Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218

Po Hu; Hui Li; Wenzhuo Sun; Hongzheng Wang; Xiaoxuan Yu; Yingjie Qing; Zhanyu Wang; Mengyuan Zhu; Jingyan Xu; Qinglong Guo; Hui Hui

doi:10.1016/j.apsb.2021.02.004

Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218

Acta Pharm Sin B. 2021 Oct;11(10):3178-3192. doi: 10.1016/j.apsb.2021.02.004. Epub 2021 Feb 11.

Authors

Po Hu¹, Hui Li¹, Wenzhuo Sun¹, Hongzheng Wang¹, Xiaoxuan Yu^{1

2}, Yingjie Qing¹, Zhanyu Wang¹, Mengyuan Zhu¹, Jingyan Xu³, Qinglong Guo¹, Hui Hui¹

Affiliations

¹ State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing 210009, China.
² Department of Pharmacology, School of Medicine & Holostic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing 210046, China.
³ Department of Hematology, the Affiliated DrumTower Hospital of Nanjing University Medical School, Nanjing 210008, China.

Abstract

The integrity of lysosomes is of vital importance to survival of tumor cells. We demonstrated that LW-218, a synthetic flavonoid, induced rapid lysosomal enlargement accompanied with lysosomal membrane permeabilization in hematological malignancy. LW-218-induced lysosomal damage and lysosome-dependent cell death were mediated by cathepsin D, as the lysosomal damage and cell apoptosis could be suppressed by depletion of cathepsin D or lysosome alkalization agents, which can alter the activity of cathepsins. Lysophagy, was initiated for cell self-rescue after LW-218 treatment and correlated with calcium release and nuclei translocation of transcription factor EB. LW-218 treatment enhanced the expression of autophagy-related genes which could be inhibited by intracellular calcium chelator. Sustained exposure to LW-218 exhausted the lysosomal capacity so as to repress the normal autophagy. LW-218-induced enlargement and damage of lysosomes were triggered by abnormal cholesterol deposition on lysosome membrane which caused by interaction between LW-218 and NPC intracellular cholesterol transporter 1. Moreover, LW-218 inhibited the leukemia cell growth in vivo. Thus, the necessary impact of integral lysosomal function in cell rescue and death were illustrated.

Keywords: AO, acridine orange; ATG, autophagy related; BAF A1, bafilomycin A1; BID, BH3-interacting domain death agonist; CCK8, Cell Counting Kit; CTSB, cathepsin B; CTSD, cathepsin D; CaN, calcineurin; Cathepsin D; Cholesterol; CsA, cyclosporine A; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DCFH-DA, 2,7-dichlorodi-hydrofluorescein diacetate; Dex, dexamethasone; EGTA, ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; FBS, fetal bovine serum; Hematological malignancies; K48, lysine 48; K63, lysine 63; LAMPs, lysosomal-associated membrane proteins; LC3, microtubule-associated protein 1 light chain 3; LCD, lysosome-dependent cell death; LMP, lysosome membrane permeabilization; LW-218; Lysophagy; Lysosomal damage; Lysosomal membrane permeabilization; Lysosome-dependent cell death; NH4Cl, ammonium chloride; NPC, Niemann-Pick type disease C; NPC1, NPC intracellular cholesterol transporter 1; OD, optical density; P62/SQSTM1, sequestosome 1; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; RAB7A, RAS-related protein RAB-7a; ROS, reactive oxygen species; RT-qPCR, real time quantitative PCR; TFEB, transcription factor EB; TRPML1, transient receptor potential mucolipin 1; shRNA, short hairpin RNA.