Because the IGF-1R, Src, and AXL signaling pathways have been implicated in tumor growth and metastasis 22, 23, 54, we compared the effects of single treatment of LL6 and linsitinib (L), dasatinib (D), and bemcentinib (B) combination (LDB) on proliferation and metastatic activities of NSCLC cells 0.05, ** 0.01, and *** 0.001, as determined using the two-tailed Student’s and and and 0.05, ** 0.01, and *** 0.001, as determined using the two-tailed Student’s (Kras 0.05, ** 0.01, and *** 0.001, as determined using the two-tailed Student’s 0.05 and 0.01, as determined using the two-tailed Student’s and and displayed no obvious toxicities and experiments. small molecule kinase inhibitor, providing a new avenue for anticancer therapies. and by concurrently targeting IGF-1R, Src, and AXL. These results suggest that LL6 is usually a useful multitarget SMKI in the treatment of cancer. Materials and Methods Cell culture Human lung cancer cell lines (A549, H1299, H1993, H1944, H226B, H226Br, H460, H522, HCC15, and PC9), a diploid human lung fibroblast cell line (Wi38), and the Lewis lung carcinoma (LLC) cell line were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) or kindly provided by Dr. John V. Heymach (MD Anderson Cancer Center, Houston, Peficitinib (ASP015K, JNJ-54781532) TX, USA). Human retinal pigment epithelial (RPE) cells were kindly provided by Dr. Jeong Hun Kim (College of Medicine, Seoul National University, Seoul, Republic of Korea). HT-22 cells were Peficitinib (ASP015K, JNJ-54781532) provided by Dr. Dong Gyu Jo (College of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea). Lung cancer cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (Welgene, Gyeongsan-si, Republic of Korea). LLC, HT-22, and Wi38 cells were maintained in DMEM supplemented with 10% FBS and antibiotics. RPE cells were maintained in DMEM/F12 supplemented with 10% FBS and antibiotics. NSCLC cell lines with acquired resistance to chemotherapy (cisplatin-resistant H1299/CsR and pemetrexed-resistant H1299/PmR and H460/PmR) and molecular targeted therapy (erlotinib-resistant PC9/ER) were generated by continuous exposure to corresponding anticancer drugs for more than six months. Cells were maintained at 37 C in a humidified atmosphere with 5% CO2. Cell lines were authenticated and validated using the AmpFLSTR identifier PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA; cat. No. 4322288) in 2013 and 2016. Peficitinib (ASP015K, JNJ-54781532) Cells that had been passaged for 6 months after receipt or resuscitation of validated cells were used in this study. Reagents Antibodies against AXL, pIGF-1R (Y1135/6), IGF-1R, pSrc (Y416), Src, pMet (Y1234/5), Met, tubulin, and cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against cleaved PARP were purchased from BD Biosciences (San Jose, CA, USA). Antibodies against pAXL (Y702) were purchased from R&D systems (Minneapolis, MN, USA). Primary antibodies against IGF-1R and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against pIR/IGF-1R (Y1162/3) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from GeneTex (Irvine, CA, USA). Linsitinib, dasatinib, and bemcentinib (R428) were purchased from Selleckchem (Houston, TX, LW-1 antibody USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. The detailed information on used primary and secondary antibodies, including vendor, catalogue number, application, and dilution ratio (or concentration) is usually listed in Table S1. Molecular docking simulations Molecular Peficitinib (ASP015K, JNJ-54781532) docking analysis was implemented using the Surflex-Dock module in Sybyl-X2.2.1 (Tripos Inc, St Louis, MO, USA) with the known crystal structure of AXL complexed with ligands (PDB ID: 5U6B). To prepare the protein, hydrogen was added and energy was minimized using Powell’s method with the Tripos force field until the root-mean-square derivation (RMSD) values were 0.05 Kcal/mol?. Initial optimization and termination of minimization were set as simplex and gradient, respectively. The new ligands were prepared using Chem3D (PerkinElmer, Waltham, MA, USA). Molecular docking simulations were conducted using the Surflex-Dock mode with the extraction of the original ligand. To generate the active site, a threshold of 0.5 ? and bloat of 0 ? were applied based on the original ligand in the crystal structure. Other parameters were used as default. The results of the docking simulation were validated by comparing the redocked structure to the original pose of the ligand. Molecular interactions between proteins and ligands were further analyzed using Discovery Studio 4.0 Visualizer (BIOVIA, San Diego, CA, USA). MTT assay Cells were seeded into 96-well.
