Ethanolic extract of Akhuni induces ROS-mediated apoptosis through ERK and AKT signalling pathways: Insights from metabolic profiling and molecular docking studies

Akhuni, an ethnic food of northeast India, induces ROS-mediated apoptosis in cancer cells. This is the first report on the anticancer potential of Akhuni. Akhuni is a traditional fermented soybean product known for its umami taste and delicacy, commonly used in Northeast India's cuisine. The current work demonstrates the antiproliferative potential of Akhuni ethanolic extract (AKET) against B16-F10 and MDA-MB-231 cancer cells and its mechanism of action supported by metabolic profiling and molecular docking. The investigation evaluated cytotoxicity, cell cycle distribution, caspase activity, apoptosis-related gene and protein expression, and oxidative stress imposed by excess reactive oxygen species (ROS) in both cell types. Phytochemical characterization of AKET was performed using HPLC. The growth of both cells is concentration-dependently inhibited after AKET treatment in MTT and flow cytometry experiments, leading to an arrest in the cell cycle at the G2 phase. Intracellular ROS levels increased in response to AKET treatment, suggesting that ROS in both cells triggered the mitochondrial pathway. Compared to the untreated cells, qRT-PCR analysis showed that AKET significantly reduced Cdk2 and Bcl-2 and increased the mRNA expression levels of Caspase-9, Bax, FasL, and Bid. Additionally, Caspase-8, Caspase-3, and the protein p53 were significantly upregulated in AKET-treated cells, as confirmed by both real-time and ELISA assays. In both the B16-F10 and MDA-MB-231 cell lines, the Western blot analysis showed that AKET caused an elevation of the expression of the Bax protein and downregulation of the Erk1/2, Akt, and Bcl2 proteins. Six isoflavones were identified from AKET through HPLC analysis. Molecular docking results indicate compounds in the AKET extract like daidzein, genistein and glycitein act as potent inhibitors of the key oncoprotein, AKT. These findings suggest that AKET has an anticancer effect through ROS-mediated ERK1/2 and AKT signalling pathways.