The Role Of PI3K/Akt/mTOR Pathway In Cadmium-induced Autophagy Of BRL 3A Cells

Autophagy is a stress-induced catabolic process involving the lysosome, which degrades long-lived proteins and cellular organelles. In addition, autophagy is involved in normal development, senescence, lifespan extension, immunity and defense against microbial invasion, and plays a key role in maintaining cellular energy homeostasis under conditions of stress. Cadmium (Cd) is a seriously environmental and occupational toxicant, targeting multiple organ systems especially liver. Some researchers showed that cadmium induces autophagy. However, the mechanisms underlying cadmium-induced autophagy were not yet completely understood. In the current experiment, BRL 3A was selected as a model. The present study surveyed whether autophagy is involved in the cytotoxicity of cadmium, and whether PI3K/Akt/mTOR pathway is involved in cadmium-induced autophagy in BRL 3A cells, which will provide theoretic evidences for future revealing the mechanism of liver damage induced by cadmium.1. Discovery of autophagy induced by cadmium in BRL 3 A cellsTo investigate the effects of cadmium on autophagy, BRL 3A cells were treated with different concentrations of cadmium (0,2,4,8,16μmol/L) for 4h. Microtubule-associated protein 1 light chain 3 (LC3), a marker of autophagy, was examined by western blot. After treatment 8μmol/L cadmium for 4 h, the presence of LC3 puncta were observed by immunofluorescence staining; autolysosomes were surveyed by MDC staining. The results showed that compared with the control group, as the concentration of cadmium increased, the levels of LC3-Ⅱ proteins significantly increased and then decreased with the highest level in 8μmol/L (P<0.05 or P<0.01). The results of immunofluorescence and MDC staining showed that the number of autophagosomes significantly increased. These results indicated that cadmium increased the level of autophagy in BRL 3A cells.2. The role of autophagy in cytotoxicity of cadmium in BRL 3A cellsTo investigate whether autophagy is involved in the cytotoxicity of cadmium, the effects of different levels of autophagy on cell viability were examined by MTT method after rapamycin (50nmol/L) and chloroquine (5μmol/L) induce an increased and decreased level of autophagy. Compared with the cadmium group, the increased level of autophagy could significantly improve the viability of cells (P<0.05), the decreased level of autophagy could reduce the viability of cells with significant difference (P<0.05). The results demonstrated that autophagy serves an important protective role against cadmium-induced cytotoxicity.3. The role of PI3K/Akt/mTOR pathway in cadmium-induced autophagyTo investigate whether PI3K/Akt/mTOR pathway is involved in cadmium-induced autophagy in BRL 3A cells, the phosphorylation levels of some key proteins in PI3K/Akt/mTOR pathway were examined by western blot after treatment with different concentrations of cadmium (0,2,4,8μmol/L) for 4h. After treatment with LY294002 alone or in combine with cadmium (8μmol/L) for 4h, the phosphorylation level of Akt and the levels of LC3-II proteins were also examined by western blot, and autolysosomes were surveyed by MDC staining. Compared with the control group, as the concentration of cadmium increased, the phosphorylation levels of PI3K, Ak, mTOR and P70S6K proteins gradually decreased with the lowest level in 8μmol/L (P<0.05 or P<0.01). Compared with the cadmium group, co-treatment with LY294002 and Cd significantly reduced the phosphorylation level of Akt and increased the levels of LC3-Ⅱ proteins (P<0.05 or P<0.01). The result of MDC staining showed that the number of autophagosomes significantly increased in LY294002 + Cd group. It was suggested that the dependence of autophagy in BRL 3A cells on the inhibition of PI3K/Akt/mTOR pathway.