apoptosis-inducing factor (aif) is targeted in ifn-α2a-induced bid‐mediated apoptosis through bak...
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Biochimica et Biophysica Acta 1823 (2012) 1378–1388
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Apoptosis-inducing factor (AIF) is targeted in IFN-α2a-induced Bid‐mediatedapoptosis through Bak activation in ovarian cancer cells
Kotaro Miyake, Joseph Bekisz, Tongmao Zhao, Christopher R. Clark, Kathryn C. Zoon ⁎Cytokine Biology Section, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, USA
Abbreviations: IFN, Interferon; Bid, BH3 interactinApoptosis-inducing factor; RNAi, RNA interference; PCD, pendonuclease G;MEFs,mouse embryonic fibroblasts; FADPARP, Poly ADP-ribose polymerase; Apaf-1, apoptotic pro3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl‐2H-tetrazoliumsis factor; TRAIL, Tumor necrosis factor related apoptosismaldehyde; PI, Propidium iodide; ΔΨm, MitochondriBismaleimidohexane⁎ Corresponding author at: Division of Intramural Resea
and InfectiousDiseases, National Institutes of Health, BldgBethesda, MD 20892, USA. Tel.: +1 301 496 3006; fax: +
E-mail address: [email protected] (K.C. Zoon).
0167-4889/$ – see front matter. Published by Elsevier Bdoi:10.1016/j.bbamcr.2012.05.031
a b s t r a c t
a r t i c l e i n f oArticle history:Received 9 December 2011Received in revised form 7 April 2012Accepted 29 May 2012Available online 7 June 2012
Keywords:IFN-αApoptosisMitochondriaAIFBidBak
Previously we have shown that interferon (IFN)-α induced apoptosis is predominantly mediated by theupregulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) via the caspase-8 pathway.It was also shown that recruitment of mitochondria in IFN-α induced apoptosis involves the cleavage ofBH3 interacting domain death agonist (Bid) to truncated Bid (tBid). In the present study, we demonstratethat tBid induced by IFN-α2a activates mitochondrial Bak to trigger the loss of mitochondrial membraneintegrity, consequently causing release of apoptosis-inducing factor (AIF) in ovarian cancer cells, OVCAR3.AIF translocates from the mitochondria to the nucleus and induces nuclear fragmentation and cell death.Both a small molecule Bid inhibitor (BI-6C9) or Bid-RNA interference (RNAi) preserved mitochondrial mem-brane potential, prevented nuclear translocation of AIF, and abrogated IFN-α2a-induced cell death. Cell deathinduced by tBid was inhibited by AIF-RNAi, indicating that caspase-independent AIF signaling is the mainpathway through which Bid mediates cell death. This was further supported by experiments showing thatBI-6C9 did not prevent the release of cytochrome c from mitochondria to cytosol, while the release of AIFwas prevented. In conclusion, IFN-α2a-induced apoptosis is mediated via the mitochondria-associated path-way involving the cleavage of Bid followed by AIF release that involves Bak activation and translocation of AIFfrom the mitochondria to the nucleus in OVCAR3 cells.
Published by Elsevier B.V.
1. Introduction
Interferons (IFNs) are members of a family of cytokines that haveantiviral, antiproliferative, and immunomodulatory properties [1].There are several types of IFNs, each of which interacts with a type-specific receptor complex. Type I IFNs, which include IFN-α, IFN-β,and IFN-ϖ, are ubiquitously induced in mammals by viruses andother factors, (e.g. dsRNA) and interact with the IFN-α receptor(IFNAR) subunits 1 and 2 [2]. Activated T lymphocytes and NK cellsproduce the single species of type II IFN (IFN-γ), which interactswith the IFN-γ receptor (IFNGR) subunits 1 and 2. Nearly every celltype expresses receptors for type I IFNs and IFN-γ [3–5]. The recentlycharacterized type III IFNs include IFN-λ1 (IL-29), IFN-λ2 (IL‐28A),
g domain death agonist; AIF,rogrammed cell death; EndoG,
D, Fas-associated death domain;tease-activating factor-1; MTT,bromide); TNF, Tumor necro-
-inducing ligand; PFA, Parafor-al membrane potential; BMH,
rch, National Institute of Allergy33Rm2N09G.2, 33NorthDrive,1 301 402 0166.
.V.
and IFN-λ3 (IL-28B), which bind to the IFN-λ receptor (IFNLR1) andthe IL-10Rβ subunit (IL-10Rβ). All IFNs exhibit species specificity[2,6].
The clinical applications of IFN-α are treatment of viral infectionssuch as hepatitis B and C, and treatment of certain malignancies suchas malignant melanoma, hairy cell leukemia, Kaposi sarcoma, chronicmyelogenous leukemia and renal cell carcinoma (in combinationwith Avastin). IFN-β has been used in the treatment of relapsing–remitting multiple sclerosis [3]. In contrast, IFN-γ is the sole type 2IFN, and is predominantly produced by T helper cell type 1 lympho-cytes and natural killer cells. IFN-γ is used clinically for the treatmentof chronic granulomatous disease and congenital osteopetrosis, but ithas been generally unsuccessful as an antitumor agent [3].
Apoptosis, a form of programmed cell death (PCD), is an essentialprocess for both development and maintenance of tissue homeostasisand was first recognized by Kerr et al. [7] It has been well establishedthat apoptosis in most cells is induced through the activation of eithermitochondrial (intrinsic) pathway or death receptor (extrinsic) path-way [8,9]. Both ways lead to cell shrinkage, membrane blebbing, chro-matin condensation and formation of apoptotic bodies [10]. Apoptoticcell death largely proceeds in either a caspase-dependent or caspase-independent manner [11]. Several studies provide evidence that, inresponse to apoptotic stimuli, mitochondria can release caspase-independent cell death effectors such as cytochrome c, Apoptosis-Inducing Factor (AIF) and endonuclease G (EndoG) [11,12].
1379K. Miyake et al. / Biochimica et Biophysica Acta 1823 (2012) 1378–1388
BH3 interacting domain death agonist (Bid), a substrate ofcaspase-8, is activated in the Fas/Apo-1 (CD95), tumor necrosis factor(TNF)-α, and tumor necrosis factor related apoptosis-inducing ligand(TRAIL) receptor-mediated cell death [13,14]. Once Bid is cleaved andactivated, it is translocated to the mitochondria as tBid and inducesthe release of proapoptotic proteins such as cytochrome c, AIF andEndoG [13,15]. The ability of Bid to induce release of cytochrome c,AIF or EndoG is mediated by the proapoptotic Bcl-2 family proteinsBak or Bax, because Bid can facilitate the insertion of Bak or Bax intothe mitochondrial membrane to form functional oligomers [15–17].Double-knockout mouse embryonic fibroblasts (MEFs) (Bak−/−and Bax−/−) are resistant to apoptosis by various agents, and micedeficient in both Bak and Bax survived anti-Fas antibody treatment[17]. There is increasing evidence to suggest the involvement of Bakand Bax in the release of cytochrome c, and it was reported that muta-tions in the Bax or Bak gene render cells resistant to apoptosis [18].
Type I IFNs have potent proapoptotic activities in cell lines rep-resenting various histologic origins, suggesting that the induction ofapoptosis is important to their overall physiological functions. IFNsinduce apoptosis indirectly by upregulating the expression of deathligands, such as TRAIL and Fas ligand, which activate the extrinsicFas-associated death domain (FADD)/Caspase-8 signaling pathway[19–21]. IFNs can also modulate mitochondrial outer membrane sta-bility, in part, by activating proapoptotic Bcl-2 members Bak andBax [22]. This leads to release of proapoptotic proteins such as cyto-chrome c, AIF and EndoG and finally results in depolarization of theinner mitochondrial membrane [23]. Pretreatment with type I IFNscan also sensitize cells to the proapoptotic effects of other therapeu-tics [24]. Although IFN-induced apoptosis is caspase-dependentand involves the mitochondria, the identities of all of the mediatorsresponsible for disruption of the mitochondria during this processare still unknown [25].
In the present study, we investigated IFN-α2a induced apoptoticsignaling in ovarian cancer cells (OVCAR3). We found that IFN-α2a in-duced apoptosis is mediated via the mitochondria-associated pathwayinvolving the cleavage of Bid followed by AIF release that involves Bakactivation and translocation of AIF from themitochondria to the nucleusin OVCAR3 cells.
2. Material and methods
2.1. Cell and reagents
OVCAR3 cells were cultured in RPMI 1640 complete medium(Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/mlpenicillin, and 100 mg/ml streptomycin at 37 °C in a humidified 5% CO2
incubator. IFN-α2a was obtained from Hoffman La Roche (Nutley, NJ).The Bid inhibitor BI-6C9 and the inhibitor of apoptosome formationNS3694 were obtained from (Sigma-Aldrich, St Louis, MO). Antibodiespurchased were as follows: rabbit polyclonal anti-Bid, caspase-3,caspase-9, poly ADP-ribose polymerase (PARP). apoptotic protease-activating factor-1 (Apaf-1), EndoG, AIF and β-actin (Cell Signaling,Danvers, MA); mouse anti‐caspase-8, cytochrome c and Heat shock pro-tein 60 (Hsp60) (BDBiosciences, San Jose, CA); rabbit polyclonal anti-Bax(Millipore, Temecula, CA) and rabbit polyclonal anti-Bak (Epitomics, Inc.,Burlingame, CA); rabbit polyclonal anti-Lamin B1 (Abcam, Cambridge,MA).
2.2. Antiproliferative assay
6×103 OVCAR3 cells pre well were seeded in 96-well plate and in-cubated for 24 h. IFN-α2a was then added for an additional 72 h andcell viability was evaluated using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl‐2H-tetrazolium bromide (MTT) as described previously[26]. Briefly, after treatment as indicated, followed by incubation
with MTT (Sigma-Aldrich) for 4 h, the stained living cells were solubi-lized with acidified isopropanol (0.04 N HCI in isopropanol) and theabsorbance was measured at 570 nm. Experiments were carried outin triplicate in duplicate plates.
2.3. RNA interference
OVCAR3 cells (1.5×106 in 10 cm dish or 6×103/well in 96-wellplate) were seeded and incubated for 24 h in antibiotic-free RPMI1640 containing 10% FBS and 2 mM L-glutamine. After incubationfor 24 h, cells were transfected with 20 nM RNA interference (RNAi)oligos complexed in Lipofectamine 2000 (Invitrogen) in Opti-MEMmedia (Invitrogen) for 24 h. IFN was then added for an additional24 h. The media was then removed and replaced with RPMI 1640containing 2% FBS, 2 mM L-glutamine, 50 U/ml penicillin G and50 mg/ml streptomycin. To determine efficiency of RNAi-mediatedgene silencing, cells were harvested and lysates were prepared andanalyzed, as described for Western blot analysis. RNAi oligo purchasedwere as follows: Bid (Oligo ID: 141377 and 141378); Bax (141354and 141355); Bak (184085 and 184087); Apaf-1 (141235); Cytochromec (147625); Caspase-9 (189012); AIF (113512 and 113513) from Invi-trogen: Apaf-1 (J003456-13); Cytochrome c (J017355-06); Caspase-9(J003309-05); ENDOG (J004426-08 and J004426-09) from ThermoFisher Scientific Dharmacon (Lafayette, CO).
2.4. Plasmid and transfection
The plasmid pDsRed2-Bid for expression of DsRed fused to theC-terminus of Bid to allow translocation analyses of full-length Bidand tBid was purchased commercially (Clontech Laboratories, Inc.,Mountain View, CA). Plasmid transfections of 3×105 OVCAR3 cellsseeded in 6-well plates were performed in antibiotic-free growth me-dium using FuGENE HD (Roche Diagnostic Corporation; Indianapolis,IN). Controls were treated with 100 μl/ml OPTI-MEM only and vehiclecontrols with 3 μl/ml FuGENE HD.
2.5. Western blot analysis
Cells were harvested as indicated, lysed in mammalian protein ex-traction (M-PER) reagent lysis buffer (Pierce Chemical, Rockford, IL)containing Halt protease inhibitor and Halt phosphatase inhibitormixtures (Thermo Fisher Scientific, Waltham, MA) for 1 hour on ice.Protein concentrations were determined by measuring the absor-bance at 280 nm, using a Nanodrop spectrophotometer (NanodropTechnologies, Wilmington, DE), and equal amounts of protein wereseparated by SDS-PAGE using 10 to 20% Tris-glycine gels, (InvitrogenCorp., Gaithersburg, MD) under reducing conditions, and transferredto nitrocellulose membranes. Membranes were blocked with 5% fat-free milk in PBS containing 0.05% Tween 20 for 1 h and incubatedwith each antibody overnight at 4 °C. Secondary goat anti-mouseand goat anti-rabbit Abs were purchased from Southern BiotechnologyAssociates (Birmingham, AL). Membranes were developed using theSuperSignal West Femto ECL kit (Thermo Fisher Scientific) andvisualized with an LAS-3000 charge-coupled device camera system(Fujifilm Medical System, Stamford, CT).
2.6. Immunostaining and confocal laser scanning microscopy
For immunofluorescence analysis, OVCAR3 cells were seeded in6-well plates at a density of 2×104cells/well. Mitochondria were sta-ined with MitoTracker (Invitrogen) according to the manufacturer'sprotocol or visualized by CellLight Reagents BacMam 2.0 (Invitrogen).After treatment, cells were fixed with 4% paraformaldehyde (PFA).The cells were permeabilized by exposure for 30 min to 0.1% TritonX-100, and were then placed in blocking solution (3% horse serum)for 30 min. Cells were then exposed to each antibody overnight at
1380 K. Miyake et al. / Biochimica et Biophysica Acta 1823 (2012) 1378–1388
4 °C, followed by incubation for 1 h with Alexa Fluor 488 or 594-labeled goat anti-rabbit IgG for 30 min at RT in the dark. The speci-ficity immunoreactivity was controlled by omission of the primaryantibody. Nuclei were counterstained with DAPI (Invitrogen). Imagesof stained cells were obtained by confocal microscopy (Leica SP5,Leica Microsystems, Exton, PA). All data were processed with LeicaLAS AF software (Version 2.2.1, Leica Microsystems).
2.7. Flow cytometry for cell death and mitochondrial membranepotential (ΔΨm) measurements
Phosphatidylserine exposure on the outer layer of the plasmamembrane was detected using the MEHBCYTO Apoptosis Kit (MBL,Nagoya, Japan) according to the manufacturer's instructions. Inbrief, 2×105 cells were pelleted following treatment and washed inPBS (pH 7.4). Next, the cells were resuspended in 100 μl of bindingbuffer containing annexin V-FITC and propidium iodide (PI) for15 min at RT in the dark. Prior to flow cytometry analysis, 400 μl ofbinding buffer were added to the cells.
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Fig. 1. Bid inhibition protects OVCAR3 cells against IFN-α2a-induced apoptosis (a–b). Biddetermined by antiproliferative assay. (c) Cleavage of caspases, PARP and Bid were assesseapoptotic population after treatment of IFN-α2a with BI-6C9 or Bid-RNAi for 24 and 72 h.early apoptotic (lower right), late apoptotic (upper right), and necrotic (upper left). Numwith no treatment. Lanes: (c) 1, No treatment; 2, IFN-α2a: 1 ng/ml; 3, IFN-α2a: 10 ng/ml;
For ΔΨm determination, Mitochondrial Membrane PotentialDetection Kit (Stratagene, La Jolla, CA, USA) was used according tothe manufacturer's instructions. Briefly, 1×106 cells were pelletedfollowing treatment, resuspended in 500 μl of 1× JC-1 reagent andincubated in a humidified 5% CO2 incubator at 37 °C for 15 min.Next, cells were pelleted and washed in 2 ml of 1× assay buffer.This step was repeated.
Finally, cells were pelleted, resuspended in 1× assay buffer, andanalyzed by flow cytometry. Analysis was performed using FlowJosoftware (Tree Star, Ashland, OR).
2.8. Determination of Bax and Bak oligomerization and activation
For detection of Bax and Bak oligomerization, 1×106 cells wereharvested, washed, and resuspended in 80 μl of 100 mM EDTA/PBS(pH7.4), incubatedwith 1 mMcross-linking agent bismaleimidohexane(BMH) for 30 min at room temperature, quenched with 100 mMdithiothreitol (Sigma-Aldrich) for 15 min, pelleted, and processed forWestern blotting.
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-RNAi or Bid inhibitor, BI-6C9 significantly attenuated IFN-α2a-induced cell death asd by immunoblot analysis after treatment for 24 h. (d) Flow cytometry analysis of theCells were stained with Annexin V and PI. Quadrants are defined as live (lower left),bers refer to the percentage of cells in each quadrant. (a–b) *pb0.05 in comparison4, IFN-α2a: 10 ng/ml+BI-6C9 (10 μM); 5, IFN-α2a: 10 ng/ml+Bid‐RNAi.
1381K. Miyake et al. / Biochimica et Biophysica Acta 1823 (2012) 1378–1388
For detection of activated Bax and Bak by flow cytometry, 1×106
cells were washed in PBS (pH 7.4) and fixed in 400 μl of 0.25% para-formaldehyde for 5 min, subsequently washed two times with2% FBS in PBS (pH 7.4), and incubated in 50 μl of staining buffer (2%FBS and 100 μg/ml digitonin in PBS (pH 7.4)) with a conformation-specific mouse monoclonal antibody against Bax and Bak for 30 minat room temperature. Then, cells were washed and resuspended in50 μl of staining buffer containing 0.25 μg Alexa Fluor 488-labeledgoat anti-rabbit IgG for 30 min in the dark. Cells were washed againand analyzed by flow cytometry. Analysis and histogram overlayswere performed using FlowJo software.
2.9. Mitochondria and nuclei isolation
Mitochondria were isolated using the Qproteome MitochondrialIsolation Kit (Qiagen, Valencia, CA, USA) following the manufacturer'sinstructions. Briefly, 2×107 cells were washed in ice-cold PBS (pH7.4) and incubated in lysis buffer with protease inhibitor (suppliedwith the kit) for 10 min on an orbital shaker at 4 °C. Samples were cen-trifuged for 10 min at 1000 g at 4 °C, and the pellet was resuspended ina disruption buffer with protease inhibitor (supplied with the kit). Cellswere disrupted on ice by 10 rounds of aspiration and ejection through a21-gauge needle. The lysate was centrifuged for 10 min at 1000 g at4 °C, and the resulting supernatant was centrifuged at 6000 g at 4 °Cto pellet mitochondria. Mitochondria were washed with ice-cold
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Fig. 2. The Bid inhibition prevents IFN-α2a-induced mitochondrial depolarization. (a) OVCABid-RNAi. Cells were collected and JC-1 was used to measure ΔΨm by flow cytometry as descin the dot plots indicate the ratios of live cells and apoptotic cells, respectively (Live cells wthree times with essentially similar results. (b) BI-6C9 or Bid-RNAi prevented the accumulatcytosol. Cells expressing a Bid-DsRed fusion protein and Mito-GFP show colocalization of mpretreatment of BI-6C9 or Bid-RNAi for 24 h. Bid (red), mitochondria (green), nuclear stainrepresents the merging of red and green.
storage buffer (supplied with the kit) and centrifuged for 20 min at6000 g at 4 °C. Cytoplasmic and nuclear proteins were isolated usingQproteome nuclear protein kit (Qiagen) following the manufacturer'sinstructions. Briefly, 1×107 cells were harvested and lysed in lysis buff-er. Twenty-five microliters of detergent solution was added to the cellsuspension and then centrifuged. The supernatant was retained as thecytosolic fraction. The remaining pellet was incubated with extractionbuffer by gentle agitation at 4 °C for 30 min and was then centrifuged.The supernatant was retained as the nuclear fraction. Samples weresnap-frozen on dry ice and stored at−80 °C.
2.10. Statistical analysis
Statistical comparisons of mean values were done by one-wayANOVA. Statistical analysis was performed using JMP version 8.0(SAS Institute, Cary, NC, USA). A P value of less than 0.05 was consid-ered to be statistically significant.
3. Results
3.1. Bid inhibition attenuates growth-inhibiting effect of IFN-α2a andinhibits IFN-α2a-induced apoptosis
To examinewhether Bid inhibition attenuates the growth-inhibitingeffect of IFN-α2a in OVCAR3 cells, an antiproliferative assay was
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R3 cells were treated with IFN-α2a for 24 and 72 h after pre-treatment with BI-6C9 orribed in Material and methods. The ratios (%) for upper right and lower right quadrantshose ΔΨm is intact; apoptotic cells whose ΔΨm is collapsed). Experiments were doneion of Bid at the mitochondria and retained the homogeneous distribution of Bid in theitochondrial staining imaged by confocal microscopy exposed to IFN-α2a for 24 h aftering (DAPI, blue), and merge of the three patterns are shown. The yellow/orange color
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Fig. 3. IFN-α2a-induced cell death is associated with Bak activation, mitochondrial translocation, and membrane insertion. OVCAR3 cells were cultured with IFN-α2a with or with-out BI-6C9 for 24 h and (a) processed for determination of Bak oligomerization by immunoblot analysis, Lanes: 1, No treatment; 2, IFN-α2a: 10 ng/ml; 3, IFN-α2a: 10 ng/ml+BI-6C9 (10 μM) or (b) Bak activation by flow cytometry analysis. Histograms show Bak-associated fluorescence in vehicle-treated (dotted line), IFN-α2a treated (black closed)IFN-α2a with BI-6C9 treated (gray closed), and numbers refer to the increase in mean fluorescence intensity. Right bar graphs represent the value of histograms for Bax andBak activation. (c) Bax and Bak translocation to Mitochondria imaged by confocal microscopy in OVCAR3 cells exposed to IFN-α2a for 24 h after pretreatment with BI-6C9 orBid-RNAi for 24 h. Bax or Bak (both green), mitochondria (red), nuclear staining (DAPI, blue), and merge of the three patterns are shown. IFN-α2a treated cells show translocationof Bak to mitochondria. BI-6C9 or Bid-RNAi prevented the translocation of Bak to mitochondria. (d)RNAi targeted for Bax and Bak were transfected into OVCAR3 cells. After 24 hincubation, the cells were exposed to IFN-α2a of indicated concentration and incubated for another 72 h. The knockdown of Bak reduced growth-inhibiting effect of IFN-α2a,whereas knockdown of Bax did not.*=pb0.05 in comparison (b) without BI-6C9 treatment or (d) with negative RNAi treatment.
1382 K. Miyake et al. / Biochimica et Biophysica Acta 1823 (2012) 1378–1388
1383K. Miyake et al. / Biochimica et Biophysica Acta 1823 (2012) 1378–1388
performed. As was previously shown, Bid-RNAi significantly reducedgrowth-inhibiting effect of IFN-α2a [14]. In addition to the RNAiapproach, the specific Bid inhibitor BI-6C9was applied to confirm the es-sential role of Bid in the growth-inhibiting effect by IFN-α2a. After cellswere incubated with Bid-RNAi or specific Bid inhibitor BI-6C9 at 37 °Cfor 24 h, IFN-α2a was added at the concentration of 1, or 10 ng/ml,followed by incubation for 72 h. As shown in Fig. 1a and b, Bid-RNAi orBI-6C9 significantly reduced the growth-inhibiting effect of IFN-α2a(Supplementary Fig. 1, BID-RNAi in the absence of IFN-α2a).
Western blot analyses for Bid, caspase-3, -8, -9, and PARP werecarried out to determine whether IFN-α2a-induced apoptosisthrough cleavage of Bid, caspase-3, -8, -9, and PARP. As shown inFig. 1c, western blot analysis revealed that Bid had been cleaved totBid after incubation with IFN-α2a, confirming our previous results[14]. Similarly, cleavage of caspase-3, -8, -9, and PARP, which aremediators of apoptosis, were observed after treatment with IFN-α2a. Bid-RNAi inhibited cleavage of Bid, however BI-6C9 did not,which is consistent with the mechanism of BI-6C9 [27].
Flow cytometry analysis by annexin V-FITC and PI stainingshowed that Bid inhibition drastically reduced IFN-α2a induced apo-ptosis at both 24 and 72 h. As shown in Fig. 1d, apoptosis was seen in28.1% of the cells at 24 h, 44.9% at 72 h with IFN-α2a and DMSO,29.4% at 24 h, 43.7% at 72 h with IFN-α2a and negative RNAi, 9.8%at 24 h, 16.5% at 72 h with IFN-α2a and BI-6C9 and 12.7% at 24 h,17.9% at 72 h with IFN-α2a and Bid-RNAi.
3.2. IFN-α2a induces Bid-dependent ΔΨm depolarization
We next examined whether IFN-α2a induces Bid-dependent ΔΨmdepolarization. As shown in Fig. 2a, IFN-α2a treatment caused a sig-nificant loss of mitochondrial membrane potential. The Bid inhibitionwith BI-6C9 or Bid-RNAi (shown previously [14]) significantly pre-vented IFN-α2a-induced breakdown of the mitochondrial membranepotential, suggesting that Bid inhibition significantly prevented Bidtranslocation to the mitochondria and consequently prevented IFN-α2a-induced collapse of the mitochondrial membrane potential inOVCAR3 cells [14].
In addition, prevention of Bid translocation to the mitochondriawas confirmed by immunostaining with confocal laser scanning mi-croscopy. Bid inhibition-related reduction of IFN-α2a cytotoxicitywas associated with a lack of Bid translocation to the mitochondriain experiments using pDsRed2-Bid vector and Mitotracker Greenstaining. Bid translocation to the mitochondria cause the merging ofDsRed2-Bid and Mitotracker Green color, resulting in appearance ofyellow/orange. As shown in Fig. 2b, Bid translocation to the mito-chondria was prevented with BI-6C9 or Bid-RNAi, indicating thatBid translocation is essential for IFN-α2a induced apoptosis.
3.3. IFN-α2a-induced apoptosis is accompanied by Bak oligomerization
We then examined the extent to which Bax and Bak had under-gone oligomerization in response to IFN-α2a, using two differenttechniques. We first investigated whether Bax and Bak underwentoligomerization using the cross-linking agent BMH. As shown inFig. 3a, cross-linked Bak protein complexes were observed inOVCAR3 cells treated with 10 ng/ml of IFN-α2a for 24 h, however,cross-linked Bax protein complexes were not. Bak oligomerizationwas prevented with BI-6C9. This result was confirmed with an activeconformation-specific monoclonal Bak antibody and flow cytometryanalysis in which activation causes a shift to the right of the resultinghistogram and bar graph valued histogram, as shown in Fig. 3b. Thesefindings were consistent with results obtained using immunostainingwith confocal laser scanning microscopy. As shown in Fig. 3c, IFN-α2atreatment induced cross-linked Bak protein complexes, which wasprevented with BI-6C9 or Bid-RNAi.
Furthermore, as shown in Fig. 3d, even though Bax‐RNAi knockeddown Bax and Bak‐RNAi knocked down Bak, only Bak-RNAi signifi-cantly attenuated IFN-α2a induced cytotoxicity in OVCAR3 cells(Supplementary Fig. 2). Collectively, these findings suggest that IFN-α2a-induced cytotoxicity is predominantly mediated by Bid translo-cation to the mitochondria, which promote Bak controlled ΔΨm.
3.4. Apoptosome formation is not required for IFN-α2a-inducedBid-mediated cell death
As shown in Fig. 4a–c, there were no significant differences in cellviability with IFN-α2a and the described gene-specific RNAi, althougheach RNAi reduced its respective protein level. In the experiment withthe diarylurea compound, NS3694 which modulates apoptosome for-mation, there was no significant difference in cell viability as well[28]. In addition, as shown in Fig. 4d, Flow cytometry analysis byannexin V-FITC and PI staining showed that there were no significantdifference in IFN-α2a-induced apoptosis with each apoptosome relatedRNAi (Caspase-9, cytochrome c and Apaf-1) or NS3694.
These findings were confirmed with immunostaining using confo-cal laser scanning microscopy. As shown in Fig. 4e, IFN-α2a-inducedapoptosome formation, which was shown as areas of colocalizationin the merge panels as yellow. Apoptosome formation was preventedwith NS3694, while BI-6C9 did not inhibit apoptosome formation.
3.5. AIF functions as a major proapoptotic factor in the IFN-α2a-inducedBid-mediated cell death through Bak oligomerization and activation
The potential role of a proapoptotic inducing protein in the execu-tion of cell death downstream of Bid-mediated mitochondrial depo-larization through Bak oligomerization, was further addressed by agene silencing approach. As shown in Fig. 5a–c, AIF-RNAi reducedprotein levels and significantly attenuated IFN-α2a-induced celldeath in OVCAR3 cells, while cytochrome c-RNAi or EndoG-RNAi didnot (Supplementary Fig. 3). Flow cytometry analysis by annexinV-FITC and PI staining showed that AIF-RNAi drastically reducedIFN-α2a-induced apoptosis at both 24 and 72 h, while neither cyto-chrome c nor EndoG-RNAi did not. As shown in Fig. 5d, apoptosiswas induced in 26.8% at 24 h, 48.4% at 72 h with IFN-α2a and nega-tive RNAi, 12.6% at 24 h, 13.5% at 72 h with IFN-α2a and AIF-RNAi,21.5% at 24 h, 42.5% at 72 h with IFN-α2a and cytochrome c-RNAiand 20.1% at 24 h, 41.9% at 72 h with IFN-α2a and EndoG-RNAi.These results suggested that AIF but not cytochrome c and EndoG isresponsible for IFN-α2a-induced Bid-mediated caspase-independentexecution of cell death.
3.6. Inhibition of Bid prevents the release of AIF from mitochondria byIFN-α2a
Finally, we confirmed the locations of AIF, cytochrome c and EndoG,which are integral inner mitochondrial membrane proteins that areoften affected by mitochondrial transmembrane potential. Cells weretreatedwith IFN-α2a for 24 h, followed by isolation using theQproteomecell compartment kit. As shown in Fig. 6a, AIF transferred frommitochon-dria to the nucleus after exposure to 10 ng/ml IFN-α2a for 24 h and AIFrelease from mitochondria to cytosol was inhibited with BI-6C9. Mean-while, cytochrome c release was not inhibited with BI-6C9. EndoGwas not released from mitochondria with IFN-α2a treatment. Hsp60,a mitochondria-associated protein, and lamin B1, a nuclear specific pro-tein, were used to show the purity of the cellular fractions.
These findingswere confirmedwith immunostaining using confocallaser scanningmicroscopy. As shown in Fig. 6b–d, IFN-α2a-induced AIFrelease from mitochondria to the nucleus, which was prevented withBI-6C9 or Bid-RNAi. Unlike AIF, cytochrome c release was not inhibitedwith BI-6C9 or Bid-RNAi. Consistent with the protein expression assay,EndoG was not released.
1 2 3 4 5
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d0.8
91.2 4.7
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71.4 17.8
8.7
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7.0
3.5
73.3 14.1
9.1
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6.5
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8.2
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55.2 29.3
11.9
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54.3 29.7
12.8
2.3
59.1 27.3
11.3
2.7
59.4 26.6
11.3
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55.3 30.0
12.6
2.3
54.8 28.7
14.2
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1384 K. Miyake et al. / Biochimica et Biophysica Acta 1823 (2012) 1378–1388
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Fig. 5. AIF involves IFN-α2a-induced Bid-mediated cell death. The RNAi gene silencing of (a) AIF did reduced growth-inhibiting effect of IFN-α2a, whereas knockdown of (b) EndoGor (c) cytochrome c did not. (d) Flow cytometry analysis of the apoptotic population after treatment of IFN-α2a with or without gene-specific RNAi for 24 and 72 h. Cells werestained with Annexin V and PI. Quadrants are defined as live (lower left), early apoptotic (lower right), late apoptotic (upper right), and necrotic (upper left). Numbers refer tothe percentage of cells in each quadrant. *pb0.05 in comparison with negative RNAi treatment.
1385K. Miyake et al. / Biochimica et Biophysica Acta 1823 (2012) 1378–1388
4. Discussion
IFN-α is among the few cytokines to have an established role as aneffective antitumor agent in some malignant diseases, notably several
Fig. 4. Apoptosome formation is not required in IFN-α2a-induced Bid‐mediated cell deatrespective RNAi was verified by immunoblot analysis. OVCAR3 cells transfected with Apaf-(b) Apaf-1, cytochrome c or caspase-9 did not attenuate IFN-α2a-induced cell death as detenot attenuate IFN-α2a-induced cell death as determined by antiproliferative assay. (d) Flowithout gene-specific RNAi for 24 and 72 h. Cells were stained with Annexin V and PI. Q(upper right), and necrotic (upper left). Numbers refer to the percentage of cells in each quamicroscopy in OVCAR3 cells exposed to IFN-α2a for 24 h after pretreatment of BI-6C9 or NS3merge of the three patterns are shown. IFN-α2a treated cells show colocalization of cytochmeaning inhibition of apoptosome formation, while there was no significant change of colonegative RNAi 20 nM; 3, negative RNAi 100 nM; 4, target RNAi 20 nM; 5, target RNAi 100 n
lymphoid malignancies [29]. The mechanism by which IFN exerts itsantitumor activity is still ill-defined, but we and others have shownthat IFN can be a potent inducer of apoptosis in some malignantcells, forming an attractive hypothesis for how it may act in cancer
h. (a) Downregulation of Apaf-1, cytochrome c and caspase-9 protein levels by their1, cytochrome c or caspase-9-RNAi were harvested and lysed for immunoblot analysis.rmined by antiproliferative assay. (c) NS3694, inhibitor of apoptosome formation, didw cytometry analysis of the apoptotic population after treatment of IFN-α2a with oruadrants are defined as live (lower left), early apoptotic (lower right), late apoptoticdrant. (e) Cytochrome c and casapase-9 release from mitochondria imaged by confocal694 for 24 h. Cytochrome c (green), caspase-9 (red), nuclear staining (DAPI, blue), androme c and caspase-9. BI-6C9 decreased colocalization of cytochrome c and caspase-9,calization of cytochrome c and caspase-9 with NS3694. Lanes: (a) 1, No treatment; 2,M.
AIF
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Fig. 6. AIF release is associated with IFN-α2a-induced Bid-mediated cell death. (a) IFN-α2a induces AIF translocation into the cytosol and nuclei from the mitochondria. Cells werecultured with IFN-α2a with or without BI-6C9 for 24 h. Cellular fractions (cytosolic, mitochondrial and nuclear) were then isolated using the Qproteome cell compartment kit. Ascontrols to confirm the fidelity of the fractionation procedure, the expression of Hsp60, lamin B1, and β-actin was also determined. Experiments were done three times withessentially similar results. (b–d) AIF, cytochrome c and EndoG release from mitochondria imaged by confocal microscopy in OVCAR3 cells exposed to IFN-α2a for 24 h AIF(green), cytochrome c (green) and EndoG (green) and nuclear staining (DAPI, blue), and merge of the two patterns are shown. IFN-α2a treated cells show release and extensivenuclear fragmentation (white arrows) of AIF and cytochrome c, whereas EndoG was not released. BI-6C9 or Bid-RNAi decreased release of AIF from mitochondria to cytosol, how-ever unaffected release of cytochrome c. Lanes: (a) 1, No treatment; 2, IFN-α2a: 10 ng/ml; 3, IFN-α2a: 10 ng/ml+BI-6C9 (10 μM).
1386 K. Miyake et al. / Biochimica et Biophysica Acta 1823 (2012) 1378–1388
[25,30,31]. Favorable results have also been observed using treatmentprotocols where IFN has been combined with different cytostaticagents [21,32,33]. IFN may be predicted to have a different mode ofactivating the apoptosis machinery in comparison with chemothera-py; this may explain the positive effects observed in such combina-tion protocols.
The present study demonstrates a key role for the proapoptotic Bcl-2family protein Bid in mitochondrial membrane permeabilization andthe subsequent nuclear translocation of AIF in OVCAR3 cells exposedto IFN-α2a. IFN-α2a-induced translocation of Bid to mitochondria,followed by mitochondrial membrane depolarization, Bak activationand AIF release to the nucleus. The pharmacological Bid inhibitorBI-6C9 and RNAi-mediated gene silencing, as shown previously both
significantly preventing mitochondrial Bid translocation, migration ofAIF to the nucleus, and, hence, IFN-α2a-induced cell death.
In order to optimize the use of IFN-α2a in the clinic, a better under-standing of the molecular background to IFN-α2a-induced apoptosisis warranted. This prompted us to define molecular events leading toIFN-α-induced apoptosis. Our results show that IFN-α2a significantlydisrupts the mitochondrial membrane potential (Fig. 2a), thus con-firming previous work [14]. We thereby presume that mitochondriaplay an important role in IFN-α-induced apoptosis. Bcl-2 proteins arethe main regulators of the mitochondrial-mediated death pathway byeither suppressing or promoting mitochondrial changes in regulatingthe release of key proteins from mitochondria. The Bcl-2 family deter-mines the cellular susceptibility of PCD [34]. How the Bcl-2 family
1387K. Miyake et al. / Biochimica et Biophysica Acta 1823 (2012) 1378–1388
proteins control the release of mitochondrial proteins is still unclear.However, it is clear that multidomain proapoptotic proteins suchas Bax and Bak participate in this process [35]. Bax activation or Bakconformational changes lead to the formation of a mitochondrial porethat facilitates the release of mitochondrial proapoptotic proteins.Therefore, in apoptotic PCD, the combined genetic ablation of bothBax and Bak confer resistance to apoptosis.
In the present study, upon treatment of IFN-α2a, Bak protein wasactivated but not Bax (Fig. 3a–c), which suggested that Bak is necessaryin the action of IFN-α2a onOVCAR3 cells. It is very interesting that thesedata well supported the hypothesis that Bax and Bak may perform dif-ferent functions in different cell death models [36], although Bax andBak are similar in protein structure and have similar function in apopto-sis [37]. However, the regulation of Bak and Bax activation is distinct, asis probably their role in IFN-α2a-induced apoptosis. Using immuno-staining with confocal laser scanning microscopy, we also demonstrat-ed that Bak activation occurred in the apoptotic response, prior to theAIF release and loss of ΔΨm, whereas Bax activation was not observed(Fig. 3c). Cells can probably die in response to IFN-α2a without activat-ing Bax since there was no significant difference in the cell viabilityassay with Bax-RNAi. Furthermore, small molecule Bid inhibitor BI-6C9 or Bid-RNAi prevented the translocation of Bak to mitochondria,but did not influence the translocation of Bax in Western blottingassay (Fig. 3a) or flow cytometric analysis (Fig. 3b), implying thatfluctuations in Bax levels are of less importance. In order to delineatethe importance of Bak in IFN-α2a-induced apoptosis, it would havebeen helpful to use Bak−/− fibroblasts. However, in contrast to manyestablished tumor cell lines and primary tumor cells, normal mouseembryo fibroblasts are not sensitive to mouse IFN-induced apoptosisand therefore these experiments cannot be preformed [22].
Another important aspect of the present study addresses the roleof AIF in mediating caspase-independent cell death downstreamof Bid activation through Bak activation. The Bak/AIF pathway is amajor caspase-independent apoptotic cascade [11,38]. AIF is a flavo-protein with both oxidoreductase and DNA-binding domains but nointrinsic DNase activity [11,39,40]. AIF can promote cell survival orinduce apoptosis. In mitochondria, AIF is involved in cellular respira-tion and is essential for cell survival [39,40].
Other proapoptotic factors such as cytochrome c, Smac/Diablo, Omi/HtrA2 and EndoG, which may execute cell death through caspase-dependent or caspase-independent mechanisms, are released withmitochondrial membrane permeabilization as well as AIF. For example,formation of the apoptosome by cytochrome c, Apaf-1, and procaspase-9 and downstream catalytic activation of caspase-3, -6, and ‐7 arewide-ly established mechanisms of caspase-dependent cell death executiondownstream of mitochondrial damage in many cells, including cancercells. Here, we detected caspase-3 activation downstream of Bid,suggesting that this pathway may also play a role in ovarian cancercell death in the present model (Fig. 1c). Caspase-3 inhibition alone,however, did not prevent IFN-α-induced cell death [14]. In addition, al-though our data strongly suggest a major role for caspase-independentcell death downstream of mitochondrial membrane permeabilization,apoptosome inhibition did not prevent IFN-α-induced cell death either(Fig. 4a–e). The release of AIF frommitochondria and its translocation tothe nucleus is oneof the events observed inOVCAR3 cells after exposureto IFN-α2a. Using Western blot analysis and immunofluoresencestaining, we showed mitochondrial release of AIF and translocation tothe nucleus, which occurred after the onset of IFN-α2a exposure. Al-though cytochrome c was released from mitochondria, Bid inhibitorBI-6C9 did not prevent the release of cytochrome c from mitochondria.On the other hand, EndoG was not released after IFN-α2a exposure(Fig. 6a–d). AIF translocation into the nucleus released from the mito-chondria exposes the role of AIF downstream of mitochondrial demisetomediate cell death independent of other established executionmech-anisms of intrinsic cell death pathways, such as apoptosome formationand activation of caspase-3. Furthermore, AIF siRNA significantly
attenuated IFN-α2a induced cell death in OVCAR3 cells, whereas cyto-chrome c or EndoG-RNAi did not (Fig. 5a–d), clearly demonstratingthat Bid cytotoxicity is predominantly mediated by mitochondrial AIFrelease and hence caspase-independent cell death pathways.
In summary, our present study demonstrates that IFN‐α2a in-duced apoptosis is mediated via the mitochondria-associated path-way which is Bid regulated, followed by AIF release that involvesBak activation and translocation to mitochondria in ovarian cancercells, OVCAR3. Further studies using different types of cancer cells,will help to clarify the signaling events underlying the induction ofapoptosis by IFN-α, and thereby provide insight for the developmentof improved ways to use IFN-α in the treatment of cancer and to iden-tify new targets for cancer therapy.
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbamcr.2012.05.031.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
We are grateful to members of Dr. Owen Schwartz's laboratory, par-ticularly S. Ganesan and S. Becker for their help with the confocalmicroscopy. We also thank the other members of the Kathryn C. Zoon'slaboratory, particularly Dr. Takaya Tsuno for many helpful discussionsand suggestions. This work was supported by the Intramural ResearchProgram of the Division of Intramural Research, National Institute ofAllergy and Infectious Diseases, National Institutes of Health.
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