title: All-trans retinoic acid arrests neuroblastoma cells in a dormant state. Subsequent nerve growth factor/brain-derived neurotrophic factor treatment adds modest benefit.
abstract: BACKGROUND
  Therapies aiming at inducing differentiation or apoptosis of neuroblastoma (NB) are an important research topic. Although retinoic acid showed promising antitumoral results, its effects against refractory disease are limited. Putative candidates for combination therapies are nerve growth factor (NGF; Tebu-Bio/Peprotech, Offenbach, Germany) and brain-derived neurotrophic factor (BDNF; Tebu-Bio/Peprotech, Offenbach, Germany) because their receptors are of prognostic clinical value in clinical neuroblastoma. Another clinical prognostic factor is the number of Schwann cells. Substances secreted by Schwann cells proved antitumoral capacities in vitro. The aim of the study was to analyze whether retinoic acid may offer an additional line of attack acting independent from Schwann cells and whether additive treatment with the neurotrophin-receptor ligands NGF/BDNF confers additional benefit.
  
  
  METHODS
  Human SHSY-5Y NB cells were cultured in vitro. After a 7-day all-trans retinoic acid (ATRA; Sigma-Aldrich Chemie, Taufkirchen, Germany) treatment (15 mumol/L of ATRA), NB proliferation was proportional to extinction in dimethyl-thiazol-diphenyltetrazoliumbromide (MTT) tests. Fluorescence-activated cell sorter (FACS) analysis for annexin and propidium iodide determined the degree of apoptosis and necrosis as well as the expression of the Schwann type cell marker S100. The S100 messenger RNA was assessed by reverse transcriptase polymerase chain reaction. In addition, the effect on NB proliferation was investigated when ATRA was combined with a 7-day treatment with NGF or BDNF (10, 50, 100 ng/mL) either before or after the 7-day ATRA treatment.
  
  
  RESULTS
  All-trans retinoic acid reduced proliferation (0.116 +/- 0.006 SEM vs 0.359 +/- 0.010 SEM in the untreated control group; P < .001). After ATRA treatment, 95% +/- 1.82% SEM were still viable, with only 2.61% +/- 1.17% SEM apoptotic and 2.38% +/- 0.69% SEM necrotic cells. All-trans retinoic acid induced a remarkable decrease in S100 expression in FACS (16.91% +/- 1.72% SEM vs 32.33% +/- 2.54% SEM in controls; P = .009). The S100 messenger RNA levels were not increased by ATRA (DeltaDeltaT values: 1.73, 2.77, and 1.43; n = 3). Both NGF and BDNF had only a modest synergistic effect when given after ATRA treatment. No effect was seen when they were administered before ATRA treatment.
  
  
  CONCLUSIONS
  All-trans retinoic proved to be a vigorous inhibitor of NB proliferation in vitro. However, because most NB cells remained viable combination therapies are required. Treatment with NGF and BDNF showed only a modest benefit and did not reflect the strong prognostic impact of tyrosine kinase receptors in clinical NB. The ATRA-induced proliferation arrest is not related to Schwann type subdifferentiation. This suggests that substances secreted by Schwann cells could be possible independent combination partners. We suggest studies using combinations of ATRA and substances secreted by Schwann cells.
authors.affiliation: Department of Pediatric Surgery, Marienhospital II-Kinderchirurgische Klinik der Ruhr-Universität Bochum, Widumerstr 8, 44627 Herne, Germany. grigorec@hotmail.com
authors.forename: Grigore
authors.initials: G
authors.lastname: Cernaianu
authors.forename: Philipp
authors.initials: P
authors.lastname: Brandmaier
authors.forename: Gabriele
authors.initials: G
authors.lastname: Scholz
authors.forename: Oliver Pelz
authors.initials: OP
authors.lastname: Ackermann
authors.forename: Ruediger
authors.initials: R
authors.lastname: Alt
authors.forename: Karin
authors.initials: K
authors.lastname: Rothe
authors.forename: Michael
authors.initials: M
authors.lastname: Cross
authors.forename: Helmut
authors.initials: H
authors.lastname: Witzigmann
authors.forename: Ralf B
authors.initials: RB
authors.lastname: Tröbs
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D006801:Humans; D020932:Nerve Growth Factor / Q000494:pharmacology*; D009447:Neuroblastoma / Q000188:drug therapy*; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D019208:Brain-Derived Neurotrophic Factor; D014212:Tretinoin; D020932:Nerve Growth Factor