title: Dihydroceramide desaturase knockdown impacts sphingolipids and apoptosis after photodamage in human head and neck squamous carcinoma cells.
abstract: BACKGROUND
  Dihydroceramide desaturase 1 (DES) is the enzyme responsible for converting dihydroceramide into ceramide in the de novo sphingolipid biosynthesis pathway. Dihydroceramide can inhibit ceramide channel formation to interfere with apoptosis. We have shown that following ceramide synthase knockdown, photodynamic therapy (PDT), a cancer treatment modality, is associated with decreased levels of ceramides and dihydroceramides in cells that are resistant to apoptosis.
  
  
  OBJECTIVE
  Here we investigated the effect of DES knockdown on the sphingolipid profile and apoptosis in human head and neck squamous carcinoma cells after PDT with the silicon phthalocyanine Pc 4.
  
  
  METHODS
  Following siRNA transfection and PDT treatment, quantitative real-time polymerase chain reaction for quantification of DES mRNA, immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectrofluorometry for caspase 3-like (DEVDase) activity, flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation, were performed.
  
  
  RESULTS
  Down-regulation of DES led to a substantial increase in levels of dihydroceramides without affecting ceramide levels. PDT-induced accumulation of individual dihydroceramides and global ceramides was increased by DES knockdown. Concomitantly, mitochondrial depolarization, DEVDase activation, late-apoptosis and cell death were attenuated by DES knockdown. Early apoptosis, however, was enhanced.
  
  
  CONCLUSIONS
  Our findings support the following: (i) dihydroceramide reduces pro-apoptotic effects of ceramide; (ii) cells adapt to DES knockdown to become more sensitive to ceramide and early-apoptosis; (iii) DES is a potential molecular target for regulating apoptotic resistance to PDT.
authors.affiliation: Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Ave., Detroit, MI 48201, USA. dseparovic@wayne.edu
authors.forename: Paul
authors.initials: P
authors.lastname: Breen
authors.forename: Nicholas
authors.initials: N
authors.lastname: Joseph
authors.forename: Kyle
authors.initials: K
authors.lastname: Thompson
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Kraveka
authors.forename: Tatyana I
authors.initials: TI
authors.lastname: Gudz
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Mehrdad
authors.initials: M
authors.lastname: Rahmaniyan
authors.forename: Jacek
authors.initials: J
authors.lastname: Bielawski
authors.forename: Jason S
authors.initials: JS
authors.lastname: Pierce
authors.forename: Eric
authors.initials: E
authors.lastname: VAN Buren
authors.forename: Gaurav
authors.initials: G
authors.lastname: Bhatti
authors.forename: Duska
authors.initials: D
authors.lastname: Separovic
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000628:therapy; D045744:Cell Line, Tumor; D002518:Ceramides / Q000378:metabolism / Q000528:radiation effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000528:radiation effects; D006258:Head and Neck Neoplasms / Q000378:metabolism / Q000628:therapy; D006801:Humans; D007211:Indoles / Q000008:administration & dosage; D058990:Molecular Targeted Therapy; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010778:Photochemotherapy; D034741:RNA, Small Interfering; D013107:Sphingolipids / Q000378:metabolism
chemical_list: D002518:Ceramides; D007211:Indoles; D034741:RNA, Small Interfering; D013107:Sphingolipids; C109343:dihydroceramide; C107448:phthalocyanine Pc 4; D010088:Oxidoreductases; C108138:dihydroceramide desaturase