Synergistic interactions between Australian propolis and Doxorubicin against the MCF-7 breast adenocarcinoma cells: Multiomics study understanding the molecular mechanisms of action

ABSTRACT: The combination of natural products with standard chemotherapeutic agents offers a promising strategy to enhance the efficacy of standard chemotherapy by reducing their dosage and side effects. The standard chemotherapeutic drug for breast and other cancer types- doxorubicin(DOX), also known as Adriamycin (an anthracycline) has several disadvantages including severe side effects and development of drug resistance. Recently, we reported the broad spectrum pharmacological activity and potential markers of Australian propolis extract (AP-1). In the present study, we explored the synergistic interactions of AP-1 with DOX against MCF-7 cells by implementing different synergy quantitation models. To identify the potential anticancer metabolites in AP-1, biochemometric and metabolomics-driven study was performed. Furthermore, we evaluated the molecular mechanisms involved by analysing the apoptotic profile (using flow cytometry and proteome array of 35 apoptotic proteins) and oxidative status (using reactive oxygen species detection) of the MCF-7 cells upon treatment with the most synergistic combination. The underlying synergistic mechanism against the MCF-7 cells was also studied using label-free quantification proteomics analysis. Five prenylated stilbenes were identified as the most discriminant metabolites in the most active AP-1 fraction, three of them previously isolated from AP-1 with IC50 values in the range 0.68-2.7 µM against the MCF-7 cells. 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 including combination index (CI), HSA, ZIP, LOEWE, and BLISS. AP-1 significantly enhanced the inhibitory effect of DOX (p < 0.05) against the MCF-7 cell proliferation in a dose-dependent manner with significant reduction of ROS production (p < 0.05; compared to DOX alone). AP-1 also enhanced the apoptotic effect of DOX significantly after 24 h of treatment with a shift of DOX-induced necrosis to apoptosis. Significant upregulation of catalase, HTRA2/Omi, FADD together with TRAIL-mediated apoptosis, DR5 and DR4 (p < 0.05) was observed which contributed to the anti-proliferative activity of AP-1 against the MCF-7 cells. The enhancement of pro-apoptotic p27, PON2 and catalase with reduction of anti-apoptotic XIAP, HSP60 and HIF-1α may be associated with the improved apoptosis in the MCF-7 cells observed in the Annexin V-7AAD flow cytometry analysis of the synergistic combination compared to the mono treatments. Furthermore, a significant increase of antioxidant proteins including catalase and PON2 was observed upon treatment with the synergistic combination which may be associated with the parallel enhancement of apoptosis. Shotgun proteomics in the synergistic combination-treated cells identified 21 significantly dysregulated proteins involving the TP53/ATM-regulated non-homologous end-joining pathway and double-strand breaks repairs, recruiting the overexpressed BRCA1 and curtailed RIF1 encoded proteins. The overexpression of UPF2 was noticed after the treatment with the synergistic combination which could assist to overcome doxorubicin resistance-associated long non-coding RNA and decline the metastasis of the MCF-7 cells. In summary, we propose a promising AP-1 and DOX synergistic combination against the MCF-7 cells and highlighted the possible synergistic mechanisms of action together with the key prenylated metabolites of AP-1. Further in vivo and clinical studies are warranted on this synergistic combination.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Muhammad Alsherbiny  

LAB HEAD: Muhammad Alsherbiny

PROVIDER: PXD026331 | Pride | 2021-07-26

REPOSITORIES: Pride