breast cancer is the most common cancer among women worldwide and it has been reported that each year about 502,000 die from the disease . breast cancer originates in the breast but metastasizes to other distant organs such as bone , lungs , and brain . global statistics showed that by the year 2020 , there will be twenty million new cancer patients every year . herbal medications are the source of many chemotherapeutic agents and it has also been demonstrated to have anti - neoplastic potential ( nigella sativa , paclitaxel , vincristine , etc . ) . thymoquinone ( tq ) is as a potential chemo preventive and chemotherapeutic compound of oil of the black seed . many investigations have shown that tq has anti - inflammation , anti - hypertensive , anti - asthmatic , anti - microbial , and anti - oxidant effects . in addition , anticancer properties of tq have also been reviewed and has been found that tq applies anticancer effects ( both in vitro and in vivo ) in various patterns of carcinogenesis . in earlier works , it was found that tq prevents the growth of different types of cancer , including breast adenocarcinoma ( multi - drug - resistant michigan cancer foundation-7 [ mcf-7]/topo , mcf-7 , mda - mb-231 , and bt-474 ) , lung cancer ( nci - h460 and a549 ) , pancreatic cancer ( mia paca-2 , hpac , and bxpc-3 ) , and prostate cancer ( lncap , c4 - 2b , and du145 ) . several lines of studies have determined that tq exhibits antimetastatic effects , and it can inhibit angiogenesis , in a way that , this compound was found to prevent c26 colon cancer cell invasion . moreover , some other studies have reported that tq was able to suppress tumor growth in pc-3 prostate cancer cells , probably through angiogenesis inhibition and it can cause to significant reduction in the number of blood vessels in the tumor . tq has shown little effect on noncancerous cells such as mouse fibroblasts , prostate epithelial cells , human normal intestinal cells , and human normal lung fibroblasts . this result shows that tq may have beneficial effects in different types of malignancy while has little effect on normal human cells . found that tq induces apoptosis in osteosarcoma cells and also decrease the number of cells in s - phase . other investigations have demonstrated that tq can induce apoptosis via p53-dependent / independent pathways . in spite of tq good anti - neoplastic activities , they found that tq induced apoptosis by up - regulating p53 and the downstream p53 target gene , p21waf1 . have shown that tq induce apoptosis by p53-independent pathway . regarding increasing incidence of breast cancer in developing countries like iran , many investigations have done on different cell line of breast cancer and many different ways have supposed for tq induced apoptosis . hence , in the present study , we hypothesized that tq may up - regulate activity of p53 gene in breast cancer cell line mcf-7 that has a wild - type p53 , and inhibit tumor cell growth . human breast cancer cell line ( mcf-7 ) was purchased from the national cell bank of iran - pasteur institute . cell line used in the present study was cultured in dulbecco 's modified eagle medium - f12 medium ( sigma ) supplemented with 10% fetal bovine serum ( sigma ) and 1% penicillin - streptomycin ( sigma ) , and incubated at 37c and in humidified atmosphere containing 5% co2 . tq was dissolved in minimum volume of dimethyl sulfoxide ( dmso ) ( 2% ) as working solution were prepared by adding of adequate water to reach the appropriate concentrations according to reported procedures . after the cells were > 80% confluent and growing exponentially in flask 75 , 10 cells ( mcf-7 ) were counted and plated in flask 25 and kept in culture medium for 24 h which were then incubated with certain concentrations of tq , based on half - maximal inhibitory concentration ( ic50 ) index , at different times ( 24 , 48 , and 72 h ) . the ic50 values for the tq in mcf-7 cells were acquired after 24 h of treatment . briefly , 10 cells ( mcf-7 ) were counted and placed into each well of a 12well microplate and were treated with various drug concentrations ( 0 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , and 80 m doses ) for 24 h , and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) survival assay was done according to the protocol , for evaluating the cell viability in all cells . a graph of viability versus drug concentration was used to calculate ic50 values for mcf-7 cell line . percentage of apoptotic cells was measured by flow cytometry following annexin v flow cytometry 1-histogram ( fl1-h ) and propidium iodide ( fl2-h ) labeling . cells were treated with tq ( 25 mol / l ) for 24 , 48 , and 72 h and so washed in phosphate - buffered saline and resuspended in binding buffer ( 10x ; 5 l ) . annexin v - fitc was added to 195 l cell suspensions , and so analysis was done according to the manufacturer 's protocol ( bms500f1/100ce annexin v - fitc apoptosis detection kit - ebioscience , usa ) . eventually , the apoptotic cells were counted by facscan flow cytometry ( becton dickinson , heidelberg , germany ) . these experiments were done in triplicate and were , independently , repeated at least 3 times . real - time polymerase chain reaction ( pcr ) was performed to quantitatively assessment the messenger rna ( mrna ) expression of p53 gene in mcf-7 cells and after treatment with tq at different times . total rna was isolated by rneasy mini kit treated by rnase - free dnase set ( qiagen ) to remove the genomic dna . total rna ( 100 ng ) was reverse - transcribed to complementary dna ( cdna ) using the revertaid first strand cdna synthesis kit ( fermentas ) according to the manufacturer 's protocol . the maxima sybr green rox qpcr master mix kit ( fermentas ) was used for real - time pcr . real - time pcr reactions were performed with using step one plus ( step one real - time pcr systems - applied biosystems ) . the pcr amplification conditions consisted of 10 min at 95c followed by 40 cycles of denaturation step at 95c for 15 s and annealing and extension for 1 min at 60c . the relative expression level of p53 was calculated by determining a ratio between the amount of p53 and that of endogenous control ( glyceraldehyde-3-phosphate dehydrogenase [ gapdh ] ) . melting curve analysis ( 60c  95c increment of 0.3c ) was used to determine the melting temperature of special amplification products and primer dimers . primer sequences for p53 and gapdh were as follow : 
 pf gapdh : caccaccatggagaaggctggpr gapdh : ccaaagttgtcatggatgaccpf p53 : cgagtcccgcggtaattcttpr p53 : tgcagaagaggtgcaagacc . pf gapdh : caccaccatggagaaggctgg pr gapdh : ccaaagttgtcatggatgacc pf p53 : cgagtcccgcggtaattctt pr p53 : tgcagaagaggtgcaagacc . repeated measure analysis of variance with post hoc test was performed to determine the statistical significance among different groups using the spss software package 20.0 ibm modeler ( 2009 ) . human breast cancer cell line ( mcf-7 ) was purchased from the national cell bank of iran - pasteur institute . cell line used in the present study was cultured in dulbecco 's modified eagle medium - f12 medium ( sigma ) supplemented with 10% fetal bovine serum ( sigma ) and 1% penicillin - streptomycin ( sigma ) , and incubated at 37c and in humidified atmosphere containing 5% co2 . tq was dissolved in minimum volume of dimethyl sulfoxide ( dmso ) ( 2% ) as working solution were prepared by adding of adequate water to reach the appropriate concentrations according to reported procedures . after the cells were > 80% confluent and growing exponentially in flask 75 , 10 cells ( mcf-7 ) were counted and plated in flask 25 and kept in culture medium for 24 h which were then incubated with certain concentrations of tq , based on half - maximal inhibitory concentration ( ic50 ) index , at different times ( 24 , 48 , and 72 h ) . the ic50 values for the tq in mcf-7 cells were acquired after 24 h of treatment . briefly , 10 cells ( mcf-7 ) were counted and placed into each well of a 12well microplate and were treated with various drug concentrations ( 0 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , and 80 m doses ) for 24 h , and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) survival assay was done according to the protocol , for evaluating the cell viability in all cells . a graph of viability versus drug concentration was used to calculate ic50 values for mcf-7 cell line . percentage of apoptotic cells was measured by flow cytometry following annexin v flow cytometry 1-histogram ( fl1-h ) and propidium iodide ( fl2-h ) labeling . cells were treated with tq ( 25 mol / l ) for 24 , 48 , and 72 h and so washed in phosphate - buffered saline and resuspended in binding buffer ( 10x ; 5 l ) . annexin v - fitc was added to 195 l cell suspensions , and so analysis was done according to the manufacturer 's protocol ( bms500f1/100ce annexin v - fitc apoptosis detection kit - ebioscience , usa ) . eventually , the apoptotic cells were counted by facscan flow cytometry ( becton dickinson , heidelberg , germany ) . these experiments were done in triplicate and were , independently , repeated at least 3 times . real - time polymerase chain reaction ( pcr ) was performed to quantitatively assessment the messenger rna ( mrna ) expression of p53 gene in mcf-7 cells and after treatment with tq at different times . total rna was isolated by rneasy mini kit treated by rnase - free dnase set ( qiagen ) to remove the genomic dna . total rna ( 100 ng ) was reverse - transcribed to complementary dna ( cdna ) using the revertaid first strand cdna synthesis kit ( fermentas ) according to the manufacturer 's protocol . the maxima sybr green rox qpcr master mix kit ( fermentas ) was used for real - time pcr . real - time pcr reactions were performed with using step one plus ( step one real - time pcr systems - applied biosystems ) . the pcr amplification conditions consisted of 10 min at 95c followed by 40 cycles of denaturation step at 95c for 15 s and annealing and extension for 1 min at 60c . the relative expression level of p53 was calculated by determining a ratio between the amount of p53 and that of endogenous control ( glyceraldehyde-3-phosphate dehydrogenase [ gapdh ] ) . melting curve analysis ( 60c  95c increment of 0.3c ) was used to determine the melting temperature of special amplification products and primer dimers . primer sequences for p53 and gapdh were as follow : 
 pf gapdh : caccaccatggagaaggctggpr gapdh : ccaaagttgtcatggatgaccpf p53 : cgagtcccgcggtaattcttpr p53 : tgcagaagaggtgcaagacc . pf gapdh : caccaccatggagaaggctgg pr gapdh : ccaaagttgtcatggatgacc pf p53 : cgagtcccgcggtaattctt pr p53 : tgcagaagaggtgcaagacc . repeated measure analysis of variance with post hoc test was performed to determine the statistical significance among different groups using the spss software package 20.0 ibm modeler ( 2009 ) . after the treatment of mcf-7 cells with mtt solution in this study , the dark blue formazan crystals were seen in cells , which indicated their metabolic activity . the reduction in the number of cells was dependent on the cell type as shown by the ic50 index . the results showed that the essential tq concentration to achieve the ic50 in mcf-7 cells at 24 h was 25 mol / l [ figure 1 ] . half - maximal inhibitory concentration assay for half - maximal inhibitory concentration analysis of thymoquinone in michigan cancer foundation-7 cancer cell lines after 24 h of treatment . the relative amount of viable cells was estimated by measuring the absorbance of the cell suspension after incubation with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay . a graph of viability versus drug concentration was used to calculate half - maximal inhibitory concentration values for michigan cancer foundation-7 cell line to establish the anti - apoptosis potential of the tq , we first investigated the effects of tq on the proliferation in mcf-7 cells . the flow cytometry results showed that tq at different time points ( 24 , 48 and 72 h ) could induce apoptosis in mcf-7 cells , and it was increased with ascending time . tq arrested mcf-7 cells proliferation ( 40% of inhibition ) in 72 h and ( approximately 30% of inhibition ) in 48 h [ figure 2a and b , p < 0.05 ] . dmso was used in the control sample ( vehicle drugs ) . using dunnett test , average of apoptotic cells at different times groups 48 h and 72 h had significant difference to the control group ( * p < 0.05 ) . ( a ) relative levels of apoptotic cells in michigan cancer foundation-7 treated with thymoquinone for different times . the percentage of apoptotic cells was measured using the annexin v - fitc flow cytometry 1-histogram and propidium iodide flow cytometry 2-histogram . dunnett 's test was used for considered comparisons ( * p < 0.05 ) ( in any figure , right upper quarter show late apoptotic cells , left upper quarter show necrosis cells , right lower quarter show early apoptotic cells and left lower quarter show normal cells ) . ( b ) cells that are annexin v - positive and propidium iodide negative are in early apoptosis as phosphatidyl serine translocation has occurred ; although , the plasma membrane remains intact . cells that are positive for both annexin v and propidium iodide either are in the late stages of apoptosis or are already dead as phosphatidyl serine translocation has occurred and the loss of plasma membrane integrity is visible . dunnett 's test was used for considered comparisons ( * p < 0.05 ) it was suggested that apoptotic induction in cancer cells by tq requires the activation of p53 gene expression . to examine this hypothesis , we used mcf-7 cell line , as cancerous cell line . we examined the inhibitory effects of 25 mol / l tq ( based on ic50 index ) at different times on the mrna expression of p53 in mcf-7 cell line . the p53 gene expression was dramatically up - regulated by ascending time , in particular , at 72 h treatment its expression was increased significantly [ figure 3 , p < 0.05 ] . using dunnett test , average of p53 gene expression at different times was compared with the control group . groups 48 h and 72 h had significant difference to the control group ( * p < 0.05 ) . results of real - time quantitative polymerase chain reaction before and after thymoquinone treatment at different times on the p53 messenger rna expression in michigan cancer foundation-7 cell . relative expression levels were obtained using the comparative ct ( ct ) method ( * p < 0.05 ) after the treatment of mcf-7 cells with mtt solution in this study , the dark blue formazan crystals were seen in cells , which indicated their metabolic activity . the reduction in the number of cells was dependent on the cell type as shown by the ic50 index . the results showed that the essential tq concentration to achieve the ic50 in mcf-7 cells at 24 h was 25 mol / l [ figure 1 ] . half - maximal inhibitory concentration assay for half - maximal inhibitory concentration analysis of thymoquinone in michigan cancer foundation-7 cancer cell lines after 24 h of treatment . the relative amount of viable cells was estimated by measuring the absorbance of the cell suspension after incubation with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay . a graph of viability versus drug concentration was used to calculate half - maximal inhibitory concentration values for michigan cancer foundation-7 cell line to establish the anti - apoptosis potential of the tq , we first investigated the effects of tq on the proliferation in mcf-7 cells . the flow cytometry results showed that tq at different time points ( 24 , 48 and 72 h ) could induce apoptosis in mcf-7 cells , and it was increased with ascending time . tq arrested mcf-7 cells proliferation ( 40% of inhibition ) in 72 h and ( approximately 30% of inhibition ) in 48 h [ figure 2a and b , p < 0.05 ] . dmso was used in the control sample ( vehicle drugs ) . using dunnett test , average of apoptotic cells at different times groups 48 h and 72 h had significant difference to the control group ( * p < 0.05 ) . ( a ) relative levels of apoptotic cells in michigan cancer foundation-7 treated with thymoquinone for different times . the percentage of apoptotic cells was measured using the annexin v - fitc flow cytometry 1-histogram and propidium iodide flow cytometry 2-histogram . dunnett 's test was used for considered comparisons ( * p < 0.05 ) ( in any figure , right upper quarter show late apoptotic cells , left upper quarter show necrosis cells , right lower quarter show early apoptotic cells and left lower quarter show normal cells ) . ( b ) cells that are annexin v - positive and propidium iodide negative are in early apoptosis as phosphatidyl serine translocation has occurred ; although , the plasma membrane remains intact . cells that are positive for both annexin v and propidium iodide either are in the late stages of apoptosis or are already dead as phosphatidyl serine translocation has occurred and the loss of plasma membrane integrity is visible . it was suggested that apoptotic induction in cancer cells by tq requires the activation of p53 gene expression . to examine this hypothesis , we used mcf-7 cell line , as cancerous cell line . we examined the inhibitory effects of 25 mol / l tq ( based on ic50 index ) at different times on the mrna expression of p53 in mcf-7 cell line . the p53 gene expression was dramatically up - regulated by ascending time , in particular , at 72 h treatment its expression was increased significantly [ figure 3 , p < 0.05 ] . using dunnett test , average of p53 gene expression at different times was compared with the control group . groups 48 h and 72 h had significant difference to the control group ( * p < 0.05 ) . results of real - time quantitative polymerase chain reaction before and after thymoquinone treatment at different times on the p53 messenger rna expression in michigan cancer foundation-7 cell . relative expression levels were obtained using the comparative ct ( ct ) method ( * p < 0.05 ) breast cancer is the most common cancer among women worldwide and it is one of the most common cancers that lead to death . therefore , many continuous searches have done for usage of new chemotherapeutic drugs in this type of cancer . over the centuries tq as an active ingredient isolated from n. sativa has tested for its efficacy against cancer . to date , many studies have demonstrated that this compound can inhibit the growth of many different types of cancer , including glioma / glioblastoma ( u87 mg ) , breast adenocarcinoma ( multi - drug - resistant mcf-7/topo , mcf-7 ) , leukemia ( hl-60 and junket ) , lung cancer ( nci - h460 ) , colorectal carcinoma ( ht-29 ) , and pancreatic cancer ( hpac and bxpc-3 ) . in the present study , we investigated the effect of tq on p53 gene expression and consequence apoptosis in breast cancer cell line . however , the assumptions indicated that the anti - tumor effect of tq may happen by one or more of the following mechanisms , antioxidant activity and cytotoxicity . the results of the present study showed that tq inhibits the viability and proliferation of breast cancer cell line by a time - dependent manner . have shown tq property as a radical scavenger with a potential role in the prevention and treatment of oxidative stress . a number of studies have shown that tq could prevent from chemical effects of cancer and inhibit tumorigenesis through different molecular mechanisms . apoptosis is one form of physiological or a key pathway for regulating homeostasis and morphogenesis of cells and is related to cancer . numerous in vitro and in vivo studies have indicated that tq has anti - proliferative effects and can induce cell cycle arrest and also induces pro - apoptotic effects in cancer cells . have reported that the apoptotic effect of tq is p53-independent in p53-null myeloblastic leukemia hl-60 cells . they also confirmed that the apoptotic effect of tq in leukemia cells is caspase - dependent . in some other studies , tq induces apoptosis through the expression of p53 gene in hct116 human colorectal carcinoma cells . thus , the results of different studies show that the anticancer activity of tq is dependent on cell type . the potencies and functional mechanisms of the tq were studied at the concentration that was confirmed by ic50 on the mcf-7 as breast cancer cell line . in recent years , a few researchers have described the therapeutic effect of tq by p53 up - regulation in different cell line ; here , we report for the first time that tq was able to up - regulate the p53 expression in mcf-7 cell line . to understand the inhibitory effect of tq in breast cancer cells , we used different molecular techniques . in the present study , we have shown that tq induce apoptosis in mcf-7 cells via time - dependent manner . in addition , real - time pcr results on mcf-7 cells , showed apoptotic induction in cancer cells by tq requires the activation of p53 gene expression as it was dramatically up - regulated by ascending time , in particular , at 72 h treatment its expression was increased significantly ( p < 0.001 ) . in this study , treatment by tq at various time showed a time - dependent increase in apoptotic cell count of the cancerous cells , as measured by flow - cytometric assay . we have found that tq caused a significant up - regulation p53 in a time - dependent manner . our results were supported by the findings of gali - muhtasib et al . , which indicated that tq is anti - neoplastic and pro - apoptotic against colon cancer cell line hct116 , and the apoptotic effects of tq are dependent on p53 . our results were matched with the findings of woo et al . which stated that tq exerted strong anti - proliferative effect in breast cancer cells . in addition , they found that tq activated caspases 8 , 9 , and 7 in a dose - dependent manner ( possible involvement of peroxisome proliferator activated receptor- [ ppar- ] pathway ) . besides , el - mahdy et al . have reported that tq exhibits antiproliferative effect , induces apoptosis and triggers the activation of caspases 8 , 9 , and 3 inmyeloblastic leukemia hl-60 cells ( p53-independent ) . taken together , our results indicated that tq inhibited breast cancer cell proliferation in a time - dependent manner . also , for the first time , we have shown that tq induce apoptosis through activation of the p53 pathway in mcf-7 cell line . further , it is required to evaluate the effect of tq on prevention and treatment of breast cancer . the results showed that the drug 's effect was significant at 48 and 72 hours . finally , tq treatment for 72 hours would have to make the most of apoptosis by expression of p53 gene and it 's resumption of activities apoptotic . considering the valuable properties of the drug , can be hope to the emergence of a new strategy in the fight against cancer .
background : nigella sativa has been a nutritional flavoring factor and natural treatment for many ailments for so many years in medical science . 
 earlier studies have been reported that thymoquinone ( tq ) , an active compound of its seed , contains anticancer properties . 
 previous studies have shown that tq induces apoptosis in breast cancer cells but it is unclear the role of p53 in the apoptotic pathway . hereby , this study reports the potency of tq on expression of tumor suppressor gene p53 and apoptosis induction in breast cancer cell line michigan cancer foundation-7 ( mcf-7).methods : mcf-7 cell line was cultured and treated with tq , and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assay was carried out for evaluating the half - maximal inhibitory concentration ( ic50 ) values after 24 h of treatment . 
 the percentage of apoptotic cells was measured by flow cytometry . 
 real - time polymerase chain reaction ( pcr ) was performed to estimate the messenger rna expression of p53 in mcf-7 cell line at different times.results:the ic50 value for the tq in mcf-7 cells was 25 m that determined using mtt assay . 
 the flow cytometry and real - time pcr results showed that tq could induce apoptosis in mcf-7 cells , and the p53 gene expression was dramatically up - regulated by ascending time , respectively . 
 hence , there was significant difference in 48 and 72 h.conclusions:our results demonstrated that tq could induce apoptosis in mcf-7 cells through up - regulation of p53 expression in breast cancer cell line ( mcf-7 ) by time - dependent manner .