Pseudolaric acid B induces apoptosis associated with the mitochondrial and PI3K/AKT/mTOR pathways in triple‑negative breast cancer

Pseudolaric acid B (PAB), a diterpene acid isolated from the root bark of Pseudolarix kaempferi, has been shown to exert strong antitumor properties. The aim of the present study was to investigate the mechanisms underlying the proposed antitumor properties of PAB in the triple-negative breast cancer cells, MDA-MB-231. The cell processes evaluated included cell proliferation by Cell Counting Kit-8 assay, colony formation and EdU assay, apoptosis by Annexin V-FITC/PI apoptosis assay, cell migration by Transwell migration assay and invasion by Transwell invasion assay. PAB significantly inhibited the proliferation of MDA-MB-231 cells through a mechanism that was considered to be associated with cell cycle arrest at the G(2)/M phase. There was decreased protein expression levels of CDK1 and cyclin B1 and increased protein expression levels of p53 and p21. However, there were no well-defined inhibitory effects on the normal breast cell line MCF10A. PAB also triggered apoptosis in a concentration-dependent manner through the mitochondrial apoptosis pathway. It caused collapse of mitochondrial membrane potential, accumulation of reactive oxygen species and release of cytochrome c, as well as upregulation of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Bax, and downregulation of Bcl-2 and Bcl-xl. The migration and invasion ability of MDA-MB-231 cells were inhibited by decreasing the expression levels of the epithelial-mesenchymal transition-related markers N-cadherin and vimentin and increasing the expression of E-cadherin. Moreover, the expression levels of PI3K (p110β), phosphorylated (p)-AKT (ser(473)) and p-mTOR (ser(2448)) were downregulated and LY294002, a PI3K inhibitor, could interact additively with PAB to induce apoptosis of MDA-MB-231 cells. Overall, the present results demonstrated that PAB induced apoptosis via mitochondrial apoptosis and the PI3K/AKT/mTOR pathway in triple-negative breast cancer. It also inhibited cellular proliferation, migration and invasion, suggesting that PAB may be a useful phytomedicine for the treatment of triple-negative breast cancer.