BACKGROUND
the mitochondrial outer membrane permeabilization  is a crucial step of the apoptotic process triggering the release of soluble apoptogenic factors from the intermembrane space such as cytochrome c and smac/diablo followed by subsequent activation of caspase cascade committing to apoptotic cell destruction  <cit> .
the momp process is often altered in cancer cells, resulting largely from the deregulated expression of bcl- <dig> family proteins  <cit> .
the bcl- <dig> family includes anti-apoptotic proteins like bcl- <dig>  bcl-xl, bcl-w, and mcl- <dig> containing all four bcl- <dig> homology domains , pro-apoptotic proteins like bax, bak and bok lacking the bh <dig> domain, and the pro-apoptotic bh3-only proteins like bim, bid, puma, bad and noxa  <cit> .
among all of these proteins, bax and bak are believed to be the “executors” which will exhibit conformational change and oligomerization upon activation and subsequently induce momp and cell death.
there are two widely embraced models for the initiation of momp: the direct activation model and the indirect activation model.
the direct model proposes that a subset of bh3-only proteins termed “activators”, namely bim, bid and puma, directly engage bax or bak, resulting in the activation and oligomerization of these two proteins.
some other bh3-only proteins, termed “sensitizers”, for example, bad and noxa, act only by displacing the activators from the anti-apoptotic proteins, allowing the activators to bind and activate bax and bak  <cit> .
the indirect model posits that bh3-only proteins activate bax and bak not by binding either one, but by antagonizing anti-apoptotic proteins that constrain bax and bak.
in this scenario, bim, bid and puma proteins are far more potent than the others, such as bad and noxa, because they can engage all the anti-apoptotic proteins, while bad and noxa selectively bind only a subset of anti-apoptotic proteins  <cit> .
newmeyer et al.
first revealed in a cell-free system that the addition of bcl- <dig> into the organelle fraction enriched in mitochondria inhibited the process of nuclear destruction, the typical morphological change when cells underwent apoptosis  <cit> .
a good many studies have cumulatively proven that anti-apoptotic bcl- <dig> members are attractive targets for anti-cancer therapy  <cit> .
numerous anti-cancer strategies based on bh <dig> peptides derived from bh3-only proteins  <cit> , anti-sense oligonucleotide or rna interference targeting anti-apoptotic bcl- <dig> family proteins  <cit> , and non-peptidic small molecules binding specifically to anti-apoptotic bcl- <dig> proteins  <cit>  have been developed.
accordingly, substantial advances have been achieved in the field of screening techniques, which are critical to the identification and verification of antagonists against bcl- <dig> anti-apoptotic proteins  <cit> .
in vitro high-throughput screening approaches utilizing technologies like fluorescence polarization  or nuclear magnetic resonance  were quite effective in the discovery and selection of lead compounds suitable for further optimization and development.
however, these methods lack the ability to mimic the intracellular environment where the interruption of protein-protein interaction actually happens.
cell-free systems using mitochondria isolated from normal and cancer cells  <cit>  have been adopted to study mitochondrial changes upon antagonizing bcl- <dig> anti-apoptotic members, which would serve as a promising tool closely imitating the intracellular initiation of momp and apoptotic core machinery to verify bh <dig> mimetics discovered by other assays.
interestingly, similar system based on isolated mitochondria was also used to characterize compounds designed to target bid to treat disorders associated with the activation of such pro-apoptotic protein  <cit> .
in this study, we set up a functional assay using mitochondria isolated from breast cancer cells, recombinant anti-apoptotic bcl- <dig> family proteins and different bh <dig> peptides.
experimental conditions under which bh <dig> peptides with selective targeting profiles induce momp either alone or in combination were determined and optimized.
in this assay, momp was allowed to be semi-quantified by measuring the release of key apoptogenic molecules  from mitochondrial intermembrane space using western blotting.
we optimized the experimental conditions by adopting the high ionic strength  buffer during permeabilization of mitochondria by bh <dig> peptides.
we believe this optimized functional assay based on momp will be a useful screening and validation tool for identifying bh <dig> mimetics selectively targeting different bcl- <dig> anti-apoptotic proteins.
methods
materials
2lmp, a subclone of mda-mb- <dig>  was kindly provided by dr. marc lippman .
normal cell lines including wi- <dig>  prec, and human breast cancer cell lines including hbl <dig>  sum <dig>  bt <dig>  mcf- <dig>  t47d, zr <dig> , mda-mb- <dig>  mda-mb- <dig>  mda-mb- <dig>  mda-mb- <dig> and mda-mb- <dig> were obtained from the american type culture collection  and cultured in medium recommended by atcc.
2lmp, mda-mb- <dig> and mda-mb- <dig> were grown in rpmi  <dig> containing l-glutamine supplemented with 10% fbs and 1% penicillin/streptomycin, maintained in antibiotic-free environment at 37°c in a 5% co <dig> atmosphere and routinely screened for mycoplasma contamination.
bh <dig> peptides were kindly provided by dr. peter p roller , including bim bh <dig> peptide, both 21-mer and 26-mer  , bid bh <dig> peptide  , bad bh <dig> peptide  , and noxa bh <dig> peptide    <cit> .
abt- <dig> was synthesized  according to the literature  <cit> .
all other chemicals used were purchased from sigma-aldrich.
protein expression and purification
human bcl- <dig> protein
the isoform  <dig> construct of the human bcl- <dig>  was used to produce n-terminal 6×his tagged recombinant protein in e. coli bl <dig> .
cells were grown in 2xyt containing antibiotics to an od <dig> of  <dig>  at 37°c.
protein expression was then induced with  <dig>  mm iptg at 20°c for  <dig> h. after centrifugation, cell pellets were resuspended in lysis buffer containing  <dig> mm tris, ph  <dig> ,  <dig> mm nacl,  <dig> % bme and leupectin/aprotin.
after sonication and centrifugation, recombinant protein was purified from the soluble fraction first using ni-nta resin , and then superdex <dig> column  in elution buffer containing  <dig> mm tris, ph  <dig> ,  <dig> mm nacl and  <dig> mm dtt.
human bcl-xl protein
gene encoding human bcl-xl protein , which has an internal deletion of 45– <dig> amino acid residues and a c-terminal truncation of 212– <dig>  was cloned into the phis-tev vector  to generate n-terminal 8xhis tagged recombinant protein in e. coli bl <dig>  the same protocols to express and purify human bcl- <dig> protein were followed.
lysis buffer contained  <dig> mm tris, ph  <dig> ,  <dig> mm nacl,  <dig> % bme and leupectin/aprotin, while protein was eluted in buffer containing  <dig> mm tris, ph <dig> ,  <dig> mm nacl and  <dig> mm dtt.
human mcl- <dig> protein
the mcl-1fragment  encoding amino acid residues of 171– <dig> was cloned into the phis-tev vector.
mcl- <dig> protein with an n-terminal 8×his tag was produced in e. coli bl <dig>  the same protocols to express and purify human bcl- <dig> protein were followed but a source q <dig> column was used in the second purification step and protein was eluted in  <dig> mm tris, ph  <dig> , with nacl gradient.
these recombinant proteins protect the mitochondria from momp by sequestering the added bim bh <dig> peptide, until bim bh <dig> is displaced by other bh <dig> molecules targeting the recombinant proteins.
fluorescence polarization  based binding assays
mcl- <dig> and bcl- <dig> protein
a 21-residue bid bh <dig> peptide   was synthesized and labeled at the n-terminus with 6-carboxyfluorescein succinimidyl ester  as the fluorescence tag .
saturation experiments determined that fam-bid binds to mcl- <dig> and bcl- <dig> protein with a kd values of  <dig>  nm and  <dig>  nm, respectively.
for competitive binding experiments, mcl- <dig> protein  or bcl- <dig> protein  were pre-incubated with fam-bid peptide  in the assay buffer .
<dig> μl of a solution in dmso of the tested bh <dig> peptide was added to the protein/fam-bid solution in black, round-bottom plates  to produce a final volume of  <dig> μl.
for each experiment, a control containing tested protein and flu-bid peptide , and another control containing only fam-bid , were included on each assay plate.
after 2– <dig> hours incubation, the polarization values in milipolarization units  were measured at an excitation wavelength of  <dig> nm and an emission wavelength of  <dig> nm using the synergy h <dig> hybrid microplate reader .
ic <dig>  the inhibitor concentration at which 50% of bound peptide is displaced, was determined from the plot using nonlinear least squares analysis and curve fitting performed using graphpad prizm  <dig> software .
the unlabeled bid bh <dig> peptide is used as the positive control.
the ki value for each bh <dig> peptide was calculated using the equation we have developed for fp-based assays  <cit> .
bcl-xl protein
for this assay, we have employed the bak bh <dig> peptide   labeled with fluorescein  instead of the fam-bid to maximize the signal.
it was determined that fam-bak has a kd value of  <dig>  nm to bcl-xl protein.
the competitive binding assay for bcl-xl was the same as that for mcl- <dig> and bcl- <dig> with  <dig> nm of bcl-xl protein and  <dig>  nm of fam-bak peptide in the following assay buffer:  <dig> mm tris-bis, ph  <dig>  and  <dig> % bovine gamma globulin.
surface plasmon resonance  based binding assay
biotin-labeled bim bh <dig> peptide   was immobilized on a streptavidin sa sensor chip, while the fc <dig> surface was used as a control surface.
the binding affinities of recombinant mcl- <dig>  bcl- <dig> and bcl-xl to immobilized bim bh <dig> peptide was determined by injecting proteins in different concentrations in hbs-ep buffer .
determination of kon, koff and kd were calculated by simultaneous non-linear regression using biaevaluation software .
bim peptide has a kd values of  <dig>  nm,  <dig>  nm and  <dig>  nm against mcl- <dig>  bcl- <dig> and bcl-xl, respectively.
the obtained results confirmed the high binding affinity of bim bh <dig> peptide against all three members from the bcl- <dig> family of proteins.
using the same bim bh <dig> peptide immobilized sa chip, spr competitive solution binding experiments were performed by pre-incubating tested proteins  with tested bh <dig> peptides for at least  <dig> minutes and then the reaction mixture was injected over the surfaces of the chip.
response units were measured at  <dig> seconds in the dissociation phase and the specific binding was calculated by subtracting the control surface  signal from the surfaces with immobilized bim bh <dig> peptide.
ic <dig> values were determined by non-linear least squares analysis using graph pad prism  <dig>  software.
isolation of breast cancer cell mitochondria
mitochondria were isolated from breast cancer cells.
cells were harvested by centrifugation at  <dig> g  for  <dig> min at 4°c and then washed with ice-cold pbs.
cell pellets were resuspended in  <dig> μl of mitochondria isolation buffer  freshly added  <dig> mm pmsf and protease inhibitor cocktail .
cell suspensions were then homogenized with  <dig> strokes in a dounce tissue grinder on ice and centrifuged at  <dig> g for  <dig> min at 4°c.
supernatants were centrifuged at  <dig> g for  <dig> min at 4°c.
pellet enriched in mitochondria was washed three times with mib, spun down at  <dig> g for  <dig> min at 4°c.
the pellet was then resuspended in appropriate volume of mitochondria reaction buffer , freshly added  <dig> mm pmsf and protease inhibitor cocktail.
in order to optimize buffer condition during permeabilization of mitochondrial outer membrane, two different  solutions were used.
low ionic strength  buffer contained  <dig> mm hepes ,  <dig> mm kcl,  <dig>  mm mgcl <dig>   <dig> mm edta, and  <dig> mm sucrose  <cit> .
high ionic strength  buffer contained  <dig> mm hepes ,  <dig> mm kcl,  <dig>  mm mgcl <dig>  and  <dig> mm sucrose  <cit> .
protein concentrations were determined using bradford protein assay .
the mitochondrial suspensions were aliquoted into  <dig> μl with  <dig> mg/ml protein concentration for permeabilization experiments.
permeabilization of mitochondrial outer membrane by bh <dig> peptides or abt-737
for experiments using peptide or abt- <dig> alone, mitochondria were incubated with solvent , different concentrations of bim, bad, noxa bh <dig> peptides, or abt- <dig> for  <dig> h in 37°c water bath.
for experiments using recombinant proteins and combination of bh <dig> peptides, mitochondria were pretreated with pbs or recombinant proteins  followed by permeabilization with solvent  or different concentrations of bh <dig> peptides.
at the end of incubation, mitochondria were pelleted at  <dig> g for  <dig> min at 4°c, supernatants were collected and briefly heated with 2 × sodium dodecyl sulfate polyacrylamide gel electrophoresis  sample buffer.
mitochondrial pellets were also dissolved and heated in 2 × sds-page sample buffer.
both the supernatant samples and mitochondrial pellet samples were subjected to immunoblotting against cytochrome c and smac/diablo proteins.
western blot analysis of released cytochrome c and smac protein
supernatant samples and mitochondrial pellet samples were subjected to protein separation by sds-page.
after transfer to polyvinylidene fluoride  membranes, cytochrome c, smac proteins and loading control  were analyzed using the following primary antibodies: anti-cytochrome c polyclonal antibody , anti-smac/diablo polyclonal antibody , anti-cox iv monoclonal antibody .
blots were then probed with species-specific horseradish peroxidase-conjugated secondary antibodies  and detected with chemiluminescence .
for supernatant samples, one blot was cut into two parts so that both cytochrome c and smac could be detected simultaneously without reprobing.
for pellet samples, the blot was probed for cytochrome c, smac, as well as cox iv that served as loading control.
RESULTS
breast cancer cells are dually or multiply-protected by anti-apoptotic bcl- <dig> family proteins
as we know, cancer cells are dually-, triply-, or multiply-protected by bcl- <dig>  bcl-xl, mcl- <dig> or other anti-apoptotic bcl- <dig> family proteins which have been less thoroughly studied.
a panel of human normal cell lines and breast cancer cell lines were analyzed by western blotting for their basal levels of three key anti-apoptotic proteins, bcl- <dig>  bcl-xl and mcl- <dig> .
2lmp cell line, a subclone of mda-mb- <dig>  is characterized by its abundant cytoplasm and rapid proliferation which are critical for providing sufficient mitochondria.
both mda-mb- <dig> and mda-mb- <dig> expressing mainly mcl- <dig> protein were suitable cell line models for studying momp induced by noxa-like molecules and the combined outcome from co-treatment with both bad-like and noxa-like molecules.
bh <dig> peptides derived from bh3-only proteins were powerful tools for setting up conditions of the mitochondria-based functional assay
binding affinities of bh <dig> peptides to bcl- <dig>  bcl-xl and mcl- <dig> recombinant proteins were determined by surface plasmon resonance  and fluorescence polarization , as shown in table  <dig>  bim  and bid bh <dig> peptides bound to all three proteins with very high affinities.
bad bh <dig> peptide bound to both bcl- <dig> and bcl-xl, but failed to find to mcl- <dig>  as expected.
noxa bh <dig> peptide selectively bound to mcl- <dig> protein only.
although there was some discrepancy between the spr and the fp data, probably due to different assay conditions, data regarding the versatility of bim bh <dig> and the selectivity of bad and noxa bh <dig> were highly consistent with the literature  <cit> .
mitochondria based-functional assay established by using these peptides will help to define how and when bim-like, bad-like or noxa-like small molecule bcl- <dig> inhibitors function on the mitochondrial level.
bh <dig> peptides were screened for their binding affinities to the three anti-apoptotic bcl- <dig> family proteins using surface plasmon resonance and fluorescence polarization methods, as described in the methods.
results are representative of at least three independent experiments.
sufficient ionic strength from kcl ensured reliable cytochrome c and smac release from mitochondria permeabilized by bh <dig> peptides
previous apoptosis studies using purified mitochondria as a cell-free system have yielded conflicting results due to less appropriate experimental conditions including low ionic strength  <cit> , physiologically irrelevant temperature  <cit>  or short incubation time  <cit> .
these studies provided substantial evidence that sufficient ionic strength and temperature of 37°c contributed to full release of cytochrome c. in our study, ionic strength-dependent release of cytochrome c was also confirmed in mitochondria permeabilized by bim bh <dig> peptide in  <dig> min in buffer containing different concentrations of kcl .
mitochondria isolated from 2lmp cells were incubated with bim bh <dig>  peptides at 37°c for  <dig> min or  <dig> min in either high ionic strength buffer containing  <dig> mm kcl or low ionic strength  buffer containing  <dig> mm kcl.
momp induced by bim bh <dig> peptides at indicated concentrations were evaluated through the analysis of released cytochrome c and smac proteins by western blotting.
as shown in figure 2b, bim bh <dig> peptide induced momp in a dose-dependent manner.
bim bh <dig> peptide induced significantly more robust release of cytochrome c and smac proteins when there was sufficient ionic strength from kcl.
preliminary fp data suggested that the binding affinity of 21-mer bim bh <dig> peptide was weaker than 26-mer bim bh <dig> peptide , we also confirmed that the induction of smac release by 21-mer bim  was weaker than that by 26-mer bim .
longer incubation resulted in more complete release of cytochrome c, as indicated by more cytochrome c being detected in the supernatant fraction and less cytochrome c remained in the pellet fraction of treatment with  <dig> nm bim for  <dig> min in his buffer , compared with similar treatment for  <dig> min .
hence, 26-mer bim bh <dig> peptide, reaction buffer containing  <dig> mm kcl, reaction temperature of 37°c and incubation time of  <dig> min were set as experimental conditions for all mitochondrial experiments.
comparing the efficiency of momp induction by different bcl- <dig> targeting agents and functional inhibition of bim bh <dig> peptide-induced momp by three different anti-apoptotic bcl- <dig> family proteins.
mitochondria isolated from 2lmp cells were permeabilized by bim, bid, bad, noxa bh <dig> peptides and abt- <dig>  a bona fide bcl- <dig> antagonist, as shown in figure 3a.
bim, bid, bad bh <dig> peptides and abt- <dig> effectively induced cytochrome c and smac release from 2lmp mitochondria.
consistent with its binding affinities to bcl- <dig>  bcl-xl and mcl- <dig> proteins, bim bh <dig> peptide was the most potent inducer among all bh <dig> peptides.
noxa alone was not able to induce cytochrome c and smac release, which is also consistent with previous reports  <cit> .
bim bh <dig> induced robust release of cytochrome c and smac proteins even at  <dig> nm.
the three major anti-apoptotic proteins were equally potent in repressing the induction of momp by  <dig> nm bim bh <dig> peptide.
even  <dig> nm of recombinant protein is enough to fully abolish bim bh3-induced cytochrome c and smac release .
titrating mcl- <dig> exogenous protein to find out the condition in which both bad and noxa bh <dig> peptides exhibit their binding selectivity
2lmp mitochondria were mixed with  <dig> nm of either bcl- <dig> or mcl- <dig> proteins.
under the inhibition of mcl- <dig>   <dig> nm of bim was unable to induce smac or cytochrome c release.
though not able to induce momp in 2lmp mitochondria when used alone, noxa bh <dig> peptide was able to overcome the protection conferred by mcl- <dig>  but not bcl- <dig>  on the other hand, bad bh <dig> peptide can overcome the protection conferred by both bcl- <dig> and mcl- <dig> proteins, as shown in figure 4a.
this was probably because the combination of bim and bad bh <dig> peptides generated enough de-repressing effect on mitochondria moderately protected by either bcl- <dig> or mcl- <dig> proteins due to the promiscuous nature of bim, while the combination of bim and noxa bh <dig> peptides could not provide such synergism when bim bh <dig> peptide was fully neutralized by bcl- <dig> protein.
since both bad and noxa bh <dig> peptides were able to restore bim bh <dig> peptide-induced momp in 2lmp mitochondria moderately protected by mcl- <dig>  the functional assay established under such concentration of recombinant protein would fail to demonstrate the selectivity and specificity of either bcl- <dig> or mcl- <dig> antagonists.
hence, appropriate concentrations of protein had to be determined to ensure discrimination between bad-like and noxa-like mitochondrial permeabilizers.
as shown in figure 4b, the synergistic effect of bad bh <dig> peptide on bim-induced momp was diminished when escalated concentrations of mcl- <dig> protein were applied, and completely abolished under  <dig> nm of mcl- <dig> protein, while the effect of noxa bh <dig> peptide was intact.
on the other hand, bad bh <dig> peptide could still successfully synergize with bim bh <dig> peptide in the induction of momp in 2lmp mitochondria protected by the same amount of either bcl- <dig> or bcl-xl proteins , indicating that a concentration of recombinant protein up to  <dig> nm was necessary to ensure reliable assay results of selectivity and specificity of bh <dig> peptides.
as it is a promiscuous bh <dig> peptide targeting all three anti-apoptotic proteins, the bim bh <dig> peptide overcame the protection of all three proteins with similar efficacy .
the combination of bad and noxa bh <dig> peptides provided synergism in the induction of momp in mitochondria dominantly protected by mcl- <dig> protein
2lmp mitochondria were pre-incubated with  <dig> nm mcl- <dig> recombinant protein.
noxa and bad bh <dig> peptides were added alone or in combination in the presence or absence of  <dig> nm of bim bh <dig> peptide as indicated.
as shown in figure 5a, neither bad nor noxa peptide alone can permeabilize the mitochondria even at  <dig> μm.
bad bh <dig> peptide, when used up to  <dig> μm, hardly recovered any release of bim bh <dig> peptide-induced cytochrome c release, while noxa bh <dig> peptide fully restored the effect of bim bh <dig> peptide repressed by overly abundant mcl- <dig> protein.
in the absence of bim bh <dig> peptides, bad and noxa bh <dig> peptides synergized with each other in permeabilizing the mitochondria dominantly protected by mcl- <dig> protein.
neither bad  nor noxa bh <dig> peptide  could overcome the repression on bim bh <dig> peptide-mediated momp by  <dig> nm mcl- <dig> protein.
however, the combination of bad and noxa bh <dig> peptides, both at  <dig> μm, significantly induced momp in mitochondria protected dominantly by mcl- <dig>  therefore, mitochondria isolated from cell lines protected mainly by mcl- <dig> were perfect models to demonstrate synergism between bad and noxa bh <dig> peptides.
both mda-mb- <dig> and mda-mb- <dig> cells expressed abundant mcl- <dig>  we hypothesized that mitochondria from these two cell lines protected dominantly by mcl- <dig> would behave in the same pattern as 2lmp mitochondria protected by overly abundant recombinant mcl- <dig> protein.
mda-mb- <dig> cells expressed high level of mcl- <dig>  moderate level of bcl-xl and undetectable bcl- <dig>  as shown in figure 5b, both bim and bad bh <dig> peptides effectively induced momp, while noxa alone, up to  <dig> μm, failed to induce momp in mitochondria isolated from this cell line, as expected.
however, in the absence of bim, combination of bad and noxa bh <dig> peptides induced momp comparable to that induced by bim bh <dig> peptide.
mda-mb- <dig> cells expressed trace amount of bcl-xl and undetectable bcl- <dig>  hence the mitochondria were protected mainly by mcl- <dig> protein.
as shown in figure 5c, bim bh <dig> peptide effectively induced momp, while noxa bh <dig> peptide alone did not induce any release of mitochondrial intermembrane space proteins.
bad bh <dig> peptide alone, up to  <dig> μm, induced the release of moderate amount of smac and barely detectable cytochrome c from mda-mb- <dig> mitochondria.
in the absence of bim, bad bh <dig> peptide and noxa bh <dig> peptide induced synergistic release of cytochrome c and smac protein from mda-mb- <dig> mitochondria, further confirming the necessity of the involvement of noxa-like molecule in permeabilizing mitochondria dominantly protected by mcl- <dig> protein .
in this study, we have optimized the condition of a mitochondria-based cell-free system to verify the bh <dig> peptides with selective targeting profile, facilitating future identification of inhibitors against bcl- <dig>  bcl-xl, or mcl- <dig>  we obtained reliable results on cytochrome c and smac release by adopting several conditions suggested by other groups and also confirmed by us.
most importantly, we have set up protocols using either 2lmp mitochondria protected by recombinant bcl- <dig> anti-apoptotic proteins or mitochondria purified from two other breast cancer cell lines dominantly protected by endogenous mcl- <dig> to demonstrate a clear synergistic effect on the induction of momp by the combination of bad and noxa bh <dig> peptides.
there are several reasons why we chose to use mitochondria isolated from cancer cells as our experimental subjects and semi-quantify cytochrome c and smac release by western blotting.
first of all, the bcl- <dig> family proteins regulates the integrity of the outer mitochondrial membrane that can be permeabilized when the balance between pro-apoptotic and anti-apoptotic bcl- <dig> proteins is interrupted by effective bh <dig> mimetics, resulting in momp followed by the release of the mitochondrial intermembrane space proteins detectable in many testing methods  <cit> .
secondly, cell-free system simplifies protein studies by eliminating the possibility of transcriptional or posttranslational modifications.
thirdly, use of western blotting to detect momp does not need dedicated laboratory equipments and can easily be carried out in almost all laboratories.
however, the disadvantages of this functional assay method include low throughput, lack of sensitivity and need for artifact-prone subcellular fractionation  <cit> .
the use of this technique may be limited to verification of bh <dig> mimetics with decent binding affinities to bcl- <dig> family proteins, hence it should be recommended to serve as a complement to screening methods with higher sensitivity, like fp or elisa.
CONCLUSIONS
the mitochondrial functional assay based on momp is a robust screening and validation tool for identifying bh <dig> mimetics with selective toxicity profile and investigating their mechanism of action on breast cancer cell lines protected by different anti-apoptotic bcl- <dig> family proteins.
it represents a reliable and predictive screening tool that is complementary to high throughput screening to further verify promising lead compounds on a functional level.
abbreviations
bad: bcl-2-associated death promoter; bcl-2: b cell lymphoma 2; bcl-xl: b-cell lymphoma-extra large; bh3: bcl- <dig> homolog domain 3; bid: bh3-interacting domain death agonist; bim: bcl-2-interacting mediator of cell death; cox iv: cytochrome c oxidase iv; fp: fluorescence polarization; mcl-1: myeloid cell leukemia 1; momp: mitochondrial outer membrane permeabilization; noxa: phorbol-12-myristate-13-acetate-induced protein 1; nmr: nuclear magnetic resonance; puma: p <dig> upregulated modulator of apoptosis; spr: surface plasmon resonance.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
jl carried out major experiments including mitochondrial purification and permeabilization, western blotting testing release of cytochrome c and smac proteins, and drafted the manuscript.
ll performed cell culture, mitochondrial purification and permeabilization, western blotting and revised manuscript.
znc accomplished peptide screening by fluorescence polarization and surface plasmon resonance, and revised manuscript.
ss participated in optimizing assay condition.
hy performed recombinant protein expression and purification.
swang and swang are the corresponding authors who performed experimental design, interpretation of concept, manuscript revision, and final approval of manuscript.
all authors read and approved the final manuscript.
authors’ information
shaomeng wang:
warner-lambert/parke-davis professor in medicine.
professor of medicine, pharmacology and medicinal chemistry.
director, cancer drug discovery program.
co-director, molecular therapeutics program.
university of michigan comprehensive cancer center.

 the mitochondrial outer membrane permeabilization is a crucial step of the apoptotic process triggering the release of soluble apoptogenic factors from the intermembrane space such as cytochrome c and smac/diablo followed by subsequent activation of caspase cascade committing to apoptotic cell destruction. the momp process is often altered in cancer cells, resulting largely from the deregulated expression of bcl- family proteins. the bcl- family includes anti-apoptotic proteins like bcl- bcl-xl, bcl-w, and mcl- containing all four bcl- homology domains, pro-apoptotic proteins like bax, bak and bok lacking the bh domain, and the pro-apoptotic bh3-only proteins like bim, bid, puma, bad and noxa. among all of these proteins, bax and bak are believed to be the “executors” which will exhibit conformational change and oligomerization upon activation and subsequently induce momp and cell death. there are two widely embraced models for the initiation of momp: the direct activation model and the indirect activation model. the direct model proposes that a subset of bh3-only proteins termed “activators”, namely bim, bid and puma, directly engage bax or bak, resulting in the activation and oligomerization of these two proteins. some other bh3-only proteins, termed “sensitizers”, for example, bad and noxa, act only by displacing the activators from the anti-apoptotic proteins, allowing the activators to bind and activate bax and bak. the indirect model posits that bh3-only proteins activate bax and bak not by binding either one, but by antagonizing anti-apoptotic proteins that constrain bax and bak. in this scenario, bim, bid and puma proteins are far more potent than the others, such as bad and noxa, because they can engage all the anti-apoptotic proteins, while bad and noxa selectively bind only a subset of anti-apoptotic proteins. newmeyer et al. first revealed in a cell-free system that the addition of bcl- into the organelle fraction enriched in mitochondria inhibited the process of nuclear destruction, the typical morphological change when cells underwent apoptosis. a good many studies have cumulatively proven that anti-apoptotic bcl- members are attractive targets for anti-cancer therapy. numerous anti-cancer strategies based on bh peptides derived from bh3-only proteins, anti-sense oligonucleotide or rna interference targeting anti-apoptotic bcl- family proteins, and non-peptidic small molecules binding specifically to anti-apoptotic bcl- proteins have been developed. accordingly, substantial advances have been achieved in the field of screening techniques, which are critical to the identification and verification of antagonists against bcl- anti-apoptotic proteins. in vitro high-throughput screening approaches utilizing technologies like fluorescence polarization or nuclear magnetic resonance were quite effective in the discovery and selection of lead compounds suitable for further optimization and development. however, these methods lack the ability to mimic the intracellular environment where the interruption of protein-protein interaction actually happens. cell-free systems using mitochondria isolated from normal and cancer cells have been adopted to study mitochondrial changes upon antagonizing bcl- anti-apoptotic members, which would serve as a promising tool closely imitating the intracellular initiation of momp and apoptotic core machinery to verify bh mimetics discovered by other assays. interestingly, similar system based on isolated mitochondria was also used to characterize compounds designed to target bid to treat disorders associated with the activation of such pro-apoptotic protein. in this study, we set up a functional assay using mitochondria isolated from breast cancer cells, recombinant anti-apoptotic bcl- family proteins and different bh peptides. experimental conditions under which bh peptides with selective targeting profiles induce momp either alone or in combination were determined and optimized. in this assay, momp was allowed to be semi-quantified by measuring the release of key apoptogenic molecules from mitochondrial intermembrane space using western blotting. we optimized the experimental conditions by adopting the high ionic strength buffer during permeabilization of mitochondria by bh peptides. we believe this optimized functional assay based on momp will be a useful screening and validation tool for identifying bh mimetics selectively targeting different bcl- anti-apoptotic proteins. methods materials 2lmp, a subclone of mda-mb- was kindly provided by dr. marc lippman. normal cell lines including wi- prec, and human breast cancer cell lines including hbl sum bt mcf- t47d, zr, mda-mb- mda-mb- mda-mb- mda-mb- and mda-mb- were obtained from the american type culture collection and cultured in medium recommended by atcc. 2lmp, mda-mb- and mda-mb- were grown in rpmi containing l-glutamine supplemented with 10% fbs and 1% penicillin/streptomycin, maintained in antibiotic-free environment at 37°c in a 5% co atmosphere and routinely screened for mycoplasma contamination. bh peptides were kindly provided by dr. peter p roller, including bim bh peptide, both 21-mer and 26-mer, bid bh peptide, bad bh peptide, and noxa bh peptide. abt- was synthesized according to the literature. all other chemicals used were purchased from sigma-aldrich. protein expression and purification human bcl- protein the isoform construct of the human bcl- was used to produce n-terminal 6×his tagged recombinant protein in e. coli bl. cells were grown in 2xyt containing antibiotics to an od of at 37°c. protein expression was then induced with mm iptg at 20°c for h. after centrifugation, cell pellets were resuspended in lysis buffer containing mm tris, ph, mm nacl, % bme and leupectin/aprotin. after sonication and centrifugation, recombinant protein was purified from the soluble fraction first using ni-nta resin, and then superdex column in elution buffer containing mm tris, ph, mm nacl and mm dtt. human bcl-xl protein gene encoding human bcl-xl protein, which has an internal deletion of 45– amino acid residues and a c-terminal truncation of 212– was cloned into the phis-tev vector to generate n-terminal 8xhis tagged recombinant protein in e. coli bl the same protocols to express and purify human bcl- protein were followed. lysis buffer contained mm tris, ph, mm nacl, % bme and leupectin/aprotin, while protein was eluted in buffer containing mm tris, ph, mm nacl and mm dtt. human mcl- protein the mcl-1fragment encoding amino acid residues of 171– was cloned into the phis-tev vector. mcl- protein with an n-terminal 8×his tag was produced in e. coli bl the same protocols to express and purify human bcl- protein were followed but a source q column was used in the second purification step and protein was eluted in mm tris, ph, with nacl gradient. these recombinant proteins protect the mitochondria from momp by sequestering the added bim bh peptide, until bim bh is displaced by other bh molecules targeting the recombinant proteins. fluorescence polarization based binding assays mcl- and bcl- protein a 21-residue bid bh peptide was synthesized and labeled at the n-terminus with 6-carboxyfluorescein succinimidyl ester as the fluorescence tag. saturation experiments determined that fam-bid binds to mcl- and bcl- protein with a kd values of nm and nm, respectively. for competitive binding experiments, mcl- protein or bcl- protein were pre-incubated with fam-bid peptide in the assay buffer. μl of a solution in dmso of the tested bh peptide was added to the protein/fam-bid solution in black, round-bottom plates to produce a final volume of μl. for each experiment, a control containing tested protein and flu-bid peptide, and another control containing only fam-bid, were included on each assay plate. after 2– hours incubation, the polarization values in milipolarization units were measured at an excitation wavelength of nm and an emission wavelength of nm using the synergy h hybrid microplate reader. ic the inhibitor concentration at which 50% of bound peptide is displaced, was determined from the plot using nonlinear least squares analysis and curve fitting performed using graphpad prizm software. the unlabeled bid bh peptide is used as the positive control. the ki value for each bh peptide was calculated using the equation we have developed for fp-based assays. bcl-xl protein for this assay, we have employed the bak bh peptide labeled with fluorescein instead of the fam-bid to maximize the signal. it was determined that fam-bak has a kd value of nm to bcl-xl protein. the competitive binding assay for bcl-xl was the same as that for mcl- and bcl- with nm of bcl-xl protein and nm of fam-bak peptide in the following assay buffer: mm tris-bis, ph and % bovine gamma globulin. surface plasmon resonance based binding assay biotin-labeled bim bh peptide was immobilized on a streptavidin sa sensor chip, while the fc surface was used as a control surface. the binding affinities of recombinant mcl- bcl- and bcl-xl to immobilized bim bh peptide was determined by injecting proteins in different concentrations in hbs-ep buffer. determination of kon, koff and kd were calculated by simultaneous non-linear regression using biaevaluation software. bim peptide has a kd values of nm, nm and nm against mcl- bcl- and bcl-xl, respectively. the obtained results confirmed the high binding affinity of bim bh peptide against all three members from the bcl- family of proteins. using the same bim bh peptide immobilized sa chip, spr competitive solution binding experiments were performed by pre-incubating tested proteins with tested bh peptides for at least minutes and then the reaction mixture was injected over the surfaces of the chip. response units were measured at seconds in the dissociation phase and the specific binding was calculated by subtracting the control surface signal from the surfaces with immobilized bim bh peptide. ic values were determined by non-linear least squares analysis using graph pad prism software. isolation of breast cancer cell mitochondria mitochondria were isolated from breast cancer cells. cells were harvested by centrifugation at g for min at 4°c and then washed with ice-cold pbs. cell pellets were resuspended in μl of mitochondria isolation buffer freshly added mm pmsf and protease inhibitor cocktail. cell suspensions were then homogenized with strokes in a dounce tissue grinder on ice and centrifuged at g for min at 4°c. supernatants were centrifuged at g for min at 4°c. pellet enriched in mitochondria was washed three times with mib, spun down at g for min at 4°c. the pellet was then resuspended in appropriate volume of mitochondria reaction buffer, freshly added mm pmsf and protease inhibitor cocktail. in order to optimize buffer condition during permeabilization of mitochondrial outer membrane, two different solutions were used. low ionic strength buffer contained mm hepes, mm kcl, mm mgcl mm edta, and mm sucrose. high ionic strength buffer contained mm hepes, mm kcl, mm mgcl and mm sucrose. protein concentrations were determined using bradford protein assay. the mitochondrial suspensions were aliquoted into μl with mg/ml protein concentration for permeabilization experiments. permeabilization of mitochondrial outer membrane by bh peptides or abt-737 for experiments using peptide or abt- alone, mitochondria were incubated with solvent, different concentrations of bim, bad, noxa bh peptides, or abt- for h in 37°c water bath. for experiments using recombinant proteins and combination of bh peptides, mitochondria were pretreated with pbs or recombinant proteins followed by permeabilization with solvent or different concentrations of bh peptides. at the end of incubation, mitochondria were pelleted at g for min at 4°c, supernatants were collected and briefly heated with 2 × sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer. mitochondrial pellets were also dissolved and heated in 2 × sds-page sample buffer. both the supernatant samples and mitochondrial pellet samples were subjected to immunoblotting against cytochrome c and smac/diablo proteins. western blot analysis of released cytochrome c and smac protein supernatant samples and mitochondrial pellet samples were subjected to protein separation by sds-page. after transfer to polyvinylidene fluoride membranes, cytochrome c, smac proteins and loading control were analyzed using the following primary antibodies: anti-cytochrome c polyclonal antibody, anti-smac/diablo polyclonal antibody, anti-cox iv monoclonal antibody. blots were then probed with species-specific horseradish peroxidase-conjugated secondary antibodies and detected with chemiluminescence. for supernatant samples, one blot was cut into two parts so that both cytochrome c and smac could be detected simultaneously without reprobing. for pellet samples, the blot was probed for cytochrome c, smac, as well as cox iv that served as loading control.  breast cancer cells are dually or multiply-protected by anti-apoptotic bcl- family proteins as we know, cancer cells are dually-, triply-, or multiply-protected by bcl- bcl-xl, mcl- or other anti-apoptotic bcl- family proteins which have been less thoroughly studied. a panel of human normal cell lines and breast cancer cell lines were analyzed by western blotting for their basal levels of three key anti-apoptotic proteins, bcl- bcl-xl and mcl-. 2lmp cell line, a subclone of mda-mb- is characterized by its abundant cytoplasm and rapid proliferation which are critical for providing sufficient mitochondria. both mda-mb- and mda-mb- expressing mainly mcl- protein were suitable cell line models for studying momp induced by noxa-like molecules and the combined outcome from co-treatment with both bad-like and noxa-like molecules. bh peptides derived from bh3-only proteins were powerful tools for setting up conditions of the mitochondria-based functional assay binding affinities of bh peptides to bcl- bcl-xl and mcl- recombinant proteins were determined by surface plasmon resonance and fluorescence polarization, as shown in table  bim and bid bh peptides bound to all three proteins with very high affinities. bad bh peptide bound to both bcl- and bcl-xl, but failed to find to mcl- as expected. noxa bh peptide selectively bound to mcl- protein only. although there was some discrepancy between the spr and the fp data, probably due to different assay conditions, data regarding the versatility of bim bh and the selectivity of bad and noxa bh were highly consistent with the literature. mitochondria based-functional assay established by using these peptides will help to define how and when bim-like, bad-like or noxa-like small molecule bcl- inhibitors function on the mitochondrial level. bh peptides were screened for their binding affinities to the three anti-apoptotic bcl- family proteins using surface plasmon resonance and fluorescence polarization methods, as described in the methods. results are representative of at least three independent experiments. sufficient ionic strength from kcl ensured reliable cytochrome c and smac release from mitochondria permeabilized by bh peptides previous apoptosis studies using purified mitochondria as a cell-free system have yielded conflicting results due to less appropriate experimental conditions including low ionic strength, physiologically irrelevant temperature or short incubation time. these studies provided substantial evidence that sufficient ionic strength and temperature of 37°c contributed to full release of cytochrome c. in our study, ionic strength-dependent release of cytochrome c was also confirmed in mitochondria permeabilized by bim bh peptide in min in buffer containing different concentrations of kcl. mitochondria isolated from 2lmp cells were incubated with bim bh peptides at 37°c for min or min in either high ionic strength buffer containing mm kcl or low ionic strength buffer containing mm kcl. momp induced by bim bh peptides at indicated concentrations were evaluated through the analysis of released cytochrome c and smac proteins by western blotting. as shown in figure 2b, bim bh peptide induced momp in a dose-dependent manner. bim bh peptide induced significantly more robust release of cytochrome c and smac proteins when there was sufficient ionic strength from kcl. preliminary fp data suggested that the binding affinity of 21-mer bim bh peptide was weaker than 26-mer bim bh peptide, we also confirmed that the induction of smac release by 21-mer bim was weaker than that by 26-mer bim. longer incubation resulted in more complete release of cytochrome c, as indicated by more cytochrome c being detected in the supernatant fraction and less cytochrome c remained in the pellet fraction of treatment with nm bim for min in his buffer, compared with similar treatment for min. hence, 26-mer bim bh peptide, reaction buffer containing mm kcl, reaction temperature of 37°c and incubation time of min were set as experimental conditions for all mitochondrial experiments. comparing the efficiency of momp induction by different bcl- targeting agents and functional inhibition of bim bh peptide-induced momp by three different anti-apoptotic bcl- family proteins. mitochondria isolated from 2lmp cells were permeabilized by bim, bid, bad, noxa bh peptides and abt- a bona fide bcl- antagonist, as shown in figure 3a. bim, bid, bad bh peptides and abt- effectively induced cytochrome c and smac release from 2lmp mitochondria. consistent with its binding affinities to bcl- bcl-xl and mcl- proteins, bim bh peptide was the most potent inducer among all bh peptides. noxa alone was not able to induce cytochrome c and smac release, which is also consistent with previous reports. bim bh induced robust release of cytochrome c and smac proteins even at nm. the three major anti-apoptotic proteins were equally potent in repressing the induction of momp by nm bim bh peptide. even nm of recombinant protein is enough to fully abolish bim bh3-induced cytochrome c and smac release. titrating mcl- exogenous protein to find out the condition in which both bad and noxa bh peptides exhibit their binding selectivity 2lmp mitochondria were mixed with nm of either bcl- or mcl- proteins. under the inhibition of mcl- nm of bim was unable to induce smac or cytochrome c release. though not able to induce momp in 2lmp mitochondria when used alone, noxa bh peptide was able to overcome the protection conferred by mcl- but not bcl- on the other hand, bad bh peptide can overcome the protection conferred by both bcl- and mcl- proteins, as shown in figure 4a. this was probably because the combination of bim and bad bh peptides generated enough de-repressing effect on mitochondria moderately protected by either bcl- or mcl- proteins due to the promiscuous nature of bim, while the combination of bim and noxa bh peptides could not provide such synergism when bim bh peptide was fully neutralized by bcl- protein. since both bad and noxa bh peptides were able to restore bim bh peptide-induced momp in 2lmp mitochondria moderately protected by mcl- the functional assay established under such concentration of recombinant protein would fail to demonstrate the selectivity and specificity of either bcl- or mcl- antagonists. hence, appropriate concentrations of protein had to be determined to ensure discrimination between bad-like and noxa-like mitochondrial permeabilizers. as shown in figure 4b, the synergistic effect of bad bh peptide on bim-induced momp was diminished when escalated concentrations of mcl- protein were applied, and completely abolished under nm of mcl- protein, while the effect of noxa bh peptide was intact. on the other hand, bad bh peptide could still successfully synergize with bim bh peptide in the induction of momp in 2lmp mitochondria protected by the same amount of either bcl- or bcl-xl proteins, indicating that a concentration of recombinant protein up to nm was necessary to ensure reliable assay results of selectivity and specificity of bh peptides. as it is a promiscuous bh peptide targeting all three anti-apoptotic proteins, the bim bh peptide overcame the protection of all three proteins with similar efficacy. the combination of bad and noxa bh peptides provided synergism in the induction of momp in mitochondria dominantly protected by mcl- protein 2lmp mitochondria were pre-incubated with nm mcl- recombinant protein. noxa and bad bh peptides were added alone or in combination in the presence or absence of nm of bim bh peptide as indicated. as shown in figure 5a, neither bad nor noxa peptide alone can permeabilize the mitochondria even at μm. bad bh peptide, when used up to μm, hardly recovered any release of bim bh peptide-induced cytochrome c release, while noxa bh peptide fully restored the effect of bim bh peptide repressed by overly abundant mcl- protein. in the absence of bim bh peptides, bad and noxa bh peptides synergized with each other in permeabilizing the mitochondria dominantly protected by mcl- protein. neither bad nor noxa bh peptide could overcome the repression on bim bh peptide-mediated momp by nm mcl- protein. however, the combination of bad and noxa bh peptides, both at μm, significantly induced momp in mitochondria protected dominantly by mcl- therefore, mitochondria isolated from cell lines protected mainly by mcl- were perfect models to demonstrate synergism between bad and noxa bh peptides. both mda-mb- and mda-mb- cells expressed abundant mcl- we hypothesized that mitochondria from these two cell lines protected dominantly by mcl- would behave in the same pattern as 2lmp mitochondria protected by overly abundant recombinant mcl- protein. mda-mb- cells expressed high level of mcl- moderate level of bcl-xl and undetectable bcl- as shown in figure 5b, both bim and bad bh peptides effectively induced momp, while noxa alone, up to μm, failed to induce momp in mitochondria isolated from this cell line, as expected. however, in the absence of bim, combination of bad and noxa bh peptides induced momp comparable to that induced by bim bh peptide. mda-mb- cells expressed trace amount of bcl-xl and undetectable bcl- hence the mitochondria were protected mainly by mcl- protein. as shown in figure 5c, bim bh peptide effectively induced momp, while noxa bh peptide alone did not induce any release of mitochondrial intermembrane space proteins. bad bh peptide alone, up to μm, induced the release of moderate amount of smac and barely detectable cytochrome c from mda-mb- mitochondria. in the absence of bim, bad bh peptide and noxa bh peptide induced synergistic release of cytochrome c and smac protein from mda-mb- mitochondria, further confirming the necessity of the involvement of noxa-like molecule in permeabilizing mitochondria dominantly protected by mcl- protein. in this study, we have optimized the condition of a mitochondria-based cell-free system to verify the bh peptides with selective targeting profile, facilitating future identification of inhibitors against bcl- bcl-xl, or mcl- we obtained reliable results on cytochrome c and smac release by adopting several conditions suggested by other groups and also confirmed by us. most importantly, we have set up protocols using either 2lmp mitochondria protected by recombinant bcl- anti-apoptotic proteins or mitochondria purified from two other breast cancer cell lines dominantly protected by endogenous mcl- to demonstrate a clear synergistic effect on the induction of momp by the combination of bad and noxa bh peptides. there are several reasons why we chose to use mitochondria isolated from cancer cells as our experimental subjects and semi-quantify cytochrome c and smac release by western blotting. first of all, the bcl- family proteins regulates the integrity of the outer mitochondrial membrane that can be permeabilized when the balance between pro-apoptotic and anti-apoptotic bcl- proteins is interrupted by effective bh mimetics, resulting in momp followed by the release of the mitochondrial intermembrane space proteins detectable in many testing methods. secondly, cell-free system simplifies protein studies by eliminating the possibility of transcriptional or posttranslational modifications. thirdly, use of western blotting to detect momp does not need dedicated laboratory equipments and can easily be carried out in almost all laboratories. however, the disadvantages of this functional assay method include low throughput, lack of sensitivity and need for artifact-prone subcellular fractionation. the use of this technique may be limited to verification of bh mimetics with decent binding affinities to bcl- family proteins, hence it should be recommended to serve as a complement to screening methods with higher sensitivity, like fp or elisa.  the mitochondrial functional assay based on momp is a robust screening and validation tool for identifying bh mimetics with selective toxicity profile and investigating their mechanism of action on breast cancer cell lines protected by different anti-apoptotic bcl- family proteins. it represents a reliable and predictive screening tool that is complementary to high throughput screening to further verify promising lead compounds on a functional level. abbreviations bad: bcl-2-associated death promoter; bcl-2: b cell lymphoma 2; bcl-xl: b-cell lymphoma-extra large; bh3: bcl- homolog domain 3; bid: bh3-interacting domain death agonist; bim: bcl-2-interacting mediator of cell death; cox iv: cytochrome c oxidase iv; fp: fluorescence polarization; mcl-1: myeloid cell leukemia 1; momp: mitochondrial outer membrane permeabilization; noxa: phorbol-12-myristate-13-acetate-induced protein 1; nmr: nuclear magnetic resonance; puma: p upregulated modulator of apoptosis; spr: surface plasmon resonance. competing interests the authors declare that they have no competing interests. authors’ contributions jl carried out major experiments including mitochondrial purification and permeabilization, western blotting testing release of cytochrome c and smac proteins, and drafted the manuscript. ll performed cell culture, mitochondrial purification and permeabilization, western blotting and revised manuscript. znc accomplished peptide screening by fluorescence polarization and surface plasmon resonance, and revised manuscript. ss participated in optimizing assay condition. hy performed recombinant protein expression and purification. swang and swang are the corresponding authors who performed experimental design, interpretation of concept, manuscript revision, and final approval of manuscript. all authors read and approved the final manuscript. authors’ information shaomeng wang: warner-lambert/parke-davis professor in medicine. professor of medicine, pharmacology and medicinal chemistry. director, cancer drug discovery program. co-director, molecular therapeutics program. university of michigan comprehensive cancer center. 
in this study, we aimed at establishing a reliable functional assay using mitochondria isolated from breast cancer cells to decipher the mode of action of bh peptides derived from bh3-only proteins. mitochondrial outer membrane permeabilization is a crucial step leading to apoptotic destruction of cancer cells. mitochondrionb cell lymphoma bcl- homolog domain mitochondrial outer membrane permeabilization but assay conditions need to be optimized for more reliable results. sufficient ionic strength was required for optimal release of cytochrome c. bad and noxa bh peptides exhibited their bona fide antagonistic effects against bcl-2/bcl-xl and mcl- proteins, respectively, whereas bim bh peptide antagonized all three anti-apoptotic bcl- members. cytochrome c and smac/diablo were tested in both supernatants and mitochondrial pellets by western blotting. in this study, high ionic strength buffer was adopted during the initiation of momp. mitochondria isolated from human breast cancer cell lines with distinct expression patterns of bcl- anti-apoptotic proteins were permeabilized by different bh peptides alone or in combination, with or without the presence of recombinant anti-apoptotic bcl- family proteins.
 mitochondrial outer membrane permeabilization is a crucial step leading to apoptotic destruction of cancer cells. bcl- family proteins delicately regulate mitochondrial outer membrane integrity through protein-protein interactions, which makes the mitochondrion an ideal cell-free system for screening molecules targeting the bcl- anti-apoptotic proteins. but assay conditions need to be optimized for more reliable results. in this study, we aimed at establishing a reliable functional assay using mitochondria isolated from breast cancer cells to decipher the mode of action of bh peptides derived from bh3-only proteins. in this study, high ionic strength buffer was adopted during the initiation of momp. mitochondria isolated from human breast cancer cell lines with distinct expression patterns of bcl- anti-apoptotic proteins were permeabilized by different bh peptides alone or in combination, with or without the presence of recombinant anti-apoptotic bcl- family proteins. cytochrome c and smac/diablo were tested in both supernatants and mitochondrial pellets by western blotting.  sufficient ionic strength was required for optimal release of cytochrome c. bad and noxa bh peptides exhibited their bona fide antagonistic effects against bcl-2/bcl-xl and mcl- proteins, respectively, whereas bim bh peptide antagonized all three anti-apoptotic bcl- members. bad and noxa peptides synergized with each other in the induction of momp when mitochondria were dually protected by both bcl-2/bcl-xl and mcl-  this method based on momp is a useful screening tool for identifying bh mimetics with selective toxicity against breast cancer cell mitochondria protected by the three major bcl- anti-apoptotic proteins. mitochondrionb cell lymphoma bcl- homolog domain mitochondrial outer membrane permeabilization 