title: Salinomycin induces cell death and differentiation in head and neck squamous cell carcinoma stem cells despite activation of epithelial-mesenchymal transition and Akt.
abstract: BACKGROUND
  Cancer stem cells (CSC) are believed to play a crucial role in cancer recurrence due to their resistance to conventional chemotherapy and capacity for self-renewal. Recent studies have reported that salinomycin, a livestock antibiotic, selectively targets breast cancer stem cells 100-fold more effectively than paclitaxel. In our study we sought to determine the effects of salinomycin on head and neck squamous cell carcinoma (HNSCC) stem cells.
  
  
  METHODS
  MTS and TUNEL assays were used to study cell proliferation and apoptosis as a function of salinomycin exposure in JLO-1, a putative HNSCC stem cell culture. MTS and trypan blue dye exclusion assays were performed to investigate potential drug interactions between salinomycin and cisplatin or paclitaxel. Stem cell-like phenotype was measured by mRNA expression of stem cell markers, sphere-forming capacity, and matrigel invasion assays. Immunoblotting was also used to determine expression of epithelial-mesenchymal transition (EMT) markers and Akt phosphorylation. Arrays by Illumina, Inc. were used to profile microRNA expression as a function of salinomycin dose.
  
  
  RESULTS
  In putative HNSCC stem cells, salinomycin was found to significantly inhibit cell viability, induce a 71.5% increase in levels of apoptosis, elevate the Bax/Bcl-2 ratio, and work synergistically with cisplatin and paclitaxel in inducing cell death. It was observed that salinomycin significantly inhibited sphere forming-capability and repressed the expression of CD44 and BMI-1 by 3.2-fold and 6.2-fold, respectively. Furthermore, salinomycin reduced invasion of HNSCC stem cells by 2.1 fold. Contrary to expectations, salinomycin induced the expression of EMT markers Snail, vimentin, and Zeb-1, decreased expression of E-cadherin, and also induced phosphorylation of Akt and its downstream targets GSK3-β and mTOR.
  
  
  CONCLUSIONS
  These results demonstrate that in HNSCC cancer stem cells, salinomycin can cause cell death and decrease stem cell properties despite activation of both EMT and Akt.
authors.affiliation: Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of California, San Diego, San Diego, CA, USA.
authors.forename: Selena Z
authors.initials: SZ
authors.lastname: Kuo
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Blair
authors.forename: Elham
authors.initials: E
authors.lastname: Rahimy
authors.forename: Alan
authors.initials: A
authors.lastname: Kiang
authors.forename: Eric
authors.initials: E
authors.lastname: Abhold
authors.forename: Jian-Bing
authors.initials: JB
authors.lastname: Fan
authors.forename: Jessica
authors.initials: J
authors.lastname: Wang-Rodriguez
authors.forename: Xabier
authors.initials: X
authors.lastname: Altuna
authors.forename: Weg M
authors.initials: WM
authors.lastname: Ongkeko
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology*; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects* / Q000235:genetics; D006258:Head and Neck Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology*; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics; D048054:Mitogen-Activated Protein Kinase 7 / Q000235:genetics; D014411:Neoplastic Stem Cells / Q000187:drug effects*; D017239:Paclitaxel / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D011714:Pyrans / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D000077195:Squamous Cell Carcinoma of Head and Neck; D051028:bcl-2-Associated X Protein / Q000235:genetics; D000095662:Polyether Polyketides
chemical_list: D002945:Cisplatin; D018960:Hyaluronan Receptors; D035683:MicroRNAs; D048054:Mitogen-Activated Protein Kinase 7; D017239:Paclitaxel; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; D011714:Pyrans; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; C497483:CD44 protein, human; C010327:salinomycin; D000095662:Polyether Polyketides; C481581:MAPK7 protein, human