Temozolomide, sirolimus and chloroquine is a new therapeutic combination that synergizes to disrupt lysosomal function and cholesterol homeostasis in GBM cells.

Sanford P.C Hsu,Yu-Shan Wang,Hsin-Ell Wang,John S Kuo,Cheng-Chung Huang,Kwan-Hwa Chi,Yi-Chun Huang,Hsin-Chien Chiang,Mau-Shin Chi,Minesh P Mehta

doi:10.18632/oncotarget.23855

Abstract

Glioblastoma (GBM) cells are characterized by high phagocytosis, lipogenesis, exocytosis activities, low autophagy capacity and high lysosomal demand are necessary for survival and invasion. The lysosome stands at the cross roads of lipid biosynthesis, transporting, sorting between exogenous and endogenous cholesterol. We hypothesized that three already approved drugs, the autophagy inducer, sirolimus (rapamycin, Rapa), the autophagy inhibitor, chloroquine (CQ), and DNA alkylating chemotherapy, temozolomide (TMZ) could synergize against GBM. This repurposed triple therapy combination induced GBM apoptosis in vitro and inhibited GBM xenograft growth in vivo. Cytotoxicity is caused by induction of lysosomal membrane permeabilization and release of hydrolases, and may be rescued by cholesterol supplementation. Triple treatment inhibits lysosomal function, prevents cholesterol extraction from low density lipoprotein (LDL), and causes clumping of lysosome associated membrane protein-1 (LAMP-1) and lipid droplets (LD) accumulation. Co-treatment of the cell lines with inhibitor of caspases and cathepsin B only partially reverse of cytotoxicities, while N-acetyl cysteine (NAC) can be more effective. A combination of reactive oxygen species (ROS) generation from cholesterol depletion are the early event of underling mechanism. Cholesterol repletion abolished the ROS production and reversed the cytotoxicity from QRT treatment. The shortage of free cholesterol destabilizes lysosomal membranes converting aborted autophagy to apoptosis through either direct mitochondria damage or cathepsin B release. This promising anti-GBM triple therapy combination severely decreases mitochondrial function, induces lysosome-dependent apoptotic cell death, and is now poised for further clinical testing and validation.

Highlights

GBM is an aggressive malignancy with high mortality, and relative resistance to radiation and other treatments [1, 2]
This study suggests that GBM is susceptible to the induction of cholesterol depletion related to the lysosome dysfunction
In comparison to mTOR inhibitor (Rapa) treatment alone, the combined Rapa and lysosome inhibitor (CQ) with or without TMZ therapy significantly increased apoptotic cell death through mitochondria damage before cathepsin released from lysosome in one type of GBM cells (GBM8401), while the addition of TMZ decidedly increased apoptotic cell death through cathepsin induced cell death in other type of cells (U87G)

Summary

Introduction

GBM is an aggressive malignancy with high mortality, and relative resistance to radiation and other treatments [1, 2]. Pre-clinical data suggest that this failure may be due to tumor metabolic adaptation toward anaerobic metabolism, and increased tumor cell invasiveness after anti-angiogenic treatment [4, 5]. These represent examples of adaptive stress-response by the tumor and therapies that induce and amplify tumor stress response, such as autophagy, unfolded protein response (UPR) and cancer metabolism, represent potential therapeutic strategies. GBM is characterized by exaggerated lipogenesis, enhanced LDL cholesterol uptake, high phagocytosis and micro-vesicle exocytosis activities, and depends very much on cholesterol homeostasis for constant membrane changes. We try to disturb the Achilles’ heel with autophagy modulation

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Results

Conclusion