The effect of alpha-v integrin inhibition on the malignant characteristics of medulloblastoma

Laboratory investigation

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Object

Hypoxia induces an aggressive phenotype in some brain tumors in part due to hypoxia-inducible factor–1α (HIF-1α) and integrin expression. The importance of hypoxia in medulloblastoma is unclear and the interaction of HIF-1α and c-Myc in medulloblastoma has not been explored. The objective of this study was to determine if hypoxia induces an aggressive phenotype in human medulloblastoma cells that constitutively express high (D283 Med) or low (DAOY) levels of c-Myc and to determine if blocking αv integrins with the monoclonal antibody intetumumab inhibits hypoxia-induced cellular stress responses.

Methods

Cells were grown at 21% and 1% O2 and in the presence or absence of intetumumab. Measures of malignancy evaluated included cell proliferation, cell migration, and expression of vascular endothelial growth factor (VEGF), αv integrins, HIF-1α, and c-Myc.

Results

Both cell lines robustly expressed αv integrins. Hypoxic DAOY cells showed significantly increased proliferation compared with normoxic controls (p < 0.05), whereas D283 Med cells did not. Both cell lines exhibited a dose-dependent decrease in proliferation when treated with intetumumab (p < 0.05). Hypoxia did not increase DAOY migration, but intetumumab significantly inhibited migration at both oxygen conditions (p < 0.05). Intetumumab significantly decreased VEGF levels in DAOY cells at both oxygen conditions (p < 0.05) and in normoxic D283 cells (p < 0.01). Neither cell line demonstrated increased HIF-1α expression in response to hypoxia. However, hypoxic D283 Med cells grown in the presence of intetumumab demonstrated significantly decreased c-Myc expression (p < 0.05).

Conclusions

Hypoxia did not clearly induce a more aggressive phenotype in medulloblastoma cells. Despite this result, intetumumab decreased medulloblastoma cell proliferation and migration and variably decreased VEGF and c-Myc expression in hypoxic conditions. Targeting αv integrins represents a promising potential adjuvant modality in the treatment of medulloblastoma, particularly subtypes that metastasize and overexpress VEGF and c-Myc.

Abbreviations used in this paper:DMEM = Dulbecco modification of Eagle medium; ELISA = enzyme-linked immunosorbent assay; FBS = fetal bovine serum; HIF = hypoxia-inducible factor; PBS = phosphate-buffered saline; VEGF = vascular endothelial growth factor.

Article Information

Address correspondence to: Edward A. Neuwelt, M.D., Department of Neurology, Blood-Brain Barrier Program, Oregon Health & Science University, 3181 Sam Jackson Park Road, L603, Portland, Oregon 97239. email: neuwelte@ohsu.edu.

Please include this information when citing this paper: published online October 19, 2012; DOI: 10.3171/2012.9.PEDS12268.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    DAOY and D283 Med cells express αv integrins. A: Graph showing DAOY cells express αv, αvβ3 and αvβ5 integrins on the cell surface in vitro. B: Graph showing D283 Med cells express αv, αvβ5 and αvβ1 integrins on the cell surface in vitro. In A and B, data represent the mean of 2 different experiments, and values are adjusted to the background level of fluorescence. Error bars = standard error. C: Representative Western blot demonstrates robust expression of αv integrins in both DAOY and D283 Med cell lines. There was no significant difference in the amount of αv integrin expression between the 2 cell lines, between cells grown at 1% or 21% O2, or cells treated with intetumumab (Int, +) or vehicle (−). RFU = relative fluorescent unit.

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    Proliferative response to hypoxia differs in DAOY and D283 Med cells and is inhibited by intetumumab. Left: Graph representing DAOY cells grown in 1% (white bars) or 21% O2 (gray bars) in the presence of 3 escalating doses of intetumumab. Cells grown in the presence of 1.5 mg/ml intetumumab showed significantly less proliferation compared with cells grown in the presence of vehicle in their respective oxygen condition. Cells grown at 1% O2 demonstrated significantly more proliferation compared with their counterparts grown at 21% O2 with the exception of cells grown in the presence of 1.5 mg/ml intetumumab. Right: Graph representing D283 Med cells grown under 1% or 21% O2 conditions at 3 escalating doses of intetumumab. Cells grown in the presence of all doses of intetumumab showed significantly less proliferation compared with cells grown in the presence of vehicle within their respective oxygen condition. There was no difference in proliferation between cells grown at 1% and 21% O2. Figures are means of 3 different experiments. Error bars = standard error. *p < 0.05, **p < 0.01; NS = not significant.

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    c-Myc expression in D283 Med cells is inhibited by the combination of hypoxia and intetumumab. A: Representative Western blot demonstrates decreased expression of c-Myc in DAOY cells compared with D283 Med cells and D283 Med cells grown at 1% O2 in the presence of intetumumab (+). B: Graph of the expression of c-Myc in 3 separate experiments normalized to tubulin, in which the expression appears greater in D283 Med cells than in DAOY cells, although this difference was not significant due to large variance. C: Graph of the expression of c-Myc in 3 separate experiments, normalized to tubulin and to the control (21% O2 vehicle). Neither hypoxic conditions nor the presence of intetumumab significantly affected the expression of c-Myc in DAOY cells. D283 Med cells grown in 1% O2 in the presence of intetumumab demonstrated significantly less c-Myc expression compared with controls. Error bars = standard error. White bars = 1% O2, gray bars = 21% O2. *p < 0.05; AU = arbitrary units.

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    Intetumumab inhibits VEGF but not HIF-1α expression in DAOY and D283 Med cells. A: Graph showing intetumumab significantly decreased VEGF production in DAOY cells grown at both oxygen conditions and D283 Med cells grown at 21% O2 in 2 separate experiments. B: Graph showing there was no significant difference in the production of HIF-1α in cells grown in the presence of intetumumab or hypoxic conditions in both DAOY and D283 Med cell lines. C: Representative Western blot demonstrates no significant difference in HIF-1α expression in both cell lines grown in various conditions. Error bars = standard error. Gray bars = 21% O2, white bars = 1% O2. *p < 0.05.

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    Cell migration is inhibited by intetumumab. Upper: DAOY cell migration in both hypoxic and normoxic conditions was significantly inhibited by intetumumab. There was no significant difference in rate of migration between the two oxygen environments. Lower: Representative photos of intetumumab inhibiting cell migration (gap closure) at 0-, 8-, and 24-hour time points. Original magnification ×50. *p < 0.05.

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