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.

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.

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Background: Hesperetin (Hst), a citrus flavanone, is widely distributed among citrus fruits, including lemons. Hst has been shown to possess bioactivity as an antioxidant, anti-inflammatory, anti-allergic, hypolipidemic, vasoprotector, and anticancer agent. This study aimed to identify potential combinations of Hst and the chemotherapeutic agent doxorubicin (Dox) as co-chemotherapy agents against 4T1 murine metastatic breast cancer cells. Results: MTT assay results showed that Hst exhibited cytotoxic effect in 4T1 cells, and its combination with Dox showed a synergistic effect based on the CI value. The combination of Hst and Dox increased G2/M phase cell cycle arrest and apoptosis induction. The combination of Hst and Dox inhibited migration and decreased MMP-9 expression in 4T1 cells. Conclusion: In conclusion, the results of this study show that Hst has potential as a Dox co-chemotherapy against 4T1 cells by inducing G2/M phase cell cycle arrest and apoptosis. More importantly, Hst reduces Dox-induced migration and decreases MMP-9 expression.

Pentagamavunone-0 (PGV-0), one of the curcumin analogue, is reported to have a cytotoxic effect on various cancer cells. This study aimed to explore the growth inhibitory effects of PGV-0 against highly-metastatic breast cancer, 4T1 cells under stress condition covering 2D and 3D speroid cytotoxic, anti-migration, and suppression of MMP-9. PGV-0 showed cytotoxic effects on 2D and 3D 4T1 cells with IC50value of 49 μM and 26 μM, respectively. In addition, PGV-0 performed anti-migratory effect. The single treatment at 25 μM PGV-0 and 50 μM showed inhibitory effect on cell migration by 54% and 51% respectively. whilst, the combination of PGV-0 at the concentration of 25 μM and 50 μM with doxorubicin significantly inhibited cell migration by 41% and 38%,  respectively. The gelatin zymography assay showed that PGV-0 decreased MMP-9 expression both in a single treatment and in combination with doxorubicin. In conclusion,  PGV-0 is potential to be developed as anti-tumorigenesis agent on highly-metastatic breast cancers.Keywords: Pentagamavunone-0 (PGV-0), anti-migration, MMP-9, 4T1 cells, spheroid