|
1
|
Sluyter R, McEwan TBD, Sophocleous RA, Stokes L. Methods for studying P2X4 receptor ion channels in immune cells. J Immunol Methods 2024; 526:113626. [PMID: 38311008 DOI: 10.1016/j.jim.2024.113626] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2023] [Revised: 01/28/2024] [Accepted: 02/01/2024] [Indexed: 02/06/2024]
Abstract
The P2X4 receptor is a trimeric ligand-gated ion channel activated by adenosine 5'-triphosphate (ATP). P2X4 is present in immune cells with emerging roles in inflammation and immunity, and related disorders. This review aims to provide an overview of the methods commonly used to study P2X4 in immune cells, focusing on those methods used to assess P2RX4 gene expression, the presence of the P2X4 protein, and P2X4 ion channel activity in these cells from humans, dogs, mice and rats. P2RX4 gene expression in immune cells is commonly assessed using semi-quantitative and quantitative reverse-transcriptase-PCR. The presence of P2X4 protein in immune cells is mainly assessed using anti-P2X4 polyclonal antibodies with immunoblotting or immunochemistry, but the use of these antibodies, as well as monoclonal antibodies and nanobodies to detect P2X4 with flow cytometry is increasing. Notably, use of an anti-P2X4 monoclonal antibody and flow cytometry has revealed that P2X4 is present on immune cells with a rank order of expression in eosinophils, then neutrophils and monocytes, then basophils and B cells, and finally T cells. P2X4 ion channel activity has been assessed mainly by Ca2+ flux assays using the cell permeable Ca2+-sensitive dyes Fura-2 and Fluo-4 with fluorescence microscopy, spectrophotometry, or flow cytometry. However, other methods including electrophysiology, and fluorescence assays measuring Na+ flux (using sodium green tetra-acetate) and dye uptake (using YO-PRO-12+) have been applied. Collectively, these methods have demonstrated the presence of functional P2X4 in monocytes and macrophages, microglia, eosinophils, mast cells and CD4+ T cells, with other evidence suggestive of functional P2X4 in dendritic cells, neutrophils, B cells and CD8+ T cells.
Collapse
Affiliation(s)
- Ronald Sluyter
- Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia.
| | - Tahnee B-D McEwan
- Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia
| | - Reece A Sophocleous
- Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia
| | - Leanne Stokes
- School of Pharmacy, University of East Anglia, Norwich, Norfolk NR4 7TJ, UK
| |
Collapse
|
|
2
|
Zhang HL, Doblin S, Zhang ZW, Song ZJ, Dinesh B, Tabana Y, Saad DS, Adam Ahmed Adam M, Wang Y, Wang W, Zhang HL, Wu S, Zhao R, Khaled B. Elucidating the molecular basis of ATP-induced cell death in breast cancer: Construction of a robust prognostic model. World J Clin Oncol 2024; 15:208-242. [PMID: 38455130 PMCID: PMC10915939 DOI: 10.5306/wjco.v15.i2.208] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/08/2023] [Revised: 12/10/2023] [Accepted: 01/12/2024] [Indexed: 02/20/2024] Open
Abstract
BACKGROUND Breast cancer is a multifaceted and formidable disease with profound public health implications. Cell demise mechanisms play a pivotal role in breast cancer pathogenesis, with ATP-triggered cell death attracting mounting interest for its unique specificity and potential therapeutic pertinence. AIM To investigate the impact of ATP-induced cell death (AICD) on breast cancer, enhancing our understanding of its mechanism. METHODS The foundational genes orchestrating AICD mechanisms were extracted from the literature, underpinning the establishment of a prognostic model. Simultaneously, a microRNA (miRNA) prognostic model was constructed that mirrored the gene-based prognostic model. Distinctions between high- and low-risk cohorts within mRNA and miRNA characteristic models were scrutinized, with the aim of delineating common influence mechanisms, substantiated through enrichment analysis and immune infiltration assessment. RESULTS The mRNA prognostic model in this study encompassed four specific mRNAs: P2X purinoceptor 4, pannexin 1, caspase 7, and cyclin 2. The miRNA prognostic model integrated four pivotal miRNAs: hsa-miR-615-3p, hsa-miR-519b-3p, hsa-miR-342-3p, and hsa-miR-324-3p. B cells, CD4+ T cells, CD8+ T cells, endothelial cells, and macrophages exhibited inverse correlations with risk scores across all breast cancer subtypes. Furthermore, Kyoto Encyclopedia of Genes and Genomes analysis revealed that genes differentially expressed in response to mRNA risk scores significantly enriched 25 signaling pathways, while miRNA risk scores significantly enriched 29 signaling pathways, with 16 pathways being jointly enriched. CONCLUSION Of paramount significance, distinct mRNA and miRNA signature models were devised tailored to AICD, both potentially autonomous prognostic factors. This study's elucidation of the molecular underpinnings of AICD in breast cancer enhances the arsenal of potential therapeutic tools, offering an unparalleled window for innovative interventions. Essentially, this paper reveals the hitherto enigmatic link between AICD and breast cancer, potentially leading to revolutionary progress in personalized oncology.
Collapse
Affiliation(s)
- Hao-Ling Zhang
- Department of Biomedical Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Penang 13200, Malaysia
| | - Sandai Doblin
- Department of Biomedical Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Penang 13200, Malaysia
| | - Zhong-Wen Zhang
- School of Public Health, Gansu University of Chinese Medicine, Lanzhou 730000, Gansu Province, China
| | - Zhi-Jing Song
- Clinical College of Chinese Medicine, Gansu University of Chinese Medicine, Lanzhou 730000, Gansu Province, China
| | - Babu Dinesh
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton AB T6G 2E1, Canada
| | - Yasser Tabana
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton AB T6G 2E1, Canada
| | - Dahham Sabbar Saad
- Department of Science, University of Technology and Applied Sciences Rustaq, Rustaq 10 P.C. 329, Oman
| | - Mowaffaq Adam Ahmed Adam
- Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182, United States
| | - Yong Wang
- Department of Pathology Center, Gansu University of Chinese Medicine, Lanzhou 730000, Gansu Province, China
| | - Wei Wang
- College of Acupuncture-moxibustion and Tuina, Gansu University of Chinese Medicine, Lanzhou 730000, Gansu Province, China
| | - Hao-Long Zhang
- Universiti Sains Malaysia, Advanced Medical and Dental Institute, Penang 13200, Malaysia
| | - Sen Wu
- Department of Biomedical Science, Universiti Sains Malaysia, Penang 13200, Malaysia
| | - Rui Zhao
- Clinical College of Chinese Medicine, Gansu University of Chinese Medicine, Lanzhou 730000, Gansu Province, China
| | - Barakat Khaled
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton AB T6G 2E1, Canada
| |
Collapse
|
|
3
|
Zhang HL, Sandai D, Zhang ZW, Song ZJ, Babu D, Tabana Y, Dahham SS, Adam Ahmed Adam M, Wang Y, Wang W, Zhang HL, Zhao R, Barakat K, Harun MSR, Shapudin SNM, Lok B. Adenosine triphosphate induced cell death: Mechanisms and implications in cancer biology and therapy. World J Clin Oncol 2023; 14:549-569. [PMID: 38179405 PMCID: PMC10762532 DOI: 10.5306/wjco.v14.i12.549] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/06/2023] [Revised: 11/08/2023] [Accepted: 11/21/2023] [Indexed: 12/22/2023] Open
Abstract
Adenosine triphosphate (ATP) induced cell death (AICD) is a critical cellular process that has garnered substantial scientific interest for its profound relevance to cancer biology and to therapeutic interventions. This comprehensive review unveils the intricate web of AICD mechanisms and their intricate connections with cancer biology. This review offers a comprehensive framework for comprehending the multifaceted role of AICD in the context of cancer. This is achieved by elucidating the dynamic interplay between systemic and cellular ATP homeostasis, deciphering the intricate mechanisms governing AICD, elucidating its intricate involvement in cancer signaling pathways, and scrutinizing validated key genes. Moreover, the exploration of AICD as a potential avenue for cancer treatment underscores its essential role in shaping the future landscape of cancer therapeutics.
Collapse
Affiliation(s)
- Hao-Ling Zhang
- Department of Biomedical Science, Advanced Medical and Dental Institute, University Sains Malaysia, Penang 13200, Malaysia
| | - Doblin Sandai
- Department of Biomedical Science, Advanced Medical and Dental Institute, University Sains Malaysia, Penang 13200, Malaysia
| | - Zhong-Wen Zhang
- School of Public Health, Gansu University of Chinese Medicine, Lanzhou 730000, Gansu Province, China
| | - Zhi-Jing Song
- Clinical College of Chinese Medicine, Gansu University of Chinese Medicine, Lanzhou 730000, Gansu Province, China
| | - Dinesh Babu
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton AB T6G 2E1, Canada
| | - Yasser Tabana
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton AB T6G 2E1, Canada
| | - Sabbar Saad Dahham
- Department of Science, University of Technology and Applied Sciences Rustaq, Rustaq 10 P.C. 329, Oman
| | - Mowaffaq Adam Ahmed Adam
- Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182, United States
| | - Yong Wang
- Pathology Center, Gansu University of Chinese Medicine, Lanzhou 730000, Gansu Province, China
| | - Wei Wang
- College of Acupuncture-Moxibustion and Tuina, Gansu University of Chinese Medicine, Lanzhou 730000, Gansu Province, China
| | - Hao-Long Zhang
- Department of Biomedical Science, Advanced Medical and Dental Institute, University Sains Malaysia, Penang 13200, Malaysia
| | - Rui Zhao
- Clinical College of Chinese Medicine, Gansu University of Chinese Medicine, Lanzhou 730000, Gansu Province, China
| | - Khaled Barakat
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton AB T6G 2E1, Canada
| | - Mohammad Syamsul Reza Harun
- Department of Biomedical Science, Advanced Medical and Dental Institute, University Sains Malaysia, Penang 13200, Malaysia
| | - Siti Nurfatimah Mohd Shapudin
- Department of Biomedical Science, Advanced Medical and Dental Institute, University Sains Malaysia, Penang 13200, Malaysia
| | - Bronwyn Lok
- Department of Biomedical Science, Advanced Medical and Dental Institute, University Sains Malaysia, Penang 13200, Malaysia
| |
Collapse
|
|
4
|
Hasheminasab SS, Conejeros I, Gärtner U, Kamena F, Taubert A, Hermosilla CR. MCT-Dependent Cryptosporidium parvum-Induced Bovine Monocyte Extracellular Traps (METs) under Physioxia. BIOLOGY 2023; 12:961. [PMID: 37508391 PMCID: PMC10376234 DOI: 10.3390/biology12070961] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/15/2023] [Revised: 06/26/2023] [Accepted: 07/03/2023] [Indexed: 07/30/2023]
Abstract
The apicomplexan protozoan parasite Cryptosporidium parvum is responsible for cryptosporidiosis, which is a zoonotic intestinal illness that affects newborn cattle, wild animals, and people all over the world. Mammalian monocytes are bone marrow-derived myeloid leukocytes with important defense effector functions in early host innate immunity due to their ATP purinergic-, CD14- and CD16-receptors, adhesion, migration and phagocytosis capacities, inflammatory, and anti-parasitic properties. The formation of monocyte extracellular traps (METs) has recently been reported as an additional effector mechanism against apicomplexan parasites. Nonetheless, nothing is known in the literature on METs extrusion neither towards C. parvum-oocysts nor sporozoites. Herein, ATP purinergic receptor P2X1, glycolysis, Notch signaling, and lactate monocarboxylate transporters (MCT) were investigated in C. parvum-exposed bovine monocytes under intestinal physioxia (5% O2) and hyperoxia (21% O2; most commonly used hyperoxic laboratory conditions). C. parvum-triggered suicidal METs were confirmed by complete rupture of exposed monocytes, co-localization of extracellular DNA with myeloperoxidase (MPO) and histones (H1-H4) via immunofluorescence- and confocal microscopy analyses. C. parvum-induced suicidal METs resulted not only in oocyst entrapment but also in hindered sporozoite mobility from oocysts according to scanning electron microscopy (SEM) analyses. Early parasite-induced bovine monocyte activation, accompanied by membrane protrusions toward C. parvum-oocysts/sporozoites, was unveiled using live cell 3D-holotomographic microscopy analysis. The administration of NF449, an inhibitor of the ATP purinergic receptor P2X1, to monocytes subjected to varying oxygen concentrations did not yield a noteworthy decrease in C. parvum-induced METosis. This suggests that the cell death process is not dependent on P2X1. Additionally, blockage of glycolysis in monocyte through 2-deoxy glucose (2-DG) inhibition reduced C. parvum-induced METosis but not significantly. According to monocyte energetic state measurements, C. parvum-exposed cells neither increased extracellular acidification rates (ECAR) nor oxygen consumption rates (OCR). Lactate monocarboxylate transporters (MCT) inhibitor (i.e., AR-C 141990) treatments significantly diminished C. parvum-mediated METs extrusion under physioxic (5% O2) condition. Similarly, treatment with either DAPT or compound E, two selective Notch inhibitors, exhibited no significant suppressive effects on bovine MET production. Overall, for the first time, we demonstrate C. parvum-mediated METosis as P2X1-independent but as an MCT-dependent defense mechanism under intestinal physioxia (5% CO2) conditions. METs findings suggest anti-cryptosporidial effects through parasite entrapment and inhibition of sporozoite excystation.
Collapse
Affiliation(s)
- Seyed Sajjad Hasheminasab
- Institute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, 35392 Giessen, Germany
| | - Iván Conejeros
- Institute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, 35392 Giessen, Germany
| | - Ulrich Gärtner
- Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, 35392 Giessen, Germany
| | - Faustin Kamena
- Laboratory for Molecular Parasitology, Department of Microbiology and Parasitology, University of Buea, Buea P.O. Box 63, Cameroon
| | - Anja Taubert
- Institute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, 35392 Giessen, Germany
| | - Carlos R Hermosilla
- Institute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, 35392 Giessen, Germany
| |
Collapse
|
|
5
|
Extracellular Adenine Nucleotides and Adenosine Modulate the Growth and Survival of THP-1 Leukemia Cells. Int J Mol Sci 2020; 21:ijms21124425. [PMID: 32580317 PMCID: PMC7352165 DOI: 10.3390/ijms21124425] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2020] [Revised: 06/19/2020] [Accepted: 06/20/2020] [Indexed: 12/26/2022] Open
Abstract
A new approach to improve the effectiveness of acute myeloid leukemia (AML) treatment is to use the properties of purinergic signaling molecules secreted into the bone marrow milieu in response to leukemic cell growth. Therefore, our study aimed to evaluate the effects of extracellular adenine nucleotides and adenosine on the growth and death parameters in the leukemic THP-1 cell line. Cells were exposed to ATP, ADP, AMP, adenosine and nonhydrolyzable analogues of ATP and ADP (ATPγS and ADPβS) in a 1–1000 μM broad concentration range. The basal mRNA expression of the P1 and P2 receptors was evaluated by real-time PCR. Changes in the processes of cell growth and death were assessed by flow cytometry analysis of proliferation, cell cycle and apoptosis. Chemotaxis toward stromal cell-derived factor-1 (SDF-1) was performed using the modified Boyden chamber assay, and chemokine receptor type 4 (CXCR4) surface expression was quantified by flow cytometry. We indicated several antileukemic actions. High micromolar concentrations (100–1000 μM) of extracellular adenine nucleotides and adenosine inhibit the growth of cells by arresting the cell cycle and/or inducing apoptosis. ATP is characterized by the highest potency and widest range of effects, and is responsible for the cell cycle arrest and the apoptosis induction. Compared to ATP, the effect of ADP is slightly weaker. Adenosine mostly has a cytotoxic effect, with the induction of apoptosis. The last studied nucleotide, AMP, demonstrated only a weak cytotoxic effect without affecting the cell cycle. In addition, cell migration towards SDF-1 was inhibited by low micromolar concentrations (10 μM). One of the reasons for this action of ATPγS and adenosine was a reduction in CXCR4 surface expression, but this only partially explains the mechanism of antimigratory action. In summary, extracellular adenine nucleotides and adenosine inhibit THP-1 cell growth, cause death of cells and modulate the functioning of the SDF-1/CXCR4 axis. Thus, they negatively affect the processes that are responsible for the progression of AML and the difficulties in AML treatment.
Collapse
|
|
6
|
Lara R, Adinolfi E, Harwood CA, Philpott M, Barden JA, Di Virgilio F, McNulty S. P2X7 in Cancer: From Molecular Mechanisms to Therapeutics. Front Pharmacol 2020; 11:793. [PMID: 32581786 PMCID: PMC7287489 DOI: 10.3389/fphar.2020.00793] [Citation(s) in RCA: 116] [Impact Index Per Article: 23.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2020] [Accepted: 05/13/2020] [Indexed: 12/18/2022] Open
Abstract
P2X7 is a transmembrane receptor expressed in multiple cell types including neurons, dendritic cells, macrophages, monocytes, B and T cells where it can drive a wide range of physiological responses from pain transduction to immune response. Upon activation by its main ligand, extracellular ATP, P2X7 can form a nonselective channel for cations to enter the cell. Prolonged activation of P2X7, via high levels of extracellular ATP over an extended time period can lead to the formation of a macropore, leading to depolarization of the plasma membrane and ultimately to cell death. Thus, dependent on its activation state, P2X7 can either drive cell survival and proliferation, or induce cell death. In cancer, P2X7 has been shown to have a broad range of functions, including playing key roles in the development and spread of tumor cells. It is therefore unsurprising that P2X7 has been reported to be upregulated in several malignancies. Critically, ATP is present at high extracellular concentrations in the tumor microenvironment (TME) compared to levels observed in normal tissues. These high levels of ATP should present a survival challenge for cancer cells, potentially leading to constitutive receptor activation, prolonged macropore formation and ultimately to cell death. Therefore, to deliver the proven advantages for P2X7 in driving tumor survival and metastatic potential, the P2X7 macropore must be tightly controlled while retaining other functions. Studies have shown that commonly expressed P2X7 splice variants, distinct SNPs and post-translational receptor modifications can impair the capacity of P2X7 to open the macropore. These receptor modifications and potentially others may ultimately protect cancer cells from the negative consequences associated with constitutive activation of P2X7. Significantly, the effects of both P2X7 agonists and antagonists in preclinical tumor models of cancer demonstrate the potential for agents modifying P2X7 function, to provide innovative cancer therapies. This review summarizes recent advances in understanding of the structure and functions of P2X7 and how these impact P2X7 roles in cancer progression. We also review potential therapeutic approaches directed against P2X7.
Collapse
Affiliation(s)
- Romain Lara
- Biosceptre (UK) Limited, Cambridge, United Kingdom
| | - Elena Adinolfi
- Department of Medical Sciences, University of Ferrara, Ferrara, Italy
| | - Catherine A Harwood
- Centre for Cell Biology and Cutaneous Research, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom
| | - Mike Philpott
- Centre for Cutaneous Research, Blizard Institute, Bart's & The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom
| | | | - Francesco Di Virgilio
- Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy
| | | |
Collapse
|
|
7
|
Filippin KJ, de Souza KFS, de Araujo Júnior RT, Torquato HFV, Dias DA, Parisotto EB, Ferreira AT, Paredes-Gamero EJ. Involvement of P2 receptors in hematopoiesis and hematopoietic disorders, and as pharmacological targets. Purinergic Signal 2020; 16:1-15. [PMID: 31863258 PMCID: PMC7166233 DOI: 10.1007/s11302-019-09684-z] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2019] [Accepted: 11/12/2019] [Indexed: 12/11/2022] Open
Abstract
Several reports have shown the presence of P2 receptors in hematopoietic stem cells (HSCs). These receptors are activated by extracellular nucleotides released from different sources. In the hematopoietic niche, the release of purines and pyrimidines in the milieu by lytic and nonlytic mechanisms has been described. The expression of P2 receptors from HSCs until maturity is still intriguing scientists. Several reports have shown the participation of P2 receptors in events associated with modulation of the immune system, but their participation in other physiological processes is under investigation. The presence of P2 receptors in HSCs and their ability to modulate this population have awakened interest in exploring the involvement of P2 receptors in hematopoiesis and their participation in hematopoietic disorders. Among the P2 receptors, the receptor P2X7 is of particular interest, because of its different roles in hematopoietic cells (e.g., infection, inflammation, cell death and survival, leukemias and lymphomas), making the P2X7 receptor a promising pharmacological target. Additionally, the role of P2Y12 receptor in platelet activation has been well-documented and is the main example of the importance of the pharmacological modulation of P2 receptor activity. In this review, we focus on the role of P2 receptors in the hematopoietic system, addressing these receptors as potential pharmacological targets.
Collapse
Affiliation(s)
- Kelly Juliana Filippin
- Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, 79070-900, Brazil
| | - Kamylla F S de Souza
- Departamento de Bioquímica, Universidade Federal de São Paulo, R. Três de Maio 100, São Paulo, SP, 04044-020, Brazil
| | | | - Heron Fernandes Vieira Torquato
- Departamento de Bioquímica, Universidade Federal de São Paulo, R. Três de Maio 100, São Paulo, SP, 04044-020, Brazil
- Universidade Braz Cubas, Av. Francisco Rodrigues Filho 1233, Mogi das Cruzes, SP, 08773-380, Brazil
| | - Dhébora Albuquerque Dias
- Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, 79070-900, Brazil
| | - Eduardo Benedetti Parisotto
- Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, 79070-900, Brazil
| | - Alice Teixeira Ferreira
- Departamento de Biofísica, Universidade Federal de São Paulo, R. Botucatu 862, São Paulo, SP, 04023-062, Brazil.
- Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição (FACFAN), Laboratório de Biologia Molecular e Culturas Celulares, Av. Costa e Silva, s/n Bairro Universitário, Campo Grande, MS, CEP: 79070-900, Brazil.
| | - Edgar J Paredes-Gamero
- Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, 79070-900, Brazil.
- Departamento de Bioquímica, Universidade Federal de São Paulo, R. Três de Maio 100, São Paulo, SP, 04044-020, Brazil.
- Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição (FACFAN), Laboratório de Biologia Molecular e Culturas Celulares, Av. Costa e Silva, s/n Bairro Universitário, Campo Grande, MS, CEP: 79070-900, Brazil.
| |
Collapse
|
|
8
|
Current Status and Future Prospects of Clinically Exploiting Cancer-specific Metabolism-Why Is Tumor Metabolism Not More Extensively Translated into Clinical Targets and Biomarkers? Int J Mol Sci 2019; 20:ijms20061385. [PMID: 30893889 PMCID: PMC6471292 DOI: 10.3390/ijms20061385] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2019] [Revised: 03/14/2019] [Accepted: 03/15/2019] [Indexed: 02/07/2023] Open
Abstract
Tumor cells exhibit a specialized metabolism supporting their superior ability for rapid proliferation, migration, and apoptotic evasion. It is reasonable to assume that the specific metabolic needs of the tumor cells can offer an array of therapeutic windows as pharmacological disturbance may derail the biochemical mechanisms necessary for maintaining the tumor characteristics, while being less important for normally proliferating cells. In addition, the specialized metabolism may leave a unique metabolic signature which could be used clinically for diagnostic or prognostic purposes. Quantitative global metabolic profiling (metabolomics) has evolved over the last two decades. However, despite the technology’s present ability to measure 1000s of endogenous metabolites in various clinical or biological specimens, there are essentially no examples of metabolomics investigations being translated into actual utility in the cancer clinic. This review investigates the current efforts of using metabolomics as a tool for translation of tumor metabolism into the clinic and further seeks to outline paths for increasing the momentum of using tumor metabolism as a biomarker and drug target opportunity.
Collapse
|
|
9
|
ATP Induces IL-1 β Secretion in Neisseria gonorrhoeae-Infected Human Macrophages by a Mechanism Not Related to the NLRP3/ASC/Caspase-1 Axis. Mediators Inflamm 2016; 2016:1258504. [PMID: 27803513 PMCID: PMC5075643 DOI: 10.1155/2016/1258504] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2016] [Revised: 08/12/2016] [Accepted: 09/06/2016] [Indexed: 12/24/2022] Open
Abstract
Neisseria gonorrhoeae (Ngo) has developed multiple immune evasion mechanisms involving the innate and adaptive immune responses. Recent findings have reported that Ngo reduces the IL-1β secretion of infected human monocyte-derived macrophages (MDM). Here, we investigate the role of adenosine triphosphate (ATP) in production and release of IL-1β in Ngo-infected MDM. We found that the exposure of Ngo-infected MDM to ATP increases IL-1β levels about ten times compared with unexposed Ngo-infected MDM (P < 0.01). However, we did not observe any changes in inflammasome transcriptional activation of speck-like protein containing a caspase recruitment domain (CARD) (ASC, P > 0.05) and caspase-1 (CASP1, P > 0.05). In addition, ATP was not able to modify caspase-1 activity in Ngo-infected MDM but was able to increase pyroptosis (P > 0.01). Notably ATP treatment defined an increase of positive staining for IL-1β with a distinctive intracellular pattern of distribution. Collectively, these data demonstrate that ATP induces IL-1β secretion by a mechanism not related to the NLRP3/ASC/caspase-1 axis and likely is acting at the level of vesicle trafficking or pore formation.
Collapse
|
|
10
|
Seo JB, Jung SR, Hille B, Koh DS. Extracellular ATP protects pancreatic duct epithelial cells from alcohol-induced damage through P2Y1 receptor-cAMP signal pathway. Cell Biol Toxicol 2016; 32:229-47. [PMID: 27197531 PMCID: PMC5493489 DOI: 10.1007/s10565-016-9331-3] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2015] [Accepted: 04/22/2016] [Indexed: 12/18/2022]
Abstract
Extracellular adenosine-5'-triphosphate (ATP) regulates cell death and survival of neighboring cells. The detailed effects are diverse depending on cell types and extracellular ATP concentration. We addressed the effect of ATP on ethanol-induced cytotoxicity in epithelial cells, the cell type that experiences the highest concentrations of alcohol. Using pancreatic duct epithelial cells (PDEC), we found that a micromolar range of ATP reverses all intracellular toxicity mechanisms triggered by exceptionally high doses of ethanol and, thus, improves cell viability dramatically. Out of the many purinergic receptors expressed in PDEC, the P2Y1 receptor was identified to mediate the protective effect, based on pharmacological and siRNA assays. Activation of P2Y1 receptors increased intracellular cyclic adenosine monophosphate (cAMP). The protective effect of ATP was mimicked by forskolin and 8-Br-cAMP but inhibited by a protein kinase A (PKA) inhibitor, H-89. Finally, ATP reverted leakiness of PDEC monolayers induced by ethanol and helped to maintain epithelial integrity. We suggest that purinergic receptors reduce extreme alcohol-induced cell damage via the cAMP signal pathway in PDEC and some other types of cells.
Collapse
Affiliation(s)
- Jong Bae Seo
- Department of Physiology and Biophysics, University of Washington, Health Sciences Bldg. Rm. G-424, Seattle, WA, 98195-7290, USA
- Department of Medicine, Division of Endocrinology and Metabolism, University of California San Diego, La Jolla, CA, 92093, USA
| | - Seung-Ryoung Jung
- Department of Physiology and Biophysics, University of Washington, Health Sciences Bldg. Rm. G-424, Seattle, WA, 98195-7290, USA
| | - Bertil Hille
- Department of Physiology and Biophysics, University of Washington, Health Sciences Bldg. Rm. G-424, Seattle, WA, 98195-7290, USA
| | - Duk-Su Koh
- Department of Physiology and Biophysics, University of Washington, Health Sciences Bldg. Rm. G-424, Seattle, WA, 98195-7290, USA.
| |
Collapse
|
|
11
|
Seeland S, Kettiger H, Murphy M, Treiber A, Giller J, Kiss A, Sube R, Krähenbühl S, Hafner M, Huwyler J. ATP-induced cellular stress and mitochondrial toxicity in cells expressing purinergic P2X7 receptor. Pharmacol Res Perspect 2015; 3:e00123. [PMID: 26038699 PMCID: PMC4448979 DOI: 10.1002/prp2.123] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2014] [Revised: 12/10/2014] [Accepted: 12/19/2014] [Indexed: 12/15/2022] Open
Abstract
Under pathological conditions, the purinergic P2X7 receptor is activated by elevated concentrations of extracellular ATP. Thereby, the receptor forms a slowly dilating pore, allowing cations and, upon prolonged stimulation, large molecules to enter the cell. This process has a strong impact on cell signaling, metabolism, and viability. This study aimed to establish a link between gradual P2X7 activation and pharmacological endpoints including oxidative stress, hydrogen peroxide generation, and cytotoxicity. Mechanisms of cellular stress and cytotoxicity were studied in P2X7-transfected HEK293 cells. We performed real-time monitoring of metabolic and respiratory activity of cells expressing the P2X7-receptor protein using a cytosensor system. Agonistic effects were monitored using exogenously applied ATP or the stable analogue BzATP. Oxidative stress induced by ATP or BzATP in target cells was monitored by hydrogen peroxide release in human mononuclear blood cells. P2X7-receptor activation was studied by patch-clamp experiments using a primary mouse microglia cell line. Stimulation of the P2X7 receptor leads to ion influx, metabolic activation of target cells, and ultimately cytotoxicity. Conversion of the P2X7 receptor from a small cation channel to a large pore occurring under prolonged stimulation can be monitored in real time covering a time frame of milliseconds to hours. Selectivity of the effects can be demonstrated using the selective P2X7-receptor antagonist AZD9056. Our findings established a direct link between P2X7-receptor activation by extracellular ATP or BzATP and cellular events culminating in cytotoxicity. Mechanisms of toxicity include metabolic and oxidative stress, increase in intracellular calcium concentration and disturbance of mitochondrial membrane potential. Mitochondrial toxicity is suggested to be a key event leading to cell death.
Collapse
Affiliation(s)
- Swen Seeland
- Actelion Pharmaceuticals Ltd Gewerbestrasse 16, 4123, Allschwil, Switzerland ; Institute for Molecular and Cell Biology, University of Applied Science Paul-Wittsack-Strasse 10, 68163, Mannheim, Germany
| | - Hélène Kettiger
- Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel Klingelbergstrasse 50, 4056, Basel, Switzerland
| | - Mark Murphy
- Actelion Pharmaceuticals Ltd Gewerbestrasse 16, 4123, Allschwil, Switzerland
| | - Alexander Treiber
- Actelion Pharmaceuticals Ltd Gewerbestrasse 16, 4123, Allschwil, Switzerland
| | - Jasmin Giller
- Actelion Pharmaceuticals Ltd Gewerbestrasse 16, 4123, Allschwil, Switzerland
| | - Andrea Kiss
- Actelion Pharmaceuticals Ltd Gewerbestrasse 16, 4123, Allschwil, Switzerland
| | - Romain Sube
- Actelion Pharmaceuticals Ltd Gewerbestrasse 16, 4123, Allschwil, Switzerland
| | - Stephan Krähenbühl
- Division of Clinical Pharmacology and Toxicology, University Hospital of Basel 4056, Basel, Switzerland
| | - Mathias Hafner
- Institute for Molecular and Cell Biology, University of Applied Science Paul-Wittsack-Strasse 10, 68163, Mannheim, Germany
| | - Jörg Huwyler
- Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel Klingelbergstrasse 50, 4056, Basel, Switzerland
| |
Collapse
|
|
12
|
Burnstock G, Di Virgilio F. Purinergic signalling and cancer. Purinergic Signal 2014; 9:491-540. [PMID: 23797685 DOI: 10.1007/s11302-013-9372-5] [Citation(s) in RCA: 257] [Impact Index Per Article: 23.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2013] [Accepted: 06/06/2013] [Indexed: 01/24/2023] Open
Abstract
Receptors for extracellular nucleotides are widely expressed by mammalian cells. They mediate a large array of responses ranging from growth stimulation to apoptosis, from chemotaxis to cell differentiation and from nociception to cytokine release, as well as neurotransmission. Pharma industry is involved in the development and clinical testing of drugs selectively targeting the different P1 nucleoside and P2 nucleotide receptor subtypes. As described in detail in the present review, P2 receptors are expressed by all tumours, in some cases to a very high level. Activation or inhibition of selected P2 receptor subtypes brings about cancer cell death or growth inhibition. The field has been largely neglected by current research in oncology, yet the evidence presented in this review, most of which is based on in vitro studies, although with a limited amount from in vivo experiments and human studies, warrants further efforts to explore the therapeutic potential of purinoceptor targeting in cancer.
Collapse
|
|
13
|
Hu XM, Tanaka S, Onda K, Yuan B, Toyoda H, Ma R, Liu F, Hirano T. Arsenic disulfide induced apoptosis and concurrently promoted erythroid differentiation in cytokine-dependent myelodysplastic syndrome-progressed leukemia cell line F-36p with complex karyotype including monosomy 7. Chin J Integr Med 2014; 20:387-93. [PMID: 24610410 DOI: 10.1007/s11655-013-1514-7] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2012] [Indexed: 11/24/2022]
Abstract
OBJECTIVE Acute myeloid leukemia progressed from myelodysplastic syndrome (MDS/AML) is generally incurable with poor prognosis for complex karyotype including monosomy 7 (-7). Qinghuang Powder (, QHP), which includes Qing Dai (Indigo naturalis) and Xiong Huang (realgar) in the formula, is effective in treating MDS or MDS/AML even with the unfavorable karyotype, and its therapeutic efficacy could be enhanced by increasing the Xiong huang content in the formula, while Xiong huang contains > 90% arsenic disulfide (As2S2). F-36p cell line was established from a MDS/AML patient with complex karyotype including -7, and was in cytokine-dependent. The present study was to investigate the effects of As2S2 on F-36p cells. METHODS Cell proliferation was measured by an 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was identified by Annexin V-staining. Cell viability was determined by a propidium iodide (PI) exclusion. Erythroid differentiation was evaluated by the expression of cell surface antigen CD235a (GpA). RESULTS After treatment with As2S2 at concentrations of 0.5 to 16 μmol/L for 72 h, As2S2 inhibited the proliferation of F-36p cells. The 50% inhibitory concentrations (IC50) of As2S2 against the proliferation of F-36p cells was 6 μmol/L. The apoptotic cells significantly increased in a dose-dependent mannar (P<0.05). The cell viabilities were significantly inhibited by As2S2 dose-dependent in a dose-dependent manner (P<0.05). Significant increases of CD235a-positive cells were concurrently observed (P<0.05) also in a dose-dependent manner. CONCLUSIONS As2S2 could inhibit proliferation and viability, induce apoptosis, and concurrently promote erythroid differentiation dose-dependently in F-36p cells. As2S2 can inhibit proliferation and viability, induce apoptosis, and concurrently promote erythroid differentiation in cytokine-dependent MDS-progressed human leukemia cell line F-36p with complex karyotype including -7. The data suggest that QHP and/or As2S2 could be a potential candidate in the treatment of MDS or MDS/AML even with unfavorable cytogenetics.
Collapse
Affiliation(s)
- Xiao-mei Hu
- Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, 192-0392, Japan
| | | | | | | | | | | | | | | |
Collapse
|
|
14
|
Baldini C, Rossi C, Ferro F, Santini E, Seccia V, Donati V, Solini A. The P2X7 receptor-inflammasome complex has a role in modulating the inflammatory response in primary Sjögren's syndrome. J Intern Med 2013; 274:480-9. [PMID: 23906036 DOI: 10.1111/joim.12115] [Citation(s) in RCA: 76] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
OBJECTIVE Innate and adaptive immunity may contribute to gland dysfunction in patients with primary Sjögren's syndrome (pSS). The P2X7 receptor (P2X7 R)-NLRP3 inflammasome complex modulates the release of the inflammatory cytokines IL-1β and IL-18. The presence of P2X7 R in salivary glands suggests an interesting scenario for the initiation and amplification of the innate immune response in pSS. Therefore, the aim of this study was to assess the role of the P2X7 R-NLRP3 inflammasome in pSS. SUBJECTS AND METHODS Twenty-one consecutive patients with pSS according to the American-European Consensus Group criteria and 15 patients with sicca syndrome (i.e. without Sjögren's syndrome, non-SS) were enrolled in this study, together with six control (CTL) subjects. Expression of the P2X7R-NLRP3 platform and IL-18 was determined by real-time PCR and western blotting in gland specimens and peripheral lymphomonocytes; data were related to patients\x92 clinical, serological and histopathological characteristics. The presence of IL-18 was determined in gland and saliva samples. RESULTS P2X7 R expression was significantly higher in salivary glands from individuals with pSS than in those from non-SS and CTL subjects. Accordingly, the gene expression levels of the inflammasome components NLRP3, ASC and caspase-1 were significantly higher in pSS gland specimens, and this was paralleled by an increased expression of mature IL-18 in pSS saliva samples. The expression of both the P2X7 R and the inflammasome components was a marker of disease-related glandular involvement, being increased in patients with anti-Ro/SSA positivity and correlated with focus score. CONCLUSION The results of this study suggest an involvement of the P2X7 R-inflammasome-caspase-1-IL-18 axis in the development of pSS exocrinopathy. This finding provides the basis for studying the complex mechanisms underlying pSS, as well as for developing novel potential therapeutic strategies.
Collapse
Affiliation(s)
- C Baldini
- Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy
| | | | | | | | | | | | | |
Collapse
|
|
15
|
Ben Yebdri F, Van Grevenynghe J, Tang VA, Goulet ML, Wu JH, Stojdl DF, Hiscott J, Lin R. Triptolide-mediated inhibition of interferon signaling enhances vesicular stomatitis virus-based oncolysis. Mol Ther 2013; 21:2043-53. [PMID: 23985699 DOI: 10.1038/mt.2013.187] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2013] [Accepted: 07/30/2013] [Indexed: 12/15/2022] Open
Abstract
Preclinical and clinical trials demonstrated that use of oncolytic viruses (OVs) is a promising new therapeutic approach to treat multiple types of cancer. To further improve their viral oncolysis, experimental strategies are now combining OVs with different cytotoxic compounds. In this study, we investigated the capacity of triptolide - a natural anticancer molecule - to enhance vesicular stomatitis virus (VSV) oncolysis in OV-resistant cancer cells. Triptolide treatment increased VSV replication in the human prostate cancer cell line PC3 and in other VSV-resistant cells in a dose- and time-dependent manner in vitro and in vivo. Mechanistically, triptolide (TPL) inhibited the innate antiviral response by blocking type I interferon (IFN) signaling, downstream of IRF3 activation. Furthermore, triptolide-enhanced VSV-induced apoptosis in a dose-dependent fashion in VSV-resistant cells, as measured by annexin-V, cleaved caspase-3, and B-cell lymphoma 2 staining. In vivo, using the TSA mammary adenocarcinoma and PC3 mouse xenograft models, combination treatment with VSV and triptolide delayed tumor growth and prolonged survival of tumor-bearing animals by enhancing viral replication. Together, these results demonstrate that triptolide inhibition of IFN production sensitizes prostate cancer cells to VSV replication and virus-mediated apoptosis.
Collapse
Affiliation(s)
- Fethia Ben Yebdri
- Department of Medicine, Lady Davis Institute-Jewish General Hospital, McGill University, Montréal, Québec, Canada
| | | | | | | | | | | | | | | |
Collapse
|
|
16
|
Scodelaro Bilbao P, Boland R. Extracellular ATP regulates FoxO family of transcription factors and cell cycle progression through PI3K/Akt in MCF-7 cells. Biochim Biophys Acta Gen Subj 2013; 1830:4456-69. [PMID: 23742826 DOI: 10.1016/j.bbagen.2013.05.034] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2013] [Revised: 05/22/2013] [Accepted: 05/24/2013] [Indexed: 12/31/2022]
Abstract
BACKGROUND Forkhead Box-O (FoxO) transcription factors regulate the expression of many genes involved in suppression. Released nucleotides can regulate intracellular signaling pathways through membrane-bound purinergic receptors, to promote or prevent malignant cell transformation. We studied the role of extracellular ATP in the modulation of Forkhead Box O (FoxO) transcription factors and of cell cycle progression in MCF-7 breast cancer cells. METHODS Western blot analysis, cell transfections with siRNA against Akt, immunocytochemistry, subcellular fractionation studies and flow cytometry analysis were performed. RESULTS ATP induced the phosphorylation of FoxO1/3a at threonine 24/32, whereas reduced the expression of FoxO1. In addition, ATP increased the expression of the cyclins D1 and D3 and down-regulated the cell cycle inhibitory proteins p21Cip1 and p27Kip1. The use of the phosphatidylinositol 3 kinase (PI3K) inhibitor, Ly294002, and/or of siRNA to reduce the expression of the serine/threonine kinase Akt showed that these effects are mediated by the PI3K/Akt signaling pathway. ATP induced the translocation of FoxO3a from the nucleus to the cytoplasm. Also, ATP increased the number of cells in the S phase of cell cycle; this effect was reverted by the use of Ly294002 and the proteasome inhibitor bortezomib. CONCLUSION Extracellular ATP induces the inactivation of FoxO transcription factors and cell cycle progression through the PI3K/Akt pathway in MCF-7 cells. GENERAL SIGNIFICANCE These findings provide new molecular basis for further understanding the mechanisms involved in ATP signal transduction in breast cancer cells, and should be considered for the development of effective breast cancer therapeutic strategies.
Collapse
Affiliation(s)
- Paola Scodelaro Bilbao
- Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan, Argentina
| | | |
Collapse
|
|
17
|
Li X, Arslan F, Ren Y, Adav SS, Poh KK, Sorokin V, Lee CN, de Kleijn D, Lim SK, Sze SK. Metabolic adaptation to a disruption in oxygen supply during myocardial ischemia and reperfusion is underpinned by temporal and quantitative changes in the cardiac proteome. J Proteome Res 2012; 11:2331-46. [PMID: 22352837 DOI: 10.1021/pr201025m] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Despite decades of intensive research, there is still no effective treatment for ischemia/reperfusion (I/R) injury, an important corollary in the treatment of ischemic disease. I/R injury is initiated when the altered biochemistry of cells after ischemia is no longer compatible with oxygenated microenvironment (or reperfusion). To better understand the molecular basis of this alteration and subsequent incompatibility, we assessed the temporal and quantitative alterations in the cardiac proteome of a mouse cardiac I/R model by an iTRAQ approach at 30 min of ischemia, and at 60 or 120 min reperfusion after the ischemia using sham-operated mouse heart as the baseline control. Of the 509 quantified proteins identified, 121 proteins exhibited significant changes (p-value<0.05) over time and were mostly clustered in eight functional groups: Fatty acid oxidation, Glycolysis, TCA cycle, ETC (electron transport chain), Redox Homeostasis, Glutathione S-transferase, Apoptosis related, and Heat Shock proteins. The first four groups are intimately involved in ATP production and the last four groups are known to be important in cellular antioxidant activity. During ischemia and reperfusion, the short supply of oxygen precipitates a pivotal metabolic switch from aerobic metabolism involving fatty acid oxidation, TCA, and phosphorylation to anaerobic metabolism for ATP production and this, in turn, increases reactive oxygen species (ROS) formation. Therefore the implication of these 8 functional groups suggested that ischemia-reperfusion injury is underpinned in part by proteomic alterations. Reversion of these alterations to preischemia levels took at least 60 min, suggesting a refractory period in which the ischemic cells cannot adjust to the presence of oxygen. Therefore, therapeutics that could compensate for these proteomic alterations during this interim refractory period could alleviate ischemia-reperfusion injury to enhance cellular recovery from an ischemic to a normoxic microenvironment. Among the perturbed proteins, Park7 and Ppia were selected for further investigation of their functions under hypoxia. The results show that Park7 plays a key role in regulating antioxidative stress and cell survival, and Ppia may function in coping with the unfolded protein stress in the I/R condition.
Collapse
Affiliation(s)
- Xin Li
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
18
|
Koppikar SJ, Choudhari AS, Suryavanshi SA, Kumari S, Chattopadhyay S, Kaul-Ghanekar R. Aqueous cinnamon extract (ACE-c) from the bark of Cinnamomum cassia causes apoptosis in human cervical cancer cell line (SiHa) through loss of mitochondrial membrane potential. BMC Cancer 2010; 10:210. [PMID: 20482751 PMCID: PMC2893107 DOI: 10.1186/1471-2407-10-210] [Citation(s) in RCA: 138] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2009] [Accepted: 05/18/2010] [Indexed: 11/14/2022] Open
Abstract
Background Chemoprevention, which includes the use of synthetic or natural agents (alone or in combination) to block the development of cancer in human beings, is an extremely promising strategy for cancer prevention. Cinnamon is one of the most widely used herbal medicines with diverse biological activities including anti-tumor activity. In the present study, we have reported the anti-neoplastic activity of cinnamon in cervical cancer cell line, SiHa. Methods The aqueous cinnamon extract (ACE-c) was analyzed for its cinnamaldehyde content by HPTLC analysis. The polyphenol content of ACE-c was measured by Folin-Ciocalteau method. Cytotoxicity analysis was performed by MTT assay. We studied the effect of cinnamon on growth kinetics by performing growth curve, colony formation and soft agar assays. The cells treated with ACE-c were analyzed for wound healing assay as well as for matrix metalloproteinase-2 (MMP-2) expression at mRNA and protein level by RT-PCR and zymography, respectively. Her-2 protein expression was analyzed in the control and ACE-c treated samples by immunoblotting as well as confocal microscopy. Apoptosis studies and calcium signaling assays were analyzed by FACS. Loss of mitochondrial membrane potential (Δψm) in cinnamon treated cells was studied by JC-1 staining and analyzed by confocal microscopy as well as FACS. Results Cinnamon alters the growth kinetics of SiHa cells in a dose-dependent manner. Cells treated with ACE-c exhibited reduced number of colonies compared to the control cells. The treated cells exhibited reduced migration potential that could be explained due to downregulation of MMP-2 expression. Interestingly, the expression of Her-2 oncoprotein was significantly reduced in the presence of ACE-c. Cinnamon extract induced apoptosis in the cervical cancer cells through increase in intracellular calcium signaling as well as loss of mitochondrial membrane potential. Conclusion Cinnamon could be used as a potent chemopreventive drug in cervical cancer.
Collapse
Affiliation(s)
- Soumya J Koppikar
- Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth University Medical College Campus, Pune, Maharashtra, India
| | | | | | | | | | | |
Collapse
|
|
19
|
Butyl benzyl phthalate suppresses the ATP-induced cell proliferation in human osteosarcoma HOS cells. Toxicol Appl Pharmacol 2010; 244:308-14. [PMID: 20114058 DOI: 10.1016/j.taap.2010.01.007] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2009] [Revised: 12/27/2009] [Accepted: 01/13/2010] [Indexed: 11/22/2022]
Abstract
Butyl benzyl phthalate (BBP), an endocrine disruptor present in the environment, exerts its genomic effects via intracellular steroid receptors and elicits non-genomic effects by interfering with membrane ion-channel receptors. We previously found that BBP blocks the calcium signaling coupled with P2X receptors in PC12 cells (Liu & Chen, 2006). Osteoblast P2X receptors were recently reported to play a role in cell proliferation and bone remodeling. In this present study, the effects of BBP on ATP-induced responses were investigated in human osteosarcoma HOS cells. These receptors mRNA had been detected, named P2X4, P2X7, P2Y2, P2Y4, P2Y5, P2Y9, and P2Y11, in human osteosarcoma HOS cells by RT-PCR. The enhancement of cell proliferation and the decrease of cytoviability had both been shown to be coupled to stimulation via different concentrations of ATP. BBP suppressed the ATP-induced calcium influx (mainly coupled with P2X) and cell proliferation but not the ATP-induced intracellular calcium release (mainly coupled with P2Y) and cytotoxicity in human osteosarcoma HOS cells. Suramin, a common P2 receptor's antagonist, blocked the ATP-induced calcium signaling, cell proliferation, and cytotoxicity. We suggest that P2X is mainly responsible for cell proliferation, and P2Y might be partially responsible for the observed cytotoxicity. BBP suppressed the calcium signaling coupled with P2X, suppressing cell proliferation. Since the importance of P2X receptors during bone metastasis has recently become apparent, the possible toxic risk of environmental BBP during bone remodeling is a public problem of concern.
Collapse
|
|
20
|
Zhang Y, Phillips GJ, Li Q, Yeung ES. Imaging localized astrocyte ATP release with firefly luciferase beads attached to the cell surface. Anal Chem 2009; 80:9316-25. [PMID: 19551993 DOI: 10.1021/ac801701w] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Extracellular adenosine triphosphate (ATP) functions as a signaling molecule in many cell regulation processes. The traditional firefly luciferase assays measure the ATP release as a signal increase with time using a luminometer. Recently, advanced cell imaging techniques using charge-coupled device (CCD) cameras have enabled two-dimensional (2D) high-resolution detection providing both spatial and temporal information. Real-time imaging of ATP release from astrocyte cells has been reported. However, the observed chemiluminescence propagation wave reflects both ATP release and diffusion in the extracellular bulk solution. The dynamic ATP efflux at the cell surface could not be accurately measured. Hence, we constructed biotinylated fused firefly luciferase proteins, immobilized the proteins on 1 microm beads, and attached the beads to the cell surface to detect ATP release from mechanically stimulated astrocyte cells. This novel detection method enables us to monitor the actual ATP concentration at the surface of single live cells. The localized ATP release was found to be prominent but lasted only <20 s, which is very different from the results obtained by free firefly luciferase detection.
Collapse
Affiliation(s)
- Yun Zhang
- Ames Laboratory, United States Department of Energy, Iowa State University, Ames, Iowa 50011, USA
| | | | | | | |
Collapse
|
|
21
|
Teixeira PCN, de Souza CAM, de Freitas MS, Foguel D, Caffarena ER, Alves LA. Predictions suggesting a participation of beta-sheet configuration in the M2 domain of the P2X(7) receptor: a novel conformation? Biophys J 2009; 96:951-63. [PMID: 19186133 DOI: 10.1016/j.bpj.2008.10.043] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2008] [Accepted: 10/15/2008] [Indexed: 11/18/2022] Open
Abstract
Scanning experiments have shown that the putative TM2 domain of the P2X(7) receptor (P2X(7)R) lines the ionic pore. However, none has identified an alpha-helix structure, the paradigmatic secondary structure of ion channels in mammalian cells. In addition, some researchers have suggested a beta-sheet conformation in the TM2 domain of P2X(2). These data led us to investigate a new architecture within the P2X receptor family. P2X(7)R is considered an intriguing receptor because its activation induces nonselective large pore formation, in contrast to the majority of other ionic channel proteins in mammals. This receptor has two states: a low-conductance channel (approximately 10 pS) and a large pore (> 400 pS). To our knowledge, one fundamental question remains unanswered: Are the P2X(7)R channel and the pore itself the same entity or are they different structures? There are no structural data to help solve this question. Thus, we investigated the hydrophobic M2 domain with the aim of predicting the fitted position and the secondary structure of the TM2 segment from human P2X(7)R (hP2X(7)R). We provide evidence for a beta-sheet conformation, using bioinformatics algorithms and molecular-dynamics simulation in conjunction with circular dichroism in different environments and Fourier transform infrared spectroscopy. In summary, our study suggests the possibility that a segment composed of residues from part of the M2 domain and part of the putative TM2 segment of P2X(7)R is partially folded in a beta-sheet conformation, and may play an important role in channel/pore formation associated with P2X(7)R activation. It is important to note that most nonselective large pores have a transmembrane beta-sheet conformation. Thus, this study may lead to a paradigmatic change in the P2X(7)R field and/or raise new questions about this issue.
Collapse
|
|
22
|
Deli T, Csernoch L. Extracellular ATP and cancer: an overview with special reference to P2 purinergic receptors. Pathol Oncol Res 2008; 14:219-31. [PMID: 18575829 DOI: 10.1007/s12253-008-9071-7] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/17/2008] [Accepted: 05/22/2008] [Indexed: 12/12/2022]
Abstract
Purinergic signal transduction mechanisms have been appreciated as a complex intercellular signalling network that plays an important regulatory role in both short- and long-term processes in practically every living cell. One of the most intriguing aspects of the field is the participation of ATP and other purine nucleotides in the determination of cell fate and the way they direct cells towards proliferation, differentiation or apoptosis, thereby possibly taking part in promoting or preventing malignant transformation. In this review, following a very brief introduction to the historical aspects of purinergic signalling and a concise overview of the structure of and signal transduction pathways coupled to P2 purinergic receptors, the current theories concerning the possible ways how extracellular ATP can alter the function of tumour cells and the effectiveness of anticancer therapies are discussed, including pharmacological, nutritional, vasoactive and 'anti-antioxidant' actions of the nucleotide. The effects of ATP on animals inoculated with human tumours and on patients with cancer are looked over next, and then an overview of the literature regarding the expression and presumed functions of P2 purinoceptors on tumour cells in vitro is presented, sorted out according to the relevant special clinical fields. The article is closed by reviewing the latest developments in the diagnostic use of P2 purinergic receptors as tumour markers and prognostic factors, while discussing some of the difficulties and pitfalls of the therapeutic use of ATP analogues.
Collapse
Affiliation(s)
- Tamás Deli
- Department of Physiology, Research Centre for Molecular Medicine, Medical and Health Science Centre, University of Debrecen, Debrecen, Hungary
| | | |
Collapse
|
|
23
|
Solini A, Cuccato S, Ferrari D, Santini E, Gulinelli S, Callegari MG, Dardano A, Faviana P, Madec S, Di Virgilio F, Monzani F. Increased P2X7 receptor expression and function in thyroid papillary cancer: a new potential marker of the disease? Endocrinology 2008; 149:389-96. [PMID: 17947359 DOI: 10.1210/en.2007-1223] [Citation(s) in RCA: 107] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Nucleotides are increasingly recognized as nonredundant extracellular signals for chemotaxis, cell growth, and cytokine release. Effects of extracellular nucleotides are mediated by P2 receptors, among which the P2X(7) subtype is attracting increasing attention for its involvement in apoptosis, cell growth, and cytokine release. Recent studies showed that P2X(7) is overexpressed in chronic lymphocytic leukemia and breast and prostate cancer. The aim of the present study was to better understand the clinical significance of P2X(7) receptor expression in normal and cancer human thyroid tissues. P2X(7) receptor message and protein expression and functional activity were tested in two cell lines (FB1 and FB2) established from either anaplastic or papillary primary thyroid cancer and in several histological samples of human papillary cancer. We show here that human thyroid papillary carcinoma, whether of the classical or follicular variant, expresses the P2X(7) receptor (P2X(7)R) to a much higher level than normal thyroid tissue. The P2X(7)R was similarly up-regulated in FB1 and FB2 cell lines. In contrast to normal thyroid cells, both cell lines responded to extracellular nucleotide stimulation with a large increase in intracellular Ca(2+) and secretion of IL-6. Ca(2+) increase was attenuated and release of IL-6 was fully blocked by P2X(7)R inhibitors. Finally, the thyroid carcinoma cell lines had at least a 3-fold higher intracellular ATP concentration and maintained at least a 3-fold higher extracellular ATP level, compared with control cells. These data suggest that an enhanced P2X(7)R function might be a feature of human thyroid cancer.
Collapse
MESH Headings
- Adenosine Triphosphate/analysis
- Adenosine Triphosphate/pharmacology
- Biomarkers, Tumor/genetics
- Biomarkers, Tumor/metabolism
- Biomarkers, Tumor/physiology
- Carcinoma, Papillary/genetics
- Carcinoma, Papillary/metabolism
- Carcinoma, Papillary/physiopathology
- Cell Line, Tumor
- Gene Expression Regulation, Neoplastic
- Humans
- Interleukin-6/metabolism
- Receptors, Purinergic P2/genetics
- Receptors, Purinergic P2/metabolism
- Receptors, Purinergic P2/physiology
- Receptors, Purinergic P2X7
- Thyroid Gland/metabolism
- Thyroid Neoplasms/genetics
- Thyroid Neoplasms/metabolism
- Thyroid Neoplasms/physiopathology
- Up-Regulation
Collapse
Affiliation(s)
- Anna Solini
- Department of Internal Medicine, University of Pisa, Via Roma 67, I-56100 Pisa, Italy.
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
24
|
Boominathan L. Some facts and thoughts: p73 as a tumor suppressor gene in the network of tumor suppressors. Mol Cancer 2007; 6:27. [PMID: 17407586 PMCID: PMC1853109 DOI: 10.1186/1476-4598-6-27] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2007] [Accepted: 04/03/2007] [Indexed: 12/30/2022] Open
Abstract
The question of whether p73 is a tumor suppressor gene, is not yet answered with full confidence. The lack of spontaneous tumor formation in p73 null mice and infrequent p73 mutations seen in a variety of cancers analyzed would straightaway negate its role as a primary tumor suppressor gene. However, accumulating evidence suggest that p73 gene and its target genes are hypermethylated in the cancer of lymphoid origin. Here I discuss some facts and thoughts that support the idea that p73 could still be a tumor suppressor gene. The tumor suppressor network in which p73 appears to be a participant involves E2F1, JunB, INK4a/p16, ARF/p19, p57kip2 and BRCA1. Knock out of each gene in E2F-1-p73-JunB-p16INK4a network of tumor suppressor proteins result in lymphoma/leukemia formation. Further, I tried to explain why lymphomas are not seen in p73 null mice and why p73 gene is not prone to frequent mutation.
Collapse
|