Fig. 6: mtFoxO3A is involved in cancer cell response to metabolic stress. | Cell Death & Disease

Fig. 6: mtFoxO3A is involved in cancer cell response to metabolic stress.

From: Uncoupling FoxO3A mitochondrial and nuclear functions in cancer cells undergoing metabolic stress and chemotherapy

Fig. 6

a HCT116-FoxO3A+/+ and HCT116-FoxO3A−/− cells were subjected to different treatments: glucose restriction (LG, 0.75 mM glucose, 24 h), metformin (MET, 10 μM, 72 h), cisplatin (CDDP, 30 μM, 48 h), irinotecan (CPT-11, 30 μM, 24 h), 5-fluorouracil (5-FU, 2 μM, 24 h) and etoposide (VP-13, 40 μM, 24 h). Relative cell viability and relative cell death were calculated. b Correlation between LG-resistance (days) and mitochondrial FoxO3A (mtFoxO3A) protein levels in different human cell lines (HCT116 and HT29 colorectal cancer cells, HEK293 embryonic kidney cell, DU145 prostate cancer cells, A549 lung cancer cells, MDA-MB-468 breast cancer cells and OVCAR3 ovarian cancer cells). a.u. arbitrary units. c HCT116-FoxO3A−/− cells were transfected with the indicated plasmids (48 h) and subjected to LG (24 h). Upper panel: relative cell viability and relative cell death. Lower panel: immunoblot analysis of total proteins. β-actin: loading control. d Transcription analysis of selected mitochondrial (ND6 and COX1) and nuclear (BIM) genes by RT-PCR in HCT116-FoxO3A−/− cells transfected with the indicated plasmids (48 h) and subjected to LG (24 h). e HCT116-FoxO3A−/− cells, transfected with the indicated plasmids (48 h), were subjected to metabolic stress with 2-DG (1 mM, 6 h). The graph reflects the quantification of tetramethylrhodamine ethyl ester (TMRE) fluorescence of active mitochondria in transfected cells. f Clonogenic assay on HCT116-FoxO3A+/+ cells cultured in LG (24 h) and treated with increasing concentrations of trametinib and/or compound C, as indicated, for 24 h. Cell growth percent inhibition at each drug concentration is presented. g Left panel: immunoblot analysis of total and mitochondrial proteins isolated from tumors (n ≥ 7 for each group) derived from HCT116-xenografted nude mice subjected to 2-DG treatment (100 mg/kg, 6 days). β-actin and HSP60 were used as total and mitochondrial loading control, respectively. Right panel: densitometric analysis of full-length and cleaved FoxO3A normalized against the mitochondrial loading control and the results of the densitometric analysis of the phosphorylated-AMPK and ERK normalized against total AMPK and ERK, respectively, and the loading control. Data are presented as mean ± SEM and significance was calculated with Student’s t test; *p < 0.05, **p < 0.01, and ***p < 0.001

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