Synergistic Antitumor Effect Of TRAIL And ADM On Human Breast Cancer Cell Line MCF-7

Objective: Breast cancer is one of the most common malignant tumorsthat do harm to our health.Tumor necrosis factor related apoptosis inducingligand(TRAIL) is a member of tumor necrosis factor(TNF) superfamily.TRAIL induces apoptosis in the majority of cancer cells without appreciableeffect in normal cells. The aim of the study was to explore the effects andpotential mechanisms of apoptosis induced by TRAIL alone or incombination with doxorubicin (ADM) on human breast cancer cell lineMCF-7.Methods: Human breast cancer cell line MCF-7 cells were maintainedin RPMI 1640 supplemented with 10% calf serum and 1%penicillin/streptomycin. The cell growth curve of MCF-7 cells was delineatedin order to analyze the cell proliferating dynamic values. The cell viabilitywas analyzed by MTT assay and the apoptosis rate was detected by flowcytometry (FCM). The expression of TRAIL receptors and the variety ofDR4, DR5 in mRNA levels in MCF-7 cells were examined by RT-PCR. Thechanges of Bax and Caspase-9 in protein levels were also determined by Western blot. Synergetic effect was analyzed by Weeb coefficient.Results:(1) Breast cancer cell line MCF-7 was treated by TRAIL (0～10μg/ml),there was no obvious correlation between cell viability and TRAILconcentration (r=-0.313,P＞0.05). IC₅₀ was above 10μg/ml, whichsuggesting that MCF-7 cells were not sensitive to TRAIL.(2) Breast cancer cell line MCF-7 was treated by ADM (0～0.5μg/ml), therewas significant negative correlation between cell viability and ADMconcentration (r=-0.941,P＜0.05). IC₅₀ was 7.0μg/ml, whichsuggesting that MCF-7 cells were sensitive to ADM.(3) Weeb coefficient analysis showed that 0.5μg/ml ADM combination with0.1, 1, 10μg/ml TRAIL had synergetic effect, respectively, and more inthe group of 0.1μg/ml TRAIL and 0.5μg/ml ADM combination. When0.05μg/ml ADM simultaneous and sequential cotreated with 0.01, 0.1, 1,10μg/ml TRAIL, cell viability was significant lower than that in TRAILalone group (P＜0.05). Compared with TRAIL alone group, cell viabilitywas no significant difference in the groups of 0.005μg/ml ADM and 0.01,0.1, 1, 10μg/ml TRAIL combination, respectively (P＞0.05).(4) The apoptosis rates of MCF-7 cells treated by 0.1μg/ml TRAIL alone and0.5μg/ml ADM alone were 9.8% and 17%, respectively. The apoptosisrates of simultaneous and sequential combination of 0.1μg/ml TRAILwith 0.5μg/ml ADM were 38.7% and 24.3%, respectively. The findingssuggested that simultaneous and sequential combination could effectivelyinduce MCF-7 cells apoptosis.(5) The result of RT-PCR showed that DR4, DR5 and DcR2 mRNA were expressed in MCF-7 cells, but DcR1 mRNA was absent. Compared withnegative control group and TRAIL alone group, 0.5μg/ml ADM and itscombination with 0.1μg/ml TRAIL could up-regulate the expression ofDR4 and DR5 mRNA in MCF-7 cells.(6) Compared with negative control group and TRAIL alone group, proteinlevels of Bax and Caspase-9 were up-regulated after combinationtreatment of TRAIL and ADM.Conclusions:(1) Human breast cancer cell line MCF-7 was not sensitive to TRAIL,butsensitive to ADM.(2) 0.5μg/ml ADM combination with 0.1μg/ml, 1μg/ml, 10μg/ml TRAIL hadsynergetic effect,respectively.0.1μg/ml TRAIL combination with0.5μg/ml ADM group had the highest synergetic effect.In somedegree, sequential combination could inhibit the prolification of MCF-7cells.(3) Compared with TRAIL and ADM alone group,apoptosis rates of breastcancer cell line MCF-7 were obviously increased in simultaneous andsequential combination groups.(4) MCF-7 cells expressed DR4,DR5 and DcR2 mRNA, but lack of DcR1mRNA expression.High expression of DcR2 may be one of themechanisms of human breast cancer cell line MCF-7 resisitant to TRAIL.(5) The combination of TRAIL and ADM could obviously up-regulate theexpression of DR4,DR5 mRNA and the protein level of Bax,Caspase-9,itmay be one of the mechanisms of the synergetic effect.