title: Carfilzomib combined with suberanilohydroxamic acid (SAHA) synergistically promotes endoplasmic reticulum stress in non-small cell lung cancer cell lines.
abstract: OBJECTIVE
  The endoplasmic reticulum (ER) stress response is a therapeutic target for pharmacologic intervention in cancer cells. We hypothesized that combining carfilzomib (CFZ), a proteasome inhibitor, and vorinostat (SAHA), a histone deacetylase (HDAC) inhibitor, would synergistically activate ER stress in non-small cell lung cancer (NSCLC) cell lines, resulting in enhanced anti-tumor activity.
  
  
  METHODS
  Five NSCLC cell lines were treated with CFZ, SAHA, or the combination and cell proliferation measured using the MTT assay. Calcusyn software was utilized to determine the combination index as a measure of synergy. Cell viability and cytotoxicity were measured using trypan blue exclusion, CellTiter, and CytoTox assays. Western blot was used to measure markers of apoptosis, ER stress, and oxidative stress-related proteins. Reactive oxygen species (ROS) was measured using the fluorophore CM-H2DCFDA.
  
  
  RESULTS
  Synergistic activity was observed for all cell lines following 48 and 72 h of combined treatment. H520 and A549 cell lines were used to assess viability and apoptosis. In both cell lines, increased death and cleaved caspase-3 were observed following combination treatment as compared with single-agent treatments. Combination therapy was associated with upregulation of ER stress-regulated proteins including activating transcription factor 4, GRP78/BiP, and C/EBP homologous protein. Both cell lines also showed increased ROS and the oxidative stress-related protein, heat shock protein 70.
  
  
  CONCLUSIONS
  Combining proteasome inhibition with HDAC inhibition enhances ER stress, which may contribute to the synergistic anticancer activity observed in NSCLC cell lines. Further preclinical and clinical studies of CFZ + SAHA in NSCLC are warranted.
authors.affiliation: Section of Hematology/Oncology, College of Medicine, University of Arizona Cancer Center, 1515 N Campbell Ave, Tucson, AZ, USA.
authors.forename: Neale T
authors.initials: NT
authors.lastname: Hanke
authors.affiliation: Section of Hematology/Oncology, College of Medicine, University of Arizona Cancer Center, 1515 N Campbell Ave, Tucson, AZ, USA.
authors.forename: Linda L
authors.initials: LL
authors.lastname: Garland
authors.affiliation: Section of Hematology/Oncology, College of Medicine, University of Arizona Cancer Center, 1515 N Campbell Ave, Tucson, AZ, USA. abaker@uacc.arizona.edu.
authors.forename: Amanda F
authors.initials: AF
authors.lastname: Baker
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology* / Q000627:therapeutic use; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000473:pathology; D009842:Oligopeptides / Q000494:pharmacology* / Q000627:therapeutic use; D018384:Oxidative Stress / Q000187:drug effects; D000077337:Vorinostat
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D009842:Oligopeptides; D000077337:Vorinostat; C524865:carfilzomib
keywords: Carfilzomib; Non-small cell lung cancer; Proteasome inhibitor; SAHA