The 17 OCCC cell lines were plated in 384 well plates with 250-2000 cells/well using an automatic cell dispenser. Cell lines were incubated with varying concentrations of AZD8055 (Axon Medchem, The Netherlands), gefitinib (Selleckchem, USA), lapatinib (Selleckchem, USA) or olaparib (Axon Medchem, The Netherlands) for 6 days. Cell viability was subsequently monitored using CellTiter-Blue cell viability assay (Promega, USA). Due to large cell density dependent variability in drug sensitivity, for each cell line the lowest and highest IC50 were removed. The cell lines CAPAN1, OE19, LOVO, OVCAR3, OVCAR4 and SKOV3 were exposed to AZD8055 to serve as sensitive and resistant controls.
Azd8055
Manufactured by Axon Medchem
Sourced in United States, Netherlands
AZD8055 is a highly potent and selective inhibitor of the mammalian target of rapamycin (mTOR) kinase. It functions by blocking the enzymatic activity of both mTOR complexes, mTORC1 and mTORC2.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Cisplatin, by Accord Healthcare (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lapatinib, by Selleck Chemicals (1 mentions)
Gefitinib, by Selleck Chemicals (1 mentions)
Celltiter blue cell viability assay, by Promega (1 mentions)
Olaparib, by Axon Medchem (1 mentions)
Rapamycin, by Merck Group (1 mentions)
P s6k1 thr389, by Cell Signaling Technology (1 mentions)
P pras40, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Polyethylene glycol 300, by Merck Group (1 mentions)
Hydroxypropyl cellulose, by Merck Group (1 mentions)
Rg7388, by Selleck Chemicals (1 mentions)
Tween 80, by Merck Group (1 mentions)
Everolimus, by Santa Cruz Biotechnology (1 mentions)
Dasatininb, by Selleck Chemicals (1 mentions)
Mk2206, by Axon Medchem (1 mentions)
4 protocols using azd8055
1
Evaluating Anticancer Drug Sensitivity
2
Molecular Mechanisms of mTOR Signaling Pathway Regulation
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Cardamonin (no 110763, purity >99%; National Institutes for Food and Drug Control, Beijing, China), rapamycin (Sigma-Aldrich Co., St Louis, MO, USA), and AZD8055 (Axonmedchem, Groningen, the Netherlands) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co.) to prepare the stock solution (2 mM, 10 μM, and 10 μM, respectively) and these solutions were stored at 4°C. Methyl thiazolyl tetrazolium (MTT) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich Co. Lipofectamine 2000 was purchased from Thermo Fisher Scientific (Waltham, MA, USA), and Raptor small interfering RNA (siRNA) was produced by GenePharma Co., Ltd (Shanghai, China). Antibodies against mTOR, p-mTOR (Ser2481), Raptor, p-Raptor (Ser792), PRAS40, p-PRAS40 (Ser183 and Thr246), S6K1, p-S6K1 (Thr389), and β-actin (Cell Signaling Technology, Beverly, MA, USA) were used at a 1:1,000 dilution. The secondary antibodies (anti-rabbit immunoglobulin G, horseradish peroxidase [HRP]-linked antibody) used for detection in all cases were from Cell Signaling Technology. The Alexa 488- and Alexa 647-conjugated secondary antibodies (goat anti-rabbit immunoglobulin G H&L) were from Abcam (Cambridge, MA, USA).
3
PDX Tumor Xenograft Treatment Study
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Mice were implanted with PDX tumours as described above. When tumours demonstrated sustained growth, mice were randomized into vehicle control or treatment groups (n = 3–7 mice/group). Cisplatin (Accord Healthcare, London, UK) (1 mg/kg or 4 mg/kg) or vehicle (saline) was administered weekly via intraperitoneal injection. AZD8055 (Axon MedChem, Groningen, Netherlands) (2.7 mg/kg or 10 mg/kg in 10% DMSO, 40% Polyethylene glycol 300 (Sigma)) or vehicle was administered daily via intraperitoneal injection. RG7388 (Selleckchem, Munich, Germany) (50 mg/kg or 75 mg/kg in 2% hydroxypropylcellulose (Sigma), 0.2% Tween-80 (Sigma)) or vehicle was administered daily via oral gavage. Tumour growth was assessed 3 times a week using caliper measurements. All mice were sacrificed after 21 days of treatment. For future analysis the tumours were resected and paraffin embedded. Tumour volumes are plotted as mean ± SEM for all groups. Two-way ANOVA with Dunnett post hoc test was used to determine the significance of all pair-wise comparisons using GraphPad Prism.
4
Cytotoxicity Assay for Cancer Cell Lines
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Cells were plated in 96-well plates at a density of 5,000 cells/well for Tera, TeraCP, and 833KE, and 7,000 cells/well for Scha and NCCIT. At 2 to 3 hours after plating cells, one of the following agents was added, cisplatin (Accord Healthcare), Everolimus (Santa Cruz Biotechnology), GDC-0941, MK-2206, AZD8055, MLN0128 (also known as TAK-228; all from Axon Medchem), and dasatininb (Selleckchem). After a 96-hour incubation, 3-(4,5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium (MTT; Sigma) was added at a concentration of 5 mg/mL for 4 hours. Medium was removed and the formazan crystals were dissolved in DMSO (Sigma). Absorbance was measured at 520 nm using an iMARK microplate absorbance reader (Bio-Rad). Relative survival was determined as the decrease in signal compared with untreated cells.
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