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.

Objective: This study aimed to explore Hesperetin (Hst) potency as a co-chemotherapeutics agent combined with Doxorubicin (Dox), particularly cytotoxic and antimetastasis effects toward MCF-7/HER2 cells. Methods: The cytotoxic effects were measured under MTT assay. The flowcytometry analysis was used to examine the cell cycle modulation and apoptosis evidence, while the effect of migration was assayed by scratch wound healing assay. Western blotting and gelatin zymography were carried out to examine the expression level of proteins, HER2, and Rac1. Results: Under MTT assay, Hst and Dox exhibited to decrease cell viability in a dose-dependent manner with the IC50 value of 377 and 0,8 µM, respectively. The combination of Hst and Dox at the respective doses of 95 and 0,2 µM showed a synergistic effect with the combination index of 0,63. Flow cytometry analysis of Hst-Dox revealed that those compounds caused cell cycle arrest at the G2/M phase and induced apoptosis. Hst also decreased HER2 and Rac1 expression, as shown by western blot. Hst inhibited lamellipodia formation and cell migration, as indicated by microscopic observation and wound healing scratch assay. The antimetastatic activity of Hst was associated with the reduction of Rac1 and MMP9 expression as measured by gelatine zymography assay. Conclusion: These results indicated that the combination of Hst and Dox-induced cell cycle arrest, apoptosis, decreased HER2, Rac1, MMP9 expression, and cell migration. Thus, Hst may have the potential to be developed as a co-chemotherapeutic agent combined with doxorubicin toward HER2 overexpressing breast cancer cells.

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. 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). 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. ChEE has potency to be developed as cardioprotector agent in chemotherapy.