BACKGROUND: Autophagy induction can increase or decrease anticancer drug 
efficacy. Anticancer drug-induced autophagy induction is poorly characterized in 
osteosarcoma (OS). In this study, we investigated the impact of autophagy 
inhibition on camptothecin (CPT)-induced cytotoxicity in OS.
METHODS: Autophagy-inhibited DLM8 and K7M3 metastatic murine OS cell lines were 
generated by infection with lentiviral shRNA directed against the essential 
autophagy protein ATG5. Knockdown of ATG5 protein expression and inhibition of 
autophagy was confirmed by immunoblot of ATG5 and LC3II proteins, respectively. 
Metabolic activity was determined by MTT assay and cell viability was determined 
by trypan blue exclusion. Acridine orange staining and immunoblotting for LC3II 
protein expression were used to determine autophagy induction. Oxidative stress 
was assessed by staining cells with HE and DCFH-DA followed by flow cytometry 
analysis. Mitochondrial membrane potential was determined by staining cells with 
TMRE followed by flow cytometry analysis. Immunoblotting was used to detect 
caspase activation, Parp cleavage and p53 phosphorylation.
RESULTS: Autophagy inhibition caused a greater deficit in metabolic activity and 
cell growth in K7M3 cells compared to DLM8 cells. K7M3 cells exhibited higher 
basal autophagy levels than DLM8 cells and non-transformed murine MCT3 
osteoblasts. Autophagy inhibition did not affect CPT-induced DNA damage. 
Autophagy inhibition decreased CPT-induced cell death in DLM8 cells while 
increasing CPT-induced cell death in K7M3 cells. Autophagy inhibition reduced 
CPT-induced mitochondrial damage and CPT-induced caspase activation in DLM8 
cells. Buthionine sulfoximine (BSO)-induced cell death was greater in 
autophagy-competent DLM8 cells and was reversed by antioxidant pretreatment. 
Camptothecin-induced and BSO-induced autophagy induction was also reversed by 
antioxidant pretreatment. Significantly, autophagy inhibition not only reduced 
CPT-induced oxidative stress but also reduced basal oxidative stress.
CONCLUSIONS: The results of this study indicate that autophagy inhibition can 
have an opposing effect on CPT-induced cytotoxicity within OS. The 
cytoprotective mechanism of autophagy inhibition observed in DLM8 cells involves 
reduced CPT-induced oxidative stress and not reduced DNA damage. Our results 
also reveal the novel finding that knockdown of ATG5 protein reduces both basal 
oxidative stress and drug-induced oxidative stress.