Chloroquine Induced Autophagy Inhibition Increases The Chemosensitivity Of Nsclc A549Cells To Paclitaxel

Objective:TO study the effect of chloroquine inhibits autophagy combined with pacilitaxel to A549cells of NSCLC,observing the combination of them if does synergistic effect or not.Further detecting autophagy-associated marker, hoping it can provide theoretical evidence to improve the therapeutic effect of NSCLC patient.Methods:Lysosomal changes induced by paclitaxel and chloroquine in A549cells which were assayed by fluorescence staining with Lyso-Tracker Red DND-99, and the viability of A549cells treated with paclitaxel and chloroquine combination was detected by using MTT assay,apoptosis was measured by AnnexinV/PI stanin through flow sytometer. Detected the concentration of autophagy marker-LC3-II by SDS-PAGE,and detected HMGB1intra/ecto-cellular through ELESA.Results:1. Inhibition rate of cell growth:With the action of different concentration of chloroquine and paclitaxel to A549cells in72hours, the survival rate decreases. Comparing to the action of paclitaxel to A549cells, the survival rate decreases obviously to2.61times (P<0.05) with the action of different concentration of chloroquine and paclitaxel.2. HMGB1rate change:With the action of different concentration of paclitaxel to A549cells in6hours or24hours, the concentration of HMGB1in cells decreases along with the increase of paclitaxel concentration and the time. However, the concentration of HMGB1in Cell supernatant tends to increase along with the increase of paclitaxel concentration and the time.3. LC3-Ⅱ rate change:With the action of different concentration of paclitaxel and chloroquine to A549cells in6hour or in24hours, the content of LC3-Ⅱ/β-actin increases obviously as the concentration of paclitaxel and chloroquine and the time increases. The tendency is more obvious when combining both.4. Cytolysosome change:With the action of different concentration of paclitaxel to A549cells in24hours, the fluorescence of cytolysosome strengthens and the quantity of acidic cytolysosome and vacuoles increase when the concentration of paclitaxel increases. It tends to be dose-dependent. When the concentration of chloroquine increases, the fluorescence of cytolysosome weakens and the quantity of acidic cytolysosome increase and the size of the acidic cytolysosome increases with the action of different concentration of chloroquine to A549cells in6hours. With the action of different concentration of paclitaxel and chloroquine to A549cells in24hours, the fluorescence of cytolysosome weakens and the quantity of punctiform acidic cytolysosome decreases. It tends to be dose-dependent. The size of cytolysosome obviously increases. The mergence and breakage occur.5. Apoptosis:With the action of different concentration of paclitaxel to A549cells24hours, the apoptosis rate increases in early stage. It tends to be dose-dependent. The tendency is more obvious when combining with chloroquine.Conclusion:Paclitaxel can make A549cells induce autophagy and increase cells apoptosis. Through chloroquine inhibited the roles of autophagolysosome could further increase inhibition and apoptosis of A549cells.Through the autophagy of A549cells,the concentration of HMGB1increases in ecto-cellular,and decreases in intra-cellular,indicates that the HMGB1acted through cells secretion or released by dead cells,which can predict autophagy.