Qualitative Analysis and Cytotoxic Activity of Ethanolic Extract of Piper betle Linn.Leaves: Induction of Apoptosis, Cell Cycle Arrest, and Caspase-3/7 Activation inHER2+ AU565 Breast Cancer Cell Line

Introduction: Piper betle, or betel vine, is a common chewing plant used for recreational purposes. Recently, there has been an increased interest in the cancer chemotherapeutic potential of dietary plants due to their antioxidant, antiinflammatory, and anti-mutagenic actions. While previous studies have demonstrated that P. betle leave extract inhibits the proliferation of breast cancer cells in vitro, the underlying mechanisms of its anti-cancer activity remain poorly understood. In this study, we investigated the effect of P. betle leaf extract on breast cancer cell lines and identified the possible mechanisms involved. Methods: Cell viability was assessed using the MTT assay. Annexin V and caspase-3/7 assays were used to determine apoptosis (programmed cell death). Major bioactive constituents in the extract were identified by using LC-MS and GC-MS techniques. Results: We found that P. betle leaves’ extract inhibited the proliferation of all the breast cancer cell lines. Mechanistic analyses revealed that P. betle mediated inhibition of cell growth is a result of cell cycle arrest and DNA fragmentation. Moreover, the expression of apoptotic genes increased, ultimately leading to the activation of caspase-3/7 and cell death. The major compounds identified were hydroxychavicol, methyl palmitate, (E)- methyl octadec-9-enoate, and coniferaldehyde. We therefore speculate that the anti-cancer effect of P. betle is largely dependent on these phytochemicals. Discussions: In line with previously reported studies, our results demonstrate, for the first time, the anti-cancer effect of P. betle extract on the AU565 a HER2+ breast cancer cell line. Together with in silico and mechanistic approaches, we established that major compounds identified in the P. betle extract could be further evaluated as potential lead molecules against breast cancer. Conclusion: In summary, we report here for the first time that P. betle extract inhibits the proliferation of the AU565 (HER2+) breast cancer cell line, which may aid in advancing treatment options, particularly for HER2+ breast cancer.