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.