title: RAF inhibition overcomes resistance to TRAIL-induced apoptosis in melanoma cells.
abstract: Mutated BRAF represents a critical oncogene in melanoma, and selective inhibitors have been approved for melanoma therapy. However, the molecular consequences of RAF inhibition in melanoma cells remained largely elusive. Here, we investigated the effects of the pan-RAF inhibitor L-779,450, which inhibited cell proliferation both in BRAF-mutated and wild-type melanoma cell lines. It furthermore enhanced apoptosis in combination with the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and overcame TRAIL resistance in melanoma cells. Enhanced apoptosis coincided with activation of mitochondrial pathways, seen by loss of mitochondrial membrane potential and release of cytochrome c, Smac (second mitochondria-derived activator of caspases), and apoptosis-inducing factor (AIF). Subsequently, caspase-9 and -3 were activated. Apoptosis induction by L-779,450/TRAIL was prevented by Bcl-2 overexpression and was dependent on Bax. Thus, activation of Bax by L-779,450 alone was demonstrated by Bax conformational changes, whereas Bak was not activated. Furthermore, the BH3-only protein Bim was upregulated in response to L-779,450. The significant roles of Smac, Bax, and Bim in this setting were proven by small interfering RNA (siRNA)-mediated knockdown experiments. L-779,450 also resulted in morphological changes indicating autophagy confirmed by the autophagy marker light chain 3-II (LC3-II). The pro-apoptotic effects of L-779,450 may explain the antitumor effects of RAF inhibition and may be considered when evaluating RAF inhibitors for melanoma therapy.
authors: Department of Dermatology and Allergy, Skin Cancer Center, University Medical Center Charité, Berlin, Germany.
authors: Anja
authors: A
authors: Berger
authors: Department of Dermatology and Allergy, Skin Cancer Center, University Medical Center Charité, Berlin, Germany.
authors: Sandra-Annika
authors: SA
authors: Quast
authors: Department of Dermatology and Allergy, Skin Cancer Center, University Medical Center Charité, Berlin, Germany.
authors: Michael
authors: M
authors: Plötz
authors: Department of Dermatology and Allergy, Skin Cancer Center, University Medical Center Charité, Berlin, Germany.
authors: Nicholas-Frederik
authors: NF
authors: Kuhn
authors: Department of Dermatology and Allergy, Skin Cancer Center, University Medical Center Charité, Berlin, Germany.
authors: Uwe
authors: U
authors: Trefzer
authors: Department of Dermatology and Allergy, Skin Cancer Center, University Medical Center Charité, Berlin, Germany. Electronic address: juergen.eberle@charite.de.
authors: Jürgen
authors: J
authors: Eberle
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects / Q000502:physiology; D000072224:Bcl-2-Like Protein 11; D049109:Cell Proliferation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D045325:HCT116 Cells; D006801:Humans; D008545:Melanoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology*; D012878:Skin Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C530671:L-779,450; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011725:Pyridines; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D051028:bcl-2-Associated X Protein; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
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