Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for 85% of all lung carcinomas

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for 85% of all lung carcinomas. M) diminished c-Met levels in H441 cells, while neither a low nor a high concentration (20 M) altered c-Met levels in A549 and H460 cells. A higher concentration of MG-132 (5 M) was required for decreasing c-Met levels in H1299 cells. Furthermore, MG-132 induced cell death in all four cell types. Among all the four cell lines, H441 cells expressed higher levels of c-Met and appeared to be the most susceptible to MG-132. MG-132 decreased c-Met mRNA levels in both H1299 and H441 cells. ONX 0914 reduced c-Met levels in H460, H1299, and H441 cells but not in A549 cells. c-Met levels were decreased the most in H441 cells treated with ONX 0914. ONX 0914 did not alter cell viability in H441; however, it did induce cell death among H460, A549, and H1299 cells. This study reveals that different proteasome inhibitors produce varied inhibitory effects in NSCLS cell lines. gene contributes to c-Met dysregulation, which promotes tumor HPGDS inhibitor 1 angiogenesis, tumor cell invasion, and metastasis8,9. c-Met dysfunction is correlated with poor clinical outcomes in NSCLC patients10C12. Therefore, c-Met has been studied extensively to elucidate its role in NSCLC. Another critical mechanism that affects NSCLC by regulating a wide range of intracellular signals is the ubiquitinCproteasome system13,14. Inhibition of proteasomes can affect cancer cells in multiple ways including inhibiting proliferation, inducing autophagy and apoptosis, and diminishing metastasis15,16. Proteasome inhibition has been used as a novel therapeutic approach in NSCLC; however, the effects of different proteasome inhibitors on NSCLC cells have not been fully investigated. The aim of this study is to determine a precise strategy for treating NSCLC by targeting c-Met with different proteasome inhibitors. MATERIALS AND METHODS Cell Culture and Reagent H1299, H441, A549, and H460 [American Type Culture Collection (ATCC), Manassas, VA, USA] were cultured in RPMI-1640 medium that was supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere of 5% CO2 and 95% air. C-Met, PARP, and cleaved caspase 3 antibodies were from Cell Signaling Technology (Danvers, MA, USA). p53 (DO-1) antibody was from Santa Cruz Biotechnology (Santa HPGDS inhibitor 1 Cruz, CA, USA). V5 antibody was from Invitrogen (Grand Island, NY, USA). -Actin antibody was from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase-conjugated goat anti-mouse secondary antibodies were obtained from Bio-Rad (Hercules, CA, USA). Goat anti-rabbit IgG (H?+?L) secondary antibody was from Invitrogen (Waltham, MA, USA). MG-132 was P21 from Calbiochem (San Diego, CA, USA). Bortezomib was from Cayman Chemical (Ann Arbor, MI, USA). ONX 0914 was purchased from ApexBio (Houston, TX, USA). Actinomycin D and cycloheximide were from Sigma-Aldrich. All of the materials used in the experiments are in the highest grades and are commercially available. Transfection of Plasmids Into NSCLC Cells Human c-Met cDNA was inserted into pcDNA3.1D/His-V5 TOPO vector. NSCLS cells grown on six-well plates (70C80% confluence) were transfected with V5-tagged c-Met (c-Met-V5) plasmids using Genjet? In Vitro DNA Transfection Reagent (SignaGen Laboratories, Inc., HPGDS inhibitor 1 Frederick, MD, USA) according to the transfection protocol, and complete media was changed after 4 h. Twenty-four hours after transfection, cells were challenged with MG-132, bortezomib, or ONX 0914 at different concentrations for an additional 48 h. Cell Lysis and Western Blot Analysis After the indicated treatments, cells were washed with cold PBS and collected in cell lysis buffer, which contains 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 5 mM -glycerophosphate, 1 mM MgCl2, 1% Triton X-100, 1 mM sodium orthovanadate, 10 g/ml of protease inhibitors, 1 g/ml of aprotinin, 1 g/ml of leupeptin, and 1 g/ml of pepstatin. The cell lysates were then sonicated on ice for 12 s, followed by centrifugation at 4C at 10,000 rotations/min for 5 min. Protein concentrations HPGDS inhibitor 1 of the samples were then determined with a Bio-Rad Protein Assay kit (Bio-Rad). Samples.

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