| Cadmium(Cd)is a recognized toxic heavy metal that is widely used in industrial and agricultural production.Recently,the potential effects of Cd exposure on environment and human health have been of great interest.Epidemiological studies and animal experiments reported a positive association between Cd exposure and tumors.Cd is thought to be a risk factor for the cause of organs damage,which including kidney,liver,brain,lung,testis,bone marrow and blood.Cd was considered as I degree carcinogen by IARC in 1993.Some studies have shown that some cancers may be related to the level of autophagy in tumor cells.Autophagy is a process,during which targeted cytoplasmic constituents are isolated from the rest of the cell within a double-membraned vesicle.Autophagy is executed by autophagy-related(ATG)genes.The autophagosome then fuses with a-lysosome and the contents are degraded and recycled.Although studies indicated that cancer and tumor metastasis may be associated w-ith the change of autophagy ability,their involvement in signaling and other molecules involved is not clearly defined.Objective: To investigate the effects of Cd on proliferation,migration and invasion of A549 cells and to clarify if the molecular mechanisms is of ROS-dependent autophagy.Methods: In our study,human lung cancer cells(A549 cells)were used as in vitro model.A549 cells were treated with different concentrations of Cd.MTT was used to determine the proliferated effects on A549 cells of different concentrations of Cd with the following pretreated reagents: 1 m M 3-MA for 2 h,1.5 m M NАC for 2h,or 50 n M Atg4 siRNA duplexes for 12 h.The cellular autophagy level was further measured by examining using fluorescence microscopy.A549 cells were seeded on glass coverslips,images were observed by a Tecnai Spirit electron microscope,the volume of the cellular acidic compartment can be visualized after AO staining,the fluorescent micrographs were taken to detect the autophagy of A549 cells.Transwell was applied to test the ability of A549 cells migration and invasion.We examined the intracellular reactive oxygen species(ROS)with 2 ’,7 ’-dihydro dichlorofluorescein(DCFH).Cell cycle analysis was performed using a flow cytometer.ATG4 gene was interfered by siRNA to determine the role of ATG4 in Cd-induced migration and invasion in A549 cells.Western blot was used to detect the intracellular protein level of Atg4,LC3 protein,Beclin-1 protein,cell cycle related protein Cyclin D1 and cell invasion related protein MMP-9.For statistical analysis,SPSS 17.0 statistical software was used.Results: Cd at a low concentration could induce proliferative effect on the growth of A549 cells.The exposure to Cd caused the content of intracellular ROS increased significantly.We found that the LC3-II protein levels increased significantly in the Cd-treated A549 cells at the concentration of 2μM and 4μM.Cadmium chloride treatmnt increases formation of autophagosomes using electron microscopy in A549 cells.Cyto-ID?Green staining showed that fluorescence intensity of autophagosomes incre-ased in A549 cells.Cd at low concentration induces ROS and autophagy in A549 cells and promotes cell proliferation,migration and invasion.NAC not only can suppress proliferation induced by Cd,but also can reduce the expression of LC3 and Atg4.Autophagy inhibitors suppress autophagy induced by Cd and reduce the ability of migration and invasion in A549 cells.Conclusion: Cd could induce autophagy of A549 cells at low concentration and results in capability improvement of cell proliferation,migration and invasion.We found that reactive oxygen species(ROS)are important mediators in autophagy induced by ATG4. |
