Transcriptomics analysis reveals distinct mechanism of breast cancer stem cells regulation in mammospheres from MCF-7 and T47D cells.

Luminal A breast cancer, constituting 70 % of breast cancer cases, presents a challenge due to the development of resistance and recurrence caused by breast cancer stem cells (BCSC). Luminal breast tumors are characterized by TP53 expression, a tumor suppressor gene involved in maintaining stem cell attributes in cancer. Although a previous study successfully developed mammospheres (MS) from MCF-7 (with wild-type TP53) and T47D (with mutant TP53) luminal breast cancer cells for BCSC enrichment, their transcriptomic profiles remain unclear. We aimed to elucidate the transcriptomic disparities between MS of MCF-7 and T47D cells using bioinformatics analyses of differentially expressed genes (DEGs), including the KEGG pathway, Gene Ontology (GO), drug-gene association, disease-gene association, Gene Set Enrichment Analysis (GSEA), DNA methylation analysis, correlation analysis of DEGs with immune cell infiltration, and association analysis of genes and small-molecule compounds via the Connectivity Map (CMap). Upregulated DEGs were enriched in metabolism-related KEGG pathways, whereas downregulated DEGs were enriched in the MAPK signaling pathway. Drug-gene association analysis revealed that both upregulated and downregulated DEGs were associated with fostamatinib. The KEGG pathway GSEA results indicated that the DEGs were enriched for oxidative phosphorylation, whereas the downregulated DEGs were negatively enriched for the p53 signaling pathway. Examination of DNA methylation revealed a noticeable disparity in the expression patterns of the PKM2, ERO1L, SLC6A6, EPAS1, APLP2, RPL10L, and NEDD4 genes when comparing cohorts with low- and high-risk breast cancer. Furthermore, a significant positive correlation was identified between SLC6A6 expression and macrophage presence, as well as MSN, and AKR1B1 expression and neutrophil and dentritic cell infiltration. CMap analysis unveiled SA-83851 as a potential candidate to counteract the effects of DEGs, specifically in cells harbouring mutant TP53. Further research, including in vitro and in vivo validations, is warranted to develop drugs targeting BCSCs.

Bioinformatics Analysis of the Genetic and Epigenetic Alterations of Bone Morphogenetic Protein Receptors in Metastatic Breast Cancer.

The leading cause of mortality in patients with breast cancer is metastasis, and bone morphogenetic protein (BMP) signaling activation regulates metastasis in breast cancer. This study explored the genetic and epigenetic modification of BMP receptor genes associated with metastatic breast cancer cells using bioinformatics. The genetic and epigenetic alterations of BMP receptors (BMPR1A, BMPR1B, BMPR2, ACVR2A, ACVR1, ACVR2B, ACVR1B, HJV, and ENG) were examined using cBioportal and methSurv, respectively. mRNA expression was analyzed using TNM plot and bcgenex, and protein expression was studied using Human Protein Atlas. Prognostic value and ROC were investigated using Kaplan-Meier (KM) and ROC plot, respectively. Finally, mutant function was predicted using several databases, including PolyPhen-2, FATHMM, Mutation Assessor, and PredictSNP. Oncoprint analysis showed genetic alterations in BMPR1A (39%), BMPR1B (13%), BMPR2 (34%), ACVR2A (14%), ACVR1 (7%), ACVR2B (13), ACVR1B (35%), HJV (40%), and ENG (33%) across the patients with breast cancer in The Metastatic Breast Cancer Project. The mRNA and protein levels of BMPR2 were increased in metastatic breast tumor tissues compared with those in normal and breast tumor tissues. BMPR1A and BMPR2 showed the highest and lowest levels of epigenetic alterations among the BMP receptors, respectively. The patients with breast cancer who had low levels of BMPR2 had a better overall survival (OS) than those with high levels of BMPR2. Functional mutation prediction showed that mutants in BMPR2 (R272L, E274K, and L685F), ACVR2A (S127L), and ACVR1B (R484H), are deleterious, probably damaging, and possess a cancer phenotype. ROC plot revealed no BMP receptors correlated with endocrine therapy sensitivity. BMPR1B, BMPR2, and ACVR2A levels were significantly linked as moderate prediction of anti-HER2, BMPR2, and ACVR1B demonstrated moderate predictive potential for chemotherapy sensitivity. This study contributed in fully comprehending the significance of genetic and epigenetic alterations in BMP receptors and BMP signaling in metastatic breast cancer cells for the development of breast cancer treatment plans.

Bioinformatics analysis reveals the potential target of rosiglitazone as an antiangiogenic agent for breast cancer therapy.

BACKGROUND: Several studies have demonstrated the antitumor activity of rosiglitazone (RGZ) in cancer cells, including breast cancer cells. However, the molecular targets of RGZ in the inhibition of angiogenesis in breast cancer cells remain unclear. This study aimed to explore the potential targets of RGZ in inhibiting breast cancer angiogenesis using bioinformatics-based analysis. RESULTS: Venn diagram analysis revealed 29 TR proteins. KEGG pathway enrichment analysis demonstrated that TR regulated the adipocytokine, AMPK, and PPAR signaling pathways. Oncoprint analysis showed genetic alterations in FABP4 (14%), ADIPOQ (2.9%), PPARG (2.8%), PPARGC1A (1.5%), CD36 (1.7%), and CREBBP (11%) in patients with breast cancer in a TCGA study. The mRNA levels of FABP4, ADIPOQ, PPARG, CD36, and PPARGC1A were significantly lower in patients with breast cancer than in those without breast cancer. Analysis of gene expression using bc-GenExMiner showed that the mRNA levels of FABP, ADIPOQ, PPARG, CD36, PPARGC1A, and CREBBP were significantly lower in basal-like and triple-negative breast cancer (TNBC) cells than in non-basal-like and non-TNBC cells. In general, the protein levels of these genes were low, except for that of CREBBP. Patients with breast cancer who had low mRNA levels of FABP4, ADIPOQ, PPARG, and PPARGC1A had lower overall survival rates than those with high mRNA levels, which was supported by the overall survival related to DNA methylation. Correlation analysis of immune cell infiltration with TR showed a correlation between TR and immune cell infiltration, highlighting the potential of RGZ for immunotherapy. CONCLUSION: This study explored the potential targets of RGZ as antiangiogenic agents in breast cancer therapy and highlighted FABP4, ADIPOQ, PPARG, PPARGC1A, CD36, and CREBBP as potential targets of RGZ. These findings require further validation to explore the potential of RGZ as an antiangiogenic agent.

Identification of potential target genes of honokiol in overcoming breast cancer resistance to tamoxifen.

Background: Honokiol (HON) inhibits epidermal growth factor receptor (EGFR) signaling and increases the activity of erlotinib, an EGFR inhibitor, in human head and neck cancers. In this study, using a bioinformatics approach and in vitro experiments, we assessed the target genes of HON against breast cancer resistance to tamoxifen (TAM). Materials and methods: Microarray data were obtained from GSE67916 and GSE85871 datasets to identify differentially expressed genes (DEGs). DEGs common between HON-treated and TAM-resistant cells were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses and protein-protein interaction (PPI) networks were constructed. Selected genes were analyzed for genetic alterations, expression, prognostic value, and receiver operating characteristics (ROC). TAM-resistant MCF-7 (MCF-7 TAM-R) cells were generated and characterized for their resistance toward TAM. A combination of HON and TAM was used for cytotoxicity and gene expression analyses. Molecular docking was performed using the Molecular Operating Environment software. Results: PPI network analysis revealed that FN1, FGFR2, and RET were the top three genes with the highest scores. A genetic alteration study of potential target genes revealed MMP16 and ERBB4 as the genes with the highest alterations among the breast cancer samples. Pathway enrichment analysis of FGFR2, RET, ERBB4, SOX2, FN1, and MMP16 showed that the genetic alterations herein were likely to impact the RTK-Ras pathway. The expression levels of RET, MMP16, and SOX2 were strongly correlated with prognostic power, with areas under the ROC curves (AUC) ​​of 1, 0.8, and 0.8, respectively. The HON and TAM combination increased TAM cytotoxicity in MCF-7 TAM-R cells by regulating the expression of potential target genes ret, ERBB4, SOX2, and FN1, as well as the TAM resistance regulatory genes including HES1, VIM, PCNA, TP53, and CASP7. Molecular docking results indicated that HON tended to bind RET, ErbB4, and the receptor protein Notch1 ankyrin domain more robustly than its native ligand. Conclusion: HON could overcome breast cancer resistance to TAM, potentially by targeting FGFR2, RET, ERBB4, MMP16, FN1, and SOX2. However, further studies are required to validate these results.

Systematic analysis of potential targets of the curcumin analog pentagamavunon-1 (PGV-1) in overcoming resistance of glioblastoma cells to bevacizumab.

BACKGROUND: Glioblastoma is one of the most aggressive and deadliest malignant tumors. Acquired resistance decreases the effectiveness of bevacizumab in glioblastoma treatment and thus increases the mortality rate in patients with glioblastoma. In this study, the potential targets of pentagamavunone-1 (PGV-1), a curcumin analog, were explored as a complementary treatment to bevacizumab in glioblastoma therapy. METHODS: Target prediction, data collection, and analysis were conducted using the similarity ensemble approach (SEA), SwissTargetPrediction, STRING DB, and Gene Expression Omnibus (GEO) datasets. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted using Webgestalt and DAVID, respectively. Hub genes were selected based on the highest degree scores using the CytoHubba. Analysis of genetic alterations and gene expression as well as Kaplan-Meier survival analysis of selected genes were conducted with cBioportal and GEPIA. Immune infiltration correlations between selected genes and immune cells were analyzed with database TIMER 2.0. RESULTS: We found 374 targets of PGV-1, 1139 differentially expressed genes (DEGs) from bevacizumab-resistant-glioblastoma cells. A Venn diagram analysis using these two sets of data resulted in 21 genes that were identified as potential targets of PGV-1 against bevacizumab resistance (PBR). PBR regulated the metabolism of xenobiotics by cytochrome P450. Seven potential therapeutic PBR, namely GSTM1, AKR1C3, AKR1C4, PTGS2, ADAM10, AKR1B1, and HSD17B110 were found to have genetic alterations in 1.2%-30% of patients with glioblastoma. Analysis using the GEPIA database showed that the mRNA expression of ADAM10, AKR1B1, and HSD17B10 was significantly upregulated in glioblastoma patients. Kaplan-Meier survival analysis showed that only patients with low mRNA expression of AKR1B1 had significantly better overall survival than the patients in the high mRNA group. We also found a correlation between PBR and immune cells and thus revealed the potential of PGV-1 as an immunotherapeutic agent via targeting of PBR. CONCLUSION: This study highlighted seven PBR, namely, GSTM1, AKR1C3, AKR1C4, PTGS2, ADAM10, AKR1B1, and HSD17B110. This study also emphasized the potential of PBR as a target for immunotherapy with PGV-1. Further validation of the results of this study is required for the development of PGV-1 as an adjunct to immunotherapy for glioblastoma to counteract bevacizumab resistance.

Integrative Bioinformatics Study of Tangeretin Potential Targets for Preventing Metastatic Breast Cancer.

Agents that target metastasis are important to improve treatment efficacy in patients with breast cancer. Tangeretin, a citrus flavonoid, exhibits antimetastatic effects on breast cancer cells, but its molecular mechanism remains unclear. Tangeretin targets were retrieved from PubChem, whereas metastatic breast cancer regulatory genes were downloaded from PubMed. In total, 58 genes were identified as potential therapeutic target genes of tangeretin (PTs). GO and KEGG pathway enrichment analyses of PTs were performed using WebGestalt (WEB-based Gene SeT AnaLysis Toolkit). The PPI network was analyzed using STRING-DB v11.0 and visualized by Cytoscape software. Hub genes were selected on the basis of the highest degree score as calculated by the CytoHubba plugin. Genetic alterations of the PTs were analyzed using cBioPortal. The prognostic values of the PTs were evaluated with the Kaplan-Meier plot. The expression of PTs across breast cancer samples was confirmed using GEPIA. The reliability of the PTs in metastatic breast cancer cells was validated using ONCOMINE. Molecular docking was performed to foresee the binding sites of tangeretin with PIK3Cα, MMP9, PTGS2, COX-2, and IKK. GO analysis showed that PTs participate in the biological process of stimulus response, are the cellular components of the nucleus and the membrane, and play molecular roles in enzyme regulation. KEGG pathway enrichment analysis revealed that PTs regulate the PI3K/Akt pathway. Genetic alterations for each target gene were MTOR (3%), NOTCH1 (4%), TP53 (42%), MMP9 (4%), NFKB1 (3%), PIK3CA (32%), PTGS2 (15%), and RELA (5%). The Kaplan-Meier plot showed that patients with low mRNA expression levels of MTOR, TP53, MMP9, NFKB1, PTGS2, and RELA and high expression of PIK3CA had a significantly better prognosis than their counterparts. Further validation of gene expression by using GEPIA revealed that the mRNA expression of MMP9 was significantly higher in breast cancer tissues than in normal tissues, whereas the mRNA expression of PTGS2 showed the opposite. Analysis with ONCOMINE demonstrated that the mRNA expression levels of MMP9 and NFKB1 were significantly higher in metastatic breast cancer cells than in normal tissues. The results of molecular docking analyses revealed the advantage of tangeretin as an inhibitor of PIK3CA, MMP9, PTGS2, and IKK. Tangeretin inhibits metastasis in breast cancer cells by targeting TP53, PTGS2, MMP9, and PIK3CA and regulating the PI3K/Akt signaling pathway. Further investigation is needed to validate the results of this study.

Bioinformatics and In Vitro Studies Reveal the Importance of p53, PPARG and Notch Signaling Pathway in Inhibition of Breast Cancer Stem Cells by Hesperetin.

Purpose: The failure of chemotherapy in breast cancer is caused by breast cancer stem cells (BCSCs), a minor population of cells in bulk mammary tumors. Previously, hesperetin, a citrus flavonoid, showed cytotoxicity in several cancer cells and increased cytotoxicity of doxorubicin and cisplatin. Hesperetin also inhibited osteogenic and adipocyte differentiation, however, a study of the effect of hesperetin on BCSCs has not yet been performed. Methods: In this study, we combined bioinformatics and in vitro works. A bioinformatic approach was performed to identify molecular targets, key proteins, and molecular mechanisms of hesperetin targeted at BCSCs, and genetic alterations among key genes. In addition, an in vitro study was carried out to measure the effects of hesperetin on BCSCs using the spheroids model of MCF-7 breast cancer cells (mammospheres). Results: Using a bioinformatics approach, we identified P53, PPARG, and Notch signaling as potential targets of hesperetin in inhibition of BCSCs. The in vitro study showed that hesperetin exhibits cytotoxicity on mammospheres, inhibits mammosphere and colony formation, and inhibits migration. Hesperetin modulates the cell cycle and induces apoptosis in mammospheres. Moreover, hesperetin treatment modulates the expression of p53, PPARG, and NOTCH1. Conclusion: Taken together, hesperetin has potential for the treatment of BCSC by targeting p53, PPARG and Notch signaling. Further investigation of the molecular mechanisms involved is required for the development of hesperetin as a BCSC-targeted drug.

The Target Differences of Anti-Tumorigenesis Potential of Curcumin and its Analogues Against HER-2 Positive and Triple-Negative Breast Cancer Cells.

Purpose: The current study aims to evaluate the in vitro cytotoxic and cell migration effects of synthetic curcumin and its analogues on HER2 and nuclear factor kappa B (NFκB) pathways, as well as the in vivo inhibitory effect on cancer growth of metastatic breast cancer. Methods: Cell viability, protein expression, and protein localization were determined in vitro using MTT assay, western blotting, and immunofluorescence, respectively. Meanwhile, scratch wound healing assay and gelatin zymography were conducted to investigate the metastasis inhibitory effect. The in vivo anti-tumor ability was evaluated in xenograft mouse model using triple-negative breast cancer (TNBC) cells. Results: Curcumin, PGV-0, and PGV-1 exhibited cytotoxic effect against HER2-overexpressing breast cancer cells. Although PGV-1 showed the best activity in the single cytotoxic assay, curcumin showed the strongest synergism with doxorubicin. Curcumin and PGV-0 inhibited membrane localization of HER2. In contrast, PGV-1 neither inhibited localization nor decreased the expression of HER2, nonetheless showed the most potent inhibition against nuclear localization of p65 indicating the different mechanisms of curcumin, PGV-0, and PGV-1. Regarding cancer metastasis, curcumin and PGV-1 showed inhibitory activities against cell migration and inhibited MMP-2 and MMP-9 protein expression. Lastly, PGV-1 was more potent compared to curcumin to suppress the tumor formation of metastatic breast cancer xenograft model in nude mice. Conclusion: Overall, our study strengthens the potency of curcumin analogue, PGV-1, for treating several types of cancer, including metastatic breast cancer.

Identification of potential therapeutic target of naringenin in breast cancer stem cells inhibition by bioinformatics and in vitro studies.

Cancer therapy is a strategic measure in inhibiting breast cancer stem cell (BCSC) pathways. Naringenin, a citrus flavonoid, was found to increase breast cancer cells' sensitivity to chemotherapeutic agents. Bioinformatics study and 3D tumorsphere in vitro modeling in breast cancer (mammosphere) were used in this study, which aims to explore the potential therapeutic targets of naringenin (PTTNs) in inhibiting BCSCs. Bioinformatic analyses identified direct target proteins (DTPs), indirect target proteins (ITPs), naringenin-mediated proteins (NMPs), BCSC regulatory genes, and PTTNs. The PTTNs were further analyzed for gene ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, protein-protein interaction (PPI) networks, and hub protein selection. Mammospheres were cultured in serum-free media. The effects of naringenin were measured by MTT-based cytotoxicity, mammosphere forming potential (MFP), colony formation, scratch wound-healing assay, and flow cytometry-based cell cycle analyses and apoptosis assays. Gene expression analysis was performed using real-time quantitative polymerase chain reaction (q-RT PCR). Bioinformatics analysis revealed p53 and estrogen receptor alpha (ERα) as PTTNs, and KEGG pathway enrichment analysis revealed that TGF-ß and Wnt/ß-catenin pathways are regulated by PTTNs. Naringenin demonstrated cytotoxicity and inhibited mammosphere and colony formation, migration, and epithelial to mesenchymal transition in the mammosphere. The mRNA of tumor suppressors P53 and ERα were downregulated in the mammosphere, but were significantly upregulated upon naringenin treatment. By modulating the P53 and ERα mRNA, naringenin has the potential of inhibiting BCSCs. Further studies on the molecular mechanism and formulation of naringenin in BCSCs would be beneficial for its development as a BCSC-targeting drug.

Identification of key genes of hesperidin in inhibition of breast cancer stem cells by functional network analysis.

Breast cancer therapy with classical chemotherapy is unable to eradicate breast cancer stem cells (BCSCs). Loss of p53 function causes growth and differentiation in cancer stem cells (CSCs); therefore, p53-targeted compounds can be developed for BCSCs-targeted drugs. Previously, hesperidin (HES), a citrus flavonoid, showed anticancer activities and increased efficacy of chemotherapy in several types of cancer in vitro and in vivo. This study was aimed to explore the key protein and molecular mechanism of hesperidin in the inhibition of BCSCs using bioinformatics and in vitro study. Bioinformatics analysis revealed about 75 potential therapeutic target proteins of HES in BCSCs (TH), in which TP53 was the only direct target protein (DTP) with a high degree score. Furthermore, the results of GO enrichment analysis showed that TH was taken part in the biological process of regulation of apoptosis and cell cycle. The KEGG pathway enrichment analysis also showed that TH is involved in several pathways, including cell cycle, p53 signaling pathway. In vitro experiment results showed that HES inhibited cell proliferation, mammosphere, and a colony formation, and migration in on MCF-7 3D cells (mammospheres). HES induced G0/G1 cell cycle arrest and apoptosis in MCF-7 cells 3D. In addition, HES treatment reduced the mRNA level of p21 but increased the mRNA level of cyclin D1 and p53 in the mammosphere. HES inhibits BCSCs in mammospheres. More importantly, this study highlighted p53 as a key protein in inhibition of BCSCs by HES. Future studies on the molecular mechanism are needed to validate the results of this study.

Functional network analysis reveals potential repurposing of ß-blocker atenolol for pancreatic cancer therapy.

BACKGROUND: The survival rate of patients with pancreatic cancer is low; therefore, continuous discovery and development of novel pancreatic cancer drugs are required. Functional network analysis is an integrated bioinformatics approach based on gene, target, and disease networks interaction, and it is extensively used in drug discovery and development. OBJECTIVE: This study aimed to identify if atenolol, a selective adrenergic inhibitor, can be repurposed for the treatment of pancreatic cancer using functional network analysis. METHODS: Direct target proteins (DTPs) and indirect target proteins (ITPs) were obtained from STITCH and STRING databases, respectively. Atenolol-mediated proteins (AMPs) were collected from DTPs and ITPs and further analyzed for gene ontology, KEGG pathway enrichment, genetic alterations, overall survival, and molecular docking. RESULTS: We obtained 176 AMPs that consisted of 10 DTPs and 166 ITPs. Among the AMPs involved in the pancreatic cancer pathways, several AMPs such as MAPK1, RELA, MAPK8, STAT1, and STAT3 were identified. Genetic alterations in seven AMPs were identified in 0.9%-16% of patients. Patients with high mRNA levels of MAPK1, RELA, STAT3, GNB1, and MMP9 had significantly worse overall survival rates compared with patients with low expression. Molecular docking studies showed that RELA and MMP9 are potential target candidates of atenolol in the treatment of patients with pancreatic cancer. CONCLUSION: In conclusion, atenolol can potentially be repurposed to target pancreatic cancer cells by modulating MMP9 and NF-κB signaling. The results of this study need to be further validated in vitro and in vivo.

Integrative Bioinformatics Analysis Reveals Potential Target Genes and TNFα Signaling Inhibition by Brazilin in Metastatic Breast Cancer Cells.

OBJECTIVE: Metastasis is the most significant cause of morbidity and mortality in breast cancer patients. Previously, a combination of brazilin and doxorubicin has been shown to inhibit metastasis in HER2-positive breast cancer cells. This present study used an integrative bioinformatics approach to identify new targets and the molecular mechanism of brazilin in inhibiting metastasis in breast cancer. METHODS: Cytotoxicity and mRNA arrays data were retreived from the DTP website, whereas genes that regulate metastatic breast cancer cells were retreived from PubMed with keywords "breast cancer metastasis". Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and Drug association analysis were carried out by using WEB-based GEne SeT AnaLysis Toolkit (WebGestalt). Construction of protein-protein interaction (PPI) network analysis was performed by STRING-DB v11.0 and Cytoscape, respectively. The genetic alterations of the potential therapeutic target genes of brazilin (PB) were analyzed using cBioPortal. RESULTS: Analysis of cytotoxicity with the public database of COMPARE showed that brazilin exerts almost the same cytotoxicity in the NCI-60 cells panel showing by similar GI50 value, in which the lowest GI50 value was observed in MDA-MB 231, a metastatic breast cancer cells. KEGG enrichment indicated several pathways regulated by brazilin such as TNF signaling pathway, cellular senescence, and pathways in cancer. We found ten drugs that are associated with PB, including protein kinase inhibitors, TNFα inhibitors, enzyme inhibitors, and anti-inflammatory agents. CONCLUSION: In conclusion, this study identified eight PB, including MMP14, PTGS2, ADAM17, PTEN, CCL2, PIK3CB, MAP3K8, and CXCL3. In addition, brazilin possibly inhibits metastatic breast cancer through inhibition of TNFα signaling. The study results study need to be validated with in vitro and in vivo studies to strengthen scientific evidence of the use of brazilin in breast cancer metastasis inhibition.

Integrative bioinformatics analysis reveals miR-494 and its target genes as predictive biomarkers of trastuzumab-resistant breast cancer.

BACKGROUND: The focus of trastuzumab resistance biomarkers in recent decades has been on epigenetic and non-coding RNA-based mechanisms. In this study, the potential of miR-494 and its target genes as predictive biomarkers for breast cancer (BC) resistance to trastuzumab was identified. The microarray data were obtained from the GEO database, including GSE101841, GSE75669, and GSE66305. Data processing was conducted using GEO2R to obtain differentially expressed genes (DEGs). RESULTS: The data analysis using GEO2R revealed that DEGs from GSE101841 and GSE75669 consisted of 3 and 135 upregulated miRNAs, respectively. On the other hand, the same analysis revealed 8 and 226 downregulated miRNAs for DEGs from GSE101841 and GSE75669, respectively. A Venn diagram showed that one miR was detectable in serum and tissue samples, namely miR-494. The miR-494 target was predicted using the miRecords database and resulted in 69 target genes. A Venn diagram between miR-494 target genes from miRecords and the mRNA array from GSE66305 revealed three potential targets of CNR1, RBM39, and ZNF207. A Kaplan-Meier survival plot showed that BC patients with a high miR-494 level and a low ZNF207 mRNA level had significantly worse overall survival. Validation of target genes in BC samples and trastuzumab-resistant and -sensitive BC cells with GEPIA and ONCOMINE highlighted the potential of CNR1, RBM39, and ZNF207 as predictive biomarkers of trastuzumab resistance in BC cells. CONCLUSION: Taken together, these results suggest that miR-494 plays a role in the mechanism of BC resistance to trastuzumab by involving its target genes CNR1, RBM39, and ZNF207.

Bioinformatics Studies Provide Insight into Possible Target and Mechanisms of Action of Nobiletin against Cancer Stem Cells.

OBJECTIVE: Nobiletin treatment on MDA-MB 231 cells reduces the expression of CXC chemokine receptor type 4 (CXCR4), which is highly expressed in cancer stem cell populations in tumor patients. However, the mechanisms of nobiletin in cancer stem cells (CSCs) remain elusive. This study was aimed to explore the potential target and mechanisms of nobiletin in cancer stem cells using bioinformatics approaches. METHODS: Gene expression profiles by public COMPARE predicting the sensitivity of tumor cells to nobiletin. Functional annotations on gene lists are carried out with The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8, and WEB-based GEne SeT Analysis Toolkit (WebGestalt). The protein-protein interaction (PPI) network was analyzed by STRING-DB and visualized by Cytoscape. RESULTS: Microarray analyses reveal many genes involved in protein binding, transcriptional and translational activity. Pathway enrichment analysis revealed breast cancer regulation of estrogen signaling and Wnt/ß-catenin by nobiletin. Moreover, three hub genes, i.e. ESR1, NCOA3, and RPS6KB1 and one significant module were filtered out and selected from the PPI network. CONCLUSION: Nobiletin might serve as a lead compound for the development of CSCs-targeted drugs by targeting estrogen and Wnt/ß-catenin signaling. Further studies are needed to explore the full therapeutic potential of nobiletin in cancer stem cells.
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Comprehensive bioinformatics study reveals targets and molecular mechanism of hesperetin in overcoming breast cancer chemoresistance.

The effectiveness of chemotherapy in breast cancer treatment can be increased using a combinatorial agent. Hesperetin has been reported to increase the sensitivity of doxorubicin in breast cancer cells; however, the underlying molecular mechanism remains unclear. This present study was conducted to identify the potential target and molecular mechanism of hesperetin in circumventing breast cancer chemoresistance using a bioinformatics approach. Microarray data obtained after hesperetin treatment in the NCI-60 cell line panel collection were retrieved from the COMPARE public library. These data were then compared with the list of the regulatory genes of breast cancer resistance obtained from PubMed and further analyzed for gene ontology and KEGG pathway enrichment, as well as protein-protein interaction network. A Venn diagram of COMPARE microarray data and the gene list from PubMed generated 56 genes (potential therapeutic target genes/PTTGs). These PTTGs participate in the biological process of the JAK-STAT cascade and are located in the nucleus, exert a molecular function in protein serine/threonine kinase activity, and regulate the erbB signaling pathway. Drug association analysis demonstrated that both hesperetin and the erbB receptor inhibitors, i.e., monoclonal antibody and tyrosine kinase inhibitor, target the same mRNA expression. Furthermore, results of the molecular docking study revealed that hesperetin is a promising inhibitor that targets ABL1, DNMT3B, and MLH1 due to the similarity of binding properties with its native ligand. In conclusion, the possible pathways and the regulatory genes identified in this study may offer new insights into the mechanism by which hesperetin overcomes breast cancer chemoresistance. A combinatorial therapy with hesperetin targeting ABL1, DNMT3B, and MLH1 may be effective in circumventing chemoresistance in breast cancer.

Anti-proliferative and Anti-metastatic Potential of Curcumin Analogue, Pentagamavunon-1 (PGV-1), Toward Highly Metastatic Breast Cancer Cells in Correlation with ROS Generation.

Purpose: Pentagamavunon-1 (PGV-1) is a curcumin analogue that shows cytotoxic activity in various cancer cells. In this study, we evaluated the effect of PGV-1 on a highly metastatic breast cancer cell line, the 4T1 cells, as an anti-metastatic and anti-proliferative agent. Methods: Cell viability was evaluated using MTT assay; while cell cycle profile, apoptosis incidence, and ROS intracellular level were determined by flow cytometry. Cell senescence was observed under senescence-associated-ß-galactosidase (SA-ß-gal) staining assay. The expression of matrixmetalloproteinase-9 (MMP-9) was determined using immunoreaction based-ELISA, while other proteins expression were detected using immunoblotting. Results: Curcumin and PGV-1 showed cytotoxic effects on 4T1 cells with IC50 value of 50 and 4 µM, respectively. The cytotoxic activity of PGV-1 was correlated to the induction of G2/M cell cycle arrest and cell senescence. Furthermore, PGV-1 increased the accumulation of intracellular ROS level. We also revealed that PGV-1 bound to several ROS-metabolizing enzymes, including glyoxalase I (GLO1), peroxiredoxin 1 (PRDX1), N-ribosyldihydronicotinamide: quinone reductase 2 (NQO2), aldo-keto reductase family 1 member c1 (AKR1C1). As an antimetastatic agent, PGV-1 showed less inhibitory effect on cell migration compared to curcumin. However, PGV-1 significantly decreased MMP-9 protein expression in a dose-dependent manner suggesting it still potent to inhibit metastatic cells. Conclusion: Overall, our findings suggest that PGV-1 is potential to be developed as an antiproliferative and anti-metastatic agent.

Current report of natural product development against breast cancer stem cells.

Chemotherapeutic agents are commonly used as neoadjuvant for breast cancer therapy. However, there is evidence of treatment failure for most of patients due to acquired resistance. Cancer stem cells, a small population of cells within tumors, contribute to cancer resistance, tumor relapse, and metastasis. Therefore breast cancer stem cells-targeted therapy is a strategic way of overcoming chemoresistance. Some natural products have been identified to target breast cancer stem cells. This article aims to discuss the current report of preclinical studies of natural products i.e., phytochemicals, compound isolated from mushroom and alkaloid from the sponge that target breast cancer stem cells. This review does not only describe biological effects of natural products i.e., its effect on the signaling pathway, but also discuss chemical structure modification as well as recent pharmaceutical technology of natural products to target breast cancer stem cells. In conclusion, those natural products posses as novel therapeutic agents for breast cancer stem cells eradication and lead compound for the development of breast cancer stem cells-targeted drug.

Combination of Potassium Pentagamavunon-0 and Doxorubicin Induces Apoptosis and Cell Cycle Arrest and Inhibits Metastasis in Breast Cancer Cells.

A salt compound of a curcumin analogue, potassium pentagamavunon-0 (K PGV-0) has been synthesized to improve solubility of pentagamavunon-0 which has been proven to have anti-proliferative effects on several cancer cells. The purpose of this study was to investigate cytotoxic activity and metastasis inhibition by K PGV- 0 alone and in combination with achemotherapeutic agent, doxorubicin (dox), in breast cancer cells. Based on MTT assay analysis, K PGV-0 showed cytotoxic activity in T47D and 4T1 cell lines with IC50 values of 94.9 µM and 49.0±0.2 µM, respectively. In general, K PGV-0+dox demonstrated synergistic effects and decreased cell viability up to 84.7% in T47D cells and 62.6% in 4T1 cells. Cell cycle modulation and apoptosis induction were examined by flow cytometry. K PGV-0 and K PGV-0+dox caused cell accumulation in G2/M phase and apoptosis induction. Regarding cancer metastasis, while K PGV-0 alone did not show any inhibition of 4T1 cell migration, K PGV-0+dox exerted inhibition. K PGV-0 and its combination with dox inhibited the activity of MMP-9 which has a pivotal role in extracellular matrix degradation. These results show that a combination of K PGV-0 and doxorubicin inhibits cancer cell growth through cell cycling, apoptosis induction, and inhibition of cell migration and MMP-9 activity. Therefore, K PGV-0 may have potential for development as a co-chemotherapeutic agent.

Cardioprotective and hepatoprotective effects of Citrus hystrix peels extract on rats model.

OBJECTIVE: To observe the combination effect of doxorubicin and Citrus hystrix (kaffir lime's) peel ethanolic extract (ChEE) on blood serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity and cardio-hepato-histopathology of female Sprague Dawley rats. METHODS: Doxorubicin and ChEE (5 rats per group) were administered in five groups of 3 rats each for 11 d. Group I: doxorubicin (dox) 4.67 mg/kg body weight; Group II: dox+ChEE 500 mg/kg body weight; Group III: dox+ChEE 1 000 mg/kg body weight; Group IV: ChEE 1 000 mg/kg body weight; Group V: untreated (control). RESULTS: ChEE repaired cardiohistopathology profile of doxorubicin induced cardiotoxicity and hepatotoxicity rats, but did not repair neither hepatohistopathology profile nor reduce serum activity of ALT and AST. CONCLUSION: ChEE has potency to be developed as cardioprotector agent in chemotherapy.