Ceritinib

Manufactured by Selleck Chemicals

Sourced in United States

Ceritinib is a small-molecule inhibitor of the anaplastic lymphoma kinase (ALK) enzyme. It is used in the laboratory setting for research purposes.

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Ceritinib (LDK378) (6 citations)

46 protocols using ceritinib

Establishing ALK-TKI Resistant Cell Lines

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To study the ALK-TKI resistant mechanisms, we established ALK-TKI-resistant cell lines. The subclones of FA34 and FA121 were incubated with crizotinib (Cr) or ceritinib (Ce) (Selleck Chemicals, Houston, TX, USA) with stepwise escalation (labelled with the suffix S) of drug concentrations (crizotinib: 0.02, 0.05, 0.2 µM; ceritinib: 0.01, 0.02, 0.04 µM), or high bolus concentrations (labelled with the suffix H) (crizotinib: 0.4 µM, ceritinib: 0.1 µM). IC50 values were determined by non-linear regression curve fitting using GraphPad Prism software 6.01. The IC₅₀ of the drug-resistant cell lines against different ALK-TKIs was determined after 20 passages. The cells that survived after prolonged exposure to TKIs were considered drug-resistant cell lines. Drug-resistant cell lines treated with first-generation ALK-TKI crizotinib were annotated with suffix Cr; resistant cell lines treated with second-generation ALK-TKI ceritinib were annotated with suffix Ce.

Kwok H.H., Ning Z., Chong P.W., Wan T.S., Ng M.H., Ho G.Y., Ip M.S, & Lam D.C. (2019). Transfer of Extracellular Vesicle-Associated-RNAs Induces Drug Resistance in ALK-Translocated Lung Adenocarcinoma. Cancers, 11(1), 104.

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Generation and Maintenance of Cell Lines

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H3122 cells were obtained from the National Cancer Institute. Generation of ceritinib-resistant H3122 cells is described in the Supplemental Experimental Procedures. H2228, HCC827, and HCC4006 were purchased from the American Type Culture Collection (ATCC). PC-9 cells were obtained from Public Health England, and KELLY neuroblastoma cells were obtained from Sigma-Aldrich. Cells were maintained in RPMI-1640 (Cellgro) with 10% fetal bovine serum (Gemini Bioproducts) and penicillin (100 units/mL) / streptomycin (100 µg/mL; Cellgro). MGH006 cells have been previously reported and were maintained in DMEM (Cellgro) with 10% fetal bovine serum, penicillin, and streptomycin (Sequist et al., 2010 (link)). All cell lines were tested to confirm the absence of mycoplasma contamination. Crizotinib, TAE684, ceritinib, erlotinib, lapatinib, and sotrastaurin were purchased from Selleck Chemicals. Blasticidin was obtained from Life Technologies. Phorbol 12-myristate 13-acetate (PMA) was purchased from Santa Cruz Biotechnology.

Wilson F.H., Johannessen C.M., Piccioni F., Tamayo P., Kim J.W., Van Allen E.M., Corsello S.M., Capelletti M., Calles A., Butaney M., Sharifnia T., Gabriel S.B., Mesirov J.P., Hahn W.C., Engelman J.A., Meyerson M., Root D.E., Jänne P.A, & Garraway L.A. (2015). A Functional Landscape of Resistance to ALK Inhibition in Lung Cancer. Cancer cell, 27(3), 397-408.

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Establishment and Characterization of Entrectinib-Resistant Cell Line

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HCC78 cell was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and cultured in RPMI-1640 medium supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C in a humidified atmosphere containing 5% CO₂. Cell line identity was authenticated by short-tandem-repeat analysis. Entrectinib-resistant HCC78 (HCC78ER) cells were newly established in our laboratory through the exposure of HCC78 cells to gradually increasing concentrations of Entrectinib (starting at 100 nM and ending with 5 μM) over 6 months. The established cells maintained resistance to Entrectinib even after the withdrawal of Entrectinib from the culture medium.
Entrectinib was provided by Ignyta, Inc./F.Hoffmann-La Roche Ltd. Crizotinib, ceritinib, lorlatinib and selumetinib were purchased from Selleckchem. All drugs were dissolved at a 10 mM concentration in dimethyl sulfoxide (DMSO) and stored in small aliquots at −20 °C until further use. Antibodies specific for p-ROS1 (Tyr1068), ROS1, p-AKT (Ser473), AKT, p-ERK1/2 (Thr202/Tyr204), ERK1/2, p-STAT3 (Tyr705), STAT3, p-p53 (Ser15), p53, p-H2AX (Ser139), H2AX and PARP were obtained from Cell Signaling Technologies. Anti-FGF3 and β-actin antibodies were obtained from Santa Cruz Biotechnology.

Ku B.M., Bae Y.H., Lee K.Y., Sun J.M., Lee S.H., Ahn J.S., Park K, & Ahn M.J. (2019). Entrectinib resistance mechanisms in ROS1-rearranged non-small cell lung cancer. Investigational New Drugs, 38(2), 360-368.

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Evaluating Larotrectinib, Ceritinib, and Fulvestrant

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Larotrectinib was purchased from AdooQ Bioscience, Irvine, CA. Ceritinib and fulvestrant were purchased from Sell-eckchem, Houston, TX. NTRK1 (30697), ALK (3633), phosphorylated AKT (4060), pan-AKT (2965), phosphorylated ERK1/2 (4370), ERK1/2 (4695), phospho-PLCγ (8713), PLCγ (5690), ERα (8644), PgR (8757), β-tubulin (2146), and glyc-eraldehyde-3-phosphate dehydrogenase (GAPDH) (2118) primary antibodies were obtained from Cell Signaling Technology, Danvers, MA.

Ross D.S., Liu B., Schram A.M., Razavi P., Lagana S.M., Zhang Y., Scaltriti M., Bromberg J.F., Ladanyi M., Hyman D.M., Drilon A., Zehir A., Benayed R., Chandarlapaty S, & Hechtman J.F. (2020). Enrichment of kinase fusions in ESR1 wild-type, metastatic breast cancer revealed by a systematic analysis of 4854 patients. Annals of oncology : official journal of the European Society for Medical Oncology, 31(8), 991-1000.

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Evaluation of Anti-Cancer Compounds

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Purchased human cancer cell lines included: (1) ES-2, HO-8910 PM and SW620 (Shanghai Institute of Biochemistry and Cell Biology); (2) HCC827, NCI-H1650, NCI-H1975, HepG2, and IMR-32 (American Type Culture Collection; Manassas, VA); and (3) OVCAR-3 (China Center for Type Culture Collection; Wuhan). All other human lines were obtained from Cobio Biosciences (Nanjing, China). Murine pro-B cell lines BaF3 and BaF3_EML4-ALK^G1202R were purchased from KYinoo (Beijing). Other BaF3-engineered cell lines were constructed internally. An osimertinib-resistant parental cell (PC-9/OR) line was established by exposing PC-9 cells to escalating concentrations of osimertinib for 3 months.
APG-2449 was synthesized by Ascentage Pharma. Alectinib, ceritinib, crizotinib, defactinib, erlotinib, lorlatinib, osimertinib, and trametinib, as well as paclitaxel and carboplatin, were purchased from Selleckchem (Houston, TX). Ensartinib was purchased from Birdo Tech (Shanghai).

Fang D.D., Tao R., Wang G., Li Y., Zhang K., Xu C., Zhai G., Wang Q., Wang J., Tang C., Min P., Xiong D., Chen J., Wang S., Yang D, & Zhai Y. (2022). Discovery of a novel ALK/ROS1/FAK inhibitor, APG-2449, in preclinical non-small cell lung cancer and ovarian cancer models. BMC Cancer, 22, 752.

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Cell Line Characterization and Drug Testing

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HR-proficient OVCAR-8 (D. Scudiero, National Cancer Institute, Frederick, MD) and OVCAR-8-DR-GFP cells (14 (link)), the BRCA1-mutant COV362 cells (Robert van Waardenburg, University of Alabama at Birmingham, Birmingham, AL), prostate cancer PC3 (Haojie Huang, Mayo Clinic), and breast cancer MDA-MB-231 cells (Zhenkun Lou, Mayo Clinic) were cultured in RPMI-1640 media (Corning) supplemented with 8% fetal bovine serum (Millipore) and maintained in a humidified 5% CO₂ incubator at 37 °C. All cells were authenticated by autosomal STR profiling (University of Arizona Genetics Core) and were free of Mycoplasma contamination as determined by testing with a MycoAlert Mycoplasma Detection Kit (catalog no. LT07-118, Lonza) every 6 months. Cell lines reinitiated from cryopreserved stocks every 3 to 6 months.
Ceritinib used for cell culture experiments as well as olaparib, veliparib, IACS-010759, and linsitinib were obtained from Selleck Chemicals. The Ceritinib used for mouse studies was provided by Novartis. Cisplatin was obtained from Teva Pharmaceutical Industries. SL0101 was purchased from Millipore.

Kanakkanthara A., Hou X., Ekstrom T.L., Zanfagnin V., Huehls A.M., Kelly R.L., Ding H., Larson M.C., Vasmatzis G., Oberg A.L., Kaufmann S.H., Mansfield A.S., Weroha S.J, & Karnitz L.M. (2021). Repurposing Ceritinib Induces DNA Damage and Enhances PARP Inhibitor Responses in High-Grade Serous Ovarian Carcinoma. Cancer Research, 82(2), 307-319.

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Investigating ALK Inhibitors and Signaling

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Crizotinib (PF-2341066), was purchased from Sigma-Aldrich (Oakville, Ontario, Canada). The HSP90 inhibitor (NVP-AUY922) and Ceritinib (LDK378) were purchased from Selleck Chemicals. Doxorubicin was purchased from LC Laboratories (Woburn, MA, USA). Each compound was dissolved in DMSO for cell culture experiments. The pcDNA3-flag-ALK wild-type and ALK^F1174L were kindly provided by Dr. Junko Takita (The University of Tokyo, Tokyo, Japan)61 (link). The EML4-ALK expression vector was a kind gift from Dr. James Dalton (University of Tennessee Health Science Center)62 (link). The NPM-ALK expression vector was a kind gift from Dr. S. Morris (St. Jude Children’s Research Hospital)63 (link). For the siRNA knockdown experiments, ALK and β-catenin specific ON-Target Plus SMARTpool small interfering RNA (siRNA) and scramble control were purchased from Thermo Scientific (Chicago, USA).

Alshareef A., Zhang H.F., Huang Y.H., Wu C., Zhang J.D., Wang P., El-Sehemy A., Fares M, & Lai R. (2016). The use of cellular thermal shift assay (CETSA) to study Crizotinib resistance in ALK-expressing human cancers. Scientific Reports, 6, 33710.

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Cytotoxicity Evaluation of Anti-cancer Drugs

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Dulbecco's modified Eagle medium (DMEM) and RPMI-1640 were obtained from Gibco BRL (Thermo Fisher Scientific Inc., Waltham, MA, USA). Doxorubicin, paclitaxel, cisplatin, topotecan, mitoxantrone, Verapamil (VRP), Fumitremorgin C (FTC), rhodamine123, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Ceritinib was purchased from selleck chemicals (Houston, TX, USA), with a molecular structure as shown in Figure 1A. Monoclonal antibodies against ABCB1, ABCG2, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), p-ERK, AKT and p-AKT were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against glyceraldehyd-3-Phosphate dehydrogenase (GAPDH) was from Kangcheng (Shanghai, China).

Hu J., Zhang X., Wang F., Wang X., Yang K., Xu M., To K.K., Li Q, & Fu L. (2015). Effect of ceritinib (LDK378) on enhancement of chemotherapeutic agents in ABCB1 and ABCG2 overexpressing cells in vitro and in vivo. Oncotarget, 6(42), 44643-44659.

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Anti-Cancer Drug Cytotoxicity Assay

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Afatinib, ceritinib, gefitinib and osimertinib were purchased from Selleck Chemicals. Carboplatin, cisplatin, docetaxel and paclitaxel were purchased from Sigma-Aldrich; Merck KGaA. Pemetrexed was purchased from Toronto Research Chemicals (37 (link),38 (link)). A549 and A549/KL-1 cells (2,000 cells/well) were seeded into 96-well plates, and the drugs were added in increasing concentrations (0, 0.01, 0.1, 1, 10 and 100 µM) at room temperature. The half maximal inhibitory concentration (IC₅₀) value was defined as the concentration of an anticancer agent required to reduce growth by 50%. The corrected absorbance of each sample compared with that of the untreated control was calculated by MTT assay using the absorbance wavelength at 570 nm.

Takegahara K., Usuda J., Inoue T., Sonokawa T., Matsui T, & Matsumoto M. (2021). Antiaging gene Klotho regulates epithelial-mesenchymal transition and increases sensitivity to pemetrexed by inducing lipocalin-2 expression. Oncology Letters, 21(5), 418.

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Inhibitors of ALK and Mps1 Kinases

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The inhibitors of ALK tyrosine kinase, crizotinib was obtained from LC Laboratories (Woburn, MA, USA), and ceritinib and TAE684 from Selleck Chemicals (Houston, TX, USA). The Mps1 inhibitor AZ3146 was obtained from Adooq Bioscience (Irvine, CA, USA), and the reversible CDK1 inhibitor RO-3306 from Selleck Chemicals. These inhibitors were dissolved in DMSO (Nacalai Tesque, Kyoto, Japan).

Munira S., Yuki R., Saito Y, & Nakayama Y. (2020). ALK Inhibitors-Induced M Phase Delay Contributes to the Suppression of Cell Proliferation. Cancers, 12(4), 1054.

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