Study On Shuganyishen Recipe For The Crosstalk Of Exosomes Mediating Tamoxifen Resistance In Breast Cancer Microenvironment

Background:The incidence of breast cancer ranks first among female malignant tumors.About 70%of breast cancers are positive for estrogen receptor(ER)/progestron receptor(PR).Tamoxifen(TAM)is still the first-line drug for comprehensive treatment of ER+/PR+breast cancer patients because of its economy,low toxicity,and long-lasting efficacy.It has been clinically used for more than 30 years and can reduce the annual mortality of breast cancer by 31%.However,about 30%-50%of patients still have recurrence and metastasis due to TAM resistance.Therefore,Revealing the resistance mechanism of tamoxifen is still a challenging hotspot in breast cancer research today.Drug resistance is inherent in cancer cells and a process of continuous selection and evolution under treatment.The core of evolution is intratumoral heterogeneity.Among the heterogeneous cell populations of tumors,cancer stem cells(CSCs)are not static cell populations,but constantly self-renew after treatment to become more aggressive and constantly changing cell populations.It is a functional cell population that drives cancer progression,and its number also increases with the emergence of drug resistance.Breast cancer stem-like cells often have a CD44+/CD24-/ER-phenotype,with epithelial-mesenchymal transition(EMT)characteristics and highly invasive characteristics with elevated ALDH.Numerous studies support that breast cancer stem cells(BCSCs)are the driving cells for ER+breast cancer to acquire TAM resistance,and they play a central role in tamoxifen resistance.However,stem cells are not static solid cells.Because stem cells and non-stem cells are undergoing dynamic changes in differentiation and dedifferentiation,it is difficult for cancer stem cells to be used as special targets.Blocking the transformation of cancer stem cells and non-stem cells is the direction we need to work hard on.Studies have shown that exosomal crosstalk in the tumor microenvironment can regulate the homeostasis of breast cancer stem cells.Exosomes(EXO)are secretory vesicles with a diameter of about 30-150nm.They are derived from the intracellular vesicle system.Many cells can secrete them.They contain a large number of microRNAs and proteins,which are important carriers for material exchange and information exchange between cells.Exosomes are considered to be a new and complex cell communication mechanism in the tumor microenvironment.They can be used as carriers of transcription factors and nucleic acids to induce epigenetic changes in recipient cells.Studies have shown that the drug resistance information of tamoxifen-resistant breast cancer cell lines can be transmitted to sensitive cell lines through exosomes.Because the exosomes of tumor cells may exist in the patient’s body fluids,it is very important to study their functions and molecular mechanisms.Studies have shown that the transport of exosomes is bidirectional.The interaction between human tumor cells and mesenchymal stem cells(MSCs)involves the exchange of exosomes.Tumor cells can change the content and characteristics of MSCs-EXO,and MSCs can also change the information carried by exosomes derived from tumor cells.The exosomes derived from tumor cells can regulate cell proliferation,apoptosis,invasion and epithelial-mesenchymal transition,inhibit immune cell activity,promote angiogenesis,and form a more suitable microenvironment for tumor survival.On the other hand,exosomes derived from stromal cells(such as mesenchymal stem cells)may affect the phenotype of tumor cells and regulate the homeostasis of tumor stem cells through the crosstalk between the transcription factors and nucleic acids they carry.MSCs are the main components in the tumor microenvironment.And bone marrow mesenchymal stem cells(BMMSCs)are the most abundant among them.Exosomes derived from BMMSCs(BMMSCs-EXO)can change the functions of tumor cells,thereby changing the tumor microenvironment and increasing tumor heterogeneity.Due to changes in the internal and external microenvironment,BMMSCs-EXO interacts with breast cancer cell-derived exosomes(BCCs-EXO),which can promote the transformation of non-stem cells into stem cells in tumor cells,thereby promoting the occurrence of tumors.Therefore,studying the effect of crosstalk between breast cancer-derived exosomes and bone marrow mesenchymal stem cell exosomes on breast cancer resistance will provide a new target for the treatment of drug-resistant breast cancer.Traditional Chinese medicine believes that stagnation of liver qi and imbalance of Chong and Ren are one of the main factors leading to breast cancer,and also an important factor for drug resistance in tumor treatment.In recent years,traditional Chinese medicine has had a significant effect in reversing the drug resistance of breast cancer.The Shugan Yishen Recipe is made up of Xiaoyaosan and Lichong decoction.It has the functions of soothing the liver and kidney,regulating the blood of Chong Ren,attacking and replenishing.It can get rid of evil without hurting the integrity.The previous research of our research group found that the TAM resistant cell line MCF7/LCC9 has stronger proliferation activity than the sensitive cell line MCF7,and the Shugan Yishen Recipe has an inhibitory effect on MCF7/LCC9,which can reverse the TAM resistance signal by inhibiting multiple resistance signals.Resistance.This study will further explore the mechanism of TAM resistance in breast cancer from the perspective of "microenvironment-breast cancer stem cells-exosomes" and the regulation of Shugan Yishen Recipe,providing a new scientific basis for the clinical application of "Shugan Yishen Recipe".Purposes:1.Clarify that Shugan Yishen Recipe can affect cells by regulating the crosstalk between BMMSCs-EXO and BCCs-EXO Biological characteristics by observing the effects of exosomal crosstalk on the proliferation,apoptosis and migration of breast cancer drug-resistant cells MCF-7/LCC9.2.By observing the effects of exosomal crosstalk on the spheroidization ability of breast cancer drug-resistant cells MCF-7/LCC9 and related protein expression,clarify that Shugan Yishen Recipe can affect drug-resistant cell stemness and reduce the transformation of breast cancer non-stem cells to stem cells and reverse tamoxifen resistance by regulating the crosstalk between BMMSCs-EXO and BCCs-EXO.Methods:1.Prepare four groups of rat drug-containing serum as intervention methods,namely the control group,TAM group,SGYS group,and TAM+SGYS combined group.2.Ultracentrifugation was used to extract exosomes derived from MCF-7/LCC9 cells and BMMSCs,respectively.3.Transmission electron microscopy(TEM)was used to observe the morphology and quantity of exosomes,nanoparticle tracking technology(NTA)was used to detect the concentration and size of exosomes,and Western blot was used to detect exosomal marker proteins CD9,CD63 and TSG101 to identify exosomes.4.Use fluorescence microscope to observe the uptake of MCF-7/LCC9-EXO by BMMSCs and the uptake of BMMSCs-EXO by MCF-7/LCC9 cells.5.The CCK8 method was used to detect the effect of exosomal crosstalk under the intervention of four drug-containing serums on the proliferation activity of MCF-7/LCC9 cells.6.Flow cytometry was used to detect the effect of exosomal crosstalk under the intervention of four drug-containing serums on MCF-7/LCC9 cell apoptosis.7.The Transwell method was used to detect the influence of exosomal crosstalk under the intervention of four drug-containing serums on the migration of MCF-7/LCC9 cells.8.The pelletization experiment was used to detect the influence of exosomal crosstalk under the intervention of four drug-containing serums on the sternness of MCF-7/LCC9 cells.9.Western blot was used to detect the effects of exosomal crosstalk intervention by four groups of drug-containing sera on the expression of tumor stem cell markers CD24 and CD44 in MCF-7/LCC9 cells and the expression of resistance-related proteins HER2 and ERα.10.Use ImageJ software for data collection and GraphPad Prism 8 software for statistical analysis of experimental data.The experimental results are expressed as"mean ± standard deviation".Differences between single-factor two groups are statistically analyzed by unpaired T-test.Statistics of differences between multiple groups were compared and analyzed by One-way ANOVA.Results:1.Electron microscopy results show that MCF7/LCC9-EXO and BMMSCs-EXO are both vesicle-like with a complete saucer structure,with typical characteristics of exosomes,and their diameters are between 30-150nm.2.The particle size results show that both MCF7/LCC9-EXO and BMMSCs-EXO are within the diameter of exosomes.3.Western blot results showed that CD9,CD63 and TSG101 were positively expressed in MCF7/LCC9-EXO and BMMSCs-EXO.4.Fluorescence microscope observed that MCF-7/LCC9-EXO and BMMSCs-EXO can crosstalk with each other.5.The effect of Shugan Yishen Recipe on the biological characteristics of breast cancer drug-resistant cells MCF-7/LCC9:(1)The results of CCK-8 detection of cell proliferation showed that the cell proliferation ability of the TAM group,SGYS group and the combination group was significantly lower than that of the control group on every time period(P<0.001),the combination group decreased the most,followed by the SGYS group,TAM group;Compared with the TAM group,the cell proliferation ability of the SGYS group and the combination group was significantly reduced on every time period(P<0.001),and the combination group was more significantly lower than that of the SGYS group,and the difference was statistically significant(P<0.01).The results show that Shugan Yishen Decoction can inhibit the proliferation of MCF-7/LCC9 cells by intervening exosomal crosstalk,and can have a synergistic effect with TAM to reverse TAM resistance.(2)Flow cytometry detection of apoptosis showed that the level of apoptosis in the experimental group was significantly higher than that of the control group,and the difference was statistically significant(P<0.001);Compared with the TAM group,the apoptosis level of the SGYS group and the combination group was also significantly increased(P<0.001).The results show that Shugan Yishen Recipe can promote MCF-7/LCC9 cell apoptosis by intervening exosomal crosstalk,and its combination with TAM can enhance the anti-tumor effect of TAM on MCF-7/LCC9 to a certain extent.(3)The results of the Transwell migration experiment showed that compared with the control group,the number of cell migration in the TAM group,the SGYS group and the combination group was less,the difference was statistically significant(P<0.05),and the number of cell migration in the SGYS group and the combination group was significant lower than the TAM group(P<0.001).The results show that the SGYS group has a more obvious inhibitory effect on cell migration,while the TAM group is not obvious.Compared with the single drug group,the combination group further enhanced the inhibition of MCF-7/LCC9 cell migration.(4)The results of the spheroidization experiment showed that compared with the control group,the number of spheroids in the TAM group,the SGYS group and the combination group was reduced,and there was statistically different(P<0.001),and the number of spheroids in the SGYS group and the combination group was significantly lower than the TAM group(P<0.001).The results show that the SGYS group can significantly inhibit the spheroidizing ability of MCF-7/LCC9 cells,reduce the stemness of the cells,further inhibit the spheroidizing ability of the cells and enhance the sensitivity of MCF-7/LCC9 to TAM by cooperating with TAM.(5)Western blot results showed that compared with the control group,the changes of CD24,CD44,HER2 and ERa in the TAM group were not statistically different(P>0.05);the expression of ERa and CD24 in the SGYS group and the combination group were significantly up-regulated(P<0.01),the expression of HER2 and CD44 were significantly down-regulated(P<0.001),and the combined group had better effects on protein expression than the single-agent group(P<0.01).The results show that Shugan Yishen Recipe can increase the expression of ERa and CD24 in MCF-7/LCC9,while reducing the expression of HER2 and CD44.Combining TAM will enhance the effect of TAM on the expression of related proteins in drug-resistant cells.Conclusions:1.Shugan Yishen Recipe can inhibit the growth of breast cancer tamoxifen-resistant MCF-7/LCC9 cells,promote cell apoptosis,and reduce cell migration by interfering with the cross-talk between BMMSCs-EXO and BCCs-EXO;2.The combined use of Shugan Yishen Recipe and tamoxifen has a synergistic enhancement effect;3.Shugan Yishen Recipe can reduce the stemness of drug-resistant cells by regulating the crosstalk of BMMSCs-EXO and BCCs-EXO,and up-regulate the expression of ERa and CD24 proteins in cells,while reducing the expression of HER2 and CD44,thereby reducing the conversion of breast cancer non-stem cells to stem cells and reversing tamoxifen resistance.