Study On The Anti-breast Cancer Effect And Mechanism Of Ruyanning Combined With Tamoxifen

Object:Breast cancer is one of the malignant tumors which occur most frequently in women. The incidence rate of breast cancer in our country presented obvious increasing trend recently. At present, it starts to decrease because of the development of chemotherapy, radiotherapy, immunotherapy and surgery for treating breast cancer. But quite a few of patients would be relapse or metastasis. However, most of terminal breast cancer patients could not suffer the second chemotherapy, radiotherapy or surgery. Endocrine therapy has been confirmed by most of doctors and patients because of its advantages, such as convenience to use, gentle side effect and long alleviation time, etc. Terminal breast cancer patients were treated with taking TAM (TAMoxifen) and Ru-yan-ning decoction in oncology department of Affiliated Hospital to Liaoning University of Traditional Chinese Medicine, and the therapy obtained good efficacy. Ru-yan-ning decoction was prescribed on the basis ofChai-hui-jia-long-gu-mu-li decoction,and its main effects are adjusting the motion of Qi, flowing the liver and nourishing the spleen, strengthening vital Qi and anticancer, resolving hard lump. The occurrence and development of cancer has close relationship with the motion of Qi, including ascending, descending, entering and leaving of Qi. We could treated various symptoms induced by malignant tumor by recuperating liver, kidney and spleen, in order to enhance the quality of life and prolong the length of life. In the area of traditional Chinese medicine, it is believed that liver pulse passing breast, and stagnant of Qi and Blood induced constraining Liver Qi to result in stagnant Qi and Blood coagulating in breast, in course of time, it induce obstructing of breast collateral to develop breast cancer. Therefore, treating breast diseases of all ages mostly focused on liver. Chai-hui-jia-long-gu-mu-li decoction in Discussion of Cold-Induced Disorders is the main decoction to harmonize Shao-yang, regulate the flow of vital Qi and adjust Liver. It could smooth the motion of Qi, freely open the ascending, descending, entering and leaving of Qi, including all things from soup to nuts. My advisor, professor Yin Dongfeng, summarized his experience to assume that cancer poison flee everywhere in the body of breast cancer patients, the mechanism is always the stagnant of the motion of Qi although syndromes occurred are variational. Therefore, Ru-Yan-Ning decoction is prescribed on the basis of Xiao-Chai-Hu decoction to smooth the motion of Qi and harmonize Liver Qi. In this description, bupleurum (Chai-Hu), as the monarch drug, matches with ministerial drug Scutellaria (Huang-Qin) to smooth the motion of Qi and harmonize the helm; Ginger (Sheng-Jiang) matches with pinellia (Ban-Xia) compose Xiao-Ban-Xia decoction which could dissipate the stagnant with acrid flavor, regulate stomach and arrest vomiting, reduce spitting and descend the rebellious Qi, and smooth the motion of Qi; Ginseng (Ren-Shen), Chinese Date (Da-Zao) and glycyrrhiza (Gan-Cao) match not only present the meaning of strengthening the vital Qi to eliminate evil Qi, but also mean that seeing liver disease is the knowledge of the liver disease could transmit to spleen, so nourishing spleen Qi is necessary. Oyster (Mu-Li) and keel (Long-Gu) are added to relieve uneasiness of mind and body. Thunberg Fritillary Bulb (Zhe-Bei-Mu), Sagittaria sagittifolia (Shan-Ci-Gu) and Zedoary (E-Zhu) assist to eliminate blood stasis, detoxify the body and resolve the lump. Making a general survey of this decoction, it has the function of ascending and descending to smooth the motion of Qi, sooth liver and strengthen spleen.The effect of Ru-Yan-Ning decoction on TAM for antitumor and its mechanism need further study. Therefore, our investigation chose human estrogen dependence breast cancer transplanting model to observe Ru-Yan-Ning decoction united TAM to effect on MCF-7 tumor bearing nude mice for antitumor and the apoptosis of it effecting on MCF-7 tumor bearing nude mice, determine the expression of MMP-2 and MMP-9 in transplanting tissues of nude mice by immunohistochemistry, detect the expression of p38MAPK and c-fos in transplanting tissues by Western blotting. Then we could discuss the inhibitory effect of Ru-Yan-Ning decoction on breast cancer metastasis and its mechanism may be related to the signal transduction passage of p38MAPK.Materials and methods:The first part: The establishment of MCF-7 tumor bearing nude mice model: after trypsinization, cultured MCF-7 cells were rinsed twice with PBS, and dyed by 1% trypan blue,and counted on the cell counting chamber. Then adjust the cell concentration to 1×10~7/ml,and filled the second breast pad into the right chest wall and filled one 0.2ml pad under the adipose layer, altogether filled 3 pads. After surgery, the tumor bearing nude mice continued to be fed in SPF environment. When tumor raised to 0.8 cm~3,the mice were treated with orthotopic transplantation. The breast cancer tissues were taken out in aseptic condition, then cut into chips about 1 mm~3 size, and transplanted under the adipose layer of second pad of right chest wall of 24 nude mice. 4 days later, tumor growth was observed, tumorigenesis rate reached to 100%. 24 MCF-7 tumor bearing nude mice were randomly divided into 4 groups (6 mice in every group):?Model group (tumor bearing nude mice),?TAM group,?Ru-Yan-Ning group,?TAM+ Ru-Yan-Ning group (i.e. union group). Model group:the mice in this group were treated with 0.9% sodium chloride inoculation fluid (0.2ml per nude mouse); TAM group: 3.6mg/kg (0.2ml per nude mouse); Ru-Yan-Ning group:the mice in the group were treated with Ru-Yan-Ning 1.149g/kg (0.2ml per nude mouse); Union group:TAM 3.6mg/kg+Ru-Yan-Ning 1.149g/kg(0.2ml per nude mouse). The mice were perfused once every day for 28d continuously. Determination indices including:?Tumor growth curve: From the 1st injection, the general situation and the growth condition were observed every two or three days. The diameters of tumor were detected under the aseptic condition and record continuously. Take the time as the abscissa,and the tumor volume as the y-coordinate. The tumor volume formula is: V=1/2×ab~2,“a”is the tumor major axis,and“b”is the tumor minor axis, and draws up the transplant tumor growth curve according to the above.?Tumor inhibition rate:4 weeks after injection medicine,the nude mice were killed by breaking the cervical vertebra off,then to measure the body weight of the killed mice with the animal balance, and measure the weight of separated tumor with the electronic balance, and then the calculate the tumor inhibition rate. The tumor inhibition rate formula: (the weight of tumor in model group- the weight of the tumor in experimental groups)/ the weight of tumor in model group×100%.?Cell apoptosis was observed by the transmission electron microscope:tumor tissues were quick frozen by ultramicrotomy, fixed by double fixation methods with the glutaric dialdehyde and the perosmic anhydride osmium,then underwent dehydration, percolation, embedding and slice,and then observed on the ultrastructure level with TEM (transmission electron microscope).?Rate of tumor cell apoptosis: It was determined by TUNNEL method. The brown granules appeared in the cell nucleus presents were positive cells. Count the number of positive cells in 100 cells under the high power field and chose 5 fields randomly in each slice to calculate AI (Apoptosis Inedx) (AI = positive cells number/cell number counted×100%). The results were analyzed by SPSS13.0 software, and take the single factor variance analysis as analysis method. The statistical data were indicated by the mean value±standard deviation, take P<0.05 as the significance difference standard. The second part:The expression of MMP-2 and MMP-9 in transplanting tumor tissues of nude mice was determined by immunohistochemistry (S-P method). Take forgone positive slice as positive control and take PBS replacing the first antibody as negative control. MMP-2 and MMP-9 results determination standard: MMP-2 and MMP-9 positive cells present the buff to sepia granules in the cell membrane or the cytoplasma. The judgment method is according to the positive intensity and area of the brown granules under the microscope. Graded every slice according to the positive intensity of positive cells, graded non-coloration, faint yellow,brownish yellow and sepia as 0, 1, 2 and 3 point respectively. Graded positive area non-coloration, coloration 0~30%,30%~60%,60%~100% as 0,1,2 and 3 point respectively. Then the results were judged according to sums of the two grades: 0 point as negative (-),?3 point as positive (+), 5 point as strong positive (++) (note: the representative regions chosen in each slice were counted under 100 time fields of vision,total 5 fields of vision, take its mean value to avoid randomness). Statistical analysis: Used SPSS13.0 software to carry on the statistics, the counting materials were indicated by the mean value±standard deviation and take P<0.05 as the significance difference standard. The third part: The expression of p38MAPK and c-fos proteins in transplanting tumor tissues in nude mice was determined by Western blotting. Statistical analysis: After scanning band on film, input it into the computer,and analyzed it with Band Scan the V5.0. The expression intensity of bands in each group was indicated by the mean of relative gray value (gray value of band in each sample/ the internal reference gray value.Results:1. The Ru-Yan-Ning inhibited the transplant tumor in nude mice to reduce the volume of tumor. The weights of tumors in each group were 2.25g±0.51g in model group, 1.51g±0.32g in TAM group, 1.71g±0.39g in the Ru-Yan-Ning group,and 1.33g±0.18g in the union group. There existed significant statistic differences between experimental groups and model group (P<0.05). Tumor inhibition rate were 32.89% in the TAM group,24.00% in the Ru-Yan-Ning group,40.89% in the union group. There existed significant statistic differences between experimental groups and model group (P<0.05);There existed significant statistic differences between the union group and TAM group ,Ru-Yan-Ning group on the volume of tumor in nude mice (P<0.05); But there had no significant statistic difference between TAM group and Ru-Yan-Ning group(P>0.05).2. Rates of cell apoptosis were 8.66%±3.11% in model group, 29.14%±8.18% in TAM the group, 22.11%±7.02% in the Ru-Yan-Ning group, and 36.60%±6.71% in the union group. Compared to model group, rates of cell apoptosis in experimental groups increased significantly (P<0.05); There existed significant statistic differences between TAM group and Ru-Yan-Ning group (P<0.05);But there had no significant statistic difference between TAM group and Ru-Yan-Ning group(P>0.05).3. MMP-2 and MMP-9 expression were highest in model group, which were 42.35 %±3.28%, 52.33%±4.76% respectively, and 28.39%±4.21%, 27.56 %±3.12% in TAM group, 31.52±3.18%?32.14%±3.98 % in Ru-Yan-Ning group, but 24.36%±3.23%, 22.29%±2.67% in union group. Compared to model group, MMP-2 and MMP-9 expression in each experimental group decreased significantly (P<0.05),but there had no significant statistic difference between TAM group and Ru-Yan-Ning group. Our investigation confirmed that TAM and Ru-Yan-Ning could down regulate the expression of MMP-2 and MMP-9,there had no significant statistic difference between the two groups on the effectiveness (P>0.05). Joint application of two medicines could play the cooperative effect.4.Phosphorylation p38MAPK protein expression in each group were 1.13±0.23 in model group, 0.70±0.13 in TAM group, 0.76±0.11 in Ru-Yan-Ning group,and 0.58±0.18 in the union group, respectively. Compared to model group,p38MAPK protein expression decreased significantly in each experimental group (P<0.05);Compared to TAM and Ru-Yan-Ning group, it existed significant differentiation in the union group (P <0.05),but there had no significant statistic difference between TAM group and Ru-Yan-Ning group(P>0.05).Conclusion:1. Ru-Yan-Ning could strengthen the inhibition effect of TAM on transplanting tumor in nude mice in vivo to reduce the tumor volume.2. Ru-Yan-Ning has the function to induce cell apoptosis of breast cancer. Jointly application with TAM could enhance inducing apoptosis effect, could strengthen TAM to induce cell apoptosis to play the role of anti-tumor.3. TAM and Ru-Yan-Ning could down regulate the expression of MMP-2 and MMP-9. Joint application of two medicines could play the cooperative effect.4. Ru-Yan-Ning could inhibit the activity of p38MAPK signal passage to reduce the content of c-fos protein, to inhibit the expression of MMP-2 and MMP-9 protein,thus to inhibit invasion and metastasis on breast cancer.