Mitochondrial Complex I Inhibitors and Forced Oxidative Phosphorylation Synergize in Inducing Cancer Cell Death
Figure 1
MDA-MB-231 cells are sensitive to rotenone in condition of glucose deprivation. (a) Proliferation curves for MDA-MB-231 cells cultured at 25 and 1 mM glucose were obtained counting cells at indicated time points. (b) Glucose amount in medium of cells cultured in 1 mM glucose was measured using enzymatic kit at indicated time points. (c) Basal OCR of MDA-MB-231 cells grown in 25 and 1 mM glucose was determined by Seahorse XF24 analyzer; data represent the average ± s.e.m. of three independent experiments (total number of samples ≥ 10), (Student’s -test). (d)–(j) MDA-MB-231 cells cultured for 48 hours in 1 mM glucose were treated for 4 hours with 3 nM rotenone. After treatment, optical microscopy images (d) and viable cell count performed by using Trypan Blue Staining (e) were obtained for untreated and treated cells. After treatment cells were also plated in normal growth medium for clonogenic assay and after ≥12 days colonies were stained (f) and counted (g). OCR of MDA-MB-231 cells cultured in low glucose was determined by Seahorse XF24 analyzer 30 minutes after injection of vehicle or 3 nM rotenone; the percentage of OCR variation after injection is reported (h). In untreated and treated cells also mitochondrial potential () indicated as ratio of mean fluorescence FL2 on mean fluorescence FL1 (see Section 2) (i) and intracellular ATP levels (j) were measured. (k)–(m) Cell count (k), clonogenic assay (l), and intracellular ATP measurement (m) were performed also in cells grown for 48 hours in 25 mM glucose and treated with rotenone as above. All data represent the average of at least three independent experiments (±s.d.); , , (Student’s -test).
