The. Tetrandrine Anti-the Mammary Tumor Effect And Its Mechanism

1 ObjectiveBreast cancer is one of most common adult female malignant tumors. About 1/3 breast cancer patients show the estrogen receptor(ER) negative, which is insensitive to the chemotherapy and the endocrine therapy. Its malignant degree is higher and prognosis is worse than ER positive breast cancer. At present there are not ideal therapeutic methods. The treatment of ER negative breast cancer is the clinical difficulty. Therefore this research studies the antitumor effect and mechanism of tetrandrine(Tet) on ER negative human breast cancer cell MDA-MB-435S in Vitro and MCF-7 in tumor bearing nude mice,which is to lay the foundation for further studies in clinical treatments.2 Methods2.1 Experiments in VitroMTT was used to detect optical density (OD) of human breast cancer cell MDA-MB-435S by intervention of Tet in seven different concentration groups in 24h,48h,72h,96h. Drawing cell growth curve,calculating cell inhibitory rate and 50% inhibitory concentration (IC50) in every group, which were used to show inhibitory effect of Tet on human breast cancer cell MDA-MB-435S in Vitro growth.Propidium iodide (PI) single-staining flow cytometry assay method was used to analyze cell cycles of human breast cancer cell MDA-MB-435S by intervention of Tet in four different concentration groups. To observe cell percentage distribution in G0/G1, G2M, S phases by intervention of different Tet concentration groups after 24h.Cell apoptosis inspection method of Annexin V-FITC,PI double labelling was used to detect the cell apoptosis effect of Tet on human breast cancer cell MDA-MB-435S in four different concentration groups after 24h and 48h.2.2 Experiments in VivoTo build transplanted tumor animal model of human breast cancer cell MCF-7 in nude mice.24 mice with transplanted tumor were randomly divided into model group, Tet group and positive control group,8 mice in each group, administration for 15 days. Detecting and recording the growth condition and volumes of transplanted tumor in every group. Nude mice were killed in the day after administration ending up, weighting the transplanted tumor and calculating tumor inhibitory rate. Light microscope and transmission electron microscopy (TEM) were used to observe histopathologic changes and ultrastructural changes of transplanted tumors in every group. Image quantitative analysis was used to observe Bcl-2, Bax, Survivin and VEGF protein expression level of transplanted tumors in every group.3 Results3.1 Experiments in VitroAs compared with normal control group (control-1) at the same time, OD value was decreased significantly(P< 0.05 or 0.01)in seven different Tet concentration groups (Tet-0.5 group, Tet-1 group, Tet-2 group, Tet-4 group, Tet-6 group, Tet-8 group, Tet-16 group),and OD value decreased more significantly as Tet concentration increased. The inhibitory rate of Tet was enlarged with the increase of the concentration in the same time, which exhibited dose-effect relationship. With the passage of time, inhibitory effect of Tet on MDA-MB-435S cell in vitro growth was enlarged, which exhibited time-dependent effect. Interaction effect of dose and time existed for the effect of Tet on MDA-MB-435S cell (P<0.01). IC50 value in 24h,48h,72h,96h were 8.748μg/ml,3.585μg/ml, 1.451μg/ml,1.117μg/ml respectively.As compared with normal control group in the same phase, cell percentage of S phase, G2M phase in MDA-MB-435S cell were decreased significantly in four different Tet concentration groups (Tet-0.5 group, Tet-1 group, Tet-2 group, Tet-4 group) after administration for 24h, with statistical significance (P< 0.05).Higher the Tet concentration, lower the cell percentage of S phase, G2M phase. As compared with normal control group in the same phase, cell percentage of Go/Gi phase were increased significantly, with statistical significance (P < 0.05). Higher the Tet concentration, higher the cell percentage of G0/G1 phase.As compared with control-1 group after administration for 24h, early apoptosis rate was increased in four different Tet concentration groups (Tet-0.5 group, Tet-1 group, Tet-2 group, Tet-4 group),with statistical significance (P< 0.05). As compared with control-1 group after administration for 48h, early apoptosis rate was increased significantly in Tet-1 group, Tet-2 group and Tet-4 group (P< 0.05).Significant positive correlation was observed between early apoptosis rate and Tet concentration after administration for 24h,48h (P<0.05). With the passage of time, early apoptosis rate were increased gradually. There were no difference in late apoptosis rate between Tet groups and control-1 group after administration for 24h(P> 0.05). As compared with control-1 group after administration for 48h, late apoptosis rate were increased gradually in Tet high-concentration groups, with statistical significance (P< 0.05). 3.2 Experiments in VivoFrom 6 days after treatment, volumes of transplanted tumor in Tet group and positive control group were significantly smaller than model group (P<0.05 or 0.01), there were no difference between Tet group and positive control group (P> 0.05).The weight of the transplanted tumor in Tet group and positive control group were significantly decreased than model group (P<0.05 or 0.01) but there were no difference between Tet group and positive control group (P> 0.05). Tumor inhibitory rate in Tet group and positive control group were 25.47% and 30.43% respectively. Transplanted tumor in Tet group and positive control group by hemotoxylin and eosin staining suggested that necrotic cells and incomplete necrotic cells were seen among the adenocarcinoma. More apoptotic cells were found in Tet group and positive control group than model group. It was found by TEM that there were many apoptotic bodies in Tet group.As compared with model group, the positive cell area, mean optical density and immunohistochemistry index of Bcl-2 in Tet group and positive control group were significantly reduced (P<0.05 or 0.01). As compared with model group, the positive cell area and immunohistochemistry index of Bax in Tet group and positive control group were significantly increased (P<0.01). There were no difference of mean optical density between Tet group and positive control group (P>0.05). As compared with model group, the positive cell area and immunohistochemistry index of Survivin in Tet group and positive control group were significantly reduced (P<0.01). There were no difference of mean optical density among three groups (P>0.05). As compared with model group, the positive cell area, mean optical density and immunohistochemistry index of VEGF in Tet group and positive control group were significantly reduced (P<0.01),but There were no difference between Tet group and positive control group (P> 0.05).4 Conclusion(1) It is first found that the effect of Tet on inhibiting ER negative breast cancer MDA-MB-435S cell proliferation is obvious, which provides the basic research data for clinical treatments on ER negative breast cancer.(2) Tet can induce the early apoptosis after administration for 24h,48h. There is significant positive correlation between early apoptosis rate and Tet concentration.It has concentration and time dependence in the range of 0.5μg /ml to 4μg/ml.(3)Blocking the cell cycle at G0/G1 phase and reducing the cell percentage of S phase to inhibit the DNA synthesis is one of the antitumor mechanisms of Tet on breast cancer cell in Vitro.(4)Tet can inhibit the growth of transplanted tumor of MCF-7 in nude mice.(5)The antitumor mechanisms of Tet on MCF-7 in tumor bearing nude mice are related to reducing Bcl-2, Survivin, VEGF protein expression and increasing Bax protein expression.