Quinacrine-Induced Autophagy in Ovarian Cancer Triggers Cathepsin-L Mediated Lysosomal/Mitochondrial Membrane Permeabilization and Cell Death.

Abstract

Simple SummaryOvarian cancer (OC) is the most common cause of cancer-related deaths among women worldwide, and its incidence has been increasing and has continued to prove resistant to a variety of therapeutics. This observation is principally disturbing given the amount of money invested in identifying novel therapies for this disease. A comparatively rapid and economical pipeline for identification of novel drugs is drug repurposing. We reported earlier that the antimalarial drug Quinacrine (QC) also has anticancer activity and here we discovered that QC significantly upregulates cathepsin L (CTSL) and promoting autophagic flux in ovarian cancer. QC-induced CTSL activation promotes lysosomal membrane permeability resulting in active CTSL release into the cytosol, which promotes Bid cleavage, mitochondrial membrane permeability, cytochrome-c release and cell death in both in-vitro and in-vivo models. Therefore, QC is a promising candidate for OC treatment.We previously reported that the antimalarial compound quinacrine (QC) induces autophagy in ovarian cancer cells. In the current study, we uncovered that QC significantly upregulates cathepsin L (CTSL) but not cathepsin B and D levels, implicating the specific role of CTSL in promoting QC-induced autophagic flux and apoptotic cell death in OC cells. Using a Magic Red® cathepsin L activity assay and LysoTracker red, we discerned that QC-induced CTSL activation promotes lysosomal membrane permeability (LMP) resulting in the release of active CTSL into the cytosol to promote apoptotic cell death. We found that QC-induced LMP and CTSL activation promotes Bid cleavage, mitochondrial outer membrane permeabilization (MOMP), and mitochondrial cytochrome-c release. Genetic (shRNA) and pharmacological (Z-FY(tBU)-DMK) inhibition of CTSL markedly reduces QC-induced autophagy, LMP, MOMP, apoptosis, and cell death; whereas induced overexpression of CTSL in ovarian cancer cell lines has an opposite effect. Using recombinant CTSL, we identified p62/SQSTM1 as a novel substrate of CTSL, suggesting that CTSL promotes QC-induced autophagic flux. CTSL activation is specific to QC-induced autophagy since no CTSL activation is seen in ATG5 knockout cells or with the anti-malarial autophagy-inhibiting drug chloroquine. Importantly, we showed that upregulation of CTSL in QC-treated HeyA8MDR xenografts corresponds with attenuation of p62, upregulation of LC3BII, cytochrome-c, tBid, cleaved PARP, and caspase3. Taken together, the data suggest that QC-induced autophagy and CTSL upregulation promote a positive feedback loop leading to excessive autophagic flux, LMP, and MOMP to promote QC-induced cell death in ovarian cancer cells.