In separate experiments, cells were pretreated with the autophagy inducer SB203580 (10 M), autophagy inhibitor wortmannin (10 M), chloroquine (30 M), bafilomycin A (10 M), and the apoptosis inhibitor Z-VAD(OMe)-FMK (20 M) for 1 hour, and then treated with ALS 5 M
October 13, 2021
In separate experiments, cells were pretreated with the autophagy inducer SB203580 (10 M), autophagy inhibitor wortmannin (10 M), chloroquine (30 M), bafilomycin A (10 M), and the apoptosis inhibitor Z-VAD(OMe)-FMK (20 M) for 1 hour, and then treated with ALS 5 M. proautophagic, and EMT-inhibitory effects on Gypenoside XVII SKOV3 and OVCAR4 cells. ALS arrested SKOV3 and OVCAR4 cells in G2/M phase and induced mitochondria-mediated apoptosis and autophagy in both SKOV3 and OVCAR4 cell lines in a concentration-dependent manner. ALS suppressed phosphatidylinositol 3-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways but activated 5-AMP-dependent kinase, as indicated by their altered phosphorylation, contributing to the proautophagic activity of ALS. Modulation of autophagy altered basal and ALS-induced apoptosis in SKOV3 and OVCAR4 cells. Further, ALS suppressed the EMT-like phenotype in both cell lines by restoring the balance between E-cadherin and N-cadherin. ALS downregulated sirtuin 1 and pre-B cell colony enhancing factor (PBEF/visfatin) expression levels and inhibited phosphorylation of AURKA in both cell lines. These findings indicate that ALS blocks the cell cycle by G2/M phase arrest and promotes cellular apoptosis and autophagy, but inhibits EMT via phosphatidylinositol 3-kinase/Akt/mTOR-mediated and sirtuin 1-mediated pathways in human epithelial ovarian cancer cells. Further studies are warranted to validate the efficacy and safety of ALS in the treatment of Gypenoside XVII ovarian cancer. maps to human chromosome 20q13 and to 17q13.1, which are loci frequently altered in human cancers. is located on chromosome 19q13.2 to 13.4, a region associated with loss of heterozygosity in ovarian cancer and pancreatic carcinomas. The expression and activity of Aurora kinases are tightly regulated, and dysregulation results in genetic instability, aneuploidy, and tumorigenesis.7,12 The gene is frequently amplified and/or overexpressed in a number of malignancies, including cancers of the ALRH bladder, breast, colon, liver, ovary, pancreas, stomach, and esophagus, and aberrant AURKA signaling is associated with malignant tumor behavior such as invasion and metastasis, advanced stage, and poor prognosis.11,13,14 Overexpression of AURKA is common in ovarian cancer, which is associated with supernumerary centrosomes, a poor response to chemotherapy, and reduced overall survival.10,15C17 AURKA has become a target of interest for the treatment of cancer, and a number of Aurora kinase inhibitors that have dual specificity for AURKA and AURKB, including MK-0457 and PHA-739358, Gypenoside XVII have been developed.11,14,18 Alisertib (MLN8237, ALS, Figure 1) is an investigational small-molecule inhibitor developed by Millennium Pharmaceuticals Inc (Boston, MA, USA) which selectively inhibits AURKA and has been shown in preclinical studies to induce cell cycle arrest, polyploidy, and mitotic catastrophe in various tumor cells, and to induce tumor regression in vivo.19C21 Currently, ALS is being tested in various Phase I and Phase II clinical trials for Gypenoside XVII advanced solid tumors and hematologic malignancies.22C27 In the present study, we aimed to uncover the underlying mechanisms for the anticancer effects of ALS in human EOC cells. Before we performed our benchmarking experiments, we ran molecular Gypenoside XVII docking assays to check how ALS bound to AURKA and AURKB and to compare the differences in the binding mode with those of other Aurora kinase inhibitors, including AMG-900, barasertib, CYC116, danusertib, MLN8054, and VX-680 (also called MK-0457), which are selective or nonselective inhibitors for AURKA.11,28 Open in a separate window Figure 1 Chemical structures of alisertib, AMG-900, barasertib, CYC116, danusertib, MLN8054, and VX-680, all of which are selective or pan inhibitors of Aurora kinase A and Aurora kinase B. Materials and methods Molecular docking In order to determine the molecular interactions between AURKA and AURKB and their inhibitors, the Discovery Studio program 3.1 designed by Accelrys Inc (San Diego, CA, USA) was used to dock ALS, AMG-900 (a potent and highly selective pan-AURKA, AURKB, and AURKC inhibitor29), barasertib (a highly selective AURKB inhibitor30), CYC116 (a potent inhibitor of AURKA and AURKB31), danusertib (an AURKA, AURKB, and AURKC inhibitor31), MLN8054 (a potent and selective inhibitor of AURKA32), and VX-680 (a pan-AURKA, AURKB, and AURKC, mostly against AURKA33) (Figure 1) into the active sites of human AURKA (Protein Data Bank [PDB] identification [ID]: 2DWB) and AURKB (PDB ID: 4AF3) as.