Experimental Study. Guben Inhibitory ¢ò On Breast Cancer Tumor Effect And Its Mechanism

Breast cancer that is harming the health of women is one of the most common malignant tumors,the incidence and the death rat of which take the first place of all kinds of malignant tumors.One would suffer from breast cancer in every 1000 adult women. There are about 7,900 newly cases of the illness annually and the trend is increasing year by year,so the prevention and treatment study on breast cancer has already turned into the mission which has no time to be delaied.The treatments including traditional operation and radiotherapy and chemotherapy have low selectivty between pathological tissue and normal tissue ,which would result in all kinds of complications or sequel. As a result the suffers have low quality of existence and behavior.The western medicine therapy of breast cancer has its limitation,so the action of TCM curing breast cancer cannot be replaced more and more.In Chinese medicine,breast cancer is a result of stagnation of blood and phlegm due to deficiency of Zang-fu organs.In fact,breast cancer sufferers, especially in late stage, did generally have symptoms with blood stagn- ation/or blood stagnation in Qi deficiency condition, so the treatment should be to strengthen qi to protect body, and activate blood to eliminate pathogenic factors, through these two ways, a new balance will be built up again. Guben Yiliu formula II(GYII),produced by Beijing City Hospital of Traditional Chinese Medicine,is an effective prescr- iption in treating cancer with syndrome of blood stasis and Qi deficie- ncy, which is generally applied in combination with chemotherapeutics. Clinic datum has proved that GYII can improve the quality of life and lessen the side effects of chemotherapeutics.Aims: To study the mechanism of GYII treating breast cancer and to provide a tentative theoretical basis for clinical therapy for breast cancer.Experiment:1.The experimental study of anti-cancer action of GYII and combination with chemotherapy on MCF-7 human breast cancerGYII aqueous extract and the medicated mouse serum of it were prepared respectively.MTT assay was used to detect the effects of GYII, Epirubicin (EPI),GYII+EPI,serum with GYII of 5%,10%,20% concentration on MCF-7 human breast cancer cell growth.Results:GYII showed depressive effect on MCF-7 cell growth.When the growth inhibition ratio of MCF-7 cell for GYII reached 30% and 50%,the concentration of crude GYII was 1.22mg/ml and 3.25mg/ml respectively. The inhibition of MCF-7 cell growth was enhanced with increasing concentration of EPI.The IC30 and IC50 were 0.095μg/ml and 0.228μg/ml repectively. The q value(combinative effect)of both GYII and EPI was between 0.85-1.15,which showed some synergetic effect of GYII + EPI.. The growth inhibition ratio of MCF-7 cell reached 20.84% and 23.91% respectively after administration of the serum containing GYII of 20% concentration 24h and 48h,which were more than 20%.This accounts for that the serum with GYII has the cytotoxic activity on tumor cells.2. Research of the treatment effect of GYII and combination with GYII and chemotherapy on EMT-6 mouse breast canerEighty BALB/C female mice with weight of 19±1g were replicated the animal model of EMT-6 mouse breast caner,which were randomly divided into eight groups:control group,EPI group[EPI](intraperitoneal injection 3mg/Kg), low dose of GYⅡ[S(GYⅡ)](13.7g/Kg·d),middle dose of GYⅡ[M(GYⅡ)](27.3g/Kg·d),high dose of GYⅡ[H(GYⅡ)] (54.6g/Kg·d), low dose of GII with EPI[S(GYⅡ)+EPI],middle dose of GII with EPI[M(GYⅡ)+EPI],high dose of GII with EPI[H(GYⅡ)+EPI],every group with ten mice.To observe GYII and combination with EPI on the transplantation tumor and the number of metastatic of EMT-6 mouse breast caner.Results:The average delay time rate of the tumors coming out in H(GYⅡ),EPI,H(GYⅡ)+EPI were 22.45%,24.49% and 28.57% respectively. The three doses of GYⅡ[S(GYⅡ),M(GYⅡ),H(GYⅡ)] had inhibitory effects on transplant EMT-6 mouse breast caner to some extent, the inhibitory rate (IR) was 13.6%,17.4% and 34.38% respectively.The weight of transplantation tumor of H(GYⅡ) group was 1.722±0.555g, less than that of control group(2.624±0.582g),p<0.01.The treatment groups decreased the volume of all the tumors.The q value of S(GYⅡ)+EPI group and M(GYⅡ)+EPI group were between 0.85-1.15,which showed additive action of GYⅡwith EPI.Both GYⅡand EPI separately had no notable effect on spontaneous lung metastasis,but combination of them reduced the number of metastasis.the number of metastasis in S(GYⅡ)+EPI group and M(GYⅡ)+EPI group were 2.9±1.0 and 2.7±1.3 respectively,less than that of control group(5.5±1.5个),p<0.05.3. Effects of GYⅡon immune function of EMT-6 mouse breast carcinoma- bearing miceThe mice which had been replicated the animal model of EMT-6 mouse breast caner were randomly divided into five groups: Control,EPI,M(GYⅡ),H(GYⅡ),M(GYⅡ)+EPI,every group with ten mice.The routine examination of peripheral blood of mice blood was detect by blood cell counter.T cell subsets of peripheral blood of mice were measured by Flow cytometry(FCM).MTT assay was used for dectectng NK cytoactive,T cell multiplication and proliferation of splenic lymphocyte.neutral red was used for peritoneal macrophage phagocytosis.Results:GYⅡcould increase RBC and HB of peripheral blood of mice and improved anemia in tumor mice,but had no effect on WBC. It could also increase thymus index in mice,however there was on effect on spleen index in mice.NK cytoactive, cell multiplication of splenic lymphocyte,phagocytosis of peritoneal macrophage expression of T cell subsets (CD3,CD4,CD8)in the peripheral blood were increased by use of GYⅡ.M(GYⅡ)group and M(GYⅡ)+EPI group had been enhanced to some extent.The average living days of EMT-6 mouse breast cancer carcinoma- bearing mice in M(GYⅡ)group and M(GYⅡ)+EPI group were 33.3±7.3 days and 33.5±6.3 days,longer than the control 23.6±6.87 days,they can prolong survival time.4.Effects of GYII and combination with GYII and EPI on cell cycle and apoptosis of solid tumor of MCF-7 human breast carcinoma-bearing mice48 BALB/C female nude mice with weight of 17±1g were replicated the ainimal model of MCF-7 human breast caner. When tumor diameter increased to nearly 0.6cm,the mice were randomly divided into six groups:Control,EPI,M(GYⅡ),H(GYⅡ),M(GYⅡ)+EPI,H(GYⅡ)+EPI, every group with eight mice.After 25 days of administration,the mice were executed,then the weight of transplant-tation tumor was calculated;Flow cytometry was applied to analyze the cell cycle and apoptosis.Results: The weight of transplantation tumor of H(GYⅡ) group was 1.59±0.39g,less than that of control group(2.49±1.35g),p<0.05.And the weight of transplantation tumor of H(GYⅡ)+ EPI group (0.70±0.55g)were less than that of both control group and EPI group(1.04±0.73g),p<0.05. The q value of GYⅡin combination with EPI was between 0.85-1.15, which showed additive action of GYⅡwith EPI in inhibitory effects on transplant MCF-7 human breast caner. GYⅡhad effects of breast cancer tissue on cell cycle,on the contrary, GYⅡin combination with EPI did not. The distribution of carcinoma cells in G0/G1 phases of cell cycle seemed tend to in-crease with the dose of GYⅡincreased.The figure of G0/G1 phases of carcinoma cells in H(GYⅡ) group was 54.74±2.11, more than that of control group(50.71±2.07),p<0.05.At the same time the distribution of carcinoma cells in G2/M phases of cell cycle had been reduced.But GYⅡhad no effects on S phases of carcinoma cells.Both GYⅡand GYⅡin combination with EPI accelerated apoptosis in breast cancer.Conclusion:GYII and the medicated mouse serum of it could inhibit growth of MCF-7 human breast cancer and have. the inhibitory effect on MCF-7 human breast cancer cells and synergistic action with EPI in vitro.In our experiments we obversed the effects of GII on transplantation tumor of EMT-6 mouse breast cancer and MCF-7 human breast cancer, and synergistic action with EPI.GYII have the effects on immune functions, living time ,improving anemia of tumor mice , blocking carcinoma cells in G0/G1 phase and accelerating them apoptosis. GYⅡin combination with EPI can reduce the number of spontaneous lung metastasis,but only GYⅡcan not.