title: Chelerythrine promotes Ca2+-dependent calpain activation in neuronal cells in a PKC-independent manner.
abstract: Chelerythrine is widely used as a broad range protein kinase C (PKC) inhibitor, but there is controversy about its inhibitory effect. Moreover, it has been shown to exert PKC-independent effects on non-neuronal cells.
  
  
  
  In this study we investigated possible off-target effects of chelerythrine on cultured cortical rodent neurons and a neuronal cell line.
  
  
  
  We found that 10μM chelerythrine, a commonly used concentration in neuronal cultures, reduces PKC and cAMP-dependent protein kinase substrates phosphorylation in mouse cultured cortical neurons, but not in rat primary cortical neurons or in a striatal cell line. Furthermore, we found that incubation with chelerythrine increases pERK1/2 levels in all models studied. Moreover, our results show that chelerythrine promotes calpain activation as assessed by the cleavage of spectrin, striatal-enriched protein tyrosine phosphatase and calcineurin A. Remarkably, chelerythrine induces a concentration-dependent increase in intracellular Ca2+levels that mediates calpain activation. In addition, we found that chelerythrine induces ERK1/2- and calpain-independent caspase-3 activation that can be prevented by the Ca2+chelator BAPTA-AM.
  
  
  
  This is the first report showing that chelerythrine promotes Ca2+-dependent calpain activation in neuronal cells, which has consequences for the interpretation of studies using this compound.
  
  
  
  Chelerythrine is still marketed as a specific PKC inhibitor and extensively used in signal transduction studies. We believe that the described off-target effects should preclude its use as a PKC inhibitor in future works.
authors.affiliation: Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain; Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain. Electronic address: anasaavedra@ub.edu.
authors.forename: Ana
authors.initials: A
authors.lastname: Saavedra
authors.affiliation: Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain; Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Fernández-García
authors.affiliation: Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Cases
authors.affiliation: Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain; Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain.
authors.forename: Mar
authors.initials: M
authors.lastname: Puigdellívol
authors.affiliation: Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain; Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Alcalá-Vida
authors.affiliation: Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain.
authors.forename: Núria
authors.initials: N
authors.lastname: Martín-Flores
authors.affiliation: Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain; Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain.
authors.forename: Jordi
authors.initials: J
authors.lastname: Alberch
authors.affiliation: Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain; Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Ginés
authors.affiliation: Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Malagelada
authors.affiliation: Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain; Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain. Electronic address: estherperez@ub.edu.
authors.forename: Esther
authors.initials: E
authors.lastname: Pérez-Navarro
mesh_terms: D000818:Animals; D053119:Benzophenanthridines / Q000494:pharmacology*; D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008565:Membrane Proteins / Q000494:pharmacology*; D051379:Mice; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D053119:Benzophenanthridines; D008565:Membrane Proteins; C410340:calpain activator; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; C016299:chelerythrine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019703:Calcineurin; D017027:Protein Tyrosine Phosphatases; D002154:Calpain; D053148:Caspase 3; D002118:Calcium
keywords: Cleaved caspase-3; ERK1/2; PKA; Spectrin breakdown products; Striatal-enriched protein tyrosine phosphatase