title: Combined efficacy of cediranib and quinacrine in glioma is enhanced by hypoxia and causally linked to autophagic vacuole accumulation.
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.
authors.affiliation: Advanced Imaging Research Center, Oregon Health and Science University, Portland, Oregon, United States of America; Department of Biomedical Engineering, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Merryl R
authors.initials: MR
authors.lastname: Lobo
authors.affiliation: Department of Molecular and Medical Genetics at Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Neurosurgery, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: G Yancey
authors.initials: GY
authors.lastname: Gillespie
authors.affiliation: Department of Pathology, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Randall L
authors.initials: RL
authors.lastname: Woltjer
authors.affiliation: Advanced Imaging Research Center, Oregon Health and Science University, Portland, Oregon, United States of America; Department of Biomedical Engineering, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Martin M
authors.initials: MM
authors.lastname: Pike
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols; D001343:Autophagy / Q000187:drug effects*; D001932:Brain Neoplasms / Q000473:pathology*; D015687:Cell Hypoxia / Q000187:drug effects; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects; D005910:Glioma / Q000473:pathology*; D051379:Mice; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011796:Quinacrine / Q000008:administration & dosage / Q000494:pharmacology*; D011799:Quinazolines / Q000008:administration & dosage / Q000494:pharmacology*; D014617:Vacuoles / Q000187:drug effects* / Q000378:metabolism; D007211:Indoles
chemical_list: D000970:Antineoplastic Agents; D051057:Proto-Oncogene Proteins c-akt; D011796:Quinacrine; D011799:Quinazolines; C500926:cediranib; D007211:Indoles