P22-13 Influence of Rubicon knockdown effect on the process of cell death by silver nanoparticles

Silver nanoparticles have specific properties of cell death induction. There are high expectations for the realization of nanomedication, i.e., attempts to apply nanoparticles to drug discovery for cancer treatment for the purpose of inducing cancer cell death. However, the mechanism of cell death induction has not yet been elucidated. In this study, we examined the effect of RNA interference with Rubicon (Run domain beclin-1-interacting and cysteine-rich domain-containing proteins), known as negative regulators of autophagy, on the induction of cell death. We investigated cell death and its molecular basis of silver nanoparticles using human lung alveolar epithelial adenocarcinoma cell A549, which was Rubicon knocked down by RNA interference. We hypothesized that the maintenance of lysosomal lumen pH and autophagosome/lysosome membrane fusion are both essential events in the process of autophagy progression in cells, and that these processes are the targets of cell death induction by silver nanoparticles. A549 cells were treated with 0–200 µg Ag/mL of citrate-capped 60 nm silver nanoparticles for cell death and autophagy-related factor expression analysis. Three types of knockdown cells were generated by gene transfer of three different Rubicon siRNAs composed of different sequences. 50 µg Ag/mL of Rubicon knockdown cells were treated with 50 µg Ag/mL and analyzed by Western blotting for factors that constitute the autophagy-lysosome system. The factors constituting the autophagy/lysosome system associated with the treatment of AgNPs were also analyzed by western blotting after treatment with a combination of bafilomycin A1 and chloroquine, which induces lysosomal disruption. Using A549 cells, we analyzed cell death induced by silver nanoparticles and found that silver nanoparticles taken into cells accumulate in lysosomes and induce cell death by increasing the pH of the lumen and disrupting the process of autophagy progression. While treatment with silver nanoparticles alone induces cell death by causing disruption of the autophagy-lysosome system, treatment of Rubicon knockdown cells with silver nanoparticles increases cell death. At the same time, no changes were observed in the expression of factors involved in lysosomal function (catapsin B, TFEB, LAMP2) or autophagy (p62, LC3B-II), and no changes in each factor were observed in Rubicon knockdown cells treated with bafilomycin A1 or chloroquine, which caused lysosomal dysfunction. No change in each factor was observed in knockdown cells. No dose-dependent effect of silver nanoparticles on the expression of STX17, one of the SNARE factors, was observed, indicating that its expression was reduced by the Rubicon knockdown effect. Silver nanoparticles disrupt the fusion process between lysosomes and autophagosomes, which is the final step of autophagy, and the reduced expression of SNAREs such as STX17 in Rubicon may be involved as the mechanism.