Topotecan

Human mesothelioma, MSTO-211H and NCI-H28 cells, were purchased from American Type Culture Collection (Manassas, VA, USA), and EHMES-1, EHMES-10 and JMN-1B cells were provided by Dr Hamada (Ehime University, Ehime, Japan). ZOL were purchased from Novartis (Basel, Switzerland), and FOH, GGOH, GGTI-298, ETO, topotecan and clodronate were from Sigma-Aldrich (St Louis, MO, USA). C3 transferase and NSC23766 were from Cytoskeleton (Denver, CO, USA) and Merck Millipore (Billerica, MA, USA), respectively. NE10790 were provided by Dr Ebetino FH (Warner Chilcott, Dundalk, Ireland). We purchased siRNA duplex targeting Cdc42 and non-specific control siRNA from Invitrogen (Carlsbad, CA, USA). Transfection of cells with the siRNA was conducted with Lipofectamine RNAiMAX (Invitrogen).

[³H]-Paclitaxel (23 Ci/mmol) was purchased from Moravek Biochemicals, Inc. (Brea, CA). Paclitaxel, doxorubicin, vincristine, cisplatin, topotecan, verapamil, Triton X-100, paraformaldehyde, DMSO, MTT were obtained from Sigma Chemical Co. (St. Louis, MO). The monoclonal antibody C219 (against ABCB1), sc-47778 (against β-Actin) and the secondary horseradish peroxidase-labeled rabbit anti-mouse IgG were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). The Alexa flour 488-conjugated goat anti-mouse IgG was purchased from Molecular Probes (Eugene, OR). WBC Diluting Fluid and Platelet Diluent were purchased from Eng Scientific Inc. (Clifton, NJ). High sensitivity mouse cardiac troponin-I ELISA KIT was purchased from Life Diagnostics, Inc. (West Chester, PA). SAView^@ (mouse/rabbit-HRP, DAB) IHC kit was obtained from Enzo Life Sciences, Inc. (Farmingdale, NY). OPSYS microplate reader was purchased from Dynex Technologies (Chantilly, VA).

Primary squamous carcinoma cell lines (University of Turku-Squamous cell carcinoma (UT-SCC)) have been established previously in the Grenman lab, Turku University, Finland and FaDu, Cal33, SAS, HSC4 and XF354 were from ATCC/LGC (Wesel, Germany). Cells were cultivated in DMEM supplemented with 10% FCS, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C at 10% CO₂. For the determination of cellular survival cells were treated with up to 1.5 μg/mL to mitomycin C (Medac, Wedel, Germany) or 1 µM topotecan (Sigma, St. Louis, MS, USA,) for 6 h or 24 h and irradiation experiments were performed using radiation doses up to 6 Gy at a dose rate of 1.2 Gy/min using a Gulmay X-ray machine (GULMAY Medical, Byfleet, UK) or UV 5mJ (BioRad Laboratories, Berkeley, CA, USA). All treatments were performed in 37 °C and 10% CO₂ atmosphere. CHK1 inhibition was achieved by using siGENOME human CHEK1 siRNA (Horizon Discovery, Cambridge, UK) up to 150 nM for 24 h or the small molecule inhibitor MK8776 (Selleck Chemicals, Houston, TX, USA) at 2 µM for 2 h.

The following antibodies were used for Western blotting or immunoprecipitation: anti-SF3B1 (Bethyl Laboratories, A300-996A-T, MBL International, D221-3), rabbit anti-cleaved Notch1 (Cell Signaling Technology, 4147S), rabbit anti–β-actin (Cell Signaling Technology, 4967S), rabbit anti-USP7 (Bethyl Laboratories, A300-033A), rabbit anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Cell Signaling Technology, 2118S), rabbit anti-CHEK2 (Bethyl Laboratories, A300-618A), mouse immunoglobulin G2b (IgG2b) (MBL International, M077-3), P5091 (Selleck Chemicals, S7132), PR619 (Sigma-Aldrich, SML0430), b-AP15 (Selleck Chemicals, S4920), ML323 (Sigma-Aldrich, SML1177), BML277 (MedChem Express, HY-13946), BML277 (Sigma-Aldrich, C3742, in vivo), SCH900776 (MedChem Express, HY-15532), camptothecin (Selleck Chemicals, S1288), flavopiridol (Sigma-Aldrich, F3055), topotecan (Sigma-Aldrich, T2705), etoposide (Sigma-Aldrich, E1383), mitoxantrone (European pharmacopoeia reference standard, M2305000), doxorubicin (Sigma-Aldrich, 44583), and E7107 and H3B-8800 (H3 Biomedicine).

Anticancer drugs and positive reversal agents: Venetoclax was purchased from ChemieTek (Indianapolis, IN, USA). Paclitaxel, mitoxantrone, topotecan, SN-38, verapamil, KO143, and cisplatin were purchased from Sigma Co (St. Louis, MO, USA). Cell culture: Bovine serum (BS), fetal bovine serum (FBS), Dulbecco’s modified eagle’s medium (DMEM) and trypsin (0.25%) were purchased from Corning Inc. (Corning, NY, USA). Cell viability assay: dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Co. Immunoblotting: Human monoclonal antibodies for ABCG2 and GAPDH were purchased from Millipore (Billerica, MA, USA). Alexa Fluor 488 conjugated secondary antibody was purchased from Thermo Fisher Scientific Inc (Rockford, IL, USA). Triton X-100, 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma Co. Other chemicals: [³H]-labeled mitoxantrone (2.5 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA, USA). All other chemicals were purchased from Sigma Co.