Experimental Study Of Ruyan Joint Exemestane Rather Antitumor Effects And Mechanism Of Breast Cancer Xenografts

Purpose:To observe the influence of Traditional Chinese medicine-Ruyanningand exemestane to the general states, the ability of independent activities andorgan coefficient of nude mouse with MCF-7breast cancer, and to find out ifare there synergistic roles of Chinese medicine-Ruyanning and exemestane. Toobserve the influence of Traditional Chinese medicine-Ruyanning and exemestaneto the pathomorphology and apoptosis of MCF-7breast cancer cell, and to findout the mechanisms of that inducing the apoptosis. To observe the influence ofTraditional Chinese medicine-Ruyanning and exemestane to the cell cycledistribution, and the protein expression of ERα and p-AKT of MCF-7breastcancer cell, and to find out the signal transduction pathway of TraditionalChinese medicine-Ruyanning and exemestane controlling the proliferation ofMCF-7breast cancer cell. To observe the influence of Traditional Chinesemedicine-Ruyanning and exemestane to the expression of the gene of Bcl-2, theprotein of Bax, p53and Survivin mRNA, which are related to the apoptosis ofMCF-7breast cancer cell, and to find out the mechanisms of that controllingthe the proliferation of MCF-7breast cancer cell.Material and method:1. Preparation of the model bilateral ovariectony:10%choral hydrate (100mg/ml) is injected in the intraperitoneal of nude mouseaccording to the standard of4ml/kg. The nude mouse is prone position on theoperating table with the fixed limbs. The skin preparation is on the middle ofthe back with the lodine disinfection. Then we slit the skin on the spine adjacentto open0.5cm, away from the costal arch the lower edge of0.5cm, Cut theperitoneum into the abdominal cavity, and dish the milky organizations from theabdominal cavity. We see the pink mulberry-like ovarian. We separate it, thenclamp, ligature, cut ovarian, check for bleeding, suture the skin and disinfect. The same method is for the contralateral ovarian. The mouse is wake up afteranesthesia in20℃. The mouse is injected in0.25ml penicillin(60000U/ml) afteroperation and disinfect with3days. The mouse is intake water and SPF fodderwith30days. Then the model is success.2. The creation of model of nude mouse with MCF-7cancer:MCF-7cell in the phase of logarithmic growth is digested with trypsin/EDTA,then is washed by PBS solution twice. That is added to the cell culture medium,directly evenly pipetted, and stained with0.4%tyrpan blue. We record the numberof viable cells (>95%) and the number of cell counting boards, then adjustingthe viable cell concentration of1×107/ml. The right chest wall of nude mouseis disinfected. We vaccinate two nude mice in right chest wall, under the secondbreast pads with cell liquid0.2ml for every mouse. After10days the tumor nudesare appear in the breast pads, which are hard, gradually increased. When theprimary tumor is0.8cm3,the tumor is removed and transplanted to40nude micewith bone marrow biopsy needle.10%choral hydrate (0.02ml/10g) is injected inthe intraperitoneal of nude mouse with tumor. Breast cancer tissue is removedunder sterile conditions, immediately placed in DMEM, cut into pieces of about1mm3into the bone marrow biopsy needle. Before vaccination the right chest wallof healthy nude mouse is disinfected. We level pierce the needle into the rightchest wall next to the second breast pads1cm, then insert the needle core. Thetumor tissue is left in the breast pads. The skin is disinfected, no need tosuture the wounds. The penicillin1.5million U is injected in theintraperitoneal once. The nude mice are continued to raise in the SPF box. After4-5days the tumor is appear. The rate of tumor formation is100%. And the shapesand sizes are more equal. After15days the tumor can grow to9±2mm. Thepathological diagnosis is invasive ductal carcinoma by HE staining, withimmunohistochemical detection of ER (+).3.Experimental groups and administration:The40successful modeling nude mice were randomly divided into four groups:①model controlling group;②group of western medicine (exemestane);③group of Ruyanning;④combined group(exemestane and Ruyanning). Each groupadministered according to the human and mouse dose conversing formula.①modelcontrolling group:0.2ml saline is gavaged for every mouse everyday.②group ofwestern medicine (exemestane): Exemestane is administration according to4mg.kgfor every mouse(equivalent to10times the amount of clinical).0.2ml is gavagedfor every mouse everyday.③group of Ruyanning: Ruyanning is according to33g.kg(equivalent to12times the amount of clinical).0.2ml is gavaged for everymouse everyday.④combined group(exemestane and Ruyanning): Exemestane isaccording to4mg.kg and Ruyanning is according to33g.kg.0.2ml is gavaged forevery mouse everyday. All are administered continuously for21days.4. Experimental drawing:After experiment every group is drawn respectively. The nude mice is executedby cervical and placed in the ice box. The tumor is stripped out completely,and placed in the ice box after weighting. After the tumor tissue is observedby eyes, the full-thickness tissue is cut, and the same time it is drawnedmulti-size avoiding necrosis. The materials are5parts.(1)cellcycle specimens: Removing a piece of tissue placed in the PBS liquid of2ml.(2)electron microscopy specimens: Tissue is cut into about1mm3,thenimmediately put into0.25%glutaraldehyde, placed in a refrigerator at4℃spare.(3)RT-PCR specimens:2-3tissue blocks about8mm3is placed in the1ml Trizol. After labeled by permanent marker they are placed immediately in-80℃refrigerator.(4)Western Bloting specimens: Tissue blocks are placeddirectly in the EP tube, frozen in-80°C refrigerator.(5)For HE,TUNEL todetect apoptosis and immune: After drawning the specimens placedin4%paraformaldehyde with4℃.5.Detection indicators:(1)To observe the diet, activity, fur color stool,urine and death of nudemice everyday. To measure the weight every other day and paint the weight changecurve. To determine the ability of activity of mice by measuring the frequencyin5minutes with Spontaneous activity tester before treatment, on the10th day of the experiment,21days. The nude mice is executed. The liver, kidney andspleen are weighted and calculated organ index. The organ index=organ weight(mg)/body weight (g).(2) The nude mice is executed by cervical after21days with medicine. Thetumor is stripped out completely and weighted. The tumor inhibition rate iscalculated. The tumor inhibition rate=(the tumor weigh of model controllinggroup-the tumor weigh of group of experiment)/the tumor weigh of modelcontrolling group×100%. Tumor tissue is fixed by4%paraformaldehyde,dehydrated by alcohol, opened by xylene, embedded in paraffin and sliced bymicrotome. The slice is5μm. Morphological changes are observed by lightmicroscopy after hematoxylin and eosin staining.The rate of apoptosis of tumorcells is detected by TUNEL assay. Tumor tissue is embedded in paraffin andsliced. The rest of the method steps are according to kit instructions. The cellswith appeared green fluorescence in the nucleus are positive cells. Observedby fluorescence microscopy, each stained slice select five400×typical highpower field randomly.100cells are observed per field.500cells are calculated.The average number of positive cells per100cells is calculated as apoptoticindex(Apoptosis Index, AI).AI=the number of positive cells/the number ofcalculating cells×100%. Further the tumor tissue is quick-frozen byultramicrotomy, fixed by glutaraldehyde and osmium tetroxide fixation,dehydrated, soaked embedded, sliced,observed by transmission electronmicroscopy (TEM)on the ultrastructual level.(3) Detecting apoptosis by annexin V (annexin V)-PI double staining: Breastcancer tissue blocks are put in4°C phosphate buffer solution. That aredigested by0.25%trypsin30min－1h after mechanically cutting into pieces.Single cell suspension can be get over the200-400mesh filter cells. Themeasuring concentration of the cells is adjusting5×105-1×106cells/ml.1ml of cells is centrifuged1000rpm,4°C for10minutes. The cells wereresuspended in200ul Binding Buffer after Supernatant discarded. That is addedto0ul Annexin V-FITC and5ul PI and mixed gently. The time of reaction is15 minutes in Dark room temperature or30minutes in4℃. It is detected in flowcytometry adding300ul Binding Buffe. The judgment of results: the upper leftquadrant: cell debris; the upper right quadrant: apoptosis late cells and deadcells; the lower left quadrant: living cells: the lower right quadrant: apoptosisearly cells.(4) The tumor is stripped out completely after21days with medicine. It isweighed and placed in the ice box. The cut specimens are placed in the EP tube,frozen in-80℃refrigerator for Western blot proteindetermination. The tissueof breast cancer after cutting into pieces is digested by0.25%trypsin30min－1h. Single cell suspension can be get over the200-400mesh filter cells. Thecells are fixed more than12hours with75％cold ethanol (-20℃precooling).It is added to1ml of PI(final concentration:50ug／ml) and RNA enzyme withenzyme-free DNA (final concentration:50ug／ml). After strained30minutes itis put into flow cytometry. The expression of apoptosis regulating gene P53,ERɑ is detected by Western blot. And the results are statistical analyze.(5)The drawning of RT-PCR is that the tissue is put in1ml Trizol, and afterremarked put in-80℃refrigerator. The expression of apoptosis regulating geneBcl-2, Bax protein are qualitatively analyze by immunohistochemical method. Theexpression of p53、Survivin gene mRNA in breast cancer tissue by RT-PCR method.Results:(1)weight: the change of nude mice weight of each group: The nude mice weightof each group is no significant difference (P=0.790) before the administration.After a week the weight of the rest of the group is decrease in addition thatthe weight of controlling group is continued to rise. The group of Ruyanningis0.63±0.028. The group of western medicine is0.62±0.019. The combined group(0.20±0.017)is lighter than the group of Ruyanning and western medicine(P﹤0.05).But there are no significant difference between them(P>0.05). Theweight of combined group is gradually upward and the average weight is higherthan the group of Ruyanning and western medicine. After15days the weight isdecrease gradually. The weight of the group of western medicine is higher than the group of Ruyanning until19days. After21days the weights: The weight ofthe group of western medicine is lightest. The controlling group is heaviest.The controlling group is higher than the group of combined, Ruyanning andwestern medicine(P<0.05). But there are no significant difference among them.In addition to the controlling group the decreasing change of other group: Thegroup of western medicine is1.61±0.12. are no difference is1.11±0.0.16.Thecombined is0.93±0.06. There are differences among them(P<0.05).The timesof autonomic activities: At the beginning the times of each group is up about52/5minutes (abbreviation52). There are no difference among them(P>0.05).After10days in addition to the controlling group the times of each group isdecreased. The group of western medicine is lower than other group(p <0.01).Butthere are no difference between the group of Ruyanning and combined(P>0.05).After21days the decreasing trend is still. The times of autonomicactivities of each group are decrease because of the tumor. The group of westernmedicine is lower than other group(p <0.01).Because of exemestane in the groupof western medicine and combined, which can influence the gastrointestinaladverse reactions leading to physical falling. And the Ruyanning can repair thedamage. The change of liver, spleen and kidney of each group: The index of liverand kidney of combined group are higher than the controlling,but there is nodifference between them(P>0.05). The index of liver,spleen and kidney of thegroup of western medicine is higher than the Ruyanning. And there is differenceof index of liver and spleen between the group of western medicine and theRuyanning(P<0.05). There are differences between the index of liver, spleenand kidney of the Ruyanning and the combined(P<0.05).The weight of tumor: Theweight of tumor of western medicine, Ruyanning and the combined are all lowerthan the controlling. There are differences with the controlling(P﹤0.01).The lightest is combined group, which is lower than the group with single drug(p﹤0.01). The weight of tumor of western medicine is lower than the Ruyanning.There are differences(P﹤0.05).The rate of inhibition to tumor of westernmedicine, Ruyanning and the combined is respectively34.2%、25.2%、46.3%. The Ruyanning can inhibit the growth of breast cancer and has synergy withexemestane.(2) pathomorphological observation: The tumor is circular, oval or lobulatedby eyes. The texture is middle. The section is pale, translucent and realqualities area. The pale necrosis is appeared in the larger tumor. Observationby light microscopy after HE staining: Tumor cells are arranged in cords orclusterswith less interstitial. There are central lacunar small pipe structurein the mass of tumor cells. The pathology is that invasive ductal carcinoma isvigorous growth, large nucleus, clear rounded or oval nucleolus, obvious mitoticand scattered necrotic foci. The controlling group is that the tumor cells aredensity, disorganized, cord-like. The tumor cells are large with abundantcytoplasm, large nucleus deeply stained, imbalance of nucleus and plasma. Thepathological mitotic is appear with significant atypia and clear cancer nests.And other groups are that tumor cells are slightly increased. The structure ofthe tumor tissue is destructed with bleeding and necrotic cells, atypical cellsreducing, some cells becoming isolating cells and fibrous tissue. There are alarge number of lymphocytes and leukocyte around the necrotic tumor cells. Thatmeans the growth of tumor cells is inhibited. Observation by electron microscopy:the controlling group: The breast cancer cell growth and metabolism are vigorous.The nucleus and cytoplasm is imbalance. The nuclear membrane is integrity withdouble nuclear membrane. There are a large number of mitochondria in thecytoplasm, scattered ribosomes and complete organs. The group of westernmedicine: There are much necrotic cells, rupturing membrane, the large numberof vacuoles in the cytoplasm, chromatin in the edge area of the nuclear membrane,mitochondria with medullary changes. The group of Ruyanning: The cells are earlyapoptotic. The Cell volume is significantly reduced or increased. Thebubble-like swelling is appear in the membrane. The nuclear heterochromatin isincrease, distributed in the periphery of the nucleus, and some with typicalapoptotic bodies. The combined group: The cells are late apoptotic. The cellmembrane is rupture. Cytoplasmic overflow is vacuolar changes, with the typical apoptotic bodies by the surrounding with membrane. There are nuclear fragmentsand organelles in small body.(3)The results of TUNEL: The cells with appeared green fluorescence in thenucleus are positive cells. That means apoptosis. Observed by fluorescencemicroscopy, each stained slice select five400×typical high power fieldrandomly.100cells are observed per field.500cells are calculated. The averagenumber of positive cells per100cells is calculated as apoptoticindex(Apoptosis Index, AI).AI=the number of positive cells/the number ofcalculating cells×100%. There are differences in AI of the controlling groupand other groups (P=0.000<0.01). The rate of apoptosis of the combined groupis highest, then the western medicine. There are differences in them and thecontrolling and single drugs(P=0.000<0.01). Detecting apoptosis by annexin V(annexin V)-PI double staining: The cell viability of controlling is higher thanother groups. There are differences in them(P<0.05).The apoptosis rate ofcombined(39.25±8.34)is higher than the western medicine and Ruyaning(P<0.05).The apoptosis rate of western medicine (31.21±7.53)is higher than the Ruyaning(26.13±6.76). There are differences(P<0.05).(4)The results of flow cytometry cell cycle: The proportion of cells inG0/G1phase of each treating group is increased, and S phase is significantlyreduced. The difference was significantly (P <0.01) comparing with the modelgroup. The proportion of cells in G0/G1phase of compared group is the most,and S phase is the least. There is difference comparing with the group of singledrug(P<0.05).But there is no difference between the group of Ruyanning andwestern medicine in the proportion of cells in G0/G1and S phase(P﹥0.05).The results of the expression of ERα、p-AKT and geneβ-actin in the tissue ofnude mouse by Western blot: The expression of ERαand p-AKT in controlling groupis high. The difference was significantly (P <0.01) comparing with other threegroups. Other three groups all can make down the expression of ERαand p-AKT.The most significant is combined group. The difference was significantly (P<0.01) comparing with the group of Ruyanning and western medicine. (5)The results of the expression of Bcl-2、Bax in tumor tissue byimmunohistochemistry: The position of the expression of Bcl-2、Bax is the mostin cytoplasm, some in membrane. The positive expression of cell is yellow, brownparticles, especially in perinuclear and membrane. The negative cell iscytoplasmic colored significantly diminishing, or even negative. In controllinggroup the expression of Bcl-2is high, Bax is low, and the ratio of Bcl-2/Baxis significantly increased. In other three groups the expression of Bcl-2islow, Bax is high, and the difference is significant comparing with thecontrolling group(p﹤0.01).The group of western medicine, Ruyanning andcombined can reduce the expression of Bcl-2, increase Bax, and reduce the ratioof Bcl-2/Bax. The combined group has more role, and the difference issignificant comparing with the group of western medicine, Ruyanning(p﹤0.01).Wecan see the process of apoptosis is blocked in the controlling group, with rapidgrowth and loss of control. Other groups can induce the apoptosis of MCF-7breastcancer cell. That maybe related to reduce the expression of Bcl-2, increaseBax, and reduce the ratio of Bcl-2/Bax.(6)The results of the expression of p53、SurvivinmRNA in tumor tissue byRT-PCR: Each group with drug all can low the expression of p53、SurvivinmRNA,lower than the controlling group(P<0.05).The compared group is most significant,having difference with the group with single drug(p﹤0.01).Conclusions:1. The Traditional Chinese medicine-Ruyanning can improve the general statesand the ability of independent activities of nude mouse with MCF-7breast cancer,and reduce the side effect of exemestane decreasing the weight of mouse.2. The Traditional Chinese medicine-Ruyanning and exemestane can inhibit thegrowth of breast cancer, which maybe related to block the procession of cellcycle and regulate the expression of gene Bcl-2、Bax to induce apoptosis.3. The Traditional Chinese medicine-Ruyanning and exemestane can inhibit theactivity of PI3K-AKT signal pathway by reducing the expression of p-AKT、ERɑin cancer tissue, and inhibit the proliferation of breast cancer cell by reducing the expression of P53、SurvivingenemRNA.