the goal of these drug cocktail regimens 
 is to achieve additive or synergistic effects among chemotherapeutics , 
 thereby maximizing summation dose - intensity with resultant enhanced 
 anticancer activities and increased patient survival .
combinations have been identified and developed both through unbiased 
 approaches and by rational design , and compounds that act on a single biochemical 
 pathway are particularly strong candidates for synergy or potentiation . 

for example , inhibitors of poly(adp - ribose)polymerase-1 ( parp-1 ) , 
 an enzyme that facilitates dna damage repair , potently synergize with 
 dna damaging agents as demonstrated in cell culture and animal models .
herein we describe an approach for potentiation not based on compounds 
 acting on two targets within a single pathway , but rather with two 
 compounds acting differentially to activate the same enzyme . during apoptosis
, the zymogen procaspase-3 is activated via proteolysis 
 to caspase-3 , and this active caspase-3 then cleaves scores of cellular 
 substrates , executing the apoptotic program .
as procaspase-3 protein levels are elevated in various tumor histologies , drug - mediated direct activation of procaspase-3 has gained significant 
 interest as a selective anticancer strategy .
furthermore , caspase-3 
 has been shown to play critical roles in cardiomyocyte hypertrophy , 
 cellular differentiation , and remodeling .
thus development 
 of a strategy to magnify the timing and level of caspase-3 activation 
 in a specific and direct manner could greatly aid the study of active 
 caspase-3 in these nonapoptotic processes . to date
, two major classes 
 of compounds have been disclosed that enhance the activity and automaturation 
 of procaspase-3 in vitro and induce apoptosis in cancer cells in culture . 

procaspase - activating compound-1 ( pac-1 , figure 1a ) enhances the activity of procaspase-3 in vitro via the chelation 
 of inhibitory zinc ions , induces apoptosis in 
 cancer cells in culture , and has shown efficacy in multiple 
 murine tumor models .
more recently , the 
 compound 1541b ( figure 1a ) was discovered to 
 promote the automaturation of procaspase-3 to caspase-3 in vitro and 
 to induce apoptotic death of cancer cells in culture .
compound 1541b appears to activate procaspase-3 via a binding - induced 
 shift in the on  off state equilibrium , or through formation of nanofibrils .
pac-1 and 
 1541b exert their activating effect on procaspase-3 by distinct biochemical 
 mechanisms , suggesting the potential for synergistic effects in cell 
 culture and in vivo .
( b ) procaspase-3 ( 100 
 nm ) was incubated with pac-1 or 1541b in zinc - supplemented caspase 
 activity buffer .
data is normalized to procaspase-3 ( 100 nm ) activity in zinc - free 
 caspase activity buffer as 100% activity .
( c ) procaspase-3 ( 350 nm ) in zinc - supplemented 
 caspase activity buffer was incubated 1541b , pac-1 , or 1541b + pac-1 , 
 and enzymatic activity was assessed with ac - devd - pna .
data is normalized 
 to 100% activity = 350 nm caspase-3 in zinc - free caspase activity 
 buffer .
error bars represent the sem of three replicates . in vitro , procaspase-3 has enzymatic activity that 
 is dramatically 
 lower than caspase-3 , with estimates ranging from reductions of 200 
 to 10 fold .
low micromolar levels 
 of zinc inhibit the activity of procaspase-3 and caspase-3 in vitro .
pac-1 , a moderate affinity zinc 
 chelator that has been shown to chelate the labile zinc pool in cells , allows procaspase-3 to once again process substrates , 
 including itself .
described herein is 
 the combination use of the two small molecule activators of procaspase-3 , 
 pac-1 and 1541b .
these compounds act synergistically to enhance procaspase-3/caspase-3 
 activity in vitro , induce rapid procaspase-3 processing and caspase-3 
 activity in cell culture , potently and rapidly cause apoptotic death 
 in a variety of cancer cell lines , and have efficacy in a murine tumor 
 model .
as zinc 
 colocalizes with cellular procaspase-3/caspase-3 , it was of interest to determine if 1541b could activate recombinantly 
 expressed procaspase-3 ( supporting figure s1a ) in the presence of zinc ; the activating effect of 1541b on procaspase-3 
 in vitro had previously only been evaluated under zinc - free conditions . as shown in figure 1b , 
 compound 1541b does not enhance intrinsic procaspase-3 enzymatic activity 
 in zinc - containing buffers , whereas pac-1 relieves zinc - mediated inhibition 
 and induces the anticipated activating effect .
this effect from pac-1 is reliant upon its zinc binding 
 ability , as an analogue that does not bind zinc ( pac-1a ) is not active in this experiment .
in addition , 
 an inactive derivative of 1541b ( compound 1541d ) has no effect in this experiment ( structures and data 
 in supporting figure s1b  d ) .
this 
 orthogonal activity of pac-1 and 1541b on procaspase-3 ( pac-1 activating 
 zinc - bound procaspase-3 , 1541b activating procaspase-3 with zinc removed ) 
 suggests synergistic potential , and indeed , the combination of pac-1 
 and 1541b , when incubated with procaspase-3 in vitro , leads to dramatic 
 caspase-3 activity far exceeding the effect of either compound alone 
 ( figure 1c ) .
consistent with the established 
 structure  activity relationships , pac-1a and 1541d as single agents and in combination were unable 
 to facilitate procaspase-3 activation in vitro ( supporting figure s1d ) . to examine pac-1 synergy with 1541b for activation 
 of procaspase-3 in cancer cells in culture ,
a panel of cancer cell 
 lines were treated with combinations of pac-1 and 1541b and the caspase-3/-7 
 activity of the cell lysates was monitored with the fluorogenic caspase 
 substrate ac - devd - afc . as seen in figure 2a , 
 cotreatment of u-937 ( human lymphoma ) , bt-549 ( human breast cancer ) , 
 and a549 ( human lung cancer ) cells with pac-1 and 1541b results in 
 markedly more dramatic and rapid increases in devdase activity than 
 treatment with either pac-1 or 1541b as single agents .
the combination 
 treatment stimulates caspase activity that rivals or surpasses that 
 induced by staurosporine ( sts , 1 m ) , one of the most rapid 
 inducers of cellular devdase activity known , and is dose dependent 
 ( supporting figure s2 ) .
pac-1/1541b combinations 
 induce rapid and dramatic procaspase-3 
 activation and maturation in cancer cell lines in culture .
( a ) cancer 
 cell lines were treated with pac-1 ( 30 m ) , 1541b ( 15 m ) , 
 or the combination , cells were lysed at various time points , and caspase-3/-7 
 activity of the lysates was evaluated with the fluorogenic ac - devd - afc 
 substrate .
( b ) western blot of various cancer cell lines after 
 treatment with combinations of pac-1 ( 30 m ) and 1541b .
treatment 
 durations of 3 , 4 , and 8 h for u-937 , bt-549 , and a549 cells , respectively , 
 were chosen on the basis of the timing of maximal caspase activity , 
 as observed in cell lysate .
to determine if the elevation of devdase activity was the 
 result 
 of enhanced cleavage of procaspase-3 to caspase-3 facilitated by compound 
 cotreatment , cells were treated with pac-1 and 1541b combinations 
 and assessed by western blotting . as shown in figure 2b , dramatic activation of procaspase-3 to caspase-3 was observed 
 in u-937 , bt-549 , and a549 cells upon treatment with the pac-1/1541b 
 combinations , whereas low / no procaspase-3 activation was observed 
 with 1541b or pac-1 alone at the times and concentrations evaluated . 

analogous results in hl-60 ( human leukemia ) , hs578 t ( human breast 
 cancer ) , and el4 ( murine lymphoma ) cells are shown in supporting figure s3 .
apoptosis through the intrinsic 
 pathway proceeds via an ordered series of events , with bcl-2 family 
 proteins acting on the mitochondria , stimulating cytochrome c release , 
 formation of the apoptosome , caspase-9 activation , and subsequent 
 cleavage of procaspase-3 to caspase-3 .
caspase-3 then cleaves 
 scores of substrates , executing the apoptotic program . to examine these events in cells treated with 
 pac-1 + 1541b , bt-549 cells were assessed for the timing of procaspase-3 
 activation relative to cytochrome c release from the mitochondria 
 by western blot of fractionated cell lysate . as shown in figure 3a , analysis of cells treated with pac-1 +
1541b 
 shows the appearance of cleaved caspase-3 bands in the cytosolic fraction 
 before cytochrome c levels diminish in the mitochondria . at these 
 compound concentrations and relatively short treatment times , 1541b 
 as
a single agent is considerably less effective at inducing procaspase-3 
 activation ( figure 3b ) , and pac-1 is not effective . 

importantly , as shown in figure 3c , staurosporine 
 induces apoptosis through the canonical intrinsic pathway , with clear 
 release of cytochrome c from the mitochondria prior to activation 
 of procaspase-3 .
rapid procaspase-3 activation is observed in cells treated 
 with 
 pac-1 + 1541b , occurring prior to release of cytochrome c from the 
 mitochondria .
( a ) bt-549 breast cancer cells were treated with pac-1 
 ( 30 m ) and 1541b ( 10 m ) over 8 h and assessed in the 
 cytosol for procaspase-3 activation , and in the mitochondria for cytochrome 
 c levels .
( b ) bt-549 cells treated with 1541b ( 10 m ) and assessed 
 for 8 h. ( c ) bt-549 cells treated with sts ( 1 m ) and assessed 
 for 10 h. bt-549 cells were assessed by 
 phase contrast microscopy and monitored 
 for the phenotypic changes associated with caspase activation and 
 apoptosis ( supporting figure s4 ) .
consistent 
 with the western blot and caspase activity data , only the combination 
 of pac-1 and 1541b demonstrated substantial morphological changes 
 indicative of apoptosis , such as blebbing .
the combination of pac-1 and 
 1541b was evaluated for the capacity 
 to induce apoptotic death in a variety of cancer cell lines in culture . 

these evaluations were performed at short incubation times , reflective 
 of the timing of caspase activation observed in figure 2 and where neither compound exerts a significant effect as 
 a single agent .
pac-1 significantly synergizes with 1541b for potent 
 proapoptotic activity in u-937 , bt-549 , and a549 cells ( figure 4a ) , and in hl-60 , hs578 t , u-87 , and el4 cells ( supporting figure s5 ; see supporting figure s6 for representative annexin v - fitc / propidium 
 iodide histograms ) ; the dashed horizontal lines in each graph mark 
 levels of cell death that would be observed from a strictly additive 
 effect of pac-1 and 1541b . while the non - blood - brain barrier permeable 
 analogue s - pac-1 possesses a comparable capacity 
 for synergy as pac-1 ( supporting figure s7 ) , synergy was not observed when either pac-1a or 1541d were used 
 in combination with 1541b or pac-1 ( supporting 
 figures s8 and s9 ) .
pac-1 and 1541b synergize to induce rapid cell 
 death in cancer 
 cell lines .
( a ) cancer cell lines were treated with the indicated 
 concentrations of pac-1 ( 0 , 15 , and 30 m ) and 1541b ( 0 , 7.5 , 
 10 m ) for six hours , and apoptotic death was assessed using 
 flow cytometry with annexin v - fitc / propidium iodide staining .
the dotted horizontal 
 lines represent the level of cell death expected from a mere additive 
 effect of pac-1 and 1541b for each drug combination .
( b ) percent cell 
 death observed after 6 h treatment of u-937 cells with pac-1 
 + 1541b combinations in matrix format .
values are heat mapped with 
 white equal to 0% cell death and red equal to 100% cell death .
( c ) combination index values 
 calculated for each combination with combosyn software ( < 1 indicates 
 synergistic interaction with values < 0.3 indicating strong synergism )
. 
 values are heat mapped with lowest values in green and highest values 
 in red . further assessment ( vs u-937 cells ) 
 of a broad range of concentrations 
 for both compounds clearly shows the dramatic synergy between these 
 two agents .
shown in figure 4b is the cell 
 death induced by the compound matrix , with synergy apparent from simple 
 comparisons of the percent cell death induced by the single agents 
 versus the combinations ; this is true at both short ( 6 h , figure 4b ) and long ( 24 h , supporting 
 figure s10 ) time points .
synergism is frequently quantified 
 in such experiments though calculation of combination indices ( ci ) . 

combinations describing synergistic interactions possess ci values 
 < 1 , while antagonistic interactions have ci value > 1 ; additive 
 effects occur when the ci value equals 1 .
when u-937 cells were treated 
 with a range of pac-1 and 1541b compound concentrations in combination , 
 synergistic interactions were observed broadly , with several combinations 
 considered strongly synergistic ( figure 4b 
 and c and supporting figure s10 ) .
the proapoptotic effect of the pac-1/1541b combination was markedly 
 reduced with the pan - caspase inhibitor q - vd - oph , consistent with the 
 involvement of caspases in the mode of cell death ( figure 5a ) .
to further investigate the connection between 
 activation of procaspase-3 and the cell death induced by the drug 
 combination , mcf-7 cells were used , a cell line that does not express 
 procaspase-3 , together with a matched 
 mcf-7 cell line where procaspase-3 is expressed via a plasmid ( figure 5b ) .
the combination of pac-1/1541b has minimal effect 
 on mcf-7 cells transfected with control plasmid ( mcf-7 vrl ) , but dramatically 
 induces apoptosis in procaspase-3 expressing mcf-7 cells ( mcf-7 c-3 ) 
 ( figure 5c ) , an effect correlated with procaspase-3 
 activation ( figure 5d ) .
pac-1 and 1541b induce 
 rapid caspase - dependent cell death in cancer 
 cell lines .
( a ) the pan - caspase inhibitor q - vd - oph ( 25 m ) protects 
 against pac-1/1541b - mediated cell death in u-937 cells .
( b ) western blot analysis of 
 mcf-7 cells transformed with empty plasmid ( mcf-7 vrl ) and mcf-7 cells 
 transformed with a plasmid containing the gene for procaspase-3 ( mcf-7 
 c-3 ) .
( c ) mcf-7 vrl and mcf-7 c-3 cells were treated with the indicated 
 concentrations of pac-1 and 1541b and apoptotic death was assessed 
 using flow cytometry with annexin v - fitc / propidium iodide staining . 

the dotted horizontal 
 lines represent the level of cell death expected from a mere additive 
 effect of pac-1 and 1541b for each drug combination .
( d ) western blot 
 of mcf-7 c-3 cells treated for 6 h with combinations of pac-1 ( 30 
 m ) and 1541b .
( e ) the combination of pac-1 and 1541b has an 
 antitumor effect in vivo .
el4 cells were injected subcutaneously into 
 c57bl/6 mice ( 10 million cells per mouse ) , the animals were split 
 into four groups and treated once - a - day for three days via ip injection 
 with vehicle ( 2-hydroxypropyl--cyclodextrin , hpcd ) , 
 1541b ( 17.5 mg / kg in hpcd ) , pac-1 ( 125 mg / kg in hpcd ) , 
 or 1541b + pac-1 ( 17.5 and 125 mg / kg , respectively , in hpcd ) . 

p - values are relative to vehicle control ; * , p < 
 0.005 ; * * , p < 5  10 .
to explore the therapeutic utility 
 of the dual procaspase-3 activation 
 strategy , the combination of pac-1 and 1541b was assessed in vivo . 

prior to beginning efficacy studies , the tolerability 
 and toxicity of 1541b as a single agent and in combination with pac-1 
 was determined ( supporting tables s1 , s2 , and 
 s3 ) . on the basis of these results , a maximum combination dosage 
 of 125 mg / kg of pac-1 followed by 17.5 mg / kg of 1541b was chosen for 
 the efficacy model .
the hematologic and nonhematologic toxicity of 
 this treatment of pac-1 and 1541b was determined following 3 consecutive 
 daily ip administrations of hpcd , pac-1 , 1541b , or combination 
 ( pac-1 + 1541b ) .
no clinically significant evidence for myelosuppression , 
 renal injury , or hepatic toxicity was identified in any of the treatment 
 cohorts ( supporting table s3 ) . for mice 
 receiving hpcd , pac-1 , or 1541b alone , all collected target 
 organs were devoid of histologic evidence for inflammation and necrosis . 
 in mice receiving combination
ip pac-1 and 1541b , pathologic changes 
 were noted in the lung parenchyma of 2 out of 3 mice .
these mice had 
 eosinophilic and histiocytic perivascular and peribronchiolar infiltrates , 
 which were mild to moderate in severity .
the clinical significance 
 of this finding is unclear , as mice in these groups were asymptomatic 
 and did not demonstrate weight loss compared to controls .
the 
 pharmacokinetic parameters of each compound were then determined ( supporting table s4 ) . following a single ip injection 
 at a dose of 125 mg / kg , pac-1 in hpcd
was rapidly absorbed 
 and achieved a maximal plasma concentration of 32720.0 ng / ml within 
 20 min postinjection .
pac-1 was distributed quickly throughout the 
 body with a mean distribution half - life of 20 min and a mean terminal 
 elimination half - life of 4.2 h. likewise , 1541b in hpcd was 
 absorbed and reached a maximal plasma concentration of 2699.9 ng / ml 
 within 20 min of intraperitoneal administration .
the distribution 
 half - life of 1541b was 20 min and the terminal elimination half - life 
 was 5.4 h. the el4 syngeneic murine lymphoma model was chosen 
 to explore the 
 therapeutic utility of pac-1 and 1541b combinations , as pac-1 and 
 1541b synergize to induce dramatic activation of procaspase-3 and 
 cell death versus this cell line ( supporting figures 
 s3c and s5d ) , and it is a challenging treatment model due to 
 its rapid doubling time ( less than 24 h ) and uniformly high tumor formation rate ( 100% ) when inoculating 
 greater than 1  10 cells subcutaneously in c57bl/6 
 mice .
pac-1 was chosen over s - pac-1 for 
 the combination studies as it has greater activity versus the el4 
 cell line in culture .
c57bl/6 mice implanted 
 with el4 cells were treated with pac-1 alone ( 125 mg / kg ) , 1541b alone 
 ( 17.5 mg / kg ) , or sequential treatments of pac-1 + 1541b ( 125 mg / kg 
 and 17.5 mg / kg , respectively ) once - a - day for three days , formulated 
 in hpcd .
once tumors in vehicle - treated mice had reached their 
 maximum allowed size of 1500 mm ( 8 days ) , all 
 mice were sacrificed , and tumors were excised and weighed . as shown 
 in figure 5e , 1541b treatment had no effect , 
 and pac-1 had a small but statistically significant effect on tumor 
 growth in this model .
while 
 there is clear benefit to anticancer strategies utilizing 
 combinations of drugs that act on different targets , the work described 
 herein demonstrates that dramatic synergy can be observed with compounds 
 that act through orthogonal mechanisms on the same biological target . 

this multitargeting approach may have particular advantages when activation 
 of an enzyme is sought .
pac-1 chelates the labile inhibitory zinc 
 from procaspase-3/caspase-3 , thus priming the enzyme for robust and 
 efficient activation by 1541b . in cell culture at concentrations where 
 neither compound significantly induces death at 612 h , dramatic 
 enhancement of cell death ( over the additive effect ) is observed with 
 the pac-1/1541b combination .
this cell death is tied to the ability 
 of the pac-1/1541b combination to induce a rapid conversion of procaspase-3 
 to caspase-3 , as shown by the western blots and the caspase-3 enzymatic 
 activity in cell lysates .
pac-1 is safe in mice and dogs , and a derivative of pac-1 , s - pac-1 , showed promising 
 activity in a phase i clinical trial of pet dogs with lymphoma , thus the observed synergy with 1541b could have 
 clinical significance .
pac-1 has been utilized as a tool for 
 studying direct procaspase-3 
 activation in various systems , and data presented 
 herein suggest that the pac-1/1541b combination will also be valuable 
 as a tool for rapidly activating procaspase-3 . as interest in activating 
 enzymes with small molecules is increasing rapidly and has considerable 
 medicinal potential
, our data suggest that targeting strategies using two small molecules 
 with different activation mechanisms could be a general approach for 
 dramatic enhancement of the intended biological effect .
procaspase-3 and 
 caspase-3 were expressed as described previously and purified with 
 qiagen nickel - nta resin . increasing concentrations 
 of pac-1 , 1541b , pac-1a and
1541d were assessed for their capacity 
 to enhance activity of 100 nm procaspase-3 in an inhibitory zinc - based 
 system ( 2 m znso4 , 50 mm hepes , 50 mm kcl , 1.5 mm 
 tcep , 0.1% chaps , ph 7.6 ) .
compounds were added to zinc - inhibited 
 pc-3 , incubated for 30 min , and activity was assessed by cleavage 
 of ac - devd - afc ( 50 m ) and was normalized to 100 nm procaspase-3 
 in zinc - free caspase activity buffer ( 50 mm hepes , 50 mm kcl , 1.5 
 mm tcep , 0.1% chaps , ph 7.6 ) .
1541b ( 20 and 25 m ) and 
 pac-1 ( 25 and 50 m ) were evaluated separately and in combination 
 for their capacity to activate 350 nm procaspase-3 over time in a 
 mildly inhibitory zinc - based system ( 750 nm znso4 , 50 mm 
 hepes , 50 mm kcl , 5 mm tcep , ph 7.6 , 0.1% tritonx-100 ) . at designated 
 time
points aliquots were assessed by cleavage of ac - devd - pna ( 200 
 m ) .
350 nm caspase-3 in a zinc - free caspase activity buffer 
 ( 50 mm hepes , 50 mm kcl , 5 mm tcep , ph 7.6 , 0.1% tritonx-100 ) was 
 used for 100% caspase-3 activity .
u-937 ( 50  000 
 cells per well ) , bt-549 ( 10  000 cells per well ) and a549 ( 10  000 
 cells per well ) were plated in 96-well plates , allowed to adhere overnight 
 ( bt-549 and a549 ) , and incubated with 1 m staurosporine ( sts ) , 
 1541b ( 7.5 , 10 , 12.5 , or 15 m ) , pac-1 ( 30 m ) , 1541b 
 ( 7.5 , 10 , 12.5 , or 15 m ) and pac-1 ( 30 m ) or dmso ( final 
 concentration 1% ) in phenol - red free rpmi media .
plates were assessed 
 for executioner caspase activity via addition of a 4 bifunctional 
 lysis activity buffer ( 200 mm hepes , 400 mm nacl , 40 mm dtt , 0.4 mm 
 edta , 1% tritonx-100 , 20 m ac - devd - afc ) .
activity is expressed as normalized to 
 minimal and maximal activity observed within the assay . for western 
 analysis one million suspension ( u-937 , hl-60 and el4 ) 
 or adherent cells at 75% confluency in 6-well plates
were treated 
 with increasing concentrations from 7.5 to 12.5 m 1541b in 
 the presence or absence of 30 m pac-1 for a duration reflective 
 of near maximal caspase activity as seen in the cell lysate experiment . 
 at the conclusion of treatment , the medium and trypsin - aided detached 
 cells were pelleted , lysed on ice in ripa buffer ( 50 mm tris base , 
 150 mm nacl , 1% tritonx-100 , 0.5% na - deoxycholate , 0.1% sds , ph7.4 , 
 with a 1:100 dilution of protease inhibitor cocktail set iii ) , and 
 clarified , and protein content was normalized by bca protein assay 
 reagent ( pierce ) .
samples were denatured , separated by sds - page ( 420% ) , 
 and transferred to a membrane for western blot analysis of pro- and 
 active caspase-3 ( cell signaling 9662 ) .
blots were stripped and reprobed 
 for -actin ( cell signaling 4970 ) as a loading control .
bt-549 cells at 75% confluency ( allowed 
 to adhere for > 24 h ) in 6-well plates were treated with 10 m 
 1541b and 30 m pac-1 , 10 m 1541b or 1 m sts for 
 up to 10 h. at each time point , medium and trypsin - aided detached 
 cells were pelleted , washed in pbs and suspended in cold digitonin 
 permeabilization buffer ( 75 mm nacl , 1 mm sodium phosphate monobasic , 
 8 mm sodium phosphate dibasic , 250 mm sucrose , 200 g / ml of 
 digitonin , protease cocktail inhibitor , ph 7.5 ) and placed on ice 
 for 5 min .
permeabilized cells were centrifuged ( 14  000 rcf , 
 5 min ) , and the supernatant ( cytosolic fraction ) was saved .
the pellet 
 ( mitochondrial fraction ) was washed with digitonin permeabilization 
 buffer , lysed in ripa buffer , and clarified .
samples were denatured , 
 separated by sds - page and transferred to a membrane for western blot 
 analysis probed for the presence of pro- and active caspase-3 ( cell 
 signaling 9662 ) , and cytochrome c ( cell signaling 4272 ) .
western blots 
 were stripped and reprobed for -actin ( cell signaling 4970 ) 
 and cox iv ( cell signaling 5247 ) as loading controls .
the induction of apoptosis was 
 measured by annexin v - fitc / propidium iodide staining and flow cytometry . 

either 1  000  000 suspension ( u-937 , hl-60 , el4 ) cells 
 or adherent cells at 75% confluency in 6-well plates were 
 treated with combinations of pac-1 and 1541b ( final concentration 
 of dmso
the entire contents of each well was then transferred 
 to flow cytometry tubes , pelleted , and suspended in 450 l of 
 annexin v - fitc binding buffer ( 10 mm hepes , 140 mm nacl , 2.5 mm cacl2 , 1% bsa , ph 7.4 ) , premixed with annexin v - fitc and propidium 
 iodide dyes . staining was assessed by flow cytometry ( 10  000 
 events per sample ) .
the annexin v - fitc ( )/propidium iodide 
 (  ) population was confirmed to be greater than 90% viable 
 and normalized as the live control .
the role of caspases in the induction 
 of apoptosis was assessed by cotreatment with 25 m q - vd - oph 
 ( calbiochem ) . all 
 experimental procedures 
 were reviewed and approved by the university of illinois institutional 
 animal care and use committee .
68 week old c57bl/6 mice were 
 used in all experiments ( charles river ) .
the maximum tolerated dose 
 ( mtd ) of 1541b was determined . beginning at 5 mg / kg , ( formulated at 
 0.5 mg / ml in hpcd , ph 5.5 ) mice ( groups of three )
mtd was 
 the highest dosage for which all mice survived and acceptable ( < 20% ) 
 weight loss was observed .
the mtds of pac-1 ( 200 mg / kg ) and 1541b 
 ( 20 mg / kg ) were used as the base unit for determining the combined 
 mtd . beginning with 1/10 of each mtd ( 20 mg / kg pac-1 + 2 mg / kg 1541b )
groups 
 of 3 mice were treated with 1/10 , 1/6 , 1/4 , 1/3 , 1/2 and 3/4 of each 
 single agent s mtd . given the tolerability of 1/2 and the toxicity 
 of 3/4 , an additional treatment of 125 mg / kg pac-1 + 17.5
ten week old , c57bl/6 female mice 
 ( n = 3/cohort ) were administered three consecutive 
 daily intraperitoneal injections of hydroxypropyl--cyclodextrin 
 ( vehicle ) , pac-1 ( 125 mg / kg ) , 1541b ( 17.5 mg / kg ) , or pac-1 + 1541b 
 ( 125 mg / kg + 17.5 mg / kg , respectively ) , and then humanely sacrificed 
 24 h later .
heparinized whole blood was collected for assessment of 
 total white blood cells , neutrophils , lymphocytes , hematocrit , platelets , 
 creatinine , blood urea nitrogen , albumin , alanine aminotransferase , 
 alkaline phosphatase , and total bilirubin .
mice were necropsied , and 
 heart , lung , kidney , liver , spleen , gastrointestinal tract and brain 
 were collected for histopathology .
tissue samples were fixed overnight 
 in 10% neutral buffered formalin , processed , and paraffin embedded 
 for histopathology using routine methods .
tissue blocks were sectioned 
 into 3 m tissue sections and stained with hematoxylin and eosin . 

all slides were systematically evaluated by a single board certified 
 veterinary pathologist ( lbb ) for evidence of acute or chronic inflammation 
 and toxicity .
all lesions were characterized , recorded , and scored 
 for severity ( minimal = 1 , mild = 2 , moderate = 3 , and severe = 4 ) .
ten week old , c57bl/6 female mice 
 were administered a single intraperitoneal dose of pac-1 at 125 mg / kg 
 or 1541b at 17.5 mg / kg , and sacrificed in cohorts of 3 at predetermined 
 time points ( 0 , 10 , 20 , 30 , 40 , 60 , 120 , 240 , 360 , 720 , and 1440 min ) . 

blood was collected and centrifuged , and plasma separated for quantification 
 of pac-1 or 1541b by hplc methods ( uiuc metabolomics center , urbana , 
 il ) .
pharmacokinetic analyses were performed , using a nonlinear regression 
 program ( winnonlin , version 5.1 , pharsight corporation , cary , nc ) .
ten million el4 cells were 
 prepared in hbss and injected subcutaneously on the right flank of 
 sedated ( ketamine / xylazine ) mice ( day 0 ) . by three hours postinjection 
 the injection bleb was no longer evident , and soft tumors appeared 
 the following morning .
mice were randomized into four treatment groups : 
 vehicle , 1541b alone , pac-1 alone , and 1541b + pac-1 .
compounds were formulated in hpcd ( 1541b 
 at 0.5 mg / ml at ph 5.5 and pac-1 at 12.5 mg / ml at ph 5.5 ) .
all mice 
 received two treatments , one hour apart : vehicle ( hpcd + hpcd ) , 
 1541b ( hpcd + 1541b ) , pac-1 ( pac-1 + hpcd ) and 1541b 
 + pac-1 ( pac-1 + 1541b ) .
after 8 days the largest tumors had achieved 
 maximal size ; mice were sacrificed , and tumors were excised and weighed .
 combination 
 anticancer therapy typically consists of drugs that 
 target different biochemical pathways or those that act on different 
 targets in the same pathway . here 
 we demonstrate a new concept in 
 combination therapy , that of enzyme activation with two compounds 
 that hit the same biological target , but through different mechanisms . 
 
 combinations of procaspase-3 activators pac-1 and 1541b show considerable 
 synergy in activating procaspase-3 in vitro , stimulate rapid and dramatic 
 maturation of procaspase-3 in multiple cancer cell lines , and powerfully 
 induce caspase - dependent apoptotic death to a degree well exceeding 
 the additive effect . 
 in addition , the combination of pac-1 and 1541b 
 effectively reduces tumor burden in a murine lymphoma model at dosages 
 for which the compounds alone have minimal or no effect . 
 these data 
 suggest the potential of pac-1/1541b combinations for the treatment 
 of cancer and , more broadly , demonstrate that differentially acting 
 enzyme activators can potently synergize to give a significantly heightened 
 biological effect . 
Introduction
Results
Discussion
Methods