| ObjectiveTriple negative breast cancer(Triple Negative Breast Cancer,TNBC)is still one of the medical challenges to be solved urgently.How to choose effective,side-effect-free and drug-resistant drugs is the bottleneck of anti-tumor therapy.Molecular target mining and the development of corresponding targeted drugs are of great significance for early diagnosis and effective treatment of triple negative breast cancer.Apoptosis resistance and metabolic abnormalities are common features of cancer,which are closely related to the mitochondrial pathway.In addition to the above two characteristics,triple negative breast cancer is usually accompanied by abnormal activation of PI3K/AKT/mTOR pathway.Sanhuang decoction(Sanhuang Decotion,SHD)is an empirical prescription for the treatment of breast cancer by a famous professor of traditional Chinese medicine Xu Zhiyin in Jiangsu Province.Previous studies have confirmed that it can significantly inhibit the activity of PI3K/AKT/mTOR pathway,but its specific mechanism still needs to be verified and discussed.In order to further reveal the mechanism of Sanhuang decoction in the treatment of triple negative breast cancer,we carried out this study.Methods1.In vivo experiment1)Effect of SHD on tumor-bearing mice with TNBC cells:25 5-week-old BALB/c-nu nude mice were selected,and their body weight was measured and fed adaptively for one week.After subcutaneous inoculation of MDA-MB-231 cells into the left axilla,the rats were randomly divided into four groups:model group(CON),high-dose Sanhuang decoction group(SHD-H),middle-dose Sanhuang decoction group(SHD-M)and low-dose Sanhuang decoction group(SHD-L).After successful establishment of the model,normal saline and Sanhuang decoction were given to the stomach.After 3 weeks of intervention,the body weight and tumor weight of mice in each group were measured,and the pathological changes of liver and kidney in each group were observed by HE staining.2)Effect of SHD on the transcription level of genes related to PI3K/AKT/mTOR pathway in tumor tissue of mice in each group:The transcription level of genes related to PI3K/AKT/mTOR pathway was detected by real-time fluorescence PCR.3)Effect of SHD on the expression of apoptosis-related proteins in tumor tissues of mice in each group:The expression of apoptosis-related phenotypic proteins Bcl-2,Bax and Cleaved-caspase-3 was detected by immunohistochemistry(IHC).TUNEL staining showed that SHD induced tumor cell apoptosis in a dose-dependent manner.2.In vitro experiment1)Effect of SHD on the survival and proliferation of MDA-MB-231 cells:The inhibitory effect of Sanhuang decoction on the survival rate of MDA-MB-231 cells was detected by MTT method,and the inhibitory effect of Sanhuang decoction on the proliferation of MDA-MB-231 cells was detected by colony formation test.2)Effect of SHD on the expression of proteins related to PI3K/AKT/mTOR pathway in MDA-MB-231 cells:Western blotting(WB)and immunofluorescence(IF)were used to detect the expression of PI3K/AKT/mTOR pathway protein.3)Effect of Sanhuang decoction on apoptosis and mitochondrial protein expression of MDA-MB-231 cells:Apoptosis was detected by Annexin V/7-ADD staining combined with flow cytometry,and the expression of apoptotic phenotypic proteins(Cleaved-caspase-3/9)and Bcl-2 family proteins(Bcl-2,Bax,Mcl-1)were detected by Western blot(WB)and immunofluorescence(IF).Western blot(WB)was used to detect the changes of Mcl-1 and Bak expression in mitochondria after intervention of Sanhuang decoction.4)SHD affected the function of mitochondria:The changes of mitochondrial membrane potential were detected by JC-1 staining combined with flow cytometry.5)SHD affected the function of mitochondria to induce apoptosis and inhibit cell survival by regulating the content of Mcl-1:The overexpression Mcl-1 was transfected with plasmid to observe and compare the survival inhibition of wild type group and overexpression group under the intervention of Sanhuang decoction.The transfection efficiency was observed and confirmed by fluorescence microscope and Western blot(WB).6)Effect of SHD on Mcl-1 transcription level of MDA-MB-231 cells:The transcription level of Mcl-1 gene under the intervention of Sanhuang decoction was detected by real-time fluorescence PCR.7)Effect of SHD on autophagy flow of MDA-MB-231 cells:After the autophagy was intervened by autophagy inhibitor,the expression of Mcl-1 and p62 was detected by Western Blot(WB).8)Effect of autophagy flow enhancement on Mcl-1 content under the intervention of SHD:After the autophagy was intervened by autophagy inhibitor,the expression of Mcl-1 and p62 was detected by western blotting(WB).9)Effect of SHD on the binding of Mcl-1 and p62 protein complex:The effect of Sanhuang decoction on the binding of Mcl-1 and p62 complex was detected by immunofluorescence(IF)co-localization and immunoprecipitation(CO-IP)combined with Western Blot(WB).10)Effect of SHD on the phosphorylation of p62 S403 site:Western Blot(WB)was used to detect whether the phosphorylation level of p62 S403 site increased after Sanhuang decoction intervention.3.Prescription miningChinese databases such as CNKI,VIP and Wanfang were used to search the related literatures about oral treatment of triple negative breast cancer with traditional Chinese medicine.After screening according to the standard of nano-platoon,the selected literature was excavated and analyzed by the auxiliary platform software of traditional Chinese medicine inheritance,combined with statistical methods such as frequency statistics and cluster analysis,to study the generality and characteristics of traditional Chinese medicine in the treatment of triple-negative breast cancer.to provide inspiration for the clinical dialectical addition and subtraction of Sanhuang decoction.4.Data miningBased on the prediction of the action target of Sanhuang decoction by network pharmacology,it was found that the possible target was related to the receptor pathway on the cell membrane surface.G protein coupled receptor(GPCR)is the most numerous and varied membrane surface receptor.The RGS protein family is the regulator of GPCR.At present,there are few studies on RGSs in breast cancer.We explore the universal expression of RGS family proteins and their correlation with the clinicopathological features of breast cancer.It is hoped that on this basis,it will be helpful to explore the mechanism of Sanhuang decoction and to develop a new prognosis model.Resunlts1.In vivo experiment1)Sanhuang decoction interfered to inhibit tumor growth and had no hepatorenal toxicity:after intervention for 3 weeks,it was found that the tumor size in the intervention group was smaller than that in the control group,and no obvious abnormality was found in liver and kidney tissues in the intervention group by HE staining,which proved that Sanhuang decoction had no hepatorenal toxicity.2)Sanhuang decoction interfered to inhibit the transcription level of genes related to PI3K/AKT/mTOR pathway:real-time fluorescence PCR detection showed that the transcription level of genes related to PI3K/AKT/mTOR pathway in tumor tissues was significantly inhibited in middle and high dose intervention groups.3)Sanhuang decoction induced apoptosis of tumor cells and necrosis of tumor tissue:the expression of Bax and Cleaved-caspase-3 increased while the expression of Bcl-2 decreased significantly by IHC assay.TUNEL staining showed that the apoptosis of tumor cells increased significantly in the intervention group.2.In vitro experiment1)Sanhuang decoction significantly inhibited the survival and proliferation of MDA-MB-231 cells:MTT assay showed that the survival and proliferation of cells in the intervention group were significantly lower than those in the control group.2)Sanhuang decoction inhibited the expression of PI3K/AKT/mTOR pathway protein in MDA-MB-231 cells:WB and IF showed that the expression level of PI3K/AKT/mTOR pathway protein decreased in intervention group.3)Sanhuang decoction induced apoptosis of MDA-MB-231 cells and affected the expression of related proteins in mitochondria:Annexin V/7-ADD staining combined with flow cytometry showed that apoptosis was significantly increased in the intervention group,while WB and IF showed that the expression of Caspase apoptosis cascade related proteins increased,the expression of Bcl-2/Mcl-1 and other anti-apoptotic proteins decreased,and the expression of pro-apoptotic genes such as Bax increased in the intervention group.WB detection of mitochondrial protein showed that the proportion of Bak/Mcl-1 in mitochondria increased in the intervention group,which confirmed that Sanhuang decoction affected the expression of mitochondrial Bak and Mcl-1 protein.4)Sanhuang decoction affected the function of mitochondria.The mitochondrial membrane potential was detected by JC-1 staining combined with flow cytometry.It was found that J-Monomer was significantly increased in the intervention group.It was confirmed that the mitochondrial function changed after the intervention of Sanhuang decoction.5)Sanhuang decoction affects the function of mitochondria to induce apoptosis and inhibit cell survival by regulating the content of Mcl-1.The successful transfection of overexpressed Mcl-1 was confirmed by WB and fluorescence microscope,and the results of MTT assay showed that Sanhuang decoction had a significant inhibitory effect on the survival of overexpressed group.6)Sanhuang decoction did not decrease the level of Mcl-1 transcription:real-time fluorescence PCR detection showed that there was no significant change in Mcl-1 transcription level in the intervention group compared with the control group.7)the increase of autophagy flow induced by Sanhuang decoction:WB detection showed that the expression of autophagy-associated protein p62 and LC3A/B increased in the intervention group,but the level of p62 protein decreased with time.8)the intervention of Sanhuang decoction to enhance the flow of autophagy led to the decrease of Mcl-1 content:after CQ blocked the fusion of autophagosome and lysosome,WB detection showed that the Mcl-1 content of inhibitor combined with Sanhuang decoction intervention group was higher than that of Sanhuang decoction intervention group.9)Sanhuang decoction intervention enhanced the binding effect of Mcl-1 and p62:IF co-localization and CO-IP combined with WB detection showed that there was a binding effect between Mcl-1 and p62,and Sanhuang decoction could enhance this effect.10)Sanhuang decoction enhanced the phosphorylation of p62S403 site:WB detection showed that the level of p62S403 phosphorylation in the intervention group was higher than that in the control group.Conclusion1)Sanhuang decoction inhibits tumor growth of triple negative breast cancer by regulating PI3K/AKT/mTOR pathway,and causes tumor tissue necrosis by inducing tumor cell apoptosis.2)Sanhuang decoction can effectively inhibit the activity of PI3K/AKT/mTOR pathway and promote apoptosis of triple negative breast cancer cell line MDA-MB-23 1.3)Sanhuang decoction inhibited PI3K/AKT/mTOR pathway,enhanced autophagy flux,promoted phosphorylation of p62 S403 site and degraded selective autophagy substrate Mcl-1.It leads to the change of mitochondrial outer membrane permeability and induces apoptosis of tumor cells via mitochondrial pathway.4)Through the study of the literature related to the treatment of TNBC with traditional Chinese medicine,it is concluded that the following kinds of prescriptions are supplemented with Sanhuang decoction:①The modified Sijunzi decoction is mainly used for the patients with spleen deficiency and phlegm obstruction syndrome;②The anti-cancer drugs of Hedyotis diffusa,Scutellaria barbata and Pleurotus ostreatus are mainly used to inhibit the progression of cancer;③The prescription is mainly composed of Ligustrum lucidum and Megan lotus,aiming at the patients with Chong-Ren disorder;④It is mainly composed of Bupleurum and Radix Paeoniae Alba,which is suitable for patients with poor mood and stagnation of liver qi.5)After mining and analyzing GEO and other databases,a risk score model consisting of seven RGS family genes,including RGS1,RGS2,RGS3,RGS9,RGS17,RGS20 and ARHGEF1,was constructed by lasso regression.The prognostic efficacy and robustness of the model were evaluated,and the results showed that the model could be used as an effective indicator of prognosis.In addition,the correlation between the model and immune infiltration and GSEA analysis showed that the model may affect the development of tumor through the perspective of immunity.This provides a new idea and basis for the follow-up study of the action mechanism of Sanhuang decoction. |
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