Functional Autophagic Flux Regulates AgNP Uptake And The Internalized Nanoparticles Determine Tumor Cell Fate By Temporally Regulating Flux

BACKGROUND: Silver nanoparticles (AgNPs) are known to induce the conserved, cellular, homeostatic process- autophagy in tumor cells. Previous studies primarily focus on the pro-survival role of autophagy post AgNP exposure in tumor cells, but seldom on its role in AgNP uptake, or on the functional significance of autophagy temporal dynamics. Our study sheds more light on the extensive crosstalk that exists between AgNP and autophagy, which can be critical to the improvement of AgNP-induced therapeutic effects. METHODS: β-cyclodextrin (β-CD) coated AgNPs of two different sizes were synthesized by nucleation method and characterized by transmission electron microscopy. Fluorescence microscopy and flow cytometry were used to probe intracellular uptake of AgNPs. Endocytic mechanism of AgNPs was classically analyzed through use of various endocytosis inhibitors. Autophagy was evaluated by immunoblot and fluorescence microscopy. Additionally, immunoblot was performed to monitor Janus Kinase (JNK) signalling, ubiquitination of proteins, expression of endo-lysosomal and apoptotic markers in correlation to AgNP-induced autophagy. RESULTS: The intra-cellular route of entry for the small NPs (~9 nm; ss-AgNPs) was different than the large NPs (~19 nm; ls-AgNPs) studied. However, irrespective of their unique route of entry an inhibition of autophagic flux by chloroquine (CQ) reduced uptake of both the AgNPs. In contrary, rapamycin (Rapa), an autophagy inducer enhanced it. Importantly, JNK activation was required for autophagy induction and AgNP uptake. Furthermore, effect of AgNPs on autophagy showed temporal dependency. An enhanced autophagic flux was noted at early time points; however, prolonged exposure resulted in inhibition of flux marked by increase in Rab7, LC3B-II and p62 proteins. Inhibition of flux was associated with lysosomal dysfunction, decreased LAMP1 expression and an increased accumulation of ubiquitinated (Ub) proteins. This resulted in heightened reactive oxygen species (ROS) and consequent cytotoxicity. CONCLUSION: In this study, we observed that a functional autophagic flux aids AgNP uptake, but AgNPs in turn, overtime, inhibits flux and endo-lysosomal function. We provide critical, novel insights into crosstalk between AgNP and autophagy which can be vital to future AgNP-based therapy development.