03/29/2021] [Indexed: 11/20/2022] Open
Abstract
How hematopoietic stem cells (HSCs) coordinate their divisional axis and whether this orientation is important for stem cell-driven hematopoiesis is poorly understood. Single-cell RNA sequencing data from patients with Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome, show that ARHGEF2, a RhoA-specific guanine nucleotide exchange factor and determinant of mitotic spindle orientation, is specifically downregulated in SDS hematopoietic stem and progenitor cells (HSPCs). We demonstrate that transplanted Arhgef2-/- fetal liver and bone marrow cells yield impaired hematopoietic recovery and a production deficit from long-term HSCs, phenotypes that are not the result of differences in numbers of transplanted HSCs, their cell cycle status, level of apoptosis, progenitor output, or homing ability. Notably, these defects are functionally restored in vivo by overexpression of ARHGEF2 or its downstream activated RHOA GTPase. By using live imaging of dividing HSPCs, we show an increased frequency of misoriented divisions in the absence of Arhgef2. ARHGEF2 knockdown in human HSCs also impairs their ability to regenerate hematopoiesis, culminating in significantly smaller xenografts. Together, these data demonstrate a conserved role for Arhgef2 in orienting HSPC division and suggest that HSCs may divide in certain orientations to establish hematopoiesis, the loss of which could contribute to HSC dysfunction in bone marrow failure.
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15
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Chavez JS, Rabe JL, Loeffler D, Higa KC, Hernandez G, Mills TS, Ahmed N, Gessner RL, Ke Z, Idler BM, Niño KE, Kim H, Myers JR, Stevens BM, Davizon-Castillo P, Jordan CT, Nakajima H, Ashton J, Welner RS, Schroeder T, DeGregori J, Pietras EM. PU.1 enforces quiescence and limits hematopoietic stem cell expansion during inflammatory stress. J Exp Med 2021; 218:211996. [PMID: 33857288 PMCID: PMC8056754 DOI: 10.1084/jem.20201169] [Citation(s) in RCA: 54] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2020] [Revised: 02/01/2021] [Accepted: 03/17/2021] [Indexed: 12/27/2022] Open
Abstract
Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.
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Affiliation(s)
- James S Chavez
- Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO
| | - Jennifer L Rabe
- Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO
| | - Dirk Loeffler
- Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, Basel, Switzerland
| | - Kelly C Higa
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO
| | - Giovanny Hernandez
- Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO
| | - Taylor S Mills
- Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO.,Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO
| | - Nouraiz Ahmed
- Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, Basel, Switzerland
| | - Rachel L Gessner
- Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO
| | - Zhonghe Ke
- Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO
| | - Beau M Idler
- Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO
| | - Katia E Niño
- Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO
| | - Hyunmin Kim
- Division of Medical Oncology, University of Colorado Anschutz Medical Campus, Aurora, CO
| | - Jason R Myers
- Genomics Research Center, University of Rochester, Rochester, NY
| | - Brett M Stevens
- Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO
| | | | - Craig T Jordan
- Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO
| | - Hideaki Nakajima
- Department of Stem Cell and Immune Regulation, Yokohama City University School of Medicine, Yokohama, Japan
| | - John Ashton
- Genomics Research Center, University of Rochester, Rochester, NY
| | - Robert S Welner
- Division of Hematology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL
| | - Timm Schroeder
- Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, Basel, Switzerland
| | - James DeGregori
- Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO.,Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO.,Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO.,Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO
| | - Eric M Pietras
- Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO.,Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO
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16
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Soni P, Ghufran MS, Olakkaran S, Puttaswamygowda GH, Duddukuri GR, Kanade SR. Epigenetic alterations induced by aflatoxin B 1: An in vitro and in vivo approach with emphasis on enhancer of zeste homologue-2/p21 axis. THE SCIENCE OF THE TOTAL ENVIRONMENT 2021; 762:143175. [PMID: 33131875 DOI: 10.1016/j.scitotenv.2020.143175] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/31/2020] [Revised: 09/21/2020] [Accepted: 10/14/2020] [Indexed: 06/11/2023]
Abstract
The potent environmental toxicant aflatoxin B1 (AFB1), is a group I carcinogen reported to induce the expression of many cancer associated proteins. Epigenetic alterations such as DNA methylation and histone modifications play vital role in AFB1-mediated carcinogenesis. These epigenetic modifications may result in the recruitment of specific proteins and transcription factors to the promoter region and regulate gene expression. Here we show that AFB1, at lower concentrations (100 and 1000 nM) induced proliferation in L-132 and HaCaT cells with activation of the Akt pathway, which ultimately steered abnormal proliferation and transmission of survival signals. We demonstrated a significant reduction in the expression of p21 with a remarkable increase in the expression of cyclin D1 that correlated with increased methylation of CpG dinucleotides in p21 proximal promoter, while cyclin D1 promoter remained unmethylated. The chromatin immunoprecipitation results revealed the enrichment of DNMT3a and H3K27me3 repressive marks on the p21 proximal promoter where EZH2 mediated H3K27me3 mark enhanced the binding of DNMT3a at the promoter and further contributed to the transcriptional inactivation. The overall study provided the novel information on the impact of AFB1 on p21 inactivation via EZH2 and promoter methylation which is known to be a vital process in proliferation. Furthermore, AFB1 induced the expression of EZH2 analogue protein E(z), cyclin D1 analogue cyclin D and decreased the expression of p21 analogue Dacapo in Drosophila melanogaster. Interestingly, the aggressiveness in their expression upon re-exposure in successive generations suggested first hand perspectives on multigenerational epigenetic memory.
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Affiliation(s)
- Priyanka Soni
- Department of Biochemistry and Molecular Biology, School of Biological Sciences, Central University of Kerala, Tejaswini Hills, Periye, Kasargod 671316, Kerala, India
| | - Md Sajid Ghufran
- Department of Biochemistry and Molecular Biology, School of Biological Sciences, Central University of Kerala, Tejaswini Hills, Periye, Kasargod 671316, Kerala, India
| | - Shilpa Olakkaran
- Department of Zoology, School of Biological Sciences, Central University of Kerala, Tejaswini Hills, Periye, Kasargod 671316, Kerala, India
| | | | - Govinda Rao Duddukuri
- Department of Biochemistry and Molecular Biology, School of Biological Sciences, Central University of Kerala, Tejaswini Hills, Periye, Kasargod 671316, Kerala, India
| | - Santosh R Kanade
- Department of Plant Science, School of Life Science, University of Hyderabad, Prof. C. R. Rao Road Gachibowli, Hyderabad 500046, India.
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17
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Nik Nabil WN, Xi Z, Song Z, Jin L, Zhang XD, Zhou H, De Souza P, Dong Q, Xu H. Towards a Framework for Better Understanding of Quiescent Cancer Cells. Cells 2021; 10:cells10030562. [PMID: 33807533 PMCID: PMC7999675 DOI: 10.3390/cells10030562] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2021] [Revised: 02/22/2021] [Accepted: 03/02/2021] [Indexed: 12/15/2022] Open
Abstract
Quiescent cancer cells (QCCs) are cancer cells that are reversibly suspended in G0 phase with the ability to re-enter the cell cycle and initiate tumor growth, and, ultimately, cancer recurrence and metastasis. QCCs are also therapeutically challenging due to their resistance to most conventional cancer treatments that selectively act on proliferating cells. Considering the significant impact of QCCs on cancer progression and treatment, better understanding of appropriate experimental models, and the evaluation of QCCs are key questions in the field that have direct influence on potential pharmacological interventions. Here, this review focuses on existing and emerging preclinical models and detection methods for QCCs and discusses their respective features and scope for application. By providing a framework for selecting appropriate experimental models and investigative methods, the identification of the key players that regulate the survival and activation of QCCs and the development of more effective QCC-targeting therapeutic agents may mitigate the consequences of QCCs.
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Affiliation(s)
- Wan Najbah Nik Nabil
- School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; (W.N.N.N.); (Z.X.); (Z.S.)
- Pharmaceutical Services Programme, Ministry of Health, Petaling Jaya 46200, Malaysia
| | - Zhichao Xi
- School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; (W.N.N.N.); (Z.X.); (Z.S.)
| | - Zejia Song
- School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; (W.N.N.N.); (Z.X.); (Z.S.)
| | - Lei Jin
- School of Medicine and Public Health, The University of Newcastle, Newcastle, NSW 2308, Australia;
| | - Xu Dong Zhang
- School of Biomedical Sciences and Pharmacy, The University of Newcastle, Newcastle, NSW 2308, Australia;
| | - Hua Zhou
- Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
| | - Paul De Souza
- School of Medicine, Western Sydney University, Sydney, NSW 2751, Australia;
| | - Qihan Dong
- Chinese Medicine Anti-Cancer Evaluation Program, Greg Brown Laboratory, Central Clinical School and Charles Perkins Centre, The University of Sydney, Sydney, NSW 2006, Australia
- Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, NSW 2050, Australia
- Correspondence: (Q.D.); (H.X.)
| | - Hongxi Xu
- Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
- Correspondence: (Q.D.); (H.X.)
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18
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Nita A, Muto Y, Katayama Y, Matsumoto A, Nishiyama M, Nakayama KI. The autism-related protein CHD8 contributes to the stemness and differentiation of mouse hematopoietic stem cells. Cell Rep 2021; 34:108688. [PMID: 33535054 DOI: 10.1016/j.celrep.2021.108688] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2020] [Revised: 10/26/2020] [Accepted: 12/30/2020] [Indexed: 01/26/2023] Open
Abstract
Chromodomain helicase DNA-binding protein 8 (CHD8) is an ATP-dependent chromatin-remodeling factor that is encoded by the most frequently mutated gene in individuals with autism spectrum disorder. CHD8 is expressed not only in neural tissues but also in many other organs; however, its functions are largely unknown. Here, we show that CHD8 is highly expressed in and maintains the stemness of hematopoietic stem cells (HSCs). Conditional deletion of Chd8 specifically in mouse bone marrow induces cell cycle arrest, apoptosis, and a differentiation block in HSCs in association with upregulation of the expression of p53 target genes. A colony formation assay and bone marrow transplantation reveal that CHD8 deficiency also compromises the stemness of HSCs. Furthermore, additional ablation of p53 rescues the impaired stem cell function and differentiation block of CHD8-deficient HSCs. Our results thus suggest that the CHD8-p53 axis plays a key role in regulation of the stemness and differentiation of HSCs.
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Affiliation(s)
- Akihiro Nita
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan
| | - Yoshiharu Muto
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan
| | - Yuta Katayama
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan
| | - Akinobu Matsumoto
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan
| | - Masaaki Nishiyama
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.
| | - Keiichi I Nakayama
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.
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19
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Abstract
In this chapter, four methods are described that can be used to assess cell cycle status in flow cytometry. The first method is based on the simultaneous analysis of cellular DNA content using a fluorescent DNA dye (propidium iodide) and of a nuclear proliferation marker (Ki-67). The second is based on the differential staining of DNA and RNA using Hoechst 33342 and Pyronin Y: this method is particularly useful to distinguish quiescent cells in G0 phase from G1 cells. Finally, two methods are described based on DNA incorporation of the synthetic nucleosides BrdU and EdU.
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Affiliation(s)
- Ramy Rahmé
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
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20
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Abstract
Cell proliferation is broadly defined as a process leading to an increase of cell number, essentially depending on a balance between cell cycle progression/cell division, cell death, and cellular senescence. Deregulation of cell proliferation is a key feature of cancer cells, making assessment of proliferation a central methodological issue in cancer research. Especially in Ewing sarcoma (EwS) that exhibit a high proliferative capacity, experimental assessment of proliferation in preclinical research plays an important role. Among the variety of applicable methods, trypan blue exclusion is described here as a robust, easy-to-perform, and cost-effective method to assess cell proliferation in an experimental setting.
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21
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Luo X, Yu Z, Yue B, Ren J, Zhang J, Mani S, Wang Z, Dou W. Obacunone reduces inflammatory signalling and tumour occurrence in mice with chronic inflammation-induced colorectal cancer. PHARMACEUTICAL BIOLOGY 2020; 58:886-897. [PMID: 32878512 PMCID: PMC8202763 DOI: 10.1080/13880209.2020.1812673] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/01/2020] [Revised: 07/07/2020] [Accepted: 08/14/2020] [Indexed: 06/11/2023]
Abstract
CONTEXT Obacunone, a limonoid abundantly found in Citrus fruits, exhibits a variety of bioactivities. OBJECTIVE To investigate the effects of obacunone on a colorectal cancer (CRC) mouse model, and clarify its potential molecular mechanisms. MATERIALS AND METHODS The male Balb/c mice were induced with azoxymethane and dextran sulfate sodium for 12 weeks. Obacunone (50 mg/kg) was administered via oral gavage three times every week until the end of the experiment. Disease indexes including body weight, spleen weight, bloody diarrhea, colon length, histopathological score, and tumor size were measured. The anti-proliferation activities of obacunone were analyzed by MTT or flow cytometry. The expression of protein and mRNA related to cell proliferation or inflammatory cytokines was determined by Western blot, q-PCR and IHC. RESULTS Obacunone significantly alleviated bloody diarrhea, colon shortening (7.35 ± 0.2128 vs. 8.275 ± 0.2169 cm), splenomegaly, histological score (9 ± 0.5774 vs. 6 ± 0.5774) and reduced tumor size (4.25 ± 0.6196 vs. 2 ± 0.5669). Meanwhile, the expression of protein and mRNA related to cell proliferation or inflammatory cytokines was remarkably decreased in tumor tissue. Obacunone inhibited the proliferation activities of colorectal cancer cells. Moreover, obacunone induced colorectal cancer cells G1 and G2 phases arrest, and suppressed the expression of cell cycle genes. CONCLUSIONS Obacunone could alleviate CRC via inhibiting inflammatory response and tumor cells proliferation. The results may contribute to the effective utilization of obacunone or its derivatives in the treatment of human CRC.
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Affiliation(s)
- Xiaoping Luo
- Shanghai Key Laboratory of Formulated Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine (SHUTCM), Shanghai, China
| | - Zhilun Yu
- Shanghai Key Laboratory of Formulated Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine (SHUTCM), Shanghai, China
| | - Bei Yue
- Shanghai Key Laboratory of Formulated Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine (SHUTCM), Shanghai, China
| | - Junyu Ren
- Shanghai Key Laboratory of Formulated Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine (SHUTCM), Shanghai, China
| | - Jing Zhang
- Shanghai Key Laboratory of Formulated Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine (SHUTCM), Shanghai, China
| | - Sridhar Mani
- Departments of Medicine and Genetics, Albert Einstein College of Medicine, Bronx, NY, USA
| | - Zhengtao Wang
- Shanghai Key Laboratory of Formulated Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine (SHUTCM), Shanghai, China
| | - Wei Dou
- Shanghai Key Laboratory of Formulated Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine (SHUTCM), Shanghai, China
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22
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Riley A, Green V, Cheah R, McKenzie G, Karsai L, England J, Greenman J. A novel microfluidic device capable of maintaining functional thyroid carcinoma specimens ex vivo provides a new drug screening platform. BMC Cancer 2019; 19:259. [PMID: 30902086 PMCID: PMC6429713 DOI: 10.1186/s12885-019-5465-z] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2018] [Accepted: 03/13/2019] [Indexed: 01/08/2023] Open
Abstract
BACKGROUND Though the management of malignancies has improved vastly in recent years, many treatment options lack the desired efficacy and fail to adequately augment patient morbidity and mortality. It is increasingly clear that patient response to therapy is unique to each individual, necessitating personalised, or 'precision' medical care. This demand extends to thyroid cancer; ~ 10% patients fail to respond to radioiodine treatment due to loss of phenotypic differentiation, exposing the patient to unnecessary ionising radiation, as well as delaying treatment with alternative therapies. METHODS Human thyroid tissue (n = 23, malignant and benign) was live-sliced (5 mm diameter × 350-500 μm thickness) then analysed or incorporated into a microfluidic culture device for 96 h (37 °C). Successful maintenance of tissue was verified by histological (H&E), flow cytometric propidium iodide or trypan blue uptake, immunohistochemical (Ki67 detection/ BrdU incorporation) and functional analysis (thyroxine [T4] output) in addition to analysis of culture effluent for the cell death markers lactate dehydrogenase (LDH) and dead-cell protease (DCP). Apoptosis was investigated by Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Differentiation was assessed by evaluation of thyroid transcription factor (TTF1) and sodium iodide symporter (NIS) expression (western blotting). RESULTS Maintenance of gross tissue architecture was observed. Analysis of dissociated primary thyroid cells using flow cytometry both prior to and post culture demonstrated no significant change in the proportion of viable cells. LDH and DCP release from on-chip thyroid tissue indicated that after an initial raised level of release, signifying cellular damage, detectable levels dropped markedly. A significant increase in apoptosis (p < 0.01) was observed after tissue was perfused with etoposide and JNK inhibitor, but not in control tissue incubated for the same time period. No significant difference in Ki-67 positivity or TTF1/NIS expression was detected between fresh and post-culture thyroid tissue samples, moreover BrdU positive nuclei indicated on-chip cellular proliferation. Cultured thyroid explants were functionally viable as determined by production of T4 throughout the culture period. CONCLUSIONS The described microfluidic platform can maintain the viability of thyroid tissue slices ex vivo for a minimum of four days, providing a platform for the assessment of thyroid tissue radioiodine sensitivity/adjuvant therapies in real time.
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Affiliation(s)
- Andrew Riley
- Faculty of Health Sciences, University of Hull, Kingston upon Hull, HU6 7RX UK
| | - Victoria Green
- Faculty of Health Sciences, University of Hull, Kingston upon Hull, HU6 7RX UK
| | - Ramsah Cheah
- Faculty of Health Sciences, University of Hull, Kingston upon Hull, HU6 7RX UK
| | - Gordon McKenzie
- Faculty of Health Sciences, University of Hull, Kingston upon Hull, HU6 7RX UK
- Hull York Medical School, University of Hull, Kingston upon Hull, HU6 7RX UK
| | - Laszlo Karsai
- Hull and East Yorkshire Hospitals NHS Trust, Kingston upon Hull, HU16 5JQ UK
| | - James England
- Hull and East Yorkshire Hospitals NHS Trust, Kingston upon Hull, HU16 5JQ UK
| | - John Greenman
- Faculty of Health Sciences, University of Hull, Kingston upon Hull, HU6 7RX UK
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