Effects And Mechanisms Of LMWH On Human Breast Cancer Cell Line MCF-7

Objective: To study the effects of LMWH on prolif- eration ,apoptosis, invasivenessof human breast cancer cell line MCF-7 and its mechanisms. Meanwhile, to investigate possible mechanism of inhibiting cell proliferation,inducing apoptosis and inhibiting cell invasiveness and feasibility therapy and chemoprevention of breast cancer.Methods: 1 MCF-7 cells were incubated in culture medium in vitro. Effect of LMWH on the proliferation of MCF-7 cells was measured by MTT colorimetric method. Morphological changes of MCF-7 cells were observed by light microscopy and transmission electron microscopy. Apoptosis and distribution of cell cycle were examined by flow cytometry. The expression of p53,Bax protein was analyzed by flow cytometric indirect immunofluorescent technigue. The expression of p53,Bax gene was analyzed by RT-PCR after treated with LMWH for 48h. 2 MTT colorimetric method was performed to cell matrigel adhesion assay the potential effect of LMWH on human breast cancer cell line MCF-7. To study the effects of LMWH on migration, invasion of human breast cancer cell line MCF-7 after treated with LMWH for 24h. Results:1 MTT colorimetric method showed that LMWH (25,50,100IU /ml) could inhibit the proliferation of MCF-7 cells. After treated with LMWH for 48h to 72h, compared with control group, the OD values of LMWH-treated groups decreased, and there was statistically significant difference between control group and every treatment group (P<0.01). Among every treatment group, there was also statistically significant difference (P<0.01). Furthermore, with the increasing concentration of LMWH and prolonging of treatment time, the OD values decreased gradually, that was, LMWH inhibited the proliferation of MCF-7 cells significantly in a dose-and time-dependent manner. When treated with LMWH, MCF-7 cells had significant morphological changes which were observed by light microscopy: The cells untreated with LMWH were diamond or pleomorphic, and they were satiation. But the cells treated with LMWH shrinked and broken, became irregular in shape, spreaded a thin and long pseudopodium in each end. Vacuolus could be observed in the kytoplasm of some cells. The cells cast-off increased. The cell ultramicrostructure changes were observed by transmission electron microscope: nucelus overshoot in acute angle outwards, chromatin concentratede highly, electron density raised and they were enriched as crescent below the nuclear membrane. We also observed karyopycnosis.When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the results were: After treated with 25,50,100IU /ml LMWH for 48h, the number of cells in G0/G1 phase increased gradually, while the number of cells in S phase decreased grudually. That was, LMWH could induce an arrest of cell cycle in G1 phase by a dose-dependent manner. In addition, after treated with 25,50,100IU /ml LMWH for 48h, the typical apoptotic peak which enhanced gradually with the increasing concentration of LMWH was observed and the apoptotic percentage was 11.6%,23.5%,30.83% seperately. Analysis on expression of p53 and Bax by FCM showed that: Treating MCF-7 cells with 25,50,100IU /ml LMWH for 48h resulted in the FI values of p53 and Bax increasing with the increasing concentration of LMWH. To the FI values of each kind of protein, there was statistically significant difference between every LMWH-treated group and control group (P<0.01). RT-PCR detection results: Before being treated with LMWH, p53 and Bax were both expressed in MCF-7 cells. Compared to the control group, the expression p53 mRNA and Bax mRNA were increased markedly in MCF-7 cells exposed to LMWH (P<0.01).2 After treated with LMWH(25,50,100IU/ml) for 60~120min the ability of adhesion in MCF-7 cells were difference(P<0.05) by MTT colorimetric method. with the increasing concentration of LMWH the OD values decreased gradually, that was, LMWH inhibited the adhesion of MCF-7 cells significantly in a dose-dependent manner. After treated with LMWH(25,50,100IU/ml) the ability of invasion and metastasis in MCF-7cells were statistically significant difference. The invaded cell numbers in metastatic experiments of MCF-7 cells untreated or treated with LMWH (25,50,100IU/ml) were statistically significant difference (P<0.01), The invaded cell numbers in invasive experiments of MCF-7 cells untreated or treated with LMWH(25,50,100IU/ml) were statistically significant difference (P<0.01).The invasive and metastatic abilities of treated group were lower than untreated group significantly. LMWH inhibited the invasive and metastatic abilities of MCF-7 cells significantly in a dose-dependent manner. The results of RT-PCR showed that the expression of TF mRNA decreased after the LMWH treatment for 24h compared to the control group. There were significant differences among the MCF-7 cells treated with differrent concentration.Conclusions:1 LMWH had the effect of anti-breast cancer cell line MCF-7, which provided a new theoretical foundation for appropriate clinical treatment and chemoprevention of breast cancer.2 LMWH had the capability of inhibiting the proliferation of MCF-7 cells in vitro in a dose-and time-dependent way.3 LMWH induced an arrest of cell cycle in G1 phase as well as apoptosis in MCF-7 cells.4 Within a certain drug concentration, LMWH can increase the expression of p53 and Bax in MCF-7 cells. We infer that LMWH's effects of inhibiting proliferation and inducing apoptosis in MCF-7 cells seem to due to up-regulation of the expression of p53 and Bax mRNA and protein.5 LMWH inhibited the adhesive,metastatic and invasive abilities of MCF-7 cells by down-regulating the expression of TF mRNA.