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.

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.