Anticancer activity of Bacopa monnieri through apoptosis induction and mitophagy-dependent NLRP3 inflammasome inhibition in oral squamous cell carcinoma

•Aqueous fraction of ethanolic extract of Bacopa monnieri (BM-AF) was found to have potent anticancer potential against oral cancer cells.•BM-AF induced Parkin dependent mitophagy in oral cancer cells.•BM-AF inhibited arecoline-induced NLRP3 inflammasome activation in both in-vitro and in-vivo oral cancer model.•BM-AF mediated NLRP3 inflammasome inhibition was mitophagy dependent. Bacopa monnieri (BM) is traditionally used in human diseases for its antioxidant, anti-inflammatory and neuroprotective effects. However, its anticancer potential has been poorly understood. The aim of this study was to explore the detailed anticancer mechanism of BM against oral cancer and to identify the bioactive BM fraction for possible cancer therapeutics. We performed bioactivity-guided fractionation and identified that the aqueous fraction of the ethanolic extract of BM (BM-AF) had a potent anticancer potential in the in vitro and in vivo oral cancer models. BM-AF inhibited cell viability, colony formation, cell migration and induced apoptotic cell death in Cal33 and FaDu cells. In addition, BM-AF at low doses promoted mitophagy and BM-AF mediated mitophagy was PARKIN dependent. In addition, BM-AF inhibited arecoline induced reactive oxygen species production in cal33 cells. In this setting, our data showed that BM-AF supressed arecoline-induced NLR family pyrin domain containing 3 (NLRP3) inflammasome activation through mitophagy in Cal33 cells. The in vivo antitumor effect of BM-AF was further validated in C57BL/6J mice through a 4-nitroquinolin-1-oxide and arecoline-induced oral cancer model. The tumor incidence was significantly reduced in BM-AF treated group. Further, data obtained from western blot and immunohistochemistry analysis showed increased expression of apoptotic markers and decreased expression of inflammasome markers in the tissue obtained from BM-AF treated mice in comparison with the control group. In conclusion, BM-AF exhibit potent anticancer activity through apoptosis induction and mitophagy-dependent inhibition of NLRP3 inflammasome activation in both in vitro and in vivo oral cancer models. Moreover, we have screened apoptosis and mitophagy-inducing compounds from this plant extract having anticancer activity against oral cancer cells.