Ition to inducing apoptosis, antineoplastic agents also can induce autophagy in cancer cells, and below specific conditions, autophagy and apoptosis appear to interact, either concurrently or sequentially, to positively or negatively determine cell fate (ten, 11). In prior work, we and others have demonstrated that the mechanisms of erlotinib antitumor activity happen by way of inhibition of EGFR phosphorylation, inhibition of downstream signaling, blockade of cell-cycle progression at G1/S, and triggering of mitochondrial-mediated intrinsic apoptotic cascades (12, 13). On the other hand, tiny is known concerning autophagy induction by EGFR inhibitors, and regardless of whether autophagy plays a part in either cell death or acts as a survival mechanism. Here we demonstrate that erlotinib, at a clinically achievable concentration (2 M), strongly induces autophagy in wild variety EGFR NSCLC cells. The degree of autophagy induction by erlotinib was higher in resistant cells than in sensitive cells, suggesting that its induction might constitute a mechanism of cytoprotection. An inhibitor of autophagy, chloroquine, potentiated the tumor inhibitory effects of erlotinib in vitro and in vivo. The information presented offer a strong rationale for exploring combinations of erlotinib with autophagy inhibitors inside the remedy of wild-type EGFR NSCLC tumors.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCell linesMaterials and MethodsChemicals and antibodies Erlotinib (OSI Pharmaceuticals, Melville, NY), was dissolved in DMSO; chloroquine (CQ) was from Sigma-Aldrich (St. Louis, MO); hydroxychloroquine (HCQ) was from Sanofi. Polyclonal antibodies utilised had been: EGFR, p-EGFR (Tyr1068), AKT, p-AKT (Ser475), (Cell Signaling, Beverly, MA); LC3 (Novus Biologicals, La Jolla, CA); monoclonal -actin (Sigma-Aldrich).Human H322, H358, H460, and A549 NSCLC cell lines have been from ATCC (Manassas, VA), and were maintained in RPMI-1640 medium with ten fetal bovine serum, penicillin and streptomycin, at 37 within a humidified atmosphere with five CO2. All four cell lines have wild-type EGFR; H322 and H358 also have wild-type K-ras genes, whilst A549 and H460 have mutant K-ras (G12S and G61H, respectively) (14). Cell proliferation Drug effects on cell proliferation were determined making use of two procedures: cells were seeded in a 96-well plate (103 per well), allowed to attach overnight, and treated with numerous concentrations of erlotinib or CQ alone, or with combinations of each agents having a 1:five molar ratio. In all experiments, medium containing 0.1 DMSO was used as a control. Just after 7 days, cell viability was determined by the MTT assay. The combination index (CI), aJ Thorac Oncol. Author manuscript; obtainable in PMC 2014 June 01.Zou et al.Pagemeasure of drug interaction (i.e. additivity, synergism) was assessed working with CalcuSyn computer software (Biosoft, Ferguson, MO).Benzene-1,2,4,5-tetraol web Alternatively, cells have been seeded in a 6-well plate (103/ well), permitted to attach overnight, and treated with 2 M erlotinib, five M CQ or ten M CQ, alone or in mixture.4-Bromo-3-nitropyridine web Just after ten days, the cells had been fixed and stained with crystal violet.PMID:23600560 Plates were scored for the number of visible colonies 2 mm. Cell-cycle analysis Cells were treated with 2 M erlotinib or ten M CQ, alone or with each other, for 24 h, and were then fixed with cold 75 ethanol overnight and stained with 1 g/ml of propidium iodide and five g/ml RNase I for 3 h. The cell-cycle distribution was assessed by FACScan evaluation (BD Biosciences, San Jose, CA). Apoptosis assay Cells we.