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Bevacizumab and intraocular tumors: an intriguing paradox.

el Filali M, Ly LV, Luyten GP, Versluis M, Grossniklaus HE, van der Velden PA, Jager MJ - Mol. Vis. (2012)

Bottom Line: We determined the effect of bevacizumab on the growth of B16F10 cells inside the eye and on B16F10 and UM cells cultured in vitro.In addition, the effect of bevacizumab on in vitro growth of B16F10 and human UM cells and on the expression of VEGF-A, GLUT-1, and HIF-1α was evaluated.Bevacizumab did not affect proliferation of B16F10 cells in vitro, while it inhibited UM cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, LUMC, Leiden, the Netherlands. m.el_filali@lumc.nl

ABSTRACT

Purpose: Bevacizumab, a humanized monoclonal antibody to vascular endothelial growth factor-A (VEGF-A), was originally developed as an anti-tumor treatment. In ocular oncology, it is being used to treat macular edema due to radiation retinopathy, but it may also be useful for the treatment of primary uveal melanoma (UM) or its metastases. We determined the effect of bevacizumab on the growth of B16F10 cells inside the eye and on B16F10 and UM cells cultured in vitro.

Methods: B16F10 melanoma cells were placed into the anterior chamber of the eye of C57Bl/6 mice and tumor growth was monitored after injection of different doses of bevacizumab or mock injection. In addition, the effect of bevacizumab on in vitro growth of B16F10 and human UM cells and on the expression of VEGF-A, GLUT-1, and HIF-1α was evaluated.

Results: Following intraocular injection of bevacizumab into murine B16 tumor-containing eyes, an acceleration of tumor growth was observed, with the occurrence of anterior chamber hemorrhages. Bevacizumab did not affect proliferation of B16F10 cells in vitro, while it inhibited UM cell proliferation. Expression analysis demonstrated that addition of bevacizumab under hypoxic conditions induced VEGF-A, GLUT-1 and HIF-1α in B16F10 cells as well as in UM cell lines and two of four primary UM tumor cultures.

Conclusions: In contrast with expectations, intraocular injection of bevacizumab stimulated B16F10 melanoma growth in murine eyes. In vitro exposure of B16 and human UM cells to bevacizumab led to paradoxical VEGF-A upregulation. The use of VEGF inhibitors for treatment of macular edema (due to radiation retinopathy) after irradiation of UM should be considered carefully, because of the possible adverse effects on residual UM cells.

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Involvement of HIF-1α in B16F10 cells after bevacizumab treatment under hypoxic conditions. Expression of HIF-1α was measured in B16F10 cells using an in-cell Western assay after cell exposure to hypoxic conditions for 24 h. Expression is demonstrated in normalized integrated intensity ((HIF-1α/cell count)*100). A significant induction of HIF-1α was observed when comparing the AVA10 treated cells with the control (p=0.03; top). qPCR analysis shows an induction of GLUT-1 expression in B16F10 cells treated with the high dose of bevacizumab (AVA20) in comparison to no treatment. Expression is demonstrated in normalized fold (bottom). AVA1=equivalent human dose: 1.25 mg/5.46 ml; AVA10=10 times the equivalent human dose: 12.5 mg/ 5.46 ml; AVA20=20 times the equivalent human dose: 25 mg/ 5.46 ml; CO=control group: culture medium.
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f5: Involvement of HIF-1α in B16F10 cells after bevacizumab treatment under hypoxic conditions. Expression of HIF-1α was measured in B16F10 cells using an in-cell Western assay after cell exposure to hypoxic conditions for 24 h. Expression is demonstrated in normalized integrated intensity ((HIF-1α/cell count)*100). A significant induction of HIF-1α was observed when comparing the AVA10 treated cells with the control (p=0.03; top). qPCR analysis shows an induction of GLUT-1 expression in B16F10 cells treated with the high dose of bevacizumab (AVA20) in comparison to no treatment. Expression is demonstrated in normalized fold (bottom). AVA1=equivalent human dose: 1.25 mg/5.46 ml; AVA10=10 times the equivalent human dose: 12.5 mg/ 5.46 ml; AVA20=20 times the equivalent human dose: 25 mg/ 5.46 ml; CO=control group: culture medium.

Mentions: As the new working hypothesis was that the observed intraocular tumor growth after bevacizumab was due to an increased production of VEGF-A, one might expect an upregulation of the pathways involved in VEGF-A induction. To investigate whether the increased VEGF-A mRNA expression after exposure to bevacizumab involved HIF-1α, an in-cell Western assay was performed. Under already hypoxic conditions, treatment with 10 times the human equivalent of bevacizumab induced an increase of HIF-1α protein in B16F10 melanoma cells in comparison to the control (Figure 5).


Bevacizumab and intraocular tumors: an intriguing paradox.

el Filali M, Ly LV, Luyten GP, Versluis M, Grossniklaus HE, van der Velden PA, Jager MJ - Mol. Vis. (2012)

Involvement of HIF-1α in B16F10 cells after bevacizumab treatment under hypoxic conditions. Expression of HIF-1α was measured in B16F10 cells using an in-cell Western assay after cell exposure to hypoxic conditions for 24 h. Expression is demonstrated in normalized integrated intensity ((HIF-1α/cell count)*100). A significant induction of HIF-1α was observed when comparing the AVA10 treated cells with the control (p=0.03; top). qPCR analysis shows an induction of GLUT-1 expression in B16F10 cells treated with the high dose of bevacizumab (AVA20) in comparison to no treatment. Expression is demonstrated in normalized fold (bottom). AVA1=equivalent human dose: 1.25 mg/5.46 ml; AVA10=10 times the equivalent human dose: 12.5 mg/ 5.46 ml; AVA20=20 times the equivalent human dose: 25 mg/ 5.46 ml; CO=control group: culture medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472924&req=5

f5: Involvement of HIF-1α in B16F10 cells after bevacizumab treatment under hypoxic conditions. Expression of HIF-1α was measured in B16F10 cells using an in-cell Western assay after cell exposure to hypoxic conditions for 24 h. Expression is demonstrated in normalized integrated intensity ((HIF-1α/cell count)*100). A significant induction of HIF-1α was observed when comparing the AVA10 treated cells with the control (p=0.03; top). qPCR analysis shows an induction of GLUT-1 expression in B16F10 cells treated with the high dose of bevacizumab (AVA20) in comparison to no treatment. Expression is demonstrated in normalized fold (bottom). AVA1=equivalent human dose: 1.25 mg/5.46 ml; AVA10=10 times the equivalent human dose: 12.5 mg/ 5.46 ml; AVA20=20 times the equivalent human dose: 25 mg/ 5.46 ml; CO=control group: culture medium.
Mentions: As the new working hypothesis was that the observed intraocular tumor growth after bevacizumab was due to an increased production of VEGF-A, one might expect an upregulation of the pathways involved in VEGF-A induction. To investigate whether the increased VEGF-A mRNA expression after exposure to bevacizumab involved HIF-1α, an in-cell Western assay was performed. Under already hypoxic conditions, treatment with 10 times the human equivalent of bevacizumab induced an increase of HIF-1α protein in B16F10 melanoma cells in comparison to the control (Figure 5).

Bottom Line: We determined the effect of bevacizumab on the growth of B16F10 cells inside the eye and on B16F10 and UM cells cultured in vitro.In addition, the effect of bevacizumab on in vitro growth of B16F10 and human UM cells and on the expression of VEGF-A, GLUT-1, and HIF-1α was evaluated.Bevacizumab did not affect proliferation of B16F10 cells in vitro, while it inhibited UM cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, LUMC, Leiden, the Netherlands. m.el_filali@lumc.nl

ABSTRACT

Purpose: Bevacizumab, a humanized monoclonal antibody to vascular endothelial growth factor-A (VEGF-A), was originally developed as an anti-tumor treatment. In ocular oncology, it is being used to treat macular edema due to radiation retinopathy, but it may also be useful for the treatment of primary uveal melanoma (UM) or its metastases. We determined the effect of bevacizumab on the growth of B16F10 cells inside the eye and on B16F10 and UM cells cultured in vitro.

Methods: B16F10 melanoma cells were placed into the anterior chamber of the eye of C57Bl/6 mice and tumor growth was monitored after injection of different doses of bevacizumab or mock injection. In addition, the effect of bevacizumab on in vitro growth of B16F10 and human UM cells and on the expression of VEGF-A, GLUT-1, and HIF-1α was evaluated.

Results: Following intraocular injection of bevacizumab into murine B16 tumor-containing eyes, an acceleration of tumor growth was observed, with the occurrence of anterior chamber hemorrhages. Bevacizumab did not affect proliferation of B16F10 cells in vitro, while it inhibited UM cell proliferation. Expression analysis demonstrated that addition of bevacizumab under hypoxic conditions induced VEGF-A, GLUT-1 and HIF-1α in B16F10 cells as well as in UM cell lines and two of four primary UM tumor cultures.

Conclusions: In contrast with expectations, intraocular injection of bevacizumab stimulated B16F10 melanoma growth in murine eyes. In vitro exposure of B16 and human UM cells to bevacizumab led to paradoxical VEGF-A upregulation. The use of VEGF inhibitors for treatment of macular edema (due to radiation retinopathy) after irradiation of UM should be considered carefully, because of the possible adverse effects on residual UM cells.

Show MeSH
Related in: MedlinePlus