title: Modulation of interactions of neuroblastoma cell lines with extracellular matrix proteins affects their sensitivity to treatment with the anti-GD2 ganglioside antibody 14G2a.
abstract: Children diagnosed with high risk neuroblastoma have poor prognosis which stimulates efforts to broaden therapies of the neoplasm. GD2-ganglioside (GD2) marks neuroblastoma cells and is a target for monoclonal antibodies. We have recently shown that some neuroblastoma cell lines are sensitive to direct cytotoxicity of the anti-GD2 mouse monoclonal antibody 14G2a (mAb). For IMR-32 and LA-N-1 cell lines, treatment with the 14G2a mAb induced evident changes in appearance such as cell rounding, aggregation, loose contact with culture plastic, or detachment. Such findings prompted us to investigate whether modulation of attachment of neuroblastoma cells to extracellular matrix (ECM) proteins can affect their sensitivity to the 14G2a mAb treatment. First, using ultra-low attachment plates, we show that survival of the IMR-32, LA-N-1, LA-N-5, CHP-134 and Kelly cells depends on attachment. Next, we compared cellular ATP levels of the cell lines treated with the 14G2a mAb using uncoated, fibronectin-, collagen IV-coated surfaces to show that the ECM proteins slightly modulate sensitivity of the cell lines to the mAb. Then, we characterized presence of selected integrin subunits or their complexes on the cell surface. Finally, we applied small molecule inhibitors of selected integrin complexes: obtustatin (inhibiting α1β1 heterodimer), BIO 1211 (inhibiting active α4β1 heterodimer), cilengitide and SB273005 (inhibitors of αVβ3, αVβ5 heterodimers) to verify their effects on attachment of cell lines, cellular ATP levels, and in some experiments activities of apoptosis-executing caspase-3 and -7, for the compounds used alone or in combination with the 14G2a mAb. We characterized levels of total FAK (focal adhesion kinase), p-FAK (Tyr397) in IMR-32 cells treated with BIO 1211, and in LA-N-5, Kelly and SK-N-SH cells treated with SB273005. Our results extend knowledge on factors influencing cytotoxicity of 14G2a.
authors.affiliation: Laboratory of Molecular Genetics and Virology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland.
authors.forename: Irena
authors.initials: I
authors.lastname: Horwacik
authors.affiliation: Laboratory of Molecular Genetics and Virology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland.
authors.forename: Hanna
authors.initials: H
authors.lastname: Rokita
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000276:immunology*; D017209:Apoptosis / Q000187:drug effects; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000008:administration & dosage; D053148:Caspase 3 / Q000096:biosynthesis; D045744:Cell Line, Tumor; D004224:Diterpenes; D016326:Extracellular Matrix Proteins / Q000276:immunology*; D005732:Gangliosides / Q000235:genetics / Q000276:immunology*; D006801:Humans; D051379:Mice; D009447:Neuroblastoma / Q000188:drug therapy* / Q000235:genetics / Q000276:immunology / Q000473:pathology; D054852:Small Molecule Libraries / Q000008:administration & dosage; D012910:Snake Venoms / Q000008:administration & dosage
chemical_list: D000911:Antibodies, Monoclonal; D019086:Bridged Bicyclo Compounds, Heterocyclic; D004224:Diterpenes; D016326:Extracellular Matrix Proteins; D005732:Gangliosides; D054852:Small Molecule Libraries; D012910:Snake Venoms; C422910:Cilengitide; C508887:retapamulin; C019403:ganglioside, GD2; D000255:Adenosine Triphosphate; D053148:Caspase 3