multidrug resistance ( mdr ) is one of the 
 main causes of the failure 
 of cancer chemotherapy .
one of the major mechanisms of drug resistance 
 is mediated by overexpression of p - glycoprotein ( p - gp , abcb1 , mdr1 ) .
p - gp is a membrane - bound protein that functions as a drug 
 efflux transporter .
this protein has two halves , each 
 containing six transmembrane domains and an atpase domain .
the drug - binding 
 site is the primary site to interact with its substrates , and its 
 complex structure allows structurally and functionally complex compounds 
 to interact with it .
drugs interact with 
 p - gp and stimulate atpase activity , which energizes drug extrusion 
 from cells , thereby reducing intracellular drug concentration . because of the wide range of compounds recognized by p - gp , mdr cells 
 expressing p - gp also exhibit cross - resistance to other drugs .
evidence 
 from in vivo and in vitro studies 
 indicates that p - gp plays an important role in drug pharmacokinetics , 
 making it a major target to circumvent drug resistance and increase 
 drug bioavailability . to overcome p - gp - mediated 
 drug resistance ,
most of the drugs were 
 developed as p - gp inhibitors to block the function of p - glycoprotein .
three generations of p - gp inhibitors have been 
 developed and have proved to be effective in vitro .
multiple reasons have 
 been suggested for these failures , including suboptimal clinical trial 
 design , heterogeneity of patient mdr phenotypes , unexpected drug toxicity , 
 poor pharmacokinetics of p - gp inhibitors , and the influence of endogenous 
 p - gp or other drug transporters .
therefore , 
 novel strategies to circumvent p - gp function in drug - resistant cancer 
 cells are actively under development .
we have focused on the effect 
 of collateral sensitivity ( cs ) , a phenomenon that occurs when cytotoxic 
 drugs are more sensitive to mdr cells than the drug - sensitive , parental 
 cells .
first described in the 1950s , cs has recently emerged as a 
 promising strategy to overcome mdr in cancer .
our laboratory identified a thiosemicarbazone 
 named nsc73306 that 
 is selectively toxic toward p - gp - expressing cells .
cytotoxicity studies suggest that the degree of sensitivity to nsc73306 
 is dependent upon the expression level of functional p - gp .
modulation of p - gp activity by p - gp inhibitors 
 or downregulation of p - gp reverses hypersensitivity to nsc73306 , suggesting 
 that the cytotoxic effect of nsc73306 is linked to p - gp function .
the molecular interactions of nsc73306 were 
 tested with other multidrug resistance related abc transporters , including 
 abcg2 .
nsc73306 does not specifically kill abcg2-expressing cells , 
 but is a potent modulator of abcg2 by virtue of its affinity as a 
 substrate for that efflux transporter . as nsc73306 can interact with other membrane proteins and 
 metals , we hypothesized that nsc73306 might be a 
 substrate of an unknown 
 solute carrier ( slc ) family uptake transporter . in this study , we 
 identified compounds that could inhibit cellular accumulation of [ h]nsc73306 . to achieve this goal , we established p - gp - expressing 
 stable cell lines in llc - pk1 porcine kidney cells .
we found that cisplatin , a substrate transported by copper 
 transporter 1 ( ctr1 , slc31a1 ) , is able to block nsc73306 
 accumulation .
nsc73306 was obtained 
 from the drug synthesis 
 and chemistry branch , developmental therapeutics program , division 
 of cancer treatment and diagnosis , national cancer institute , nih . 

[ h]nsc73306 ( specific activity : 40 ci / mmol ) was obtained 
 from american radiolabeled chemicals ( st .
bodipy - verapamil , 
 fumitremorgin c ( ftc ) ( inhibitor of abcg2/bcrp ) , and mk571 ( inhibitor 
 of multiple mrps ) were kindly provided by dr .
( 2r)-anti-5-[3-[4-(10,11-dichloromethanodibenzo - suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy ] 
 quinoline trihydrochloride ( dcpq ) was kindly provided by dr .
3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide ( mtt ) , 
 doxorubicin , verapamil , cisplatin , carboplatin , oxaliplatin , pyrilamine , 
 quinidine , copper chloride , triethylamine , cimetidine , trimethyoprim , 
 3-o - methylglucose , sodium azide ( nan3 ) , 
 and sodium fluoride ( naf ) , sodium deoxycholate , and dithiothreitol 
 ( dtt ) were purchased from sigma - aldrich ( st . louis , mo ) .
medium 199 , dulbecco s 
 modified eagle medium ( dmem ) , trypsin / edta solution , lipofectamine2000 , 
 sirna - oligofectamine , and geneticin were obtained from life technologies 
 ( carlsbad , ca ) .
monoclonal anti - gapdh and polyclonal anti - human ctr1 ( f-190 ) 
 antibodies were purchased from santa cruz ( santa cruz , ca ) .
monoclonal 
 mouse anti - p - gp c219 antibody was obtained from dako ( carpinteria , 
 ca ) .
the ctr1 sirnas and the ctrl_allstars_1 sirna were purchased 
 from qiagen ( valencia , ca ) .
llc - pk1 , hek-293 , and 
 cos7 cell lines 
 were obtained from the american type culture collection ( manassas , 
 va ) .
llc - pk1 parental cells were grown at 37 c in medium 199 
 with 4.5 g / l glucose ( gibco , carlsbad , ca ) supplemented with 3% ( v / v ) 
 fetal bovine serum ( biowhittaker , walkersville , md ) with l - glutamine , penicillin , and streptomycin .
the llc - mdr1-wt and llc - mdr1-eq 
 cell lines were maintained in the above medium plus 500 g / ml 
 geneticin ( invitrogen , carlsbad , ca ) .
the parental human epidermoid 
 carcinoma cell line kb-3 - 1 ( a hela subclone ) and two independent cisplatin - resistant 
 derivatives were studied : kb - cp.5 ( selected in a single step at 0.5 
 g of cisplatin / ml ) and kb - cp20 ( selected with stepwise increases 
 up to 20 g of cisplatin / ml ) .
the parental 
 liver carcinoma cell line bel-7404 and its cisplatin - resistant derivatives 
 bel-7404-cp1 ( selected in a single step at 1 g of cisplatin / ml ) 
 and bel-7404-cp20 ( selected with stepwise increases up to 20 g 
 of cisplatin / ml ) were studied . with the 
 exception of the llc - pk1 cell lines ,
all cell lines were grown at 
 37 c in 5% co2 , using dulbecco s modified 
 eagle medium ( dmem ) with 4.5 g / l glucose , supplemented with l - glutamine , penicillin , streptomycin , and 10% fetal bovine serum .
briefly , 2  10 cells were grown in each well of 
 a 24-well culture plate ( corning , corning , ny ) for 24 h. before initiation 
 of the assay , cells were washed with a transport buffer containing 
 125 mm nacl , 20 mm nahco3 , 3 mm kcl , 1.8 mm cacl2 , 1 mm kh2po4 , 1.2 mm mgso4 , 10 
 mm d - glucose , and 10 mm hepes ( ph 7.4 ) and preincubated in 
 the same buffer for 5 min .
subsequently , 200 l of transport 
 buffer containing [ h]nsc73306 ( 1 ci / ml ) and , where 
 relevant , other small molecules was simultaneously added . to stop 
 the experiment ,
drugs were removed and the cells were washed three 
 times with ice - cold pbs .
cells were dissolved in 100 l of 0.3 
 m naoh for 3 h following neutralization with an equal volume of 0.3 
 m hcl .
an aliquot of sample ( 100 l ) was taken and mixed with 
 scintillation cocktail ( 10 ml ) ( bio - safe ii ) , and the disintegrations 
 per minute were measured using a beckman coulter ls-6500 liquid scintillation 
 analyzer .
each assay was repeated at least 3 times . for calcium - dependent 
 transport assays ,
calcium was replaced with magnesium in the transport 
 buffer . for sodium - dependent transport assays , nacl or nahco3
transfection of pcmv6 or 
 pcmv6-hctr1-gfp vectors was performed by lipofectamine2000 transfection 
 reagent according to the manufacturer s instructions .
briefly , 
 dna and lipofectamine2000 were mixed in opti - mem medium at room temperature 
 for 30 min .
the mixture was added to the cells and incubated for 24 
 h. transfection efficiency was estimated by western blot analysis 
 using anti - ctr1 antibody .
four distinct small 
 interfering rnas ( sirnas ) were designed to target human ctr1/slc31a1 
 transcripts based on predictors of on- and off - target activity ( qiagen ) . 

the sense sequences of sirnas were as follows : hs_slc31a1_8 , cagcatgagctgctatagaat ; 
 hs_slc31a1_9 , agcctactggattgaggttaa ; 
 hs_slc31a1_10 , ccgagagagcctgctgcgtaa ; 
 hs_slc31a1_7 , atcgacttgcattcccactta . 

individual sirnas were reconstituted in nuclease - free water to achieve 
 a 10 m solution .
the ctrl_allstars_1 sirna ( 1 nmol ) negative 
 control was reconstituted to 10 m according to the manufacturer s 
 protocol .
briefly , prior to transfection , sirna - oligofectamine 
 ( invitrogen ) complexes were established according to the manufacturer s 
 protocol and then added to each well to achieve a final concentration 
 of 50 nm sirna and 0.5% oligofectamine .
after incubation at 37 c 
 in 5% co2 for 20 h , medium was removed from the cells and 
 replaced with assay medium .
cell survival was 
 measured by the mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium ) 
 assay following the published protocol .
briefly , cells were seeded on 96-well plates and allowed to incubate 
 for 24 h. drugs were then added to the cells and incubated for an 
 additional 72 h. the remaining viable cells converted mtt to a purple 
 formazan product , and after solubilization the absorbance at 570 nm 
 was recorded .
cytotoxicity ( ic50 ) was defined as the drug 
 concentration that reduced cell viability to 50% of the untreated 
 control .
the ic50 values were calculated by graphpad prism 
 5 software ( la jolla , ca ) .
cells grown 
 on tissue culture 
 plates were washed three times with ice - cold pbs and solubilized in 
 ice - cold extraction buffer ( 50 mm triscl ph7.5 , 150 mm naoh , 
 0.5% np-40 , 1 mm dtt , 0.25% sodium deoxycholate , 1 mm edta ph8 , 1 
 mm naf , 1 mm sodium vanadate , and 1 mm pmsf ) .
total protein was released 
 from the cells by repeated freeze  thawing and centrifugation 
 ( 16,000 rpm , 4 c , 5 min ) . in each sample ,
protein concentration 
 was estimated by the bradford method , and protein ( 25 g total ) 
 was denatured and allowed to separate in 10% sds  page .
the 
 acrylamide gel was transferred to a nitrocellulose membrane using 
 the iblot system ( invitrogen ) .
the membrane was first treated with 
 blocking solution for 4 h ( tbs - t supplemented with 5% nonfat dry milk ) 
 and then incubated with anti - ctr1 antibody ( 1:2,000 ) or anti - gapdh 
 antibody ( 1:25,000 ) in 1% tbs - t at 4 c overnight .
the following 
 day , the membrane was washed with tbs - t , and incubated with hrp - conjugated 
 igg secondary antibody ( 1:20,000 ) for 90 min .
statistical significance of the 
 experimental results was calculated by the two - sample t test using prism 5 ( graphpad , la jolla , ca ) .
to determine the rate of initial drug uptake , llc - pk1 
 ( p - gp nonexpressing ) and llc - mdr1-wt ( wild type p - gp expressing ) cells 
 were incubated with increasing concentrations of [ h]nsc73306 
 for 5 min
. the initial drug accumulation rate of llc - pk1 cells was 
 1.36  0.2 pmol mg min , and for p - gp - expressing llc - mdr1-wt cells it was 1.58  0.1 
 pmol mg min .
there was no 
 statistically significant difference in the initial drug accumulation 
 rates between these two cell lines ( figure s1 in the supporting information ) .
this lack of difference between p - gp - expressing 
 and parental cell lines was also confirmed in kb-3 - 1 and kb - v1 cells 
 ( figure 3d ) . as there was no significant difference in [ h]nsc73306 
 accumulation between llc - pk1 and llc - mdr1-wt cells , we screened for 
 small molecules that inhibit the uptake of nsc73306 into llc - pk1 cells . 

llc - pk1 cells were incubated with [ h]nsc73306 for 5 min 
 in the presence of various compounds ( figure 1 ) .
we found that cisplatin ( 100 m ) , cyclosporin a ( 1 mm ) , 
 and verapamil ( 1 mm ) significantly inhibited [ h]nsc73306 
 uptake ( figure 1a ) .
however , we found that 
 the p - gp inhibitors tariquidar ( 100 nm ) and dcpq ( 100 nm ) , the mrp 
 inhibitor mk571 ( 50 m ) , the bcrp inhibitor ftc ( 20 m ) , 
 and the slc uptake transporter inhibitors pyrilamine ( 1 mm ) , quinidine 
 ( 1 mm ) , tetraethylammonium ( tea ) ( 1 mm ) , cimetidine ( 1 mm ) , and trimethoprim 
 ( 1 mm ) had no effect on [ h]nsc73306 uptake ( figure 1a ) .
the p - gp substrates doxorubicin or paclitaxel 
 also did not inhibit [ h]nsc73306 uptake .
other than drug 
 transporter substrate / modulators , we tested whether [ h]nsc73306 
 uptake is dependent on sodium or calcium .
uptake studies were performed 
 in sodium - free or calcium - free transport buffers .
a significant drop in [ h]nsc73306 accumulation was observed 
 when  cold  nsc73306 was added , suggesting saturable 
 uptake of [ h]nsc73306 .
( a ) [ h]nsc73306 ( 25 pmol / ml ) 
 was incubated with llc - pk1 cells for 5 min following cell lysis and 
 scintillation counting .
the relative [ h]nsc73306 accumulation 
 after cells were treated with [ h]nsc73306 and dmso was 
 calculated .
( b ) [ h]nsc73306 accumulation in 
 llc - pk1 cells with various concentrations of verapamil ( up to 1 mm ) . 

( c ) [ h]nsc73306 accumulation in llc - pk1 cells treated 
 with various concentrations of cyclosporin a ( up to 1 mm ) .
( d ) time 
 course [ h]nsc73306 accumulation assay in the presence 
 of cyclosporin a ( 0.5 mm ) ( black line ) . for control experiments ,
( a ) determination of ic50 of cisplatin after [ h]nsc73306 accumulation in llc - pk1 cells and kb-3 - 1 cells .
cells 
 were treated with various concentrations of cisplatin for either 5 
 min ( llc - pk1 cells ) or 3 min ( kb-3 - 1 cells ) .
( b ) inhibition of accumulation 
 of [ h]nsc73306 in llc - pk1 and llc - pk1-mdr1 cells .
cells 
 were incubated with cisplatin ( gray bars ) or dmso ( black bars ) for 
 5 min , and the relative [ h]nsc73306 accumulation in the 
 dmso - treated llc - pk1 cells was calculated .
( c ) [ h]nsc73306 
 uptake can be inhibited by cisplatin but not carboplatin or oxaliplatin . 

cells were preincubated ( gray ) or coincubated ( black ) with [ h]nsc73306 and platinum drugs .
each point in the plot represents 
 an average cell number from 3 independent experiments .
cell lysates were extracted after transfection 
 for 24 h. the presence of endogenous ctr1 and ctr1-gfp proteins was 
 detected by immunoblotting using a polyclonal anti - ctr1 antibody . 

accumulation of drug in control cells and ctr1-overexpressing 
 cells was estimated . at each time point , the drug accumulation relative 
 to time 0 was determined .
( c ) knockdown of ctr1 in kb-3 - 1 and kb - v1 
 cells .
immunoblot by ctr1 antibody showing the presence of ctr1 in 
 mock - transfected kb-3 - 1 and kb - v1 and ctr1 sirna - transfected kb-3 - 1 
 and kb - v1 cells
. results of the same immunoblot with low exposure 
 and high exposure are shown .
( d ) drug accumulation assay showing 
 [ h]nsc73306 accumulation in kb-3 - 1 and kb - v1 with ctr1 
 knockdown cells .
the cells were incubated with [ h]nsc73306 
 for 3 min , and the relative drug accumulation values with mock - transfected 
 cells were determined .
results are mean  sd of 3 independent 
 experiments . to characterize further 
 the inhibitory effect of cyclosporin a , 
 verapamil , and cisplatin , the half maximal inhibitory concentrations 
 ( ic50s ) of [ h]nsc73306 cell uptake were determined . 

the ic50 of verapamil against [ h]nsc73306 uptake 
 was 0.7  0.1 mm ( figure 1b ) , and that 
 of cyclosporin a was 0.5  0.05 mm ( figure 1c ) .
the time course of [ h]nsc73306 uptake revealed that , 
 in the presence of cyclosporin a ( 0.5 mm ) , [ h]nsc73306 
 uptake was lower at all time points ( compared with dmso control ) and 
 the maximum reduction in [ h]nsc73306 uptake was 62% ( figure 1d ) .
there were no significant differences between 
 inhibitor - treated llc - pk1 and llc - mdr1-wt cells ( not shown ) .
these 
 results showed that the ic50 values of verapamil and cyclosporin 
 a are much higher than the amount required to inhibit p - gp function 
 ( 20 m ) , suggesting that inhibition of nsc73306 uptake 
 by these compounds may involve other mechanisms that are yet to be 
 identified .
cisplatin inhibited [ h]nsc73306 uptake 
 in a concentration - dependent 
 manner ( figure 2a ) .
the ic50 of 
 cisplatin to inhibit [ h]nsc73306 uptake was 77  
 2 m for llc - pk1 cells and 128  40 m for kb-3 - 1 
 cells , suggesting that this inhibitory effect is not a cell - specific 
 effect ( figure 2a ) .
uptake of [ h]nsc73306 in llc - pk1 and llc - mdr1-wt cells in the presence and absence 
 of cisplatin were evaluated .
coincubation of 100 m cisplatin 
 and [ h]nsc73306 equally reduced [ h]nsc73306 
 uptake in both cell lines ( figure 2b ) .
in addition 
 to cisplatin , a pt - containing anticancer agent , we sought to explore 
 whether other pt - based compounds could share similar inhibitory effects . 
 both 1 mm carboplatin and 250 m oxaliplatin had no statistically 
 significant effect on [ h]nsc73306 uptake ( figure 2c ) , suggesting that the inhibitory effect of [ h]nsc73306 on platinum drug uptake is dependent on the nature 
 of the platinum compound . also , preincubation of cells with cisplatin , 
 carboplatin , and oxaliplatin did not affect the ic50 ( figure 2c ) .
the incomplete inhibition of uptake is consistent 
 with a transporter - facilitated component of uptake , and a passive 
 component . since
[ h]nsc73306 uptake and function could be modulated 
 by cisplatin , we 
 decided to investigate whether these two compounds share a common 
 uptake pathway .
given cisplatin s association with ctr1-mediated 
 uptake , we hypothesized that ctr1 might play a role and therefore examined 
 the effect of this cu transporter on the cellular accumulation of 
 nsc73306 .
cos7 african green monkey kidney cells were used 
 for this experiment because they have low endogenous ctr1 expression .
cells were transfected with gfp - tagged human 
 ctr1 cdna , resulting in expression of recombinant protein ( figure 3a ) . in cells overexpressing ctr1 , higher [
h]nsc73306 uptake was observed ( figure 3b ) . 
 at 45 min , [ h]nsc73306 uptake was 36% higher in ctr1-overexpressing 
 cells ( figure 3b ) . in another experiment , we 
 examined drug accumulation in cells treated with ctr1 sirna .
we used 
 kb-3 - 1 ( p - gp negative ) and kb - v1 ( p - gp positive ) human adenocarcinoma 
 cell lines because they express human ctr1 protein ( vide infra , figure 7a ) .
western blot analysis confirmed 
 that transfection of ctr1 sirna reduced ctr1 levels in kb-3 - 1 and 
 kb - v1 cells ( figure 3c ) .
drug uptake experiments 
 showed that ctr1 sirna treatment of kb-3 - 1 and kb - v1 cells reduced 
 [ h]nsc73306 accumulation by approximately the same amount 
 ( figure 3d ) .
we further wanted to determine 
 if cisplatin - resistant cell lines showed cross - resistance to nsc73306 , 
 as cisplatin resistance is associated with a decrease in ctr1 protein 
 in small cell lung cancer cells ( sclcs ) .
western blot analysis showed that the kb - cp20 cell line , which is 
 highly resistant to cisplatin , expresses more ctr1 protein than the 
 kb-3 - 1 cells ( figure 4a , left ) .
however , cisplatin - sensitive 
 kb-3 - 1 cells express amounts of ctr1 comparable to cisplatin - resistant 
 kb - cp.5 cells . in the liver cancer cell line bel-7404-cp20 , which 
 is highly resistant to cisplatin , ctr1 overexpression was also observed . 

ctr1 was not detected in the parental cell line , bel-7404 , or in its 
 subline bel-7404-cp1 , with lower - level cisplatin resistance ( figure 4a , right ) .
( a ) immunoblot 
 showing the level of ctr1 protein in kb-3 - 1 , kb - cp.5 , kb - cp20 , bel-7404 , 
 bel-7404-cp1 , and bel-7404-cp20 cells
( b ) [ h]nsc73306 accumulation 
 in kb-3 - 1 and kb - cp20 cells .
( c ) [ h]nsc73306 accumulation 
 in kb-3 - 1 , kb - cp20 , and kb - cp20 cells treated with scrambled sirna 
 or ctr1 sirna .
( d ) sensitivity of kb-3 - 1 , kb - cp.5 , and kb - cp20 cells 
 to nsc73306 .
cells were incubated with increasing concentrations of 
 nsc73306 for 72 h at 37 c .
the function of ctr1 with respect to nsc73306 in cisplatin - resistant 
 cells was examined by drug uptake assays .
kb - cp20 cells showed a 2.1-fold 
 increase in [ h]nsc73306 uptake relative to the parent 
 line kb-3 - 1 ( figure 4b ) . to confirm that the 
 increase in drug uptake was caused by upregulation of ctr1
, we also 
 determined the uptake of [ h]nsc73306 in kb - cp20 cells 
 transfected with ctr1 sirna .
in ctr1 knockdown kb - cp20 cells , a significant 
 reduction of nsc73306 uptake was observed ( figure 4c ) .
ctr1 knockdown cells showed drug accumulation comparable 
 to that of kb-3 - 1 cells .
control kb - cp20 cells transfected with scrambled 
 sirna also had decreased drug accumulation , suggesting that some of 
 the reduction in nsc73306 uptake may be caused by the sirna transfection 
 method .
since ctr1 levels are upregulated in kb and bel-7404 cisplatin - resistant 
 cells , we examined whether the cellular toxicity of nsc73306 could 
 be enhanced by the high levels of ctr1 in cisplatin - resistant cells . 

as noted earlier , cytotoxicity assays showed that the nsc73306 ic50 values of the kb-3 - 1 , kb - cp.5 , and kb - cp20 cells were not 
 significantly different , suggesting that the cytotoxic effect of nsc73306 
 in cisplatin - resistant cells is not dependent on total intracellular 
 nsc73306 levels ( figure 4d ) .
the correlation of p - gp expression and function with 
 nsc73306 cytotoxicity in llc - pk1 cells was assessed , using a wild - type 
 p - gp - expressing stable cell line ( llc - mdr1-wt ) and a functional mutant 
 p - gp - expressing stable cell line ( llc - mdr1-eq ) .
the ic50 values of nsc73306 to llc - pk1 , llc - mdr1-wt , 
 and llc - mdr1-eq cells were 4.7  2.0 m , 0.9  0.2 
 m , and 4.5  1.0 m , respectively ( figure 5a ) .
the selectivity ratio ( sr ) of nsc73306 killing 
 llc - mdr1-wt cells over parental llc - pk1 cells was 5.4-fold ( figure 5a ) , which was comparable to other cell line pairs 
 that we reported .
sensitivity of nsc73306 
 requires functional p - gp and loss of nsc73306 
 sensitivity in llc - mdr1-wt cells upon cisplatin treatment .
llc - pk1 cells 
 were treated with increasing concentrations of nsc73306 in 37 c 
 for 72 h following mtt assay .
( b ) the llc - pk1 and llc - mdr1-wt cells were treated 
 with various concentrations of nsc73306 .
llc - mdr1-wt cells were treated 
 with 0 , 5 , 10 , or 40 m of cisplatin .
each point in the plot represents 
 an average cell number from 3 independent experiments .
we further examined whether cisplatin could influence 
 the hypertoxicity 
 of nsc73306 in cells expressing p - gp .
llc - mdr1-wt cells were treated 
 with increasing concentrations ( 5 m , 10 m , and 40 m ) 
 of cisplatin for 72 h. as expected , in the absence of cisplatin , llc - mdr1-wt 
 cells showed 4-fold lower ic50 values for nsc73306 
 than the llc - pk1 cells ( figure 5b ) .
however , 
 coincubation of cisplatin in llc - mdr1-wt cells reversed their hypersensitivity 
 to nsc73306 in a dose - dependent manner . in the presence of 40 m 

cisplatin , the ic50 of llc - mdr1-wt cells was 76% 
 that of llc - pk1 cells ( figure 5b ) . to determine 
 if ctr1 could be regulated by nsc73306 , we analyzed 
 ctr1 levels after drug treatment .
hek293 human embryonic kidney cells , 
 known to express ctr1 , were treated with 
 increasing concentrations of nsc73306 .
as negative feedback mechanisms 
 of ctr1 in response to cu and cisplatin have been reported , we sought to explore whether nsc73306 could 
 also reduce ctr1 expression .
as control experiments , cells exposed 
 to cisplatin ( cddp ) for 1 h or copper ( cucl2 ) for 3 h produced 
 lower ctr1 levels , as expected ( figure 6a ) . 

after 24 h , treatment with nsc73306 reduced the ctr1 level in a dose - dependent 
 manner ( figure 6b ) .
as gapdh levels were slightly 
 affected by nsc73306 , the relative ctr1 level was calculated by densitometry 
 analysis .
reduction in ctr1 levels was observed at even the lowest 
 nsc73306 concentration tested ( 1 m ) . in cells treated with 
 10 m nsc73306 ,
the ctr1 level was reduced by 63% , which was 
 comparable to the effect of cisplatin ( 60% reduction ) .
ctr1 
 levels in cells treated with nsc73306 for 3 h were not affected ( figure 
 s2 in the supporting information ) .
( a ) hek293 cells were treated with different concentrations of nsc73306 
 at 37 c for 24 h following cell lysis and immunoblotting . as 
 a positive control ,
cells were treated with cisplatin ( 5 m ) 
 and cucl2 ( 100 m ) for 3 h. the presence of ctr1 
 was detected by polyclonal anti - ctr1 antibody .
the same blot was labeled 
 with gapdh antibody , serving as a loading control .
( b ) the ctr1 level 
 in each sample was analyzed by imagej software and displayed as a 
 bar chart .
change in ctr1 level relative to control was determined 
 by densitometry analysis . statistical significance between nsc73306-treated 
 cells and the control cells
sd 
 of 3 independent experiments . to 
 examine whether overexpression of p - gp modulates ctr1 levels , 
 we tested for the presence of ctr1 in drug - resistant cell lines and 
 recombinant cell lines .
we found that ctr1 protein levels were not 
 significantly different between kb-3 - 1 and kb - v1 cells , the latter 
 expressing a high level of p - gp ( figure 7a ) . in llc - pk1 , llc - mdr1-wt , and llc - mdr1-eq cells , 
 ctr1 levels were also unaltered ( figure 7b ) . 

conversely , p - gp levels did not change in kb - v1 cells overexpressing 
 ctr1-gfp , as determined by western blot analysis with anti - p - gp monoclonal 
 antibody ( c219 ) ( figure 7c ) .
immunoblots showing ctr1 , p - gp , 
 and gapdh levels in ( a ) kb-3 - 1 and kb - v1 cells and ( b ) ctr1 and gapdh 
 levels in llc - pk1 , llc - mdr1-wt , and llc - mdr1-eq cells .
( c ) transient 
 overexpression of the ctr1 gene does not change p - gp level .
protein 
 lysate from p - gp negative kb-3 - 1 cells , p - gp positive kb - v1 cells , 
 and kb - v1 cells overexpressing ctr1-gfp protein were analyzed in an 
 immunoblot labeled with anti - p - gp antibody ( c219 ) . since cellular ctr1 levels 
 affect intracellular levels of nsc73306 and vice versa , we examined 
 whether the cytotoxicity of nsc73306 could be affected by ctr1 levels . 

transfection of kb-3 - 1 
 and kb - v1 cells with ctr1 sirna resulted in a 25% reduction of ctr1 
 protein .
scrambled sirna and ctr1 sirna - transfected cells were incubated 
 with nsc73306 for 72 h , and then ic50 values were determined 
 by mtt assay . as expected , mock control and control scrambled sirna - transfected 
 kb - v1 cells showed higher sensitivity to nsc73306 than similarly treated 
 kb-3 - 1 cells .
ctr1-sirna treatment only slightly decreased ic50 values in both kb-3 - 1 ( from 10.9  0.04 m to 
 7.9  0.02 m ) and kb - v1 cells ( from 3.3  0.04 m 
 to 1.7  0.04 m ) ( table 1 ) .
this 
 slight decrease might be due to the toxicity mediated by sirna transfection . 
 in another experiment , we sought to compare the effect of nsc73306 
 in kb-3 - 1 and hek293 cells overexpressing ctr1 .
we used hek293 cells 
 that express low levels of p - gp instead of kb - v1 cells , because overexpression 
 of ctr1 in kb - v1 cells resulted in cell death ( not shown ) . compared 
 with the kb-3 - 1 cells , hek293 cells showed higher sensitivity to nsc73306 
 ( ic50 10.9  0.04 m vs 4.42  0.03 m ) , 
 possibly due to endogenous p - gp expression .
however , when compared 
 with the mock and ctr1-gfp transfected hek293 cells , they showed similar 
 cytotoxicity .
in addition , the ic50s were evaluated for 
 cells incubated with nsc73306 for an extended period of time and we 
 found no significant differences .
similarly , nsc73306 cytotoxicity 
 was not significantly different in mock - transfected kb-3 - 1 cells and 
 ctr1-gfp - transfected kb-3 - 1 cells ( table 1 ) . 

these results indicate that while ctr1 levels affect uptake of nsc73306 
 ( figure 3 ) , in the absence of p - gp , they do 
 not correlate with toxicity of nsc73306 .
cells were incubated with increasing concentrations 
 of nsc73306 at 37 c for 72 h. ic50 values were determined 
 by survival curves obtained using mtt assays .
in 
 this work , a search was conducted for molecules that could inhibit 
 cellular uptake of nsc73306 , a compound that specifically kills p - gp - expressing 
 cells . at physiological ph , nsc73306 is neutral , lipophilic ( clogp 
 = 2.02 ) and likely capable of passive diffusion .
we found that its 
 uptake in the nm range is saturable and inhibited by cisplatin , verapamil , 
 and cyclosporin a. therefore , we hypothesized that nsc73306 could 
 be transported by the cu and cisplatin uptake transporter ctr1 ( verapamil 
 and cyclosporin a are not known to be substrates of ctr1 ) , and we 
 show here that cellular uptake of the drug is modulated by levels 
 of ctr1 .
ctr1 is a cu uptake transporter that mediates cu homeostasis 
 and 
 is expressed in many human tissues .
it is essential for embryonic development , and knockout 
 of both alleles of ctr1 in the mouse produces an embryonic lethal 
 phenotype .
the functional unit of ctr1 is 
 a homotrimeric complex with each monomer containing 190 amino acid 
 residues , spanning the cell membrane .
electron crystallographic 
 analysis of human ctr1 shows that the complex forms a cone - shaped 
 structure with a narrower extracellular pore ( 8  ) and 
 a wider intracellular pore ( 22  ) .
human ctr1 has also been found to transport silver and regulates platinum ( pt ) drug uptake ( reviewed 
 by liu et al . ) . as such , ctr1 influences 
 the cytotoxicity of pt - based drugs including cisplatin , carboplatin , 
 and oxaliplatin .
yeast and mammalian 
 cells that do not express ctr1 are more resistant to cu and pt drugs , 
 whereas overexpression of ctr1 increases drug uptake and increases 
 drug sensitivity .
exposure of 
 cells to cisplatin causes a rapid reduction in ctr1 protein , and this 
 can be triggered by a low concentration of the drug .
although it is known that ctr1 mediates cisplatin uptake , 
 the mechanism of pt translocation into cells by ctr1 remains to be 
 defined .
our results indicate that [ h]nsc73306 uptake 
 is inhibited 
 by cisplatin and by cold nsc73306 in the micromolar range ( 77  
 2 m and 7.1  1 m ) . however , to inhibit [ h]nsc73306 uptake , 10-fold higher concentrations of cisplatin 
 were required as compared to cold nsc73306 , suggesting that the drug 
 uptake pathway of nsc73306 may partially overlap with the uptake pathway 
 of cisplatin .
also , [ h]nsc73306 uptake can be inhibited 
 by cisplatin but not by other pt drugs such as carboplatin and oxaliplatin . 

others have reported that ctr1 is able to transport cisplatin , carboplatin , 
 and oxaliplatin , although the kinetics of these compounds are different .
there is still no consensus on exactly how pt drugs or cu enter 
 the cell via ctr1 .
several reports have strongly suggested that the 
 coordination of cisplatin with ctr1 is very different from its coordination 
 with cu .
human ctr1 
 includes a long stretch of extracellular domain rich in methionine 
 and histidine .
mutagenesis and truncation studies have revealed that 
 several amino acid residues critical to regulation of cu uptake do 
 not affect pt drug transport . because of this , alternative modes of 
 pt drug transport by ctr1 have been proposed .
proposed that the methionine residues of the extracellular domain 
 of ctr1 cooperatively assist pt drug uptake .
the results of our drug inhibition experiments suggest that the 
 transport of nsc73306 via ctr1 might be different from pt or cu transport 
 mechanisms .
also , uptake studies and cytotoxicity studies of copper 
 and pt drugs are not included .
further analysis will be needed to 
 determine the functional effects of ctr1 transport of nsc73306 and 
 pt drugs or cu , and also the critical amino acids / motifs required 
 for nsc73306 transport .
ctr1 has been known to regulate itself 
 post - translationally in 
 response to a sudden surge of substrates .
our experiments confirmed that nsc73306 could reduce ctr1 expression 
 in a dose - dependent manner .
time - course experiments showed 
 that the effect of nsc73306 is slower than that of cisplatin or cu . 

however , the molecular mechanism of this downregulation by nsc73306 
 is not clear . in mammalian cells , cisplatin- and cu - induced downregulation 
 of ctr1
involves non - clathrin - mediated endocytosis followed by formation 
 of ubiquitinated intermediates and degradation in the 26s proteasome . in yeast , the copper ion - sensing transcription factor mac1p post - translationally 
 controls the degradation of yeast ctr1 , ctr1p . however , our findings suggest that ctr1-mediated nsc73306 
 uptake in mammalian cells is regulated by a post - translational mechanism 
 involving drug - stimulated degradation of the transporter .
this 
 study confirms that nsc73306 requires the presence of functional 
 p - gp in order to increase cytotoxicity to the cell .
our results demonstrate 
 that there is no direct relationship between ctr1 expression and p - gp 
 expression . in the llc - mdr1-wt cells ,
no statistical significance 
 was found when we compared [ h]nsc73306 uptake in drug - sensitive 
 kb-3 - 1 and llc - pk1 and drug - resistant kb - v1 and llc - mdr1-wt cell lines . 

these results indicate that [ h]nsc73306 import is independent 
 of p - gp , a drug efflux transporter , and that the intracellular level 
 of nsc73306 does not correlate with cytotoxicity .
this can be understood 
 if one considers that the [ h]nsc73306 uptake assay is 
 done using low nm concentrations of nsc73306 , while its toxicity occurs 
 in the low m range ( a difference of 3 orders of magnitude ) , 
 where uptake is likely to be diffusion - limited and not carrier dependent . 

this is consistent with a previous publication showing that nsc73306 
 interacts with the major abc multidrug transporter abcg2 .
although nsc73306 alone can not sensitize abcg2-expressing 
 cells , it can reduce abcg2-mediated drug transport of other known 
 abcg2 substrates including mitoxantrone . in the current study
, we found that a slc transporter such as ctr1 
 could influence cellular drug uptake of nsc73306 .
the concentration 
 of nsc73306 in the cell lines we tested was affected by the presence 
 of cisplatin or a change in ctr1 protein level . however , in the absence 
 of p - gp , ctr1 itself could not change cellular sensitivity to nsc73306 . 

together with abcg2 , ctr1 is involved in the drug transport pathway , 
 but not the cell killing pathway of the drug nsc73306 . in this 
 study ,
elevated ctr1 levels were found in two cell lines 
 highly resistant to cisplatin , kb - cp20 ( cervical adenocarcinoma , a 
 hela subclone ) and bel-7404-cp20 ( hepatoma ) cells .
these cells have 
 different cellular origins , suggesting that the effect is not cell - type 
 specific .
however , we did not observe changes in ctr1 expression in 
 cell lines only moderately resistant to cisplatin , which is in agreement 
 with a previous study .
our results indicate 
 that expression of ctr1 in cells shows three responses to cisplatin . 

short - term drug exposure results in decreased ctr1 levels resulting 
 from rapid degradation . but ctr1 levels do not vary after long - term 
 drug exposure and selection of cisplatin - resistant cells compared 
 with control parental cells .
western blot analysis 
 confirmed that ctr1 levels in low - level cisplatin - resistant cells 
 were very similar to those of the parental cells , suggesting that 
 inhibition of drug uptake by shutting down drug transporters is replaced 
 by another signaling mechanism , perhaps because a supply of cu is 
 essential .
the exact mechanism that leads to higher ctr1 levels in 
 highly cisplatin - resistant cells is not clear , but may be the result 
 of mislocalization or a defect in post - translational modification 
 that allows cells with such high levels of resistance to survive in 
 cisplatin .
we have shown that these cells 
 accumulate less pt than parent cells , consistent with the expected 
 resistance phenotype , but not consistent with the expected facilitation 
 of pt uptake by ctr1 .
we can not rule 
 out the possibility that the ctr1 protein found in kb - cp20 and bel-7404-cp20 
 cells is folded differently , so as to change its function , perhaps 
 as a result of altered post - translational modification . while ctr1 levels were significantly increased in two highly cisplatin - resistant 
 cell lines ( kb - cp20 and bel-7404-cp20 ) , mtt assays showed that the 
 kb - cp20 cells were not sensitized to nsc73306 , in keeping with our 
 other results showing no correlation between ctr1 levels and nsc73306 
 cytotoxicity . in summary
, this study demonstrates that uptake 
 of the thiosemicarbazone 
 nsc73306 is affected by levels of ctr1 , an slc transporter .
our data 
 strongly suggest that , at nm concentrations , nsc73306 transport is 
 dependent on ctr1 level and can be inhibited by the ctr1 substrate 
 cisplatin .
the connection between p - gp , ctr1 , and nsc73306 provides 
 important clues to the mechanism of p - gp sensitizing drugs that confer 
 collateral sensitivity .
 acquired 
 drug resistance in cancer continues to be a challenge 
 in cancer therapy , in part due to overexpression of the drug efflux 
 transporter p - glycoprotein ( p - gp , mdr1 , abcb1 ) . 
 nsc73306 
 is a thiosemicarbazone compound that displays greater toxicity against 
 cells expressing functional p - gp than against other cells . here 
 , we 
 investigate the cellular uptake of nsc73306 , and examine its interaction 
 with p - gp and copper transporter 1 ( ctr1 , slc31a1 ) . 
 overexpression of p - gp sensitizes llc - pk1 cells to nsc73306 . 
 cisplatin 
 ( ic50 = 77 m ) , cyclosporin a ( ic50 = 
 500 m ) , and verapamil ( ic50 = 700 m ) inhibited 
 cellular accumulation of [ 3h]nsc73306 . cellular hypertoxicity 
 of nsc73306 to p - gp - expressing cells was inhibited by cisplatin in 
 a dose - dependent manner . 
 cells transiently expressing the cisplatin 
 uptake transporter ctr1 ( slc31a1 ) showed increased 
 [ 3h]nsc73306 accumulation . 
 in contrast , ctr1 knockdown 
 decreased [ 3h]nsc73306 accumulation . 
 the presence of nsc73306 
 reduced ctr1 levels , similar to the negative feedback of ctr1 levels 
 by copper or cisplatin . 
 surprisingly , although cisplatin is a substrate 
 of ctr1 , we found that ctr1 protein was overexpressed in high - level 
 cisplatin - resistant kb - cp20 and bel7404-cp20 cell lines . 
 we confirmed 
 that the ctr1 protein was functional , as uptake of nsc73306 was increased 
 in kb - cp20 cells compared to their drug - sensitive parental cells , 
 and downregulation of ctr1 in kb - cp20 cells reduced [ 3h]nsc73306 
 accumulation . 
 these results suggest that nsc73306 is a transport substrate 
 of ctr1 . 
Introduction
Experimental Section
Results
Discussion