Downregulation of TIGAR sensitizes the antitumor effect of physapubenolide through increasing intracellular ROS levels to trigger apoptosis and autophagosome formation in human breast carcinoma cells.
Physapubenolide (PB) is a cytotoxic withanolide isolated from Physalis angulata that was used as a traditional Chinese medicine. In this study, we investigated the role of TIGAR and ROS in PB-induced apoptosis and autophagosome formation in human breast carcinoma MDA-MB-231 and MCF-7 cells. PB induced apoptosis by decreasing mitochondrial membrane potential and elevating the Bax/Bcl-2 protein expression ratio in MDA-MB-231 and MCF-7 cells. Caspase inhibitor Z-VAD-FMK treatment partly blocked PB induced cytotoxicity, suggesting that apoptosis serves as an important role in the anti-proliferative effect of PB. Meanwhile, PB induced autophagosome formation, as characterized by increased acridine orange-stained positive cells, accumulation of punctate LC3B fluorescence and a greater number of autophagic vacuoles under electron microscopy. Furthermore, PB inhibited autophagic flux as reflected by the overlapping of mCherry and GFP fluorescence when MDA-MB-231 cells were transfected with GFP-mCherry-LC3 plasmid. Depletion of LC3B, ATG5 or ATG7 reduced PB-induced cytotoxicity, indicating that autophagosome associated cell death participated in the anti-cancer effect of PB. Moreover, PB-induced apoptosis and autophagosome formation were linked to the generation of intracellular ROS, and pre-treatment with the antioxidant NAC obviously mitigated the effects. Interestingly, PB treatment slightly increased TIGAR expression at low concentrations but decreased TIGAR expression drastically at high concentrations. Downregulation of TIGAR by small interfering RNA augmented low concentrations of PB-induced apoptosis and autophagosome formation, which contributed to the observed anti-cancer effect of PB and were reversed by NAC pre-treatment. Consistently, in MDA-MB-231 or MCF-7 xenograft mouse model, PB suppressed tumor growth through ROS induced apoptosis and autophagosome associated cell death accompanied with the downregulation of TIGAR. Taken together, these results indicate that downregulation of TIGAR increased PB-induced apoptosis and autophagosomes associated cell death through promoting ROS generation in MDA-MB-231 and MCF-7 cells.
['Ma|Ting|T|', 'Zhang|Yi|Y|', 'Zhang|Chao|C|', 'Luo|Jian-Guang|JG|', 'Kong|Ling-Yi|LY|']
D000818:Animals D000972:Antineoplastic Agents, Phytogenic D017209:Apoptosis D051017:Apoptosis Regulatory Proteins D000071182:Autophagosomes D001343:Autophagy D001943:Breast Neoplasms D049109:Cell Proliferation D002470:Cell Survival D004305:Dose-Response Relationship, Drug D015536:Down-Regulation D005260:Female D005434:Flow Cytometry D006801:Humans D047908:Intracellular Signaling Peptides and Proteins D061986:MCF-7 Cells D008807:Mice, Inbred BALB C D008819:Mice, Nude D010744:Phosphoric Monoester Hydrolases D032321:Physalis D034741:RNA, Small Interfering D017382:Reactive Oxygen Species D014410:Tumor Stem Cell Assay D054358:Withanolides
TIGAR Physapubenolide ROS Apoptosis Autophagy Physapubenolide (PubChem CID: 101317813)