| Objective:To observe the effects of Ruyan Ning and Yiqi Xiaoji combined with tamoxifen(TAM) to the general state and the level of estrogen in tumor-bearing nude mice,to understand whether compound traditional chinese medicine combined withtamoxifen (TAM) had positive effect or not. To observe the influence of CompoundChinese medicine combined with tamoxifen (TAM) to induce apoptosis of MCF-7breast cancer cells in tumor-bearing nude mice and discuss the possible mechanismInhibition and pathological. To observe Compound Chinese medicine combined withTAM to affect cell cycle distribution of MCF-7breast cancer cell and the relatedregulatory protein CyclinD1,the influence to the expression of apoptosis relatedgenes Bcl-2and Bax protein, understanding the active mechanism of CompoundChinese medicine combined with TAM to inhibite proliferation of MCF-7breastcancer cell. According to observe the the influence of Compound Chinesemedicine combined with TAM to the expression of ERK1/2, ERɑ protein and HER-2gene mRNA,discussed the signal pathway of Compound Chinese medicine combinedwith TAM to inhabite the proliferation of MCF-7breast cancer cell.Methods:1.The establishment of MCF-7tumor-bearing nude mice model:MCF-7cells in logarithmic growth phase were digested by trypsin/EDTA, thenwashed2times with PBS, cell culture medium was added, blow and beat it directlyto the uniform, stained by0.4%trypan blue, take the record of the number ofliving cells (>95%), counting cells on the cell counting board, adjust thedensity of living cell1×107/ml, BALB/C Nude mice were injected MCF-7on thesecond breast pad of the right chest wall subcutaneously after disinfected theright chest wall of nude mice with iodophor, vaccinated cytosol0.2ml each nudemice, then went on raising in a laminar flow hood. Tumor nodules could been seen at the inoculation site of breast pads10-15days after vaccination, thecharacter of tumor nodules was hard and gradually increasing. The longer thetime,the faster the tumor nodules, that the primary model was recognized to besuccessful. When the primary tumor grew to0.8cm3, to removed the tumor by sterilesurgery, placed in DMEM medium, cut into pieces of about1mm3, then transferredto breast pads of the remaining40nude mice taking use of bone marrow needle,4days later you could see growth of transplanted tumor, successful rate of tumorformation was100%.The diagnosis of pathology was the infiltrating ductalcarcinoma identified by HE staining.2.Divided groups of experiment: Forty mice were randomly divided intocontrol group, tamoxifen (TAM) group, Ru combined group (the Ruyanning Recipe+TAM) and Yi combined group (YiQi Xiaoji Recipe+TAM), after establishing themodel of MCF-7cancer nude mice successfully. The Control group was givendistilled water0.2ml each one,TAM group was given TAM3.6mg.kg-1body weight(This doseage was equivalent to nine times as the clinical human’s), traditionalChinese medicine Ruyan Ning Recipe was given according to33.3g.kg-1body weight(equivalent to12times as the amount of clinical human’s dosage), Yiqi XiaojiRecipe was given35.3g.kg-1body weight (equivalent to12times as the clinicalhuman’s dosage), Traditional Chinese medicine+TAM conbining group was giventraditional Chinese medicine of Ruyan Ning Recipe or Yiqi Xiaoji Recipe andTAM3.6mg kg-1body weight, gavaging once a day for consecutive21days.3. Detection index:(1)The conditions of drink and food, weight of nude mice, mental state anddeath were observed and recorded every other day,drew the curve of changingweight of the mice. Take the record of autonomic activities of mice within fiveminutes before experiment, on the first,twenty-first day separately using ZZ6autonomic locomotor tester. Blood was taken approximately0.5-1ml by the removalof eyeball before mice were sacrificed,the blood was placed in a tube, then putit aside for1-2hours at room temperature, centrifuged them2500r/min for10minutes, separated supernatant serum to get ready for ELISA test. The levels of E2and progesterone (PROG referred to as P) were detected by enzyme-linkedimmunosorbent assay (ELISA) in the serum of mouse.when the mice were sacrificed,removed the uterus and weighed, calculated the index of organ. Uterine organindex=uterine weight(mg)/body weight(g).(2)Allthe mice were sacrificed through pull off neck after given drug for21days, tumor were taken completely and weighed in each group respectively.Then the inhibition rate of tumor were calculated,The formula of tumorinhibition rate was accordance with the tumor weight of control group-tumorweight in experimental group/tumor weight of the control group×100%; Tumortissue was fixed by4%formaldehyde, graded dehydration by ethanol, xylene madeit transparent, dipped and embeded by paraffin,slicing machine for slicing atthickness of5μm, observed the pathological changes by light microscopy afterHE staining;The apoptosis rate of tumor cells was detected by TUNNEL method,tissue was embedded with paraffin, sectioned in routine, and the rest of thesteps are carried out according to instructions about Apoptosis Assay Kit,Greenfluorescence that was observed within the nucleus were positive cells byfluorescence microscopy, each stained slice was selected randomly five400×high power typical fields, each field were observed in100cells,500cells inall, calculated the average of positive cells per100,the result was regardedas the apoptotic index (AI). AI=the number of positive cells/the number of cellsthat counted×100%; ultramicrotomy and fast-frozen were taken to deal with tumortissue, using the double glutaral dehyde and osmium tetroxide to fix, dehydrated,soaked, embedded, sliced, then observed by transmission electron microscopy(TEM) on the ultrastructural level to determine whether apoptosis.(3) All the mice were sacrificed after given drug for21days, tumor weretaken completely and weighed in each group respectively, placed on the ice box,and cut the specimen into small pieces,placed it in EP tube in-80℃refriger-ator for protein detection. Flow cytometry cell cycle: breast cancer tissue wascut into pieces and digested with0.25%trypsin for30min-1h, filtered by200-400mesh to obtain single suspension cell, fixed by75%cold ethanol for more than 12h, added PI (final concentration50ug/ml) and RNA enzyme of no DNA enzyme(finalconcentration50ug/ml)1ml,staining30min-1h before Flow cytometry detection;the expression of apoptotic regulated gene Bcl-2and Bax were detected in theway of immunohistochemistry (IHC)in qualitative analysis; then detected byWestern blot in quantitative cell analysis,detecting CyclinD1proteinsexpression using Western blot method, and the results for statistical analysis.(4)The specimen of RT-PCR was to placed into1mlTrizol, tagged,then placedit in-80℃refrigerator immediately. To detected the expression of ERK1/2,ERɑ protein of MCF-7breast cancer cell in the way of Western blot, RT-PCR methodwas taken to detect the expression of HER-2mRNA.Results:(1)Weight of mice: The decreased weight of nude mice in TAM group was themost obvious(3.18±0.061),Ru combined group followed by(2.5±0.042), Yicombined group（1.3±0.019）,the difference was significant among groups(p<0.01),The weight of mice in the three treatment groups was lower than the controlgroup（p﹤0.05）;The number of autonomic activity: Two combined groups were betterthan TAM group significantly on the tenth, twenty-first day of experiment(p<0.01);The index of the uterine: The index of the uterine in Ru combined groupwas highest,TAM group was second, the Yi combined group followed, they weresignificantly higher than the control group(p<0.01);The results of estrogen’s levels: TAM group can reduce the level of E2and P,comparing with the controlgroup, there were significant differences(p<0.01),the level E2in Ru combinedgroup was significantly lower than TAM group(p<0.01),but there was meaninglessbetween Yi combined group’s and TAM group about the level of E2(p>0.05), whilethe progesterone’s level in two combined groups were significantly lower thancontrol group(p<0.01).(2)The tumor weight: The weight of tumor in TAM group, combined group andYi combined group were lower than the control group, comparing with the controlgroup,there was a significant difference(p<0.01),Ru combined group was significantly lower than the TAM group (p<0.01), but the TAM group compared withYi combined group, Ru combined group compared with Yi combined group there wasno significant difference (p>0.05).The rates of tumor inhibition in TAM groupwas32.9%, Ru combined group was44.9%,Yi combined group was37.1%; The resultsof TUNEL: Apoptosis index (AI) in control group was higher than other TAM groups(p<0.01), AI in Ru combined group was the highest, the Yi combination groupwas second, there was no significant difference statistically between the twogroups. But comparing to TAM group,the differences were significant (p<0.01);Pathological diagnosis was invasive ductal carcinoma by HE staining, in thecontrol group, realm of cancer nests could be seen clearly, tumor cells grewstrongly, packed densely, lots of mitosis and heteromorphism could be seenvisibly in cells,while in the treated groups, the volume of tumor cells increasedslightly, the structure of tumor tissue was damaged, some cells shrinked toisolated cells, a large number of necrotic and apoptotic cells was seen. Tumorcells of necrosis were surrounded by a lot of lymphocytes and leukocyte(WBC),All show that the growth of tumor cells was inhibited in the treated groups;Theresults of pathomorphology: The pathomorphological change on the ultrastructural level observed by transmission electron microscopy (TEM) found thatMCF-7cells in control group have strong ability for growth and metabolism,big cell volume and nucleus, the ratio of nucleus and cytoplasm imbalance, richin organelles, while in each treatment group could find apoptotic changes, andsee a typical apoptotic manifestations in late stage-a large number of apoptoticbodies, in the TAM group under the electron microscope can be seen a small amountof necrotic cells.(3)Results of cell cycle detected by flow cytometry: In each treatmentgroup,the proportion of cells in G0/G1phase increased, S phase was sharplyreduced compared with the control group (P<0.01), inhibited the transformationfrom G0/G1phase to S phase in a definite range, but there were not statisticallydifferent between Ru combined group and Yi combined groups about cell ratio inthe G0/G1phase and S phase (P>0.05); Bcl-2,Bax detection by immunohistochemical method: The positive expression of Bcl-2and Bax was mainly localized in thecytoplasm, and some in the membrane.Bcl-2staining in the control group，thecytoplasm stained yellow, brown or dark brown, while in each treated groupcytoplasm coloration was significantly weakened, light yellow and some cellseven negative. Bax staining showed cytoplasm stained light yellow in the controlgroup, while in each treated group cytoplasm coloration was significantlyenhanced, especially in the part of peri nuclear and membrane significantly,yellow dying heavier. The semi-quantitative of Bcl-2and Bax by Western blot:The expression of Bcl-2protein was high, Bax with low expression, the ratioof Bcl-2/Bax was significantly increased in the control group compared withthe three treatment groups (p<0.01); The treated group could down-regulate theexpression of Bcl-2protein and up-regulate the expression of Bax protein,Bcl-2/Bax ratio was decreased to induce apoptosis of MCF-7breast cancer cellstogether. The expression results of Bcl-2and Bax detected by Western blot werebasically the same as results detected by immunohistochemistry.The expressionof cell cycle regulatory CyclinD1detected by Western blot: CyclinD1proteinin the control group were highly expressed, CyclinD1protein while in the treatedgroup expression levels were significantly decreased comparing with the controlgroup (p<0.01); in Ru combined group the expression of CyclinD1protein wasleast,the Yi combined group second, the results indicating that in eachtreatment group cell cycle blocked at G0/G1phase was related to down-regulateexpression of CyclinD1protein.(4)The results of expression of ERK1/2and ERɑ protein: The expression ofERK1/2, ERɑ protein in treated groups was significantly lower than in the controlgroup (p<0.01); the two combined groups could reduce the expression of ERK1/2and ERɑ compared with TAM group (p<0.01), but no significant difference the twocombination groups (p>0.05). HER-2gene mRNA expression detected by RT-PCR: Theexpression of HER-2gene mRNA in control group was higher than other groupsobviously (p<0.01); Ru combined group reduced the expression of HER-2gene mRNAmost of three groups. HER-2gene mRNA expression in TAM group was less than the two combined groups (p<0.01), while no significant difference between the twocombination groups (p>0.05).Conclusions:1. Compound Chinese medicine can improve the general state of tumor-bearingnude mice,reducing loss of the weight brought by TAM, improve the ability ofnude mice to move.2. Compound Chinese medicine combined with TAM can significantly reduce thelevels of E2, progesterone.3. Compound Chinese medicine has a function inhibite growth of tumor,thepossible mechanism was closely related to inhibite cell cycle and induceapoptosis.combined groups is the most obvious, illustrate that traditionalChinese medicine can collaborate TAM to tumor inhibitation.4. Compound Chinese medicine combined with TAM can inhibite breast cancer cellproliferation,it may be related to inhibite the activity of the MAPK signalingpathway to down-regulate ERK1/2, ERɑ protein, and it maybe by inhibiting theexpression of HER-2gene mRNA in breast cancer tissue to inhibite the activityof the MAPK signaling pathway.5.Compound Chinese medicine can enhance efficacy of TAM,but compares theefficacy,there is no significant difference between the Ruyan ning Recipe andYi Qi Xiaoji Recipe. |
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