Introduction: Lung cancer is the leading cause of cancer-related death. Resistance to treatment contributes to poor outcome. A form of resistance to antineoplastic agents involves the sequestration of chemotherapy inside lysosomes followed by its delivery outside the cell through exocytosis. Lysosomal membrane permeabilization (LMP) not only releases the trapped drug, but also allows the translocation of lysosomal proteases into the cytosol, triggering cell demise. Since lysosomes in cancer cells are more susceptible to LMP than normal cells, we hypothesize that targeting LMP in cisplatin-resistant cells may provide a successful strategy to restore sensitivity to chemotherapy.
Materials and Methods: Cisplatin-resistant (cisR) A549 lung cancer cells, obtained by treating parental A549 cells with incremental concentrations of cisplatin, were kindly provided by Dr. Martin Barr. To detect LMP, lysosomes were loaded with FITC-dextran 40KDa and immunofluorescence images were obtained by confocal microscopy. To confirm LMP, cells were homogenized, cytosolic and lysosomal fractions were separated by ultracentrifugation, and cell lysates were immunoblotted with specific antibodies against cathepsin D, α-tubulin and LAMP-1. Cell viability was evaluated using CellTiter blue assay. To measure apoptosis, cells were stained for Annexin-V/Propidium iodide, or FITC-conjugated caspase 3 substrate, and mounted on the Cellometer vision CBA platform, an automated image-based fluorescence microscope equipped with bio-analysis system.
Results: cisR A549 cells loaded with FITC-dextran, and incubated with different concentrations of chloroquine (25-100μM) for 24 hours demonstrated increased green haziness in the cytosol and loss of the punctate pattern of the dextran-loaded lysosomes, indicative of dextran leakage following LMP. Release of cathepsins into the cytosol due to LMP was confirmed by the increase in cathepsin D signal in the cytosolic extract on western blot. Next, cisR A549 cells were treated with chloroquine, cisplatin or the drug mix. Cell viability assay revealed a synergistic effect of the combination, with a combination index of 0.70 for cisplatin 25μM and chloroquine 100μM at 48 hours. Notably, there is significant increase in Annexin V stained apoptotic cells, accompanied by a corresponding increase in caspase 3 activity, in the combination. Interestingly, inhibition of lysosomal proteases using E64 was cytoprotective for cells treated with cisplatin and chloroquine, suggesting that chloroquine-induced cell death is LMP mediated.
Conclusion: Chloroquine re-sensitizes cisR A549 cells to cisplatin in an LMP-mediated manner. Our preliminary results support the concept that targeting LMP may provide a viable therapeutic strategy to restore sensitivity to chemotherapy in refractory lung cancer. Screening for other repurposed LMP inducer drugs is ongoing.
Citation Format: Magdalena Circu, James Cardelli, Glenn Mills, Martin Barr, Hazem E. El-Osta. Chloroquine-induced lysosomal membrane permeabilization restores sensitivity to cisplatin in refractory lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3511.
- ©2016 American Association for Cancer Research.