growth factors like platelet - derived growth factor ( pdgf ) and stem cell factor ( scf ) are naturally occurring substances capable of stimulating cellular growth , proliferation and differentiation . normal development and function of the testicular somatic cells ( e.g. leydig cells ) are controlled by growth factors like scf , pdgf and their interaction with tyrosine kinase receptors ( pdgfr and c - kit ) ( 15 ) . imatinib mesylate ( glivec , sti 571 ; novartis pharma , switzerland ) , an antitumor agent has been widely used to treat multiple cancers , most notably philadelphia chromosome - positive ( ph+ ) chronic myelogenous leukemia and gastrointestinal stromal tumors . like all tyrosine kinase inhibitors , imatinib works by preventing a tyrosine kinase enzyme and therefore inhibits growth factor induced receptor phosphorylation and blocks the pdgf / pdgfr and pi3-k / akt pathway ( 69 ) . although such drugs could be targeted with a high degree of selectivity towards the tumor cells , mentioned signaling pathways perform critical functions in normal cells , especially during development . for instance , pdgf is a central mediator of leydig cells development and these cells supply supply the testosterone necessary for the completion of spermatogenesis and maintenance of male reproductive function ( 2 , 10 , 11 ) . to the best of our knowledge , there is no study showing the effect of imatinib on growth factor levels in normal leydig cells . however , heim et al . demonstrated that germline stem cell grown in imatinib led to decreased numbers of differentiated spermatogonia and reduced culture growth consistent with the known requirement for c - kit in survival and proliferation of spermatogonia ( 12 ) . besides testosterone reduction has been reported by some investigators following imatinib therapy ( 13 , 14 ) . on the other hand , some reports have documented findings contradictory to those previously described in the literature and the effect of imatinib on fertility potential is controversial . for instance , unaffected spermatogenesis in a leukemic mouse ( 15 ) and successful pregnancies involving men with chronic myeloid leukaemia and imatinib therapy have been reported ( 16 ) . therefore , in this study , an attempt was made to determine cellular viability , pdgf and scf levels in normal mouse leydig cells exposed to imatinib to address this issue . imatinib mesylate was kindly gifted by fararyand - shimi company , iran ; 1 mm stock solution was prepared in distilled water and stored at 20c . in this study , imatinib was used at increasing concentrations from 0 ( control ) , 2.5 , 5 , 10 and 20 m for 2 , 4 and 6 days . imatinib concentrations were chosen according to previous studies . in the study of soares et al . , the decrease in cell viability was observed only with high concentrations of imatinib mesylate ( 1525 mm ) ( 17 ) . besides , lower imatinib concentrations ( lower than 1 m ) have been reported which led to an underestimation of imatinib anti - tyrosine kinas activity ( 18 ) . the mouse tm3 leydig cells line of musmusculus normal testis were provided from the genetic laboratory of tehran university and cultured in 90% dmem - f12 medium ( paa , uk ) , supplemented with 5% fetal bovine serum ( paa , uk ) , 5% horse serum ( paa , uk ) and 100 unit / ml penicillin . cells were incubated at 37c in a 5% humidified co2-enriched atmosphere . to determine the effect of imatinib , cells were treated with 0 ( control ) , 2.5 , 5 , 10 and 20 m imatinib for 2 , 4 and 6 days . tm3 cells ( 5000 per well ) were treated with 0 ( control ) , 2.5 , 5 , 10 and 20 m imatinib in 100 l of cultured media for 2 , 4 and 6 days . after treatments , 10 l of mtt reagent was added per well and the plates were incubated at 37c for 3 hr . mtt - containing media were then removed and the reduced formazan dye was solubilized by adding 100 l of crystal dissolving solution to each well . cell survival rate was calculated as follows : ( od values of the experimental samples / od values of the control ) 100% . the ic50 value was determined on the basis of dose - response curves from the mtt assay using the compusyn software . to determine the concentration of pdgf and scf in different groups , 50 l cultured media were assayed using a human / mouse pdgf - aa immunoassay and mouse scf immunoassay kits ( r&d systems , usa ) , respectively . the normality of data distribution was checked with kolmogorov - smirnov test and homogeneity of variance was assessed by levene s test . subgroup analyses were performed using one - way analysis of variance ( anova ) and the kruskal - wallis test ( anova on ranks ) for parametric and nonparametric data , respectively . imatinib mesylate was kindly gifted by fararyand - shimi company , iran ; 1 mm stock solution was prepared in distilled water and stored at 20c . in this study , imatinib was used at increasing concentrations from 0 ( control ) , 2.5 , 5 , 10 and 20 m for 2 , 4 and 6 days . imatinib concentrations were chosen according to previous studies . in the study of soares et al . , the decrease in cell viability was observed only with high concentrations of imatinib mesylate ( 1525 mm ) ( 17 ) . besides , lower imatinib concentrations ( lower than 1 m ) have been reported which led to an underestimation of imatinib anti - tyrosine kinas activity ( 18 ) . the mouse tm3 leydig cells line of musmusculus normal testis were provided from the genetic laboratory of tehran university and cultured in 90% dmem - f12 medium ( paa , uk ) , supplemented with 5% fetal bovine serum ( paa , uk ) , 5% horse serum ( paa , uk ) and 100 unit / ml penicillin . cells were incubated at 37c in a 5% humidified co2-enriched atmosphere . to determine the effect of imatinib , cells were treated with 0 ( control ) , 2.5 , 5 , 10 and 20 m imatinib for 2 , 4 and 6 days . cellular viability in different groups was determined using mtt proliferation assay kit ( cayman chemical , usa ) . tm3 cells ( 5000 per well ) were treated with 0 ( control ) , 2.5 , 5 , 10 and 20 m imatinib in 100 l of cultured media for 2 , 4 and 6 days . after treatments , 10 l of mtt reagent was added per well and the plates were incubated at 37c for 3 hr . mtt - containing media were then removed and the reduced formazan dye was solubilized by adding 100 l of crystal dissolving solution to each well . cell survival rate was calculated as follows : ( od values of the experimental samples / od values of the control ) 100% . the ic50 value was determined on the basis of dose - response curves from the mtt assay using the compusyn software . to determine the concentration of pdgf and scf in different groups , 50 l cultured media were assayed using a human / mouse pdgf - aa immunoassay and mouse scf immunoassay kits ( r&d systems , usa ) , respectively . the normality of data distribution was checked with kolmogorov - smirnov test and homogeneity of variance was assessed by levene s test . subgroup analyses were performed using one - way analysis of variance ( anova ) and the kruskal - wallis test ( anova on ranks ) for parametric and nonparametric data , respectively . by increasing drug concentration in cultured media from 0 to 20 m , cellular viability decreased significantly in each separate day . differences in cellular viability between groups treated with 0 and 20 m in all days and also 0 and 10 m and 0 and 5 m on day 6 were significant ( p<0.05 ) as shown by tukey s post hoc tests ( figure 1 ) . the effective concentration of imatinib to reduce cellular viability by 50% ( ic50value ) on day 4 calculated by compusyn software was 6.42 m . cellular viability in leydig cells treated with increasing concentrations of imatinib on different days . in all days of experiments , * p<0.05 , * * p<0.01 according to tukey s post hoc tests increasing drug concentration in cultured media from 0 to 20 m increased pdgf level , which was statistically significant on days 4 and 6 , and only between groups treated with 0 and 20 m imatinib according to tukey s post hoc tests ( figure 2 ) . however , scf level did not change significantly following treatment with increasing drug concentration ( table 1 ) . pdgf levels in leydig cells treated with increasing concentrations of imatinib on different days . on days 4 and 6 , pdgf levels * p<0.05 according to tukey s post hoc tests scf levels in leydig cells treated with increasing concentrations of imatinib on different days data represent mean valuessd of three replicates in each subgroup . scf : stem cell factor increasing duration of treatment from 2 to 6 days had no obvious effect on cellular viability , pdgf and scf levels by increasing drug concentration in cultured media from 0 to 20 m , cellular viability decreased significantly in each separate day . differences in cellular viability between groups treated with 0 and 20 m in all days and also 0 and 10 m and 0 and 5 m on day 6 were significant ( p<0.05 ) as shown by tukey s post hoc tests ( figure 1 ) . the effective concentration of imatinib to reduce cellular viability by 50% ( ic50value ) on day 4 calculated by compusyn software was 6.42 m . cellular viability in leydig cells treated with increasing concentrations of imatinib on different days . in all days of experiments , * p<0.05 , * * p<0.01 according to tukey s post hoc tests increasing drug concentration in cultured media from 0 to 20 m increased pdgf level , which was statistically significant on days 4 and 6 , and only between groups treated with 0 and 20 m imatinib according to tukey s post hoc tests ( figure 2 ) . however , scf level did not change significantly following treatment with increasing drug concentration ( table 1 ) . pdgf levels in leydig cells treated with increasing concentrations of imatinib on different days . on days 4 and 6 , pdgf levels * p<0.05 according to tukey s post hoc tests scf levels in leydig cells treated with increasing concentrations of imatinib on different days data represent mean valuessd of three replicates in each subgroup . increasing duration of treatment from 2 to 6 days had no obvious effect on cellular viability , pdgf and scf levels . the main finding of this study was that the imatinib mesylate , an anticancer drug and a selective tyrosine kinases inhibitor , decreased cellular viability of normal testicular leydig cells , which was associated with the stimulation of pdgf levels . the effect of imatinib on cell viability and pdgf level was sustained and occurred in a concentration - but not a time - dependent manner . functional pdgfr- , pdgfr- , c - kit and their ligands , pdgf - aa , pdgf - bb and scf are expressed in normal leydig cells and they play a crucial role in leydig cell development and function , including testosterone secretion ( 10 , 1922 ) . pdgfs and pdgfrs mrnas of leydig cells were readily detected by both northern analysis and rt - pcr and an identical pattern of protein expression was also confirmed by immunohistochemistry ( 19 ) . imatinib mesylate , as a signal transduction inhibitor , reduces tyrosine kinases activity in leydig cells by inhibition of c - kit , pdgfr- and pdgfr- receptors ( 23 ) . according to our results however , scf level was unaffected by imatinib exposure . to the best of our knowledge , there has been no study concerning the effect of imatinib on pdgf and scf levels in normal leydig cells . testicular mrna levels of pdgf and scf showed no changes by the postnatal short term treatment with imatinib ( 2 ) . the distinction in pdgf level is likely due to differences in the model of the study , the method , target site and concentration of drug application in the experimental conditions . the increase in pdgf level in our study might be due to feedback inhibition of growth factor downstream signaling pathways which have also been shown in our previous work ( 24 ) . inhibition of this path by decreasing pdgfr phosphorylation has been reported in tumor cells like mouse melanoma ( 25 ) and human neuroblastoma cells ( 26 ) , with different effects on cellular growth and viability . the altered pdgf response to different concentrations of imatinib might be mediated by the extent of inhibition within the cell tyrosine kinas signaling pathways . the reason for unaffected scf levels by imatinib treatment in our study was probably due to higher and more constant imatinib concentrations which were required to suppress scf / c - kit - dependent proliferation or survival , according to previously published data ( 18 ) . other studies in testes with disrupted spermatogenesis showed decreased levels of c - kit and the augmented expression of scf as a protective mechanism of sertoli cells through a counteracting mechanism ( 22 , 27 ) . although the suppression of viability in our studies ( ic50=6.42 m , 4 days of imatinib treatment ) occurred at concentrations nearly similar to the ic50 values reported for imatinib - mediated apoptosis induction and proliferation inhibition ( ic50=5 m ) and in the approximately same order than the concentrations achieved for the inhibition of leydig cell tumor growth in vitro ( ic50=5 m ) ( 23 ) , it was higher than concentrations required in studies on chronic myeloid leukemia ( ic50=1 m ) ( 28 , 29 ) and lower than that required in studies on neuroblastoma ( 26 ) . according to previous reports , the mean maximal plasma concentration of oral imatinib ( 400 mg / body ) reached 4.6 m in the patients of chronic myeloid leukemia . the mean concentration was kept at 1.46 m even 24 hr after the administration ( 30 , 31 ) . it has also been reported that 110 m of imatinib in cultured cells is almost equivalent to the clinical dose ( 31 ) . these data indicate that the doses employed in our in vitro study , especially lower doses , are clinically relevant and suitable for in vitro imatinib tyrosine kinas inhibition . according to our data , imatinib decreased cellular viability of normal testicular leydig cells in a concentration - but not a time - dependent manner . reported that imatinib caused concentration - dependent decreases in the viability of leydig tumor cells , which of course have more tyrosine kinase pathways activity , comparing to normal cells ( 23 , 32 ) . inhibition of cell growth and viability by imatinib are achieved through reduction of proliferation and induction of apoptosis ( 33 ) . a further mechanism may include an antiangiogenic effect of imatinib observed in various cancers ( 34 , 35 ) and the inhibition of vascular endothelial growth factor ( 26 ) through a pdgf inhibition - mediated mechanism . although no study concerning the effect of imatinib on normal leydig cells viability was found , decreased testosterone production has been reported repeatedly as an adverse effect of imatinib therapy ( 13 , 14 , 36 , 37 ) . moreover , loss of adult leydig cell population , reductions in testicular size and testosterone production , and the subsequent loss of spermatogenesis and fertility have been reported in pdgfa deficient mice ( 10 ) . these data point to imatinib s influence on leydig cells function including testosterone production , which might affect fertility potential . decreased number of differentiated spermatogonia has also been reported in imatinib treated germline stem cells ( 12 ) . nonetheless , schultheis et al . showed that imatinib at therapeutic doses has no impact on folliculogenesis or spermatogenesis in a leukaemia mouse model ( 15 ) . the difference between this in vivo study and our study may be related to hormonal regulation of pdgf , scf and c - kit in response to gnrh ( 22 ) . altogether , it appears that imatinib may cause reproductive impairment and might reduce fertility potential by decreasing cellular viability through pdgf signaling pathways . in conclusion , this study shows that exposure of normal leydig cells to imatinib resulted in a sustained suppression of cellular viability and enhancement of pdgf levels of normal testicular leydig cells in a concentration- dependent manner . whilst multiple cellular signaling pathways involving in imatinib - induced cellular toxicity could not be ruled out , it is hypothesized that pdgf mediated mechanisms may be involved in this phenomenon . since pdgf and its receptors are important for the development and function of the leydig cells , which in turn are involved in regular spermatogenesis , their abnormal levels caused by some factors like imatinib seem to be associated with reproductive disorders and even infertility . whilst it is hypothesized that imatinib may reduce fertility potential by decreasing cellular viability through pdgf signaling pathways , future studies focusing on the mechanism of imatinib action , especially pdgf signaling pathways and testosterone production are required to establish the precise nature of their interaction .
background : growth factors play an essential role in the development of tumor and normal cells like testicular leydig cells . 
 treatment of cancer with anti - cancer agents like imatinib mesylate may interfere with normal leydig cell activity , growth and fertility through failure in growth factors production or their signaling pathways . 
 the purpose of the study was to determine cellular viability and the levels of , platelet derived growth factor ( pdgf ) and stem cell factor ( scf ) in normal mouse leydig cells exposed to imatinib , and addressing the effect of imatinib on fertility potential.methods:the mouse tm3 leydig cells were treated with 0 ( control ) , 2.5 , 5 , 10 and 20 m imatinib for 2 , 4 and 6 days . 
 each experiment was repeated three times ( 15 experiments in each day).the cellular viability and growth factors levels were assessed by mtt and elisa methods , respectively . for statistical analysis , one - way anova with tukey s 
 post hoc and kruskal - wallis test were performed . 
 a p - value less than 0.05 was considered statistically significant.results:with increasing drug concentration , cellular viability decreased significantly ( p<0.05 ) and in contrast , pdgf levels increased ( p<0.05 ) . 
 different imatinib concentrations had no significant effect on scf level . 
 increasing the duration of treatment from 2 to 6 days had no obvious effect on cellular viability , pdgf and scf levels.conclusion:imatinib may reduce fertility potential especially at higher concentrations in patients treated with this drug by decreasing cellular viability . 
 the effect of imatinib on leydig cells is associated with pdgf stimulation . of course future studies 
 can be helpful in exploring the long term effects of this drug .