Pseudolaric Acid B Induces Apoptosis Associated With Mitochondrial And PI3K/AKT/mTOR Pathways In Triple-Negative Breast Cancer

Objective:The anticancer effect and related mechanism of Pseudolaric acid B in triple negative breast cancer were investigated.This study aims to find new therapeutic targets for the treatment of triple negative breast cancer and provide theoretical basis for the development of new drugs.Methods:The effect of Pseudolaric acid B on the activity of MDA-MB-231 cells was determined by CCK-8 assay.Ed U cell proliferation assay and colony formation assay were used to detect the effect of Pseudolaric acid B on cell proliferation.The effects of Pseudolaric acid B on cell apoptosis rate,cell cycle distribution,mitochondrial membrane potential and intracellular reactive oxygen species（ROS）were studied by flow cytometry.The effect of Pseudolaric acid B on cell migration was detected by wound healing assay and Transwell migration assay.Transwell invasion assay was used to detect the effect of Pseudolaric acid B on cell invasion.The effects of Pseudolaric acid B on the expression of proliferation-related genes（p53,p21,Cyclin B1,CDK1）were detected by Western Blot.The effects of Pseudolaric acid B on protein levels of Caspase-3,Caspase-9,PARP,Bax,Bcl-2,Bcl-xl and Cytochrome c were determined by Western Blot.The effects of Pseudolaric acid B on the expression of marker proteins such as N-cadherin,Vimentin and E-cadherin in Epithelial-mesenchymal transformation were detected by Western Blot.The effects of Pseudolaric acid B on the signaling pathway PI3K/AKT/mTOR were detected by Western Blot.Results:The results of CCK-8 assay showed that MDA-MB-231 cell viability was inhibited in a concentration-dependent and time-dependent manner.Ed U cell proliferation assay and colony formation assay showed that the cell proliferation ability was inhibited by Pseudolaric acid B.Flow cytometry results showed that Pseudolaric acid B could induce cell apoptosis,arrest the cell cycle in G2/M phase,reduce the level of mitochondrial membrane potential and significantly increase the level of reactive oxygen species in MDA-MB-231 cells.Both the wound healing assay and Transwell migration assay showed that the cell migration ability was inhibited by Pseudolaric acid B.Transwell invasion assay showed that Pseudolaric acid B inhibited the invasion ability of tumor cells.The results of Western Blot showed that Pseudolaric acid B inhibited cell proliferation by up-regulating the expressions of p53 and p21 and down-regulating the expressions of CDK1 and Cyclin B1.Western Blot results showed that Pseudolaric acid B acted on the targets of the mitochondrial apoptosis pathway to induce tumor cell apoptosis,such as promoting the release of Cytochrome c from the mitochondria into the cytoplasm,up-regulating the protein expression of Cleaved caspase-3,Cleaved caspase-9,Cleaved PARP,Bax and down-regulating the protein levels of Bcl-2 and Bcl-xl.The results of Western Blot showed that Pseudolaric acid B could inhibit the expression of N-cadherin and Vimentin and promote the expression of E-cadherin,thus regulating the Epithel-mesenchymal transformation process.The results of Western Blot demonstrated that Pseudolaric acid B inhibited the activity of PI3K/AKT/mTOR signaling pathway by down-regulating the protein expression of PI3K（p110?）,p-AKT(ser⁴⁷³)and p-mTOR(ser²⁴⁴⁸),thus playing an anticancer role.Conclusion:Pseudolaric acid B can inhibit the proliferation of MDA-MB-231 cells,and its mechanism may be related to the regulation of cyclin checkpoint to induce cell cycle arrest.Pseudolaric acid B can induce apoptosis of MDA-MB-231 cells,and its mechanism may be related to activation of mitochondrial apoptosis pathway and inhibition of PI3K/AKT/mTOR pathway.Pseudolaric acid B can inhibit the invasion and migration of MDA-MB-231 cells,and its mechanism may be related to the regulation of Epithelial-mesenchymal transformation process,suggesting that Pseudolaric acid B plays an effective anticancer role in triple negative breast cancer,and may be a new plant drug for the treatment of triple negative breast cancer.