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Triple-negative breast cancer (TNBC), a highly aggressive and heterogeneous subtype of breast cancer, lacks effective treatment options. Sophora flavescens Aiton, a Chinese medicinal plant, is often used in traditional Chinese medicine to treat cancer. Matrine (MAT) is an alkaloid extracted from Sophora flavescens. It has good anticancer effects, and thus can be explored as a new therapeutic agent in TNBC research. We performed bioinformatics analysis to analyze the differentially expressed genes between normal breast tissues and TNBC tissues, and comprehensive network pharmacology analyses. The activity and invasion ability of TNBC cells treated with MAT were analyzed. Apoptosis and cell cycle progression were determined using cytometry. We used Monodansylcadaverine (MDC) staining to determine the condition of autophagosomes. Finally, the expression levels of the key target proteins of the PI3K/AKT pathway were determined using western blotting. The proliferation and invasion ability of MDA-MB-231 and MDA-MB-468 can be effectively inhibited by MAT. The results of flow cytometry indicated that MAT arrested the TNBC cell cycle and induced apoptosis. In addition, we confirmed that MAT inhibited the expression of BCL-2 while up-regulating the expression of cleaved caspase-3. Moreover, enhanced intensity of MDC staining and high LC3-II expression were observed, which confirmed that MAT induced autophagy in TNBC cells. Western blotting showed that MAT inhibited the PI3K/AKT pathway and downregulated the expressions of PI3K, AKT, p-AKT, and PGK1. This study provides feasible methods, which include bioinformatics analysis and in vitro experiments, for the identification of compounds with anti-TNBC properties. MAT inhibited the PI3K/AKT signaling pathway, arrested cell cycle, as well as promoted cell apoptosis and autophagy. These experiments provide evidence for the anti-TNBC effect of MAT and identified potential targets against TNBC. [ABSTRACT FROM AUTHOR]

Background. Breast cancer (BC) is the most common malignant tumor among females. Although there are multiple treatments for breast cancer, many patients still face the dilemma of drug resistance after multiline treatment. It would be greatly helpful for clinical work to identify additional and improved prognostic predictors. Y-box binding protein-1 (YB-1) is a member of the cold shock protein family, and patients with overexpression of YB-1 have a worse prognosis. Methods. This study collected 48 specimens from 48 patients with breast cancer and analyzed the clinicopathological characteristics of the patients. Immunohistochemistry, immunofluorescence, cell viability analysis, tumor spheroid formation and cell morphology, cell invasion, cycle analysis, qRT-PCR, Western blot, and tumorigenicity in BALB/c nude mice were performed to verify the results. Results. We found that patients with overexpression of YB-1 were related to lymph node metastasis and the patients' age tended to be young. Because of the short follow-up time, a survival analysis could not be performed. Based on the results of in vitro and in vivo experiments, this study indicated that breast cancer cells with overexpression of YB-1 had stronger proliferation, migration, and invasion abilities than cells with low expression of YB-1. Compared with cells with low expression of YB-1, the proliferation, migration, and invasion abilities of YB-1 overexpressed cells were not significantly affected by adriamycin. Conclusion. This suggested that breast cancer cells with overexpression of YB-1 were resistant to adriamycin. Therefore, YB-1 is associated with lymph node metastasis of breast cancer cell. YB-1 could be a prognostic, predictive factor and a novel therapeutic target of BC. [ABSTRACT FROM AUTHOR]

Background: Extensive amounts of archived formalin fixed paraffin embedded (FFPE) human tumor tissues are the ultimate resource to investigate signaling network underlying tumorigenesis in human. Yet, their usage is severely limited for lacking of suitable protein techniques. In this study, a quantitative, objective, absolute, and high throughput immunoblot method, quantitative dot blot (QDB), was explored to address this issue by investigating the putative relationship between estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) pathways in breast cancer tumorigenesis. Methods: In this descriptive observational retrospective study, ER, PR, Her2, and Ki67 protein levels were measured absolutely and quantitatively in 852 FFPE breast cancer tissues using the QDB method. ER, PR, and Her2 levels were charted on the X, Y, and Z-axes to observe samples distribution in a 3D scatterplot. Results: A "seesaw" relationship between ER/PR and Her2 pathways was observed in ER–PR–Her2 space, characterized by the expression levels of these 3 proteins. Specimens with strong expressions of ER/PR proteins were found spreading along the ER/PR floor while those with strong Her2 expression were found wrapping around the Her2 axis. Those lacking strong expressions of all 3 proteins were found accumulating at the intersection of the ER, PR, and Her2 axes. Few specimens floated in the ER–PR–Her2 space to suggest the lack of co-expression of all 3 proteins simultaneously. Ki67 levels were found to be significantly reduced in specimens spreading in the ER–PR space. Conclusions: The unique distribution of specimens in ER–PR–Her2 space prior to any clinical intervention provided visual support of bidirectional talk between ER/PR and Her2 pathways in breast cancer specimens. Clinical interventions to suppress these 2 pathways alternatively warrant further exploration for breast cancer patients accordingly. Our study also demonstrated that the QDB method is an effective tool to analyze archived FFPE cancer specimens in biomedical research. [ABSTRACT FROM AUTHOR]

Background: Ki67 is a biomarker of proliferation to be used in immunohistochemistry (IHC)-based surrogate assay to determine the necessity of cytotoxic therapy for Luminal-like breast cancer patients. cyclinD1 is another frequently used biomarker of proliferation. A retrospective study was performed here to investigate if these two biomarkers may be combined to improve the prognosis of Luminal-like patients. Methods: Both Ki67 and cyclinD1 protein levels were measured absolutely and quantitatively using Quantitative Dot Blot method in 143 Luminal-like specimens. Optimized cutoffs for these two biomarkers were developed to evaluate their prognostic roles using Kaplan–Meier overall survival (OS) analysis. Results: cyclinD1 was found as an independent prognostic factor from Ki67 in univariate and multivariate OS analyses. At optimized cutoffs (cyclinD1 at 0.44 μmol/g and Ki67 at 2.31 nmol/g), the subgroup with both biomarkers below the cutoffs (n = 65) had 10-year survival probability at 90% in comparison to those with both biomarkers above the cutoffs (n = 18) with 8-year survival probability at 26% (log-rank test, p < 0.0001). This finding was used to modify the surrogate assay using IHC-based cyclinD1 scores, with p -value decreased from 0.031 to 0.00061 or from 0.1 to 0.02, when the Ki67 score of 14 or 20% was used as cutoff, respectively, in the surrogate assay. Conclusion: The current study supports the prospective investigation of cyclinD1 relevance in the clinic. [ABSTRACT FROM AUTHOR]

Background: Immunohistochemistry (IHC)-based surrogate assay is the prevailing method in daily clinical practice to determine the necessity of chemotherapy for Luminal-like breast cancer patients worldwide. It relies on Ki67 scores to separate Luminal A-like from Luminal B-like breast cancer subtypes. Yet, IHC-based Ki67 assessment is known to be plagued with subjectivity and inconsistency to undermine the performance of the surrogate assay. A novel method needs to be explored to improve the clinical utility of Ki67 in daily clinical practice. Materials and Methods: The Ki67 protein levels in a cohort of 253 specimens were assessed with IHC and quantitative dot blot (QDB) methods, respectively, and used to assign these specimens into Luminal A-like and Luminal B-like subtypes accordingly. Their performances were compared with the Kaplan–Meier, univariate, and multivariate survival analyses of the overall survival (OS) of Luminal-like patients. Results: The surrogate assay based on absolutely quantitated Ki67 levels (cutoff at 2.31 nmol/g) subtyped the Luminal-like patients more effectively than that based on Ki67 scores (cutoff at 14%) (Log rank test, p = 0.00052 vs. p = 0.031). It is also correlated better with OS in multivariate survival analysis [hazard ratio (HR) at 6.89 (95% CI: 2.66–17.84, p = 0.0001) vs. 2.14 (95% CI: 0.89–5.11, p = 0.087)]. Conclusions: Our study showed that the performance of the surrogate assay may be improved significantly by measuring Ki67 levels absolutely, quantitatively, and objectively using the QDB method. [ABSTRACT FROM AUTHOR]

Background: As the original compound of many podophyllotoxin derivatives, podophyllotoxin has a beneficial antitumor effect. The mechanism of podophyllotoxin activity in triple-negative breast cancer still needs to be explored. Methods: We used cell proliferation assay, scratch and transwell experiments, and cell cycle and apoptosis analyses to observe the intervention effect of podophyllotoxin on breast cancer. Furthermore, we analyzed the differences between GSE31448, GSE65194, and GSE45827 in the Gene Expression Omnibus database (GEO) and explored the differential genes using a STRING database. Centiscape2.2, MCODE cluster analysis and KEGG pathway analysis were used to identify the most significant gene differences. Next, we utilized BATMAN-TCM and TCMSP databases for further screening to identify key genes. Finally, quantitative RT-PCR (qRT-PCR) and Western blotting were performed to detect the expression of key targets. Results: Our research confirmed that podophyllotoxin could not only inhibit the migration and invasion of triple-negative breast cancer but also affect the cell cycle and induce apoptosis. In total, 566 differential genes were obtained by using the GEO database. After topological network analysis, cluster analysis, and molecular docking screening, we finally identified PLK1, CCDC20, and CDK1 as key target genes. The results of the qRT-PCR assay showed that the mRNA levels of PLK1, CDC20, and CDK1 decreased, while the expression of upstream P53 increased, after drug induction. The Gene Set Enrichment Analysis (GSEA) and conetwork analysis showed that PLK1 is a more critical regulatory factor. Further Western blotting analysis revealed that there was a negative regulatory relationship between the key gene PLK1 and P53 on the protein level. The results were presented as the mean ± standard deviation of triplicate experiments and P<0.05 was considered to indicate a statistically significant difference. Conclusion: Podophyllotoxin has an intervention effect on the development of triple-negative breast cancer. The expression of PLK1, CDC20, and CDK1 in the cell cycle pathway is inhibited by regulating P53. Our research shows that natural drugs inhibit tumor activity by regulating the expression of cyclins, and the combination of natural drugs and modern extensive database analysis has a wide range of potential applications in the development of antitumor therapies. [ABSTRACT FROM AUTHOR]

Developing immunoassay for absolute quantitation of protein biomarkers in Formalin Fixed Paraffin Embedded (FFPE) samples promises improved objectivity, consistency and accuracy in daily clinical practice. The feasibility of Quantitative Dot Blot (QDB) method for this purpose was explored in this study. We were able to measure HER2 protein levels using 0.5 µg/sample total protein lysate extracted from 2 × 5 µm FFPE slices absolutely and quantitatively using QDB method in 332 breast cancer FFPE samples. HER2 levels measured using two clinically validated antibodies for immunohistochemistry respectively were highly correlated (r = 0.963). We also achieved area under the curve (AUC) at 0.9998 ± 0.0002 (p < 0.0001, n = 224) with IHC analysis, and 0.9942 ± 0.0031 (p < 0.0001, n = 319) with combined results from IHC and Fluorescence in situ hybridization (FISH) analyses when analyzed with Receiver Operative Characteristics analysis (ROC) respectively. When the results were converted dichotomously with optimized cutoffs from ROC analyses, we achieved 99.5% concordance with IHC; and 96.9% with combined results from both IHC and FISH analyses. Therefore, we were able to demonstrate QDB method as the first immunoassay platform for absolute quantitation of protein biomarkers in FFPE samples to meet the need of daily clinical practice, especially for local laboratories or laboratories in developing countries. [ABSTRACT FROM AUTHOR]

There has been increasing attention on immune-oncology for its impressive clinical benefits in many different malignancies. However, due to molecular and genetic heterogeneity of tumors, the activities of traditional clinical and pathological criteria are far from satisfactory. Immune-based strategies have re-ignited hopes for the treatment and prevention of breast cancer. Prognostic or predictive biomarkers, associated with tumor immune microenvironment, may have great prospects in guiding patient management, identifying new immune-related molecular markers, establishing personalized risk assessment of breast cancer. Therefore, in this study, weighted gene co-expression network analysis (WGCNA), single-sample gene set enrichment analysis (ssGSEA), multivariate COX analysis, least absolute shrinkage, and selection operator (LASSO), and support vector machine-recursive feature elimination (SVM-RFE) algorithm, along with a series of analyses were performed, and four immune-related genes (APOD , CXCL14 , IL33 , and LIFR) were identified as biomarkers correlated with breast cancer prognosis. The findings may provide different insights into prognostic monitoring of immune-related targets for breast cancer or can be served as reference for the further research and validation of biomarkers. [ABSTRACT FROM AUTHOR]

To circumvent the limitations associated with sandwich ELISA for tissue biomarker quantitation, Quantitative Dot Blot method (QDB) was proposed using antibodies clinically validated for immunohistochemistry (IHC), as this method requires only one primary antibody in the analysis. The protein levels of four breast cancer tissue biomarkers, including Estrogen Receptor (ER), Progesterone Receptor (PR), Ki67 and Her2, were absolutely quantitated successfully in 190 frozen breast tissue biopsies, and the results were further verified with provided IHC results. We propose QDB method as an alternative platform to Sandwich ELISA for absolute quantitation of tissue biomarkers with significantly reduced developing effort and time. • QDB as alternative immunoassay platform for cellular and tissue proteins. • QDB-based assay measures tissue biomarkers absolutely and quantitatively. • An ELISA-like assay with significantly reduced developing time and effort. • Only one primary antibody in QDB compared with two in ELISA. • Using clinically validated IHC antibodies (IVD/ASR) directly in QDB. • Suitable with both native and denatured samples. [ABSTRACT FROM AUTHOR]

Background: Astragalus polysaccharides (APS), natural plant compounds, have recently emerged as a promising strategy for cancer treatment, but little is known concerning their effects on breast cancer (BC) tumorigenesis. Methods: We obtained breast cancer genetic data from The Cancer Genome Atlas (TCGA) database, network pharmacology to further clarify its biological properties. Survival analysis and molecular docking techniques were implemented for the final screening to obtain key target information. Our experiments focused on the detection of intervention effects of APS on BC cells (MCF-7 and MDA-MB-231), and quantitative RT-PCR (qRT-PCR) was used to assess the expression of key targets. Results: A total of 1,439 differentially expressed genes (DEGs) were identified by TCGA and used to build disease networks. Module analysis, gene ontology and pathway analysis revealed characteristic of the DEGs network. Topological properties were used to identify key targets, survival analysis and molecular docking finally found that the targets of APS regulation of BC cells may be CCNB1, CDC6, and p53. Through cell viability, migration and invasion assays, we found that APS interferes with the development of breast cancer in MCF7 and MDA-MB-231 cells in a dose-dependent manner. Furthermore, qRT-PCR verification suggested that the expression of CCNB1 and CDC6 in breast cancer cells was significantly downregulated in response to APS, while expression of the tumor suppressor gene P53 was significantly increased. Conclusion: Results of this study suggest therapeutic potential for APS in BC treatment, possibly through interventions with CCNB1, CDC6, and P53. Furthermore, these findings illustrate the feasibility of using network pharmacology to connect large-scale target data as a way to discover the mechanism of natural products interfering with disease. [ABSTRACT FROM AUTHOR]

With the rapid development of next-generation sequencing technology, several studies have shown that ncRNAs can act as competitive endogenous RNAs (ceRNAs) and are involved in various biological processes, such as proliferation, differentiation, apoptosis, and migration of breast cancer (BC) cells, and plays an important role in BC progression as a molecular target for its diagnosis, treatment, prognosis, and differentiation of subtypes and age groups of BC patients. Based on the description of ceRNA-related biological functions, this study screened and sorted the sequencing analysis and experimental verification conclusions of BC-related ceRNAs and found that the ncRNAs mediated ceRNA networks can promote the development of BC by promoting the expression of genes related to BC proliferation, drug resistance, and apoptosis, inducing the production of epithelial-mesenchymal transition (EMT) to promote metastasis and activating cancer-related signaling pathways. [Display omitted] • ncRNA-mediated ceRNA networks is involved in various biological processes of breast cancer. • ncRNA-mediated ceRNA networks play a crucial role in diagnosis of breast cancer. • ncRNA-mediated ceRNA networks may facilitate the development of new anti-tumor drugs and clinical interventions of breast cancer. [ABSTRACT FROM AUTHOR]