Abstract

Objectives Compared to normal cells, malignant cells have a high degree of aerobic glycolysis, also known as the Warburg effect. Therefore, supplementing photodynamic therapy (PDT), an established cancer therapy, with metabolic inhibitors can augment the mitochondrial damage by depleting ATP. To assess the combined impact of the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) and PDT on apoptosis and autophagy in human breast cancer cells, and examine the molecular basis. Methods Calcium-AM/PI double staining was used to evaluate cell viability. Reactive oxygen species (ROS), mitochondria membrane potential (MMP), nuclear morphology, and autophagosomes were measured using specific fluorescent markers. In addition, translocation of the apoptosis inducing factor (AIF) from the mitochondria to nucleus was imaged by confocal laser scanning microscopy, and DNA fragmentation was measured using PI staining and comet assay. PGC-1α expression, oxidative phosphorylation, ATP levels, and autophagy related proteins were detected by qRT-PCR, seahorse bioscience XF P extracellular flux analyzer, and Western blotting, respectively. Results Compared to with either monotherapy, 2-DG+PDT resulted in significantly higher cytotoxicity in the three breast cancer cell lines (MDA-MB-231, MCF-7, and 4T1), which was consistent with tumor growth regression trends seen in the 4T1 xenograft model. A synergistic augmentation of mitochondrial dysfunction (in terms of ROS generation, MMP loss, and PGC-1α down-regulation) and ATP depletion was seen in cells receiving 2-DG and PDT. In addition, nuclear translocation of AIF and the subsequent DNA damage indicated that the cytotoxic effects were mediated by a caspase-independent mechanism, which was relieved by the ROS scavenger N-acetylcysteine. Autophagy via the AMP-activated protein kinase (AMPK) was also observed following 2-DG+PDT, and reversed upon pre-treatment with the autophagy inhibitor 3-methyladenine. Conclusions The anti-cancer effects of