RESUMEN
Prognosis remains extremely poor for malignant glioma. Targeted therapeutic approaches, including single agent anti-angiogenic and proteasome inhibition strategies, have not resulted in sustained anti-glioma clinical efficacy. We tested the anti-glioma efficacy of the anti-angiogenic receptor tyrosine kinase inhibitor cediranib and the novel proteasome inhibitor SC68896, in combination and as single agents. To assess anti-angiogenic effects and evaluate efficacy we employed 4C8 intracranial mouse glioma and a dual-bolus perfusion MRI approach to measure Ktrans, relative cerebral blood flow and volume (rCBF, rCBV), and relative mean transit time (rMTT) in combination with anatomical MRI measurements of tumor growth. While single agent cediranib or SC68896 treatment did not alter tumor growth or survival, combined cediranib/SC68896 significantly delayed tumor growth and increased median survival by 2-fold, compared to untreated. This was accompanied by substantially increased tumor necrosis in the cediranib/SC68896 group (p<0.01), not observed with single agent treatments. Mean vessel density was significantly lower, and mean vessel lumen area was significantly higher, for the combined cediranib/SC68896 group versus untreated. Consistent with our previous findings, cediranib alone did not significantly alter mean tumor rCBF, rCBV, rMTT, or Ktrans. In contrast, SC68896 reduced rCBF in comparison to untreated, but without concomitant reductions in rCBV, rMTT, or Ktrans. Importantly, combined cediranib/SC68896 substantially reduced rCBF, rCBV. rMTT, and Ktrans. A novel analysis of Ktrans/rCBV suggests that changes in Ktrans with time and/or treatment are related to altered total vascular surface area. The data suggest that combined cediranib/SC68896 induced potent anti-angiogenic effects, resulting in increased vascular efficiency and reduced extravasation, consistent with a process of vascular normalization. The study represents the first demonstration that the combination of cediranib with a proteasome inhibitor substantially increases the anti-angiogenic efficacy produced from either agent alone, and synergistically slows glioma tumor growth and extends survival, suggesting a promising treatment which warrants further investigation.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Animales , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioma/irrigación sanguínea , Glioma/patología , Ratones , Quinazolinas/administración & dosificación , Semicarbazonas/administración & dosificaciónRESUMEN
We have previously reported that the in vivo anti-glioma efficacy of the anti-angiogenic receptor tyrosine kinase inhibitor cediranib is substantially enhanced via combination with the late-stage autophagy inhibitor quinacrine. The current study investigates the role of hypoxia and autophagy in combined cediranib/quinacrine efficacy. EF5 immunostaining revealed a prevalence of hypoxia in mouse intracranial 4C8 glioma, consistent with high-grade glioma. MTS cell viability assays using 4C8 glioma cells revealed that hypoxia potentiated the efficacy of combined cediranib/quinacrine: cell viability reductions induced by 1 µM cediranib +2.5 µM quinacrine were 78±7% (hypoxia) vs. 31±3% (normoxia), p<0.05. Apoptosis was markedly increased for cediranib/quinacrine/hypoxia versus all other groups. Autophagic vacuole biomarker LC3-II increased robustly in response to cediranib, quinacrine, or hypoxia. Combined cediranib/quinacrine increased LC3-II further, with the largest increases occurring with combined cediranib/quinacrine/hypoxia. Early stage autophagy inhibitor 3-MA prevented LC3-II accumulation with combined cediranib/quinacrine/hypoxia and substantially attenuated the associated reduction in cell viability. Combined efficacy of cediranib with bafilomycin A1, another late-stage autophagy inhibitor, was additive but lacked substantial potentiation by hypoxia. Substantially lower LC3-II accumulation was observed with bafilomycin A1 in comparison to quinacrine. Cediranib and quinacrine each strongly inhibited Akt phosphoryation, while bafilomycin A1 had no effect. Our results provide compelling evidence that autophagic vacuole accumulation plays a causal role in the anti-glioma cytotoxic efficacy of combined cediranib/quinacrine. Such accumulation is likely related to stimulation of autophagosome induction by hypoxia, which is prevalent in the glioma tumor microenvironment, as well as Akt signaling inhibition from both cediranib and quinacrine. Quinacrine's unique ability to inhibit both Akt and autophagic vacuole degradation may enhance its ability to drive cytotoxic autophagic vacuole accumulation. These findings provide a rationale for a clinical evaluation of combined cediranib/quinacrine therapy for malignant glioma.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Autofagia/efectos de los fármacos , Neoplasias Encefálicas/patología , Glioma/patología , Quinacrina/farmacología , Quinazolinas/farmacología , Vacuolas/efectos de los fármacos , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinacrina/administración & dosificación , Quinazolinas/administración & dosificación , Vacuolas/metabolismoRESUMEN
BACKGROUND: Despite malignant glioma vascularity, anti-angiogenic therapy is largely ineffective. We hypothesize that efficacy of the antiangiogenic agent cediranib is synergistically enhanced in intracranial glioma via combination with the late-stage autophagy inhibitor quinacrine. METHODS: Relative cerebral blood flow and volume (rCBF, rCBV), vascular permeability (K(trans)), and tumor volume were assessed in intracranial 4C8 mouse glioma using a dual-bolus perfusion MRI approach. Tumor necrosis and tumor mean vessel density (MVD) were assessed immunohistologically. Autophagic vacuole accumulation and apoptosis were assessed via Western blot in 4C8 glioma in vitro. RESULTS: Cediranib or quinacrine treatment alone did not alter tumor growth. Survival was only marginally improved by cediranib and unchanged by quinacrine. In contrast, combined cediranib/quinacrine reduced tumor growth by >2-fold (P < .05) and increased median survival by >2-fold, compared with untreated controls (P < .05). Cediranib or quinacrine treatment alone did not significantly alter mean tumor rCBF or K(trans) compared with untreated controls, while combined cediranib/quinacrine substantially reduced both (P < .05), indicating potent tumor devascularization. MVD and necrosis were unchanged by cediranib or quinacrine treatment. In contrast, MVD was reduced by nearly 2-fold (P < .01), and necrosis increased by 3-fold (P < .05, one-tailed), in cediranib + quinacrine treated vs untreated groups. Autophagic vacuole accumulation was induced by cediranib and quinacrine in vitro. Combined cediranib/quinacrine treatment under hypoxic conditions induced further accumulation and apoptosis. CONCLUSION: Combined cediranib/quinacrine treatment synergistically increased antivascular/antitumor efficacy in intracranial 4C8 mouse glioma, suggesting a promising and facile treatment strategy for malignant glioma. Modulations in the autophagic pathway may play a role in the increased efficacy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Permeabilidad Capilar/efectos de los fármacos , Glioma/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Quinacrina/uso terapéutico , Quinazolinas/uso terapéutico , Animales , Biomarcadores de Tumor/metabolismo , Western Blotting , Sinergismo Farmacológico , Femenino , Glioma/irrigación sanguínea , Glioma/patología , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Carga Tumoral , Células Tumorales CultivadasRESUMEN
We previously observed that 17ß-estradiol (E2) augments ischemic borderzone vascular density 10 days after focal cerebral ischemia-reperfusion in rats. We now evaluated the effect of E2 on vascular remodeling, lesional characteristics, and motor recovery up to 30 days after injury. Peri-lesional vascular density in tissue sections from rats treated with 0.72 mg E2 pellets was higher compared to 0.18 mg E2 pellets or placebo (P) pellets: vascular density index, 1.9 ± 0.2 (0.72 mg E2) vs. 1.4 ± 0.2 (0.18 mg E2) vs. 1.5 ± 0.4 (P), p=0.01. This was consistent with perfusion magnetic resonance imaging (MRI) measurements of lesional relative cerebral blood flow (rCBF): 1.89 ± 0.32 (0.72 mg E2) vs. 1.32 ± 0.19 (P), p=0.04. Post-ischemic angiogenesis occurred in P-treated as well as E2-treated rats. There was no treatment-related effect on lesional size, but lesional tissue was better preserved in E2-treated rats: cystic component as a % of total lesion, 30 ± 12 (0.72 mg E2) vs. 29 ± 17 (0.18 mg E2) vs. 61 ± 29 (P), p=0.008. Three weeks after right middle cerebral artery territory injury, rats treated with 0.72 mg E2 pellets used the left forelimb more than P-treated or 0.18 mg E2-treated rats: limb use asymmetry score, 0.09 ± 0.43 (0.72 mg E2) vs. 0.54 ± 0.12 (0.18 mg E2) vs. 0.54 ± 0.40 (P), p=0.05. We conclude that treatment with 0.72 mg E2 pellets beginning one week prior to ischemia/reperfusion and continuing through the one-month recovery period results in augmentation of lesional vascularity and perfusion, as well as improved motor recovery.