Ana Saavedra1, Sara Fernández-García2, Silvia Cases3, Mar Puigdellívol2, Rafael Alcalá-Vida2, Núria Martín-Flores3, Jordi Alberch2, Silvia Ginés2, Cristina Malagelada3, Esther Pérez-Navarro4. 1. 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. 2. 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. 3. Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain. 4. 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.
Abstract
BACKGROUND: 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. METHODS: In this study we investigated possible off-target effects of chelerythrine on cultured cortical rodent neurons and a neuronal cell line. RESULTS: 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. CONCLUSIONS: 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. GENERAL SIGNIFICANCE: 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.
BACKGROUND: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. METHODS: In this study we investigated possible off-target effects of chelerythrine on cultured cortical rodent neurons and a neuronal cell line. RESULTS: 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. CONCLUSIONS: 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. GENERAL SIGNIFICANCE: 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: Ashwini Saxena; Giselli Scaini; Daniela V Bavaresco; Camila Leite; Samira S Valvassori; André F Carvalho; João Quevedo Journal: Mol Neuropsychiatry Date: 2017-10-07
Authors: Moutoshi Chakraborty; S M Fajle Rabby; Dipali Rani Gupta; Mahfuzur Rahman; Sanjoy Kumar Paul; Nur Uddin Mahmud; Abdullah Al Mahbub Rahat; Ljupcho Jankuloski; Tofazzal Islam Journal: Microorganisms Date: 2022-06-09