Azd9291

Manufactured by Selleck Chemicals

Sourced in United States

AZD9291 is a small molecule tyrosine kinase inhibitor. It is designed to target specific mutations in the epidermal growth factor receptor (EGFR) gene.

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Osimertinib-AZD9291 (6 citations)

26 protocols using azd9291

Evaluating EGFR Inhibitor Efficacy Under Hypoxia

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Cell survival was assayed by clonogenic assays, cell proliferation assays, or monolayer growth. For clonogenic assays, after the H1975 cells were exposed to normoxia or hypoxia for 4 weeks, cells were seeded at 500/well in 6-well plates and treated with AZD9291 or WZ4002 (Selleckchem) at the indicated concentrations. Colonies were washed with 0.9% saline solution and stained with crystal violet 10–14 days later. Colonies consisting of >50 cells were counted. Colony formation was normalized to plating efficiency of the untreated cells. Errors bars in the survival analysis are based on three independent experiments. For cell proliferation assay, after the H1975 cells were exposed to normoxia or hypoxia for 4 weeks, 1X10⁵ cells were seeded in 100 mm dish and treated with AZD9291 or WZ4002 (Selleckchem) at 1 μM. Cell growth inhibition by AZD9291 and WZ4002 in H1975 N and H1975 H cells were measured by serial cell counts after 3, 6 and 9 days treatment. To quantify survival by monolayer growth, cells were seeded at a defined density in 6-well plates and treated with AZD9291 at the indicated concentrations for 5 days. Cells were stained with crystal violet for monolayer visualization and then the stain was dissolved in 70% ethanol and quantified by plate reader.

Lu Y., Liu Y., Oeck S., Zhang G.J., Schramm A, & Glazer P.M. (2020). Hypoxia induces resistance to EGFR inhibitors in lung cancer cells via upregulation of FGFR1 and the MAPK pathway. Cancer research, 80(21), 4655-4667.

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AZD9291-Resistant NSCLC Cell Lines

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The AZD9291-resistant NSCLC cell lines PC9/AZD9291 and HCC827/AZD9291, as well as the respective sensitive cell lines PC9 and HCC827, were kindly provided by Dr Tianxiang Chen (Shanghai Chest Hospital, Shanghai, China). These four cell lines were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). For resistance maintenance, 1 µmol/l AZD9291 (Selleck Chemicals, Houston, TX, USA) was also added to the culture of the two resistant cell lines. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂.

Fei X., Wang G., Shen H, & Gu X. (2019). Placenta-specific 8 is a potential novel target for osimertinib resistance in non-small cell lung cancer. Oncology Letters, 18(1), 955-961.

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Investigating EGFR and FGF Signaling Pathways

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The following antibodies were used: phospho EGFR Y1068 (Abcam AB5644),
phospho EGFR Y1068 (Cell Signaling 3777), EGFR (Cell Signaling 2646), EGFR (Cell
Signaling 4267), phospho ERK1/2 T202/Y204 (Cell Signaling 9101), phospho ERK1/2,
T202/Y204 (Cell Signaling 4370), ERK1/2 (Cell Signaling 9102), phospho AKT S473
(Cell Signaling 4060), AKT1/2/3 (Santa Cruz sc-8312), BIM (Cell Signaling 2933),
Actin (Cell Signaling 4970), Actin-HRP conjugated (Cell Signaling 12262), FGFR1
(Cell Signaling 9740), and FGFR3 (Cell Signaling 4574), phospho FRS2α
Y436 (Cell Signaling 3861), E-Cadherin (Cell Signaling 3195), N-Cadherin (Cell
Signaling 13116), Zeb1 (Cell Signaling 3396), Vimentin (Cell Signaling 5741).
Gefitinib, WZ4002, AZD9291, and BGJ398 (all from Selleck) were dissolved in DMSO
to a final concentration of 10 mmol/liter and stored at −20 °C.
The 18 drugs tested in the long-term assay are listed in Supplemental Table 3.

Raoof S., Mulford I.J., Frisco-Cabanos H., Nangia V., Timonina D., Labrot E., Hafeez N., Bilton S.J., Drier Y., Ji F., Greenberg M., Williams A., Kattermann K., Damon L., Sovath S., Rakiec D.P., Korn J.M., Ruddy D.A., Benes C.H., Hammerman P.S., Piotrowska Z., Sequist L.V., Niederst M.J., Barretina J., Engelman J.A, & Hata A.N. (2019). Targeting FGFR overcomes EMT-mediated resistance in EGFR mutant non-small cell lung cancer. Oncogene, 38(37), 6399-6413.

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Apoptosis Modulation in Cancer Cells

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Apigenin, ABT-263, PD0325901, MK-2206 and AZD9291 were purchased from Selleck (Selleck Chemicals, Houston, USA). Fetal bovine serum (FBS) was obtained from BI (Biological Industries, Shanghai, China). Antibodies of cleaved PARP (#5625), cleaved caspase3 (#9661), Mcl-1 (#39224), Bcl-xL (#2764), Survivin (#2808), Bim (#2933), Noxa (#14766), STAT3 (#12640), p-STAT3 (Y705) (#9145), p-EGFR (Y1068) (#2234), EGFR (#4267), p-ERK1/2 (T202/Y204) (#4370), ERK1/2 (#4695), p-AKT (S473) (#4060) and p-FoxO3a (#9465) were obtained from Cell Signaling Technologies (Beverly, MA, USA). The antibody of Bcl-2 (#AB40639) was from Aboci (Aboci, MD, USA). Bax (#23931-1-AP), β-actin (#60008-1-lg), β-tubulin (#10094-1-AP) and FoxO3a (#10849-1-AP) was obtained from Proteintech (Wuhan, Hubei, China). AKT1 (#sc-5298) was purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, California). 4′,6-Diamidino-2-phenylindole (DAPI) were obtained from Solarbio (Beijing, China).

Zhan Y., Wang Y., Qi M., Liang P., Ma Y., Li T., Li H., Dai C., An Z., Qi Y., Wu H, & Shao H. (2019). BH3 mimetic ABT-263 enhances the anticancer effects of apigenin in tumor cells with activating EGFR mutation. Cell & Bioscience, 9, 60.

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Cell Culture and Reagent Protocol for NSCLC Cell Lines

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PC9 cells were kindly provided by J. Engelman (Massachusetts General Hospital Cancer Center and Harvard Medical School). H1975 cells were kindly provided by Kwok Kin Wong (Dana-Farber Cancer Institute and Harvard Medical School). 293T cells were purchased from American Type Culture Collection (ATCC). PC9 cells/H1975 cells and 293T cells were maintained in RPMI 1640 medium (ATCC modification) and DMEM, respectively (Thermo Fisher Scientific, Inc.), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (GE Healthcare HyClone), 100 IU/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. Gefitinib, erlotinib, and AZD9291 were purchased from Selleck Chemicals. Palbociclib was a gift from K. Cichowski. The antibodies used were Tubulin (1:2500; Cell Signaling, 2128S), GAPDH (1:2500; Cell Signaling, 8884), PBRM1 (1:500; Bethyl Laboratories, A301-591A-M), p21 (1:1000; Cell Signaling, 2947S), phospho-EGFR (Tyr1068; 1:1000; Cell Signaling, 2236S), EGFR (1:1000; Cell Signaling, 2232S), phospho-ERK1/2 (T202/Y204; 1:1000; Cell Signaling, 4370P), total ERK1/2 (1:1000; Cell Signaling, 4695P), phospho-AKT (S473; 1:1000; Cell Signaling, 4060P), total AKT (1:1000; Cell Signaling, 2920S), ETV1 (1:1000; Abcam, ab184120), ETV5 (1:1000; Abcam, ab54704), and CIC (1:5000; a gift from H. Zoghbi).

Liao S., Davoli T., Leng Y., Li M.Z., Xu Q, & Elledge S.J. (2017). A genetic interaction analysis identifies cancer drivers that modify EGFR dependency. Genes & Development, 31(2), 184-196.

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Lung Cancer Cell Line Characterization

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All human lung cancer cell lines used in this study were obtained or established in our previous studies [20 (link)–25 ]. All cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 1× penicillin / streptomycin solution (Mediatech, Inc., Manassas, VA) at 37°C / 5% CO₂. T790M-specific EGFR-TKI, AZD9291 and cytotoxic agents (vinorelbine and cisplatin) were purchased from Selleck Chemicals (Houston, TX). H1975-AZD cells, SW900-VNR cells, and H647-CDDP cells were developed via chronic, repeated exposure to AZD9291, vinorelbine and cisplatin, respectively, as described previously [20 (link)]. All experiments using acquired resistance cells, including the tissue microarray (TMA) preparation, were performed following removal of drug exposure to avoid the direct effects of drugs on PD-L1 expression.

Suda K., Rozeboom L., Rivard C.J., Yu H., Ellison K., Melnick M.A., Hinz T.K., Chan D., Heasley L.E., Politi K., Mitsudomi T, & Hirsch F.R. (2017). Therapy-induced E-cadherin downregulation alters expression of programmed death ligand-1 in lung cancer cells. Lung cancer (Amsterdam, Netherlands), 109, 1-8.

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Targeted Cancer Therapies Evaluation Protocol

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AZD9291, gefitinib, afatinib, and crizotinib were purchased from Selleckchem. SHR‐A1403, the naked anti‐c‐Met monoclonal antibody c‐Met mAb and free toxin SHR152852, were provided by Jiangsu Hengrui Medicine Co. Ltd.31 DyLight 488 N‐hydroxysuccinimide (NHS) ester was purchased from Thermo‐Fisher Scientific. Sulforhodamine B was purchased from Sigma‐Aldrich.
Antibodies against EGFR, phospho‐EGFR (Tyr1173), c‐Met, phospho‐c‐Met (Tyr1234/1235), STAT3, phospho‐STAT3 (Tyr705), AKT, phospho‐AKT (Ser473), ERK1/2, phospho‐ERK1/2 (Thr202/Tyr204), and GAPDH were purchased from Cell Signaling. β‐Tubulin antibody was purchased from Sigma‐Aldrich.

Tong M., Gao M., Xu Y., Fu L., Li Y., Bao X., Fu H., Quan H, & Lou L. (2019). SHR‐A1403, a novel c‐mesenchymal‐epithelial transition factor (c‐Met) antibody‐drug conjugate, overcomes AZD9291 resistance in non‐small cell lung cancer cells overexpressing c‐Met. Cancer Science, 110(11), 3584-3594.

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STING Pathway Activation and Inhibition

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2′3′-cGAMP (tlrl-nacga23-02) was purchased from InvivoGen. Ibrutinib (BTK inhibitor; no. S2680), PF-562271 (FAK inhibitor; no. S2890), PP2 (Src inhibitor; no. S7008), saracatinib (Fyn inhibitor; no. S1006), amuvatinib (c-Kit inhibitor; no. S1244), ruxolitinib (Jak1/2 inhibitor; no. S1378), alectinib (ALK inhibitor; no. S2762), gefitinib (EGFR inhibitor; no. S1025), AZD9291 (EGFR inhibitor; no. S7297), and R406 (Syk inhibitor; no. S2194) were obtained from Selleck. Polyethyleneimine (PEI; no. 195444) was purchased from Mpbio, and the transfection procedure was as previously described (16 (link), 33 (link)). Lipofectamine 2000 (no. 11668019) was obtained from Invitrogen. Antibodies against EGFR (no. 4267), GFP (no. 2956), phospho-Syk (Tyr525/526) (no. 2710), Syk (no. 13198), tubulin (no. 2146), and actin (no. 3700) were from Cell Signaling, and hemagglutinin (HA) antibody (ab18181) was purchased from Abcam. STING antibody (A3575) and GST (AE001) antibodies were obtained from Abclonal Technology.

Wang C., Sharma N., Veleeparambil M., Kessler P.M., Willard B, & Sen G.C. (2021). STING-Mediated Interferon Induction by Herpes Simplex Virus 1 Requires the Protein Tyrosine Kinase Syk. mBio, 12(6), e03228-21.

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Characterization of Erlotinib-Resistant NSCLC Cell Lines

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MGH121, MGH121 Res # 1 and PC9 cells were cultured in RPMI with 10% serum. 293FT cells were cultured in DMEM with 10% serum. PC9 cells were a gift from Pasi Janne, 293FT cells are from Invitrogen. MGH121 cells were generated from a pleural effusion of an erlotinib-resistant NSCLC patient on July 19, 2011 and were originally developed in ACL4 supplemented with 10% serum. Once completed the cell line was sequenced to confirm that it matched the patient effusion sample. Experiments involving the 293FT cells were completed within 6 months of purchasing from Invitrogen and did not undergo any further testing. PC9 cells were verified by STR analysis within 6 months to 1 year of experimentation. Gefitinib, Afatinib, WZ4002, CO-1686 and AZD-9291 were purchased from Selleck and re-suspended in DMSO. pEGFR antibody (pY1068) was from Abcam, total EGFR was from Santa Cruz Biotechnology. pERK (T202/Y204), total ERK, pS6 (S240/244), total S6, Actin, pAKT (T308) and total AKT were purchased from Cell Signaling Technologies. All antibodies were used at a concentration of 1:1000.

Niederst M.J., Hu H., Mulvey H.E., Lockerman E.L., Garcia A.R., Piotrowska Z., Sequist L.V, & Engelman J.A. (2015). The allelic context of the C797S mutation acquired upon treatment with third generation EGFR inhibitors impacts sensitivity to subsequent treatment strategies. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(17), 3924-3933.

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Comparative Analysis of Tyrosine Kinase Inhibitors

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Total 11 TKIs were tested in this study. DMSO was used as a solvent for the TKIs and also as the negative vehicle control. Pyrimethamine 5 μM was used for positive control. Afatinib (BIBW2992), AG1478, dacomitinib (PF299), erlotinib (OSI-420), gefitinib (ZD1839), lapatinib, neratinib, AZD9291 (osimertinib), pelitinib, and nintedanib (BIBF 1120) were purchased from Selleck Chemicals (Houston, Texas, USA). Dimethyl sulfoxide (DMSO), pyrimethamine, and sunitinib malate (SU 11248) were purchased from Sigma Aldrich (St. Louis, Missouri, USA).
Bovine serum albumin was purchased from Bovogen Biologicals (Melbourne, Australia). Antibodies against β-actin were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). FITC-conjugated anti-mouse IgG antibody, TRTIC-conjugated anti-rabbit IgG antibody, and horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies were purchased from Sigma Aldrich. Mouse Tg563 monoclonal antibody was cloned in our laboratory. PDCD4 (D29C6) XP® anti-rabbit monoclonal antibody was purchased from Cell Signaling.

Kim Y.H., Bhatt L., Ahn H.J., Yang Z., Lee W.K, & Nam H.W. (2017). Suppressors for Human Epidermal Growth Factor Receptor 2/4 (HER2/4): A New Family of Anti-Toxoplasmic Agents in ARPE-19 Cells. The Korean Journal of Parasitology, 55(5), 491-503.

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