Sirtuin-3 modulates Bak- and Bax-dependent apoptosis

Abstract

Sirtuin-3 exhibits properties of a tumor suppressor partly emanating from its ability to control the state of mitochondrial metabolism, with depletion of sirt-3 increasing tumor cell survival. In the present study we demonstrate that depletion of sirtuin-3 brings about an anti-apoptotic phenotype via stimulating cyclophilin-D activity, which promotes the binding of hexokinase II to the mitochondria, thereby preventing Bak/Bax dependent mitochondrial injury and cell death. By contrast, increased expression of sirtuin-3 decreases cyclophilin-D activity, resulting in detachment of hexokinase II from the mitochondria and potentiation of Bak- and Bax-induced mitochondrial injury and loss of cell viability.

Fig. 1.

Sirt-3 modulates Bak/Bax dependent apoptosis induced by Mcl-1 and Bcl-X_L depletion. (A) HeLa cells were transfected with 50 nM of a non-targeting control siRNA or siRNAs targeting Mcl-1 or Bcl-X_L, either alone or in tandem. Following 24 hours incubation, the cells were harvested and the levels of Mcl-1, Bcl-X_L and tubulin were determined by western blotting. The results are typical of three independent experiments. (B) HeLa cells were transfected with 50 nM of the indicated siRNAs, either individually or in tandem. Following 48 hours, the cells were harvested and the degree of apoptosis determined by staining with Yo-Pro-1 as described in Materials and Methods. Values are the means of three independent experiments with the error bars indicating standard deviations. (C) HeLa cells were transfected with 50 nM of a non-targeting control siRNA or siRNAs targeting Bak, Bax, sirtuin-3, Mcl-1 or Bcl-X_L, either alone or in tandem. Following 24 hours incubation, the cells were harvested and the levels of Bak, Bax, Mcl-1, Bcl-X_L and tubulin were determined by western blotting. The results are typical of three independent experiments. (D) HeLa cells were transfected with 50 nM of the indicated siRNAs. Following 24 hours incubation, the cells from four wells were harvested and the mitochondrial fraction isolated. The isolated mitochondria were incubated with 0.1 mM of the cross-linking reagent BMH for 30 minutes. The samples were then run out on 12% SDS-PAGE gels and Bak oligomerization was assessed by western blotting. (E) HeLa cells were plated on 12 mm coverslips (Fisher Scientific) at 5.0×10⁴ cells and allowed to attach overnight. The next day, cells were transfected with the indicated siRNAs. Following 24 hours incubation, the cells were washed twice with PBS, fixed and immunostained with anti-Bax clone 6A7 as described in Materials and Methods. Quantification was done by drawing a region of interest around each cell in the acquired images (200–250 cells). The intensity was determined by SlideBook and is expressed as the percentage of Bax immunostaining induced by Triton X-100.

Fig. 2.

Depletion of sirt-3 prevents Bak/Bax dependent mitochondrial injury, caspase activation and phosphatidylserine externalization. (A) HeLa cells were transfected with 50 nM of a non-targeting control siRNA or siRNAs targeting Mcl-1, Bcl-X_L, Bak, Bax, sirtuin-3; either alone or in tandem. Forty-eight hours after siRNA transfection, 200 nM TMRM and 200 nM of Mito-Tracker Green was added to each well and the cells were incubated at 37°C for 30 minutes. After incubation, floating and attached cells were collected and washed twice with ice cold PBS. The cells were suspended in ice cold PBS and analyzed immediately using flow cytometry as described in Materials and Methods. Values are the means of three independent experiments with the error bars indicating standard deviations. (B) HeLa cells were transfected with 50 nM of a non-targeting control siRNA or siRNAs targeting Mcl-1, Bcl-X_L, Bak, Bax, or sirtuin-3, either alone or in tandem. Forty-eight hours after siRNA transfection, 200 nM of MitoSOX or 10 µM DCFDA was added to each well and the cells were incubated for 30 minutes at 37°C. After incubation, floating and attached cells were collected and washed twice with ice cold PBS. After the final wash, cells were suspended in ice cold PBS and analyzed immediately by flow cytometry as described in Materials and Methods. Values are the means of three independent experiments with the error bars indicating standard deviations. (C) HeLa cells were transfected with 50 nM of a non-targeting control siRNA or siRNAs targeting Mcl-1, Bcl-X_L, Bak, Bax, sirtuin-3; either alone or in tandem. Following 24 hours incubation, the cells were harvested. The samples were separated by SDS-PAGE and immunoblotted onto PVDF membranes to detect PARP cleavage. Alternatively, caspase activity was determined using NucView 488 Caspase-3 activity kit as described in Materials and Methods. Values are the means of three independent experiments with the error bars indicating standard deviations. (D) HeLa cells were transfected with 50 nM of a non-targeting control siRNA or siRNAs targeting Mcl-1, Bcl-X_L, Bak, Bax, sirtuin-3; either alone or in tandem. Following 48 hours incubation, the cells were harvested. Floating and attached cells were collected and resuspended in 100 µl of binding buffer at 1.0×10⁶ cells/ml. FITC-Annexin-V (5 µl) was added to determine phosphatidylserine (PS) externalization, and the cells were incubated for 15 minutes at room temperature. PS positive cells were determined by flow cytometry. Values are the means of three independent experiments with the error bars indicating standard deviations.

Fig. 3.

Sirt-3 is required for Bak/Bax dependent apoptosis in MDA-MB-231 breast cancer cells. (A) MDA-MB-231 cells were transfected with 50 nM of the indicated siRNAs, either individually or in tandem. Following 48 hours, the cells were harvested and the degree of apoptosis determined by staining with Yo-Pro-1 as described in Materials and Methods. Values are the means of three independent experiments with the error bars indicating standard deviations. For annexin V staining, floating and attached cells were collected and re-suspended in 100 µl of binding buffer at 1.0×10⁶ cells/ml. FITC-Annexin-V (5 µl) was added to determine phosphatidylserine (PS) externalization, and the cells were incubated for 15 minutes at room temperature. PS positive cells were determined by flow cytometry. Values are the means of three independent experiments with the error bars indicating standard deviations. (B) MDA-MB-231 cells were transfected with 50 nM of a non-targeting control siRNA or siRNAs targeting Mcl-1, Bcl-X_L, Bak, Bax, sirtuin-3; either alone or in tandem. Forty-eight hours after siRNA transfection, 200 nM TMRM and 200 nM of Mito-Tracker Green was added to each well and the cells were incubated at 37°C for 30 minutes. After incubation, floating and attached cells were collected and washed twice with ice cold PBS. The cells were suspended in ice cold PBS and analyzed immediately using flow cytometry as described in Materials and Methods. Values are the means of three independent experiments with the error bars indicating standard deviations. (C) MDA-MB-231 cells were transfected with 50 nM of a non-targeting control siRNA or siRNAs targeting Mcl-1, Bcl-X_L, Bak, Bax, sirtuin-3; either alone or in tandem. Forty-eight hours after siRNA transfection, 200 nM MitoSOX or 10 µM DCFDA was added to each well and the cells were incubated for 30 minutes at 37°C. After incubation, floating and attached cells were collected and washed twice with ice cold PBS. After the final wash, cells were suspended in ice cold PBS and analyzed immediately by flow cytometry as described in Materials and Methods. Values are the means of three independent experiments with the error bars indicating standard deviations. (D) MDA-MB-231 cells were plated on 12 mm coverslips (Fisher Scientific) at 5.0×10⁴ cells and allowed to attach overnight. The next day, cells were transfected with the indicated siRNAs. Following 24 hours incubation, the cells were washed twice with PBS, fixed and immunostained with anti-Bax clone 6A7 as described in Materials and Methods. Quantification was done by drawing a region of interest around each cell in the acquired images. The intensity was determined by SlideBook and is expressed as the percentage of Bax immunostaining induced by Triton X-100. The results are mean of three independent experiments ± s.d.

Fig. 4.

The protective effect of sirt-3 depletion is dependent on hexokinase II binding to mitochondria. (A) HeLa cells were transfected with 50 nM of a non-targeting control siRNA or siRNA targeting Bak/Bax or sirtuin-3. Following 48 hours incubation, HeLa cells were loaded with 200 nM of TMRM for 30 minutes. The cells were then washed twice and placed in respiratory buffer. The cells were mounted on a heated stage kept at 37°C. Digitonin (2.5 µg/ml final concentration) was then added to permeabilize the plasma membrane and TMRM fluorescence was monitored over a 20 minute time course. Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM at the 2-minute time point and 5 µM of CCCP was added at the 18 minute time point. The result is the average of three independent experiments. (B) HeLa cells were plated and then transfected with siRNAs targeting Bak/Bax or sirt-3. Following 48 hours, the cells were loaded with MitoSOX and mounted on a heated microscopy stage. Digitonin (2.5 µg/ml) was then added to permeabilize the plasma membrane. Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM. Time-lapse microscopy was conducted over a 20 minute time course with MitoSOX fluorescence intensity assessed as described in Materials and Methods. The results are mean of three independent experiments±s.d. (C) Following time lapse microscopy, cells from four wells for each condition were pooled and mitochondria isolated. Cytochrome c content was determined by western blotting using mouse anti-cytochrome c antibody (BD Pharmingen) at 1:1000 dilution. The results are representative of three independent experiments. (D) HeLa cells were transfected with 50 nM of a non-targeting control siRNA, siRNA targeting sirtuin-3 or siRNA targeting sirt-3 and hexokinase II in tandem. Following 48 hours incubation, the cells were loaded with 200 nM of TMRM for 30 minutes. The cells were mounted on a heated microscopy stage kept at 37°C. The cells were then washed twice and placed in respiratory buffer. Where indicated, the cells were pre-treated with 10 µM of clotrimazole (CTZ) for 10 minutes. Digitonin (2.5 µg/ml) was then added to permeabilize the plasma membrane and TMRM fluorescence was monitored over a 20 minute time course. Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM at 2 minutes and 5 µM of CCCP was added at the 18 minute time point. The result is the average of three independent experiments. (E) HeLa cells were transfected with 50 nM of a non-targeting control siRNA, siRNA targeting sirtuin-3 or siRNAs targeting sirt-3 and hexokinase II in tandem. Following 48 hours incubation, the cells were loaded with MitoSOX for 30 minutes and then mounted on a heated microscopy stage kept at 37°C. Cells were then washed twice and placed in respiratory buffer. Where indicated, the cells were pre-treated with 10 µM of clotrimazole (CTZ) for 10 minutes. Digitonin at 2.5 µg/ml was then added to permeabilize the plasma membrane. Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM at 2 minutes. Time-lapse microscopy was conducted over a 20 minute time course with MitoSOX fluorescence intensity assessed as described in Materials and Methods. The results are mean of three independent experiments±s.d. (F) Following time lapse microscopy, cells from four wells for each condition were harvested and mitochondria isolated. Cytochrome c content was detected by western blotting using mouse anti-cytochrome c antibody (BD Pharmingen) at 1:1000 dilution. The results are representative of three independent experiments.

Fig. 5.

Cyclophilin-D is required for sirt-3 depletion to protect against mitochondrial injury. (A) HeLa cells were transfected with 50 nM of a non-targeting control siRNA, siRNA targeting sirtuin-3 or siRNAs targeting sirt-3 and CyP-D in tandem. Following 48 hours incubation, the cells were loaded with 200 nM of TMRM for 30 minutes. The cells were then mounted on a heated microscopy stage kept at 37°C. The cells were then washed twice and placed in respiratory buffer. Where indicated, the cells were pre-treated with 10 µM of cyclosporin A (CsA) for 10 minutes. Digitonin (2.5 µg/ml) was then added to permeabilize the plasma membrane. TMRM fluorescence was monitored over a 20 minute time course. Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM at 2 minutes and 5 µM of CCCP was added at the 18 minute time point. The result is the average of three independent experiments. (B) HeLa cells were transfected with 50 nM of a non-targeting control siRNA, siRNA targeting sirtuin-3 or siRNAs targeting sirt-3 and cyclophilin-D in tandem. Following 48 hours incubation, the cells were loaded with MitoSOX for 30 minutes. The cells were then washed twice, placed in respiratory buffer and mounted on a heated microscopy stage kept at 37°C. Where indicated, the cells were pre-treated with 10 µM of CsA (CsA) for 10 minutes. Digitonin at 2.5 µg/ml was then added to permeabilize the plasma membrane. Recombinant truncated Bid (t-Bid) was added at a final concentration of 5 µM at 2 minutes. Time-lapse microscopy was conducted over a 20 minute time course with MitoSOX fluorescence intensity assessed as described in Materials and Methods. The results are mean±s.d. of three independent experiments. (C) Following time lapse microscopy, cells from four wells per condition were harvested and mitochondria isolated. Cytochrome c content was detected by western blotting using mouse anti-cytochrome c antibody (BD Pharmingen) at 1:1000 dilution. The results are representative of three independent experiments. (D,E) HeLa cells were transfected with 50 nM of a non-targeting control siRNA, siRNAs targeting Bak/Bax or siRNAs targeting Bak/Bax and CyP-D or HXK II in tandem. Following 48 hours incubation, the cells were loaded with 200 nM of TMRM for 30 minutes. The cells were washed twice, placed in respiratory buffer and mounted on a heated microscopy stage kept at 37°C. Where indicated, the cells were pre-treated with 10 µM of cyclosporin A (CsA) or clotrimazole (CTZ) for 10 minutes. Digitonin (2.5 µg/ml) was then added to permeabilize the plasma membrane. TMRM fluorescence was monitored over a 20 minute time course. Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM at 2 minutes and 5 µM of CCCP was added at the 18 minute time point. The results are the average of three independent experiments. (F) HeLa cells were transfected with 50 nM of a non-targeting control siRNA, siRNAs targeting Bak/Bax or siRNAs targeting Bak/Bax and CyP-D or HXK II in tandem. Following 48 hours incubation, the cells were loaded with MitoSOX for 30 minutes. The cells were then washed twice, placed in respiratory buffer and mounted on a heated microscopy stage kept at 37°C. Where indicated, the cells were pre-treated with 10 µM of cyclosporin A (CsA) or clotrimazole (CTZ) for 10 minutes. Digitonin (2.5 µg/ml) was then added to permeabilize the plasma membrane. Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM at 2 minutes. Time-lapse microscopy was conducted over a 20 minute time course with MitoSOX fluorescence intensity assessed as described in Materials and Methods. The results are the mean±s.d. of three independent experiments. (G) Following time lapse microscopy, cells from four wells for each condition were harvested and mitochondria isolated. Cytochrome c content was determined by western blotting using mouse anti-cytochrome c antibody (BD Pharmingen) at 1:1000 dilution. The results are representative of three independent experiments. (H) HeLa cells were transfected with 50 nM of the indicated siRNAs, either individually or in tandem. Following 48 hours incubation, the cells were permeabilized with digitonin (2.5 µg/ml). Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM at 18 minutes. The cells were then harvested from four wells per condition and the mitochondria fraction isolated. The isolated mitochondria were incubated with 0.1 mM of the cross-linking reagent BMH for 30 minutes. The samples were then run out on 12% SDS-PAGE gels and Bak oligomerization was assessed by western blotting. The results are representative of three independent experiments.

Fig. 6.

Sirt-3 depletion protects against cisplatin induced cytotoxicity by regulating the binding of hexokinase II to the mitochondria. (A) HeLa cells were transfected with 50 nM of the indicated siRNAs. Following 24 hours incubation, the cells were treated with 30 µM of cisplatin. Following 24 hours exposure to cisplatin, the cells from four wells per condition were harvested and the mitochondrial fraction isolated. The isolated mitochondria were incubated with 0.1 mM of the cross-linking reagent BMH for 30 minutes. The samples were then run out on 12% SDS-PAGE gels and Bak oligomerization was assessed by western blotting. The result is representative of three independent experiments. (B) HeLa cells were transfected with 50 nM of the indicated siRNAs. Following 24 hours incubation, the cells were treated with 30 µM of cisplatin. At the time points indicated, the cells were harvested and viability determined utilizing Yo-Pro-1 as described in Materials and Methods. Values are the mean of three independent experiments with the error bars indicating standard deviations. (C) HeLa cells were transfected with 50 nM of the indicated siRNAs. Following 24 hours incubation, the cells were treated with 30 µM of cisplatin. After 24 hours exposure to cisplatin, the cells from four wells for each condition were harvested and the mitochondrial fraction isolated. The isolated mitochondria were incubated with 0.1 mM of the cross-linking reagent BMH for 30 minutes. The samples were then run out on 12% SDS-PAGE gels and Bak oligomerization was assessed by western blotting. The results are representative of three independent experiments. (D) HeLa cells were transfected with 50 nM of siRNAs targeting sirt-3 and Bid, either separately or in tandem with siRNAs targeting HXK II or CyP-D. Following 24 hours incubation, the cells were treated with 30 µM of cisplatin in the absence or presence of 10 µM of CsA or 10 µM of CTZ. At the time points indicated, the cells were harvested and viability determined utilizing Yo-Pro-1 as described in Materials and Methods. Values are the mean of three independent experiments with the error bars indicating standard deviations.

Fig. 7.

Sirt-3 is required for Bak/Bax dependent apoptosis in U2OS osteosarcoma cells. (A) U2OS cells were transfected with 50 nM of a non-targeting control siRNA or siRNA targeting Bak/Bax or sirtuin-3. Following 48 hours incubation, U2OS cells were loaded with 200 nM of TMRM for 30 minutes. The cells were then washed twice and placed in respiratory buffer. The cells were mounted on a heated stage kept at 37°C. Digitonin (2.5 µg/ml final concentration) was then added to permeabilize the plasma membrane and TMRM fluorescence was monitored over a 20 minute time course. Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM at the 2 minute time point and 5 µM of CCCP was added at the 18 minute time point. The result is the average of three independent experiments. (B) U2OS cells were plated and then transfected with siRNAs targeting Bak/Bax or sirt-3. Following 48 hours, the cells were loaded with MitoSOX and mounted on a heated microscopy stage. Digitonin (2.5 µg/ml) was then added to permeabilize the plasma membrane. Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM. Time-lapse microscopy was conducted over a 20 minute time course with MitoSOX fluorescence intensity assessed as described in Materials and Methods. The results are mean of three independent experiments±s.d. (C) U2OS cells were transfected with 50 nM of a non-targeting control siRNA, siRNA targeting sirtuin-3 or siRNA targeting sirt-3 and hexokinase II in tandem. Following 48 hours incubation, the cells were loaded with 200 nM of TMRM for 30 minutes. The cells were mounted on a heated microscopy stage kept at 37°C. The cells were then washed twice and placed in respiratory buffer. Where indicated, the cells were pre-treated with 10 µM of clotrimazole (CTZ) for 10 minutes. Digitonin (2.5 µg/ml) was then added to permeabilize the plasma membrane and TMRM fluorescence was monitored over a 20 minute time course. Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM at 2 minutes and 5 µM of CCCP was added at the 18 minute time point. The result is the average of three independent experiments. (D) U2OS cells were transfected with 50 nM of a non-targeting control siRNA, siRNA targeting sirtuin-3 or siRNAs targeting sirt-3 and CyP-D in tandem. Following 48 hours incubation, the cells were loaded with 200 nM of TMRM for 30 minutes. The cells were then mounted on a heated microscopy stage kept at 37°C. The cells were then washed twice and placed in respiratory buffer. Where indicated, the cells were pre-treated with 10 µM of cyclosporin A (CsA) for 10 minutes. Digitonin (2.5 µg/ml) was then added to permeabilize the plasma membrane. TMRM fluorescence was monitored over a 20 minute time course. Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM at 2 minutes and 5 µM of CCCP was added at the 18 minute time point. The result is the average of three independent experiments. (E) U2OS cells were transfected with 50 nM of a non-targeting control siRNA, siRNAs targeting Bak/Bax or siRNAs targeting Bak/Bax or HXK II in tandem. Following 48 hours incubation, the cells were loaded with 200 nM TMRM for 30 minutes. The cells were then washed twice, placed in respiratory buffer and mounted on a heated microscopy stage kept at 37°C. Where indicated, the cells were pre-treated with 10 µM of cyclosporin A (CsA) for 10 minutes. Digitonin (2.5 µg/ml) was then added to permeabilize the plasma membrane. Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM at 2 minutes. Time-lapse microscopy was conducted over a 20 minute time course with TMRM fluorescence intensity assessed as described in Materials and Methods. The result is the average of three independent experiments. (F) U2OS cells were transfected with 50 nM of siRNAs targeting sirt-3 and Bid, either separately or in tandem with siRNAs targeting HXK II or CyP-D. Following 24 hours incubation, the cells were treated with 30 µM of cisplatin in the absence or presence of 10 µM of CsA or 10 µM of CTZ. At the time points indicated, the cells were harvested and viability determined utilizing Yo-Pro-1 as described in the Materials and Methods. Values are the mean of three independent experiments with the error bars indicating standard deviations.

Fig. 8.

Increased sirt-3 expression sensitizes cells to cisplatin induced cytotoxicity. (A) HeLa cells were incubated in DMEM containing glucose (4.5 g/l) or transferred to DMEM containing galactose at 4.5 g/l. Following 24 hours incubation, HeLa cells were transfected with 50 nM of non-targeting siRNA or siRNA targeting sirt-3. After 48 hours incubation, the cells were loaded with 200 nM of TMRM for 30 minutes. The cells were washed twice, placed in respiratory buffer and mounted on a heated microscopy stage kept at 37°C. Digitonin (2.5 µg/ml) was then added to permeabilize the plasma membrane and TMRM fluorescence was monitored over a 20 minute time course. Recombinant truncated Bid (t-Bid) was added at a concentration of 5 µM at 2 minutes and 5 µM of CCCP was added at the 18 minute time point. The result is the average of three independent experiments. (B) HeLa cells were transferred to DMEM containing galactose at 4.5 g/l. Following 24 hours incubation, HeLa cells were transfected with 50 nM of siRNA targeting sirt-3 or Bid, either separately or in tandem with siRNA against HXK II. The cells were incubated for another 24 hours. Where indicated, the cells were pre-treated for 30 minutes with 10 µM of CsA. The cells were then treated with 15 µM of cisplatin and cell viability determined at the time points indicated. Values are the means of three independent experiments with the error bars indicating standard deviations. (C) U2OS cells were transfected with 250 nM of the indicated plasmids. Following 24 hours incubation, the cells were harvested and the mitochondrial fraction isolated from four wells for each condition. The isolated mitochondria were incubated with 0.1 mM of the cross-linking reagent BMH for 30 minutes. The samples were then run out on 12% SDS-PAGE gels and Bak oligomerization was assessed by western blotting. (D) U2OS cells were co-transfected with 250 nM of the indicated plasmids and 250 nM of EGFP. Following 48 hours, the cells were harvested and the number of EGFP positive cells stained with Yo-Pro-1 determined as described in Materials and Methods. Values are the means of three independent experiments with the error bars indicating standard deviations. (E) Sirtuin-3 exerts opposing effects on apoptosis and necrosis by mediating the localization and activity of cyclophilin-D and hexokinase II.