Quinacrine‐induced autophagy in ovarian cancer triggers cathepsin‐l mediated lysosomal/mitochondrial membrane permeabilization and cell death

We previously reported that the antimalarial compound quinacrine (QC) induces autoph-agy 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 pro-moting QC‐induced autophagic flux and apoptotic cell death in OC cells. Using a Magic Red® ca-thepsin 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 cy-tosol 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 chloro-quine. 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.