Objective: We aim to enhance the effectiveness of curcumin analog PGV-1 through co-treatment with diosmin, a citrus flavonoid, on 4T1 cells and evaluate the molecular targets underlying its effect on the cell cycle.
Methods: Cytotoxic effects were performed by MTT assay against 4T1 cells. The May Grünwald-Giemsa staining was used to observe cell cycle arrest. The senescence was assayed with SA-ß-gal staining. Bioinformatic studies were utilized to discover protein targets of PGV-1 and diosmin on triple-negative breast cancer (TNBC) using SwissTargetPrediction, then exploration of protein targets was performed using the TCGA dataset via the UALCAN website. Kaplan-Meier was performed using GraphPad with data from the TCGA dataset via Oncoln. Using MOE 2010, we conducted the binding affinity between PGV-1 and diosmin to protein targets.
Results: PGV-1 and diosmin showed cytotoxic effect with IC50 values of 9 µM and 389 µM, respectively, and the combined cytotoxic assay exhibited a synergistic effect with a combination index (CI) of <1. PGV-arrested 4T1 cells in pro-metaphase and induced mitotic catastrophe, while the combination of diosmin with PGV-1 increased the number of mitotic catastrophes. The SA-ß-gal assay revealed that both compounds were capable of inducing senescence in 4T1 cells. Study bioinformatics and molecular docking showed that PGV-1 and diosmin target cell cycle regulatory proteins in TNBC, namely CDK1, KIF11, and AURKA. Thus, the combination of diosmin and PGV-1 modulating the cell cycle that causes senescence and catastrophic death of 4T1 cancer cells is related to the inhibition of these cell cycle proteins.
Conclusion: Diosmin enhances the cytotoxic effect of PGV-1 synergistically on 4T1 cancer cells, which correlates to the increasing senescence and mitotic catastrophe. The synergistic effect of the co-treatment is likely to target AURKA, CDK1, and KIF11. The combination of PGV-1 and diosmin performs a potential as a combinatorial anticancer drug for TNBC.
Keywords: 4T1; Co-treatment diosmin and PGV-1; Mitotic catastrophe; Mitotic regulatory proteins; senescence.