Antiproliferative effects of mitochondria-targeted cationic antioxidants and analogs: Role of mitochondrial bioenergetics and energy-sensing mechanism

Abstract

One of the proposed mechanisms for tumor proliferation involves redox signaling mediated by reactive oxygen species such as superoxide and hydrogen peroxide generated at moderate levels. Thus, the antiproliferative and anti-tumor effects of certain antioxidants were attributed to their ability to mitigate intracellular reactive oxygen species (ROS). Recent reports support a role for mitochondrial ROS in stimulating tumor cell proliferation. In this study, we compared the antiproliferative effects and the effects on mitochondrial bioenergetic functions of a mitochondria-targeted cationic carboxyproxyl nitroxide (Mito-CP), exhibiting superoxide dismutase (SOD)-like activity and a synthetic cationic acetamide analog (Mito-CP-Ac) lacking the nitroxide moiety responsible for the SOD activity. Results indicate that both Mito-CP and Mito-CP-Ac potently inhibited tumor cell proliferation. Both compounds altered mitochondrial and glycolytic functions, and intracellular citrate levels. Both Mito-CP and Mito-CP-Ac synergized with 2-deoxy-glucose (2-DG) to deplete intracellular ATP, inhibit cell proliferation and induce apoptosis in pancreatic cancer cells. We conclude that mitochondria-targeted cationic agents inhibit tumor proliferation via modification of mitochondrial bioenergetics pathways rather than by dismutating and detoxifying mitochondrial superoxide.

Keywords: 2-DG; Antioxidant; Mitochondria; ROS; Superoxide; Tumor cell proliferation.

Fig 1. Chemical structures of Mito-CP and Mito-CP-Ac

Fig. 2. Superoxide dismutation activity of Mito-CP and Mito-CP-Ac as determined by DIPPMPO-superoxide adduct formation

(A) EPR spectra collected during EPR spin trapping of superoxide radical anion. Control incubations contained DIPPMPO (25 mM), KO₂ in DMSO was slowly infused over 10 min into aqueous phosphate buffer (10 mM, pH 7.4) containing dtpa (100 µM). Where indicated, incubations contained Mito-CP (1 mM), Mito-CP-Ac (1 mM), Tempol (1 mM) or CP (1 mM). (B) Same as panel (A) but spectra were recorded at a lower receiver gain, so as to compare the concentrations of different nitroxides used. (C) Quantitative analyses of the DIPPMPO-^•OOH adduct formed in various incubations, using the EPR intensity of the low field line, as indicated by the arrows in panel A. *** p < 0.001 vs. control, ## p < 0.01 vs. Mito-CP. (D) Quantitative comparison of different nitroxides using the double integration of the spectra shown in panel B. (E) MS spectra (top) and HPLC traces (bottom) of synthesized Mito- CP and Mito-CP-Ac. (F) Intracellular concentrations of Mito-CP and Mito-CP-Ac in MiaPaCa-2 cells treated with 1 µM Mito-CP or Mito-CP-Ac for 24 h.

Fig. 3. Effects of Mito-CP or Mito-CP-Ac on colony formation, cell proliferation and intracellular ATP levels

(A) MiaPaCa-2 and MCF-10A cells were treated with Mito-CP (top) or Mito-CP-Ac (bottom) for 24 h and the colonies formed were counted after additional incubation. ** - p < 0.01 treatment vs. correspond control.^## - p < 0.01, comparing MiaPaCa-2 with MCF-10A under the same treatment conditions.. (B) Effects of Mito-CP (top) or Mito-CP-Ac (bottom) at different concentrations on cell proliferation in MiaPaCa-2 cells. (C) Both Mito-CP and Mito-CP-Ac equally synergize with 2-DG to decrease intracellular ATP levels in MiaPaCa-2 cells. The cells seeded in 96-well plates were treated with 2-DG in the presence and absence of 1 µM of the Mito-CP or Mito-CP-Ac for 3 h. Data are represented as a percentage of control (untreated) cells after normalization to total protein for each well. The calculated absolute values of ATP after normalization to total protein for each well for MiaPaCa-2 control cells were 41 ± 3 nmol ATP/mg protein. *,^# - p < 0.05 for Mito-CP and Mito-CP-Ac vs. 2-DG alone, respectively. Data shown are the mean ± SD, n = 6 (panel A), n = 4 (panels B and C).

Fig. 4. Effects of Mito-CP and Mito-CP-Ac on proliferation in multiple cancer cell lines

Cell proliferation in PANC-1, MCF-7, MDA-MB-231, A431, and 253J cells was monitored in real-time with the continuous presence of indicated treatments until the end of each experiment. Data shown are the mean ± SD, n = 4.

Fig. 5. Dose- and time-dependent effects of Mito-CP and Mito-CP-Ac on cell bioenergetic status, as shown in two-dimensional map of oxygen consumption rate (OCR) and proton production rate (PPR) measured in MiaPaCa-2 cells

(A) Two-dimensional bioenergetics map of OCR and ECAR at different concentrations of Mito-CP (left panel) and Mito-CP-Ac (right panel). (B) Effect of Mito-CP or Mito-CP-Ac in real time on OCR and PPR in MiaPaCa-2 cells. OCR and PPR were monitored in real time with Seahorse analyzer, and arrows indicate the time point of injection of compounds of interests. (C) Effect of 24 h treatment with Mito-CP or Mito-CP-Ac on OCR in MiaPaCa-2 cells.

Fig. 6. Effect of Mito-CP and Mito-CP-Ac on intracellular level of citric acid and activation of AMPK in MiaPaCa-2 cells

(A) LC-MS/MS chromatograms corresponding to citrate (MRM 191.00>86.95). (B) Quantitative analysis of the effect of Mito-CP and Mito-CP-Ac on intracellular citrate levels. **- p < 0.01 vs. control. Cells were treated with 0.1 µM Mito-CP or Mito-CP-Ac for 24 h and analyzed as described in Materials and Methods section. (C) (top) The representative blot of three independent biological replicates of lysates obtained from MiaPaCa-2 cells stimulated for 5, 10, 15, or 30 min with equivalent doses of 1 µM Mito-CP or Mito-CP-Ac. (bottom) Densitometric analysis of replicate immunoblots obtained after stimulation for 5 min with either Mito-CP or Mito-CP-Ac. *- p < 0.05 vs. unstimulated controls (Un). Cells stimulated with oligomycin served as a positive control (+). Data shown are the mean ± SD, n = 3.

Fig. 7. Effect of Mito-CP and Mito-CP-Ac on activation of FOXM1 in MiaPaCa-2 cells

(A) The representative blot of three independent biological replicates of lysates obtained from MiaPaCa-2 cells stimulated for 12 h with indicated doses of Mito-CP or Mito-CP-Ac. (B) Densitometric analysis of replicate immunoblots obtained with either Mito-CP or Mito-CP-Ac. **- p < 0.01 vs. unstimulated controls (Un). Data shown are the mean ± SD, n = 3.