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First published online November 21, 2007
doi: 10.1242/10.1242/jcs.011163

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Mitochondrial electron-transport-chain inhibitors of complexes I and II induce autophagic cell death mediated by reactive oxygen species

¹ Manitoba Institute of Cell Biology, 675 McDermot Avenue, Winnipeg, Manitoba R3E 0V9, Canada
² Department of Human Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada
³ CancerCare Manitoba, 675 McDermot Avenue, Winnipeg, Manitoba, Canada
⁴ Biochemistry and Medical Genetics, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

View larger version (16K): [in this window] [in a new window]   Fig. 1. Rotenone and TTFA induce cell death in HEK 293 and U87 cells over a 72-hour time course. Cell death was quantified as stated in the Materials and Methods section. HEK 293 (A) and U87 (B) cells were treated with 50.0 µM rotenone (R; complex-I inhibitor) or 0.5 mM TTFA (T; complex-II inhibitor) over a 72-hour time course. Error bars represent s.e. from three independent experiments.

View larger version (47K): [in this window] [in a new window]   Fig. 2. Rotenone and TTFA induce autophagy. HEK 293 and U87 cells were treated as described in Fig. 1. (A) The percentage of cells with AVOs (autophagosomes and autolysosomes) was determined by flow cytometry. Rates of AVO formation induced by rotenone (R) and TTFA (T) are indicated in (i) HEK 293 and (ii) U87 cells over a 72-hour time course. (iii) Effect of 3-MA (2.0 mM) on the formation of AVOs that were induced by rotenone or TTFA after a treatment of 48 hours in U87 and HEK 293 cells. P values less than 0.05 represent significant difference between conditions, as indicated. (B) Electron-microscopy pictures were taken of HEK 293 cells that were untreated (control) or treated with TTFA (0.5 mM) for 48 hours. Arrows represent double-membrane vacuoles in TTFA-treated cells (enlarged image). N represents the nucleus. (C) Formation of GFP-LC3-labeled vacuoles (dots). The percentage of cells with GFP-LC3-labeled vacuoles (dots) in (i) HEK 293 and (ii) U87 cells following rotenone or TFFA treatment over a 48-hour time course was determined. Error bars represent s.e. of three independent experiments. (iii) Representative pictures from three independent experiments of U87 cells treated with GFP alone; GFP-LC3 alone; GFP-LC3 and rotenone; and GFP-LC3, rotenone and 3-MA (2.0 mM) were captured by an Olympus microscope and coolsnap camera. The nucleolus was stained with DAPI (blue). (iv) HEK 293 and U87 cells were treated with rotenone or TTFA in the presence or absence of 3-MA (2.0 mM). (D) The conversion of LC3-I to LC3-II was determined in (i) HEK 293 and (ii) U87 cells treated with rotenone or TTFA for 6, 16 or 24 hours in the presence or absence of the lysosomal inhibitor NH4Cl (30 mM). Cells were lysed and western blotted for expression of LC3. Blots were stripped and re-probed with anti-actin antibody for equal loading. (E) Beclin 1 expression was determined after rotenone and TTFA treatment in HEK 293 and U87 cells after 48 hours of treatment. The cells were lysed and western blotted for beclin 1 and actin was used as a loading control.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Rotenone and TTFA induce autophagic cell death in HEK 293 and U87 cells. Cells were treated with 50.0 µM rotenone (R); 0.5 mM TTFA (T); 3-MA, 2.0 mM (autophagy inhibitor); and/or zVAD, 0.1 mM (caspase inhibitor). (A) Effect of autophagy and apoptosis inhibitors on rotenone- or TTFA-induced cell death after treatment for 48 hours was determined. HEK 293 or U87 cells were treated with TTFA or rotenone alone or with each in combination with 3-MA, zVAD or both. Etoposide (100 µM) was used as an apoptotic stimuli. The amount of cell death was determined by membrane permeabilization, as described in the Materials and Methods section. (B) The amount of apoptotic cell death was determined by sub-G1 peak or TUNEL analysis. HEK 293 or U87 cells were treated with rotenone, TTFA or etoposide and the percentage of apoptotic cells was determined. (C) Effect of siRNAs against the autophagic genes beclin 1 and ATG5 on rotenone- or TTFA-induced autophagy, cell death and apoptosis in U87 cells after a treatment of 48 hours was determined. U87 cells were not transfected (non siRNA) or were transfected with scrambled siRNA or siRNAs against beclin 1 or ATG5. (i) Cells were lysed and western blotted for beclin 1 and ATG5. Blots were stripped and re-probed with anti-actin antibody for equal loading. (Cii,Ciii,Di,Dii) The effects of siRNA against beclin 1 and ATG5, and of scrambled siRNA, on AVO formation (Cii), GFP-LC3-labeled vacuoles (Ciii), cell death (Di) and apoptosis (formation of sub-G1 peaks) (Dii) following rotenone or TTFA treatment were determined. Error bars represent s.e. from three independent experiments. * Represents significant difference from control conditions (P<0.05). # (A) Represents a lack of significant difference from control conditions (P>0.05).

View larger version (29K): [in this window] [in a new window]   Fig. 4. ROS scavenger tiron decreases autophagy and autophagic cell death induced by rotenone and TTFA in HEK 293 and U87 cells. Cells were treated with 50.0 µM rotenone (R), 0.5 mM TTFA (T), and/or tiron (1.0 mM). (A) ROS generation after (i) HEK 293 and (ii) U87 cells were treated with rotenone or TTFA over a 72-hour time course. (B) HEK 293 and U87 cells were treated with tiron alone or in combination with rotenone or TTTF. The percentages of (i) ROS generation, (ii) AVO formation and (iii) GFP-LC3-labeled vacuoles (dots) were determined after 48 hours. (C) Expression of beclin 1 (i) and conversion of LC3-I to LC3-II (ii) were determined by western blotting after 48 hours in the presence or absence of tiron. Actin was used as a loading control. NH4Cl was used as a lysosomal inhibitor. (Di) Cell death was determined by membrane permeabilization following rotenone or TTFA treatment in the presence or absence of tiron in HEK 293 and U87 cells after 48 hours. (Dii) Apoptosis (formation of sub-G1 peaks) was determined in HEK 293 and U87 cells treated as above. Error bars represent s.e. from three independent experiments. P values less than 0.05 represent significant difference between conditions, as indicated.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Overexpression of SOD2 in HeLa cells decreases autophagy and autophagic cell death induced by rotenone and TTFA. Wild-type (wt) and SOD2-overexpressing (SOD2) HeLa cells were treated with rotenone (R, 50.0 µM) or TTFA (T, 0.5 mM) as indicated. (A) ROS generation was determined following 48 hours of rotenone or TTFA treatment. DMSO is a solvent control. (B) AVO formation (i) was determined after 48 hours of treatment with rotenone or TTFA and representative pictures of HeLa (wt and SOD2) cells with GFP-LC3-labeled vacuoles (green dots, ii) were obtained by a fluorescent microscope. In HeLa (wt) cells: a, control; b, rotenone; c, TTFA. In HeLa (SOD2) cells: d, control; e, rotenone; f, TTFA. The nucleolus was stained with DAPI (blue). (C) Beclin 1 expression (i) and conversion of LC3-I to LC3-II (ii) in the presence or absence of NH4Cl (30 mM) was determined by western blot. Actin was used as a loading control. (D) Cell death was determined after cells were treated with rotenone or TTFA in the presence or absence of 3-MA (2.0 mM) and/or zVAD (0.1 mM). * Represents significant difference between rotenone or TTFA treatment alone and combined treatment with 3-MA and/or zVAD (P<0.05). @ Represents significant differences between wt and SOD2 cells treated with rotenone or TTFA (P<0.05). # Represents a lack of significant difference between rotenone or TTFA treatment alone and the combined treatment with 3-MA in SOD2 cells (P>0.05). (E) Apoptosis (formation of sub-G1 peak and TUNEL assay) was determined after treatment with rotenone, TTFA or etoposide (Et, apoptotic stimuli). # Represents significant differences between etoposide treatment and control wt cells. * Represents a lack of significant differences between wt and SOD2 cells treated with rotenone or TTFA alone (P>0.05). Error bars represent s.e. from three independent experiments. P values less than 0.05 represent significant difference between conditions, as indicated.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Silencing SOD2 expression by siRNA increases autophagy and autophagic cell death induced by rotenone or TTFA in HeLa cells. (A) HeLa cells were transfected with scrambled or SOD2 siRNAs. The cells were lysed and western blotted for SOD2. The blot was stripped and re-probed for actin. (B-F) After HeLa cells were treated with rotenone (R, 50.0 µM) or TTFA (T, 0.5 mM) for 24 hours, (B) ROS generation, (C) AVO formation, (D) formation of GFP-LC3-labeled vacuoles (dots), (E) cell death and (F) apoptosis (formation of sub-G1 peaks) were determined as described in the Materials and Methods section. Error bars represent s.e. from three independent experiments. * Represents significant difference from control conditions (P<0.05). # Represents a lack of significant difference from control conditions.

View larger version (25K): [in this window] [in a new window]   Fig. 7. Blockage of autophagy response failed to affect ROS generation induced by rotenone or TTFA. (A) HEK 293 cells were treated with rotenone (R, 50 µM) or TTFA (T, 0.5 mM) in the presence or absence of 3-MA (2.0 mM), and ROS generation was determined. (B) Expression of beclin 1 and ATG5 was silenced by siRNAs, and cells were treated with rotenone or TTFA as described in Fig. 1. ROS generation was determined as described above. * Represents a lack of significant difference from control conditions (P>0.05).

View larger version (28K): [in this window] [in a new window]   Fig. 8. Rotenone and TTFA do not induce autophagy in primary mouse astrocytes. Normal mouse astrocytes were treated with rotenone (R, 50.0 µM) or TTFA (T, 0.5 mM) over a 48-hour time course. (A) ROS generation and (B) autophagy [AVO formation and formation of GFP-LC3-labeled vacuoles (dots)] were determined as described in the Materials and Methods section. (C) Expression of beclin 1 (i) and conversion of LC3-I and LC3-II (ii) were determined by western blotting. As a positive control for conversion of LC3-I to LC3-II, astrocytes were starved of glucose and pyruvate for 3 days. Astrocytes were also incubated in the presence or absence of NH4Cl (30 mM) and conversion of LC3-I to LC3-II was determined. (D) Cell death was determined by membrane permeabilization (i) and apoptosis was determined by the formation of sub-G1 peaks (ii). Error bars represent s.e. from three independent experiments. * Represents significant difference from control conditions (P<0.05).