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1
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Gonciarz W, Kozlowska L, Róg J, Chmiela M. Untargeted metabolomic profiling for identifying systemic signatures of helicobacter pylori infection in a guinea pig model. Sci Rep 2025; 15:12889. [PMID: 40234702 PMCID: PMC12000522 DOI: 10.1038/s41598-025-98016-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2024] [Accepted: 04/08/2025] [Indexed: 04/17/2025] Open
Abstract
Infections caused by the Gram-negative bacterium Helicobacter pylori (H. pylori) can lead to gastritis, gastric or duodenal ulcers, and even gastric cancer in humans. Investigating quantitative changes in soluble biomarkers associated with H. pylori infection offers a promising method for monitoring the progression of the infection, inflammatory response and potentially systemic consequences. This study aimed to identify, using an experimental model of H. pylori infection in guinea pigs, the specific metabolomic biomarkers in the serum of H. pylori-infected (32) versus uninfected (32) animals. The H. pylori status was confirmed through histological, molecular, and serological examinations. Metabolomic profiling was conducted using UPLC-QTOF/MS methods. The metabolomic biomarkers significantly associated with H. pylori infection were selected based on volcano plots and traditional univariate receiver operating characteristics (ROC). This study identified 12 unique metabolites significantly differentiating H. pylori-infected guinea pigs from uninfected ones. In summary, the metabolomic profiling of serum samples, in combination with ROC characteristics of the data, enhances the monitoring of H. pylori infection and related inflammatory responses in guinea pigs experimentally infected with these bacteria, with potential applications in humans for prediction the infection course and its systemic effects.
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Affiliation(s)
- Weronika Gonciarz
- Department of Immunology and Infectious Biology, Faculty of Biology and Environmental Protection, University of Lodz, 12/16 Banacha St., 90‑237, Lodz, Poland.
| | - Lucyna Kozlowska
- Laboratory of Human Metabolism Research, Department of Dietetics, Institute of Human Nutrition Sciences, Warsaw University of Life Sciences, 02-776, Warsaw, Poland
| | - Joanna Róg
- Laboratory of Human Metabolism Research, Department of Dietetics, Institute of Human Nutrition Sciences, Warsaw University of Life Sciences, 02-776, Warsaw, Poland
| | - Magdalena Chmiela
- Department of Immunology and Infectious Biology, Faculty of Biology and Environmental Protection, University of Lodz, 12/16 Banacha St., 90‑237, Lodz, Poland
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2
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Balena T, Staley K. Neuronal Death: Now You See It, Now You Don't. Neuroscientist 2024:10738584241282632. [PMID: 39316584 DOI: 10.1177/10738584241282632] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/26/2024]
Abstract
Fatally injured neurons may necrose and rupture immediately, or they may initiate a programmed cell death pathway and then wait for microglial phagocytosis. Biochemical and histopathologic assays of neuronal death assess the numbers of neurons awaiting phagocytosis at a particular time point after injury. This number varies with the fraction of neurons that have necrosed vs initiated programmed cell death, the time elapsed since injury, the rate of phagocytosis, and the assay's ability to detect neurons at different stages of programmed cell death. Many of these variables can be altered by putatively neurotoxic and neuroprotective interventions independent of the effects on neuronal death. This complicates analyses of neurotoxicity and neuroprotection and has likely contributed to difficulties with clinical translation of neuroprotective strategies after brain injury. Time-resolved assays of neuronal health, such as ongoing expression of transgenic fluorescent proteins, are a useful means of avoiding these problems.
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Affiliation(s)
- Trevor Balena
- Department of Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA
| | - Kevin Staley
- Department of Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA
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3
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Preedy MK, White MRH, Tergaonkar V. Cellular heterogeneity in TNF/TNFR1 signalling: live cell imaging of cell fate decisions in single cells. Cell Death Dis 2024; 15:202. [PMID: 38467621 PMCID: PMC10928192 DOI: 10.1038/s41419-024-06559-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2023] [Revised: 02/07/2024] [Accepted: 02/13/2024] [Indexed: 03/13/2024]
Abstract
Cellular responses to TNF are inherently heterogeneous within an isogenic cell population and across different cell types. TNF promotes cell survival by activating pro-inflammatory NF-κB and MAPK signalling pathways but may also trigger apoptosis and necroptosis. Following TNF stimulation, the fate of individual cells is governed by the balance of pro-survival and pro-apoptotic signalling pathways. To elucidate the molecular mechanisms driving heterogenous responses to TNF, quantifying TNF/TNFR1 signalling at the single-cell level is crucial. Fluorescence live-cell imaging techniques offer real-time, dynamic insights into molecular processes in single cells, allowing for detection of rapid and transient changes, as well as identification of subpopulations, that are likely to be missed with traditional endpoint assays. Whilst fluorescence live-cell imaging has been employed extensively to investigate TNF-induced inflammation and TNF-induced cell death, it has been underutilised in studying the role of TNF/TNFR1 signalling pathway crosstalk in guiding cell-fate decisions in single cells. Here, we outline the various opportunities for pathway crosstalk during TNF/TNFR1 signalling and how these interactions may govern heterogenous responses to TNF. We also advocate for the use of live-cell imaging techniques to elucidate the molecular processes driving cell-to-cell variability in single cells. Understanding and overcoming cellular heterogeneity in response to TNF and modulators of the TNF/TNFR1 signalling pathway could lead to the development of targeted therapies for various diseases associated with aberrant TNF/TNFR1 signalling, such as rheumatoid arthritis, metabolic syndrome, and cancer.
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Affiliation(s)
- Marcus K Preedy
- Laboratory of NF-κB Signalling, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore, 138673, Singapore
- Division of Molecular and Cellular Function, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Michael Smith Building, D3308, Dover Street, Manchester, M13 9PT, England, UK
| | - Michael R H White
- Division of Molecular and Cellular Function, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Michael Smith Building, D3308, Dover Street, Manchester, M13 9PT, England, UK.
| | - Vinay Tergaonkar
- Laboratory of NF-κB Signalling, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore, 138673, Singapore.
- Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), 8 Medical Drive, MD7, Singapore, 117596, Singapore.
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4
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Zhao A, Varady S, O'Kelley-Bangsberg M, Deng V, Platenkamp A, Wijngaard P, Bern M, Gormley W, Kushkowski E, Thompson K, Tibbetts L, Conner AT, Noeckel D, Teran A, Ritz A, Applewhite DA. From network analysis to experimental validation: identification of regulators of non-muscle myosin II contractility using the folded-gastrulation signaling pathway. BMC Mol Cell Biol 2023; 24:32. [PMID: 37821823 PMCID: PMC10568788 DOI: 10.1186/s12860-023-00492-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2023] [Accepted: 09/29/2023] [Indexed: 10/13/2023] Open
Abstract
The morphogenetic process of apical constriction, which relies on non-muscle myosin II (NMII) generated constriction of apical domains of epithelial cells, is key to the development of complex cellular patterns. Apical constriction occurs in almost all multicellular organisms, but one of the most well-characterized systems is the Folded-gastrulation (Fog)-induced apical constriction that occurs in Drosophila. The binding of Fog to its cognizant receptors Mist/Smog results in a signaling cascade that leads to the activation of NMII-generated contractility. Despite our knowledge of key molecular players involved in Fog signaling, we sought to explore whether other proteins have an undiscovered role in its regulation. We developed a computational method to predict unidentified candidate NMII regulators using a network of pairwise protein-protein interactions called an interactome. We first constructed a Drosophila interactome of over 500,000 protein-protein interactions from several databases that curate high-throughput experiments. Next, we implemented several graph-based algorithms that predicted 14 proteins potentially involved in Fog signaling. To test these candidates, we used RNAi depletion in combination with a cellular contractility assay in Drosophila S2R + cells, which respond to Fog by contracting in a stereotypical manner. Of the candidates we screened using this assay, two proteins, the serine/threonine phosphatase Flapwing and the putative guanylate kinase CG11811 were demonstrated to inhibit cellular contractility when depleted, suggestive of their roles as novel regulators of the Fog pathway.
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Affiliation(s)
- Andy Zhao
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA
| | - Sophia Varady
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA
| | | | - Vicki Deng
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA
| | - Amy Platenkamp
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA
| | - Petra Wijngaard
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA
| | - Miriam Bern
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA
| | - Wyatt Gormley
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA
| | - Elaine Kushkowski
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA
| | - Kat Thompson
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA
| | - Logan Tibbetts
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA
| | - A Tamar Conner
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA
| | - David Noeckel
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA
| | - Aidan Teran
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA
| | - Anna Ritz
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA.
| | - Derek A Applewhite
- Reed College Department of Biology, 3203 SE Woodstock Blvd, Portland, OR, 97202, USA.
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5
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Balena T, Lillis K, Rahmati N, Bahari F, Dzhala V, Berdichevsky E, Staley K. A Dynamic Balance between Neuronal Death and Clearance in an in Vitro Model of Acute Brain Injury. J Neurosci 2023; 43:6084-6107. [PMID: 37527922 PMCID: PMC10451151 DOI: 10.1523/jneurosci.0436-23.2023] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2023] [Revised: 06/15/2023] [Accepted: 07/20/2023] [Indexed: 08/03/2023] Open
Abstract
In in vitro models of acute brain injury, neuronal death may overwhelm the capacity for microglial phagocytosis, creating a queue of dying neurons awaiting clearance. Neurons undergoing programmed cell death are in this queue, and are the most visible and frequently quantified measure of neuronal death after injury. However, the size of this queue should be equally sensitive to changes in neuronal death and the rate of phagocytosis. Using rodent organotypic hippocampal slice cultures as a model of acute perinatal brain injury, serial imaging demonstrated that the capacity for microglial phagocytosis of dying neurons was overwhelmed for 2 weeks. Altering phagocytosis rates (e.g., by changing the number of microglia) dramatically changed the number of visibly dying neurons. Similar effects were generated when the visibility of dying neurons was altered by changing the membrane permeability for stains that label dying neurons. Canonically neuroprotective interventions, such as seizure blockade, and neurotoxic maneuvers, such as perinatal ethanol exposure, were mediated by effects on microglial activity and the membrane permeability of neurons undergoing programmed cell death. These canonically neuroprotective and neurotoxic interventions had either no or opposing effects on healthy surviving neurons identified by the ongoing expression of transgenic fluorescent proteins.SIGNIFICANCE STATEMENT In in vitro models of acute brain injury, microglial phagocytosis is overwhelmed by the number of dying cells. Under these conditions, the assumptions on which assays for neuroprotective and neurotoxic effects are based are no longer valid. Thus, longitudinal assays of healthy cells, such as serial assessment of the fluorescence emission of transgenically expressed proteins, provide more accurate estimates of cell death than do single-time point anatomic or biochemical assays of the number of dying neurons. More accurate estimates of death rates in vitro will increase the translatability of preclinical studies of neuroprotection and neurotoxicity.
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Affiliation(s)
- Trevor Balena
- Department of Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 02114
| | - Kyle Lillis
- Department of Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 02114
| | - Negah Rahmati
- Department of Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 02114
| | - Fatemeh Bahari
- Department of Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 02114
| | - Volodymyr Dzhala
- Department of Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 02114
| | - Eugene Berdichevsky
- Department of Electrical and Computer Engineering, Lehigh University, Bethlehem, Pennsylvania 18015
| | - Kevin Staley
- Department of Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 02114
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6
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Salek F, Mirzaei H, Khandaghi J, Javadi A, Nami Y. Apoptosis induction in cancer cell lines and anti-inflammatory and anti-pathogenic properties of proteinaceous metabolites secreted from potential probiotic Enterococcus faecalis KUMS-T48. Sci Rep 2023; 13:7813. [PMID: 37188770 DOI: 10.1038/s41598-023-34894-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2023] [Accepted: 05/09/2023] [Indexed: 05/17/2023] Open
Abstract
Potential probiotic Enterococcus faecalis KUMS-T48, isolated from a kind of Iranian traditional dairy product (Tarkhineh), was assessed for its anti-pathogenic, anti-inflammatory and anti-proliferative properties against HT-29 and AGS cancer cell lines. This strain showed strong effects on Bacillus subtilis and Listeria monocytogenes and moderate effect on Yersinia enterocolitica, while indicated weak effect on Klebsiella pneumoniae and Escherichia coli. Also, neutralizing the cell-free supernatant and treating it with catalase and proteinase K enzymes reduced the antibacterial effects. Similar to Taxol, the cell-free supernatant of E. faecalis KUMS-T48 inhibited the in vitro proliferation of both cancer cells in a dose-dependent manner, but unlike Taxol, they had no activity against normal cell line (FHs-74). Pronase-treatment of the CFS of E. faecalis KUMS-T48 abrogated its anti-proliferative capacity, thereby showing the proteinaceous nature of the cell-free supernatant. Further, induction of apoptosis-based cytotoxic mechanism by E. faecalis KUMS-T48 cell-free supernatant is related to anti-apoptotic genes ErbB-2 and ErbB-3, which is different from Taxol's apoptosis induction (intrinsic mitochondria apoptosis pathway). Also, as evidenced by a decline in interleukin 1β inflammation-promoting gene expression and a rise in the anti-inflammatory interleukin-10 gene expression in the HT-29 cell line, probiotic E. faecalis KUMS-T48 cell-free supernatant demonstrated a significant anti-inflammatory impact.
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Affiliation(s)
- Faezeh Salek
- Department of Food Hygiene, Faculty of Veterinary Medicine, Tabriz Medical Sciences, Islamic Azad University, Tabriz, Iran
| | - Hamid Mirzaei
- Department of Food Hygiene, Faculty of Veterinary Medicine, Tabriz Medical Sciences, Islamic Azad University, Tabriz, Iran
- Department of Food Biotechnology, Biotechnology Research Center, Tabriz Branch, Islamic Azad University, Tabriz, Iran
| | - Jalil Khandaghi
- Department of Food Biotechnology, Biotechnology Research Center, Tabriz Branch, Islamic Azad University, Tabriz, Iran
- Department of Food Science and Technology, Sarab Branch, Islamic Azad University, Sarab, Iran
| | - Afshin Javadi
- Department of Food Hygiene, Faculty of Veterinary Medicine, Tabriz Medical Sciences, Islamic Azad University, Tabriz, Iran
- Department of Food Biotechnology, Biotechnology Research Center, Tabriz Branch, Islamic Azad University, Tabriz, Iran
| | - Yousef Nami
- Department of Food Biotechnology, Branch for Northwest and West Region, Agricultural Biotechnology Research Institute of Iran, Agricultural Research, Education and Extension Organization (AREEO), Tabriz, Iran.
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7
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Balena T, Lillis K, Rahmati N, Bahari F, Dzhala V, Berdichevsky E, Staley K. A dynamic balance between neuronal death and clearance after acute brain injury. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.02.14.528332. [PMID: 36824708 PMCID: PMC9948967 DOI: 10.1101/2023.02.14.528332] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/16/2023]
Abstract
After acute brain injury, neuronal apoptosis may overwhelm the capacity for microglial phagocytosis, creating a queue of dying neurons awaiting clearance. The size of this queue should be equally sensitive to changes in neuronal death and the rate of phagocytosis. Using rodent organotypic hippocampal slice cultures as a model of acute perinatal brain injury, serial imaging demonstrated that the capacity for microglial phagocytosis of dying neurons was overwhelmed for two weeks. Altering phagocytosis rates, e.g. by changing the number of microglia, dramatically changed the number of visibly dying neurons. Similar effects were generated when the visibility of dying neurons was altered by changing the membrane permeability for vital stains. Canonically neuroprotective interventions such as seizure blockade and neurotoxic maneuvers such as perinatal ethanol exposure were mediated by effects on microglial activity and the membrane permeability of apoptotic neurons, and had either no or opposing effects on healthy surviving neurons. Significance After acute brain injury, microglial phagocytosis is overwhelmed by the number of dying cells. Under these conditions, the assumptions on which assays for neuroprotective and neurotoxic effects are based are no longer valid. Thus longitudinal assays of healthy cells, such as assessment of the fluorescence emission of transgenically-expressed proteins, provide more accurate estimates of cell death than do single-time-point anatomical or biochemical assays. More accurate estimates of death rates will increase the translatability of preclinical studies of neuroprotection and neurotoxicity.
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8
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Rosemary essential oil and its components 1,8-cineole and α-pinene induce ROS-dependent lethality and ROS-independent virulence inhibition in Candida albicans. PLoS One 2022; 17:e0277097. [DOI: 10.1371/journal.pone.0277097] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Accepted: 10/18/2022] [Indexed: 11/17/2022] Open
Abstract
The essential oil from Rosmarinus officinalis L., a composite mixture of plant-derived secondary metabolites, exhibits antifungal activity against virulent candidal species. Here we report the impact of rosemary oil and two of its components, the monoterpene α-pinene and the monoterpenoid 1,8-cineole, against Candida albicans, which induce ROS-dependent cell death at high concentrations and inhibit hyphal morphogenesis and biofilm formation at lower concentrations. The minimum inhibitory concentrations (100% inhibition) for both rosemary oil and 1,8-cineole were 4500 μg/ml and 3125 μg/ml for α-pinene, with the two components exhibiting partial synergy (FICI = 0.55 ± 0.07). At MIC and 1/2 MIC, rosemary oil and its components induced a generalized cell wall stress response, causing damage to cellular and organelle membranes, along with elevated chitin production and increased cell surface adhesion and elasticity, leading to complete vacuolar segregation, mitochondrial depolarization, elevated reactive oxygen species, microtubule dysfunction, and cell cycle arrest mainly at the G1/S phase, consequently triggering cell death. Interestingly, the same oils at lower fractional MIC (1/8-1/4) inhibited virulence traits, including reduction of mycelium (up to 2-fold) and biofilm (up to 4-fold) formation, through a ROS-independent mechanism.
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9
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Prasad A, Khatua A, Mohanta YK, Saravanan M, Meena R, Ghosh I. Low-dose exposure to phytosynthesized gold nanoparticles combined with glutamine deprivation enhances cell death in the cancer cell line HeLa via oxidative stress-mediated mitochondrial dysfunction and G0/G1 cell cycle arrest. NANOSCALE 2022; 14:10399-10417. [PMID: 35819245 DOI: 10.1039/d2nr02150a] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
Cancer cells use nutrients like D-glucose (Glc) and L-glutamine (Q) more efficiently for their development. This increased nutritional dependency of malignant cells has been commonly employed in various in vitro and in vivo models of anticancer therapies. This study utilized a combination of a low dose (25 μg mL-1) of S2, a phytosynthesized gold nanoparticle (AuNP) that was previously proven to be non-toxic, and deprivation of extracellular glutamine as an anticancer strategy in the human cervical cancer cell line HeLa. We discovered that 24 h Q deprivation led to a less significant decrease in the viability of HeLa cells while a low dose of S2 caused a non-significant reduction in the viability of HeLa cells. However, combining these two treatments resulted in highly significant inhibition of cell growth, as measured by the MTT test and morphological examination. Glutamine starvation in HeLa cells was found to induce cellular uptake of S2 via clathrin-mediated endocytosis, thus facilitating the improved antitumor effects of the combined treatment. Flow cytometry-based assays using fluorescent probes H2DCFDA and MitoSOX Red confirmed that this combination therapy involved the development of oxidative stress conditions owing to a surplus of cytosolic reactive oxygen species (cytoROS) and mitochondrial superoxide (mtSOX) generation. Furthermore, the investigated combinatorial treatment also indicated mitochondrial inactivity and disintegration, as evidenced by the drop in the mitochondrial membrane potential (Δψm) and the decrease in the mitochondrial mass (mtMass) in a flow-cytometric assay utilizing the probes. Tetramethylrhodamine ethyl ester and MitoTracker Green FM, respectively. Cell cycle arrest in the G0/G1 phase, induction of cell death via apoptosis/necrosis, and inhibition of migration capacities of HeLa cells were also seen after the combined treatment. Thus, this research provides insight into a new combinatorial approach for reducing the dose of nanoparticles and increasing their efficacy to better inhibit the growth of human cervical cancer cells by leveraging their extracellular glutamine dependence.
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Affiliation(s)
- Abhinav Prasad
- Biochemistry and Environmental Toxicology Laboratory, Lab#103, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
| | - Ashapurna Khatua
- Nanotoxicology Laboratory, Lab#312, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
| | - Yugal Kishore Mohanta
- Department of Applied Biology, School of Biological Sciences University of Science and Technology Meghalaya, Ri-Bhoi-793101, India.
| | - Muthupandian Saravanan
- AMR and Nanotherapeutics Laboratory, Department of Pharmacology, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences (SIMATS), Chennai 600077, India.
| | - Ramovatar Meena
- Nanotoxicology Laboratory, Lab#312, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
| | - Ilora Ghosh
- Biochemistry and Environmental Toxicology Laboratory, Lab#103, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
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10
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Platenkamp A, Detmar E, Sepulveda L, Ritz A, Rogers SL, Applewhite DA. The Drosophila melanogaster Rab GAP RN-tre cross-talks with the Rho1 signaling pathway to regulate nonmuscle myosin II localization and function. Mol Biol Cell 2020; 31:2379-2397. [PMID: 32816624 PMCID: PMC7851959 DOI: 10.1091/mbc.e20-03-0181] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/04/2022] Open
Abstract
To identify novel regulators of nonmuscle myosin II (NMII) we performed an image-based RNA interference screen using stable Drosophila melanogaster S2 cells expressing the enhanced green fluorescent protein (EGFP)-tagged regulatory light chain (RLC) of NMII and mCherry-Actin. We identified the Rab-specific GTPase-activating protein (GAP) RN-tre as necessary for the assembly of NMII RLC into contractile actin networks. Depletion of RN-tre led to a punctate NMII phenotype, similar to what is observed following depletion of proteins in the Rho1 pathway. Depletion of RN-tre also led to a decrease in active Rho1 and a decrease in phosphomyosin-positive cells by immunostaining, while expression of constitutively active Rho or Rho-kinase (Rok) rescues the punctate phenotype. Functionally, RN-tre depletion led to an increase in actin retrograde flow rate and cellular contractility in S2 and S2R+ cells, respectively. Regulation of NMII by RN-tre is only partially dependent on its GAP activity as overexpression of constitutively active Rabs inactivated by RN-tre failed to alter NMII RLC localization, while a GAP-dead version of RN-tre partially restored phosphomyosin staining. Collectively, our results suggest that RN-tre plays an important regulatory role in NMII RLC distribution, phosphorylation, and function, likely through Rho1 signaling and putatively serving as a link between the secretion machinery and actomyosin contractility.
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Affiliation(s)
| | - Elizabeth Detmar
- Department of Biology & Integrative Program for Biological and Genome Sciences, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280
| | - Liz Sepulveda
- Department of Biology, Reed College, Portland, OR 97202
| | - Anna Ritz
- Department of Biology, Reed College, Portland, OR 97202
| | - Stephen L Rogers
- Department of Biology & Integrative Program for Biological and Genome Sciences, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280
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11
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Jiang L, Poon IKH. Methods for monitoring the progression of cell death, cell disassembly and cell clearance. Apoptosis 2020; 24:208-220. [PMID: 30684146 DOI: 10.1007/s10495-018-01511-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Cell death through apoptosis, necrosis, necroptosis and pyroptosis, as well as the clearance of dead cells are crucial biological processes in the human body. Likewise, disassembly of dying cells during apoptosis to generate cell fragments known as apoptotic bodies may also play important roles in regulating cell clearance and intercellular communication. Recent advances in the field have led to the development of new experimental systems to identify cells at different stages of cell death, measure the levels of apoptotic cell disassembly, and monitor the cell clearance process using a range of in vitro, ex vivo and in vivo models. In this article, we will provide a comprehensive review of the methods for monitoring the progression of cell death, cell disassembly and cell clearance.
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Affiliation(s)
- Lanzhou Jiang
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Ivan K H Poon
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia.
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12
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Ahmed AHR, Dereli-Korkut Z, Lee JH, Piracha S, Gilchrist ML, Jiang X, Wang S. Apoptosis detection via automated algorithms to analyze biomarker translocation in reporter cells. Biotechnol Bioeng 2020; 117:1470-1482. [PMID: 31956989 DOI: 10.1002/bit.27280] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2019] [Revised: 11/26/2019] [Accepted: 01/11/2020] [Indexed: 11/10/2022]
Abstract
Rapid, efficient, and robust quantitative analyses of dynamic apoptotic events are essential in a high-throughput screening workflow. Currently used methods have several bottlenecks, specifically, limitations in available fluorophores for downstream assays and misinterpretation of statistical image data analysis. In this study, we developed cytochrome-C (Cyt-C) and caspase-3/-8 reporter cell lines using lung (PC9) and breast (T47D) cancer cells, and characterized them from the response to apoptotic stimuli. In these two reporter cell lines, the spatial fluorescent signal translocation patterns served as reporters of activations of apoptotic events, such as Cyt-C release and caspase-3/-8 activation. We also developed a vision-based, tunable, automated algorithm in MATLAB to implement the robust and accurate analysis of signal translocation in single or multiple cells. Construction of the reporter cell lines allows live monitoring of apoptotic events without the need for any other dyes or fixatives. Our algorithmic implementation forgoes the use of simple image statistics for more robust analytics. Our optimized algorithm can achieve a precision greater than 90% and a sensitivity higher than 85%. Combining our automated algorithm with reporter cells bearing a single-color dye/fluorophore, we expect our approach to become an integral component in the high-throughput drug screening workflow.
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Affiliation(s)
- A H Rezwanuddin Ahmed
- Biomedical Engineering Department, City College, City University of New York, New York City, New York
| | - Zeynep Dereli-Korkut
- Biomedical Engineering Department, City College, City University of New York, New York City, New York
| | - Joanne Haeun Lee
- Biomedical Engineering Department, City College, City University of New York, New York City, New York
| | - Sidra Piracha
- Biomedical Engineering Department, City College, City University of New York, New York City, New York
| | - M Lane Gilchrist
- Chemical Engineering Department, City College, City University of New York, New York City, New York
| | - Xuejun Jiang
- Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York City, New York
| | - Sihong Wang
- Biomedical Engineering Department, City College, City University of New York, New York City, New York
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13
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Satar NA, Fakiruddin KS, Lim MN, Mok PL, Zakaria N, Fakharuzi NA, Abd Rahman AZ, Zakaria Z, Yahaya BH, Baharuddin P. Novel triple‑positive markers identified in human non‑small cell lung cancer cell line with chemotherapy-resistant and putative cancer stem cell characteristics. Oncol Rep 2018; 40:669-681. [PMID: 29845263 PMCID: PMC6072294 DOI: 10.3892/or.2018.6461] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2017] [Accepted: 04/03/2018] [Indexed: 02/06/2023] Open
Abstract
Through the specific identification and direct targeting of cancer stem cells (CSCs), it is believed that a better treatment efficacy of cancer may be achieved. Hence, the present study aimed to identify a CSC subpopulation from adenocarcinoma cells (A549) as a model of non-small cell lung cancer (NSCLC). Initially, we sorted two subpopulations known as the triple-positive (EpCAM+/CD166+/CD44+) and triple-negative (EpCAM−/CD166−/CD44−) subpopulation using fluorescence-activated cell sorting (FACS). Sorted cells were subsequently evaluated for proliferation and chemotherapy-resistance using a viability assay and were further characterized for their clonal heterogeneity, self-renewal characteristics, cellular migration, alkaline dehydrogenase (ALDH) activity and the expression of stemness-related genes. According to our findings the triple-positive subpopulation revealed significantly higher (P<0.01) proliferation activity, exhibited better clonogenicity, was mostly comprised of holoclones and had markedly bigger (P<0.001) spheroid formation indicating a better self-renewal capacity. A relatively higher resistance to both 5-fluouracil and cisplatin with 80% expression of ALDH was observed in the triple-positive subpopulation, compared to only 67% detected in the triple-negative subpopulation indicated that high ALDH activity contributed to greater chemotherapy-resistance characteristics. Higher percentage of migrated cells was observed in the triple-positive subpopulation with 56% cellular migration being detected, compared to only 19% in the triple-negative subpopulation on day 2. This was similarly observed on day 3 in the triple-positive subpopulation with 36% higher cellular migration compared to the triple-negative subpopulation. Consistently, elevated levels of the stem cell genes such as REX1 and SSEA4 were also found in the triple-positive subpopulation indicating that the subpopulation displayed a strong characteristic of pluripotency. In conclusion, our study revealed that the triple-positive subpopulation demonstrated similar characteristics to CSCs compared to the triple-negative subpopulation. It also confirmed the feasibility of using the triple-positive (EpCAM+/CD166+/CD44+) marker as a novel candidate marker that may lead to the development of novel therapies targeting CSCs of NSCLC.
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Affiliation(s)
- Nazilah Abdul Satar
- Regenerative Medicine Cluster, Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia, 13200 Penang, Malaysia
| | - Kamal Shaik Fakiruddin
- Stem Cell Laboratory, Haematology Unit, Cancer Research Centre, Institute for Medical Research (IMR), 50588 Kuala Lumpur, Malaysia
| | - Moon Nian Lim
- Stem Cell Laboratory, Haematology Unit, Cancer Research Centre, Institute for Medical Research (IMR), 50588 Kuala Lumpur, Malaysia
| | - Pooi Ling Mok
- Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor
| | - Norashikin Zakaria
- Regenerative Medicine Cluster, Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia, 13200 Penang, Malaysia
| | - Noor Atiqah Fakharuzi
- Stem Cell Laboratory, Haematology Unit, Cancer Research Centre, Institute for Medical Research (IMR), 50588 Kuala Lumpur, Malaysia
| | - Ahmad Zuhairi Abd Rahman
- Stem Cell Laboratory, Haematology Unit, Cancer Research Centre, Institute for Medical Research (IMR), 50588 Kuala Lumpur, Malaysia
| | - Zubaidah Zakaria
- Stem Cell Laboratory, Haematology Unit, Cancer Research Centre, Institute for Medical Research (IMR), 50588 Kuala Lumpur, Malaysia
| | - Badrul Hisham Yahaya
- Regenerative Medicine Cluster, Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia, 13200 Penang, Malaysia
| | - Puteri Baharuddin
- Stem Cell Laboratory, Haematology Unit, Cancer Research Centre, Institute for Medical Research (IMR), 50588 Kuala Lumpur, Malaysia
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14
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Gwalani LA, Orange JS. Single Degranulations in NK Cells Can Mediate Target Cell Killing. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2018; 200:3231-3243. [PMID: 29592963 PMCID: PMC6020067 DOI: 10.4049/jimmunol.1701500] [Citation(s) in RCA: 81] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/30/2017] [Accepted: 03/01/2018] [Indexed: 11/19/2022]
Abstract
NK cells are cytotoxic lymphocytes important in defense against viral infection and cancer. NK cells mediate cytotoxicity predominantly through directed secretion of lytic granules, which are specialized lysosome-related organelles, containing effector molecules such as perforin and granzymes. Although many requirements for lytic granule transport to, and secretion at, the NK cell lytic synapse are known, the minimum number of degranulation events required by an NK cell to kill its target is unknown. We performed high-resolution four-dimensional confocal microscopy of human NK-target cell conjugates to quantify NK cell degranulation (using a degranulation indicator, LAMP-1-pHluorin) as well as target cell death. Despite containing almost 200 granules, we found that an individual NK cell needed only two to four degranulation events, on average, to mediate target cell death. Although NK cells released approximately one-tenth of their total lytic granule reserve upon a single target, they required just over one-hundredth of their total lytic granules to kill a target cell. Importantly, the kinetics of NK cell killing correlated to the size of and the amount of effector molecules contained within lytic granules, as well as the temporal, but not spatial, organization of degranulation events. Thus, our study answers a fundamental question as to how many degranulation events it takes for a human NK cell to kill its target.
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Affiliation(s)
- Lavesh A Gwalani
- Department of Pathology and Immunology, Baylor College of Medicine, and Center for Human Immunobiology, Texas Children's Hospital, Houston, TX 77030
| | - Jordan S Orange
- Department of Pathology and Immunology, Baylor College of Medicine, and Center for Human Immunobiology, Texas Children's Hospital, Houston, TX 77030
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15
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Harwig MC, Viana MP, Egner JM, Harwig JJ, Widlansky ME, Rafelski SM, Hill RB. Methods for imaging mammalian mitochondrial morphology: A prospective on MitoGraph. Anal Biochem 2018; 552:81-99. [PMID: 29505779 DOI: 10.1016/j.ab.2018.02.022] [Citation(s) in RCA: 67] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2017] [Revised: 02/06/2018] [Accepted: 02/22/2018] [Indexed: 12/22/2022]
Abstract
Mitochondria are found in a variety of shapes, from small round punctate structures to a highly interconnected web. This morphological diversity is important for function, but complicates quantification. Consequently, early quantification efforts relied on various qualitative descriptors that understandably reduce the complexity of the network leading to challenges in consistency across the field. Recent application of state-of-the-art computational tools have resulted in more quantitative approaches. This prospective highlights the implementation of MitoGraph, an open-source image analysis platform for measuring mitochondrial morphology initially optimized for use with Saccharomyces cerevisiae. Here Mitograph was assessed on five different mammalian cells types, all of which were accurately segmented by MitoGraph analysis. MitoGraph also successfully differentiated between distinct mitochondrial morphologies that ranged from entirely fragmented to hyper-elongated. General recommendations are also provided for confocal imaging of labeled mitochondria (using mito-YFP, MitoTracker dyes and immunostaining parameters). Widespread adoption of MitoGraph will help achieve a long-sought goal of consistent and reproducible quantification of mitochondrial morphology.
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Affiliation(s)
- Megan C Harwig
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, 53226, United States
| | - Matheus P Viana
- Visual Analytics and Comprehension Group, IBM Research, Brazil; Department of Developmental and Cell Biology and Center for Complex Biological Systems, University of California Irvine, Irvine, CA, 92697, United States
| | - John M Egner
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, 53226, United States
| | | | - Michael E Widlansky
- Division of Cardiovascular Medicine, Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, 53226, United States
| | - Susanne M Rafelski
- Department of Developmental and Cell Biology and Center for Complex Biological Systems, University of California Irvine, Irvine, CA, 92697, United States; Assay Development, Allen Institute for Cell Science, Seattle, WA, 98109, United States
| | - R Blake Hill
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, 53226, United States.
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16
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Buschhaus JM, Gibbons AE, Luker KE, Luker GD. Fluorescence Lifetime Imaging of a Caspase-3 Apoptosis Reporter. CURRENT PROTOCOLS IN CELL BIOLOGY 2017; 77:21.12.1-21.12.12. [PMID: 29227553 PMCID: PMC5729923 DOI: 10.1002/cpcb.36] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Caspase-3 is a proteolytic enzyme that functions as a key effector in apoptotic cell death. Determining activity of caspase-3 provides critical information about cancer cell viability and response to treatment. To measure apoptosis in intact cells and living mice, a fluorescence imaging reporter that detects caspase-3 activity by Förster resonance energy transfer (FRET) was used. Changes in FRET by fluorescence lifetime imaging microscopy (FLIM) were measured. Unlike FRET measurements based on fluorescence intensity, lifetime measurements are independent of reporter concentration and scattering of light in tissue, making FLIM a robust method for imaging in 3D environments. Apoptosis of breast cancer cells in 2D culture, spheroids, and in vivo murine breast tumor xenografts in response to a variety of genetic and pharmacologic methods implicated in apoptosis of cancer cells was studied. This approach for quantifying apoptosis of cancer cells is based on caspase-3 activity at single-cell resolution using FLIM. © 2017 by John Wiley & Sons, Inc.
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Affiliation(s)
- Johanna M. Buschhaus
- Center for Molecular Imaging, Department of Radiology, University of Michigan, Ann Arbor, MI, USA
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA
| | | | - Kathryn E. Luker
- Center for Molecular Imaging, Department of Radiology, University of Michigan, Ann Arbor, MI, USA
| | - Gary D. Luker
- Center for Molecular Imaging, Department of Radiology, University of Michigan, Ann Arbor, MI, USA
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA
- Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA
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17
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Neely AM, Zhao G, Schwarzer C, Stivers NS, Whitt AG, Meng S, Burlison JA, Machen TE, Li C. N-(3-Oxo-acyl)-homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts. Cell Microbiol 2017; 20. [PMID: 28876505 DOI: 10.1111/cmi.12787] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2017] [Revised: 08/23/2017] [Accepted: 09/04/2017] [Indexed: 12/19/2022]
Abstract
N-(3-Oxododecanoyl)-l-homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum-sensing molecule for bacteria-bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12-triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in "initiator" caspases or "effector" caspases. Our data indicate that C12 selectively induces the mitochondria-dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both "initiator" and "effector" caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis.
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Affiliation(s)
- Aaron M Neely
- Molecular Targets Program, James Graham Brown Cancer Center, Departments of Medicine, Pharmacology and Toxicology, University of Louisville, Louisville, KY, USA
| | - Guoping Zhao
- Molecular Targets Program, James Graham Brown Cancer Center, Departments of Medicine, Pharmacology and Toxicology, University of Louisville, Louisville, KY, USA.,Institute of Technical Biology and Agriculture Engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, China
| | - Christian Schwarzer
- Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
| | - Nicole S Stivers
- Molecular Targets Program, James Graham Brown Cancer Center, Departments of Medicine, Pharmacology and Toxicology, University of Louisville, Louisville, KY, USA
| | - Aaron G Whitt
- Molecular Targets Program, James Graham Brown Cancer Center, Departments of Medicine, Pharmacology and Toxicology, University of Louisville, Louisville, KY, USA
| | - Shuhan Meng
- Molecular Targets Program, James Graham Brown Cancer Center, Departments of Medicine, Pharmacology and Toxicology, University of Louisville, Louisville, KY, USA
| | - Joseph A Burlison
- Structural Biology Program, James Graham Brown Cancer Center, Department of Medicine, University of Louisville, Louisville, KY, USA
| | - Terry E Machen
- Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
| | - Chi Li
- Molecular Targets Program, James Graham Brown Cancer Center, Departments of Medicine, Pharmacology and Toxicology, University of Louisville, Louisville, KY, USA
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18
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19
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Peintner L, Borner C. Role of apoptosis in the development of autosomal dominant polycystic kidney disease (ADPKD). Cell Tissue Res 2017; 369:27-39. [PMID: 28560694 DOI: 10.1007/s00441-017-2628-6] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2017] [Accepted: 04/19/2017] [Indexed: 02/06/2023]
Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is a widespread genetic disorder in the Western world and is characterized by cystogenesis that often leads to end-stage renal disease (ESRD). Mutations in the pkd1 gene, encoding for polycystin-1 (PC1) and its interaction partner pkd2, encoding for polycystin-2 (PC2), are the main drivers of this disease. PC1 and PC2 form a multiprotein membrane complex at cilia sites of the plasma membrane and at intracellular membranes. This complex mediates calcium influx and stimulates various signaling pathways regulating cell survival, proliferation and differentiation. The molecular consequences of pkd1 and pkd2 mutations are still a matter of debate. In particular, the ways in which the cysts are initially formed and progress throughout the disease are unknown. The mechanisms proposed to play a role include enhanced cell proliferation, increased apoptotic cell death and diminished autophagy. In this review, we summarize our current understanding about the contribution of apoptosis to cystogenesis and ADPKD. We present the animal models and the tools and methods that have been created to analyze this process. We also critically review the data that are in favor or against the involvement of apoptosis in disease generation. We argue that apoptosis is probably not the sole driver of cystogenesis but that a cooperative action of cell death, compensatory cell proliferation and perturbed autophagy gradually establish the disease. Finally, we propose novel strategies for uncovering the mode of action of PC1 and PC2 and suggest means by which their dysfunction or loss of expression lead to cystogenesis and ADPKD development.
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Affiliation(s)
- Lukas Peintner
- Institute of Molecular Medicine and Cell Research, Albert Ludwigs University of Freiburg, Stefan Meier Strasse 17, 79104, Freiburg, Germany
| | - Christoph Borner
- Institute of Molecular Medicine and Cell Research, Albert Ludwigs University of Freiburg, Stefan Meier Strasse 17, 79104, Freiburg, Germany. .,Spemann Graduate School of Biology and Medicine, Albert Ludwigs University of Freiburg, Albertstrasse 19a, 79104, Freiburg, Germany.
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20
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Wang Q, Zhao H, Zheng T, Wang W, Zhang X, Wang A, Li B, Wang Y, Zheng Q. Otoprotective effects of mouse nerve growth factor in DBA/2J mice with early-onset progressive hearing loss. J Neurosci Res 2017; 95:1937-1950. [PMID: 28345280 DOI: 10.1002/jnr.24056] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2016] [Revised: 02/17/2017] [Accepted: 03/02/2017] [Indexed: 12/12/2022]
Abstract
As it displays progressive hair-cell loss and degeneration of spiral ganglion neurons (SGNs) characterized by early-onset progressive hearing loss (ePHL), DBA/2J is an inbred mouse strain widely used in hearing research. Mouse nerve growth factor (mNGF), as a common exogenous nerve growth factor (NGF), has been studied extensively for its ability to promote neuronal survival and growth. To determine whether mNGF can ameliorate progressive hearing loss (PHL) in DBA/2J mice, saline or mNGF was given to DBA/2J mice of either sex by daily intramuscular injection from the 1st to the 9th week after birth. At 5, 7, and 9 weeks of age, in comparison with vehicle groups, mNGF groups experienced decreased auditory-evoked brainstem response (ABR) thresholds and increased distortion product otoacoustic emission (DPOAE) amplitudes, the prevention of hair cell loss, and the inhibition of apoptosis of SGNs. Downregulation of Bak/Bax and Caspase genes and proteins in cochleae of mice receiving the mNGF treatment was detected by real-time PCR, Western blot, and immunohistochemistry. This suggests that the Bak-dependent mitochondrial apoptosis pathway may be involved in the otoprotective mechanism of mNGF in progressive hearing loss of DBA/2J mice. Our results demonstrate that mNGF can act as an otoprotectant in the DBA/2J mice for the early intervention of PHL and, thus, could become of great value in clinical applications. © 2017 Wiley Periodicals, Inc.
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Affiliation(s)
- Qingzhu Wang
- Transformative Otology and Neuroscience Center, Binzhou Medical University, Yantai, 264003, Shandong, PR China.,Department of Otolaryngology/Head and Neck Surgery, Institute of Otolaryngology, Affiliated Hospital of Binzhou Medical University, Binzhou, 256600, Shandong, PR China
| | - Hongchun Zhao
- Department of Otolaryngology/Head and Neck Surgery, Institute of Otolaryngology, Affiliated Hospital of Binzhou Medical University, Binzhou, 256600, Shandong, PR China
| | - Tihua Zheng
- Transformative Otology and Neuroscience Center, Binzhou Medical University, Yantai, 264003, Shandong, PR China
| | - Wenjun Wang
- Transformative Otology and Neuroscience Center, Binzhou Medical University, Yantai, 264003, Shandong, PR China.,Department of Otolaryngology/Head and Neck Surgery, Institute of Otolaryngology, Affiliated Hospital of Binzhou Medical University, Binzhou, 256600, Shandong, PR China.,Department of Otolaryngology-HNS, Case Western Reserve University, Cleveland, OH, 44106, USA
| | - Xiaolin Zhang
- Department of Otolaryngology/Head and Neck Surgery, Institute of Otolaryngology, Affiliated Hospital of Binzhou Medical University, Binzhou, 256600, Shandong, PR China
| | - Andi Wang
- Department of Otolaryngology-HNS, Case Western Reserve University, Cleveland, OH, 44106, USA
| | - Bo Li
- Transformative Otology and Neuroscience Center, Binzhou Medical University, Yantai, 264003, Shandong, PR China
| | - Yanfei Wang
- Transformative Otology and Neuroscience Center, Binzhou Medical University, Yantai, 264003, Shandong, PR China.,Department of Otolaryngology/Head and Neck Surgery, Institute of Otolaryngology, Affiliated Hospital of Binzhou Medical University, Binzhou, 256600, Shandong, PR China
| | - Qingyin Zheng
- Department of Otolaryngology-HNS, Case Western Reserve University, Cleveland, OH, 44106, USA
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21
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Cheng Y, He L, Prasad V, Wang S, Levy RJ. Anesthesia-Induced Neuronal Apoptosis in the Developing Retina: A Window of Opportunity. Anesth Analg 2016; 121:1325-35. [PMID: 26465931 DOI: 10.1213/ane.0000000000000714] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
BACKGROUND Anesthetics cause widespread apoptosis in the developing brain, resulting in neurocognitive abnormalities. However, it is unknown whether anesthesia-induced neurotoxicity occurs in humans because there is currently no modality to assess for neuronal apoptosis in vivo. The retina is unique in that it is the only portion of the central nervous system that can be directly visualized noninvasively. Thus, we aimed to determine whether isoflurane induces apoptosis in the developing retina. METHODS CD-1 male mouse pups underwent 1-hour exposure to isoflurane (2%) or air. After exposure, activated caspase-3, caspase-9, and caspase-8 were quantified in the retina, cytochrome c release from retinal mitochondria was assessed, and the number and types of cells undergoing apoptosis were identified. Retinal uptake and the ability of fluorescent-labeled annexin V to bind to cells undergoing natural cell death and anesthesia-induced apoptosis in the retina were determined after systemic injection. RESULTS Isoflurane activated the intrinsic apoptosis pathway in the inner nuclear layer (INL) and activated both the intrinsic and extrinsic pathways in the ganglion cell layer. Immunofluorescence demonstrated that bipolar and amacrine neurons within the INL underwent physiologic cell death in both cohorts and that amacrine cells were the likely targets of isoflurane-induced apoptosis. After injection, fluorescent-labeled annexin V was readily detected in the INL of both air-exposed and isoflurane-exposed mice and colocalized with activated caspase-3-positive cells. CONCLUSIONS These findings indicate that isoflurane-induced neuronal apoptosis occurs in the developing retina and lays the groundwork for development of a noninvasive imaging technique to detect anesthesia-induced neurotoxicity in infants and children.
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Affiliation(s)
- Ying Cheng
- From the *Division of Anesthesiology and Pain Medicine, Children's National Medical Center, The George Washington University School of Medicine and Health Sciences, Washington, DC; and †Department of Biostatistics, Columbia University, Mailman School of Public Health, New York, New York
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22
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Moreno‐Ortega AJ, Buendia I, Mouhid L, Egea J, Lucea S, Ruiz‐Nuño A, López MG, Cano‐Abad MF. CALHM1 and its polymorphism P86L differentially control Ca²⁺homeostasis, mitogen-activated protein kinase signaling, and cell vulnerability upon exposure to amyloid β. Aging Cell 2015; 14:1094-102. [PMID: 26416646 PMCID: PMC4693463 DOI: 10.1111/acel.12403] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/20/2015] [Indexed: 11/30/2022] Open
Abstract
The mutated form of the Ca2+ channel CALHM1 (Ca2+ homeostasis modulator 1), P86L‐CALHM1, has been correlated with early onset of Alzheimer's disease (AD). P86L‐CALHM1 increases production of amyloid beta (Aβ) upon extracellular Ca2+ removal and its subsequent addback. The aim of this study was to investigate the effect of the overexpression of CALHM1 and P86L‐CALHM, upon Aβ treatment, on the following: (i) the intracellular Ca2+ signal pathway; (ii) cell survival proteins ERK1/2 and Ca2+/cAMP response element binding (CREB); and (iii) cell vulnerability after treatment with Aβ. Using aequorins to measure the effect of nuclear Ca2+ concentrations ([Ca2+]n) and cytosolic Ca2+ concentrations ([Ca2+]c) on Ca2+ entry conditions, we observed that baseline [Ca2+]n was higher in CALHM1 and P86L‐CALHM1 cells than in control cells. Moreover, exposure to Aβ affected [Ca2+]c levels in HeLa cells overexpressing CALHM1 and P86L‐CALHM1 compared with control cells. Treatment with Aβ elicited a significant decrease in the cell survival proteins p‐ERK and p‐CREB, an increase in the activity of caspases 3 and 7, and more frequent cell death by inducing early apoptosis in P86L‐CALHM1‐overexpressing cells than in CALHM1 or control cells. These results suggest that in the presence of Aβ, P86L‐CALHM1 shifts the balance between neurodegeneration and neuronal survival toward the stimulation of pro‐cytotoxic pathways, thus potentially contributing to its deleterious effects in AD.
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Affiliation(s)
- Ana José Moreno‐Ortega
- Servicio de Farmacología Clínica Instituto de Investigación Sanitaria Hospital Universitario de la Princesa Madrid Spain
- Instituto Teófilo Hernando Universidad Autónoma de Madrid Madrid Spain
- Departamento de Farmacología y Terapéutica Facultad de Medicina Universidad Autónoma de Madrid Madrid Spain
| | - Izaskun Buendia
- Instituto Teófilo Hernando Universidad Autónoma de Madrid Madrid Spain
- Departamento de Farmacología y Terapéutica Facultad de Medicina Universidad Autónoma de Madrid Madrid Spain
| | - Lamia Mouhid
- Departamento de Farmacología y Terapéutica Facultad de Medicina Universidad Autónoma de Madrid Madrid Spain
| | - Javier Egea
- Servicio de Farmacología Clínica Instituto de Investigación Sanitaria Hospital Universitario de la Princesa Madrid Spain
- Instituto Teófilo Hernando Universidad Autónoma de Madrid Madrid Spain
| | - Susana Lucea
- Instituto Teófilo Hernando Universidad Autónoma de Madrid Madrid Spain
- Departamento de Farmacología y Terapéutica Facultad de Medicina Universidad Autónoma de Madrid Madrid Spain
| | - Ana Ruiz‐Nuño
- Servicio de Farmacología Clínica Instituto de Investigación Sanitaria Hospital Universitario de la Princesa Madrid Spain
- Instituto Teófilo Hernando Universidad Autónoma de Madrid Madrid Spain
| | - Manuela G. López
- Servicio de Farmacología Clínica Instituto de Investigación Sanitaria Hospital Universitario de la Princesa Madrid Spain
- Instituto Teófilo Hernando Universidad Autónoma de Madrid Madrid Spain
- Departamento de Farmacología y Terapéutica Facultad de Medicina Universidad Autónoma de Madrid Madrid Spain
| | - María F. Cano‐Abad
- Servicio de Farmacología Clínica Instituto de Investigación Sanitaria Hospital Universitario de la Princesa Madrid Spain
- Instituto Teófilo Hernando Universidad Autónoma de Madrid Madrid Spain
- Departamento de Farmacología y Terapéutica Facultad de Medicina Universidad Autónoma de Madrid Madrid Spain
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23
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Yin J, Miao P. Apoptosis Evaluation by Electrochemical Techniques. Chem Asian J 2015; 11:632-41. [DOI: 10.1002/asia.201501045] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2015] [Indexed: 01/22/2023]
Affiliation(s)
- Jian Yin
- CAS Key Lab of Bio-Medical Diagnostics; Suzhou Institute of Biomedical Engineering and Technology; Chinese Academy of Sciences; Suzhou 215163 P.R. China
| | - Peng Miao
- CAS Key Lab of Bio-Medical Diagnostics; Suzhou Institute of Biomedical Engineering and Technology; Chinese Academy of Sciences; Suzhou 215163 P.R. China
- University of Chinese Academy of Sciences; Beijing 100049 P.R. China
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24
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Salvianolic acid A attenuates TNF-α- and d-GalN-induced ER stress-mediated and mitochondrial-dependent apoptosis by modulating Bax/Bcl-2 ratio and calcium release in hepatocyte LO2 cells. Naunyn Schmiedebergs Arch Pharmacol 2015; 388:817-30. [DOI: 10.1007/s00210-015-1116-3] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2014] [Accepted: 03/13/2015] [Indexed: 02/07/2023]
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25
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Song IS, Jeong JY, Jeong SH, Kim HK, Ko KS, Rhee BD, Kim N, Han J. Mitochondria as therapeutic targets for cancer stem cells. World J Stem Cells 2015; 7:418-427. [PMID: 25815125 PMCID: PMC4369497 DOI: 10.4252/wjsc.v7.i2.418] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/27/2014] [Revised: 09/25/2014] [Accepted: 11/03/2014] [Indexed: 02/06/2023] Open
Abstract
Cancer stem cells (CSCs) are maintained by their somatic stem cells and are responsible for tumor initiation, chemoresistance, and metastasis. Evidence for the CSCs existence has been reported for a number of human cancers. The CSC mitochondria have been shown recently to be an important target for cancer treatment, but clinical significance of CSCs and their mitochondria properties remain unclear. Mitochondria-targeted agents are considerably more effective compared to other agents in triggering apoptosis of CSCs, as well as general cancer cells, via mitochondrial dysfunction. Mitochondrial metabolism is altered in cancer cells because of their reliance on glycolytic intermediates, which are normally destined for oxidative phosphorylation. Therefore, inhibiting cancer-specific modifications in mitochondrial metabolism, increasing reactive oxygen species production, or stimulating mitochondrial permeabilization transition could be promising new therapeutic strategies to activate cell death in CSCs as well, as in general cancer cells. This review analyzed mitochondrial function and its potential as a therapeutic target to induce cell death in CSCs. Furthermore, combined treatment with mitochondria-targeted drugs will be a promising strategy for the treatment of relapsed and refractory cancer.
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Zeng W, Wang X, Xu P, Liu G, Eden HS, Chen X. Molecular imaging of apoptosis: from micro to macro. Theranostics 2015; 5:559-82. [PMID: 25825597 PMCID: PMC4377726 DOI: 10.7150/thno.11548] [Citation(s) in RCA: 76] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2015] [Accepted: 02/18/2015] [Indexed: 12/21/2022] Open
Abstract
Apoptosis, or programmed cell death, is involved in numerous human conditions including neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer, and is often confused with other types of cell death. Therefore strategies that enable visualized detection of apoptosis would be of enormous benefit in the clinic for diagnosis, patient management, and development of new therapies. In recent years, improved understanding of the apoptotic machinery and progress in imaging modalities have provided opportunities for researchers to formulate microscopic and macroscopic imaging strategies based on well-defined molecular markers and/or physiological features. Correspondingly, a large collection of apoptosis imaging probes and approaches have been documented in preclinical and clinical studies. In this review, we mainly discuss microscopic imaging assays and macroscopic imaging probes, ranging in complexity from simple attachments of reporter moieties to proteins that interact with apoptotic biomarkers, to rationally designed probes that target biochemical changes. Their clinical translation will also be our focus.
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Wecksler AT, Hwang SH, Wettersten HI, Gilda JE, Patton A, Leon LJ, Carraway KL, Gomes AV, Baar K, Weiss RH, Hammock BD. Novel sorafenib-based structural analogues: in-vitro anticancer evaluation of t-MTUCB and t-AUCMB. Anticancer Drugs 2014; 25:433-46. [PMID: 24525589 DOI: 10.1097/cad.0000000000000079] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
In the current work, we carried out a mechanistic study on the cytotoxicity of two compounds, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-N-methyl-benzamide (t-AUCMB) and trans-N-methyl-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzamide (t-MTUCB), that are structurally similar to sorafenib. These compounds show strong cytotoxic responses in various cancer cell lines, despite significant differences in the induction of apoptotic events such as caspase activation and lactate dehydrogenase release in hepatoma cells. Both compounds induce autophagosome formation and LC3I cleavage, but there was little observable effect on mTORC1 or the downstream targets, S6K1 and 4E-binding protein. In addition, there was an increase in the activity of upstream signaling through the IRS1/PI3K/Akt-signaling pathway, suggesting that, unlike sorafenib, both compounds induce mammalian target of rapamycin (mTOR)-independent autophagy. The autophagy observed correlates with mitochondrial membrane depolarization, apoptosis-inducing factor release, and oxidative stress-induced glutathione depletion. However, there were no observable changes in the endoplasmic reticulum-stress markers such as binding immunoglobulin protein, inositol-requiring enzyme-α, phosphorylated eukaryotic initiation factor 2, and the lipid peroxidation marker, 4-hydroxynonenal, suggesting endoplasmic reticulum-independent oxidative stress. Finally, these compounds do not have the multikinase inhibitory activity of sorafenib, which may be reflected in their difference in the ability to halt cell cycle progression compared with sorafenib. Our findings indicate that both compounds have anticancer effects comparable with sorafenib in multiple cell lines, but they induce significant differences in apoptotic responses and appear to induce mTOR-independent autophagy. t-AUCMB and t-MTUCB represent novel chemical probes that are capable of inducing mTOR-independent autophagy and apoptosis to differing degrees, and may thus be potential tools for further understanding the link between these two cellular stress responses.
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Affiliation(s)
- Aaron T Wecksler
- Departments of aEntomology and Nematology bNeurobiology, Physiology and Behavior, University of California Davis, Davis cUC Davis Comprehensive Cancer Center dDepartment of Internal Medicine, Davis Medical Center, Division of Nephrology, University of California eDepartment of Biochemistry and Molecular Medicine, University of California Davis School of Medicine fUS Department of Veterans' Affairs Medical Center, Sacramento, California, USA
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Yan X, Zhang L, Guo J, Cao Y, Shang E, Tang Y, Ding A, Duan JA. Processing of kansui roots stir-baked with vinegar reduces kansui-induced hepatocyte cytotoxicity by decreasing the contents of toxic terpenoids and regulating the cell apoptosis pathway. Molecules 2014; 19:7237-54. [PMID: 24896263 PMCID: PMC6271383 DOI: 10.3390/molecules19067237] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2013] [Revised: 05/26/2014] [Accepted: 05/28/2014] [Indexed: 12/23/2022] Open
Abstract
Euphorbia kansui is a Traditional Chinese Medicine widely used for the treatment of oedema, ascites and asthma. However, its serious hepatotoxicity hinders its safe clinical application. The process of stir-baking with vinegar is regularly used to reduce the toxicity of kansui. Up till now, the exact mechanism of the reduction in hepatotoxicity of kansui stir-baked with vinegar has been poorly defined. In this study, decreased contents of five diterpene and one triterpene in kansui (GS-1) after stir-baking with vinegar (GS-2) was investigated by UPLC-QTOF/MS. Flow cytometry and Hoechst staining were used to show that the stir-baking with vinegar process reduces kansui-induced cell apoptosis. Furthermore, the result also indicated that kansui stir-baked with vinegar protects LO2 cells from apoptosis by increasing the cell mitochondrial membrane potential (ΔΨm), decreasing the release of cytochrome c and inhibiting the activities of caspase-9 and caspase-3 as evidenced by means of high content screening (HCS), ELISA and western blotting. These results suggested that the stir-baking vinegar could reduce the hepatotoxicity of kansui by effectively decreasing the contents of toxic terpenoids and inhibiting the intrinsic pathway of hepatocyte cell apoptosis. In conclusion, the study provided significant data for promoting safer and better clinical use of this herb.
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Affiliation(s)
- Xiaojing Yan
- Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, China.
| | - Li Zhang
- Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, China.
| | - Jianming Guo
- Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, China.
| | - Yudan Cao
- Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, China.
| | - Erxin Shang
- Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, China.
| | - Yuping Tang
- Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, China.
| | - Anwei Ding
- Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, China.
| | - Jin-Ao Duan
- Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, China.
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Dason JS, Smith AJ, Marin L, Charlton MP. Cholesterol and F-actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane. J Physiol 2013; 592:621-33. [PMID: 24297851 DOI: 10.1113/jphysiol.2013.265447] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Synaptic vesicles (SVs) and their proteins must be recycled for sustained synaptic transmission. We tested the hypothesis that SV cholesterol is required for proper sorting of SV proteins during recycling in live presynaptic terminals. We used the reversible block of endocytosis in the Drosophila temperature-sensitive dynamin mutant shibire-ts1 to trap exocytosed SV proteins, and then examined the effect of experimental treatments on the distribution of these proteins within the presynaptic plasma membrane by confocal microscopy. SV proteins synaptotagmin, vglut and csp were clustered following SV trapping in control experiments but dispersed in samples treated with the cholesterol chelator methyl-β-cyclodextrin to extract SV cholesterol. There was accumulation of phosphatidylinositol (4,5)-bisphosphate (PIP2) in presynaptic terminals following SV trapping and this was reduced following SV cholesterol extraction. Reduced PIP2 accumulation was associated with disrupted accumulation of actin in presynaptic terminals. Similar to vesicular cholesterol extraction, disruption of actin by latrunculin A after SV proteins had been trapped on the plasma membrane resulted in the dispersal of SV proteins and prevented recovery of synaptic transmission due to impaired endocytosis following relief of the endocytic block. Our results demonstrate that vesicular cholesterol is required for aggregation of exocytosed SV proteins in the presynaptic plasma membrane and are consistent with a mechanism involving regulation of PIP2 accumulation and local actin polymerization by cholesterol. Thus, alteration of membrane or SV lipids may affect the ability of synapses to undergo sustained synaptic transmission by compromising the recycling of SV proteins.
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Affiliation(s)
- Jeffrey S Dason
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada, M5S 1A8.
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Li XH, de Castro-Borges W, Parker-Manuel S, Vance GM, Demarco R, Neves LX, Evans GJO, Wilson RA. The schistosome oesophageal gland: initiator of blood processing. PLoS Negl Trop Dis 2013; 7:e2337. [PMID: 23936568 PMCID: PMC3723592 DOI: 10.1371/journal.pntd.0002337] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2013] [Accepted: 06/15/2013] [Indexed: 11/19/2022] Open
Abstract
BACKGROUND Although the ultrastructure of the schistosome esophageal gland was described >35 years ago, its role in the processing of ingested blood has never been established. The current study was prompted by our identification of MEG-4.1 expression in the gland and the observation of erythrocyte uncoating in the posterior esophagus. METHODOLOGY/PRINCIPAL FINDINGS The salient feature of the posterior esophagus, characterized by confocal and electron microscopy, is the enormous increase in membrane surface area provided by the plate-like extensions and basal invaginations of the lining syncytium, with unique crystalloid vesicles releasing their contents between the plates. The feeding process was shown by video microscopy to be divided into two phases, blood first accumulating in the anterior lumen before passing as a bolus to the posterior. There it streamed around a plug of material revealed by confocal microscopy as tethered leucocytes. These were present in far larger numbers than predicted from the volume of the lumen, and in varying states of damage and destruction. Intact erythrocytes were detected in the anterior esophagus but not observed thereafter, implying that their lysis occurred rapidly as they enter the posterior. Two further genes, MEGs 4.2 and 14, were shown to be expressed exclusively in the esophageal gland. Bioinformatics predicted that MEGs 4.1 and 4.2 possessed a common hydrophobic region with a shared motif, while antibodies to SjMEG-4.1 showed it was bound to leucocytes in the esophageal lumen. It was also predicted that MEGs 4.1 and 14 were heavily O-glycosylated and this was confirmed for the former by 2D-electrophoresis and Western blotting. CONCLUSIONS/SIGNIFICANCE The esophageal gland and its products play a central role in the processing of ingested blood. The binding of host antibodies in the esophageal lumen shows that some constituents are antibody targets and could provide a new source of vaccine candidates.
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Affiliation(s)
- Xiao-Hong Li
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Key Laboratory of Parasitology and Vector Biology, Ministry of Health, Shanghai, People's Republic of China.
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Iyer D, Ray RD, Pappas D. High temporal resolution fluorescence measurements of a mitochondrial dye for detection of early stage apoptosis. Analyst 2013; 138:4892-7. [PMID: 23831722 DOI: 10.1039/c3an01142a] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
In the present study, early stage apoptosis is explored with high temporal resolution. In addition to monitoring early apoptosis induction in single cells by ultrasensitive confocal fluorescence microscopy (UCFM), the mitochondrial protein release kinetics was explored. The current study shows development and optimization of a novel, rapid apoptosis assay to explore the earliest changes in cells by the intrinsic apoptosis pathway. We show that early apoptotic changes in the mitochondria begin nearly simultaneously with the addition of an apoptosis-inducing drug, such as staurosporine. With a temporal resolution of five minutes, this non-invasive analytical technique can elucidate the earliest apoptotic events in living cells. Moreover, our results show that the mitochondrial inter-membrane proteins are not involved in the extrinsic pathway of Ramos cells mediated by an anti-CD95 antibody. Additional techniques such as light microscopy and flow cytometry were employed to confirm the results obtained by ultrasensitive confocal fluorescence microscopy. The results of this study help to understand the earliest mechanisms of apoptosis induction in cells, enabling new methods of drug testing and dose-response analyses.
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Affiliation(s)
- Divya Iyer
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA
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Spiclomazine induces apoptosis associated with the suppression of cell viability, migration and invasion in pancreatic carcinoma cells. PLoS One 2013; 8:e66362. [PMID: 23840452 PMCID: PMC3688794 DOI: 10.1371/journal.pone.0066362] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2012] [Accepted: 05/03/2013] [Indexed: 12/14/2022] Open
Abstract
The effective treatment for pancreatic carcinoma remains critically needed. Herein, this current study showed that spiclomazine treatment caused a reduction in viability in pancreatic carcinoma cell lines CFPAC-1 and MIA PaCa-2 in vitro. It was notable in this regard that, compared with pancreatic carcinoma cells, normal human embryonic kidney (HEK-293) and liver (HL-7702) cells were more resistant to the antigrowth effect of spiclomazine. Biochemically, spiclomazine treatment regulated the expression of protein levels in the apoptosis related pathways. Consistent with this effect, spiclomazine reduced the mitochondria membrane potential, elevated reactive oxygen species, and activated caspase-3/9. In addition, a key finding from this study was that spiclomazine suppressed migration and invasion of cancer cells through down-regulation of MMP-2/9. Collectively, the proposed studies did shed light on the antiproliferation effect of spiclomazine on pancreatic carcinoma cell lines, and further clarified the mechanisms that spiclomazine induced apoptosis associated with the suppression of migration and invasion.
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Abstract
Podocytes are highly specialized epithelial cells that line the urinary surface of the glomerular capillary tuft. To maintain kidney filtration, podocytes oppose the high intraglomerular hydrostatic pressure, form a molecular sieve, secrete soluble factors to regulate other glomerular cell types, and provide synthesis and maintenance of the glomerular basement membrane. Impairment of any of these functions after podocyte injury results in proteinuria and possibly renal failure. Loss of glomerular podocytes is a key feature for the progression of renal diseases, and detached podocytes can be retrieved in the urine of patients with progressive glomerular diseases. Thus, the concept of podocyte loss as a hallmark of progressive glomerular disease has been widely accepted. However, the nature of events that promote podocyte detachment and whether detachment is preceded by any kind of podocyte cell death, such as apoptosis, necroptosis, or necrosis, still remains unclear and is discussed in this review.
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Affiliation(s)
- Pierre-Louis Tharaux
- PARCC Paris Cardiovascular Centre, Institut National de la Santé et de la Recherche Médicale, Paris, France.
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Eupomatenoid-5 Isolated from Leaves of Piper regnellii Induces Apoptosis in Leishmania amazonensis. EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE 2013; 2013:940531. [PMID: 23573160 PMCID: PMC3618946 DOI: 10.1155/2013/940531] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/15/2013] [Accepted: 02/26/2013] [Indexed: 11/24/2022]
Abstract
Leishmania spp. are protozoa responsible for leishmaniasis, a neglected disease that kills up to 50,000 people every year. Current therapies mainly rely on antimonial drugs that are inadequate because of their poor efficacy and safety and increased drug resistance. An urgent need exists to find new and more affordable drugs. Our previous study demonstrated the antileishmanial activity of eupomatenoid-5, a neolignan obtained from leaves of Piper regnellii var. pallescens. The aim of the present study was to clarify the mode of action of eupomatenoid-5 against L. amazonensis. We used biochemical and morphological techniques and demonstrated that eupomatenoid-5 induced cell death in L. amazonensis promastigotes, sharing some phenotypic features observed in metazoan apoptosis, including increased reactive oxygen species production, hypopolarization of mitochondrial potential, phosphatidylserine exposure, decreased cell volume, and G0/G1 phase cell cycle arrest.
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Jeong SH, Song IS, Kim HK, Lee SR, Song S, Suh H, Yoon YG, Yoo YH, Kim N, Rhee BD, Ko KS, Han J. An analogue of resveratrol HS-1793 exhibits anticancer activity against MCF-7 cells via inhibition of mitochondrial biogenesis gene expression. Mol Cells 2012; 34:357-65. [PMID: 23104437 PMCID: PMC3887771 DOI: 10.1007/s10059-012-0081-7] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2012] [Revised: 07/24/2012] [Accepted: 09/07/2012] [Indexed: 12/20/2022] Open
Abstract
Resveratrol is a phytoalexin and polyphenol derived from grapes, berries, and peanuts. It has been shown to mediate death of a wide variety of cancer cells. Although resveratrol is considered an important potential chemotherapeutic agent, it is required at high doses to achieve a biologically or physiologically significant effect, which may be impractical for treating cancer. Thus, a more stable and potent derivative of resveratrol, with more effective tumoricidal activity, must be developed. A novel resveratrol analog, HS-1793, has recently been synthesized and was determined to exhibit a greater decrease in cancer cell viability than resveratrol. However, the underlying mechanism of HS-1793-induced cancer cell death remains unknown. We thus investigated the mechanism by which HS-1793 induces cell death and assessed whether this occurs through a mitochondrial-mediated mechanism. Using the MCF-7 breast cancer cell line, we determined that HS-1793 treatment significantly increased cell death at a relatively low dose compared with resveratrol. HS-1793 treatment more significantly decreased mitochondrial membrane potential, cellular ATP concentration, and cellular oxygen consumption rate than resveratrol treatment. At the molecular level, HS-1793 treatment down-regulated the expression of major mitochondrial biogenesis-regulating proteins, including mitochondrial transcriptional factor A (TFAM), Tu translation elongation factor (TUFM), and single-stranded DNA-binding protein. We conclude that HS- 1793 acts by regulating the expression of TFAM and TUFM, leading to a block in normal mitochondrial function, which sensitizes cancer cells to cell death. We therefore propose that HS-1793 can be a useful chemosensitization agent, which together with other such agents can efficiently target cancer cells.
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Affiliation(s)
- Seung Hun Jeong
- National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan 614-735, Korea
| | - In Sung Song
- National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan 614-735, Korea
| | - Hyoung Kyu Kim
- National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan 614-735, Korea
| | - Sung Ryul Lee
- National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan 614-735, Korea
| | | | | | | | | | - Nari Kim
- National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan 614-735, Korea
| | - Byoung Doo Rhee
- National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan 614-735, Korea
| | - Kyung Soo Ko
- National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan 614-735, Korea
| | - Jin Han
- National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan 614-735, Korea
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Kominami K, Nakabayashi J, Nagai T, Tsujimura Y, Chiba K, Kimura H, Miyawaki A, Sawasaki T, Yokota H, Manabe N, Sakamaki K. The molecular mechanism of apoptosis upon caspase-8 activation: quantitative experimental validation of a mathematical model. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2012; 1823:1825-40. [PMID: 22801217 DOI: 10.1016/j.bbamcr.2012.07.003] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/31/2012] [Revised: 06/29/2012] [Accepted: 07/06/2012] [Indexed: 12/22/2022]
Abstract
Caspase-8 (CASP8) is a cysteine protease that plays a pivotal role in the extrinsic apoptotic signaling pathway via death receptors. The kinetics, dynamics, and selectivity with which the pathway transmits apoptotic signals to downstream molecules upon CASP8 activation are not fully understood. We have developed a system for using high-sensitivity FRET-based biosensors to monitor the protease activity of CASP8 and its downstream effector, caspase-3, in living single cells. Using this system, we systematically investigated the caspase cascade by regulating the magnitude of extrinsic signals received by the cell. Furthermore, we determined the molar concentration of five caspases and Bid required for hierarchical transmission of apoptotic signals in a HeLa cell. Based on these quantitative experimental data, we validated a mathematical model suitable for estimation of the kinetics and dynamics of caspases, which predicts the minimal concentration of CASP8 required to act as an initiator. Consequently, we found that less than 1% of the total CASP8 proteins are sufficient to set the apoptotic program in motion if activated. Taken together, our findings demonstrate the precise cascade of CASP8-mediated apoptotic signals through the extrinsic pathway.
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Affiliation(s)
- Katsuya Kominami
- Department of Animal Development and Physiology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
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Liu Z, Li D, Yu L, Niu F. Gallic acid as a cancer-selective agent induces apoptosis in pancreatic cancer cells. Chemotherapy 2012; 58:185-94. [PMID: 22739044 DOI: 10.1159/000337103] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2011] [Accepted: 02/05/2012] [Indexed: 12/16/2022]
Abstract
BACKGROUND Gallic acid (GA) is a plant phenol isolated from water caltrop which is reported to have anti-inflammatory and anti-cancer effects. In this study, the antiproliferative effect of GA on human pancreatic cancer cell lines CFPAC-1 and MiaPaCa-2 as well as hepatocytes HL-7702 as normal cells was examined. Particularly, the mechanism of GA-induced apoptosis in MiaPaCa-2 cells in vitro was further studied. METHODS Cell viability was measured using SRB assay, and apoptosis was detected by Hoechst staining and annexin V-PI staining assays. Mitochondrial membrane potential was detected by rhodamine-123 staining. Flow cytometry analysis was employed to detect the apoptosis-related events. RESULTS GA inhibited the proliferation of CFPAC-1 and MiaPaCa-2 cells in a time- and dose-dependent manner, with IC(50)S of 102.3 ± 2.4 and 135.2 ± 0.6 µM at 48 h, respectively. GA treatment led to the increased proportion of cell apoptosis from 12.5 ± 0.72 to 78.3 ± 2.48% at the concentrations of 6.25 and 25.0 µg/ml, which was evidenced again by chromatins staining assay. Also, GA activated caspase-3, caspase-9, and reactive oxygen species, elevated Bax expression and [Ca(2+)](i) and reduced mitochondrial membrane potential (ΔΨm) in MiaPaCa-2 cells. Remarkably, when compared with human normal cells HL-7702 (IC(50) >100 µg/ml), GA showed selective toxicity for cancer cells. CONCLUSIONS GA can function as a cancer-selective agent by inducing apoptosis in MiaPaCa-2 cells via the mitochondria-mediated pathways. To the best of our knowledge, GA should open up new opportunities for the therapy of pancreatic cancer.
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Affiliation(s)
- Zuojia Liu
- School of Public Health, Jilin University, Changchun, PR China
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Liu Z, Li D, Zhao W, Zheng X, Wang J, Wang E. A potent lead induces apoptosis in pancreatic cancer cells. PLoS One 2012; 7:e37841. [PMID: 22745658 PMCID: PMC3380052 DOI: 10.1371/journal.pone.0037841] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2011] [Accepted: 04/28/2012] [Indexed: 02/05/2023] Open
Abstract
Pancreatic cancer is considered a lethal and treatment-refractory disease. To obtain a potent anticancer drug, the cytotoxic effect of 2-(benzo[d]oxazol-3(2H)-ylmethyl)- 5-((cyclohexylamino)methyl)benzene-1,4-diol, dihydrochloride (NSC48693) on human pancreatic cancer cells CFPAC-1, MiaPaCa-2, and BxPC-3 was assessed invitro. The proliferation of CFPAC-1, MiaPaCa-2, and BxPC-3 is inhibited with IC50 value of 12.9±0.2, 20.6±0.3, and 6.2±0.6 µM at 48 h, respectively. This discovery is followed with additional analysis to demonstrate that NSC48693 inhibition is due to induction of apoptosis, including Annexin V staining, chromatins staining, and colony forming assays. It is further revealed that NSC48693 induces the release of cytochrome c, reduces mitochondrial membrane potential, generates reactive oxygen species, and activates caspase. These results collectively indicate that NSC48693 mainly induces apoptosis of CFPAC-1, MiaPaCa-2, and BxPC-3 cells by the mitochondrial-mediated apoptotic pathway. Excitingly, the study highlights an encouraging inhibition effect that human embryonic kidney (HEK-293) and liver (HL-7702) cells are more resistant to the antigrowth effect of NSC48693 compared to the three cancer cell lines. From this perspective, NSC48693 should help to open up a new opportunity for the treatment of patients with pancreatic cancer.
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Affiliation(s)
- Zuojia Liu
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, China
| | - Dan Li
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, China
| | - Wenjing Zhao
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, China
| | - Xiliang Zheng
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, China
| | - Jin Wang
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, China
- Department of Chemistry and Physics, State University of New York, Stony Brook, New York, United States of America
- * E-mail: (EW); (JW)
| | - Erkang Wang
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, China
- * E-mail: (EW); (JW)
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Acinetobacter calcoaceticus-baumannii complex strains induce caspase-dependent and caspase-independent death of human epithelial cells. Curr Microbiol 2012; 65:319-29. [PMID: 22684803 PMCID: PMC3401494 DOI: 10.1007/s00284-012-0159-7] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2012] [Accepted: 05/15/2012] [Indexed: 12/25/2022]
Abstract
We investigated interactions of human isolates of Acinetobacter calcoaceticus–baumannii complex strains with epithelial cells. The results showed that bacterial contact with the cells as well as adhesion and invasion were required for induction of cytotoxicity. The infected cells revealed hallmarks of apoptosis characterized by cell shrinking, condensed chromatin, and internucleosomal fragmentation of nuclear DNA. The highest apoptotic index was observed for 4 of 10 A.calcoaceticus and 4 of 7 A. baumannii strains. Moreover, we observed oncotic changes: cellular swelling and blebbing, noncondensed chromatin, and the absence of DNA fragmentation. The highest oncotic index was observed in cells infected with 6 A.calcoaceticus isolates. Cell-contact cytotoxicity and cell death were not inhibited by the pan-caspase inhibitor z-VAD-fmk. Induction of oncosis was correlated with increased invasive ability of the strains. We demonstrated that the mitochondria of infected cells undergo structural and functional alterations which can lead to cell death. Infected apoptotic and oncotic cells exhibited loss of mitochondrial transmembrane potential (ΔΨm). Bacterial infection caused generation of nitric oxide and reactive oxygen species. This study indicated that Acinetobacter spp. induced strain-dependent distinct types of epithelial cell death that may contribute to the pathogenesis of bacterial infection.
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Asphahani F, Thein M, Wang K, Wood D, Wong SS, Xu J, Zhang M. Real-time characterization of cytotoxicity using single-cell impedance monitoring. Analyst 2012; 137:3011-9. [PMID: 22498491 DOI: 10.1039/c2an16079j] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Cellular impedance sensors have attracted great attention as a powerful characterization tool for real-time, label-free detection of cytotoxic agents. However, impedance measurements with conventional cell-based sensors that host multiple cells on a single electrode neither provide optimal cell signal sensitivity nor are capable of recording individual cell responses. Here we use a single-cell based platform to monitor cellular impedance on planar microelectrodes to characterize cellular death. In this study, individual cells were selectively patterned on microelectrodes with each hosting one live cell through ligand-mediated natural cell adhesion. Changes in cellular morphology and cell-electrode adherence were monitored after the patterned cells were treated with varying concentrations of hydrogen peroxide, sodium arsenite, and disodium hydrogen arsenate, three potent toxicants related to neurotoxicity and oxidative stress. At low toxicant concentrations, impedance waveforms acquired from individual cells showed variable responses. A time- and concentration-dependent response was seen in the averaged single-cell impedance waveform for all three toxicants. The apoptosis and necrosis characterizations were performed to validate cell impedance results. Furthermore, time constants of apoptosis and necrosis in response to toxicant exposure were analytically established using an equivalent circuit model that characterized the mechanisms of cell death.
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Affiliation(s)
- Fareid Asphahani
- Department of Materials Science & Engineering, University of Washington, Seattle, Washington 98195-2120, USA
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41
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Xue X, You S, Zhang Q, Wu Y, Zou GZ, Wang PC, Zhao YL, Xu Y, Jia L, Zhang X, Liang XJ. Mitaplatin increases sensitivity of tumor cells to cisplatin by inducing mitochondrial dysfunction. Mol Pharm 2012; 9:634-44. [PMID: 22289032 DOI: 10.1021/mp200571k] [Citation(s) in RCA: 78] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Tumor resistance to chemotherapy is the major obstacle to employ cisplatin, one of the broadly used chemotherapeutic drugs, for effective treatment of various tumors in the clinic. Most acknowledged mechanisms of cancer resistance to cisplatin focus on increased nuclear DNA repair or detoxicity of cisplatin. We previously demonstrated that there was a unique metabolic profile in cisplatin-resistant (CP-r) human epidermoid adenocarcinoma KB-CP 20 and hepatoma BEL 7404-CP 20 cancer cells. In this study, we further defined hyperpolarized mitochondrial membrane potentials (Δψ(m)) in CP-r KB-CP 20 and BEL 7404-CP 20 cells compared to the cisplatin-sensitive (CP-s) KB-3-1 and BEL 7404 cells. Based on the mitochondrial dysfunction, mitaplatin was designed with two mitochondrial-targeting moieties [dichloroacetate (DCA) units] to the axial positions of a six-coordinate Pt(IV) center to sensitize cisplatin resistance. It was found that mitaplatin induced more apoptosis in CP-r KB-CP 20 and BEL 7404-CP 20 cells than that of cisplatin, DCA and cisplatin/DCA compared on an equal molar basis. There was more platinum accumulation in mitaplatin-treated CP-r cells due to enhanced transmembrane permeability of lipophilicity, and mitaplatin also showed special targeting to mitochondria. Moreover, in the case of treatment with mitaplatin, the dramatic collapse of Δψ(m) was shown in a dose-dependent manner, which was confirmed by FACS and confocal microscopic measurements. Reduced glucose utilization of CP-r cells was detected with specifically inhibited phosphorylation of pyruvate dehydrogenase (PDH) at Ser-232, Ser-293, and Ser-300 of the E1α subunit when treated with mitaplatin, which was indicated to modulate the abnormal glycolysis of resistant cells. The present study suggested novel mitochondrial mechanism of mitaplatin circumventing cisplatin resistance toward CP-r cells as a carrier across membrane to produce CP-like cytotoxicity and DCA-like mitochondria-dependent apoptosis. Therefore, mitochondria targeting compounds would be more vulnerable and selective to overcome cisplatin resistance due to the unique metabolic properties of CP-r cancer cells.
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Affiliation(s)
- Xue Xue
- CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology of China, Beijing 100190, PR China
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Reis Y, Bernardo-Faura M, Richter D, Wolf T, Brors B, Hamacher-Brady A, Eils R, Brady NR. Multi-parametric analysis and modeling of relationships between mitochondrial morphology and apoptosis. PLoS One 2012; 7:e28694. [PMID: 22272225 PMCID: PMC3260148 DOI: 10.1371/journal.pone.0028694] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2010] [Accepted: 11/14/2011] [Indexed: 12/20/2022] Open
Abstract
Mitochondria exist as a network of interconnected organelles undergoing constant fission and fusion. Current approaches to study mitochondrial morphology are limited by low data sampling coupled with manual identification and classification of complex morphological phenotypes. Here we propose an integrated mechanistic and data-driven modeling approach to analyze heterogeneous, quantified datasets and infer relations between mitochondrial morphology and apoptotic events. We initially performed high-content, multi-parametric measurements of mitochondrial morphological, apoptotic, and energetic states by high-resolution imaging of human breast carcinoma MCF-7 cells. Subsequently, decision tree-based analysis was used to automatically classify networked, fragmented, and swollen mitochondrial subpopulations, at the single-cell level and within cell populations. Our results revealed subtle but significant differences in morphology class distributions in response to various apoptotic stimuli. Furthermore, key mitochondrial functional parameters including mitochondrial membrane potential and Bax activation, were measured under matched conditions. Data-driven fuzzy logic modeling was used to explore the non-linear relationships between mitochondrial morphology and apoptotic signaling, combining morphological and functional data as a single model. Modeling results are in accordance with previous studies, where Bax regulates mitochondrial fragmentation, and mitochondrial morphology influences mitochondrial membrane potential. In summary, we established and validated a platform for mitochondrial morphological and functional analysis that can be readily extended with additional datasets. We further discuss the benefits of a flexible systematic approach for elucidating specific and general relationships between mitochondrial morphology and apoptosis.
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Affiliation(s)
- Yara Reis
- Division of Theoretical Bioinformatics, German Cancer Research Center and Institute of Pharmacy and Molecular Biotechnology, Bioquant, University of Heidelberg, Heidelberg, Germany
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Fulda S, Kroemer G. Mitochondria as therapeutic targets for the treatment of malignant disease. Antioxid Redox Signal 2011; 15:2937-49. [PMID: 21644835 DOI: 10.1089/ars.2011.4078] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
SIGNIFICANCE Mitochondria exert vital functions during normal physiology and are also centrally involved in the regulation of various modes of cell death. Thus, engaging the mitochondrial apoptosis pathway presents an attractive possibility to activate lethal effectors in cancer cells. RECENT ADVANCES Compounds that directly target mitochondria offer the advantage to initiate mitochondrial outer membrane permeabilization independently of upstream signal transduction elements that are frequently impaired in human cancers. As a consequence, mitochondrion-targeted agents may bypass some forms of drug resistance. CRITICAL ISSUES An ever-increasing number of compounds, including natural compounds and rationally designed drugs, has been shown to directly act on mitochondria. FUTURE DIRECTIONS Forthcoming insights into the fine regulation of mitochondrial apoptosis will likely open future perspectives for cancer drug development.
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Affiliation(s)
- Simone Fulda
- Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt am Main, Germany.
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Visagie MH, Joubert AM. In vitro effects of 2-methoxyestradiol-bis-sulphamate on reactive oxygen species and possible apoptosis induction in a breast adenocarcinoma cell line. Cancer Cell Int 2011; 11:43. [PMID: 22152028 PMCID: PMC3251537 DOI: 10.1186/1475-2867-11-43] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2011] [Accepted: 12/12/2011] [Indexed: 01/20/2023] Open
Abstract
Background In the search for anticancer agents, a promising 17-β-estradiol metabolite, 2-methoxyestradiol (2ME2) was found that exerts antiproliferative in vitro and in vivo activity. Since 2ME2 has limited biological accessibility and rapid metabolic degradation, the purpose of this study was to investigate the in vitro influence exerted by an analogue of 2ME2 namely 2-methoxyestradiol-bis-sulphamate (2MEBM) in a breast adenocarcinoma cell line (MCF-7). Methods This was conducted by investigating 2MEBM's in vitro influence on cell cycle progression, mitochondrial membrane potential and possible production of reactive oxygen species (ROS) generation. In vitro effects of 2MEBM on cell cycle progression was demonstrated by means of flow cytometry using propidium iodide. Hydrogen peroxide and superoxide production was investigated using 2,7-dichlorofluorescein diacetate and hydroethidine, respectively. The probable reduction in the mitochondrial membrane potential was demonstrated using a MitoCapture™ kit. Results Cell cycle progression revealed the presence of a sub-G1 apoptotic peak. Reduction of mitochondrial membrane potential after exposure to 2MEBM was demonstrated and an increase in ROS production was also observed. Conclusion This study verified that 2MEBM exposure resulted in apoptosis induction, increased ROS production and reduced mitochondrial membrane potential in a tumorigenic breast epithelial cell line. Data obtained from this project contributes to the unravelling of the in vitro signal transduction of 2MEBM in tumorigenic cell lines.
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Affiliation(s)
- Michelle H Visagie
- Department of Physiology, University of Pretoria, P,O, Box 2034, Pretoria, 0001, South Africa.
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Miller MA, Barkal L, Jeng K, Herrlich A, Griffith LG, Lauffenburger DA. Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous determination of multiple protease activities. Integr Biol (Camb) 2011; 3:422-38. [PMID: 21180771 PMCID: PMC3173501 DOI: 10.1039/c0ib00083c] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Matrix metalloproteinases (MMPs) and A Disintegrin and Metalloproteinases (ADAMs) are two related protease families that play key roles in matrix remodeling and growth factor ligand shedding. Directly ascertaining the proteolytic activities of particular MMPs and ADAMs in physiological environments in a non-invasive, real-time, multiplex manner remains a challenge. This work describes Proteolytic Activity Matrix Analysis (PrAMA), an integrated experimental measurement and mathematical analysis framework for simultaneously determining the activities of particular enzymes in complex mixtures of MMPs and ADAMs. The PrAMA method interprets dynamic signals from panels of moderately specific FRET-based polypeptide protease substrates to deduce a profile of specific MMP and ADAM proteolytic activities. Deconvolution of signals from complex mixtures of proteases is accomplished using prior data on individual MMP/ADAM cleavage signatures for the substrate panel measured with purified enzymes. We first validate PrAMA inference using a compendium of roughly 4000 measurements involving known mixtures of purified enzymes and substrates, and then demonstrate application to the live-cell response of wildtype, ADAM10-/-, and ADAM17-/- fibroblasts to phorbol ester and ionomycin stimulation. Results indicate PrAMA can distinguish closely related enzymes from each other with high accuracy, even in the presence of unknown background proteolytic activity. PrAMA offers a valuable tool for applications ranging from live-cell in vitro assays to high-throughput inhibitor screening with complex enzyme mixtures. Moreover, our approach may extend to other families of proteases, such as caspases and cathepsins, that also can lack highly-specific substrates.
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Affiliation(s)
- Miles A. Miller
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139
| | - Layla Barkal
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139
| | - Karen Jeng
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139
| | - Andreas Herrlich
- Whitehead Institute for Biomedical Research, Cambridge, MA, 02139
| | - Linda G. Griffith
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139
| | - Douglas A. Lauffenburger
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139
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Liu X, Lin D, Ma W. Quantitative analysis of intracellular calcium and mitochondrial kinetic fluorescence changes in GSNO-induced thymocyte early apoptosis. J Fluoresc 2011; 21:1285-92. [PMID: 21210193 DOI: 10.1007/s10895-010-0811-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2010] [Accepted: 12/29/2010] [Indexed: 11/27/2022]
Abstract
A fluorescence microscopy imaging technique was applied to observe the single-cell kinetic changes of intracellular Ca(2+) concentration ([Ca(2+)](i)) and mitochondrial membrane potential (ΔΨ(m)) during the early stage of S-nitrosoglutathione (GSNO)-induced thymocytes apoptosis. The kinetic features of [Ca(2+)](i) and ΔΨ(m) were quantitatively analyzed and compared by fitting the fluorescence intensity data. The mathematical parameter, inflection point which indicated the time point when [Ca(2+)](i) or ΔΨ(m) changed the most rapidly, was proposed to analyze the fitting curve. The results revealed that the inflection point of [Ca(2+)](i) always appeared prior to that of ΔΨ(m) during apoptosis induced by a certain GSNO concentration. Both the [Ca(2+)](i) and ΔΨ(m) changed in a GSNO concentration-dependent manner. Another parameter, half-max effect point was also employed and displayed the similar results. Such quantitative analyses of real-time observations at the single-cell level are useful for interpreting the sequence of the biological events operating in GSNO-induced thymocyte apoptosis.
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Affiliation(s)
- Xiaochen Liu
- Key Laboratory for Atomic and Molecular Nanosciences of Education Ministry, Department of Physics, Tsinghua University, Beijing 100084, China
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Wang X, Olberding KE, White C, Li C. Bcl-2 proteins regulate ER membrane permeability to luminal proteins during ER stress-induced apoptosis. Cell Death Differ 2011; 18:38-47. [PMID: 20539308 PMCID: PMC2947581 DOI: 10.1038/cdd.2010.68] [Citation(s) in RCA: 89] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2009] [Revised: 04/13/2010] [Accepted: 05/03/2010] [Indexed: 01/25/2023] Open
Abstract
Endoplasmic reticulum (ER) stress-induced apoptosis may arise from multiple environmental and pharmacological causes, but the precise mechanism(s) involved are not completely known. Members of Bcl-2 protein family are important regulators of apoptosis. In this study, we report that in a process dependent on the proapoptotic Bcl-2 members Bax and Bak, exogenously expressed fluorescent protein localized to the ER lumen is released into the cytosol in cells undergoing ER stress. Upon ER stress induction, endogenous ER luminal proteins are also released into the cytosol in a similar manner accompanied by translocation and anchorage of Bax to the ER membrane. In addition, Bax and truncated-Bid (tBid) mediate a global increase in ER membrane permeability to ER luminal proteins in vitro. Importantly, antiapoptotic Bcl-X(L) antagonizes the effects of proapoptotic Bcl-2 proteins on ER membrane permeability. Consistent with Bax translocation to the ER membrane in whole apoptotic cells, there is also increased tight association of Bax with the ER membrane correlated with the increase in ER membrane permeability in vitro. Overall, these data suggest that the regulation of ER membrane permeability by Bcl-2 proteins could be an important molecular mechanism of ER stress-induced apoptosis.
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Affiliation(s)
- X Wang
- Molecular Targets Group, James Graham Brown Cancer Center, Departments of Medicine, and Pharmacology and Toxicology, University of Louisville, Louisville, KY 40202, USA
| | - K E Olberding
- Molecular Targets Group, James Graham Brown Cancer Center, Departments of Medicine, and Pharmacology and Toxicology, University of Louisville, Louisville, KY 40202, USA
| | - C White
- Department of Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA
| | - C Li
- Molecular Targets Group, James Graham Brown Cancer Center, Departments of Medicine, and Pharmacology and Toxicology, University of Louisville, Louisville, KY 40202, USA
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Bexiga MG, Varela JA, Wang F, Fenaroli F, Salvati A, Lynch I, Simpson JC, Dawson KA. Cationic nanoparticles induce caspase 3-, 7- and 9-mediated cytotoxicity in a human astrocytoma cell line. Nanotoxicology 2010; 5:557-67. [DOI: 10.3109/17435390.2010.539713] [Citation(s) in RCA: 107] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
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49
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Fulda S. Exploiting mitochondrial apoptosis for the treatment of cancer. Mitochondrion 2010; 10:598-603. [DOI: 10.1016/j.mito.2010.05.001] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2009] [Revised: 03/10/2010] [Accepted: 05/12/2010] [Indexed: 01/11/2023]
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50
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Busuttil V, Droin N, McCormick L, Bernassola F, Candi E, Melino G, Green DR. NF-kappaB inhibits T-cell activation-induced, p73-dependent cell death by induction of MDM2. Proc Natl Acad Sci U S A 2010; 107:18061-6. [PMID: 20921405 PMCID: PMC2964227 DOI: 10.1073/pnas.1006163107] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
NF-κB is a key transcription factor involved in the regulation of T-cell activation and proliferation upon engagement of the T-cell receptor (TCR). T cells that lack the IκB kinase (IKKβ) are unable to activate NF-κB, and rapidly undergo apoptosis upon activation. NF-κB activation following T-cell receptor engagement induces the expression of Mdm2 through interaction with NF-κB sites in its P1 promoter, and enforced expression of Mdm2 protected T cells deficient for NF-κB activation from activation-induced cell death. In T cells with intact NF-κB signaling, ablation or pharmacologic inhibition of Mdm2 resulted in activation-induced apoptosis. Mdm2 coprecipitates with p73 in activated T cells, and apoptosis induced by inhibition of Mdm2 was p73-dependent. Further, Bim was identified as a p73 target gene required for cell death induced by Mdm2 inhibition, and a p73-responsive element in intron 1 of Bim was characterized. Our results demonstrate a pathway for survival of activated T cells through NF-κB-induced Mdm2, which blocks Bim-dependent apoptosis through binding and inhibition of p73.
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Affiliation(s)
- Valere Busuttil
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105
| | - Nathalie Droin
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105
- Hematopoiesis Unit, Institut National de la Santé et de la Recherche Médicale U1009, Institut de Recherche Intégrée en Cancérologie de Villejuif, Institut Gustave Roussy, 94805 Villejuif, France
| | - Laura McCormick
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105
| | - Francesca Bernassola
- Department of Experimental Medicine and Biochemical Sciences, Biochemistry Laboratory, Istituto Dermopatico dell'Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Università degli Studi di Roma “Tor Vergata,” 00133 Rome, Italy; and
| | - Eleonora Candi
- Department of Experimental Medicine and Biochemical Sciences, Biochemistry Laboratory, Istituto Dermopatico dell'Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Università degli Studi di Roma “Tor Vergata,” 00133 Rome, Italy; and
| | - Gerry Melino
- Department of Experimental Medicine and Biochemical Sciences, Biochemistry Laboratory, Istituto Dermopatico dell'Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Università degli Studi di Roma “Tor Vergata,” 00133 Rome, Italy; and
- Medical Research Council Toxicology Unit, University of Leicester, Leicester LE1 9HN, United Kingdom
| | - Douglas R. Green
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105
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