Modulating lysosomal function through lysosome membrane permeabilization or autophagy suppression restores sensitivity to cisplatin in refractory non-small-cell lung cancer cells

Abstract

Lung cancer is the leading cause of cancer-related deaths. Most patients develop resistance to platinum within several months of treatment. We investigated whether triggering lysosomal membrane permeabilization (LMP) or suppressing autophagy can restore cisplatin susceptibility in lung cancer with acquired chemoresistance. Cisplatin IC50 in A549Pt (parental) and A549cisR (cisplatin resistant) cells was 13 μM and 47 μM, respectively. Following cisplatin exposure, A549cisR cells failed to elicit an apoptotic response. This was manifested by diminished Annexin–V staining, caspase 3 and 9, BAX and BAK activation in resistant but not in parental cells. Chloroquine preferentially promoted LMP in A549cisR cells, revealed by leakage of FITC-dextran into the cytosol as detected by immunofluorescence microscopy. This was confirmed by increased cytosolic cathepsin D signal on Immunoblot. Cell viability of cisplatin-treated A549cisR cells was decreased when co-treated with chloroquine, corresponding to a combination index below 0.8, suggesting synergism between the two drugs. Notably, chloroquine activated the mitochondrial cell death pathway as indicated by increase in caspase 9 activity. Interestingly, inhibition of lysosomal proteases using E64 conferred cytoprotection against cisplatin and chloroquine co-treatment, suggesting that chloroquine-induced cell death occurred in a cathepsin-mediated mechanism. Likewise, blockage of caspases partially rescued A549cisR cells against the cytotoxicity of cisplatin and chloroquine combination. Cisplatin promoted a dose-dependent autophagic flux induction preferentially in A549cisR cells, as evidenced by a surge in LC3-II/α-tubulin following pre-treatment with E64 and increase in p62 degradation. Compared to untreated cells, cisplatin induced an increase in cyto-ID-loaded autophagosomes in A549cisR cells that was further amplified by chloroquine, pointing toward autophagic flux activation by cisplatin. Interestingly, this effect was less pronounced in A549Pt cells. Blocking autophagy by ATG5 depletion using siRNA markedly enhances susceptibility to cisplatin in A549cisR cells. Taken together, our results underscore the utility of targeting lysosomal function in overcoming acquired cisplatin refractoriness in lung cancer.

MicroRNA‐1 overexpression increases chemosensitivity of non‐small cell lung cancer cells by inhibiting autophagy related 3‐mediated autophagy

“…Following experiments further demonstrated that LC3B‐II/LC3B‐I ratio was strikingly increased and p62 level was remarkably reduced in DDP resistant NSCLC cells compared with their parental counterparts. In other words, autophagic activity was enhanced in DDP resistant NSCLC cells, which is in agreement with prior studies (Ren et al, ; Circu et al, ). However, Sirichanchuen et al () demonstrated that LC3 protein expression and autophagosome numbers were reduced in DDP resistant H460 cells than that in parental H460.…”

Proteomic Signature of Neuroblastoma Cells UKF-NB-4 Reveals Key Role of Lysosomal Sequestration and the Proteasome Complex in Acquiring Chemoresistance to Cisplatin

“…From the available literature, it is obvious that the mechanism conferring chemoresistance to CDDP is complex, highly multifactorial and often specific for distinct types of cells 5,[17][18][19][27][28][29] . In the present study, we performed a comparative analysis of proteomic signatures of UKF- Despite this phenomenon has not yet been described for CDDP-chemoresistance in Nbl, it is a generally accepted mechanism for other types of cancers [32][33][34] . Similarly, to those studies, we found that UKF-NB-4 CDDP cells acquire a specific phenotype resulting in a pronounced lysosomal enrichment and up-regulation of V-ATPases that are required for proper lysosomal acidification to allow efficient sequestration of drugs, including CDDP 25 .…”

Mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology

“…It is widely accepted that chloroquine and hydroxychloroquine accumulate in lysosomes (lyso somotropism) and inhibit their function. In vitro, chloro quine can destabilize lysosomal membranes and promote the release of lysosomal enzymes inside cells 76 . Although evidence of this latter mechanism is scarce, the ability of these drugs to interfere with lysosomal activity has been repeatedly documented [77][78][79] .…”