Figure 1.
Growth inhibition by the ATM inhibitor KU-55933 and metformin.
(A) MCF-7 (LKB+/+) and (B) HeLa (LKB−/−) cancer cells in exponential stages of growth were seeded into 96-well plates with 10% FBS and after 24 hrs exposed to increasing concentrations of KU-55933 (ATM inhibitor) in media containing 1% FBS for 72 hrs. Cell growth was estimated by Alamar Blue dye reduction (resazurin (3 µM)). Data are presented as mean ± S.E.M. from 3 independent experiments done in triplicate. (C–F) MCF-7 HepG2, HeLa and MCF-10A cells were growth inhibited by KU-55933 and metformin. Cells were seeded into 96-well plates in the presence of 1% FBS and after 24 hrs treated with KU-55933 (10 µM) or metformin (5 mM). Data are presented as mean ± S.E.M. from 4 independent experiments done in triplicate. * indicates a result significantly different from that obtained in the absence of KU-55933 or metformin as determined by 2-way ANOVA (P<0.0001). (G) MCF-7 cells were transfected with 50 nM ATM-siRNA or with control siRNA. Twenty-four hours after transfection, cells were treated with KU-55933 (10 µM) or metformin (5 mM) and incubated for 48 hrs in RPMI containing 1% FBS. Cell growth in each well was measured by counting cells using Trypan blue. Results using cell number or Alamar blue as endpoints yielded the same conclusions. Columns, mean of 3 independent experiments carried out in triplicate (n = 9); bars, S.E.M. (H) After transfecting MCF-7 cells with 50 nM ATM-siRNA or with control siRNA, cells were lysed and prepared for immunoblot analyses using antibodies against ATM. ß-actin is shown as a loading control.
Figure 2.
Effects of KU-55933 and metformin on metabolism in MCF-7 cells.
Cells were exposed to KU (10 µM) or metformin (5 mM) for 72 hrs. (A) The effect of KU-55933 or metformin on viable cell number was measured by counting cells able to exclude Trypan blue. Cell number was significantly reduced by KU-55933 (*P = 0.0042) and by metformin (**P = 0.0011). (B) Lactate production was significantly increased in cells treated with KU-55933 (*P = 0.0218) or metformin (**P = 0.0012). (C) Glucose consumption was increased with exposure to either KU-55933 (*P = 0.0463) or metformin (**P = 0.0058) treated cells. (D) Both KU-55933 and metformin decreased ATP levels in MCF-7 cells. Results are the mean ± S.E (n = 4). (KU-55933 compared to control *P = 0.0015 and metformin compared to control ** P = 0.0005). (E) Cells were incubated with JC-1 (2 µM), or H2O2 (100 µM, used to activate ATM by oxidative stress), or rotenone (1 µM), or FCCP (1 µM). Mitochondrial membrane potential was probed with JC-1 and visualized by flow cytometry. Loss of mitochondrial membrane potential (ΔΨ) is indicated by a decrease in FL2/FL1 fluorescence intensity ratio (see Figure S1 for flow cytometry data set). Results are expressed as mean ± S.E.M. (n = 4). KU-55933 (*P = 0.0003) and metformin (** P<0.0001) both significantly decreased ΔΨ. (F) Total cellular respiration (black bars, left y-axis) of MCF-7 cells treated with KU-55933 or metformin was compared with untreated cells. Results are the mean ± S.E.M. (KU-55933 compared to control *P = 0.0045, and metformin compared to control ** P = 0.0496). Uncoupled respiration was determined in the presence of oligomycin. The percentage of uncoupled respiration was calculated as: (uncoupled respiration/total mitochondrial respiration), and is shown by hatched bars, right y-axis. (G) KU-55933 or metformin treatment increased cell death (see Figure S2 for flow cytometry data set). Bars represent percentage of necrotic cells. Results are expressed as the mean ± S.E.M. (n = 3) in duplicate (KU-55933 compared to control *P = 0.0005, and metformin compared to control **P = 0.0299). (H) KU-55933 or metformin treatment resulted in increased apoptosis (see Figure S2 for flow cytometry data set). Bars represent percentage of apoptotic cells. Results are expressed as the mean ± S.E.M. (n = 3) in duplicate (KU-55933 compared to control *P<0.0001, and metformin compared to control **P = 0.0458).
Figure 3.
Inhibition of ATM by KU-55933 decreases SCO2 expression in MCF-7 cells.
MCF-7 cells were exposed to KU-55933 (10 µM) for the indicated time. After harvesting, cells were lysed and prepared for immunoblot analyses using antibodies against SCO2, phospho-ATM (Ser1981), ATM, phosphorylated p53 (Ser15), p53, phospho-S6 (Ser235/236), S6rp, phospho-AMPK (Thr172) and AMPK. ß-actin is shown as a loading control. The results are representative of three individual experiments.
Figure 4.
Effects of KU-55933 and metformin on cell number, lactate production, glucose consumption and SCO2 levels in different cancer cell lines.
Cells were exposed to KU (10 µM) or metformin (5 mM) for 72 hrs. (A) The effect of KU-55933 or metformin on cell number was measured by counting cells able to exclude Trypan blue. Cell number was significantly reduced by KU-55933 (*P = 0.0394) and by metformin (**P = 0.0058). KU-55933 and metformin stimulated lactate production. Lactate production was significantly increased in cells treated with KU-55933 (*P = 0.0012) or metformin (**P = 0.0222). Glucose consumption was increased with exposure to either KU-55933 (*P = 0.0034) or metformin (**P = 0.0385) treated cells. (B) MCF-7, HeLa and HepG2 cells were exposed to KU-55933 (10 µM) or metformin (5 mM) for the indicated time. After harvesting, cells were lysed and prepared for immunoblot analyses using antibodies against SCO2, phospho-AMPK (Thr172). ß-actin is shown as a loading control. The results are representative of three individual experiments.
Figure 5.
Effects of KU-55933 on HCT116 p53+/+ and HCT116 p53−/− cells.
Cells were seeded into 96-well plates with 10% FBS and after 24 hrs. exposed to KU-55933 (10 µM) or metformin (5 mM) in DMEM containing 1% FBS for 72 hrs. (A) Cell growth was estimated by Alamar Blue dye reduction. Results are presented as mean ± S.E.M. from 3 independent experiments in triplicate. HCT116 p53+/+ cell growth was significantly inhibited by both KU-55933 (*P<0.0001) and metformin (** P = 0.0013). For HCT116 p53−/− cells, KU-55933 significantly inhibited growth (*P = 0.0002), but this effect was not seen with metformin exposure (P = 0.223). (B) Under the above conditions, after harvesting, cells were lysed and prepared for immunoblot analyses using antibodies against phosphorylated p53 (Ser15), and SCO2. ß-actin is shown as a loading control.
Figure 6.
Effects of KU-55933 and metformin on TCA metabolites.
(A) TCA metabolites were measured by NMR. P values for changes in TCA metabolites are shown in Table S1 (B) Interpretation of metabolic changes observed. [a] ATM is hypothesized to have a role in oxidative phosphorylation, effecting respiratory complex II. Therefore, the ATM inhibitor KU-55933 leads not only to reduced ATP production, but also to accumulation of succinate. [b] KU-55933 also may reduce oxidative phosphorylation by a mechanism involving SCO2, as discussed in the text. [c] Metformin acts to inhibit oxidative phosphorylation, but prior evidence together with our findings of decreased NAD+ suggest a site of action involving respiratory complex I. [d] Both KU-55933 and metformin exposure lead to increased glucose uptake and lactate production, consistent with a compensatory increase in glycolysis following decreased oxidative phosphorylation. [e] Our observations provide evidence for reduced concentrations of TCA cycle intermediates with exposure to either KU-55933 or metformin, but we postulate different reasons for this: metformin may reduce TCA cycle activity because of a reduction in supply of complex I-generated NAD+, while KU-55933 may act to inhibit conversion of succinate to fumarate. ATM, Ataxia Telangiectasia Mutated protein; SCO2, Synthesis of Cytochrome C Oxidase 2; AMPK, AMP-activated protein kinase; TSC1/TSC2, Tuberous Sclerosis 1/Tuberous Sclerosis 2; mTOR, Mammalian Target of Rapamycin complex 1; rpS6, ribosomal protein S6.
Figure 7.
Subcellular localization of ATM.
Total MCF-7 cell lysate and MCF-7 cells fractionated into cytoplasmic, nuclear and mitochondrial extracts were immunoblotted with ATM antibody, α-Tubulin (cytoplasmic marker), Ki67 (nuclear marker) and VDAC (mitochondrial marker). The results indicate ATM immunoreactivity in mitochondrial extracts that are negative for cytoplasmic and nuclear markers.