2‐deoxy‐D‐glucose augments photodynamic therapy induced mitochondrial caspase‐independent apoptosis and energy‐mediated autophagy

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 XFP 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 2‐DG+PDT are mediated by both mitochondria triggered apoptosis and AMPK‐mediated autophagy. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.

Autophagy Regulation and Photodynamic Therapy: Insights to Improve Outcomes of Cancer Treatment

“…Corroborating this finding, Ce6-PDT also activates AMPK that is further enhanced upon glycolysis inhibition by 2-DG, with consequent tumor regression in vitro and in vivo ( 167 ). The AMPK hyperactivation relates to high ATP depletion, which leads to an increase in Beclin 1 and LC3 lipidation that could be lessened by 3-MA ( 167 ). Especially in caspase-3 mutated cancer cells resistant to either multidrug (e.g.…”

“…Indeed, through AMPK activity abrogation by compound C or VPS34 inhibition (3-MA mediated) the cell survival might be partially rescued, possibly due to the autophagy-independent mitochondrial photodamage (104). Corroborating this finding, Ce6-PDT also activates AMPK that is further enhanced upon glycolysis inhibition by 2-DG, with consequent tumor regression in vitro and in vivo (167). The AMPK hyperactivation relates to high ATP depletion, which leads to an increase in Beclin 1 and LC3 lipidation that could be lessened by 3-MA (167).…”

“…However, one should initially consider the main differences between AMCD and AACD in tumor cells succumbing to photooxidative stress. Unlike AACD ( 35 , 59 , 62 , 128 , 141 , 167 , 170 ), AMCD may require the autophagic machinery to intrinsically regulate apoptosis ( 56 , 104 , 147 , 155 , 168 ), as summarized in Table 2 .…”

“…The type of activation mechanism (e.g. AMPK) regardless of the type of PS (i.e., 5-ALA or Ce6) also leads to pro-death autophagy ( 104 , 167 ). Although this interpretation is correct, it is still not possible to correlate pro-death autophagy with PS type and subcellular localization.…”

“…Some reports have shown that depending on the stress level, the autophagic apparatus might intrinsically contribute to other cell death programs, like apoptosis or necroptosis (35,56,59,62,104,128,141,144,145,147,155,(166)(167)(168)(169)(170), see Table 2. In spite of not inducing cell death per si, the RCD routine autophagymediated cell death (AMCD) may be significantly rescued by chemical autophagic inhibition (e.g.…”