title: Effect of sulfasalazine on human neuroblastoma: analysis of sepiapterin reductase (SPR) as a new therapeutic target.
abstract: BACKGROUND
  Neuroblastoma (NB) is an aggressive childhood malignancy in children up to 5 years of age. High-stage tumors frequently relapse even after aggressive multimodal treatment, and then show therapy resistance, typically resulting in patient death. New molecular-targeted compounds that effectively suppress tumor growth and prevent relapse with more efficacy are urgently needed. We and others previously showed that polyamines (PA) like spermidine and spermine are essential for NB tumorigenesis and that DFMO, an inhibitor of the key PA synthesis gene product ODC, is effective both in vitro and in vivo, securing its evaluation in NB clinical trials. To find additional compounds interfering with PA biosynthesis, we tested sulfasalazine (SSZ), an FDA-approved salicylate-based anti-inflammatory and immune-modulatory drug, recently identified to inhibit sepiapterin reductase (SPR). We earlier presented evidence for a physical interaction between ODC and SPR and we showed that RNAi-mediated knockdown of SPR expression significantly reduced native ODC enzyme activity and impeded NB cell proliferation.
  
  
  METHODS
  Human NB mRNA expression datasets in the public domain were analyzed using the R2 platform. Cell viability, isobologram, and combination index analyses as a result of SSZ treatment with our without DFMO were carried out in NB cell cultures. Molecular protein-ligand docking was achieved using the GRAMM algorithm. Statistical analyses were performed with the Kruskal-Wallis test, 2log Pearson test, and Student's t test.
  
  
  RESULTS
  In this study, we show the clinical relevance of SPR in human NB tumors. We found that high SPR expression is significantly correlated to unfavorable NB characteristics like high age at diagnosis, MYCN amplification, and high INSS stage. SSZ inhibits the growth of NB cells in vitro, presumably due to the inhibition of SPR as predicted by computational docking of SSZ into SPR. Importantly, the combination of SSZ with DFMO produces synergistic antiproliferative effects in vitro.
  
  
  CONCLUSIONS
  The results suggest the use of SSZ in combination with DFMO for further experiments, and possible prioritization as a novel therapy for the treatment of NB patients.
authors.affiliation: Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, 301 Michigan Street, NE, Grand Rapids, MI, 49503, USA. yco@hawaii.edu.|Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, Hilo, HI, 96720, USA. yco@hawaii.edu.|Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI, 96822, USA. yco@hawaii.edu.
authors.forename: Lisette P
authors.initials: LP
authors.lastname: Yco
authors.affiliation: Department of Pediatric Oncology/Hematology, Sophia Children's Hospital, Erasmus University Medical Center, Rotterdam, GE, 3015, The Netherlands. dirkgeerts@gmail.com.
authors.forename: Dirk
authors.initials: D
authors.lastname: Geerts
authors.affiliation: Pacific Biosciences Research Center, University of Hawaii at Manoa, Honolulu, HI, 96822, USA. gmocz@hawaii.edu.
authors.forename: Gabor
authors.initials: G
authors.lastname: Mocz
authors.affiliation: Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, AZ, 1105, The Netherlands. koster.jan@gmail.com.
authors.forename: Jan
authors.initials: J
authors.lastname: Koster
authors.affiliation: Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, 301 Michigan Street, NE, Grand Rapids, MI, 49503, USA. andre.bachmann@hc.msu.edu.|Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, Hilo, HI, 96720, USA. andre.bachmann@hc.msu.edu.|Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI, 96822, USA. andre.bachmann@hc.msu.edu.
authors.forename: André S
authors.initials: AS
authors.lastname: Bachmann
mesh_terms: D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D063646:Carcinogenesis / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002648:Child; D002675:Child, Preschool; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007223:Infant; D008297:Male; D058990:Molecular Targeted Therapy; D009447:Neuroblastoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D011073:Polyamines / Q000378:metabolism; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012460:Sulfasalazine / Q000008:administration & dosage*
chemical_list: D011073:Polyamines; D011506:Proteins; C031017:ornithine decarboxylase antizyme; D012460:Sulfasalazine; D000429:Alcohol Oxidoreductases; C022630:sepiapterin reductase