title: Two death pathways induced by sorafenib in myeloma cells: Puma-mediated apoptosis and necroptosis.
abstract: OBJECTIVE
  Sorafenib is a multikinase inhibitor that targets the MAPK pathway and is currently used for the treatment of hepatocellular and renal carcinoma. Recently, it has been shown that sorafenib is also cytotoxic to multiple myeloma (MM) cells. Here, we have further analyzed the mechanism of sorafenib-induced death in MM cells.
  
  
  METHODS
  Cell death induced by sorafenib in MM cell lines and in plasma cells from MM patients was evaluated by analysis of gene expression by RT-MLPA and quantitative PCR, protein levels and functionality by Western blot and flow cytometry and gene silencing with siRNA.
  
  
  RESULTS
  Cell death was characterized by phosphatidylserine exposure, ΔΨm loss, cytochrome c release and caspase activation, hallmarks of apoptosis. DL50 at 24 h ranged from 6 to 10 µM. Ex vivo treatment with 20 µM sorafenib induced apoptosis in around 80 % myeloma cells from six multiple myeloma patients. Sorafenib induced caspase-dependent degradation of Bcl-xL and Mcl-1 proteins, destabilizing the mitochondria and speeding up the development of apoptosis. Sorafenib treatment increased levels of Puma at mRNA and protein level and gene silencing with siRNA confirmed a relevant role for Puma in the induction of apoptosis. Co-treatment with the pan-caspase inhibitor Z-VAD-fmk prevented cell death to a variable degree depending on the cell line. In RPMI 8226 cells, Z-VAD-fmk prevented most of sorafenib-induced death. However, death in MM.1S was only prevented by co-incubation with both Z-VAD-fmk and the RIP1K inhibitor necrostatin-1, indicating that under conditions of inefficient caspase activation, sorafenib induces death by necroptosis.
  
  
  CONCLUSIONS
  Our results demonstrate a key role for Puma in the triggering of sorafenib-induced apoptosis and that this drug can also induce death by necroptosis in multiple myeloma cells.
authors.affiliation: Departamento de Bioquimica, Biologia Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009, Zaragoza, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Ramírez-Labrada
authors.forename: N
authors.initials: N
authors.lastname: López-Royuela
authors.forename: V
authors.initials: V
authors.lastname: Jarauta
authors.forename: P
authors.initials: P
authors.lastname: Galán-Malo
authors.forename: G
authors.initials: G
authors.lastname: Azaceta
authors.forename: L
authors.initials: L
authors.lastname: Palomera
authors.forename: J
authors.initials: J
authors.lastname: Pardo
authors.forename: A
authors.initials: A
authors.lastname: Anel
authors.forename: I
authors.initials: I
authors.lastname: Marzo
authors.forename: J
authors.initials: J
authors.lastname: Naval
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D061945:Caspase Inhibitors / Q000494:pharmacology; D020169:Caspases / Q000737:chemistry; D049109:Cell Proliferation / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D009101:Multiple Myeloma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D009336:Necrosis; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077157:Sorafenib; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D061945:Caspase Inhibitors; D010671:Phenylurea Compounds; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D009536:Niacinamide; D000077157:Sorafenib; D020169:Caspases