Effect of chloroquine on cathepsin D distribution in breast cancer cells

PEREYRA L; PERALTA S; VARGAS-ROIG LM; SOSA MA; CARVELLI L

Virtual

Congreso; IV Reunión Conjunta de Sociedades de Biología de la República Argentina; 2020

Sociedad de Biología de Cuyo

Breast cancer causes high mortality worldwide. Cells of some tumors have increased the lysosomal biogenesis as a response to its altered metabolism. These events affect lysosomal integrity and/or functionality, where increased levels of lysosomal proteases such as cathepsin D (CatD) are observed. In most cell types, lysosomal proteins are selectively transported from the trans Golgi network (TGN) to lysosomes by the mannose-6-phosphate receptors (CD-MPR and CI-MPR). In acidic compartments (e.g. late endosomes), the enzyme-MPR complexes are dissociated and receptors can recycle back. Alterations in the lysosomal membrane release CatD into the cytoplasm, triggering apoptotic processes. Thus, lysosomes are considered as potential therapeutic targets for antitumor drugs. Usually, acidotropic amines accumulate in lysosomes and could induce an increase in lysosomal membrane permeability, leading to an escape of enzymes into the cytoplasm. Chloroquine (CQ) is an acidotropic amine known to affect lysosomal acidification and inhibits autophagy. The aim of this study was to evaluate the effect of CQ on the distribution of CatD and CD-MPR in human mammary tumor cells MCF-7. Cultures incubated with CQ at different times were processed for CatD and CD-MPR detection by indirect immunofluorescence. After 6 h of incubation with CQ, CD-MPR is mostly distributed in the cytoplasm, indicating that the increased lysosomal pH prevents the recycling of CD-MPR to the Golgi. In turn, the TGN becomes disorganized and co-localizes with CD-MPR. However, CatD exhibits an apparent increase in perinuclear staining compared to control cells. This effect could be due to a delay in the transport of the newly synthesized enzymes to lysosomes. Surprisingly, at 12 h of incubation, the perinuclear CatD signal is redistributed throughout the cytoplasm, indicating that it would have reached the lysosomes. At 18 h, CD-MPR and CatD fully recover their initial location, denoting that the CQ effect is lost over time. From these preliminary results, we conclude that CQ affects the endo-lysosomal system, altering the normal transport of CatD, although the mechanism by which CQ effect is reversed should be elucidated. .