| Breast cancer has always been an important problem troubling women’s health.The IARC has reported in 2020 that the prevalence of cancer is high(up to 11.7%),making it more common than lung cancer.The side effects of existing breast cancer therapies remain inevitable.The potential of traditional Chinese medicine to treat breast cancer is immense.Therefore,it is of great importance to seek new Chinese medicine with anti-cancer effect in the field of cancer.SNH is the chemical synthesis of houttuynia,which has anti-inflammatory,anti-fungal and anti-cancer properties.Studies suggested that SNH can exert anti-inflammatory effects by promoting the generation of ROS,which is considered as the product of oxygen consumption and cell metabolism.Excessive ROS may disrupt mitochondrial functions.In general,endogenous ROS are maintained within a normal range.Within the normal range,the increase of ROS will contribute to the occurrence and development of the disease,but excessive or exact low amount of ROS out of the normal range will show anti-cancer impacts.The criticality of the PI3K-AKT pathway’s abnormal activation in the emergence,metastasis,and drug resistance is undeniable,which provides a new direction in breast cancer.The biological function of PI3K-AKT pathway can be modulated by ROS.SNH has been shown to be effective in non-small cell lung cancer,but it has not yet been studied in breast cancer.Therefore,this study sought to uncover the therapeutic effect of SNH on breast cancer,as well as its internal mechanism of anti-cancer effect,in order to create a novel approach for cancer diagnosis and drug research.The experiment scheme was as follows:1.Bioinformatics Analysis:Venn diagram was applied to illustrate the DEGs shared between two expression profiles related to breast cancer(GSE139038 and GSE109169)from GEO Date Sets.GO and KEGG analysis of these DEGs were conducted.Cytoscape software was employed to thoroughly analyze the interaction network of certain significant genes.The molecular structure of SNH was assessed by Pub Chem.And the potential target genes of SNH were predicted through Swiss Target Prediction.The expression analysis and prognosis analysis of a target gene were conducted on GEPIA and Kaplan-Meier Plotter,individually.2.In vitro experiment:human breast cancer cells MCF-7 and canine breast cancer cells CMT-1211 were selected.Multiple experimental methods were utilized to detect the influence of SNH on the viability and apoptosis of CMT-1211 and MCF-7,as well as the ROS contents.The effect of SNH on mitochondrial morphology of MCF-7 and CMT-1211was detected by transmission electron microscopy.The effect of SNH on PDK1-AKT-GSK3βpathway was detected by Western blot and immunofluorescence.3.In vivo experiment:The tumor model was conducted using CMT-1211.The 5groupsc contained Docetaxel group(8mg/kg,i.p),SNH low-dose group(SNH,20mg/kg,i.p),SNH high-dose group(SNH,40mg/kg,i.p),Control group(HP-β-CD,0.1m L(30%w/v)/mouse,i.p),Mock group.The weight changes and tumor volume changes were measured.After a 22-day period of treatment,tumor tissue and organs of mice were collected.H&E staining was performed on each organ tissue.Immunofluorescence and Western blot were applied to detect the expressions of some proteins in tumor tissues.The main experiment results are as follows:1.Through bioinformatics analysis,137 common DEGs were found in the gene expression profiles of GSE139038 and GSE109169.The 137 DEGs were mainly involved in biological regulation,including PI3K signaling,cell proliferation and angiogenesis regulation.The cellular components involved included extracellular regions,extracellular matrix,endoplasmic reticulum lumen,perinucleolar region,cell surface,etc.The molecular functions involved include chemokine activity,zinc ion binding,protein isomerization activity,etc.Analysis of KEGG revealed that these DEGs were mainly implicated in AMPK signaling,PPAR signaling,PI3K-AKT signaling,etc.In addition,two potential target genes of SNH were consistent with 137 DEGs,namely MMP13 and MMP1.2.The CCK-8 results demonstrated that SNH had a significant effect on viability of CMT-1211 and MCF-7.Moreover,multiple experimental methods revealed that SNH could impede the migration and invasiveness of CMT-1211 and MCF-7.Flow cytometry revealed that the rate of apoptosis in breast cancer cells augmented with SNH concentration,and the expression of apoptosis-related proteins(Bcl-2,BAX,cleaved Caspase9,cleaved PARP)also indicated the occurrence of apoptosis in breast cancer cells during SNH treatment.In addition,ROS levels in breast cancer cells treated with SNH were detected by flow cytometry.The results showed that SNH could induce excessive accumulation of ROS in cells,and reactive oxygen scavger NAC could partially reduce excessive accumulation of ROS.Transmission electron microscopy showed that SNH could cause mitochondrial damage in breast cancer cells.Immunofluorescence Western blot results suggested that SNH could inactived PDK1-AKT-GSK3βpathway by promoting the excessive accumulation of ROS,thus playing an anticancer role.3.Ten days after inoculation with CMT-1211 cells,mice successfully developed tumors with a volume of about 200mm~3.After 22 days of treatment,SNH obviously slowed down the growth rate of tumor,and H&E staining revealed that SNH could significantly impede metastasis of breast tumors in the liver and lung.Western blot and immunofluorescence further revealed that SNH decreased expression of MMP1,and increased cleaved PARP and BAX expression in tumor tissue compared with control group.SNH also inhibited AKT phosphorylation in tumor tissues.Conclusion:The occurrence of breast cancer produces a large number of DEGs,which are mainly linked to cancer-related pathways.SNH can promote the excessive accumulation of ROS in MCF-7 and CMT1211 cells,leading to mitochondrial dysfunction,and inhibit the activation of PDK1-AKT1-GSK3βpathway,thereby inducing cell apoptosis.Additionally,it can significantly inhibit the growth of breast tumor in vivo.Therefore,SNH has the potential to treat breast cancer. |
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