AB77. Tetrandrine blocks autophagic flux by preventing lysosomal acidification in 786-O cells

Introduction and objectives: Autophagy is a highly regulated catabolic process that in cancer cells maintains metabolism and energy balances, both of which protect malignant cells from stress. Lysosomes are acidic organelles that play a crucial role in degrading intracellular macromolecules and organelles during the final stage of autophagy. Tetrandrine (Tet) is a natural product isolated from the root of Stephania tetrandra S. Moore. Recently, Tet was reported to trigger apoptosis and cell cycle arrest in human renal carcinoma cells. However, the detailed antitumor mechanism of Tet remains unclear. Here, we show that Tet is a potent and rapid lysosomal deacidification agent and single-agent Tet exhibits powerful antitumor activity in vitro. Methods: 786-O cells were used to evaluate the anti-tumor effects of Tet. The lipidation levels of microtubuleassociated protein 1 light chain 3 (LC3) and expression levels of sequestosome 1 (p62) were detected by Western blotting. Apoptosis, mitochondrial membrane potential (ΔΨm) were determined using flow cytometry. LC3 turnover assays were performed to assess autophagy flux. Oxidative phosphorylation (OXPHOS) was measured using the Seahorse XF24 analyzer. Lysosomal pH was detected using LysoSensor DND-160. Results: Tet exhibits powerful antitumor activity in vitro. Tet induces significant apoptosis and decrease of ΔΨm in 786-O cells. In the presence of Tet, apoptosis was preceded by a robust accumulation of autophagosomes and increased level of LC3-II. However, Tet simultaneously increased the level of p62 and decreased LC3 turnover, indicating the blockade of autophagic flux in the degradation stage. Moreover, the blockade of autophagy is mainly caused by its effect on neutralizing lysosomal acidity. Tetmediated inhibition of autophagy leads to insufficient substrates for tricarboxylic acid (TCA) cycle, impaired oxidative phosphorylation, reduction of ATP and activation of AMPKα . Blunting autophagosome formation with 3-methyladenine or genetically knocking down Beclin-1 did not rescue cells upon Tet treatment. By contrast, the addition of methyl pyruvate to supplement TCA substrates partially protected Tet-treated 786-O cells. Taken together, our results suggest that Tet blocks autophagic flux by promoting lysosomal deacidification, leading to energy depletion and caspase-mediated apoptosis. Conclusions: Our research recommends the evaluation of Tet as a potential cancer chemotherapeutic drug, based on its demonstrated ability to inhibit autophagy.