Crizotinib pf 02341066

Manufactured by Selleck Chemicals

Sourced in United States

Crizotinib/PF-02341066 is a tyrosine kinase inhibitor used in laboratory research. It is a chemical compound with the molecular formula C21H22Cl2FN5O.

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PF-02341066 Crizotinib

9 protocols using crizotinib pf 02341066

Evaluating Combinatorial Drug Therapy for NSCLC

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PC9-1 cells were plated in duplicate in six-well plates with erlotinib-free media at 40,000 cells per well (Supplementary Fig. 2b). Media was changed to drug-containing media (described below) after allowing cells to adhere overnight. Plates were treated with various drugs either singly in erlotinib-free media or in combination with erlotinib media. The following drugs were used: 0.1 μM WZ8040 (Selleckchem, Cat.#S1179), 0.1 μM WZ3146 (Selleckchem, Cat.#S1170), 0.1 μM SGX-523 (Selleckchem, Cat.#S1112), 0.0316 μM Crizotinib (PF-02341066; Selleckchem, Cat.#S1068) and 0.02 μM trichostatin A (Selleckchem, Cat.#S1045). One pair of wells was used as controls with no drug in either erlotinib-free media or erlotinib media. The cells were grown for 14 days, with media being changed every 3 days. Cells were imaged every 3 days at × 10 magnification with phase-contrast on a Nikon Ti Eclipse.

Ramirez M., Rajaram S., Steininger R.J., Osipchuk D., Roth M.A., Morinishi L.S., Evans L., Ji W., Hsu C.H., Thurley K., Wei S., Zhou A., Koduru P.R., Posner B.A., Wu L.F, & Altschuler S.J. (2016). Diverse drug-resistance mechanisms can emerge from drug-tolerant cancer persister cells. Nature Communications, 7, 10690.

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Inhibitor Assays on MHCC97-H and COS7 Cells

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MHCC97-H cells were obtained from the Liver Cancer Institute and Zhongshan Hospital of Fudan University, Shanghai. COS7 cells are a monkey kidney fibroblast-like cell line obtained from Dr. John Kuriyan at UC-Berkeley. COS7 and MHCC97-H cells were cultured in DMEM supplemented with 10% FBS and streptomycin/penicillin. Inhibitor assays were treated for 6–24 hours in serum-free media containing capmatinib/INCB28060 (Selleckchem), crizotinib/PF-02341066 (Selleckchem), gefitinib/ZD1839 (Selleckchem), or lapatinib (Selleckchem).

Frazier N.M., Brand T., Gordan J.D., Grandis J, & Jura N. (2018). Overexpression-mediated activation of MET in the Golgi promotes HER3/ERBB3 phosphorylation. Oncogene, 38(11), 1936-1950.

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Kinase Inhibitors in Cancer Research

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Kinase inhibitors used were AT9283 (cat. no. S1134, Selleckchem), crizotinib (PF-02341066) (cat. no. S1068, Selleckchem), ponatinib (AP24534) (cat. no. S1490, Selleckchem), bosutinib (SKI-606) (cat. no. S1014, Selleckchem), BRITE-0600690 (BRITE-690, inactive compound), and BRITE-0600719 (BRITE-719, compound 1s, active compound)^{31 (link)}.

Bok S., Shin D.Y., Yallowitz A.R., Eiseman M., Cung M., Xu R., Li N., Sun J., Williams A.L., Scott J.E., Su B., Shim J.H, & Greenblatt M.B. (2020). MEKK2 mediates aberrant ERK activation in neurofibromatosis type I. Nature Communications, 11, 5704.

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Multiplex Cytokine Profiling of Choriocarcinoma

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Using a Luminex® xMAP® platform in a magnetic bead format, we simultaneously studied the following analytes from the culture supernatants of MUG-Chor1 and UM-Chor1 cells: epidermal growth factor (EGF), tumour necrosis factor alpha (TNFα), vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF2). No cross-reactivity was noted between the antibodies for an analyte or between any of the other analytes in this panel. Treatment was performed with 4 μM crizotinib (PF-02341066; SelleckChem, Houston, TX, USA), 0.16 μM afatinib (BIBW2992; SelleckChem), or with a simultaneous co-dosing of crizotinib and afatinib for 72 h. For detection, we used a commercially available Procarta Plex (Thermo Fisher, Waltham, MA, USA) on a Bioplex200 system (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturers´ instructions. Measurement of mean fluorescence intensities was performed using Bio-Plex Manager software, version 4.1 (Bio-Rad Laboratories, Hercules, CA, USA) with a 5-parametric curve fitting.

Scheipl S., Barnard M., Lohberger B., Zettl R., Brcic I., Liegl-Atzwanger B., Rinner B., Meindl C, & Fröhlich E. (2021). Drug combination screening as a translational approach toward an improved drug therapy for chordoma. Cellular Oncology (Dordrecht), 44(6), 1231-1242.

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Inhibitor Assays on MHCC97-H and COS7 Cells

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Frazier N.M., Brand T., Gordan J.D., Grandis J, & Jura N. (2018). Overexpression-mediated activation of MET in the Golgi promotes HER3/ERBB3 phosphorylation. Oncogene, 38(11), 1936-1950.

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Comprehensive Western Blot Protocol for MET Signaling

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All antibodies used for Western blot analysis were diluted in 5% v/v BSA in TBS-T prior to use. Primary antibodies were used at 1:1000 dilution, while secondary antibodies were used at 1:10,000 dilution. All antibodies except anti-ERM/ETV5 (Abcam, cat #ab102010, Cambridge, United Kingdom) and anti-DUSP6 (Santa Cruz Biotechnology, cat #sc-37707, Dallas, TX, USA) were purchased from Cell Signaling Technology (Danvers, MA, USA): MET (8198S), p-MET Tyr1234 (3077S), p-MET Tyr1349 (3121S), AKT (4691S)), p-AKT Ser473 (4060L), p44/42 MAPK (137F5), p-p44/42 MAPK Thr202/Tyr204 (9101S), MEK1/2 (9122S), p-MEK1/2 Ser217/221 (9121S), STAT3 (12640S), p-STAT3 Y705 (9145S), SRC (2108S), p-SRC Y416 (59548S), Ras (8955S), cMYC (5605S), cFOS (2250S), p-cFOS S32 (5348S), FRA1 (5281S), p-FRA1 S265 (5841S), Vinculin (13901S), β-Actin HRP-linked (12620S), Histone H3 (4499S), Anti-Rabbit IgG HRP linked (7074S), and Anti-Mouse IgG HRP linked (7076S). MET kinase inhibitors crizotinib (PF-02341066), cabozantinib (BMS-907351), and capmatinib (INCB28060) were purchased from SelleckChem (Houston, TX, USA) and dissolved in DMSO (Thermo Fisher Scientific, cat #BP231-100) prior to use. Hepatocyte Growth Factor recombinant human protein (PHG0324) was purchased from Thermo Scientific and reconstituted according to manufacturer’s instruction prior to use.

Lu D., Nagelberg A., Chow J.L., Chen Y.T., Michalchuk Q., Somwar R, & Lockwood W.W. (2022). MET Exon 14 Splice-Site Mutations Preferentially Activate KRAS Signaling to Drive Tumourigenesis. Cancers, 14(6), 1378.

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Evaluating TRK Inhibitor Cytotoxicity

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Cells were seeded (3,000–5,000 cells per well) into laminin or laminin–fibronectin-coated 96-well plates and treated with different TRK inhibitors at concentration ranging from 0 to 20 μmol/L for 8 days. The drugs used were entrectinib (RXDX-101, Selleckchem), crizotinib (PF-02341066, Selleckchem), and milciclib (PHA-848125, Selleckchem). Each assay was performed in three independent biological replicates of three technical replicates each. Cell viability was assessed with CellTiter-Glo using a FLUOstar Omega plate reader (BMG, LABTECH). Data was analyzed and IC₅₀ values were calculated using GraphPad Prism software.

Clarke M., Mackay A., Ismer B., Pickles J.C., Tatevossian R.G., Newman S., Bale T.A., Stoler I., Izquierdo E., Temelso S., Carvalho D.M., Molinari V., Burford A., Howell L., Virasami A., Fairchild A.R., Avery A., Chalker J., Kristiansen M., Haupfear K., Dalton J.D., Orisme W., Wen J., Hubank M., Kurian K.M., Rowe C., Maybury M., Crosier S., Knipstein J., Schüller U., Kordes U., Kram D.E., Snuderl M., Bridges L., Martin A.J., Doey L.J., Al-Sarraj S., Chandler C., Zebian B., Cairns C., Natrajan R., Boult J.K., Robinson S.P., Sill M., Dunkel I.J., Gilheeney S.W., Rosenblum M.K., Hughes D., Proszek P.Z., Macdonald T.J., Preusser M., Haberler C., Slavc I., Packer R., Ng H.K., Caspi S., Popović M., Kotnik B.F., Wood M.D., Baird L., Davare M.A., Solomon D.A., Olsen T.K., Brandal P., Farrell M., Cryan J.B., Capra M., Karremann M., Schittenhelm J., Schuhmann M.U., Ebinger M., Dinjens W.N., Kerl K., Hettmer S., Pietsch T., Andreiuolo F., Driever P.H., Korshunov A., Hiddingh L., Worst B.C., Sturm D., Zuckermann M., Witt O., Bloom T., Mitchell C., Miele E., Colafati G.S., Diomedi-Camassei F., Bailey S., Moore A.S., Hassall T.E., Lowis S.P., Tsoli M., Cowley M.J., Ziegler D.S., Karajannis M.A., Aquilina K., Hargrave D.R., Carceller F., Marshall L.V., von Deimling A., Kramm C.M., Pfister S.M., Sahm F., Baker S.J., Mastronuzzi A., Carai A., Vinci M., Capper D., Popov S., Ellison D.W., Jacques T.S., Jones D.T, & Jones C. (2020). Infant high grade gliomas comprise multiple subgroups characterized by novel targetable gene fusions and favorable outcomes. Cancer discovery, 10(7), 942-963.

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Proteomic Analysis of HCC Cells

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HCC cells were lysed with RIPA peptide lysis buffer (Beyotime Biotechnology, China) containing 1 % protease inhibitors (Thermo Scientific, USA). Equal amounts of proteins were loaded and resolved using 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Antibodies for CD9, CD63, GAPDH, caspase-9, caspase-3, PARP, c-Met, p-Met, AKT, p-Akt, VEGFR2 and p-VEGFR2 were obtained from Cell Signaling Technology (Beverly, MA, USA). The Met inhibitor crizotinib (PF-02341066) and the p-Akt inhibitor MK-2206 2HCl were purchased from Selleck (Selleck Chemicals, China). After incubation with horseradish peroxidase-conjugated secondary antibodies, protein bands were visualized using enhanced chemiluminescence (Millipore, USA).

Qu Z., Wu J., Wu J., Luo D., Jiang C, & Ding Y. (2016). Exosomes derived from HCC cells induce sorafenib resistance in hepatocellular carcinoma both in vivo and in vitro. Journal of Experimental & Clinical Cancer Research : CR, 35, 159.

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Isolation and Maintenance of Murine ERMS and Satellite Cells

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Murine ERMS cells and satellite cells were isolated as previously described (Crepaldi et al., 2007 (link)). ERMS cells were maintained in DMEM supplemented with 10% FBS. Satellite cells were maintained as previously described (Crepaldi et al., 2007 (link)). SU-DHL-1 and TS cells (a subclone of SUP-M2) were kindly provided by R. Chiarle and maintained in RPMI 1640 supplemented with 10% FBS. Methods of characterization from the cell bank include karyotyping and DNA fingerprinting. All cell lines were regularly tested with MycoAlert (Lonza, Walkersville, MD) to ascertain that cells were not infected with mycoplasma. Met inhibitor PHA-665752 was used at 250 nmol/L. Crizotinib (PF-02341066) (Selleckchem, Houston, TX) was used at 250 nmol/L. LY-294002 was used at 10 µmol/L. Selumetinib (AZD6244) (Selleckchem) was used at 1 µmol/L. DMSO was used as a control.

Morena D., Maestro N., Bersani F., Forni P.E., Lingua M.F., Foglizzo V., Šćepanović P., Miretti S., Morotti A., Shern J.F., Khan J., Ala U., Provero P., Sala V., Crepaldi T., Gasparini P., Casanova M., Ferrari A., Sozzi G., Chiarle R., Ponzetto C, & Taulli R. (2016). Hepatocyte Growth Factor-mediated satellite cells niche perturbation promotes development of distinct sarcoma subtypes. eLife, 5, e12116.

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