austocystin d was tested for cytotoxicity after 72 h treatment in a panel of cell lines . as indicated in table 1 , austocystin d displayed potent cytotoxicity , with gi50 values below 10 nm for three cell lines , and marked selectivity , with greater than 10  000-fold selectivity between the most sensitive cell line ( mcf7 ) and the least sensitive cell line ( mes - sa ) . the selectivity of austocystin d was not easily explained by tissue origin , as demonstrated by a comparison of mcf7 and mcf10a breast cell lines , where a selectivity of greater than 1000-fold was observed for mcf7 cells ( figure 2a ) . furthermore , the selectivity and activity was unlike that of chemotherapeutic agents such as doxorubicin and etoposide , both 50-fold more toxic to mcf10a cells , or the structurally related aflatoxin b1 , which did not display selectivity or potency , with gi50 values in each cell line above 5 m ( figure 2 ) . the differential selectivity to that of doxorubicin and etoposide indicates that austocystin d may have the potential to overcome resistance to these agents , and the differential potency to aflatoxin b1 indicates that these structurally related natural products ( figure 1 ) may not share a related mechanism of action or cellular activity . austocystin d potency and selective cytotoxicity is distinct from doxorubicin , etoposide , and aflatoxin b1 . dose - dependent growth inhibition of tissue - matched transformed , mcf7 , and nontransformed , mcf10a , cells after 72 h treatment with ( a ) austocystin d , ( b ) doxorubicin , ( c ) etoposide , and ( d ) aflatoxin b1 . vertical bars represent sem , representative experiments shown . since austocystin d was previously shown to display selective cytotoxicity to cells overexpressing the abc transporter mdr1 , we investigated the possibility that austocystin d interacts directly with this transporter . mdr1 drug efflux activity is accompanied by atp hydrolysis and therefore can be monitored in vitro by measuring the production of inorganic phosphate from membranes containing mdr1.(6 ) similar to known mdr1 substrates , verapamil and vinblastine,(1 ) austocystin d stimulated atpase activity in this assay , suggesting that it is also likely to be an mdr1 substrate ( figure 3a ) . furthermore , cyclosporin a , a known inhibitor of the substrate - stimulated mdr1 atpase activity,(7 ) fully inhibited austocystin d - stimulated atpase activity . ( a ) effect of austocystin d ( ausd ) and mdr1 substrates verapamil ( ver ) and vinblastine ( vin ) on the atp hydrolysis by membranes containing mdr1 in the presence ( dashed lines ) or absence ( solid lines ) of 1 m mdr1 inhibitor cyclosporin a ( csa ) . austocystin d , verapamil , and vinblastine each activated mdr1 atp hydrolysis that was inhibitable by cyclosporin a. ( b ) effect of 10 m austocystin d , 40 m verapamil , and 10 m cyclosporin a on the retention of calcein in hct-15 cells . austocystin d behaved similarly to verapamil and cyclosporin a to inhibit calcein - am efflux and thereby increase intracellular calcein fluorescence relative to vehicle ( dmso)-treated cells . ( c , d ) effect of austocystin d and verapamil on ( c ) calcein - am efflux and ( d ) growth in hct-15 cells . vertical bars represent sem , representative experiment shown . to determine whether austocystin d also modulated drug efflux activity in cells , we measured the abundance of the fluorescent dye calcein in live cells.(8 ) in this assay , the nonfluorescent calcein acetoxymethyl ester ( calcein - am ) is added to cells and is hydrolyzed to calcein by cellular esterases . calcein - am is an mdr1 and mrp1 family substrate and thus is actively effluxed from cells expressing those transport proteins , resulting in low levels of retained fluorescence.(9 ) mdr1 and mrp1 family substrates or inhibitors prevent calcein - am efflux , leading to high levels of retained fluorescence . similar to mdr1 substrate verapamil and inhibitor cyclosporin a , austocystin d inhibited efflux of calcein - am , indicating that it can also modulate drug efflux activity in cells ( figure 3b ) . although austocystin d modulated abc transporter activity in vitro and in cells , this activity does not account for the cytotoxicity of austocystin d. verapamil and austocystin d both act as mdr1 substrates with similar cellular activity , yet verapamil is more than 1000-fold less cytotoxic than austocystin d ( figure 3c , d ) . furthermore , we evaluated a panel of cell lines for abc transporter activity in relation to austocystin d sensitivity . we measured calcein - am efflux as an indication of the presence of functional mdr1 or mrp1 family members and hoechst 33342 efflux as an indication of the presence of functional bcrp family members . as indicated in table 2 , sw620 cells lacked activity for known abc transporters yet were sensitive to austocystin d. taken together , the lack of correlation between verapamil and austocystin d cytotoxicity and abc transporter activity provides strong evidence that the cytotoxicity of austocystin d is not due to its interaction with abc transporters . < 50% of mcf7 cells displayed bcrp activity . because of its structural similarity to aflatoxin b1 , a cyp - activated mutagen,(11 ) we investigated whether the selective cytotoxic activity of austocystin d was due to cyp - activated dna damage . austocystin d and aflatoxin b1 share a vinyl ether moiety , critical in aflatoxin b1 for its biological activity ( figure 1 ) . this moiety in aflatoxin b1 undergoes epoxidation catalyzed by cyp enzymes , and the resulting epoxide forms dna adducts.(11 ) to detect cellular dna damage , we established an in - cell western assay measuring the phosphorylation levels of histone h2ax . histone h2ax is phosphorylated by the kinase atm at serine 139 in response to dna double strand breaks . this signal occurs in response to dna - damaging agents such as bleomycin and camptothecin and is a sensitive readout of dna damage response pathways.(14 ) in this assay , we observed that known dna damage agents camptothecin , doxorubicin , and bleomycin induced dose - dependent phosphorylation of histone h2ax after 4 h of drug treatment ( figure 4a , figure s1a , b ) . we subsequently determined that austocystin d induced the phosphorylation of histone h2ax in cell lines for which it was cytotoxic ( figure 4a , b ) . austocystin d caused a cell - line selective induction of the dna damage response that matched its cell - line selective cytotoxicity . ( a ) in - cell western showing the effect of austocystin d , doxorubicin , bleomycin , and dmso ( vehicle ) on levels of phosphorylated histone h2ax ( ph2ax ) after 4 h treatment of hct-15 cells . ( b ) in - cell western data showing the effect of austocystin d on ph2ax in sw620 , hct-15 , and hela cells following 1 h of treatment . vertical bars represent sem . to investigate the likelihood that austocystin d required cyp activation to induce the dna damage response and be cytotoxic , we treated cells with both ketoconazole , a cell - permeable inhibitor of cyp 3a4 activity,(5 ) and austocystin d. ketoconazole fully prevented austocystin d cytotoxicity and induction of the dna damage response , demonstrating that cyp activity is required for austocystin d s antiproliferative activity ( figure 5a , b ) . this effect was specific to austocystin d and did not result from general inhibition of the dna damage response ( figure 5c ) . inhibition of cyp or hydrogenation of austocystin d abrogates austocystin d dna damage and cytotoxicity . ( a ) inhibitory effect of the cyp inhibitor ketoconazole on hct-15 cell growth inhibition of 0.5 m ( light blue ) , 1 m ( blue ) , and 2 m ( dark blue ) austocystin d. ( b ) inhibitory effect of 10 m ketoconazole on austocystin d - induced phosphorylation of histone h2ax in hct-15 cells following 1 h ketoconazole pretreatment and 4 h austocystin d treatment . ( c ) specific inhibition of 10 m ketoconazole ( ket ) on 10 m austocystin d - induced phosphorylation of histone h2ax in sw620 cells in comparison to 20 m camptothecin . ( d , e ) comparison of austocystin d and dihydro - austocystin d for ( d ) growth inhibition after 72 h treatment of mcf7 cells and ( e ) induction of phosphorylation of histone h2ax after 4 h in mcf7 cells . the epoxide forms a direct covalent interaction with guanine nucleotides , causing dna damage.(11 ) we sought to probe the involvement of an austocystin d epoxide intermediate by hydrogenation of the vinyl ether , since the aflatoxin b1 epoxide intermediate is known to be short - lived under physiological conditions,(15 ) and we therefore expected the austocystin d epoxide to also be short - lived . we reduced the vinyl ether of austocystin d , yielding dihydro - austocystin d(16 ) ( 3 , figure 1 ) and observed that dihydro - austocystin d displayed only very weak cytotoxicity and induction of the dna damage response , further supporting the importance of metabolic activation for the mechanism of action of austocystin d ( figure 5d , e ) . to support the cellular data indicating that austocystin d induced dna damage following cyp activation , we applied an in vitro reconstituted dna damage assay . in this assay , supercoiled plasmid dna is incubated with compound in the presence or absence of human liver microsomes , a rich source of cyp enzymes , and an nadph regenerating system to generate insitu the nadph that is required for the cyp activity . the resulting nicked dna can be detected by agarose gel electrophoresis . in this assay , austocystin d caused direct dna damage at 100 nm , consistent with the compound s gi50 in cells ( figure 6 and table 1 ) . this in vitro dna damage activity required cyp activity , as increased nicked dna was observed only in the presence of both liver microsomes and the nadph regenerating system required for activity of cyp enzymes in vitro . ( a ) dna agarose gel showing the effect of 1 m austocystin d on converting supercoiled plasmid dna to nicked dna after treatment with different additives . ( b ) dna agarose gel showing the potency of austocystin d - induced dna nicking . austocystin d was originally discovered more than 30 years ago in an effort to identify and characterize secondary metabolites in maize meal cultures of aspergillus ustus ( bainier ) thom . and church.(16 ) more recently , it was reported to act as a selectively cytotoxic agent to certain cancer cell lines with an apparent selectivity toward cells expressing mdr1.(3 ) we sought to elucidate the mechanism of action of austocystin d to uncover a potentially useful approach to selectively target chemoresistant cancer cells that act by expression of abc transporters . our approach involved the investigation of two possible leads : that abc transporter activity might be required for austocystin d antiproliferative activity and that austocystin d might act by a mechanism similar to aflatoxin b1 , a well - characterized structurally related compound that is activated by cyp enzymes to induce dna damage . we began by confirming the cell - line selectivity of austocystin d and found it to be remarkably potent and selective , displaying over 1000-fold selectivity between sensitive and resistant cell lines . in comparison to the chemotherapeutic agents doxorubicin and etoposide , austocystin d displayed a reverse sensitivity pattern between the breast cancer cell line mcf7 and the proliferative nontransformed breast cell line mcf10a with the advantage of being more selective for the cancer cell line . austocystin d was also only weakly active against the normal human umbilical vein endothelial cell line ( huvec ) , suggesting the possibility of selectively targeting cancer cells , particularly in the setting of resistance to standard chemotherapy . further characterization of a subset of cell lines revealed that abc transporter activity did not correlate with austocystin d antiproliferative activity . for example , sw620 cells were highly sensitive to austocystin d with a gi50 of 27 nm but did not display abc transporter activity . to investigate the possibility that austocystin d induced dna damage in cells , we measured the induction of phosphorylation of histone h2ax , which occurs in response to dna damage . austocystin d induced histone h2ax phosphorylation , and this activity fully correlated with its cellular cytotoxicity . the histone h2ax phosphorylation and antiproliferative activity could be fully inhibited by treatment with ketoconazole , a cyp inhibitor with highest potency toward cyp 3a4 , or reduction of austocystin d to dihydro - austocystin d , a form that was not expected to undergo cyp - catalyzed epoxidation like aflatoxin b1.(11 ) therefore , a mechanism emerged in which austocystin d is activated by cyp enzymes to induce dna damage leading to cell death . further support for this mechanism of action came from in vitro experiments in which we directly observed dna damage in the presence of liver microsomes expected to contain cyp enzymes . while austocystin d and aflatoxin b1 share a similar mode of action in vitro and in the ames test,(4 ) namely , cyp - activated dna damage , the compounds differ dramatically in both potency and cell - line selectivity . in the comparison between mcf7 and mcf10a , aflatoxin b1 displayed little selectivity and poor potency , while austocystin d was highly selective and potent , being 1000-fold more active against mcf7 cells . although both compounds contain a vinyl ether moiety , the remainder of the structures may encode cyp enzyme specificity or sensitivity , or differences in dna recognition . alternatively , the reactivity of the vinyl ether or epoxide may vary between the compounds , leading to differences in rate of epoxide formation or covalent interaction with cellular dna . one possibility for the difference in activity between austocystin d and aflatoxin b1 is the absence of a tertiary alcohol in aflatoxin b1 adjacent to the site of epoxidation . this hydroxyl is present in aflatoxin m1 , a metabolite of aflatoxin b1.(19 ) neal et al . directly compared the cytotoxicity of aflatoxins b1 and m1 and reported differences ; the human b lymphoblastoid cell line mcl-5 that stably expresses cyps 1a2 , 2a6 , 3a4 , and 2e1 and microsomal epoxide hydrolase was sensitive only to aflatoxin b1 , while the untransfected line , chol , was sensitive only to aflatoxin m1.(20 ) a comparison of the biological activity of austocystin d to aflatoxin m1 will help reveal the importance of the tertiary alcohol to differences between austocystin d and aflatoxins . in addition , an investigation into cyp selectivity and hepatoxicity will help reveal distinctions between austocystin d and aflatoxins and the potential clinical utility of austocystin d. despite its initial discovery as an mdr+ cell selective agent , we have determined here that the unusual selectivity of austocystin d is mediated by cyp activation , rather than by a direct interaction with abc transporters . this unanticipated finding is likely explained by coordinate expression of cyps and abc transporters by the pregnane xenobiotic receptor ( pxr ) or the constitutive androstane receptor ( car ) nuclear hormone receptors . these receptors induce the expression of a coordinated cellular detoxification program in response to xenobiotic small molecules , and each is known to upregulate both abc transporters and cyp enzymes . thus , our findings support a model whereby transcriptional upregulation of abc transporters by pxr / car can lead to a multiple drug resistance phenotype and that this transcriptional program also induces tumoral expression of cyp enzymes . this model is also supported by the observations that pxr agonists lead to multiple drug resistance and that chemotherapeutics prone to multiple drug resistance induce pxr - mediated transcription . these findings suggest a context - dependent cytotoxicity strategy for the design of new therapeutics for multiple drug resistant cancers , where the co - regulation of mdr and cyp enzymes creates a vulnerability to cyp - activated prodrugs that are directly activated in the tumor tissue . previous cyp - activated anticancer agents , such as cyclophosphamide , have relied on metabolic activation of the drug in the liver . this is typically associated with systemic toxicity as the activated drug enters circulation.(25 ) in the present work , we show that austocystin d has significant cancer cell autonomous activity , suggesting that the molecule or derivatives could be suitably activated directly within tumors . due to the coordinate expression of cyps and abc transporters such as mdr1 , the natural product austocystin d or compounds with a similar mechanism of action have the potential to target multiple drug resistant tumors , and thus may address a significant clinical need . austocystin d was isolated as previously described,(27 ) and dihydro - austocystin d was synthesized as described in the supporting information and has previously been synthesized.(16 ) all other compounds were obtained from commercial suppliers . huvec cells were obtained from lonza , and other cell lines were obtained from atcc . cells were plated in 96-well plates at densities ranging from 2500 to 10  000 cells / well 24 h prior to the addition of compounds . compound stocks in 100% dmso were 3-fold serially diluted in 100% dmso in 96-well u - bottom plates to generate 1000 dose curves . these were subsequently diluted 1:100 in rpmi-1640 medium and then diluted 1:10 into the wells plated with cells for a final dmso concentration of 0.1% . the cell - titer blue viability assay ( promega ) gi50 values for compounds were determined relative to dmso control samples measured at 0 h ( 0% growth ) and 72 h ( 100% growth ) . drug - stimulated mdr1 atpase activity was determined by measuring inorganic phosphate release.(6 ) human mdr1 and control membranes ( bd biosciences ) were prepared in assay buffer ( 50 mm tris - mes ph 6.8 , 1 mm egta , 1 mm dithiothreitol , 25 mm kcl , 2.5 mm sodium azide ) with and without cyclosporin a ( 50 m final concentration ) . compounds were added to membranes and incubated for 10 min at 37 c , and the atpase reaction was initiated by the addition of atp . the final concentrations of membrane , atp , and dmso in the reactions were 200 g / ml , 4 mm , and 1% , respectively . the reactions were stopped by the addition of sds solution containing antifoam a ( final concentration of 3% ) after 0 and 60 min incubation periods . two equal volumes of 35 mm ammonium molybdate in 15 mm zinc acetate:/10% ascorbic acid ( 1:4 ) were subsequently added followed by a 20 min incubation at 37 c and measurement of absorbance at 800 nm . the released inorganic phosphate was determined by comparison to a phosphate standard curve at 800 nm . the mdr1-specific atpase activity was calculated by comparing atpase activity from mdr1 membranes to that of control membranes . mdr1/mrp cellular activity was determined by measuring calcein - am efflux.(28 ) cells were plated in triplicate wells at a density of 5  10 cells per six - well 2 days prior to the assay . for the assay , cells were treated with compounds for 60 min at 37 c followed by the addition of calcein - am ( 0.25 m ) . after 30 min at 37 c , the cells were washed twice with pbs , trypsinized , suspended in cold pbs , and analyzed with a facs calibur equipped with a 488 nm argon laser ( bectondickinson ) . in figure 3c , the mean fluorescence of each histogram was calculated for austocystin d and verapamil at the indicated doses in duplicate , and the average of two samples was plotted with graphpad prism5 , as above . dmso was set to 100% abc transporter activity , and 100 m verapamil was set as 0% abc transporter activity for normalization . bcrp cellular activity was determined by measuring hoechst 33342 efflux.(10 ) cells were trypsinized from semiconfluent 150 cm plates , resuspended at 10 cells / ml in media ( rpmi-1640 , 10% fbs ) , and incubated with 5 g / ml hoechst 33342 at 37 c . after 45 min , cells were washed once with cold hanks balanced saline solution supplemented with 1 mm hepes and 2% fcs ( hbss+ ) and resuspended in media ( rpmi-1640 , 10% fbs ) with 10 m fumitremorgin c at 37 c . after 20 min , cells were washed with cold hbss+ , resuspended in cold hbss+ containing 2 g / ml propidium iodide ( pi ) to exclude nonviable cells , and analyzed with a facs lsrii ( bd biosciences ) equipped with 360 nm uv and 488 nm argon lasers . cells were plated in 96-well black - walled plates at 10  000 cells / well 24 h prior to the addition of compounds . compound stocks in 100% dmso were 3-fold serially diluted in 100% dmso in 96-well u - bottom plates to generate 1000 dose curves . these were subsequently diluted 1:100 in rpmi-1640 medium and then diluted 1:10 into the wells plated with cells for a final dmso concentration of 0.1% . after 4 h at 37 c , cells were fixed in 4% paraformaldehyde for 20 min , washed briefly in 0.1% tween-20 , and permeabilized in 0.1% triton - x 100 in pbs . after 20 min , cells were incubated in blocking buffer ( licor biosciences , odyssey blocking buffer ) for 1 h and incubated overnight with antiphospho - h2ax antibody ( cell signaling technology , phospho - h2a.x ser139 ) at 1:400 in blocking buffer . the cells were subsequently washed and incubated with goat - anti - rabbit ir800cw ( licor biosciences ) at 1:800 in blocking buffer and draq5 ( biostatus limited ) at 1:10  000 . signals were detected with a licor odyssey infrared scanner ( licor biosciences ) at 700 and 800 nm . each well was normalized within the well by taking the ratio of 800 nm/700 nm . ratios of duplicate or triplicate wells were averaged and normalized to the in - plate controls 0.1% dmso ( 0% induction ) and 10 m etoposide ( 100% induction ) . , cells were pretreated with 10 m ketoconazole for 1 h prior to the 4 h treatment with austocystin d or camptothecin . supercoiled plenti6 plasmid dna ( 120 g / ml ) was incubated at 37 c with compound ( 100 nm , 500 nm , or 1 m ; 1% dmso final concentration ) in the absence or presence of human liver microsomes ( 0.25 mg / ml ; cellzdirect ) and an nadph regenerating system ( 1.5 mm nadp , 12.5 mm glucose-6-phosphate , 0.25 u / ml glucose-6-phosphate dehydrogenase , 100 mm phosphate ph 7.5 , 5 mm mgcl2 ) . after 4 h , the reaction was stopped by addition of an equal volume of phenol / chloroform ( 1:1 ) solution . after vigorous vortexing , the emulsion was separated by centrifugation , and the dna was recovered from the aqueous phase by using a gel extraction kit ( qiagen ) . the purified dna was then subjected to electrophoresis in tae buffer using a 0.8% agarose gel , and the bands were photographed using biorad gel doc .
the natural product austocystin d was identified as a potent cytotoxic agent with in vivo antitumor activity and selectivity for cells expressing the multidrug resistance transporter mdr1 . 
 we sought to elucidate the mechanism of austocystin d s selective cytotoxic activity . here 
 we show that the selective cytotoxic action of austocystin d arises from its selective activation by cytochrome p450 ( cyp ) enzymes in specific cancer cell lines , leading to induction of dna damage in cells and in vitro . 
 the potency and selectivity of austocystin d is lost upon inhibition of cyp activation and does not require mdr1 expression or activity . 
 furthermore , the pattern of cytotoxicity of austocystin d was distinct from doxorubicin and etoposide and unlike aflatoxin b1 , a compound that resembles austocystin d and is also activated by cyp enzymes to induce dna damage . 
 theses results suggest that austocystin d may be of clinical benefit for targeting or overcoming chemoresistance .