BACKGROUND
ghrelin was identified in human and rat stomach in  <dig> by kojima et al.
<cit>  as endogenous ligand for growth hormone secretagogue receptor type 1a .
it belongs to the g protein coupled receptor family and is widely distributed along various tissues.
ghs-r has two variants of splicing: functional type 1a and truncated type 1b, which arises from an alternative splicing and its function is still unclear
<cit> .
the ghs-r activation in pituitary results in considerably increased secretion of growth hormone .
this is the main effect of ghrelin activity in humans and in animals.
ghrelin is a highly conserved peptide among various species
<cit> .
it is mainly produced by endocrine x/a-like cells of stomach’s submucosal layer and, secreted directly into the blood
<cit> .
however, the presence of ghrelin has also been detected in other areas of gastrointestinal tract, as well as in many other tissues
<cit> .
in healthy dogs, ghrelin regulates feeding behavior and energy metabolism.
plasma ghrelin levels increase before feeding time, and decrease after eating.
these changes are associated with the insulin and glucose concentration
<cit> .
some studies on dogs suggested role of ghrelin in the development of adipositas due to higher circulating levels of ghrelin in obese dog than in normal or lean animals
<cit> .
the ghrelin expression  has also been observed in multiple endocrine and non-endocrine tumors and related cell lines in humans, such as: pituitary adenomas, thyroid follicular cancer and parathyroid adenomas, pancreatic-related endocrine tumors, oral squamous cell carcinoma, gastric carcinoids, colon cancer, renal carcinoma, bronchial carcinoid, testicular and ovarian tumors, adrenocortical tumors, prostate cancer and breast cancer
<cit> .
the co-expression of ghrelin and its receptor in various tumors and cancer cell lines may indicate their autocrine/paracrine role in the tumor growth and development.
thus, the effect of ghrelin on cancer cells was investigated in multiple in vitro experiments in humans and rats, however the result are contradictory
<cit> .
the role of ghrelin/ghs-r axis in tumor initiation, progression and metastasis in animals is still unclear.
as far as we realize, there are no published studies about the expression and function of ghrelin in canine tumors.
our previous studies have shown that cell lines with the highest proliferative potential have high expression of ghs-r
<cit> .
moreover, we found its up-regulation in metastatic cancer cell lines  what may indicate the role of ghrelin and ghs-r in metastasis
<cit> .
this study was therefore designed to characterize the expression of ghrelin and its receptor in canine mammary cancer tissues and cancer cell lines.
we have also examined the effect of ghrelin on cancer cells proliferation, apoptosis, migration and invasion.
methods
cell culture
the cell lines used for this study have previously been given an account of król et al.
<cit> .
two adenocarcinoma cell lines isolated from the canine mammary gland tumors  and two cell lines isolated from their lung metastases  were kindly donated by prof. dr. maciej ugorski and dr. joanna polanska from wroclaw university .
the cell lines were cultivated in rpmi- <dig> medium containing 10% heat-inactivated fetal bovine serum ,  <dig> u/ml penicillin,  <dig> μg/ml streptomycin,  <dig>  μg/ml amphotericin b  and grown in tissue culture flasks  in an atmosphere of 5% co <dig> and 95% humidified air at 37°c, and routinely subcultured every second day.
tissue samples
canine mammary tumor tissue sections were derived from the archives of the department of pathology and veterinary diagnostics, faculty of veterinary medicine, warsaw university of life sciences –wuls .
the samples were surgically obtained during the mastectomy from  <dig> female dogs of various breeds.
the tissue samples were fixed in 8% neutral buffered formalin and routinely embedded in paraffin.
eighteen canine mammary tumors were snap frozen using liquid nitrogen and archived at -80°c.
most of the cases’ information about the presence/absence of metastases was known.
the tumors that gave metastases were surgically removed together with the metastatic site.
the presence of neoplastic cells on the metastatic site was histologically confirmed.
the immunohistochemical examination of cytokeratin, vimentin, smooth muscle actin, s <dig> protein and p <dig> protein expression was performed .
histological diagnoses were achieved on haematoxylin and eosin  stained slides according to the world health organization  histological classification and mammary tumors of the dog and cat classification
<cit> .
tumor grading was based on the misdorp
<cit>  classification and was evaluated in respect to tubule formation, degree of differentiation and mitotic index as: the 1st, the 2nd and the 3rd grade of malignancy.
the tumors were classified as benign adenomas or malignant carcinomas .
the tumors were divided into five categories: benign adenomas , malignant adenocarcinomas: of the 1st grade of malignancy , of the 2nd grade of malignancy , of the 3rd grade of malignancy  and metastatic  .
immunohistochemistry
four micrometer  sections from paraffin blocks containing tumor tissue were baked in 37°c overnight.
after dewaxing in xylene and rehydration in ethanol, for antigen retrieval, the slides were placed in  <dig>  m citrate buffer, ph  <dig>  and boiled in the decloaking chamber.
the cell lines were cultured on lab-tek  4-chamber culture slides for  <dig> hrs and subjected to the immunohistochemical analysis after ethanol  fixation .
the samples were incubated in the peroxidase blocking reagent  for  <dig> min at room temperature prior to the antibody incubation.
after  <dig> min incubation in 5% bovine serum albumin , the following primary antibodies were used : rabbit polyclonal anti-ghrelin  diluted 1: <dig> and incubated one hour at room temperature; rabbit polyclonal anti-growth hormone secretagogue receptor  diluted  <dig> μg/ml and incubated overnight at +4°c.
for the staining the envision kit  was used.
to develop a colored product, the  <dig> ′-diaminobenzidine  substrate was used .
finally, the hematoxyline was used for nuclei counterstaining.
for each immunohistochemical experiment of paraffin slides, positive controls were set aside: canine stomach  and canine pituitary .
for each experiment of paraffin slides and cell lines the negative control was stained without use of primary antibodies.
three consecutive tissue sections and four slides of each cell line were analyzed.
ten to  <dig> pictures of each slide were taken  using olympus microscopy bx <dig>  only the regions of cancer cells and stroma were analyzed in the primary tumors whereas the necrotic regions were avoided.
the number of ghrelin positive cells  and the colorimetric intensity of the ghs-r expression reflected as ihc-stained antigen spots  were counted by a computer-assisted image analyzer .
the antigen spot color intensity is expressed as a mean pixel integrated optical density  on a 1- <dig>  scale.
real-time qpcr
total rna from the frozen canine mammary tissues and cell lines suspensions 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 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.
the ghsr and ghrl primers were designed using primer <dig> software  and checked using oligo calculator  and primer-blast .
the used sequences for ghsr were as follow: 1) ttccacgtgggcgatatttatt and tggcagcactgaggtagaagagg  and cctggtatcctttgtcctcttct and ctttcatccttcagagtggagag .
the optimal annealing time for the first pair was  <dig> sec, whereas optimal annealing temperature was 59°c.
the optimal annealing time for the second pair was  <dig> sec, whereas optimal annealing temperature was 63°c.
the used sequences for ghrl were as follow: tcaggtgagtgcttctaggg and cactcgggaagtttcttcaa.
the optimal annealing time was  <dig> sec, whereas optimal temperature was 61°c.
rps <dig> and hprt genes were used as a non-regulated reference for the normalization of target gene expression .
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 are expressed as ΔΔct values .
the experiment was conducted three times.
cell viability assay
cell viability  was quantified by mtt assay.
cells were seeded into 96-well plate  at the concentration of  <dig> ×  <dig> cells per well.
when cells reached 60-70% confluence, the medium was replaced with medium containing 10% fbs and n-octanoylated ghrelin peptide  at the concentrations of  <dig>   <dig>   <dig> and  <dig> nm.
to protect ghrelin from inactivation, the culture medium contained also  <dig> nm serine protease inhibitor, 4- benzenesulphonyl fluoride hydrochloride
<cit> .
the cultures were incubated for  <dig> hours.
in order to determine the mechanism by which ghrelin increased proliferation/viability of the cells , they were additionally pre-treated for  <dig> hr with the  <dig> and  <dig> μm of ghs-r-specific antagonist -ghrp <dig> .
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 five times .
cell proliferation assay
brdu cell proliferation assay was carried out according to the brdu labeling protocol .
briefly, canine mammary cancer cells where cultivated in culture flask to reach 60-70% confluence.
the medium was then removed and replaced medium containing 10% fbs with  <dig> nm aebsf and:  ghrelin at the concentration of  <dig>   <dig>   <dig> and  <dig> nm or  ghs-r-specific peptide antagonist -ghrp <dig>  for  <dig> hrs.
then,  <dig> μm of brdu  was added to culture medium for one hour.
the cells were washed twice with pbs, harvested by trypsinization, suspended in pbs and fixed in ice-cold ethanol .
the cells were then stored at -20°c for  <dig> hrs and then washed with wash buffer  and suspended in denaturing solution  for  <dig> min at room temperature  to denature dna.
the cells were then washed again with wash buffer and resuspended in  <dig>  m sodium borate  of ph  <dig> , for  <dig> minutes at rt, to neutralize any residual acid.
the cells were then washed with wash buffer and incubated with fitc-conjugated anti-brdu antibodies  and isotype control  for  <dig> min at rt.
cells were then incubated with  <dig> μl/ml of propidium iodide   for  <dig> min at rt.
all the samples were analyzed using flow cytometry .
the experiment was conducted in three replicates.
sirna transfection
the sirna transfection procedure on canine mammary cancer cells has been given an account of in our previous paper
<cit> .
however, the procedure was optimized for cmt-w1m cell line.
thus the cell density, transfection reagent toxicity and transfection efficacy has been assessed .
the canine  ghsr sequence was obtained from gene bank with accession number .
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 at last two duplexes were chosen for further experiments  .
each duplex was used at  <dig> different concentrations  the mixture of both duplexes was also used .
the rna interference has been assessed by real-time qpcr.
transfected and control cmt-w1m cells ) were scraped, and the total rna from the cell suspension samples was isolated.
because during optimization of the sirna transfection procedure for cmt-w1m cell line any significant changes in cells viability between control cells and cells treated with only transfection reagent has been observed , the mock-transfected control has been omitted.
apoptosis assay
the annexin v-fitc and propidium iodide  dual staining was applied for apoptosis analysis.
normal cells and cells treated with  ghrelin at the concentration of  <dig>   <dig>   <dig> and  <dig> nm and  <dig> nm aebsf,  inhibitor of ghrelin receptor -ghrp <dig> at the concentration of  <dig> μm,  <dig> μm,  <dig> μm,  <dig> μm and  <dig> μm for  <dig> hrs  transfection reagent + non-coding rna  and  ghsr sirna, were harvested by trypsinization and together with the cells floated in medium were stained using an annexin v kit , according to the manufacturer’s protocol.
the cells were immediately  analyzed by flow cytometry .
early apoptotic cells with exposed phosphatidylserine but intact cell membranes bound to annexin v-fitc but 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, and each experiment was performed three times .
in vitro wound healing assay
scratch assay was conducted to assess the influence of ghrelin on canine mammary carcinoma cell motility.
the cells were seeded in a high density at  <dig> mm petri dishes .
after  <dig> hrs, the medium was removed and replaced medium containing 10% fbs with  <dig> nm aebsf and  ghrelin at concentration of  <dig>   <dig>   <dig> and  <dig> nm or  specific ghs-r inhibitor [d-lys3-ghrp <dig> at concentration of  <dig> um,  <dig> μm,  <dig> um,  <dig> μm and  <dig> μm for  <dig> hrs.
when the cells reached full confluence, the monolayers were wounded by scratching the surface as uniformly and straight as possible with a pipette tip  at least three times.
the images of cells invading the scratch were captured at indicated time points  using phase contrast microscopy .
the pictures have been analyzed using a computer-assisted image analyzer .
the migration rate was expressed as percentage of scratch closure and was calculated as follows: % of scratch closure = a-b/a, where  is a distance between edges of the wound, and  is the distance which remained cell-free during cell migration to close the wound
<cit> .
the values are the means of three independent wound fields from three independent experiments .
invasion and migration assay
the bd biocoat™ 24-multiwell invasion system  pre-coated with bd matrigel™ matrix were used according to the manufacturer’s protocol.
the insert plates were prepared by rehydrating the bd matrigel™ matrix layer with phosphate buffered saline  for two hours at 37°c.
the rehydration solution was then carefully removed, and  <dig> μl of cell suspension was added to the apical chambers .
cell suspension was prepared by trypsinizing cell monolayers  and resuspending the cells in rpmi  <dig> medium containing  <dig> % fbs,  <dig> nm of aebsf and ghrelin at the concentration of  <dig>   <dig> and  <dig> nm.
then  <dig> μl of chemoattractant  was added to the each of the basal chambers.
assay plates were incubated for  <dig> hours at standard culturing conditions.
followed incubation medium was carefully removed from apical chamber and insert system was transferred into a second 24-well plate containing  <dig> μl of  <dig>  μg/ml calcein am in hanks’ balanced salt solution .
plates were incubated one hour at standard culturing conditions.
then the fluorescence of invaded cells was measured at excitation wavelength  <dig> nm and emission wavelength  <dig> nm using florescent plate reader with bottom reading capabilities infinite  <dig> pro tecan™ .
all samples were assayed in triplicate, and each experiment was conducted three times .
to evaluate migratory potential the bd falcon™ fluoroblock™ 24-multiwell insert plates   was used.
the determination protocol for the canine mammary cancer cells migration was the same as the invasion assay, with the exception that no matrigel was used and rehydrating of the plate was omitted.
in order to determine fluorescence of cells invaded through membrane coated by matrigel fluorescence microscopic analysis using olympus microscopy bx <dig> at x <dig> magnifications was applied.
statistical analysis
the statistical analysis was conducted using prism version  <dig>  software .
the one-way anova and tukey hsd  post-hoc test, dunnett’s test and t-test were applied as well as regression analysis.
the p-value <  <dig>  was regarded as significant whereas p-value <  <dig>  and p-value <  <dig>  as highly significant.
the data was expressed as means +/- s.e.m.
unless otherwise stated.
the in vitro wound healing assay was analyzed using two-way rm anova and bonferroni post-hoc test.
RESULTS
expression of ghrelin and ghs-r in canine mammary tumors and in canine mammary carcinoma cell lines
the expression of ghrelin and growth hormone secretagogue receptor at mrna level was detected in adenocarcinomas of the 1st, the 2nd and the 3rd grade of malignancy .
no significant differences in ghrelin mrna level have been observed between these groups , however mrna level of growth hormone secretagogue receptor was significantly lower  in tumors of the 3rd grade of malignancy compared to the tumors of the 2nd grade of malignancy .
the expression of ghrelin peptide was detected in both benign and malignant canine mammary tumors.
the analysis showed that ghrelin immunoreactivity was significantly lower  in benign tumors  comparing to the tumors that gave local or distant metastases   .
the number of ghrelin-positive cells increased in the group of non-metastatic tumors from benign to the most malignant samples, however no significant differences between these groups have been observed .
immunohistochemistry also demonstrated expression of growth hormone secretagogue receptor  in all benign and malignant canine mammary tumors.
significantly lower expression  of this antigen was found in tumors of the 3rd grade of malignancy  than in tumors of the 2nd grade of malignancy or in metastatic tumors  .
no significant differences have been observed between the other tumor groups.
real-time pcr analysis showed ghrelin and growth hormone secretagogue receptor mrna expression in all the examined cell lines .
growth hormone secretagogue receptor expression was significantly higher in the cell lines isolated from mammary carcinoma metastases to the lungs .
immunohistochemical examination confirmed expression of both peptides in all of the examined cell lines .
significantly lower expression  of ghrelin antigen was detected in cell line isolated from primary canine mammary adenocarcinoma  than in the cell line isolated from its metastasis to the lungs  .
the immunoreactivity of ghrelin was similar in another pair of the cell lines  .
however, ghs-r immunoreactivity was significantly lower  in both canine mammary carcinomas isolated from primary tumors than in cell lines isolated from their metastases to the lungs: 5230 ±  <dig>  and 6729 ±  <dig>  in cmt-w <dig> and cmt-w1m cell lines, respectively and 6189 ±  <dig>  and 7792 ±  <dig>  in cmt-w <dig> and cmt-w2m cell lines, respectively .
ghrelin enhances viability and proliferation of canine mammary cancer cells
ghrelin treatment increased cells viability  and cells proliferation  in three of the examined cell lines except the cmt-w2m cell line.
the viability of the cmt-w <dig> cells was significantly increased by approximately 14% when treated with  <dig> nm n-octanoylated ghrelin  and 12% when treated with  <dig> nm ghrelin  compared to control cells .
ghrelin treatment at dose of  <dig> nm and  <dig> nm also increased cell viability in the cmt-w1m cell line  .
the highest response for ghrelin treatment was observed in the cmt-w <dig> cell line.
the viability of the cmt-w <dig> cells was significantly increased when treated with ghrelin at the concentration of  <dig> nm ,  <dig> nm ,  <dig> nm  and  <dig> nm  by approximately 25%, 19%, 18% and 16%, respectively .
metabolic activity of the cmt-w2m cells was not significantly changed after incubation with ghrelin .
the pre-treatment of cells for  <dig> hr with specific inhibitor of ghs receptor  completely abolished the cells response for ghrelin  what confirmed that in examined cell lines ghrelin acts via ghs-r.
the role of ghrelin on cellular proliferation was confirmed by brdu incorporation assay.
ghrelin treatment at  <dig> nm and  <dig> nm doses increased proliferation of cancer cells to  <dig>  ±  <dig> % and  <dig>  ±  <dig> %  in cmt-w <dig> cell line and to  <dig>  ±  <dig> % and  <dig>  ±  <dig> %  in cmt-w1m cell line .
grelin treatment at the concentration of  <dig> nm caused increase of proliferation to  <dig>  ±  <dig> %  in cmt-w <dig> cell line, however treatment with specific ghs-r inhibitor at  <dig> μm dose induced anti-proliferative effect, which was not observed in other examined cell lines .
cell proliferation was not affected by ghrelin in the cmt-w2m cell line .
in both experiments it was impossible to create a dose–response curve.
the results indicated that smaller doses of ghrelin  significantly increased cancer cells proliferation, however any dose-dependent effect was not observed .
ghsr rna interference
based on the mtt assay results, the optimal cell number for transfection experiment was  <dig>  <dig> per well on 96-well plate.
culture of  <dig>  <dig> cells per well at the beginning of the experiment ensured 60% confluence  at the day of transfection.
transfection reagent toxicity evaluation revealed that in case of cmt-w <dig> cell line the best viability results were reached using lipofectamine rnaimax .
the highest viability of the cells  was reached with lipofectamine rnaimax at the concentration of  <dig>  μl per well.
the confocal microscopy observations and subsequent computer-assisted image analysis  revealed that lipofectamine rnaimax at the concentration of  <dig>  μl per  <dig>  <dig> cells per well proved a highly effective transfection reagent ensuring the transfection efficiency at the level of 90%.
consequently, experiments were conducted under the same conditions.
the control cells used for further experiments were cultured under the transfection conditions with tranfection reagent + non-coding rna.
the real-time qpcr and immunohistochemistry results confirmed the effect of ghsr gene silencing.
the lowest expression of ghsr  was found in the cells treated with the mixture of ghsr <dig> and ghsr <dig> sequences .
only about 15-28% reduction of ghsr expression, comparing against the control cells, was observed in case of ghsr <dig> sequence given at two concentrations .
we observed  <dig> % and  <dig> % reduction of expression compared to control cells after the ghsr <dig> and ghsr2′ interference, respectively .
the immunohistochemical studies revealed higher efficacy of gene silencing at the protein level than at the mrna level.
the most effective was ghsr <dig> sequence causing  <dig> % reduction in protein levels compared against the control cells.
ghrs1′ sequence caused  <dig> % of protein expression reduction, whereas ghsr <dig> sequence caused  <dig> % reduction.
ghsr2′ and mixture of ghsr <dig> and ghsr <dig> sequences caused  <dig> % and  <dig> % reduction of protein expression.
all these results have been statistically significant at the level of p <  <dig> .
for apoptosis analysis the ghsr <dig> sirna duplex was used.
ghrelin is an anti-apoptotic factor in canine carcinoma cell lines
for analysis of ghrelin influence on apoptosis, the annexin v and pi double staining was applied.
the number of apoptotic cells represented as a percentages of annexin-v-positive cells was significantly decreased after the ghrelin treatment at the dose of  <dig> nm  in the cmt-w <dig> cell line  and in the cmt-w <dig> cell line   .
anti-apoptotic effect of ghrelin was higher in the cell lines isolated from lung metastases of mammary cancer.
in control conditions  <dig> % and  <dig> % of cells has been identified as apoptotic in cmt-w1m and cmt-w2m cell line, respectively.
after ghrelin treatment at the concentration of  <dig> nm and  <dig> nm the number of apoptotic cells decreased to 4% and  <dig> %  in the cmt-w1m cell line and to  <dig> % and  <dig> %  in the cmt-w2m cell line.
the statistical analysis showed that differences were highly significant  .
among all of the examined cell lines only in cmt-w1m cell line incubation with  <dig> μm ghs-r-specific peptide inhibitor  for 24hrs significantly increased the number of apoptotic cells  .
in order to investigate if this effect was caused by toxicity of this peptide, we knocked-down the ghsr expression by rnai to confirm the functional importance of the ghs-r in apoptosis in cmt-w1m cell line.
our results showed that sirna treatment significantly increased number of early apoptotic cells in cmt-w1m cell line  up to  <dig> % .
cancer cells treatment with ghs-r inhibitor increased number of early apoptotic cells highly significantly  up to  <dig> % .
these differences may be caused by not completely knock-down of ghsr expression .
ghrelin promotes migration and invasion of canine mammary carcinoma cells
the wound healing assay showed that ghrelin treatment at the concentration of  <dig> nm increased migratory abilities of the cmt-w <dig>  cmt-w1m and cmt-w <dig> cell lines , however, no effect on cmt-w2m cells has been observed .
cmt-w <dig> cells incubated with ghrelin  almost completely closed the wound  in  <dig> hours after scratching, whereas cmt-w <dig> control cells after  <dig> hrs closed only  <dig>  ±  <dig> % of the wound .
similarly, cmt-w1m cells incubated with ghrelin  closed the wound at the level of  <dig>  ±  <dig> % in  <dig> hours after scratching, whereas cmt-w1m control cells after  <dig> hrs closed only  <dig>  ±  <dig> % of the wound .
also, cmt-w <dig> cells incubated with ghrelin  closed the wound at the level of  <dig>  ±  <dig> % in  <dig> hours after scratching, whereas cmt-w <dig> control cells after  <dig> hrs closed only  <dig>  ±  <dig> % of the wound .
administration of ghs-r-specific peptide inhibitor  at  <dig> μm dose decreased the motility of the cmt-w <dig> cells, whereas had no effect on motility of the cmt-w1m or cmt-w <dig> cells even at the concentration of  <dig> μm.
cmt-w <dig> cells incubated with ghrelin receptor inhibitor  closed  <dig>  ±  <dig> % and  <dig>  ±  <dig> % of the wound in  <dig> and  <dig> hrs after scratching  .
cmt-w2m control cells and ghrelin  treated cells closed  <dig>  ±  <dig> % and  <dig>  ±  <dig> % of the wound  in  <dig> hours after scratching, however the difference was not statistically significant .
cmt-w2m completely  closed the wound after  <dig> hours after scratching in control conditions and after ghrelin treatment.
ghs-r-specific peptide inhibitor  also had no effect on motility of those cells .
cell migration assessed in boyden chambers was increased by incubation with  <dig> nm of ghrelin in cmt-w <dig> cell line and in the other cell lines  by incubation with  <dig> and  <dig> nm of ghrelin .
cancer cell invasion was increased by ghrelin treatment for  <dig> hours.
ghrelin was the most effective at the  <dig> nm concentration in cmt-w <dig>  cmt-w1m, cmt-w2m, whereas it was the most effective in cmt-w <dig> cell line at  <dig> nm concentration .
the analysis conducted using olympus microscopy bx <dig> confirmed the results .
discussion
the co-expression of ghrelin and ghs-r has previously been observed in various neoplasms and related cancer cell lines in humans .
it may suggest their role in neoplastic cell growth and development through autocrine/paracrine mechanism.
however, as far as we realize, there are no published reports regarding similar data on tumors in domestic animals.
this is the first report of ghrelin and its receptor expression in canine mammary tumor tissues and cell lines isolated from the canine primary carcinomas  and from their lung metastases .
we have demonstrated the expression of ghrelin in benign and malignant canine mammary tumors  and in all of the examined cancer cell lines .
interestingly, we observed significantly higher expression of ghrelin in the distant metastases than in the primary carcinomas, which suggest that ghrelin might be involved in mechanism of metastasis .
a recent study on human colorectal cancers indicated that ghrelin might initiate malignant transformation and might contribute to the cancer spread
<cit> .
it was also hypothesized that ghrelin mediates the physiological responses of tissues which surround the tumor, to malignant changes
<cit> .
the presence of ghs-r mrna has been previously detected in all of the examined canine cancer cell lines
<cit> .
similarly to mrna expression patterns, ghs-r protein showed significantly higher expression in cell lines isolated from lung metastases of mammary cancer  what may indicate its role in metastasis .
moreover, we have demonstrated that expression of ghrelin receptor varies in canine mammary tumors depending on the histological grade of malignancy .
the ghs-r expression level was higher in well or moderately differentiated cancers of the 1st and the 2nd grade of malignancy compared to poorly-differentiated cancers of the 3rd grade of malignancy.
this intriguing finding overlaps with those reported previously, that expression and functionality of specific binding sites for ghs  in various types of breast and thyroid carcinomas was maintained in better-differentiated tumors and was decreased in less-differentiated neoplasms
<cit> .
similar expression patterns of ghs-r were observed in human gastric and colorectal cancers
<cit> , where the ghs-r expression level was lowest in undifferentiated tumors and correlated inversely with histological grade of malignancy and tnm stage.
moreover, patients with more weight loss showed lower expression of ghs receptor
<cit> .
therefore, decreased ghrelin receptor expression seemed to correlate with poor prognostic factors such as poor differentiation, advanced stage and malnutrition.
the next step of this study was to assess a role of ghrelin in cancer cell biology.
our results revealed that ghrelin stimulates canine mammary cancer cells proliferation and inhibits apoptosis, possibly via an autocine/paracrine mechanism.
although the role of ghrelin in cancer cells was investigated in multiple experiments using human and rat cancer cell lines, the results were equivocal.
some investigators have found ghrelin as antiproliferative factor
<cit> , but other have found it a growth stimulator of cancer cells
<cit> .
cassoni and co-workers described an inhibition of cell proliferation in human thyroid, breast and lung cancer by ghrelin
<cit> .
authors found that ghrelin given for  <dig> hrs at relatively high doses  decreased cell proliferation.
however, after incubation of these cells with ghrelin for  <dig> hrs, the effect was abolished
<cit> .
volante et al.
<cit>  have confirmed antiproliferative effect of ghrelin in thyroid cancer cells showing significant growth reduction after  <dig> hrs and  <dig> hrs of incubation with ghrelin at the concentration of 100- <dig> nm.
interestingly, cassoni et al.
<cit>  have showed dose-dependent effect of ghrelin treatment in prostate cancer cells.
contrary to the mentioned reports the pro-proliferative effect of ghrelin in several human cancer cell lines has also been well documented.
ghrelin at doses ranging from  <dig> to  <dig> nm, which encompasses normal circulating ghrelin levels increased cellular proliferation after  <dig> h of incubation in breast and prostate cancers
<cit> .
ghrelin treatment significantly stimulated cells growth in the highly metastatic  breast cancer cell line up to 36% above control.
the most significant proliferative response to ghrelin treatment occurred at the concentrations of  <dig> and  <dig> nm in the mda-mb- <dig> cell line, and 1– <dig> nm in the mda-mb- <dig> cell line
<cit> .
thus, these results show that ghrelin given in high doses inhibits cancer cell proliferation, whereas given at low doses increases proliferation.
our results also indicated that ghrelin given at the dose of  <dig> or  <dig> nm increased canine mammary cancer cells proliferation in three of four examined cell lines , whereas higher doses caused not significant effect also in three of four examined cell lines.
in one cell line, ghrelin did not cause any affect.
to clarify the role of ghrelin in cell proliferation the cell cycle analysis was conducted
<cit> .
this study revealed that although ghrelin did not change the proportion of cells in the g0/g <dig>  s or g2/m phases of cell cycle, it caused a decrease in the number of apoptotic cells in sub-g <dig> phase.
in fact ghrelin was reported to be the antiapoptotic factor in colonic cancer cells
<cit>  as well as in many other types of cells like adipocytes
<cit> , osteoblastic cells
<cit> , or human endothelial cells
<cit> .
the antiapoptotic activity of ghrelin is also supported by our findings .
we have showed that ghrelin decreases the number of apoptotic cells in all of the examined canine mammary cancer cell lines .
because in cmt-w1m cell line we observed a significant increase in number of apoptotic cells after the treatment with ghrelin inhibitor, our next step was to knock-down the ghsr gene to check if the toxic effect was caused by the inhibitor itself or by blocking the ghs-r. our results showed that treatment of cmt-w1m cell line with ghsr-specific sirna also increased the number of apoptotic cells  .
an increase in number of apoptotic cells was slight, however significant, which can be explained by efficacy of gene silencing.
in our experiment only 85% of cells remained transfected.
recently, a few studies have focused on the involvement of ghrelin in cancer cells motility and ability to metastasis.
duxbury and co-workers
<cit>  investigated the role of ghrelin in metastatic potential of poorly differentiated human pancreatic cancer cells.
they found that ghrelin at  <dig> nm concentration increased not only the proliferation rate of cancer cells but also cellular motility and invasiveness .
another study showed the promotion of proliferation and invasiveness of human colorectal cancer cells by ghrelin in an autocrine and paracrine manner.
this effect was almost completely abolished when the cells were pre-treated with either ghs-r antagonist  or a neutralizing ghrelin – specific antibody
<cit> .
furthermore dixit et al.
<cit>  revealed that ghrelin acting via functional ghs-r1a caused increase in intracellular calcium mobilization and led to actin polymerization and membrane ruffling, which resulted in an increase of migration and invasion of carcinoma cells.
our study also showed a role of ghrelin in cancer cells migration and invasion.
we have demonstrated that ghrelin given at the concentration of  <dig> nm increased the migration ability of canine carcinoma cell lines cmt-w <dig> and cmt-w1m, evaluated by wound healing test .
moreover ghrelin given at the concentration of  <dig> and/or  <dig> nm significantly increased migration and invasion of all the examined canine carcinoma cell lines .
the fact that the examined carcinoma cell lines respond in different manner to the various ghrelin doses may be related with differences in endogenous ghrelin production levels.
differences between effective ghrelin doses that stimulate cells proliferation or survival and doses that increase cells migration can be explained by the fact that these different cellular activities are mediated by different cellular pathways.
literature data show that ghrelin increases cellular migration acting on camkii, ampk, and nf-kb pathways
<cit> , whereas increases cellular proliferation and survival acting on erk1/2
<cit> .
thus, probably various ghrelin doses are required to activate various cellular pathways.
CONCLUSIONS
we have showed that ghrelin and ghs-r are differentially expressed in canine mammary tumors.
the lowest expression of ghs-r in tumors of the 3rd grade of malignancy may by a potential prognostic factor.
taken together, the data demonstrated that exposure to low doses of ghrelin stimulate cellular proliferation, inhibit apoptosis and promote motility and invasion of canine mammary carcinoma cells.
however, further studies are required to investigate the molecular mechanism responsible for ghrelin action in cancer cells.
our study indicates that inhibition of ghrelin/ghs-r axis may be a potential therapeutic target of mammary cancers in dogs in the future.
competing interest
the authors declare that they have no competing interests.
authors’ contributions
km: mtt assays, brdu assay, migration and invasion assays, wound healing assay, immunohistochemistry, statistical analysis, manuscript and figures preparation; kmp: sirna transfection, real-time qpcr; ejo: sirna transfection, real-time qpcr; id: pathological examination of mammary carcinomas; jm: rna isolation, immunohistochemistry; tm: research design; mk: research design, experiments design, apoptosis tests, facs analyses, manuscript preparation, figures preparation.
all authors read and approved the final manuscript.

 ghrelin was identified in human and rat stomach in by kojima et al. as endogenous ligand for growth hormone secretagogue receptor type 1a. it belongs to the g protein coupled receptor family and is widely distributed along various tissues. ghs-r has two variants of splicing: functional type 1a and truncated type 1b, which arises from an alternative splicing and its function is still unclear. the ghs-r activation in pituitary results in considerably increased secretion of growth hormone. this is the main effect of ghrelin activity in humans and in animals. ghrelin is a highly conserved peptide among various species. it is mainly produced by endocrine x/a-like cells of stomach’s submucosal layer and, secreted directly into the blood. however, the presence of ghrelin has also been detected in other areas of gastrointestinal tract, as well as in many other tissues. in healthy dogs, ghrelin regulates feeding behavior and energy metabolism. plasma ghrelin levels increase before feeding time, and decrease after eating. these changes are associated with the insulin and glucose concentration. some studies on dogs suggested role of ghrelin in the development of adipositas due to higher circulating levels of ghrelin in obese dog than in normal or lean animals. the ghrelin expression has also been observed in multiple endocrine and non-endocrine tumors and related cell lines in humans, such as: pituitary adenomas, thyroid follicular cancer and parathyroid adenomas, pancreatic-related endocrine tumors, oral squamous cell carcinoma, gastric carcinoids, colon cancer, renal carcinoma, bronchial carcinoid, testicular and ovarian tumors, adrenocortical tumors, prostate cancer and breast cancer. the co-expression of ghrelin and its receptor in various tumors and cancer cell lines may indicate their autocrine/paracrine role in the tumor growth and development. thus, the effect of ghrelin on cancer cells was investigated in multiple in vitro experiments in humans and rats, however the result are contradictory. the role of ghrelin/ghs-r axis in tumor initiation, progression and metastasis in animals is still unclear. as far as we realize, there are no published studies about the expression and function of ghrelin in canine tumors. our previous studies have shown that cell lines with the highest proliferative potential have high expression of ghs-r. moreover, we found its up-regulation in metastatic cancer cell lines what may indicate the role of ghrelin and ghs-r in metastasis. this study was therefore designed to characterize the expression of ghrelin and its receptor in canine mammary cancer tissues and cancer cell lines. we have also examined the effect of ghrelin on cancer cells proliferation, apoptosis, migration and invasion. methods cell culture the cell lines used for this study have previously been given an account of król et al.. two adenocarcinoma cell lines isolated from the canine mammary gland tumors and two cell lines isolated from their lung metastases were kindly donated by prof. dr. maciej ugorski and dr. joanna polanska from wroclaw university. the cell lines were cultivated in rpmi- medium containing 10% heat-inactivated fetal bovine serum, u/ml penicillin, μg/ml streptomycin, μg/ml amphotericin b and grown in tissue culture flasks in an atmosphere of 5% co and 95% humidified air at 37°c, and routinely subcultured every second day. tissue samples canine mammary tumor tissue sections were derived from the archives of the department of pathology and veterinary diagnostics, faculty of veterinary medicine, warsaw university of life sciences –wuls. the samples were surgically obtained during the mastectomy from female dogs of various breeds. the tissue samples were fixed in 8% neutral buffered formalin and routinely embedded in paraffin. eighteen canine mammary tumors were snap frozen using liquid nitrogen and archived at -80°c. most of the cases’ information about the presence/absence of metastases was known. the tumors that gave metastases were surgically removed together with the metastatic site. the presence of neoplastic cells on the metastatic site was histologically confirmed. the immunohistochemical examination of cytokeratin, vimentin, smooth muscle actin, s protein and p protein expression was performed. histological diagnoses were achieved on haematoxylin and eosin stained slides according to the world health organization histological classification and mammary tumors of the dog and cat classification. tumor grading was based on the misdorp classification and was evaluated in respect to tubule formation, degree of differentiation and mitotic index as: the 1st, the 2nd and the 3rd grade of malignancy. the tumors were classified as benign adenomas or malignant carcinomas. the tumors were divided into five categories: benign adenomas, malignant adenocarcinomas: of the 1st grade of malignancy, of the 2nd grade of malignancy, of the 3rd grade of malignancy and metastatic. immunohistochemistry four micrometer sections from paraffin blocks containing tumor tissue were baked in 37°c overnight. after dewaxing in xylene and rehydration in ethanol, for antigen retrieval, the slides were placed in m citrate buffer, ph and boiled in the decloaking chamber. the cell lines were cultured on lab-tek 4-chamber culture slides for hrs and subjected to the immunohistochemical analysis after ethanol fixation. the samples were incubated in the peroxidase blocking reagent for min at room temperature prior to the antibody incubation. after min incubation in 5% bovine serum albumin, the following primary antibodies were used: rabbit polyclonal anti-ghrelin diluted 1: and incubated one hour at room temperature; rabbit polyclonal anti-growth hormone secretagogue receptor diluted μg/ml and incubated overnight at +4°c. for the staining the envision kit was used. to develop a colored product, the ′-diaminobenzidine substrate was used. finally, the hematoxyline was used for nuclei counterstaining. for each immunohistochemical experiment of paraffin slides, positive controls were set aside: canine stomach and canine pituitary. for each experiment of paraffin slides and cell lines the negative control was stained without use of primary antibodies. three consecutive tissue sections and four slides of each cell line were analyzed. ten to pictures of each slide were taken using olympus microscopy bx only the regions of cancer cells and stroma were analyzed in the primary tumors whereas the necrotic regions were avoided. the number of ghrelin positive cells and the colorimetric intensity of the ghs-r expression reflected as ihc-stained antigen spots were counted by a computer-assisted image analyzer. the antigen spot color intensity is expressed as a mean pixel integrated optical density on a 1- scale. real-time qpcr total rna from the frozen canine mammary tissues and cell lines suspensions 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 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. the ghsr and ghrl primers were designed using primer software and checked using oligo calculator and primer-blast. the used sequences for ghsr were as follow: 1) ttccacgtgggcgatatttatt and tggcagcactgaggtagaagagg and cctggtatcctttgtcctcttct and ctttcatccttcagagtggagag. the optimal annealing time for the first pair was sec, whereas optimal annealing temperature was 59°c. the optimal annealing time for the second pair was sec, whereas optimal annealing temperature was 63°c. the used sequences for ghrl were as follow: tcaggtgagtgcttctaggg and cactcgggaagtttcttcaa. the optimal annealing time was sec, whereas optimal temperature was 61°c. rps and hprt genes were used as a non-regulated reference for the normalization of target gene expression. 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 are expressed as ΔΔct values. the experiment was conducted three times. cell viability assay cell viability was quantified by mtt assay. cells were seeded into 96-well plate at the concentration of × cells per well. when cells reached 60-70% confluence, the medium was replaced with medium containing 10% fbs and n-octanoylated ghrelin peptide at the concentrations of and nm. to protect ghrelin from inactivation, the culture medium contained also nm serine protease inhibitor, 4- benzenesulphonyl fluoride hydrochloride. the cultures were incubated for hours. in order to determine the mechanism by which ghrelin increased proliferation/viability of the cells, they were additionally pre-treated for hr with the and μm of ghs-r-specific antagonist -ghrp. 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 five times. cell proliferation assay brdu cell proliferation assay was carried out according to the brdu labeling protocol. briefly, canine mammary cancer cells where cultivated in culture flask to reach 60-70% confluence. the medium was then removed and replaced medium containing 10% fbs with nm aebsf and: ghrelin at the concentration of and nm or ghs-r-specific peptide antagonist -ghrp for hrs. then, μm of brdu was added to culture medium for one hour. the cells were washed twice with pbs, harvested by trypsinization, suspended in pbs and fixed in ice-cold ethanol. the cells were then stored at -20°c for hrs and then washed with wash buffer and suspended in denaturing solution for min at room temperature to denature dna. the cells were then washed again with wash buffer and resuspended in m sodium borate of ph, for minutes at rt, to neutralize any residual acid. the cells were then washed with wash buffer and incubated with fitc-conjugated anti-brdu antibodies and isotype control for min at rt. cells were then incubated with μl/ml of propidium iodide for min at rt. all the samples were analyzed using flow cytometry. the experiment was conducted in three replicates. sirna transfection the sirna transfection procedure on canine mammary cancer cells has been given an account of in our previous paper. however, the procedure was optimized for cmt-w1m cell line. thus the cell density, transfection reagent toxicity and transfection efficacy has been assessed. the canine ghsr sequence was obtained from gene bank with accession number. 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 at last two duplexes were chosen for further experiments. each duplex was used at different concentrations the mixture of both duplexes was also used. the rna interference has been assessed by real-time qpcr. transfected and control cmt-w1m cells ) were scraped, and the total rna from the cell suspension samples was isolated. because during optimization of the sirna transfection procedure for cmt-w1m cell line any significant changes in cells viability between control cells and cells treated with only transfection reagent has been observed, the mock-transfected control has been omitted. apoptosis assay the annexin v-fitc and propidium iodide dual staining was applied for apoptosis analysis. normal cells and cells treated with ghrelin at the concentration of and nm and nm aebsf, inhibitor of ghrelin receptor -ghrp at the concentration of μm, μm, μm, μm and μm for hrs transfection reagent + non-coding rna and ghsr sirna, were harvested by trypsinization and together with the cells floated in medium were stained using an annexin v kit, according to the manufacturer’s protocol. the cells were immediately analyzed by flow cytometry. early apoptotic cells with exposed phosphatidylserine but intact cell membranes bound to annexin v-fitc but 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, and each experiment was performed three times. in vitro wound healing assay scratch assay was conducted to assess the influence of ghrelin on canine mammary carcinoma cell motility. the cells were seeded in a high density at mm petri dishes. after hrs, the medium was removed and replaced medium containing 10% fbs with nm aebsf and ghrelin at concentration of and nm or specific ghs-r inhibitor [d-lys3-ghrp at concentration of um, μm, um, μm and μm for hrs. when the cells reached full confluence, the monolayers were wounded by scratching the surface as uniformly and straight as possible with a pipette tip at least three times. the images of cells invading the scratch were captured at indicated time points using phase contrast microscopy. the pictures have been analyzed using a computer-assisted image analyzer. the migration rate was expressed as percentage of scratch closure and was calculated as follows: % of scratch closure = a-b/a, where is a distance between edges of the wound, and is the distance which remained cell-free during cell migration to close the wound. the values are the means of three independent wound fields from three independent experiments. invasion and migration assay the bd biocoat™ 24-multiwell invasion system pre-coated with bd matrigel™ matrix were used according to the manufacturer’s protocol. the insert plates were prepared by rehydrating the bd matrigel™ matrix layer with phosphate buffered saline for two hours at 37°c. the rehydration solution was then carefully removed, and μl of cell suspension was added to the apical chambers. cell suspension was prepared by trypsinizing cell monolayers and resuspending the cells in rpmi medium containing % fbs, nm of aebsf and ghrelin at the concentration of and nm. then μl of chemoattractant was added to the each of the basal chambers. assay plates were incubated for hours at standard culturing conditions. followed incubation medium was carefully removed from apical chamber and insert system was transferred into a second 24-well plate containing μl of μg/ml calcein am in hanks’ balanced salt solution. plates were incubated one hour at standard culturing conditions. then the fluorescence of invaded cells was measured at excitation wavelength nm and emission wavelength nm using florescent plate reader with bottom reading capabilities infinite pro tecan™. all samples were assayed in triplicate, and each experiment was conducted three times. to evaluate migratory potential the bd falcon™ fluoroblock™ 24-multiwell insert plates was used. the determination protocol for the canine mammary cancer cells migration was the same as the invasion assay, with the exception that no matrigel was used and rehydrating of the plate was omitted. in order to determine fluorescence of cells invaded through membrane coated by matrigel fluorescence microscopic analysis using olympus microscopy bx at x magnifications was applied. statistical analysis the statistical analysis was conducted using prism version software. the one-way anova and tukey hsd post-hoc test, dunnett’s test and t-test were applied as well as regression analysis. the p-value <  was regarded as significant whereas p-value <  and p-value <  as highly significant. the data was expressed as means +/- s.e.m. unless otherwise stated. the in vitro wound healing assay was analyzed using two-way rm anova and bonferroni post-hoc test.  expression of ghrelin and ghs-r in canine mammary tumors and in canine mammary carcinoma cell lines the expression of ghrelin and growth hormone secretagogue receptor at mrna level was detected in adenocarcinomas of the 1st, the 2nd and the 3rd grade of malignancy. no significant differences in ghrelin mrna level have been observed between these groups, however mrna level of growth hormone secretagogue receptor was significantly lower in tumors of the 3rd grade of malignancy compared to the tumors of the 2nd grade of malignancy. the expression of ghrelin peptide was detected in both benign and malignant canine mammary tumors. the analysis showed that ghrelin immunoreactivity was significantly lower in benign tumors comparing to the tumors that gave local or distant metastases. the number of ghrelin-positive cells increased in the group of non-metastatic tumors from benign to the most malignant samples, however no significant differences between these groups have been observed. immunohistochemistry also demonstrated expression of growth hormone secretagogue receptor in all benign and malignant canine mammary tumors. significantly lower expression of this antigen was found in tumors of the 3rd grade of malignancy than in tumors of the 2nd grade of malignancy or in metastatic tumors. no significant differences have been observed between the other tumor groups. real-time pcr analysis showed ghrelin and growth hormone secretagogue receptor mrna expression in all the examined cell lines. growth hormone secretagogue receptor expression was significantly higher in the cell lines isolated from mammary carcinoma metastases to the lungs. immunohistochemical examination confirmed expression of both peptides in all of the examined cell lines. significantly lower expression of ghrelin antigen was detected in cell line isolated from primary canine mammary adenocarcinoma than in the cell line isolated from its metastasis to the lungs. the immunoreactivity of ghrelin was similar in another pair of the cell lines. however, ghs-r immunoreactivity was significantly lower in both canine mammary carcinomas isolated from primary tumors than in cell lines isolated from their metastases to the lungs: 5230 ± and 6729 ±  in cmt-w and cmt-w1m cell lines, respectively and 6189 ±  and 7792 ± in cmt-w and cmt-w2m cell lines, respectively. ghrelin enhances viability and proliferation of canine mammary cancer cells ghrelin treatment increased cells viability and cells proliferation in three of the examined cell lines except the cmt-w2m cell line. the viability of the cmt-w cells was significantly increased by approximately 14% when treated with nm n-octanoylated ghrelin and 12% when treated with nm ghrelin compared to control cells. ghrelin treatment at dose of nm and nm also increased cell viability in the cmt-w1m cell line. the highest response for ghrelin treatment was observed in the cmt-w cell line. the viability of the cmt-w cells was significantly increased when treated with ghrelin at the concentration of nm, nm, nm and nm by approximately 25%, 19%, 18% and 16%, respectively. metabolic activity of the cmt-w2m cells was not significantly changed after incubation with ghrelin. the pre-treatment of cells for hr with specific inhibitor of ghs receptor completely abolished the cells response for ghrelin what confirmed that in examined cell lines ghrelin acts via ghs-r. the role of ghrelin on cellular proliferation was confirmed by brdu incorporation assay. ghrelin treatment at nm and nm doses increased proliferation of cancer cells to  ±  % and  ±  % in cmt-w cell line and to  ±  % and  ±  % in cmt-w1m cell line. grelin treatment at the concentration of nm caused increase of proliferation to  ±  % in cmt-w cell line, however treatment with specific ghs-r inhibitor at μm dose induced anti-proliferative effect, which was not observed in other examined cell lines. cell proliferation was not affected by ghrelin in the cmt-w2m cell line. in both experiments it was impossible to create a dose–response curve. the results indicated that smaller doses of ghrelin significantly increased cancer cells proliferation, however any dose-dependent effect was not observed. ghsr rna interference based on the mtt assay results, the optimal cell number for transfection experiment was per well on 96-well plate. culture of cells per well at the beginning of the experiment ensured 60% confluence at the day of transfection. transfection reagent toxicity evaluation revealed that in case of cmt-w cell line the best viability results were reached using lipofectamine rnaimax. the highest viability of the cells was reached with lipofectamine rnaimax at the concentration of μl per well. the confocal microscopy observations and subsequent computer-assisted image analysis revealed that lipofectamine rnaimax at the concentration of μl per cells per well proved a highly effective transfection reagent ensuring the transfection efficiency at the level of 90%. consequently, experiments were conducted under the same conditions. the control cells used for further experiments were cultured under the transfection conditions with tranfection reagent + non-coding rna. the real-time qpcr and immunohistochemistry results confirmed the effect of ghsr gene silencing. the lowest expression of ghsr was found in the cells treated with the mixture of ghsr and ghsr sequences. only about 15-28% reduction of ghsr expression, comparing against the control cells, was observed in case of ghsr sequence given at two concentrations. we observed % and % reduction of expression compared to control cells after the ghsr and ghsr2′ interference, respectively. the immunohistochemical studies revealed higher efficacy of gene silencing at the protein level than at the mrna level. the most effective was ghsr sequence causing % reduction in protein levels compared against the control cells. ghrs1′ sequence caused % of protein expression reduction, whereas ghsr sequence caused % reduction. ghsr2′ and mixture of ghsr and ghsr sequences caused % and % reduction of protein expression. all these results have been statistically significant at the level of p < . for apoptosis analysis the ghsr sirna duplex was used. ghrelin is an anti-apoptotic factor in canine carcinoma cell lines for analysis of ghrelin influence on apoptosis, the annexin v and pi double staining was applied. the number of apoptotic cells represented as a percentages of annexin-v-positive cells was significantly decreased after the ghrelin treatment at the dose of nm in the cmt-w cell line and in the cmt-w cell line. anti-apoptotic effect of ghrelin was higher in the cell lines isolated from lung metastases of mammary cancer. in control conditions % and % of cells has been identified as apoptotic in cmt-w1m and cmt-w2m cell line, respectively. after ghrelin treatment at the concentration of nm and nm the number of apoptotic cells decreased to 4% and % in the cmt-w1m cell line and to % and % in the cmt-w2m cell line. the statistical analysis showed that differences were highly significant. among all of the examined cell lines only in cmt-w1m cell line incubation with μm ghs-r-specific peptide inhibitor for 24hrs significantly increased the number of apoptotic cells. in order to investigate if this effect was caused by toxicity of this peptide, we knocked-down the ghsr expression by rnai to confirm the functional importance of the ghs-r in apoptosis in cmt-w1m cell line. our results showed that sirna treatment significantly increased number of early apoptotic cells in cmt-w1m cell line up to %. cancer cells treatment with ghs-r inhibitor increased number of early apoptotic cells highly significantly up to %. these differences may be caused by not completely knock-down of ghsr expression. ghrelin promotes migration and invasion of canine mammary carcinoma cells the wound healing assay showed that ghrelin treatment at the concentration of nm increased migratory abilities of the cmt-w cmt-w1m and cmt-w cell lines, however, no effect on cmt-w2m cells has been observed. cmt-w cells incubated with ghrelin almost completely closed the wound in hours after scratching, whereas cmt-w control cells after hrs closed only  ±  % of the wound. similarly, cmt-w1m cells incubated with ghrelin closed the wound at the level of  ±  % in hours after scratching, whereas cmt-w1m control cells after hrs closed only  ±  % of the wound. also, cmt-w cells incubated with ghrelin closed the wound at the level of  ±  % in hours after scratching, whereas cmt-w control cells after hrs closed only  ±  % of the wound. administration of ghs-r-specific peptide inhibitor at μm dose decreased the motility of the cmt-w cells, whereas had no effect on motility of the cmt-w1m or cmt-w cells even at the concentration of μm. cmt-w cells incubated with ghrelin receptor inhibitor closed  ±  % and  ±  % of the wound in and hrs after scratching. cmt-w2m control cells and ghrelin treated cells closed  ±  % and  ±  % of the wound in hours after scratching, however the difference was not statistically significant. cmt-w2m completely closed the wound after hours after scratching in control conditions and after ghrelin treatment. ghs-r-specific peptide inhibitor also had no effect on motility of those cells. cell migration assessed in boyden chambers was increased by incubation with nm of ghrelin in cmt-w cell line and in the other cell lines by incubation with and nm of ghrelin. cancer cell invasion was increased by ghrelin treatment for hours. ghrelin was the most effective at the nm concentration in cmt-w cmt-w1m, cmt-w2m, whereas it was the most effective in cmt-w cell line at nm concentration. the analysis conducted using olympus microscopy bx confirmed the results. discussion the co-expression of ghrelin and ghs-r has previously been observed in various neoplasms and related cancer cell lines in humans. it may suggest their role in neoplastic cell growth and development through autocrine/paracrine mechanism. however, as far as we realize, there are no published reports regarding similar data on tumors in domestic animals. this is the first report of ghrelin and its receptor expression in canine mammary tumor tissues and cell lines isolated from the canine primary carcinomas and from their lung metastases. we have demonstrated the expression of ghrelin in benign and malignant canine mammary tumors and in all of the examined cancer cell lines. interestingly, we observed significantly higher expression of ghrelin in the distant metastases than in the primary carcinomas, which suggest that ghrelin might be involved in mechanism of metastasis. a recent study on human colorectal cancers indicated that ghrelin might initiate malignant transformation and might contribute to the cancer spread. it was also hypothesized that ghrelin mediates the physiological responses of tissues which surround the tumor, to malignant changes. the presence of ghs-r mrna has been previously detected in all of the examined canine cancer cell lines. similarly to mrna expression patterns, ghs-r protein showed significantly higher expression in cell lines isolated from lung metastases of mammary cancer what may indicate its role in metastasis. moreover, we have demonstrated that expression of ghrelin receptor varies in canine mammary tumors depending on the histological grade of malignancy. the ghs-r expression level was higher in well or moderately differentiated cancers of the 1st and the 2nd grade of malignancy compared to poorly-differentiated cancers of the 3rd grade of malignancy. this intriguing finding overlaps with those reported previously, that expression and functionality of specific binding sites for ghs in various types of breast and thyroid carcinomas was maintained in better-differentiated tumors and was decreased in less-differentiated neoplasms. similar expression patterns of ghs-r were observed in human gastric and colorectal cancers, where the ghs-r expression level was lowest in undifferentiated tumors and correlated inversely with histological grade of malignancy and tnm stage. moreover, patients with more weight loss showed lower expression of ghs receptor. therefore, decreased ghrelin receptor expression seemed to correlate with poor prognostic factors such as poor differentiation, advanced stage and malnutrition. the next step of this study was to assess a role of ghrelin in cancer cell biology. our results revealed that ghrelin stimulates canine mammary cancer cells proliferation and inhibits apoptosis, possibly via an autocine/paracrine mechanism. although the role of ghrelin in cancer cells was investigated in multiple experiments using human and rat cancer cell lines, the results were equivocal. some investigators have found ghrelin as antiproliferative factor, but other have found it a growth stimulator of cancer cells. cassoni and co-workers described an inhibition of cell proliferation in human thyroid, breast and lung cancer by ghrelin. authors found that ghrelin given for hrs at relatively high doses decreased cell proliferation. however, after incubation of these cells with ghrelin for hrs, the effect was abolished. volante et al. have confirmed antiproliferative effect of ghrelin in thyroid cancer cells showing significant growth reduction after hrs and hrs of incubation with ghrelin at the concentration of 100- nm. interestingly, cassoni et al. have showed dose-dependent effect of ghrelin treatment in prostate cancer cells. contrary to the mentioned reports the pro-proliferative effect of ghrelin in several human cancer cell lines has also been well documented. ghrelin at doses ranging from to nm, which encompasses normal circulating ghrelin levels increased cellular proliferation after h of incubation in breast and prostate cancers. ghrelin treatment significantly stimulated cells growth in the highly metastatic breast cancer cell line up to 36% above control. the most significant proliferative response to ghrelin treatment occurred at the concentrations of and nm in the mda-mb- cell line, and 1– nm in the mda-mb- cell line. thus, these results show that ghrelin given in high doses inhibits cancer cell proliferation, whereas given at low doses increases proliferation. our results also indicated that ghrelin given at the dose of or nm increased canine mammary cancer cells proliferation in three of four examined cell lines, whereas higher doses caused not significant effect also in three of four examined cell lines. in one cell line, ghrelin did not cause any affect. to clarify the role of ghrelin in cell proliferation the cell cycle analysis was conducted. this study revealed that although ghrelin did not change the proportion of cells in the g0/g s or g2/m phases of cell cycle, it caused a decrease in the number of apoptotic cells in sub-g phase. in fact ghrelin was reported to be the antiapoptotic factor in colonic cancer cells as well as in many other types of cells like adipocytes, osteoblastic cells, or human endothelial cells. the antiapoptotic activity of ghrelin is also supported by our findings. we have showed that ghrelin decreases the number of apoptotic cells in all of the examined canine mammary cancer cell lines. because in cmt-w1m cell line we observed a significant increase in number of apoptotic cells after the treatment with ghrelin inhibitor, our next step was to knock-down the ghsr gene to check if the toxic effect was caused by the inhibitor itself or by blocking the ghs-r. our results showed that treatment of cmt-w1m cell line with ghsr-specific sirna also increased the number of apoptotic cells. an increase in number of apoptotic cells was slight, however significant, which can be explained by efficacy of gene silencing. in our experiment only 85% of cells remained transfected. recently, a few studies have focused on the involvement of ghrelin in cancer cells motility and ability to metastasis. duxbury and co-workers investigated the role of ghrelin in metastatic potential of poorly differentiated human pancreatic cancer cells. they found that ghrelin at nm concentration increased not only the proliferation rate of cancer cells but also cellular motility and invasiveness. another study showed the promotion of proliferation and invasiveness of human colorectal cancer cells by ghrelin in an autocrine and paracrine manner. this effect was almost completely abolished when the cells were pre-treated with either ghs-r antagonist or a neutralizing ghrelin – specific antibody. furthermore dixit et al. revealed that ghrelin acting via functional ghs-r1a caused increase in intracellular calcium mobilization and led to actin polymerization and membrane ruffling, which resulted in an increase of migration and invasion of carcinoma cells. our study also showed a role of ghrelin in cancer cells migration and invasion. we have demonstrated that ghrelin given at the concentration of nm increased the migration ability of canine carcinoma cell lines cmt-w and cmt-w1m, evaluated by wound healing test. moreover ghrelin given at the concentration of and/or nm significantly increased migration and invasion of all the examined canine carcinoma cell lines. the fact that the examined carcinoma cell lines respond in different manner to the various ghrelin doses may be related with differences in endogenous ghrelin production levels. differences between effective ghrelin doses that stimulate cells proliferation or survival and doses that increase cells migration can be explained by the fact that these different cellular activities are mediated by different cellular pathways. literature data show that ghrelin increases cellular migration acting on camkii, ampk, and nf-kb pathways, whereas increases cellular proliferation and survival acting on erk1/2. thus, probably various ghrelin doses are required to activate various cellular pathways.  we have showed that ghrelin and ghs-r are differentially expressed in canine mammary tumors. the lowest expression of ghs-r in tumors of the 3rd grade of malignancy may by a potential prognostic factor. taken together, the data demonstrated that exposure to low doses of ghrelin stimulate cellular proliferation, inhibit apoptosis and promote motility and invasion of canine mammary carcinoma cells. however, further studies are required to investigate the molecular mechanism responsible for ghrelin action in cancer cells. our study indicates that inhibition of ghrelin/ghs-r axis may be a potential therapeutic target of mammary cancers in dogs in the future. competing interest the authors declare that they have no competing interests. authors’ contributions km: mtt assays, brdu assay, migration and invasion assays, wound healing assay, immunohistochemistry, statistical analysis, manuscript and figures preparation; kmp: sirna transfection, real-time qpcr; ejo: sirna transfection, real-time qpcr; id: pathological examination of mammary carcinomas; jm: rna isolation, immunohistochemistry; tm: research design; mk: research design, experiments design, apoptosis tests, facs analyses, manuscript preparation, figures preparation. all authors read and approved the final manuscript. 
for apoptosis analysis the annexin v and propidium iodide dual staining was applied whereas cell proliferation was evaluated by mtt assay and brdu incorporation test. they are often co-expressed in multiple human tumors and related cancer cell lines what can indicate that the ghrelin/ghs-r axis may have an important role in tumor growth and progression. higher expression of ghrelin and its receptor was detected in cancer cell lines isolated from metastases than in cell lines isolated from primary tumors. ghrelin is a natural ligand of the growth hormone secretagogue receptor. growth hormone secretagogue receptor inhibitor as well as rna interference enhances early apoptosis. canine mammary carcinomaghrelingrowth hormone secretagogue receptor however, a role of ghrelin in canine tumors remains unknown. the expression of ghrelin and its receptor in canine mammary cancer tissues and cell lines was examined using real-time qpcr and immunohistochemistry. in vitro experiments demonstrated that exposure to low doses of ghrelin stimulates cellular proliferation, inhibits apoptosis and promotes motility and invasion of canine mammary cancer cells. the presence of ghrelin and ghs-r in all of the examined canine mammary tumors may indicate their biological role in cancer growth and development. the influence of ghrelin on cancer cells migration and invasion was assessed using boyden chamber assays and wound healing assay.
 ghrelin is a natural ligand of the growth hormone secretagogue receptor. they are often co-expressed in multiple human tumors and related cancer cell lines what can indicate that the ghrelin/ghs-r axis may have an important role in tumor growth and progression. however, a role of ghrelin in canine tumors remains unknown. thus, the aim of our study was two-fold: to assess expression of ghrelin and its receptor in canine mammary cancer and to examine the effect of ghrelin on carcinoma cells proliferation, apoptosis, migration and invasion. the expression of ghrelin and its receptor in canine mammary cancer tissues and cell lines was examined using real-time qpcr and immunohistochemistry. for apoptosis analysis the annexin v and propidium iodide dual staining was applied whereas cell proliferation was evaluated by mtt assay and brdu incorporation test. the influence of ghrelin on cancer cells migration and invasion was assessed using boyden chamber assays and wound healing assay.  the highest expression of ghrelin was observed in metastatic cancers whereas the lowest expression of ghrelin receptor was detected in tumors of the 3rd grade of malignancy. higher expression of ghrelin and its receptor was detected in cancer cell lines isolated from metastases than in cell lines isolated from primary tumors. in vitro experiments demonstrated that exposure to low doses of ghrelin stimulates cellular proliferation, inhibits apoptosis and promotes motility and invasion of canine mammary cancer cells. growth hormone secretagogue receptor inhibitor as well as rna interference enhances early apoptosis.  the presence of ghrelin and ghs-r in all of the examined canine mammary tumors may indicate their biological role in cancer growth and development. our experiments conducted in vitro confirmed that ghrelin promotes cancer development and metastasis. canine mammary carcinomaghrelingrowth hormone secretagogue receptor 