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 Ca2+ 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.
We previously demonstrated that cadmium (Cd) is able to induce autophagic cell death through a calcium-extracellular signal-regulated kinase pathway. Here, the object of this study is to investigate the role of glycogen synthase kinase-3beta (GSK-3beta) in the induction of autophagy. After treatment with Cd, MES-13 mesangial cells were determined to have undergone autophagy based on the formation of acidic vesicular organelles and autophagosomes as well as on the processing of microtubule-associated protein 1 light chain 3, using flow cytometry with acridine orange staining, electron microscopy, and immunoblot, respectively. Use of the GSK-3beta inhibitor SB 216763 or the small interfering RNA technique to knockdown the expression of GSK-3beta resulted in a decrease of Cd-induced autophagy. In contrast, overexpression of GSK-3beta by transient transfection potentiated Cd toxicity toward the mesangial cells, suggesting that GSK-3beta plays a crucial role in regulating Cd-induced autophagy. Moreover, a decrease of the phosphorylated level at Ser9 of GSK-3beta was observed by immunoblot after treatment with Cd, indicating GSK-3beta was activated by Cd. This phenomenon was reversed by the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC), demonstrated that ROS might activate GSK-3beta. In fact, intracellular hydrogen peroxide (H(2)O(2)) was 2.6-fold elevated after 3 h of exposure to Cd. Both Cd-induced ROS bursts and autophagy were reduced by NAC and vitamin E. In summary, this study demonstrated that, in MES-13 mesangial cells, Cd-induced autophagy was mediated through the ROS-GSK-3beta signaling pathway.
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