| Objective: Breast cancer is one of the most common malignant tumors in women,and it has been the critical disease harming female health.Chemotherapy plays an important role in the systemic therapy of breast cancer focusing on surgery. Accompanied with other therapies,chemotherapy can effectively inhibit the invision and metastasis of breast cancer,making the life of the patients longer and the quality of life better.However,the disseminated nature of breast cancer and the development of drug resistance are the primary causes of failure of current therapies.By the time a tumor is detected,there is a high probability that metastatic lesions will be present,and many of these will already contain a resistant sub-population of cells.Therefore,the study and investigation of new anticancer agents are always the focus of people s attention. Nordihydroguaiaretic acid(NDGA) is a strong lipoxygenase inhibitor which can block the lipoxygenase metabolism of arachidonic acid.Recently,NDGA has been found to have functions which can prevent the development of many tumors such as breast cancer,hepatic carcinoma,colon cancer and glioma ,and can decrease the drug resistance at the same time.But the mechanism is not clear. In this study,we will culture human breast cancer MCF-7 cells in vitro and examine the apoptosis,proliferation, 5-LOX,Bcl-2,telomerase,VEGF and CD44V6 of the cell line in order to investigate the biologic characteristic influence of MCF-7 cells interrupted by NDGA,throughwhich we can elucidate the antineoplastic mechanisms of lipoxygenase inhibitors for the clinical application.Material and Methods: A human breast cancer cell line,MCF-7,was cultured in vitro and NDGA was divided into 1,10,100μmol/L at concentration.Cells in logarithmic growth phase were selected and examined. (1)The proliferative dynamic indexes including cell growth curve and cell survival rate were observed by means of MTT asssy.(2)Morphological variation was investigated by using invert phase-contrast microscopy and scanning electron microscopy. (3 )Cell cycle analysis, apoptosis rate,Bcl-2 and CD44V6 were evaluated by flow cytometry(FCM).(4)The mRNA expressions of 5-lipoxygenase,telomerase and VEGF were measured by reverse transcription-polymerase chain reaction(RT-PCR).Results:(1)The cell growth curve showed NDGA at various concentrations inhibited the growth of MCF-7 cells in a time-and dose-dependent manner.(2)After treatment with NDGA at different concentrations( 1,10,100,1000μmol/L) for 48h,the cell survival rate of MCF-7 cells was 66.43 ± 3.01%,45.48 ± 1.02%,33.21 ± 0.97% and 20.02 ± 0.36% respectively,presenting a trend of decrease.The value of IC50 was about 8.13μmol/L.(3)Under the invert phase-contrast microscope,the cell morphology obviously changed while treated with different concentrations of NDGA. Some cells became much more round and brighter, shrinking with the membrane crimpled like burrs. The cytoplasm contained highly concentrated chromosomes which attached to the nuclear membrane and came into the shape of clump,crescent or fragment.Many cells fell off the wall of medium and were suspensive in the culture medium.(4)Under the scanning electron microscope,MCF-7 cells showed typical apoptotic morphological changes after treatment with 100μmol/L NDGA for 48h,characterized by cellular roundness,volume shrinkage and membranouscrinkle.The changes of microvillus and filopodium were very distinct.The filopodia were broken,and the microvilla were curled and shortened.Some MCF-7 cells produced apoptotic bodies in the form of bud,characterized by many globules protuberating from the cellular surface. And some apoptotic bodies had fallen off from the cellular surface.(5)After MCF-7 cells had been exposed to different concentrations of NDGA for 48h,the flow cytometry analysis showed the apoptotic peak,the apoptotic ratio and the cell proportion of S phase increased following the enhanced concentration in NDGA groups,significantly higher than the control group (PO.001).(6) After 48 hours of incubation ,the flow cytometry analysis revealed the expressions of Bcl-2 and CD44V6 in MCF-7 cells were significant in NDGA groups compared with the control group(P<0.001).Correlative analysis showed the relation between Bcl-2 and CD44V6 with concentration was significantly negative linear correlation.The correlation parameter was -0.569 and -0.581 respectively.(7)The expressions of 5-LOX mRNA,hTERT mRNA and VEGF mRNA in MCF-7 cells were examined by RT-PCR after NDGA treatment for 48h.Different concentrations of NDGA down-regulated all the expressions of mRNA with dose-related effect and were significant compared with the control group(P<0.001).Conclusion:(1)NDGA can inhibit the growth of human breast cancer MCF-7 cell line in a time-and dose-dependent manner, and block the cell cycle in S phase at the same time.The mechanism may be implemented through inducing apoptosis and preventing proliferention.(2)Because NDGA has a dramatical effect on the morphology of MCF-7 cells, especially on the filopodia and microvilla,we can conclude NDGA might prevent the adhesion and invasion of the cells.(3)The expression of 5-LOX mRNA was stongly positive in MCF-7 cells.One of the mechanisms of NDGA inhibiting proliferation and inducing apoptosis is likely to... |
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