Abstract
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.
Highlights
Malignant gliomas are the most frequently occurring primary malignant brain tumors in adults
Growth factor receptor pathways, such as epidermal growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor (VEGFR), and others can be excessively activated due to overexpression or mutation of the receptors or ligands [3, 4, 5]
receptor tyrosine kinase (RTK) signaling plays a critical role in tumorigenic processes, driving proliferation, survival, and angiogenesis through modulation of key downstream pathways such as PI3K/Akt/mTOR, Ras/Raf/MEK/MAPK, and others [3, 4, 5]
Summary
Malignant gliomas are the most frequently occurring primary malignant brain tumors in adults. Growth factor receptor pathways, such as epidermal growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor (VEGFR), and others can be excessively activated due to overexpression or mutation of the receptors or ligands [3, 4, 5] Such aberrant growth factor signaling can drive glioma growth by promoting proliferation, apoptotic resistance, invasion, angiogenesis, and other processes. Relevant RTK inhibitors have been tested in a number of clinical studies, and these agents have shown significant clinical success in many types of tumors, they have not been able to effectively improve clinical survival for GBM [3, 4, 5]. In the context of treatment-induced increased autophagic flux in tumor cells, an appropriate modulation of this process could enhance the efficacy of the anticancer treatment
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