ELM Secretion Inhibition in Lung Cancer

The human non-small cell lung cancer cell line H460 and mouse Lewis lung cancer cell line LLC1 were obtained from ATCC. Cells were grown in DMEM medium with glutamax-I supplemented with 10% (v/v) FBS and Pen/Strep in an atmosphere of 95% air and 5% CO₂ at 37°C. H460 cells were transfected with the plasmid encoding hCD63-GFP, pCT-CD63-GFP (SBI, USA). Stable H460-hCD63-GFP cells were selected and maintained in puromycin-containing medium at 1 µg/ml. Dimethyl amiloride (DMA) (Sigma, MO) was used to inhibit ELM secretion in H460-hCD63-GFP cells [40] (link). Cells were treated with 1 µmol or 10 µmol DMA for 48 h, and the amount of ELMs secreted into medium in the presence of DMA was determined and compared to untreated controls using an acetylcholinesterase activity assay. The trypan blue exclusion assay was used to determine the cytotoxity of DMA using a Beckman Coulter Vi-Cell (Beckman Coulter Inc., CA).