| Autor: |
Pagliero RJ; 1 Department of Cell Biology, University Medical Center Utrecht (UMCU) , Utrecht, the Netherlands ., D'Astolfo DS; 1 Department of Cell Biology, University Medical Center Utrecht (UMCU) , Utrecht, the Netherlands .; 2 KNAW-Hubrecht Institute , Utrecht, the Netherlands ., Lelieveld D; 1 Department of Cell Biology, University Medical Center Utrecht (UMCU) , Utrecht, the Netherlands ., Pratiwi RD; 1 Department of Cell Biology, University Medical Center Utrecht (UMCU) , Utrecht, the Netherlands ., Aits S; 3 Cell Death and Metabolism Unit, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research Center , Copenhagen, Denmark ., Jaattela M; 3 Cell Death and Metabolism Unit, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research Center , Copenhagen, Denmark ., Martin NI; 4 Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University , Utrecht, the Netherlands ., Klumperman J; 1 Department of Cell Biology, University Medical Center Utrecht (UMCU) , Utrecht, the Netherlands ., Egan DA; 1 Department of Cell Biology, University Medical Center Utrecht (UMCU) , Utrecht, the Netherlands . |
| Abstrakt: |
The lysosomal cell death (LCD) pathway is a caspase 3-independent cell death pathway that has been suggested as a possible target for cancer therapy, making the development of sensitive and specific high-throughput (HT) assays to identify LCD inducers highly desirable. In this study, we report a two-step HT screening platform to reliably identify such molecules. First, using a robust HT primary screen based on propidium iodide uptake, we identified compounds that kill through nonapoptotic pathways. A phenotypic image-based assay using a galectin-3 (Gal-3) reporter was then used to further classify hits based on lysosomal permeabilization, a hallmark of LCD. The identification of permeabilized lysosomes in our image-based assay is not affected by changes in the lysosomal pH, thus resolving an important limitation in currently used methods. We have validated our platform in a screen by identifying 24 LCD inducers, some previously known to induce LCD. Although most LCD inducers were cationic amphiphilic drugs (CADs), we have also identified a non-CAD LCD inducer, which is of great interest in the field. Our data also gave new insights into the biology of LCD, suggesting that lysosomal accumulation and acid sphingomyelinase inhibition are not sufficient or necessary for the induction of LCD. Overall, our results demonstrate a robust HT platform to identify novel LCD inducers that will also be very useful for gaining deeper insights into the molecular mechanism of LCD induction. |
