Tetrandrine blocks autophagic flux and induces apoptosis via energetic impairment in cancer cells

W Qiu,Y Ren,M Su,W He,F Xie,J Ai,J Zhang,Y Gong,R Guan,Y Guo

doi:10.1038/cddis.2014.84

Abstract

Lysosomes are acidic organelles that have a crucial role in degrading intracellular macromolecules and organelles during the final stage of autophagy. Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid, was reported as an autophagy activator. Here, in contrast with previous studies, we show that Tet is a potent lysosomal deacidification agent and is able to block autophagic flux in the degradation stage. Single-agent Tet induces significant apoptosis both in vitro and in xenograft models. In the presence of Tet, apoptosis was preceded by a robust accumulation of autophagosomes and an increased level of microtubule-associated protein 1 light chain 3, type II (LC3-II). However, Tet increased the level of sequestosome 1 and decreased the turnover of LC3, indicating the blockade of autophagic flux in the degradation stage. As blockade of autophagic flux decreases the recycling of cellular fuels, Tet reduces the uptake of glucose in cancer cells. These effects lead to insufficient substrates for tricarboxylic acid (TCA) cycle and impaired oxidative phosphorylation. Blunting autophagosome formation using 3-methyladenine or genetic knockdown of Beclin-1 failed to rescue cells upon Tet treatment. By contrast, addition of methyl pyruvate to supplement TCA substrates protected Tet-treated tumor cells. These results demonstrate that energetic impairment is required in Tet-induced apoptosis. Tet, as a potent lysosomal inhibitor, is translatable to the treatment of malignant tumor patients.

Highlights

Tetrandrine (Tet) is a natural product isolated from the root of Stephania tetrandra S
In order to compare whether Tet has same effects on different cell lines, Cell Counting Kit-8 (CCK-8) assays were conducted using three human cancer cell lines: PC-3, 786-O, and PANC-1
The inhibitory concentration 50 of Tet in the 11 established tumor cell lines, determined after 48 h of incubation with the drug, ranged from 2.62 to 8.03 mM (Figure 1b). These results suggested that Tet had broad antitumor effects in various cell lines

Summary

Introduction

Tetrandrine (Tet) is a natural product isolated from the root of Stephania tetrandra S. The structure of Tet contains two nitrogen atoms, which make the solution of it alkaline Most weak bases, such as chloroquine (CQ) and its derivatives, can be protonated and accumulates in lysosomes, resulting in the deacidification of their lumens.[15,16] The lumen of the lysosome contains B60 types of soluble hydrolases.[17] These enzymes are active in acidic environments and, as such, are responsible for the lysosomal hydrolysis of many different substrates, including during autophagy.[17] Autophagic flux can be blocked when the acidity of lysosome is reduced by CQ or other weak bases. We specially focus on the effects of Tet on lysosomal acidification and autophagic flux in tumor cells. We found that Tet is a potent lysosomal deacidification agent that blocks autophagic flux. Our results provide evidence for the clinical use of Tet as a lysosome inhibitor in treating malignant tumors

Methods

Results

Conclusion