BACKGROUND
in both women and female dogs, the most prevalent type of malignant neoplasm is the spontaneous mammary tumor.
in dogs, half of these are malignant.
there is a threefold incidence in the canine, with middle aged, non-spayed female dogs being the most affected  <cit> .
the early ovariectomy is thought to reduce the risk of mammary cancer development  <cit> , however, the high morbidity and mortality rate due to ineffective treatment strategies makes this problem as being still actual.
the treatment of choice for the canine patients is surgical mastectomy.
unfortunately, it is often recognized - unsatisfactory as in case of invasive cancer at the time of surgery the micro-metastases are present.
moreover, clinical studies reveal that after regional mastectomy 77% of dogs developed a new malignancy  <cit> .
there are neither large studies conducting to prove to the benefit of chemotherapy in dogs nor established chemotherapy treatment protocols available.
moreover, the effectiveness of each individual chemotherapeutic agent and drug resistance of canine mammary cancer have not yet been characterized.
the available data is conflicting, possibly due to the small number of studies performed, or the insufficient number of dogs used in these studies.
some veterinarians adapt human chemotherapy protocols; however these treatment modalities are often unsatisfactory due to similarities observed in recurrence time, time to metastasis and overall survival  <cit> .
the resistance to a specific cytotoxic drug is ensured by the activity of efflux pumps that belong to the abc-transporters super family.
the most important in breast cancer are: pgp , bcrp , mrp <dig>  and mrp <dig> .
for example, in human cancer over-expression of pgp is linked to resistance to vinca alkaloids, anthracyclines, epipodophyllotoxins and also tubulin polymerizing drugs whereas mrp <dig> is responsible for resistance to vincristine and vinblastine, methothrexate, anthracyclines, etoposide, paclitaxel and irinotecan  <cit> .
unfortunately, expression of efflux pumps and their specific substrates have not been assessed in dogs yet.
there is just one document published showing that bcrp can fully protect canine mammary cancer cells against doxorubicin  <cit> .
that is why the aim of our study was to assess expression of pgp, bcrp, mrp <dig> and mrp <dig> in five canine mammary cancer cell lines and to investigate their role in cancer resistance to vinblastine, cisplatin and cyclophosphamide which are used in breast cancer treatment  <cit> .
the examined drugs were selected based on the available data in the field of veterinary oncology.
they presented positive results of cisplatin and cyclophosphamide treatment in canine mammary cancer  <cit>  whereas vinblastine was selected due to its general tolerance  <cit> .
moreover, these anticancer agents belong to various groups, thus we used them to observe efflux pumps activity during treatment with drugs from different groups.
we also treated canine mammary cancer cells with sirnas specific for these efflux pumps to knock-down their expression.
treatment of cancer cells by using classical inhibitors of efflux pumps often fails  <cit> , thus we used a novel approach to reduce their activity.
our results have pointed out that treatment of patients with the supportive use of the sirnas specific for efflux pumps may help with improving results of chemotherapy.
despite the useful function of rnai therapeutics for disease treatment, it, yet - still requires the development of clinically suitable, safe and effective drug delivery vehicles, there are some promising data from ongoing clinical trials that give hope for their practical application in the future  <cit> .
methods
cell lines
the cell lines used for the study have been previously used in other published research  <cit> .
two canine mammary adenocarcinoma cell lines , anaplastic cancer cell line , simple carcinoma cell line  and spindle-cell mammary tumor cell line  had been examined.
cells were cultured under optimal conditions: in rpmi- <dig> medium enriched with 10%  heat-inactivated fetal bovine serum , penicillin-streptomycin , and fungizone  , in an atmosphere of 5% co <dig> and 95% humidified air at 37°c.
sirna transfection
the sirna transfection procedure used in canine mammary cancer cells was described in details in our previously published studies  <cit> .
the cell density, transfection reagent toxicity and transfection efficacy were optimized according to the procedure described in our previous manuscript:  <cit> .
the canine  pgp, bcrp, mrp <dig> and mrp <dig> sequences were obtained from gene bank with accession numbers: nm_ <dig>  dq <dig> , nm_ <dig>  xm_ <dig> , respectively.
the sirna duplexes were designed by http://www.sigmaaldrich.com/life-science/custom-oligos/sirna-oligos/sirna-design-service.html.
the results were confirmed using two independent algorithms: dharmacon  and ambion and two duplexes were used for further experiments  .
for each gene silencing the mixture of both duplexes was used .
all the experiments with transfected cells were conducted 24– <dig> hrs after the transfection.
sirna sequences used to pgp, bcrp, mrp <dig> and mrp <dig> genes silencing in canine mammary cancer cell lines.
the canine  pgp, bcrp, mrp <dig> and mrp <dig> sequences were obtained from gene bank with accession numbers: nm_ <dig>  dq <dig> , nm_ <dig>  xm_ <dig> , respectively.
the sirna duplexes were designed byhttp://www.sigmaaldrich.com/life-science/custom-oligos/sirna-oligos/sirna-design-service.html.
the results were confirmed using two independent algorithms: dharmacon  and ambion.
reverse-transcriptase qpcr
total rna was isolated using a total rna kit  according to the manufacturer‘s protocol.
isolated rna samples were dissolved in rnase-free water.
the quantity of isolated rna was measured using nanodrop .
the mean concentration of rna was  <dig> ng/μl, and a260/ <dig> ratio was between  <dig>  and  <dig> .
the samples with adequate amounts of rna were treated with dnasei to eliminate dna contamination.
the samples were subsequently purified using rneasy minielute cleanup kit .
finally rna samples were analyzed on a bioanalyzer  to measure final rna quality and integrity.
only rna with rin  >  <dig> was used for the further analyses.
primers used to detect the expression of pgp, bcrp, mrp <dig> and mrp <dig> genes were designed using primer <dig> software  and checked using oligo calculator  and primer-blast .
the used sequences are listed in table  <dig>  rps <dig> and hprt genes were used as non-regulated references for the normalization of target gene expression  <cit> .
quantitative rt-pcr was performed using fluorogenic sybr green and the sequence detection system, fast  <dig> .
data analysis was carried out using the  <dig> fast system sds software version  <dig> .
<dig>  .
the results were analyzed using comparative ct method  <cit> .
relative transcript abundance of the gene equals Δct values .
relative changes in transcript were calculated as ΔΔct values .
the experiment was conducted three times.
primer sequences used in this study and their annealing optimal temperature and time.
the mrna sequences of key genes were obtained from ncbi database.
primers were designed using primer <dig> software  and checked using oligo calculator  and primer-blast .
primers sequences are listed in table  <dig>  hprt and rps <dig> genes were used as non-regulated reference genes for normalization of target gene expression  <cit> .
flow cytometry determination of rhodamine- <dig> accumulation
examined cancer cells   were incubated for  <dig> h at 37°c in the  <dig> mmol of rhodamine- <dig> .
after the incubation time, cells were washed twice, re-suspended in ice cold pbs and kept at 4°c in the dark until analysis in the flow cytometer.
at least  <dig>  <dig> cells per sample were counted and analyzed by flow cytometry .
cells shown in forward scatter and side scatter were gated and acquired through the fluorescence channel.
the amount of fluorescence was plotted as a histogram within the gate.
data acquisition was performed using bd facs diva software  to determine mean fluorescence intensity values.
this procedure has been published before  <cit> .
results represent the average of at least three independent experiments.
the overlay histogram was created using flowing software , available at http://www.flowingsoftware.com.
cell viability assay  and ic <dig> determination
cell viability  was quantified by mtt assay.
cells were seeded into 96-well plate  at the density which ensured their 50-70% confluence at the day of experiment and: 1) treated with vinblastine , cisplatin  or cyclophosphamide  , 2) transfected with pgp, bcrp, mrp <dig> and mrp <dig> -specific sirna and then treated with the anticancer drugs as given above, 3) treated with non-coding sirna  and cyctotoxic drugs as given above.
these concentrations of anticancer drugs were determined in course of our preliminary experiments  and chosen as appropriate to determine ic <dig> doses in all of the examined cell lines.
then, cells were incubated in  <dig>  mg/ml tetrazolium salt mtt diluted in phenol red-free rpmi  <dig> medium  for  <dig> hrs at 37°c.
to complete solubilization of the formazan crystals,  <dig> μl of dmso  was added to each well.
cells viability was quantified by measuring photometric absorbance at  <dig> nm in multi well plate reader infinite  <dig> pro tecan™ .
all the samples were examined in triplicate, each experiment was conducted seven times .
based on these results cytotoxicity was determined.
it was expressed as a mean percentage decrease relative to unexposed control ± s.d.
control values were set at 100% viability.
cytotoxicity data were fitted to a sigmoidal curve and a four parameter logistic model was used to calculate ic <dig>  which is the concentration of agent which reduces cell growth by 50% under the experimental conditions .
this analysis was performed using graphpad prism  <dig>  .
this method of ic <dig> analysis has been previously published  <cit> .
apoptosis assay
the annexin v-fitc and propidium iodide  dual staining was applied for apoptosis analysis.
control cells  and cells treated with  anticancer drug at ic <dig> dose and  transfected with pgp, bcrp, mrp <dig> and mrp3-specific sirna moreover treated with anticancer drug at ic <dig> dose were harvested by trypsinization.
these cells, as well as the cells floating in medium  were stained with an annexin v kit  according to the manufacturer’s protocol.
the cells then were analyzed by flow cytometry  within  <dig> hr after staining.
early apoptotic cells with exposed phosphatidylserine but intact cell membranes bound to annexin v-fitc however excluded pi.
cells in late apoptotic stages were labeled with both annexin v-fitc and pi, whereas necrotic cells were labeled with pi only.
all samples were assayed in triplicate.
the experiment was conducted at least twice.
statistical analysis
the analysis for statistical purposes was conducted using prism version  <dig>  software .
the one-way anova and tukey hsd  post-hoc test were applied as well as regression analysis.
determination of ic <dig> test has been used.
the p-value < <dig>  was recognized as significant, whereas, p-value < <dig>  and p-value < <dig>  as highly significant.
the data was expressed as means +/− s.d.
for molarity calculations, the molarity calculator  on-line platform was used.
RESULTS
pgp, bcrp, mrp <dig> and mrp <dig> are expressed in canine mammary cancer cells and their expression level changes due to anticancer drug in vitro treatment
rt-qpcr analysis revealed that all of the examined cell lines express pgp, bcrp, mrp <dig> and mrp <dig> .
the pgp expression ranged between  <dig>  in cmt-u <dig> cell line and  <dig>  in cmt-w <dig> cell line.
the bcrp expression was the lowest in cmt-u <dig> cell lines  whereas the highest in cmt-w <dig> cell line .
the highest expression of mrp <dig> was observed in cmt-w <dig> cell line  whereas the lowest expression was noticed in cmt-u <dig> cell line .
cmt-w <dig> cell line exposed the lowest expression of mrp <dig> .
the highest expression of mrp <dig> was detected in cmt-u <dig> cell line .
expression of efflux pumps in canine mammary cell lines in control conditions, after scrambled sirna treatment and pgp, bcrp, mrp <dig> and mrp <dig> -specific sirna treatment as well as after treatment with vinblastine, cisplatin and cyclophosphamide at ic <dig> doses.
p <  <dig>  was marked as*, p <  <dig>  was marked as **, and p <  <dig>  was marked as ***.
one-way anova followed by tukey hsd post-hoc test were applied.
increase in expression was marked as red text, decreased in expression was marked as green text, whereas no change in transcript level was marked as black text.
treatment of cancer cells using scrambled sirna did not cause any significant effect on examined gene expression , whereas treatment of these cells with pgp, bcrp, mrp <dig> or mrp <dig> -specific sirnas caused significant decrease in transcript level of targeted genes  .
the efficacy of silencing reaction was very high, as there was observed 88-99% decrease in pgp transcript level, 14-100% in bcrp, 94-99% in mrp <dig> and 22-100% in mrp <dig> expression .
interestingly, we observed that pgp expression increased significantly in all the examined cell lines after viblastine and cisplatin treatment at ic <dig> doses, whereas decreased after cyclophosphamide treatment at ic <dig> dose .
vinblastine treatment increased mrp <dig> expression in all of the examined cell lines with exception of cmt-w <dig> cell line where its expression increased also after treatment with cyclophosphamide at ic <dig> dose .
in case of bcrp, we observed increased tendency in its expression after cyclophosphamide treatment and decreased in its expression after vinblastine treatment at ic <dig> dose .
we are as well able to point out the increase in mrp <dig> expression in all the examined cell lines after treatment with cisplatin at ic <dig> dose .
cancer cell treatment with pgp, bcrp, mrp <dig> and mrp <dig> –specific sirna decreases efflux pumps activity enhancing rhodamine- <dig> accumulation
the rhodamine- <dig> efflux assay was conducted to confirm our results of gene expression silencing and their functional inhibition.
it showed that after knock-down of pgp, bcrp, mrp <dig> and mrp <dig> expression level, the rhodamine-; <dig> accumulation significantly increased and that shows decreased activity of the efflux pumps .
treatment of examined cells with scrambled sirna did not cause any significant effect .
we observed that sirna treatment of cmt-u <dig> caused significant but slight effect on rhodamine- <dig> accumulation .
in other cell lines the effect was higher , for example in p <dig> cell line the mean fluorescence related to rhodamine- <dig> accumulation in control cells was only  <dig>  however, after the treatment with bcrp-specific sirna is was  <dig> .
the highest effect of rhodamine- <dig> accumulation after sirna treatment was observed in cmt-w <dig> cell line .
cancer cell treatment with sirna specific to efflux pumps decreases drug-resistance
the mtt assay of cells viability after treatment with increasing doses of anticancer drugs showed significant variations in ic <dig> doses between the examined cell lines.
all ic <dig> doses are listed in table  <dig>  vinblastine ic <dig> dose in cmt-u <dig> cell line was  <dig>  <dig> nmol, whereas after pgp and mrp <dig> knock-down it was only  <dig> nmol and  <dig> nmol, respectively .
in cmt-w <dig> cell line vinblastine ic <dig> was  <dig>  <dig> nmol, whereas after pgp and mrp <dig> silencing it was  <dig> nmol and  <dig> nmol, respectively .
we showed that after pgp and mrp <dig> silencing ic <dig> was significantly lower in all the examined cell lines ranging 203– <dig> nmol and 144– <dig> nmol, respectively .
significant variations were also observed in case of ic <dig> doses of cisplatin – the lowest ic <dig> dose was in cmt-u <dig> cell line  , whereas the highest was in p <dig> cell line  .
cisplatin ic <dig> was significantly decreased in all the examined cell lines after silencing of pgp, bcrp, mrp <dig> and mrp <dig> .
regarding cyclophosphamide, we also noticed significant variations in ic <dig> values.
it was the lowest in cmt-w <dig> cell line , when on the other hand the highest in cmt-w <dig> cell line  .
treatment of cells with bcrp-specific sirna decreased cyclophosphamide ic <dig> in all of the examined cell lines  .
ic <dig> doses  of anticancer drugs: vinblastine, cisplatin and cyclophosphamide in canine mammary cancer cell lines in control conditions and after silencing of efflux pumps expression.
p <  <dig>  was marked as *, p <  <dig>  was marked as **, and p <  <dig>  was marked as ***.
one-way anova followed by tukey hsd post-hoc test was applied.
cancer cell treatment with anticancer drug and sirna specific to efflux pumps increases apoptosis
the annexin v assay was used to examine the influence of pgp, bcrp, mrp <dig> and mrp <dig> expression knock-down on apoptosis induced by vinblastine, cisplatin and cyclophosphamide .
due to any changes in cells viability based on transfection procedure were observed in mtt assay, this kind of control has been omitted in apoptosis assay.
we used similar approach in our previous study  <cit> .
annexin v analysis revealed that treatment of cells using pgp and mrp <dig> -specific sirna together with vinblastine  significantly increased number of apoptotic cells, compared to apoptosis caused by anticancer drug which was given as a single agent  .
the most significant effect was pointed out in cmt-w <dig> cell line:  <dig> % and  <dig> % increase in number of apoptotic cells after pgp and mrp <dig> silencing and vinblastine treatment, respectively .
similarly, in cmt-w <dig> cell line  <dig> % and  <dig> % increase in number of apoptotic cells was shown after pgp and mrp <dig> silencing and vinblastine treatment, respectively .
in case of cisplatin treatment at ic <dig> dose, an increase in number of apoptotic cells was noticed in each cell line after silencing of all the efflux pumps: pgp, bcrp, mrp <dig> and mrp <dig>  the mostly significant result was observed in p <dig> cell line, where 40%, 41%, 42% and 40% increase in number of apoptotic cells  was visible, respectively .
in cmt-u <dig> cell line the highly significant  effect on apoptosis was as well as in case of cisplatin treatment in cells transfected with sirna specific to: pgp, bcrp and mrp <dig> , whereas significant effect  was observed in cells transfected with mrp <dig> -specific sirna  .
in all the examined cell lines increase in number of apoptotic cells related to cyclophosphamide treatment at ic <dig> dose was undoubtedly visually recognized after bcrp knock-down .
additionally, in cmt-w <dig> cell line the significant increase in number of apoptotic cells was observed due to cyclophosphamide treatment following transfection with bcrp, mrp <dig> and mrp <dig> -specific sirna .
discussion
cancer cells retain the important mechanism of self-protection through the activity of multiple drug resistance transporters.
the multidrug resistance is frequently associated with over-expression of two or more membrane pumps that efflux anticancer drug from the cytoplasm.
this protects tumor cells against the drug effects and its correlated molecular processes  <cit> .
expression of efflux pumps is usually higher in tumors that originate from tissues that normally show their expression and it is always higher in the tumors than in normal cells  <cit> .
unfortunately, the expression and role of proteins that mediate drug resistance in canine cancer cells has not yet been recognized.
as increased efflux is such a significant contributor to a multidrug resistance in cancer cells, current research is aimed at blocking or inhibiting this specific mechanism.
that is why the aim of our study was to assess expression of four the most important efflux pumps: pgp, bcrp, mrp <dig> and mrp <dig> in canine mammary cancer cells as well as to investigate their role in resistance to: cisplatin, cyclophosphamide and vinblastine.
because treatment of cancer cells with the use of classical inhibitors of efflux pumps often fails  <cit> , we used a novel approach, that is: specific rnai to knock-down their expression.
examination of each efflux pump expression in control conditions and after treatment with anticancer drug was also quite a novel approach.
we observed significant differences in pgp transcript level in control conditions and after treatment with anticancer drug.
in all of the examined cell lines the pgp expression increased due to vinblastine and cisplatin treatment whereas decreased due to cyclophosphamide treatment .
it ought to be outlined that these results are in accordance with ic <dig> doses .
in case of cisplatin treatment, expression of all four efflux pumps increased, and thus, any relationship between their transcript level and ic <dig> doses was very complicated to calculate .
we also observed that cyclophosphamide treatment caused increase in bcrp expression in all of the examined cell lines.
these results are in accordance with previous findings that cytotoxic drugs can initiate or increase expression of efflux pumps in cancer cells  <cit> .
moreover, the expression level of these efflux pumps correlates with response to anticancer therapy and patient follow up  <cit> .
based on our results, we have come up with the conclusion that examination of efflux pumps expression before initiation of chemotherapy could help to predict response to anticancer drugs and could be helpful in assessment of their proper doses.
however, more studies in this field are required.
the rnai gene silencing was highly successful , reaching even 100% of gene knock-down in case of bcrp in cmt-w <dig> cell line and mrp <dig> in case of cmt-u <dig> cell line.
the results of gene knock-down were confirmed by the assay with rhodamine- <dig> efflux.
to assess whether efflux pumps silencing has an influence on cancer cell susceptibility to cytostatic drugs, the ic <dig> doses were determined in transfected cells .
our results showed that vinblastine ic <dig> doses significantly decreased in all the examined cell lines after pgp and mrp <dig> silencing .
examination of cisplatin ic <dig> in transfected cells pointed that it was significantly lower in all of them when compared to control cells .
in case of cyclophosphamide, bcrp sirna treatment significantly decreased ic <dig> dose in all of the examined cell lines .
comparison of ic <dig> doses of anticancer drugs given in vitro and their maximum possible plasma concentrations showed that in case of vinblastine the plasma concentration was much lower than the ic <dig> doses  <cit> .
it means that the examined cancer cell lines are vinblastine-resistant and therefore this anticancer drug may be ineffective in canine mammary cancer treatment.
however, more studies in this field should be conducted.
in case of cisplatin, the ic <dig> doses in the three examined cell lines  were lower than the reachable plasma concentrations  <cit> .
however, after bcrp, mrp <dig> or mrp <dig> knock-down, the cisplatin ic <dig> doses were similar or lower than the maximum possible plasma drug concentration in all of the examined cell lines.
it means that knock-down of efflux pumps expression during chemotherapy can reverse cancer drug-resistance.
the ic <dig> doses of cyclophosphamide in all of the examined cell lines were significantly lower than its maximum possible plasma concentration  <cit> .
however, in all the cases given above, targeting of abc efflux pumps using sirna significantly decreased ic <dig> doses of anticancer drugs.
these results were also confirmed by apoptosis assay .
in summary: knock-down of efflux pumps expression during chemotherapy could allow decreased doses of anticancer drugs thereby reducing the risk of their side effects.
however, more studies in this field are required as well as large clinical trials to prove this hypothesis.
our research is a completely new approach in the field of veterinary oncology.
the results show that targeting of abc-efflux pumps using rnai can be promising strategy in cancer treatment.
however, these in vitro studies should be confirmed using in vivo animal models.
CONCLUSIONS
the most important findings of our study are:  the expression level of efflux pump reflects the resistance to specific anticancer drug thus it should be assessed before the initiation of chemotherapy;  in canine mammary cancer pgp and mrp <dig> are responsible for vinblastine resistance, pgp, bcrp, mrp <dig> and mrp <dig> are cisplatin transporters whereas bcrp is a transporter of cyclophosphamide;  treatment of cells using sirna targeting efflux pumps significantly increases cancer susceptibility to anticancer drug and allows to decrease the effective dose of anticancer drug.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
kp research design, experimental design, ic <dig> determination, facs analyses, rhodamine- <dig> accumulation assay, sirna transfections, jm apoptosis analysis, km apoptosis analysis, tm manuscript preparation, mk research design, experimental design, facs analyses, sirna transfection, rt-qpcr, manuscript preparation.
all authors read and approved the final manuscript.

 in both women and female dogs, the most prevalent type of malignant neoplasm is the spontaneous mammary tumor. in dogs, half of these are malignant. there is a threefold incidence in the canine, with middle aged, non-spayed female dogs being the most affected. the early ovariectomy is thought to reduce the risk of mammary cancer development, however, the high morbidity and mortality rate due to ineffective treatment strategies makes this problem as being still actual. the treatment of choice for the canine patients is surgical mastectomy. unfortunately, it is often recognized - unsatisfactory as in case of invasive cancer at the time of surgery the micro-metastases are present. moreover, clinical studies reveal that after regional mastectomy 77% of dogs developed a new malignancy. there are neither large studies conducting to prove to the benefit of chemotherapy in dogs nor established chemotherapy treatment protocols available. moreover, the effectiveness of each individual chemotherapeutic agent and drug resistance of canine mammary cancer have not yet been characterized. the available data is conflicting, possibly due to the small number of studies performed, or the insufficient number of dogs used in these studies. some veterinarians adapt human chemotherapy protocols; however these treatment modalities are often unsatisfactory due to similarities observed in recurrence time, time to metastasis and overall survival. the resistance to a specific cytotoxic drug is ensured by the activity of efflux pumps that belong to the abc-transporters super family. the most important in breast cancer are: pgp, bcrp, mrp and mrp. for example, in human cancer over-expression of pgp is linked to resistance to vinca alkaloids, anthracyclines, epipodophyllotoxins and also tubulin polymerizing drugs whereas mrp is responsible for resistance to vincristine and vinblastine, methothrexate, anthracyclines, etoposide, paclitaxel and irinotecan. unfortunately, expression of efflux pumps and their specific substrates have not been assessed in dogs yet. there is just one document published showing that bcrp can fully protect canine mammary cancer cells against doxorubicin. that is why the aim of our study was to assess expression of pgp, bcrp, mrp and mrp in five canine mammary cancer cell lines and to investigate their role in cancer resistance to vinblastine, cisplatin and cyclophosphamide which are used in breast cancer treatment. the examined drugs were selected based on the available data in the field of veterinary oncology. they presented positive results of cisplatin and cyclophosphamide treatment in canine mammary cancer whereas vinblastine was selected due to its general tolerance. moreover, these anticancer agents belong to various groups, thus we used them to observe efflux pumps activity during treatment with drugs from different groups. we also treated canine mammary cancer cells with sirnas specific for these efflux pumps to knock-down their expression. treatment of cancer cells by using classical inhibitors of efflux pumps often fails, thus we used a novel approach to reduce their activity. our results have pointed out that treatment of patients with the supportive use of the sirnas specific for efflux pumps may help with improving results of chemotherapy. despite the useful function of rnai therapeutics for disease treatment, it, yet - still requires the development of clinically suitable, safe and effective drug delivery vehicles, there are some promising data from ongoing clinical trials that give hope for their practical application in the future. methods cell lines the cell lines used for the study have been previously used in other published research. two canine mammary adenocarcinoma cell lines, anaplastic cancer cell line, simple carcinoma cell line and spindle-cell mammary tumor cell line had been examined. cells were cultured under optimal conditions: in rpmi- medium enriched with 10% heat-inactivated fetal bovine serum, penicillin-streptomycin, and fungizone, in an atmosphere of 5% co and 95% humidified air at 37°c. sirna transfection the sirna transfection procedure used in canine mammary cancer cells was described in details in our previously published studies. the cell density, transfection reagent toxicity and transfection efficacy were optimized according to the procedure described in our previous manuscript:. the canine pgp, bcrp, mrp and mrp sequences were obtained from gene bank with accession numbers: nm_ dq, nm_ xm_, respectively. the sirna duplexes were designed by http://www.sigmaaldrich.com/life-science/custom-oligos/sirna-oligos/sirna-design-service.html. the results were confirmed using two independent algorithms: dharmacon and ambion and two duplexes were used for further experiments. for each gene silencing the mixture of both duplexes was used. all the experiments with transfected cells were conducted 24– hrs after the transfection. sirna sequences used to pgp, bcrp, mrp and mrp genes silencing in canine mammary cancer cell lines. the canine pgp, bcrp, mrp and mrp sequences were obtained from gene bank with accession numbers: nm_ dq, nm_ xm_, respectively. the sirna duplexes were designed byhttp://www.sigmaaldrich.com/life-science/custom-oligos/sirna-oligos/sirna-design-service.html. the results were confirmed using two independent algorithms: dharmacon and ambion. reverse-transcriptase qpcr total rna was isolated using a total rna kit according to the manufacturer‘s protocol. isolated rna samples were dissolved in rnase-free water. the quantity of isolated rna was measured using nanodrop. the mean concentration of rna was ng/μl, and a260/ ratio was between and. the samples with adequate amounts of rna were treated with dnasei to eliminate dna contamination. the samples were subsequently purified using rneasy minielute cleanup kit. finally rna samples were analyzed on a bioanalyzer to measure final rna quality and integrity. only rna with rin  >  was used for the further analyses. primers used to detect the expression of pgp, bcrp, mrp and mrp genes were designed using primer software and checked using oligo calculator and primer-blast. the used sequences are listed in table  rps and hprt genes were used as non-regulated references for the normalization of target gene expression. quantitative rt-pcr was performed using fluorogenic sybr green and the sequence detection system, fast. data analysis was carried out using the fast system sds software version.. the results were analyzed using comparative ct method. relative transcript abundance of the gene equals Δct values. relative changes in transcript were calculated as ΔΔct values. the experiment was conducted three times. primer sequences used in this study and their annealing optimal temperature and time. the mrna sequences of key genes were obtained from ncbi database. primers were designed using primer software and checked using oligo calculator and primer-blast. primers sequences are listed in table  hprt and rps genes were used as non-regulated reference genes for normalization of target gene expression. flow cytometry determination of rhodamine- accumulation examined cancer cells were incubated for h at 37°c in the mmol of rhodamine-. after the incubation time, cells were washed twice, re-suspended in ice cold pbs and kept at 4°c in the dark until analysis in the flow cytometer. at least cells per sample were counted and analyzed by flow cytometry. cells shown in forward scatter and side scatter were gated and acquired through the fluorescence channel. the amount of fluorescence was plotted as a histogram within the gate. data acquisition was performed using bd facs diva software to determine mean fluorescence intensity values. this procedure has been published before. results represent the average of at least three independent experiments. the overlay histogram was created using flowing software, available at http://www.flowingsoftware.com. cell viability assay and ic determination cell viability was quantified by mtt assay. cells were seeded into 96-well plate at the density which ensured their 50-70% confluence at the day of experiment and: 1) treated with vinblastine, cisplatin or cyclophosphamide, 2) transfected with pgp, bcrp, mrp and mrp -specific sirna and then treated with the anticancer drugs as given above, 3) treated with non-coding sirna and cyctotoxic drugs as given above. these concentrations of anticancer drugs were determined in course of our preliminary experiments and chosen as appropriate to determine ic doses in all of the examined cell lines. then, cells were incubated in mg/ml tetrazolium salt mtt diluted in phenol red-free rpmi medium for hrs at 37°c. to complete solubilization of the formazan crystals, μl of dmso was added to each well. cells viability was quantified by measuring photometric absorbance at nm in multi well plate reader infinite pro tecan™. all the samples were examined in triplicate, each experiment was conducted seven times. based on these results cytotoxicity was determined. it was expressed as a mean percentage decrease relative to unexposed control ± s.d. control values were set at 100% viability. cytotoxicity data were fitted to a sigmoidal curve and a four parameter logistic model was used to calculate ic which is the concentration of agent which reduces cell growth by 50% under the experimental conditions. this analysis was performed using graphpad prism. this method of ic analysis has been previously published. apoptosis assay the annexin v-fitc and propidium iodide dual staining was applied for apoptosis analysis. control cells and cells treated with anticancer drug at ic dose and transfected with pgp, bcrp, mrp and mrp3-specific sirna moreover treated with anticancer drug at ic dose were harvested by trypsinization. these cells, as well as the cells floating in medium were stained with an annexin v kit according to the manufacturer’s protocol. the cells then were analyzed by flow cytometry within hr after staining. early apoptotic cells with exposed phosphatidylserine but intact cell membranes bound to annexin v-fitc however excluded pi. cells in late apoptotic stages were labeled with both annexin v-fitc and pi, whereas necrotic cells were labeled with pi only. all samples were assayed in triplicate. the experiment was conducted at least twice. statistical analysis the analysis for statistical purposes was conducted using prism version software. the one-way anova and tukey hsd post-hoc test were applied as well as regression analysis. determination of ic test has been used. the p-value < was recognized as significant, whereas, p-value < and p-value < as highly significant. the data was expressed as means +/− s.d. for molarity calculations, the molarity calculator on-line platform was used.  pgp, bcrp, mrp and mrp are expressed in canine mammary cancer cells and their expression level changes due to anticancer drug in vitro treatment rt-qpcr analysis revealed that all of the examined cell lines express pgp, bcrp, mrp and mrp. the pgp expression ranged between in cmt-u cell line and in cmt-w cell line. the bcrp expression was the lowest in cmt-u cell lines whereas the highest in cmt-w cell line. the highest expression of mrp was observed in cmt-w cell line whereas the lowest expression was noticed in cmt-u cell line. cmt-w cell line exposed the lowest expression of mrp. the highest expression of mrp was detected in cmt-u cell line. expression of efflux pumps in canine mammary cell lines in control conditions, after scrambled sirna treatment and pgp, bcrp, mrp and mrp -specific sirna treatment as well as after treatment with vinblastine, cisplatin and cyclophosphamide at ic doses. p <  was marked as*, p < was marked as **, and p <  was marked as ***. one-way anova followed by tukey hsd post-hoc test were applied. increase in expression was marked as red text, decreased in expression was marked as green text, whereas no change in transcript level was marked as black text. treatment of cancer cells using scrambled sirna did not cause any significant effect on examined gene expression, whereas treatment of these cells with pgp, bcrp, mrp or mrp -specific sirnas caused significant decrease in transcript level of targeted genes. the efficacy of silencing reaction was very high, as there was observed 88-99% decrease in pgp transcript level, 14-100% in bcrp, 94-99% in mrp and 22-100% in mrp expression. interestingly, we observed that pgp expression increased significantly in all the examined cell lines after viblastine and cisplatin treatment at ic doses, whereas decreased after cyclophosphamide treatment at ic dose. vinblastine treatment increased mrp expression in all of the examined cell lines with exception of cmt-w cell line where its expression increased also after treatment with cyclophosphamide at ic dose. in case of bcrp, we observed increased tendency in its expression after cyclophosphamide treatment and decreased in its expression after vinblastine treatment at ic dose. we are as well able to point out the increase in mrp expression in all the examined cell lines after treatment with cisplatin at ic dose. cancer cell treatment with pgp, bcrp, mrp and mrp –specific sirna decreases efflux pumps activity enhancing rhodamine- accumulation the rhodamine- efflux assay was conducted to confirm our results of gene expression silencing and their functional inhibition. it showed that after knock-down of pgp, bcrp, mrp and mrp expression level, the rhodamine-; accumulation significantly increased and that shows decreased activity of the efflux pumps. treatment of examined cells with scrambled sirna did not cause any significant effect. we observed that sirna treatment of cmt-u caused significant but slight effect on rhodamine- accumulation. in other cell lines the effect was higher, for example in p cell line the mean fluorescence related to rhodamine- accumulation in control cells was only however, after the treatment with bcrp-specific sirna is was. the highest effect of rhodamine- accumulation after sirna treatment was observed in cmt-w cell line. cancer cell treatment with sirna specific to efflux pumps decreases drug-resistance the mtt assay of cells viability after treatment with increasing doses of anticancer drugs showed significant variations in ic doses between the examined cell lines. all ic doses are listed in table  vinblastine ic dose in cmt-u cell line was nmol, whereas after pgp and mrp knock-down it was only nmol and nmol, respectively. in cmt-w cell line vinblastine ic was nmol, whereas after pgp and mrp silencing it was nmol and nmol, respectively. we showed that after pgp and mrp silencing ic was significantly lower in all the examined cell lines ranging 203– nmol and 144– nmol, respectively. significant variations were also observed in case of ic doses of cisplatin – the lowest ic dose was in cmt-u cell line, whereas the highest was in p cell line. cisplatin ic was significantly decreased in all the examined cell lines after silencing of pgp, bcrp, mrp and mrp. regarding cyclophosphamide, we also noticed significant variations in ic values. it was the lowest in cmt-w cell line, when on the other hand the highest in cmt-w cell line. treatment of cells with bcrp-specific sirna decreased cyclophosphamide ic in all of the examined cell lines. ic doses of anticancer drugs: vinblastine, cisplatin and cyclophosphamide in canine mammary cancer cell lines in control conditions and after silencing of efflux pumps expression. p <  was marked as *, p < was marked as **, and p <  was marked as ***. one-way anova followed by tukey hsd post-hoc test was applied. cancer cell treatment with anticancer drug and sirna specific to efflux pumps increases apoptosis the annexin v assay was used to examine the influence of pgp, bcrp, mrp and mrp expression knock-down on apoptosis induced by vinblastine, cisplatin and cyclophosphamide. due to any changes in cells viability based on transfection procedure were observed in mtt assay, this kind of control has been omitted in apoptosis assay. we used similar approach in our previous study. annexin v analysis revealed that treatment of cells using pgp and mrp -specific sirna together with vinblastine significantly increased number of apoptotic cells, compared to apoptosis caused by anticancer drug which was given as a single agent. the most significant effect was pointed out in cmt-w cell line: % and % increase in number of apoptotic cells after pgp and mrp silencing and vinblastine treatment, respectively. similarly, in cmt-w cell line % and % increase in number of apoptotic cells was shown after pgp and mrp silencing and vinblastine treatment, respectively. in case of cisplatin treatment at ic dose, an increase in number of apoptotic cells was noticed in each cell line after silencing of all the efflux pumps: pgp, bcrp, mrp and mrp the mostly significant result was observed in p cell line, where 40%, 41%, 42% and 40% increase in number of apoptotic cells was visible, respectively. in cmt-u cell line the highly significant effect on apoptosis was as well as in case of cisplatin treatment in cells transfected with sirna specific to: pgp, bcrp and mrp, whereas significant effect was observed in cells transfected with mrp -specific sirna. in all the examined cell lines increase in number of apoptotic cells related to cyclophosphamide treatment at ic dose was undoubtedly visually recognized after bcrp knock-down. additionally, in cmt-w cell line the significant increase in number of apoptotic cells was observed due to cyclophosphamide treatment following transfection with bcrp, mrp and mrp -specific sirna. discussion cancer cells retain the important mechanism of self-protection through the activity of multiple drug resistance transporters. the multidrug resistance is frequently associated with over-expression of two or more membrane pumps that efflux anticancer drug from the cytoplasm. this protects tumor cells against the drug effects and its correlated molecular processes. expression of efflux pumps is usually higher in tumors that originate from tissues that normally show their expression and it is always higher in the tumors than in normal cells. unfortunately, the expression and role of proteins that mediate drug resistance in canine cancer cells has not yet been recognized. as increased efflux is such a significant contributor to a multidrug resistance in cancer cells, current research is aimed at blocking or inhibiting this specific mechanism. that is why the aim of our study was to assess expression of four the most important efflux pumps: pgp, bcrp, mrp and mrp in canine mammary cancer cells as well as to investigate their role in resistance to: cisplatin, cyclophosphamide and vinblastine. because treatment of cancer cells with the use of classical inhibitors of efflux pumps often fails, we used a novel approach, that is: specific rnai to knock-down their expression. examination of each efflux pump expression in control conditions and after treatment with anticancer drug was also quite a novel approach. we observed significant differences in pgp transcript level in control conditions and after treatment with anticancer drug. in all of the examined cell lines the pgp expression increased due to vinblastine and cisplatin treatment whereas decreased due to cyclophosphamide treatment. it ought to be outlined that these results are in accordance with ic doses. in case of cisplatin treatment, expression of all four efflux pumps increased, and thus, any relationship between their transcript level and ic doses was very complicated to calculate. we also observed that cyclophosphamide treatment caused increase in bcrp expression in all of the examined cell lines. these results are in accordance with previous findings that cytotoxic drugs can initiate or increase expression of efflux pumps in cancer cells. moreover, the expression level of these efflux pumps correlates with response to anticancer therapy and patient follow up. based on our results, we have come up with the conclusion that examination of efflux pumps expression before initiation of chemotherapy could help to predict response to anticancer drugs and could be helpful in assessment of their proper doses. however, more studies in this field are required. the rnai gene silencing was highly successful, reaching even 100% of gene knock-down in case of bcrp in cmt-w cell line and mrp in case of cmt-u cell line. the results of gene knock-down were confirmed by the assay with rhodamine- efflux. to assess whether efflux pumps silencing has an influence on cancer cell susceptibility to cytostatic drugs, the ic doses were determined in transfected cells. our results showed that vinblastine ic doses significantly decreased in all the examined cell lines after pgp and mrp silencing. examination of cisplatin ic in transfected cells pointed that it was significantly lower in all of them when compared to control cells. in case of cyclophosphamide, bcrp sirna treatment significantly decreased ic dose in all of the examined cell lines. comparison of ic doses of anticancer drugs given in vitro and their maximum possible plasma concentrations showed that in case of vinblastine the plasma concentration was much lower than the ic doses. it means that the examined cancer cell lines are vinblastine-resistant and therefore this anticancer drug may be ineffective in canine mammary cancer treatment. however, more studies in this field should be conducted. in case of cisplatin, the ic doses in the three examined cell lines were lower than the reachable plasma concentrations. however, after bcrp, mrp or mrp knock-down, the cisplatin ic doses were similar or lower than the maximum possible plasma drug concentration in all of the examined cell lines. it means that knock-down of efflux pumps expression during chemotherapy can reverse cancer drug-resistance. the ic doses of cyclophosphamide in all of the examined cell lines were significantly lower than its maximum possible plasma concentration. however, in all the cases given above, targeting of abc efflux pumps using sirna significantly decreased ic doses of anticancer drugs. these results were also confirmed by apoptosis assay. in summary: knock-down of efflux pumps expression during chemotherapy could allow decreased doses of anticancer drugs thereby reducing the risk of their side effects. however, more studies in this field are required as well as large clinical trials to prove this hypothesis. our research is a completely new approach in the field of veterinary oncology. the results show that targeting of abc-efflux pumps using rnai can be promising strategy in cancer treatment. however, these in vitro studies should be confirmed using in vivo animal models.  the most important findings of our study are: the expression level of efflux pump reflects the resistance to specific anticancer drug thus it should be assessed before the initiation of chemotherapy; in canine mammary cancer pgp and mrp are responsible for vinblastine resistance, pgp, bcrp, mrp and mrp are cisplatin transporters whereas bcrp is a transporter of cyclophosphamide; treatment of cells using sirna targeting efflux pumps significantly increases cancer susceptibility to anticancer drug and allows to decrease the effective dose of anticancer drug. competing interests the authors declare that they have no competing interests. authors’ contributions kp research design, experimental design, ic determination, facs analyses, rhodamine- accumulation assay, sirna transfections, jm apoptosis analysis, km apoptosis analysis, tm manuscript preparation, mk research design, experimental design, facs analyses, sirna transfection, rt-qpcr, manuscript preparation. all authors read and approved the final manuscript. 
in both women and female dogs, the most prevalent type of malignant neoplasm is the spontaneous mammary tumor. that has become the aim of our study, to assess the expression of pgp, bcrp, mrp and mrp in canine mammary cancer cell lines and to investigate their role in cancer resistance to vinblastine, cisplatin and cyclophosphamide with using rnai approach. moreover, there are neither large studies conducting to prove of the benefit from the chemotherapy in dogs nor established chemotherapy treatment protocols available. the treatment of choice for the canine patients is surgical mastectomy. the results suggested that in canine mammary cancer, the vinblastine efflux was mediated by pgp and mrp proteins, cisplatin efflux was mediated by all four examined efflux pumps, whereas cyclophosphamide resistance was related to bcrp activity. our results have indicated the treatment of cells involving use of the sirna targeting efflux pumps could be a beneficial approach in the future. additionally, the effectiveness of each individual chemotherapeutic agent and drug resistance of canine mammary cancer have not yet been characterized. rnai silencing of these efflux pumps significantly decreased ic doses of the examined drugs in canine mammary carcinoma cells.
 in both women and female dogs, the most prevalent type of malignant neoplasm is the spontaneous mammary tumor. in dogs, half of these are malignant. the treatment of choice for the canine patients is surgical mastectomy. unfortunately, it often fails in high-risk, locally invasive mammary tumors as of during the time of the surgery the micro-metastases are present. moreover, there are neither large studies conducting to prove of the benefit from the chemotherapy in dogs nor established chemotherapy treatment protocols available. additionally, the effectiveness of each individual chemotherapeutic agent and drug resistance of canine mammary cancer have not yet been characterized. that has become the aim of our study, to assess the expression of pgp, bcrp, mrp and mrp in canine mammary cancer cell lines and to investigate their role in cancer resistance to vinblastine, cisplatin and cyclophosphamide with using rnai approach.  the results suggested that in canine mammary cancer, the vinblastine efflux was mediated by pgp and mrp proteins, cisplatin efflux was mediated by all four examined efflux pumps, whereas cyclophosphamide resistance was related to bcrp activity. rnai silencing of these efflux pumps significantly decreased ic doses of the examined drugs in canine mammary carcinoma cells.  our results have indicated the treatment of cells involving use of the sirna targeting efflux pumps could be a beneficial approach in the future. multidrug resistancechemotherapycanine mammary cancervinblastinecisplatincyclophosphamidepgpbcrpmrp1mrp3 