RESUMEN
Angiogenesis is essential to tumor progression, and the precise imaging of the angiogenic marker vascular endothelial growth factor receptor 2 (VEGFR-2) may provide an accurate evaluation for angiogenesis during a therapeutic response. With the use of molecular magnetic resonance imaging (mMRI), an in vitro cell assay indicated significantly decreased T1 relaxation values when tumor endothelial cells (TEC), which positively expressed VEGFR-2 (Western blot), were in the presence of the VEGFR-2 probe compared to TEC alone (P < 0.001). For in vivo mMRI evaluations, we assessed VEGFR-2 levels in untreated and OKN-007-treated GL261 mouse gliomas. Regarding treatment response, OKN-007 was also able to significantly decrease tumor volumes (P < 0.01) and increase survival (P < 0.001) in treated animals. Regarding in vivo detection of VEGFR-2, OKN-007 was found to significantly decrease the amount of VEGFR-2 probe (P < 0.05) compared to an untreated control group. Fluorescence imaging for the VEGFR-2 probe indicated that there was colocalization with the endothelial marker CD31 in an untreated tumor bearing mouse and decreased levels for an OKN-007-treated animal. Immuno-fluorescence imaging for VEGFR-2 indicated that OKN-007 treatment significantly decreased VEGFR-2 levels (P < 0.0001) when compared to untreated tumors. Immuno-electron microscopy was used with gold-labeled anti-biotin to detect the anti-VEGFR-2 probe within the plasma membrane of GL261 tumor endothelial cells. This is the first attempt at detecting in vivo levels of VEGFR-2 in a mouse GL261 glioma model and assessing the anti-angiogenic capability of an anticancer nitrone. The results indicate that OKN-007 treatment substantially decreased VEGFR-2 levels in a GL261 glioma model, and can be considered as an anti-angiogenic therapy in human gliomas.
RESUMEN
Free radicals are associated with glioma tumors. Here, we report on the ability of an anticancer nitrone compound, OKN-007 [Oklahoma Nitrone 007; a disulfonyl derivative of α-phenyl-tert-butyl nitrone (PBN)] to decrease free radical levels in F98 rat gliomas using combined molecular magnetic resonance imaging (mMRI) and immunospin-trapping (IST) methodologies. Free radicals are trapped with the spin-trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), to form DMPO macromolecule radical adducts, and then further tagged by immunospin trapping by an antibody against DMPO adducts. In this study, we combined mMRI with a biotin-Gd-DTPA-albumin-based contrast agent for signal detection with the specificity of an antibody for DMPO nitrone adducts (anti-DMPO probe), to detect in vivo free radicals in OKN-007-treated rat F98 gliomas. OKN-007 was found to significantly decrease (P < 0.05) free radical levels detected with an anti-DMPO probe in treated animals compared to untreated rats. Immunoelectron microscopy was used with gold-labeled antibiotin to detect the anti-DMPO probe within the plasma membrane of F98 tumor cells from rats administered anti-DMPO in vivo. OKN-007 was also found to decrease nuclear factor erythroid 2-related factor 2, inducible nitric oxide synthase, 3-nitrotyrosine, and malondialdehyde in ex vivo F98 glioma tissues via immunohistochemistry, as well as decrease 3-nitrotyrosine and malondialdehyde adducts in vitro in F98 cells via ELISA. The results indicate that OKN-007 effectively decreases free radicals associated with glioma tumor growth. Furthermore, this method can potentially be applied toward other types of cancers for the in vivo detection of macromolecular free radicals and the assessment of antioxidants.
