Ethanolic extract of Otostegia fruticosa induces ROS-dependent apoptosis and reduces migration of MDA-MB-231 cells in vitro

Breast cancer (BC) has a highly aggressive and metastatic nature. Specifically, triple-negative breast cancer (TNBC) is highly complicated to treat and recover from. Nowadays, treatment strategies focus on the beneficial nature of the herbal source. Otostegia fruticosa gained focus on treating various cancers due to its potential against cancer. This study aimed to investigate the apoptotic and metastasis properties of Otostegia fruticosa against triple-negative breast cancer cells (MDA-MB-231). The ethanol extract of Otostegia fruticosa exhibited cytotoxic effect on MDA-MB-231 cells, with an IC 50 value of 23 µg/mL. various tests, including MTT, morphological analysis, fluorescence staining, and Trypan blue exclusion, invasion, migration ability were employed to evaluate the effects of Otostegia fruticosa on cell viability and apoptosis. The results showed that Otostegia fruticosa induced apoptosis in MDA-MB-231 cells, characterized by the accumulation of reactive oxygen species (ROS), mitochondrial damage, and loss of nuclear potential. Furthermore, Otostegia fruticosa treatment reduced cell viability due to excessive ROS generation, as evidenced by DCFH-DA staining. Functional assays revealed that Otostegia fruticosa impaired cellular mobility, invasion, and colony-forming ability in MDA-MB-231 cells. The Otostegia fruticose significantly suppresses invasion property of breast cancer cells about 50% and significantly reduced migrated cells time dependent manner. Gene expression analysis by qRT-PCR and immunofluorescence showed altered expressions of genes linked to apoptotic (Caspase-3, 8, 9, and Bcl2, Bax, Bid, Cytochrome c, PTEN) and metastasis (MMP-9) protein. Notably, Otostegia fruticosa downregulated MMP9 and OPN protein expressions, while upregulating caspase-9 and Cyt-c. Western blot analysis validated these findings, highlighting the involvement of the p-Akt and OPN downregulation. Altogether, this study demonstrates that Otostegia fruticosa induces apoptosis and inhibits metastasis in MDA-MB-231 cells by regulating caspase-3, 8, and 9, Bcl2, Bax, and Bid. Meanwhile, it potentially arrests the translation level of MMP-9 and OPN proteins. In addition, cellular migration and invasion were significantly downregulated, and Otostegia fruticosa possesses the anti-tumor effect against the TNBC cells (MDA-MB-231).