Enhanced Sensitivity of Nonsmall Cell Lung Cancer with Acquired Resistance to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors to Phenformin: The Roles of a Metabolic Shift to Oxidative Phosphorylation and Redox Balance

Figure 3

Reactivation of mitochondria function in acquired EGFR-TKI-resistant lung cancer. (a) OCR values were measured by XFp analyzer with cell mito stress kit. Oligomycin (1.5 μM), FCCP (0.5 μM), and mixture of rotenone (Ro, 0.5 μM) and antimycin A (AA, 0.5 μM) were treated at indicated time points. Data represent (, vs. parental cell line). (b) Relative contribution of glycolysis and OXPHOS to ATP production. Using XFp real-time ATP rate assay kit, ATP production rate from glycolysis and OXPHOS was simultaneously determined in HCC827 and HCC827 GR cells. Data represent means (, vs. mito ATP production rate in HCC827). (c) Mitochondrial membrane potential. HCC827 and HCC827 GR cells were incubated with 100 nM TMRM for 30 min, and fluorescence signals were detected by IncuCyte ZOOM. Total integrated intensity of TMRM (red fluorescence) was normalized with cell confluence (outlined with yellow line). Data represent (, vs. HCC827). (d) Intracellular lactate levels. HCC827 and HCC827 GR cells were treated with 100 μM phenformin for 24 h, and lactate levels in cell lysates were determined by LC-MS/MS. Intracellular lactate level was normalized with total protein amounts. Data represent (, , significant difference between the two indicated groups). (e) Number and size of mitochondria (red arrows) in HCC827 and HCC827 GR cells were analyzed by TEM. (f) Protein level of OXPHOS subunits (ATP5A, UQCRC2, SDHB, COX II, and NDUFB8) was detected by immunoblotting.

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