breast cancer is the most common malignant neoplasm among women in the world . due to globally uncontrolled growth in recent years , it is very important . in united states one of every eight women is facing this disease . only in 2013 although the incidence of cancer in asia is low , its death toll is more than that in western countries . in iran , breast cancer has the highest incidence among malignancies in women . it has been found that the incidence of breast cancer in iran is less than that in developed countries . radiotherapy and chemotherapy could damage healthy tissues too , so that for considering healthy tissues tolerance radiation and drug doses have to be kept at a limited level . it could be used to cure vomiting , dental and gingival pain , insomnia , depression , seizures , cognitive disorders , diaphoretic , eupeptic , expectorant , and aphrodisiac and in the treatment of hepatic disorders , flatulence , spasm , asthma , coughs , bronchitis , colds , fever , cardiovascular disorders , cancer , and many other diseases . therefore , it could be used in combination with chemotherapy drugs to reduce the toxic effects of drugs . because of paclitaxel 's ability to bind to the beta tubulin subunit , it increases the production of microtubules and prevents them from depolymerization . this event consequently disrupts the cell mitosis and stops cells in the g2 phase leading to apoptosis . the most important adverse effect and dose - limiting prescription of paclitaxel is the deactivation of the hematopoietic system . this is manifested by reduction of neutrophils ( neutropenia ) , reduction of leukocytes ( leukopenia ) , and anemia . paclitaxel may cause some cardiovascular complications such as low blood pressure ( hypotension ) , heart rate ( bradycardia ) , and high blood pressure ( hypertension ) . nausea and vomiting are the other side effects of paclitaxel [ 12 , 13 ] . dna is a critical target for ionizing radiation since it includes information to encode biomolecules , so that its damage such as single strand break ( ssb ) , double strand break ( dsb ) , and dimerization could threat the cell viability strongly [ 14 , 15 ] . the term of apoptosis was first established by kerr in 1972 to describe the physiological cell death based on morphological changes and to make differentiation from necrosis . this process is performed during embryonic stage and tumor suppression . according to increasing prevalence of breast cancer and the adverse effects of treatment programs including resistance to chemotherapy drug and damage to healthy tissues during chemotherapy and radiotherapy , this study aimed to asses synergistic apoptotic effect of crocin and paclitaxel and crocin and gamma radiation , respectively , on mcf-7 cell line . mcf-7 cell line , noninvasive estrogen receptor ( er ) positive , was prepared from the pasteur institute ( tehran , iran ) . trypsin , crocin , 3-(4,5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium ( mtt ) , and propidium iodide ( pi ) were purchased from sigma ( germany ) . dulbecco 's modified eagle 's medium ( dmem ) high glucose with 5% fetal bovine serum was purchased from gibco ( usa ) . rabbit monoclonal antibody of caspase-7 , caspase-9 , and parp and mouse monoclonal antibody of p53 were prepared from cell signaling ( usa ) . mcf-7 cells were cultured in the dmem high glucose medium with 10% heat - inactivated fetal bovine serum , 100  units / ml penicillin , and 100  mg / ml streptomycin and maintained in a humidified atmosphere at 37c and 5% co2 . 25000 cells were seeded in the 24-well plate with 1  ml of culture medium for 24  h. different concentrations of 1.5  mg / ml , 2.5  mg / ml , 3.5  mg / ml , 4.5  mg / ml , and 6  mg / ml for the crocin and 0.01  m / ml , 0.03  m / ml , 0.05  m / ml , 0.1  m / ml , 0.5  m / ml , and 1  m / ml for the paclitaxel were examined separately to get ic50 ( inhibition concentration ) for mcf-7 at the minimum possible time . on this base the incubation times for crocin were used 24  h , 48  h , and 72  h and for paclitaxel 48  h. then the medium was being removed and cells were incubated with 100  l ( 5  mg / ml dissolved in pbs ) mtt and 900  l medium culture at 37c for 4  h. for each well the supernatant was discarded , 500  l dmso was added , and the mixture was suspended . the light absorbance ( a ) was measured at 570  nm wavelength using elisa . finally survival rate calculated as follows:(1)survival  rate  of  tumor  cells  % = experimental  group  a  valuecontrol  group  a  value100%.all above steps were done for crocin and paclitaxel separately . as a result concentrations of 2.5  mg / ml for crocin and 0.01  m / ml for doxorubicin four groups of mcf-7 were selected to study three methods of treatments as follows : control group , second group irradiated by 2  gy gamma radiation ( cobalt 60 source ) , the third group which received 2.5  mg / ml crocin with 0.01  m / ml paclitaxel , and finally the fourth group which was studied by 2.5  mg / ml crocin with 2  gy gamma radiation for the combined group ; meanwhile , we had several control groups . apoptotic cells were revealed by the flow cytometry using pi staining to detect the so - called sub - g1 peak . for this assay mcf-7 cells were cultured in a 6-well plate ( 70000 cells per well ) and treated with 2.5  mg / ml crocin for 24  h , 48  h , and 72  h. the second group of cells was treated with 0.01  m / ml of paclitaxel and the third group was irradiated with 2  gy gamma . to evaluate the combined therapy , the fourth group was treated by 2.5  mg / ml crocin and 0.01  m / ml paclitaxel for 48  h and the fifth group was treated by 2  gy gamma and 2.5  mg / ml crocin with 24  h incubation . then for all groups beside the control group the flow cytometry analysis was being done . then treated cells were detached by trypsinization , washed with pbs , centrifuged in 4000  rpm for 5 minutes , and added to ripa ( lysis buffer ) . in the next step all samples were put in ice for 30 minutes and vortexed every 5 minutes until those would have been homogenized well the supernatant was taken out and the concentration of protein was being measured by bradford method . protein samples were divided into smaller amounts and kept at 80c . to do western blot at first running buffer and transfer buffer were prepared and then three steps were being followed up : ( a ) proteins were separated according to molecular weight by gel electrophoresis and ( b ) transferred to nitrocellulose membrane and ( c ) the desired protein was specified with the primary antibody and shown with the secondary antibody . mtt showed crocin decreased cell viability of mcf-7 cell line with increase of dose and time . ic50 for this cell line was obtained with 3.5  mg / ml crocin after 48  h treatment ( p < 0.05 ) ( figure 1 ) . ic50 was measured for treatment with 0.1  m / ml paclitaxel after 48 hours ( p < 0.05 ) ( figure 1 ) . this study revealed that the combined therapy of 0.01  mol / ml paclitaxel and 2.5  mg / ml crocin after 48  h could cause ic50 for mcf-7 cell line ( p < 0.05 ) ( figure 2 ) . also the combined therapy of 2  gy gamma radiation with 2.5  mg / ml crocin revealed 50% cell death ( p < 0.05 ) ( figure 3 ) . that was greater than 22% cell death for using 2  gy radiations alone . therefore , nuclear dna content of the cells is less than that in normal cells . nuclear - containing could be evaluated and specified in hypodiploid cells by dna binding fluorochromes such as pi . this study revealed that treatment with 2.5  mg / ml crocin in 24 , 48 , and 72 hours could cause increase of apoptosis in mcf-7 cell line with time ( figure 4 ) . also it was found that the combined therapy of 2.5  mg / ml crocin and 0.01  m / ml paclitaxel could increase apoptosis significantly more than that in the single therapy ( p < 0.05 ) ( figure 4 ) . this study showed that the combined therapy of 2  gy gamma radiation with crocin could raise apoptosis in mcf-7 cell line from 21% ( related to using 2  gy gamma radiation alone ) to 46.6% ( p < 0.05 ) ( figure 4 ) . western blot test showed combined therapy of mcf-7 cell line with crocin and paclitaxel or crocin and radiation could increase the expression of 4 proteins including caspase-7 and caspase-9 , p53 , and parp more than that in single therapy ( p < 0.05 ) ( figure 5 ) . as mentioned earlier , breast cancer is the most common malignant neoplasm among women in the world . despite using the routine therapeutic approaches including surgery , chemotherapy , and radiotherapy in patients with cancer , in addition , the destructive effects of chemotherapy and radiation on normal cells are other important concerns . nowadays trends have been focused on natural products such as saffron with anticancer properties to get the better treatment for cancer . showed that constituents of saffron ( crocin , crocetin , and safranal ) could stop the proliferation of mcf-7 cell line . other scientists have approved this antiproliferative effect of crocin , saffron carotenoids pigment on hepg2 cell , and colorectal cancer cells with no harmful effect on normal cells too [ 1315 ] . our study evaluated survival rate of mcf-7 cells in treatment with paclitaxel , crocin , radiation and used mtt assay . our research showed that crocin could inhibit the proliferation of mcf-7 cells in a dose and time dependent manner . for example , when the concentration of crocin increased from 1.5  mg / ml to 6  mg / ml in 48  h , the survival rate of mcf-7 cells reduced from 75% to 23% and the survival rate of mcf-7 cells reduced from 80% to 17% in treatment of cells with paclitaxel from 0.01  m / ml to 1  m / ml in 48  h. paclitaxel reduced the survival rate in a dose dependent manner . they showed the combination of crocin and cisplatin to have the strong effect of killing and suppressing of invasion cells , and it could be even more due to the expression of caspases-8 and -3 . caspases in cascade activation could activate another caspase through a chain reaction strengthening and intensifying the route of apoptosis and rapid cell death . these enzymes are in three groups : initiator ( caspases-2 , -8 , -9 , and -10 ) , broker ( caspases-3 , -6 , and -7 ) , and inflammatory ( caspases-1 , -4 , and -5 ) [ 1921 ] . puma gene is directly induced by p53 in response to dna injury . expression of puma causes the release of cytochrome c of mitochondria stimulating cell death [ 23 , 24 ] . in the inner or mitochondrial pathway of apoptosis , cellular stresses such as dna damage , radiation , or chemical carcinogens activate p53 . according to the type and severity of toxicity , the former allows dna repair and the latter causes cell loss [ 27 , 28 ] . caspase-3 and caspase-7 are the most effective proteases for parp cleavage . as a result , therefore , in this study pi flow cytometry was used to evaluate mcf-7 cell line apoptosis and western blot was used to study the expression of proteins involved in apoptosis . saunders et al . demonstrated apoptosis in mcf-7 cells using 020  ng / ml paclitaxel . our study evaluated the combined therapy of paclitaxel - crocin and paclitaxel - radiation for mcf-7 cell line and found the synergistic effect of those methods . so that 2.5  mg / ml crocin and 0.01  m / ml paclitaxel for 48 hours could cause 54.01% of cells in subg1 phase . this value for the combined therapy of 2.5  mg / ml crocin and 2  gy gamma radiation was obtained 46.6% . the results showed that the expression of apoptotic proteins in the combination groups was significantly higher than the single groups . the combined therapy of crocin and paclitaxel or crocin and gamma radiation has synergistic effect for increasing apoptosis and survival rate reduction of mcf-7 cell line . therefore , it could be suggested to do more study for new breast cancer treatment . in this combined therapy concentration of crocin , drug , and treatment duration are the important factors to get higher apoptosis and lower survival rate in mcf-7 cell line .
background . chemotherapy , radiotherapy , and surgery are routine treatments of breast cancer . 
 however , these methods could only improve the living survival . nowadays 
 the combined therapy including herbals such as crocin is to study for improving breast cancer treatment . 
 the purpose of this study was to evaluate the effects of crocin , paclitaxel , and radiation on mcf-7 cell . methods . to evaluate the effect of crocin , paclitaxel , and radiation on survival rate of mcf-7 cells mtt assay was done . to investigate the apoptotic effect of experimental groups pi - flow cytometry 
 was used and expression of apoptotic proteins ( caspase-7 , caspase-9 , parp , and p53 ) was studied by western blot . results . 
 this study revealed that the combined therapy of 0.01mol / ml paclitaxel and 2.5 mg / ml crocin after 48  h could cause ic50 for mcf-7 cell line . 
 this study showed that the combined therapy of 2  gy gamma radiation with crocin could rise apoptosis in mcf-7 cell line from 21% ( related to using 2  gy gamma radiation alone ) to 46.6% . 
 conclusion . 
 crocin and paclitaxel and crocin and gamma radiation had synergistic effect on mcf-7 cell line to get more significant apoptosis .