Journal: Journal of Medicinal Chemistry

Article Title: New Mitochondria-Targeted Fisetin Derivative Compromises Mitophagy and Limits Survival of Drug-Induced Senescent Breast Cancer Cells

doi: 10.1021/acs.jmedchem.4c01664

Figure Lengend Snippet: Mito-fisetin (mF3)-mediated changes in mitochondrial parameters (A,B) and mitophagic pathway (C) in tamoxifen-induced noncancerous and breast cancer cells. Senescent cells were treated with fisetin (F) and mito-fisetin (mF3) (1 or 5 μM) for 24 h (A,C) or 5 μM F or mF3 for 6 h (B). (A) Depolarization of the MMP (Δψ m ) was assayed using a dedicated fluorogenic probe and flow cytometry. The effects in nonsenescent proliferating cells are also shown. (B) Mitochondrial function was assayed as real-time measurements of mitochondrial oxidative phosphorylation (OXPHOS) as selected OCR parameters (pmol/min or %), namely, basal respiration, ATP production, proton leak (stimulation with oligomycin), maximal respiration (stimulation with the uncoupler FCCP), and spare respiratory capacity (stimulation with rotenone and antimycin A). Uncoupling efficiency [%] and nonmitochondrial oxygen consumption [pmol/min] are also presented. A summarizing scheme showing mF3-mediated effects on mitochondrial function is also provided. (A,B) Bars indicate SD, n = 3, *** p < 0.001, ** p < 0.01, * p < 0.05 compared to the corresponding untreated control (CTR, proliferating cells or CTR SEN, senescent cells) (ANOVA and Dunnett’s a posteriori test), ### p < 0.001, # p < 0.05 compared to fisetin treatment (F) (ANOVA and Tukey’s a posteriori test). (C) Cytoplasmic levels of selected markers of mitophagy and autophagy (PINK1, Parkin, ubiquitin, LC3B, ULK1, VPS34, beclin 1, BNIP3, and Rab9) were studied using dedicated antibodies, an immunostaining protocol, and imaging cytometry. Protein levels are presented as relative fluorescent units (RFU). Box and whisker plots are shown, n = 3, *** p < 0.001, ** p < 0.01, * p < 0.05 compared to the corresponding untreated proliferating control (CTR) (ANOVA and Dunnett’s a posteriori test), ∧∧∧ p < 0.001, ∧∧ p < 0.01, ∧ p < 0.05 compared to the corresponding untreated senescent control (CTR SEN) (ANOVA and Dunnett’s a posteriori test), ### p < 0.001, ## p < 0.01, # p < 0.05 compared to fisetin treatment (F) (ANOVA and Tukey’s a posteriori test).

Article Snippet: The following primary and secondary antibodies were used, namely, anti-PINK1 (PA5-86941, 1:200), anti-Parkin (PA5–13399, 1:100), anti-Ubi1 (13–1600, 1:100), anti-LC3B (PA5–32254, 1:500), anti-ULK1 (20986–1-AP, 1:100), anti-BECN1 (TA502527, 1:100), anti-VPS34 (38-2100, 1:100), anti-BNIP3 (710728, 1:250), anti-Rab9 (MA3-067, 1:200), anti-SOD1 (PA1–30195, 1:200), anti-SOD2 (MA1-106, 1:200), anticaspase 3 (PA5-77887, 1:100), anticaspase 9 (PA5-17913, 1:100), antiphospho-AMPK alpha-1,2 (Thr183, Thr172) (PA5-17831, 1:200), anti-HSP90 (MA-110373, 1:200), anti-IL-8 (M801, 1:500), antimouse IgG conjugated to Texas Red-X (T-6390, 1:1000), antirabbit IgG conjugated to PE-cyanine5.5 (L43018, 1:1000), and antimouse IgG conjugated to FITC (F2761, 1:1000) (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Flow Cytometry, Control, Immunostaining, Imaging, Cytometry, Whisker Assay