Perturbation of autophagy by basic lipophilic compounds : correlation of LC3 and p62 abundance employing U2OS-GFP cells

Includes bibliographical references (p. 44-47).Many psychotropic (i.e. anti depressants, psychotics and serotonin selective re-uptake inhibitors) compounds are deemed weakly basic lipophilic/lysosomotropic compounds, with lysosomal trapping being an important mechanism of their distribution. Weakly basic lipophilic compounds accumulate in acidic intracellular organelles (i.e. lysosomes, endosomes, and phagosomes) via pH partitioning, resulting in cytotoxicity. Multiple mechanisms regulate autophagy (digestion of cellular constituents), with the lysosome being the major component of the autophagic pathway. Drugs with specific physiochemical properties promote drug-induced toxicities due to lysosomal accumulation. Previously published data has demonstrated that jointly administered psychotropic drugs can mutually inhibited lysosomal uptake due to being simultaneously trapped. This action can lead to a larger increase in the internal pH of the lysosome altering intra-lysosomal compound concentrations and shifting drug distribution to lysosome poor organs (i.e. heart), leading to cytotoxic effects. We hypothesized that psychotropic agents with lysosomotrophic properties have the potential to disrupt autophagy and potentially lead to toxicity. We examined the link between lysosomal accumulation and autophagic dysfunction in osteosarcoma cells (U2OS), by examining the effects of eleven weakly basic lipophilic compounds known to accumulate in lysosomes. Their affects on autophagy were determined by using specific autophagy markers Microtubule Associated Protein 1 Light Chain 3 (LC3), Sequestosome 1 (p62) and p70[superscrip S6K]. Microtubule Associated Protein 1 Light Chain 3 and p62 were employed to induce autophagy and inhibit or perturb autophagolysosomal degradation. To gain greater insight regarding the pathway regulating autophagy, p70[suprscript S6K] a main juncture of Mammalian Target of Rapamycin (mTOR) was examined. All treatment compounds resulted in at least a 3-fold increase above solvent control in LC3 immunofluorescence and p62 accumulation. Treatment with lipophilic compounds did not inhibit phosphorylation of p70[superscript S6K] as observed via immunofluorescent staining. The data presented indicates lysosomotropic compounds may contribute to the perturbation of autophag