Metabolomic Identification of Anticancer Metabolites of Australian Propolis and Proteomic Elucidation of Its Synergistic Mechanisms with Doxorubicin in the MCF7 Cells

The combination of natural products with standard chemotherapeutic agents offers a promising strategy to enhance the efficacy or reduce the side effects of standard chemotherapy. Doxorubicin (DOX), a standard drug for breast cancer, has several disadvantages, including severe side effects and the development of drug resistance. Recently, we reported the potential bioactive markers of Australian propolis extract (AP−1) and their broad spectrum of pharmacological activities. In the present study, we explored the synergistic interactions between AP−1 and DOX in the MCF7 breast adenocarcinoma cells using different synergy quantitation models. Biochemometric and metabolomics‐driven analysis was performed to identify the potential anticancer metabolites in AP−1. The molecular mechanisms of synergy were studied by analysing the apoptotic profile via flow cytometry, apoptotic proteome array and measuring the oxidative status of the MCF7 cells treated with the most synergistic combination. Furthermore, label‐free quantification proteomics analysis was performed to decipher the underlying synergistic mechanisms. Five prenylated stilbenes were identified as the key metabolites in the most active AP−1 fraction. Strong synergy was observed when AP−1 was combined with DOX in the ratio of 100:0.29 (w/w) as validated by different synergy quantitation models implemented. AP−1 significantly enhanced the inhibitory effect of DOX against MCF7 cell proliferation in a dose‐dependent manner with significant inhibition of the reactive oxygen species (p < 0.0001) compared to DOX alone. AP−1 enabled the reversal of DOX-mediated necrosis to programmed cell death, which may be advantageous to decline DOX‐related side effects. AP−1 also significantly enhanced the apoptotic effect of DOX after 24 h of treatment with significant upregulation of catalase, HTRA2/Omi, FADD together with DR5 and DR4 TRAIL-mediated apoptosis (p < 0.05), contributing to the antiproliferative activity of AP−1. Significant upregulation of pro‐apoptotic p27, PON2 and catalase with downregulated anti‐apoptotic XIAP, HSP60 and HIF‐1α, and increased antioxidant proteins (catalase and PON2) may be associated with the improved apoptosis and oxidative status of the synergistic combination‐treated MCF7 cells compared to the mono treatments. Shotgun proteomics identified 21 significantly dysregulated proteins in the synergistic combination‐treated cells versus the mono treatments. These proteins were involved in the TP53/ATM‐regulated non‐homologous end‐joining pathway and double‐strand breaks repairs, recruiting the overexpressed BRCA1 and suppressed RIF1 encoded proteins. The overexpression of UPF2 was noticed in the synergistic combination treatment, which could assist in overcoming doxorubicin resistance‐associated long non‐coding RNA and metastasis of the MCF7 cells. In conclusion, we identified the significant synergy and highlighted the key molecular pathways in the interaction between AP−1 and DOX in the MCF7 cells together with the AP−1 anticancer metabolites. Further in vivo and clinical studies are warranted on this synergistic combination.