| Background and significance:Breast cancer is the most common malignant tumor in women all over the world,and its mortality ranks first among female cancers.More than 90%of breast cancer deaths are related to recurrence and metastasis.As a selective estrogen receptor modulator,tamoxifen(Tam)has always been a first-line drug in the endocrine therapy of ER+breast cancer and has been widely used in the prevention and treatment of breast cancer at all stages.However,30%～40%of patients with initial tolerance will develop secondary tamoxifen resistance.Compared with normal cancer cells,drug-resistant cancer cells have faster migration and stronger invasiveness,so tamoxifen-resistant breast cancer is more prone to metastasis,and the five-year survival rate of drug-resistant patients is only about 30%.Many studies have shown that epithelial-mesenchymal transformation(EMT)is the common pathway through which tumor cells acquire the ability to migrate and invade and tamoxifen acquire drug resistance.Therefore,drugs that can intervene and reverse the EMT process are sought to inhibit the migration and invasion of breast cancer cells and reduce the risk of metastasis of tamoxifen-resistant breast cancer.It is very important to reduce the mortality rate and prolong the survival time of patients with drug-resistant breast cancer.In ancient Books of Traditional Chinese medicine,the disease with similar clinical manifestations of breast cancer is called "ruyan" or "naiyan".Physicians of all dynasties attach importance to the role of fuzheng prescriptions in the treatment.Tutor professor Li Zhong also emphasized the method of "fuzhenggushe" in the treatment of cancer,and qijiafuzheng formula(QJFZ)is based this idea.This study will take qijiafuzheng formula as the research object to explore the effect of this prescription on the metastasis of tamoxifenresistant breast cancer,and explore its possible mechanism based on EMT.Objective:(1)Systematic evaluation and Meta analysis were used to explore the effectiveness of fuzheng Traditional Chinese medicine in preventing postopcrative recurrence and metastasis of breast cancer.(2)To establish and verify tamoxifen-resistant breast cancer cell(Mcf-7-tamR)and explore the effects of QJFZ on proliferation,invasion and migration of Mcf-7-tamR cells.(3)To establish a breast cancer model of Mcf-7 tamoxifen transplanted subcutaneously in nude mice,to explore the effects of QJFZ on the growth,lymphatic metastasis and angiogenesis of drug-resistant breast cancer,and to explore its possible mechanism from the perspective of EMT.(4)Explore the medication rules of the tutor in the clinical treatment of breast cancer and summarize the medication ideas of the tutor.Research methods:(1)Seven databases including CNKI,Wanfang Database,VIP Database,CBM,PubMed,Embase and Cochrane Library were searched,and Cochrane(RCT)Version 6.3 was used for risk bias assessment.Literature screening and content extraction were strictly carried out.Revman 5.4 software was used for statistical analysis,forest map was used to represent effect estimators,I2 test was used to determine heterogeneity,and funnel plot was used to analyze publication bias.(2)Mcf-7-tamR cells were established by low-concentration step-up induction method,cell morphology was observed by inverted microscope,cell resistance was verified by CCK-8 method,cell scratched test and invasion test were used to compare the cell migration and invasion ability of Mcf-7 and Mcf-7-tamR.Immunofluorescence and Western-blot were used to detect the expression levels of EMT marker proteins in Mcf-7 and Mcf-7-tamR cells,and Q-PCR was used to detect the expression levels of EMT-related mRNA in McF-7 and Mcf-7tamR cells.(3)The EMT marker protein and mRNA expression of Mcf-7-tamR cells were observed by intragastric administration of rats with high,medium and low doses of normal saline and QJFZ Formula to extract drug-containing serum and intervene Mcf-7-tamR cells.(4)The Mcf-7-tamr breast cancer model was established by subcutaneous transplantation in nude mice,and the body weight,tumor volume,tumor tissue HE stains of different groups of nude mice were recorded regularly,and the effects of QJFZ on the growth of transplanted tumor were observed.HE stains of ipsilateral lymph nodes in nude mice was used to evaluate the effect of QJFZ formula on tumor lymphatic metastasis.Photoacoustic 3D imaging was used to quantitatively evaluate the vascular density of different groups of tumor tissues,and the expression levels of VEGF-A and VEGF-C protein in different groups were detected by Western-blot,to evaluate the effect of Qijiafuzheng formula on tumor tissue angiogenesis.Western-blot was used to detect the expression of EMT marker protein,TGF-β1/Samd signaling pathway and AKT protein in each group,and to evaluate the intervention effect of Qijiafuzheng formula on EMT and its pathway.(5)All prescriptions of Professor Li Zhong for breast cancer treatment in Dongzhimen International Department outpatient clinic from January 2015 to December 2020 were collected,and drug frequency statistics,classification statistics,basic characteristics statistics of high-frequency drug use,prescription law analysis and core prescription mining were conducted by using ancient and modern medical case cloud platform;SPSS26.0 was used for cluster analysis and complex network analysis,and tree graph and community structure were used to represent the compatibility relationship between drugs.Results:(1)After retrieval and sorting,a total of 16 literatures were included.Revman 5.4 software was used to analyze the combined effect size and conduct heterogeneity and sensitivity analysis.The results showed that:I~2=0,P>0.1,indicating homogeneity of the included studies.The combined effect statistic Z=7.36(P<0.00001),OR=0.37(95%CI:0.29-0.49),indicating that taking fuzheng Traditional Chinese medicine can reduce the recurrence and metastasis rate of postoperative breast cancer.(2)Mcf-7-tamR cell line was successfully established by the parent cell Mcf-7 induced by the gradual addition of low concentration Tam.There were significant differences between the drug-resistant cells and the parent cells in morphology.Mcf-7-tamr cells were flat,mainly angular or polygonal,with large volume and irregular arrangement.While Mcf-7 cells are oval or long spindle shaped,uniform in size and regular in arrangement.Cell scratch test showed that wound healing percentage of Mcf-7-tamr cells was higher than that of Mcf-7 cells at 24 h and 48 h(P<0.05),suggesting that migration rate of Mcf-7-tamr cells was higher than that of Mcf-7 cells.The cell invasion assay showed that the number of transmembrane cells Mcf-7-tamr passing through Transwell 24h later was significantly higher than that of Mcf-7(p<0.05),suggesting that the invasion ability of Mcf-7-tamr cells was greater than that of Mcf-7 cells.Compared with Mcf-7 cells,the expression of E-cadherin in Mcf-7-tamr cells was decreased,the expression of N-cadherin was increased,and the expression of Vimentin was increased(P<0.05).The mRNA expressions of the three proteins showed the same trend,suggesting the occurrence of EMT in Mcf-7-tamr cells.(3)CCK-8 showed that QJFZ at low,medium and high doses could inhibit the proliferation of Mcf-7-tamr cells,and the higher the concentration was,the stronger the inhibition was.Cell scratch experiment showed that the model group(drug-free serum)had the fastest cell migration rate,with the wound healing percentage of 26.28%at 24h and 80.80%at 48h,followed by the QJFZ low-dose group,followed by the QJFZ medium-dose group,and the QJFZ high-dose group had the slowest cell migration rate.The percentage of wound healing at 24h was 6.63%,and that at 48h was 20.82%,with statistical difference among the four groups(P<0.05).Cell invasion experiment showed that the model group(drug-free serum)had the strongest cell invasion,and the number of transmembrane cells was 642.83±39.46 at 24h.The number of transmembrane cells in the low-dose group was 464.50± 29.10 at 24h.The number of transmembrane cells in the medium dose group was 229.25 ±14.62 at 24h.The cell invasion ability of the high dose group was the weakest,and the number of transmembrane cells in the high dose group was 120.58 ± 17.82 at 24h.There were statistical differences among model,low dose,medium dose and high dose groups(P<0.05).Compared with the model group,different doses of Qiefuzheng formula could increase the expression of e-cadherin protein and mRNA,decrease the expression of N-cadherin protein and mRNA,and decrease the expression of Vimentin protein and mRNA.The high-dose group had a better effect on the reversal of EMT process in Mcf-7-tamr cells than the lowdose group.(4)QJFZ can inhibit the growth of tamoxifen-resistant breast cancer.The weight and volume of subcutaneous transplanted tumors in nude mice treated with low,medium and high doses of QJFZ were smaller than those in model group and tamoxifen group(P<0.05),and the weight and volume of tumors in the high dose group were lower than those in the low dose group(P<0.05).HE staining of tumor tissue showed large area of multifocal necrosis of tumor cells in the medium-dose group of QJFZ,and there were a small number of tumor cell necrosis foci in the tamoxifen group,and almost no necrosis foci in the model group.Ipsilateral lymph node and HE staining showed that suspected tumor cells were found in ipsilateral lymph nodes in the tamoxifen group,with the possibility of lymph node metastasis,while no suspected tumor cells were found in the medium-dose QJFZ group.The protein expression levels of VEGF-A and VEGF-C in QJFZ low-dose group,QJFZ medium-dose group and QJFZ high-dose group were lower than those in model group and tamoxifen group(P<0.05),and the higher the concentration of QJFZ,the lower the expression levels were.Photoacoustic imaging showed that the vascular density of tumor tissues in QJFZ low-dose,medium-dose and high-dose groups was lower than that in model group and tamoxifen group(P<0.05).The protein level of E-cadherin in qifu low-dose,medium-dose and high-dose groups was higher than that in model group and tamoxifen group(P<0.05),while the protein level of N-cadherin was lower than that in model group and tamoxifen group(P<0.05).The levels of TGF-β1,P-SMad2/3 and P-Akt protein in the QJFZ medium-dose group were lower than those in the model group(P<0.05).Therefore,it was suggested that QJFZ might reverse the EMT process of McF-7-tamr cells by intervening TGF-β/Smad and PI3K/AKT signaling pathways.(5)A total of 4988 breast cancer prescriptions were included,and the top 5 Chinese herbs with the highest frequency were Astragalus membranaceus(99.46%),Trionychia chinensis(97.69%),Herba nobilis(81.74%),Plum plum(80.05%)and Fritillaria thunbergii(79.29%).The medicinal taste is mainly sweet,bitter and bitter.The highest confidence of drug pair association rule was Astragalus membranaceus+turtle shell.Complex network analysis was used to extract the core drug combinations in breast cancer prescription with edge weight>150,including Astragalus membranaceus,Trichoderma vinegar,Herba sibilis,plum,etc.Conclusion:(1)Fuzheng prescriptions can effectively reduce the risk of postoperative recurrence and metastasis of breast cancer.(2)Compared with the parent Mcf-7,Mcf-7-tamr cells had a faster migration rate and stronger invasion ability,and developed EMT.QJFZ can reduce the migration and invasion ability of Mcf-7-tamr cells,increase the levels of e-cadherin protein and mRNA,decrease the levels of N-cadherin protein and mRNA,and partially reverse the EMT process.(3)Qijia Fuzheng Decoction can inhibit the growth of Mcf-7-tamr breast cancer transplanted subcutaneously in nude mice,promote multifocal necrosis of tumor cells,reduce the risk of lymph node metastasis,inhibit the vascular density of tumor tissues,and reduce the expression of VEGF-A and VEGF-C proteins.Increasing E-cadherin level and decreasing Ncadherin level may reverse the EMT process of Mcf-7-tamr cells by intervening TGF-β/Smad and PI3K/AKT signaling pathways.(4)The core idea of professor Li Zhong for the cancer treatment is "fuzheng and gushe",and QJFZ is prescribed by this idear.The core drugs in the prescription are huangqi,biejia,xianhecao,wumei which reflect the characteristics of the use of "fuzheng and gushe",and are also the most frequently used drugs in tutor’s clinical breast cancer prescription. |
PDF Full Text Request