Molecular Mechanisms Of Aptamer AS1411-Induced Autophagy In Human Lung Adenocarcinoma A549 Cells

Nowadays, lung cancer has the highest morbidity and mortality in the world.Moreover, the treatment for it is relatively poor. Lung cancer has become one of the primary causes of death in China along with the growing of population, aging of people, and the impact of environment pollution, so it is urgently required to develop new drug candidates for treatment of lung cancer with high efficiency and specificity.Aptamers are single-stranded oligonucleic acid molecules. Now, aptamers have become burgeoning cancer drug candidates for their vast advantages, such as high specificity and affinity with target, low immunogenicity, ease of modification and production. The first-in-human anticancer aptamer AS1411 can induce remarkable death for various cancer cell types since it can specific target nucleolin which is biomarker overexpressed on the external surface of many cancer cells. However,most pharmacological studies of AS1411 are focus on the cell cycle and cell apoptosis, there is no article discussing the influence of AS1411 on cell autophagy.This study chooses human lung adenocarcinoma epithelial A549 cells as the research object. We used immunofluorescence assay to verify that the nucleolin can mediate AS1411 internalizing into A549 cells, and investigated the effects of concentration and action period of AS1411 on A549 cells morphology and viability.Then the results of Western Blotting and EGFP-LC3 fluorescent spot assay certified that AS1411 can induce A549 cells autophagy.To study the molecular mechanism of autophagy, we analyzed the activity of mTORC1 which is the central molecule in autophagy signal. Western Blotting showed that AS1411 can inhibit the activity of p70S6K1 and 4E-BP1 which are the two main substrates of mTORC1. The results indicated that AS1411 can inhibit themTORC1 activity, then induce the autophagy. Besides, we also found that Akt can be activated after AS1411 treatment for 72 h, which relates to the block of m TORC1/IRS1 and p70S6K1/ IRS1 negative feedback.Autophagy is a double-edged sword for cancer cells, thus, after studying the mechanism of autophagy, it is essential to investigate the nature of the drug-induced autophagy. Using autophgay inhibitor LY294002, we conducted the CCK-8 assay,the result showed that the drug induced autophagy rescues A549 cells from death.After propidium iodide(PI) stained, A549 cells cycle was examined using flow cytometry, the result showed that AS1411 can increase in G2 phase cells of A549,which is associated with the inhibition of mTORC1 and protein synthesis process.In conclusion, AS1411 can hinder A549 cells protein synthesis process, induce protective autophagy and increase G2 phase cells by mediating mTORC1 inhibition.