title: Involvement of mitochondrial and B-RAF/ERK signaling pathways in berberine-induced apoptosis in human melanoma cells.
abstract: The natural isoquinoline alkaloid berberine exhibits a wide spectrum of biological activities including antitumor activity, but its mechanism of action remains to be fully elucidated. Here, we report that berberine induced apoptosis in human melanoma cells, through a process that involved mitochondria and caspase activation. Berberine-induced activation of a number of caspases, including caspases 3, 4, 7, 8, and 9. Pan-caspase inhibitor, z-VAD-fmk, and caspase-8 and caspase-9 inhibitors prevented apoptosis. Berberine also led to the generation of the p20 cleavage fragment of BAP31, involved in directing proapoptotic signals between the endoplasmic reticulum and the mitochondria. Treatment of SK-MEL-2 melanoma cells with berberine induced disruption of the mitochondrial transmembrane potential, release of cytochrome c and apoptosis-inducing factor from the mitochondria to the cytosol, generation of reactive oxygen species (ROS), and a decreased ATP/ADP ratio. Overexpression of bcl-xL by gene transfer prevented berberine-induced cell death, mitochondrial transmembrane potential loss, and cytochrome c and apoptosis-inducing factor release, but not ROS generation. N-acetyl-L-cysteine inhibited the production of ROS, but did not abrogate the berberine-induced apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) phosphorylation, by using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and reduction of B-RAF levels by silencing RNA induced cell death of SK-MEL-2 cells, and diminished the berberine concentration required to promote apoptosis. These data show that berberine-induced apoptosis in melanoma cells involves mitochondria and caspase activation, but ROS generation was not essential. Our results indicate that inhibition of B-RAF/ERK survival signaling facilitates the cell death response triggered by berberine.
authors.affiliation: Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas - Universidad de Salamanca, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Burgeiro
authors.forename: Consuelo
authors.initials: C
authors.lastname: Gajate
authors.forename: El Habib
authors.initials: el H
authors.lastname: Dakir
authors.forename: Janny A
authors.initials: JA
authors.lastname: Villa-Pulgarín
authors.forename: Paulo J
authors.initials: PJ
authors.lastname: Oliveira
authors.forename: Faustino
authors.initials: F
authors.lastname: Mollinedo
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001599:Berberine / Q000494:pharmacology*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D045304:Cytochromes c / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology*; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D008545:Melanoma / Q000503:physiopathology*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000187:drug effects*; D048493:Proto-Oncogene Proteins B-raf / Q000096:biosynthesis / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D000227:Adenine Nucleotides; D000972:Antineoplastic Agents, Phytogenic; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D001599:Berberine; D045304:Cytochromes c; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D020169:Caspases