Neuroblastoma survival and death: an in vitro model of hypoxia and metabolic stress
| Autor: | Kartik Prabhakaran, Jeff C. Hoehner, Deborah A. Sampson |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Vascular Endothelial Growth Factor A
Pathology medicine.medical_specialty Time Factors Cell Survival Metabolite Immunocytochemistry Apoptosis Biology Severity of Illness Index Cell Line Neuroblastoma chemistry.chemical_compound Proliferating Cell Nuclear Antigen medicine Humans Hypoxia TUNEL assay Hypoxia (medical) Immunohistochemistry Oxygen tension Vascular endothelial growth factor Vascular endothelial growth factor A Proto-Oncogene Proteins c-bcl-2 chemistry Cancer research Surgery medicine.symptom Cell Division |
| Zdroj: | Journal of Surgical Research. 116:288-296 |
| ISSN: | 0022-4804 |
| DOI: | 10.1016/j.jss.2003.08.008 |
| Popis: | Heterogeneous oxygen tension and access to metabolites in solid tumors may produce variability in response to adjuvant therapy. To better understand these microenvironmental features, we examined survival and proliferation of neuroblastoma (NB) cells in an in vitro model of hypoxia and metabolite deprivation. Human NB cells (SH-SY5Y) were subjected to a "self-generated" diffusion gradient of nutrient and oxygen deprivation in a modified in vitro "sandwich model." In this model, the extent of both hypoxia and metabolite deprivation were individually altered, and the effects of each were studied. Cellular proliferation was confirmed by proliferating cell nuclear antigen (PCNA) immunocytochemistry and morphology and hypoxia by vascular endothelial growth factor (VEGF) and pimonidazole immunocytochemistry. We examined apoptotic cell death using TUNEL analysis, assaying for plasma membrane transfer of phosphotidylserine and the presence of the anti-apoptotic protein Bcl-2 using immunocytochemistry. As predicted, cellular survival diminished with increasing duration and severity of hypoxia and metabolite deprivation; oxygen deprivation was determined to be the more important contributory factor to early survival and proliferation. PCNA immunocytochemistry confirmed decreasing fractions of proliferating cells as a function of distance from oxygen and metabolites. VEGF and Bcl-2 immunoreactivity increased with prolonged exposure and increased extent of oxygen/metabolite deprivation. TUNEL analysis and phosphotidylserine transfer demonstrated cellular death of hypoxic and metabolite-deprived NB cells in a manner consistent with a mitochondrial apoptotic pathway. This in vitro model demonstrates that increasing the severity of hypoxia and metabolite deprivation results in diminished proliferation and greater apoptotic death, observations analogous to that of clinical NB tumors. |
| Databáze: | OpenAIRE |
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