title: Metabolic characteristics of 13-cis-retinoic acid (isotretinoin) and anti-tumour activity of the 13-cis-retinoic acid metabolite 4-oxo-13-cis-retinoic acid in neuroblastoma.
abstract: OBJECTIVE
  Isotretinoin (13-cis-retinoic acid; 13-cRA) is a differentiation inducer used to treat minimal residual disease after myeloablative therapy for high-risk neuroblastoma. However, more than 40% of children develop recurrent disease during or after 13-cRA treatment. The plasma concentrations of 13-cRA in earlier studies were considered subtherapeutic while 4-oxo-13-cis-RA (4-oxo-13-cRA), a metabolite of 13-cRA considered by some investigators as inactive, were greater than threefold higher than 13-cRA. We sought to define the metabolic pathways of 13-cRA and investigated the anti-tumour activity of its major metabolite, 4-oxo-13-cRA.
  
  
  METHODS
  Effects of 13-cRA and 4-oxo-13-cRA on human neuroblastoma cell lines were assessed by DIMSCAN and flow cytometry for cell proliferation, MYCN down-regulation by reverse transcription PCR and immunoblotting, and neurite outgrowth by confocal microscopy. 13-cRA metabolism was determined using tandem MS in human liver microsomes and in patient samples.
  
  
  RESULTS
  Six major metabolites of 13-cRA were identified in patient samples. Of these, 4-oxo-13-cRA was the most abundant, and 4-oxo-13-cRA glucuronide was also detected at a higher level in patients. CYP3A4 was shown to play a major role in catalysing 13-cRA to 4-oxo-13-cRA. In human neuroblastoma cell lines, 4-oxo-13-cRA and 13-cRA were equi-effective at inducing neurite outgrowth, inhibiting proliferation, decreasing MYCN mRNA and protein, and increasing the expression of retinoic acid receptor-β mRNA and protein levels.
  
  
  CONCLUSIONS
  We showed that 4-oxo-13-cRA is as active as 13-cRA against neuroblastoma cell lines. Plasma levels of both 13-cRA and 4-oxo-13-cRA should be evaluated in pharmacokinetic studies of isotretinoin in neuroblastoma.
authors.affiliation: Cancer Center, Texas Tech University Health Sciences Center, Lubbock, TX, USA; Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, TX, USA.
authors.forename: Poonam
authors.initials: P
authors.lastname: Sonawane
authors.forename: Hwang Eui
authors.initials: HE
authors.lastname: Cho
authors.forename: Ashujit
authors.initials: A
authors.lastname: Tagde
authors.forename: Dattesh
authors.initials: D
authors.lastname: Verlekar
authors.forename: Alice L
authors.initials: AL
authors.lastname: Yu
authors.forename: C Patrick
authors.initials: CP
authors.lastname: Reynolds
authors.forename: Min H
authors.initials: MH
authors.lastname: Kang
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000097:blood / Q000493:pharmacokinetics* / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016543:Central Nervous System Neoplasms / Q000097:blood / Q000188:drug therapy / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D006801:Humans; D015474:Isotretinoin / Q000097:blood / Q000493:pharmacokinetics* / Q000494:pharmacology; D008807:Mice, Inbred BALB C; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D000071447:N-Myc Proto-Oncogene Protein; D016501:Neurites / Q000187:drug effects; D009447:Neuroblastoma / Q000097:blood / Q000188:drug therapy / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014212:Tretinoin / Q000031:analogs & derivatives* / Q000097:blood / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; C082925:retinoic acid receptor beta; C002202:4-oxoretinoic acid; D014212:Tretinoin; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D015474:Isotretinoin