multidrug resistance ( mdr ) remains the major obstacle to success in a wide variety of advanced malignancies , particularly in solid cancers .
the overexpression of p - gp is one of the important mechanisms involved in mdr [ 13 ] .
p - gp - mediated cellular uptake and the efflux of anticancer drugs from cancer cells are responsible for the cell elimination of the drugs which in turn decreases their therapeutic effect .
therefore , one of the best methods of reversing mdr and increasing the efficacy of anticancer drugs is to use the potent p - gp inhibitors modulating p - gp function and/or expression .
it is an important step to use assays to evaluate the effect of candidate drugs as inhibitors of p - gp function in vivo after the drugs were found to have an mdr - reversing effect in vitro . for nonsurgical solid cancer patients ,
there are currently no direct assays measuring intratumoral drug concentrations or the alteration of p - gp function and/or expression .
thus , alternative approaches to evaluate the degree of functional inhibition of p - gp in vivo will be a useful aid in the development of mdr modulators .
p - gp is also expressed in normal tissues in which they seem to have an important role in the protection against xenobiotics .
several authors reported that p - gp was expressed in lymphocytes including cd3 , cd4 , and cd8 t cells , as well as the cd56 natural killer ( nk ) cells , with the highest expression levels and activities observed in cd56 cells followed by cd8 cells [ 5 , 6 ] .
however , the physiological roles that p - gp plays in these cells are unclear .
the p - gp in the lymphocytes appears functionally identical to that observed in multidrug resistant cells ; they have the same substrate and antagonist specificities [ 5 , 7 , 8 ] .
since the expression and function of p - gp in cd56 cells are the highest , is rho123 accumulation widely used in cd56 populations as a surrogate indicator to evaluate the degree of functional inhibition of p - gp in clinical trials of p - gp inhibitors , for example , the work of tariquidar   and zosuquidar .
as mouse nk cells do not express cd56 , we used rho123 accumulation in cd8 cells as a surrogate indicator to evaluate the reversal activity by p - gp reversors in the mouse mdr tumor - bearing model .
the search for mdr modulators has extended to the natural products and their derivatives ; natural source compounds have become the most widely used of fourth - generation p - gp inhibitors because they are less toxic and more potent than the disappointing first- and second - generation mdr modulators [ 1214 ] .
ch , manufactured by salification from cepharanthine , which is a biscoclaurine alkaloid , extracted from stephania cepharantha hayata has a variety of biological activities ( figure 1 ) [ 1517 ] .
recently , it has been reported that ch has an mdr - reversal effect , and p - gp inhibition is one of the reversal mechanisms of mdr in vitro [ 18 , 19 ] ; however , the effect was not evaluated in vivo .  
we present here a method to detect the effect of the inhibitor of the p - gp function on rho123 accumulations of the cd8 cells in whole blood from the mdr tumor - bearing mouse .
this method was first used to develop and validate the efficacy of ch against p - gp functions in the mouse model for potential use in subsequent clinical evaluations .
the experiments were carried out on 24-month - old balb / c male mice , obtained from the henan experimental animal center ( zhengzhou , china ) .
the animals had been raised from birth in a specific pathogen - free environment and were handled according to the institutional guidelines complying with chinese legislation .
hca / fap variants were developed from the parental hca hepatocarcinoma cells , which were obtained from keygen biotechnology co. ltd .
( nanjing , china ) . by step - by - step , continuous exposure to increasing doses of fap ,
the overexpression of p - gp in the variant was confirmed by the western blot analysis and the cells that have been previously described .
ch was supplied by the henan academy of medical and pharmaceutical sciences ( zhengzhou , china ) .
adriamycin ( adr ) was purchased from hisun pharmaceutical co. , ltd ( zhejiang , china ) .
cisplatin ( cddp ) was purchased from qilu pharmaceutical co. , ltd ( shandong , china ) .
5-flurouracil ( 5-fu ) was purchased from xudong haipu pharmaceutical co. , ltd ( shanghai , china ) .
rho123 was purchased from sigma - aldrich ( st . louis , mo , usa ) and dissolved in pbs at a concentration of 1  mg / ml . the ficoll - histopaque mouse lymphocyte separation medium was obtained from solarbio science & technology co. , ltd ( beijing , china ) .
the pe antimouse cd8 antibody ( clone 53 - 6.7 ) and pe rat igg2 ,    isotype ctrl were obtained from biolegend ( biolegend corp .
hca / fap cells were collected from the ascitic fluid of balb / c mice harboring 57 day - old ascitic tumor .
the 1  10  hca / fap cells were injected intramuscularly in the right axilla of balb / c male mice selected for the experiment on day 0 .
the next day , the animals were randomized and divided into different groups ; each group comprised 10  mice .   to study the effects of rho123 administration on peripheral blood cd8 cells , the retention of fluorescence in these cells was investigated as described previously   with some modifications and a dose - response curve established .
on the 8th day after the hca / fap injection , the mice received a single intravenous ( i.v . ) injection of rho123 .
the doses used were 0.5 , 1.0 , 2.5 , 5.0 , and 7.5  mg / kg , and the volume of administration was 10  l / g .
one hour after administration , peripheral blood was collected and cell suspension was prepared as described below .    for the investigation of the effects of p - gp inhibitors ex vivo ,
blood samples were collected as described below on the 8th day after hca / fap injection .
ch was then added at final concentrations of 10.0 , 5.0 , and 2.5  m ; ver was added at a final concentration of 5.0  m , an equal volume of the vehicle was added as control .
the samples were incubated at 37c for one hour before rho123 was added at a final concentration of 50  ng / ml , and the tubes were incubated further at 37c for one hour .
cell suspension was prepared as described below .   to study p - gp function in vivo
, rho123 was injected with or without ch or ver as described before   with small modifications on the 8th day after hca / fap injection .
briefly , mice , respectively , received a single intravenous ( i.v . ) injection of the vehicle as control ; 2.5 , 5.0 , and 10.0  mg / kg of ch or 2.5  mg / kg of ver followed one hour later by a single i.v .
after one hour , blood samples were collected and cell suspension was prepared as described below .
peripheral blood was collected from the orbital sinus of each mouse in a tube containing k2edta as an anticoagulant .
mononuclear cells were obtained by the separation of anticoagulated blood by a ficoll - histopaque density - gradient centrifugation and transferred to 1.5  ml eppendorf tubes and kept on ice .
cells were washed twice with ice - cold phosphate buffered saline ( pbs , ph 7.4 ) .
after that , the pellet was gently resuspended in ice - cold pbs and adjusted to a concentration of 1  10  cells / ml . single - cell suspensions were incubated with either the pe - conjugated antimouse cd8 antibody or pe - conjugated rat igg2 ,  isotype ctrl as a negative control . after staining for 30 minutes in the darkness at 4c
, the cells were washed twice with ice - cold pbs and then resuspended in pbs and kept on ice in the dark until analyzed as previously described . a life - gate based on forward scatter ( fsc ) and side scatter ( ssc ) parameters were made to analyze only viable cells ; other gates were made to determine the subpopulations .
amplifier settings for fsc and side ssc were used in linear mode and those for fluorescence channels were used in a logarithmic mode .
fluorescence compensation was manually set for fl1 channel ( rho123 ) and fl2 channel ( pe ) with single rho123-stained cells and pe - stained cells separately .
multicolor flow cytometry analyses were used to evaluate the proportions of the cd8 population and the mean fluorescence intensity ( mfi ) of rho123 in the population .
cells were analyzed on epics - xl mcl , and data were analyzed with expo32 adc software ( beckman coulter , fullerton , calif , usa ) . to evaluate the antitumor effect of fap chemotherapy protocol plus various concentrations of ch or ver in vivo as previously described .
twenty four - hours after the hca / fap injection , the mice were randomly divided into different groups and treated as follows : 
 group i : control ( normal saline i.v . injected consecutively from day 1 to 9),group ii : 10  mg / kg of ch alone i.v . injected from day 1 to 8 ,
group iii : fap chemotherapy : 4.2  mg / kg of adr and 85.0  mg / kg of 5-fu i.v . injected on day 1 , 2.8  mg / kg of cddp i.v . injected from day 1 to 8 ,
group iv : 2.5  mg / kg of ch i.v . injected from day 1 to 8 with fap chemotherapy , group v : 5.0  mg / kg of ch i.v . injected from day 1 to 8 with fap chemotherapy , group vi : 10.0  mg / kg of ch i.v . injected from day 1 to 8 with fap chemotherapy , group vii
: 2.5  mg / kg of ver i.v . injected from day 1 to 8 with fap chemotherapy . 
  
control ( normal saline i.v . injected consecutively from day 1 to 9 ) ,   10  mg / kg of ch alone i.v . injected from day 1 to 8 ,    fap chemotherapy : 4.2  mg / kg of adr and 85.0  mg / kg of 5-fu i.v . injected on day 1 , 2.8  mg / kg of cddp i.v . injected from day 1 to 8 ,    2.5  mg / kg of ch i.v . injected from day 1 to 8 with fap chemotherapy ,   group v : 5.0  mg / kg of ch i.v . injected from day 1 to 8 with fap chemotherapy ,   10.0  mg / kg of ch i.v . injected from day 1 to 8 with fap chemotherapy ,   2.5  mg / kg of ver i.v . injected from day 1 to 8 with fap chemotherapy .  
the tumor length ( l ) and width ( w ) were measured , and the tumor weight ( wr ) was calculated as follows : 
 ( 1)wr  = 12  l    w2 . 
							
the percent of tumor growth inhibition was calculated on day 8 by comparing the average values of the treated groups with those of the tumor - bearing control group .
for all the experiments , data are presented as the mean  sd . statistical comparison between the groups with different doses or concentrations
tests for significant differences among the groups were done using one - way anova with multiple comparisons ( fisher 's pairwise comparisons ) using spss 13.0 ( ibm software , somers , ny , usa ) .
administration of rho123 ( 0.57.5  mg / kg ) in mice produced a dose - dependent fluorescence fashion in peripheral blood cd8 cells ( figure 2 ) .
all doses were well tolerated without significant toxicity . the lower dose of rho123 , 2.5  mg / kg ,
was selected to investigate the effects of ch on the accumulation of rho123 in peripheral blood cd8 cells . using this dose ,
rho123 accumulations were increased after administration of either ver ( positive control ) or ch and presented a clear dose - dependent relationship with ch ( 2.510  mg / kg ) compared with vehicle control , as shown in figure 3 .
the efficacy of the p - gp reversors was not equal ; ver was less efficient and potent than ch .
there has similar result in ex vivo blood samples ; the mfis of cd8 + cells in the samples treated with the vehicle , 5.0  m of ver and 2.5 , 5.0 and 10.0  m of ch were 14.4  0.3 , 20.0  0.3 , 22.9  0.5 , 25.4  1.0 and 29.8  0.9 , respectively .
the mfi was related to the presence of ch in a concentration pattern in ex vivo blood samples , as shown in figure 4 .
this demonstrated that p - gp is active in cd8 cells and can be modulated by ch in vivo and ex vivo .
2.5  mg / kg of ver or 2.5 , 5.0 , and 10.0  mg / kg of ch were evaluated in the hca / fap tumor - bearing mouse .
the fap combination chemotherapy plus 10.0  mg / kg of ch provided the maximum antitumor activity of the treatments studied .
compared with the fap chemotherapy group , the antitumor effect was increased in a dose - dependent manner when the fap combined chemotherapy plus various concentrations of ch was used , as shown in table 1 .
hepatocellular carcinoma ( hcc ) represents the third leading cause of cancer - related death .
chemotherapy consisting of fap was commonly used to treat hcc in clinical situations ; however , hcc is often resistant to these drugs . therefore , one of the key approaches is to overcome drug resistance in the success of chemotherapy against hcc .
previous studies of p - gp inhibitors such as ver , cyclosporine a , and its analog psc833 [ 2628 ] have been limited because of its serious toxicity at the time of dosage for reversing drug resistance .
an important step to develop the mdr reversors targeting p - gp is to determine the inhibition level of p - gp in the preclinical animal model .
an ideal approach is to detect the real concentrations of the investigated drug directly in vivo , but it is almost impossible , even in the animal model . if there is no related data on the inhibition level of p - gp , it is difficult to interpret the efficacy of p - gp inhibitors in pre- or clinical trials .
recently , the functional study of p - gp was of great concern . among lymphocytes in the mouse
, cd8 cells expressed the highest levels of p - gp followed by cd4 > cd3 [ 6 , 21 , 2932 ] . since blood samples are easy to be collected , we selected peripheral blood cd8 cells as a surrogate indicator to investigate the alteration of p - gp activities before and after the administration of p - gp inhibitors and then evaluate the reversal effect of the drug in a solid - tumor - bearing mouse
. rho123 is a fluorescent substrate and has been shown to be a sensitive reagent for the detection of p - gp function by flow cytometry [ 14 , 33 ] .
some authors reported that rho123 was a substrate for mrp and p - gp , but leite et al .   reported that classical mrp1 reversors were unable to enhance rho123 accumulation in lymphocytes suggesting that mrp1 might not be active or rho123 is not a good substrate for mrp1 expressed in these cells
therefore , the alteration of rho123 fluorescence intensities in these cells responded to the alteration of the p - gp function . in this study , we demonstrated that the mean fluorescence intensities of cd8 cells were related to the dose of rho123 in a dose - dependent manner suggesting that it is a good choice to use the accumulation of rho123 in cd8 cells as a surrogate indicator for the study of p - gp inhibitors .
we further studied the modulation effect of ch in vivo and ex vivo ; the results showed that the mean fluorescence intensities of cd8 cells were related to ch in a dose- or concentration - dependent manner either in vivo or ex vivo .
this suggested that the activities of p - gp were inhibited by ch . in our previous study , it was shown that the enhancement of cytotoxicity of fap against cultured hca / fap cells by ch occurs in vitro ; the efflux rate of rho123 in hca / fap cells was 2 times less than that of hca cells after 1 hour explosure to ch plus fap .
mice is 0.47 times of clinical dosing of fap protocol , that is , 85.0  mg / kg of 5-fu , 4.2  mg / kg of adr , and 2.8  mg / kg of cddp . in the present study , we established an hca / fap solid tumor mouse model and assessed the antitumor effect of fap combination protocol plus ch .
the i.v . dosing for a mouse is 42.50  mg / kg of 5-fu , 2.10  mg / kg of adr , 1.40  mg / kg of cddp .
dose of 10.0  mg / kg ch partly inhibited the tumor growth , but the effect was weak ; this is consistent with conclusions drawn from other authors ' reports : a single i.v .
dose of 5.0  mg / kg ch has no antitumor effect ( data not shown ) . when fap combined chemotherapy plus various concentrations of ch was used , the antitumor effect was increased in a dose - dependent manner .
therefore , with this correlation with the results of the inhibition of p - gp function by ch in cd8 cells , we think that the antitumor activity recovered by ch in a dose - dependent manner was related to the inhibition of the p - gp function .
all of the efficacious combination protocols with ch appeared to be well tolerated , as indicated by the minimal changes in body weights of the various treatment groups ( as shown in table 2 ) . since an ld50 of ver for the i.v .
administration in mouse was reported to be 7.6  mg / kg , but a single i.v .
dose of 5.0  mg / kg ver caused the death of 60% of animal in the treated balb / c mice ( data not shown ) , we selected the dose of 2.5  mg / kg as a positive control .
there is no mortality in the fap treated group ; we think the 20% mortality in the fap plus ver treated group was caused by the increasing toxicity when ver combined with adr .  
although one can argue that what occurs at the level of peripheral blood cd8 cells may not accurately reflect the real inhibition level of p - gp in the solid tumor in mouse , tc - sestamibi scans performed in some studies have shown effective p - gp inhibition in both normal tissues and solid tumors [ 9 , 38 ] , suggesting the inhibition level of p - gp was modulated simultaneously .
we have to note that no model is perfect and there are obvious differences between humans and mice ; the balb / c mouse is unique for its features , making even more difficult extrapolations to human conditions .   with regard to the results obtained , we conclude that ch is a potent p - gp inhibitor for clinical application .
although limitations may exist in the surrogate assay in cd8 cells in the mouse model , we are convinced that this assay can provide us with much useful information in screening potential p - gp inhibitors in vivo .
 the purpose of this study was the use of rhodamine 123 ( rho123 ) accumulation in peripheral blood cd8+cells as a surrogate indicator to evaluate the modulating effect of p - glycoprotein ( p - gp ) inhibitors in the multidrug resistance ( mdr ) tumor - bearing mouse model . 
 rho123 was administered to mice , and the fluorescence level in cd8 + cells was measured . 
 cepharanthine hydrochloride ( ch ) and verapamil ( ver ) , two p - gp inhibitors , were administered to mice 1 hour prior to rho123 administration in vivo or added to peripheral blood 1 hour prior to rho123 addition ex vivo . 
 the tumor inhibition effect of 5-fluorouracil / adriamycin / cisplatin ( fap ) protocol plus ch was also investigated . 
 a concentration- or dose - response relationship was shown between the concentration and dose of ch and rho123 accumulation or the antitumor activity . in conclusion 
 , the measurement of rho123 accumulation in cd8 + cells provides a surrogate assay for the screening of candidate p - gp inhibitors in preclinical trials , and ch is effective in modulating p - gp - mediated mdr in vivo . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion