Autophagy is a crucial process for cells to maintain homeostasis and survival 
through degradation of cellular proteins and organelles, including mitochondria 
and endoplasmic reticula (ER). We previously demonstrated that temozolomide 
(TMZ), an alkylating agent for brain tumor chemotherapy, induced reactive oxygen 
species (ROS)/extracellular signal-regulated kinase (ERK)-mediated autophagy to 
protect glioma cells from apoptosis. In this study, we investigated the role of 
mitochondrial damage and ER stress in TMZ-induced cytotoxicity. Mitochondrial 
depolarization and mitochondrial permeability transition pore (MPTP) opening 
were observed as a prelude to TMZ-induced autophagy, and these were followed by 
the loss of mitochondrial mass. Electron transport chain (ETC) inhibitors, such 
as rotenone (a complex I inhibitor), sodium azide (a complex IV inhibitor), and 
oligomycin (a complex V inhibitor), or the MPTP inhibitor, cyclosporine A, 
decreased mitochondrial damage-mediated autophagy, and therefore increased 
TMZ-induced apoptosis. TMZ treatment triggered ER stress with increased 
expression of GADD153 and GRP78 proteins, and deceased pro-caspase 12 protein. 
ER stress consequently induced autophagy through c-Jun N-terminal kinases (JNK) 
and Ca(2+) signaling pathways. Combination of TMZ with 4-phenylbutyrate (4-PBA), 
an ER stress inhibitor, augmented TMZ-induced cytotoxicity by inhibiting 
autophagy. Taken together, our data indicate that TMZ induced autophagy through 
mitochondrial damage- and ER stress-dependent mechanisms to protect glioma 
cells. This study provides evidence that agents targeting mitochondria or ER may 
be potential anticancer strategies.