Cell Adhesion and ROS Production of Synthetic GL-9 Peptide in A549 Lung Carcinoma Cells

GL-9 is a synthetic peptide with GASRMWYFL sequence, which was used to investigate invitro attachment and its effect on A549 cell line (epithelial human lung cancer). To evaluatethe adhesion of the peptide to desired cells, cell attachment technique was used. In thisexperiment, peptide at 12, 50ïg/ml were added to 96-well plates and stored overnight at 4ºC. Then a certain amount of RPMI-1640 2% and A549cells were added to the plate. After 1h, the plates were washed with PBS(0.1%) and the attached cells were stained with crystalviolet(0.5x) and photographed with optical microscope. Finally, by the use of aceticacid(10%), the crystal violet was washed and the cells absorption was recorded at 595nm. Toinvestigate releasing of reactive oxygen species(ROS), the ROS measurement method wasused. In this attempt, a certain amount of cells were cultured in 6 well plates with peptideconcentrations of 12, and 50 ïg/ml for 24 and 48 h. Then DCF-DA probe(10ïM) was added tothe culture and the cells were incubated for 30 minutes, after washing with PBS(0.1%), theflorescence emission was checked out through a flow cytometer. Cell attachment in controlsamples was 28.8%; while,the cell attachment in treated cells varied from 102% to 103%representing the binding affinity of the peptide to A549 cell line. Generally, increased amountof ROS secretion is related with Caspase activation and cell death via apoptosis. Treatmentof A549 cells with GL-9 at different concentrations and time in the presence of DCF-DA probeinduced ROS secretion in time-dependent manner from 13.2% in 24h to 73.2% in 48hcompared with 0.6% in control. The present study showed the induced adhesion ability andcytotoxic effect of synthetic peptide GL-9 on A549.

Keywords:

A549 cell line , GL-9 Synthetic Peptide , Cell attachment , ROS measurement , cytotoxic Effect