Lysosome swelling precedes lysosomal membrane permeabilization in breast cancer cells

LB-51

Introduction:
 Recent studies demonstrated that lysosomal membrane permeabilization (LMP) resulted in the release of lysosomal enzymes into the cytosol, which induced mitochondrial cytochrome c release and apoptosis (1). In this study, we have investigated the role of LMP in breast cancer aggressiveness to test its future potential for breast cancer treatment. LMP was induced with the V-ATPase inhibitor Bafilomycin A1 (Baf A1) and/or the fluoroquinolone ciprofloxacin (CPX) (2, 3). We observed changes in cellular and lysosome morphology in the human breast cancer cell lines MCF-12A, MCF-7 and MD-MBA-231.
 Methods:
 We used non-malignant MCF-12A, weakly aggressive MCF-7, and highly aggressive MD-MBA-231 cells. Live cells were treated with 0.1 μM Baf A1 or 500-750 μg/ml CPX or a combination of 0.1 μM Baf A1 and 500 μg/ml CPX. Lysosomes were fluorescently labeled with Alexa-Fluor-488 bound to 10 kDa dextran. Dynamic changes in cell morphology were observed by phase contrast microscopy, and dynamic changes in lysosomes by fluorescence microscopy. These studies were corroborated by analogous confocal fluorescence microscopy detecting lysosomal CD63 immunofluorescence staining.
 Results:
 Dynamic phase contrast microscopy showed that both Baf A1 and CPX led to changes in cell morphology as characterized by cell shrinkage, blebbing, and loss of characteristic cell shape. Both LMP-inducing agents caused cell death in a time-dependent manner. Upon treatment, we either observed increased numbers of lysosomes, or enlarged lysosomes, or both. At later time points, we observed disappearance of dextran-labeling from lysosomes in treated cells, which was a sign of LMP.
 Discussion:
 We have shown for the first time that an enlargement of lysosomal vesicles and/or an increase in lysosome number preceded LMP. Recent studies demonstrated that in breast carcinoma cells, Baf A1 inhibited proton transport causing an alkalization in the lysosomal compartments (4). Such a change in the lysosomal pH could indirectly result in water influx into lysosomes and increase their volumes. This process was time-dependent as it resulted in increased numbers of large lysosomes over time, and eventually led to LMP, that resulted in cell death, probably by apoptosis. We will further investigate the mechanism of lysosome swelling preceding LMP in breast cancer cells, and its role in cell death pathways.
 References:
 (1) Kroemer, G. and M. Jaattela (2005), Nat Rev Cancer 5, 886-97.
 (2) Boya, P., et al. (2003), J Exp Med 197, 1323-34.
 (3) Nakashima, S., et al. (2003), J Biochem 134, 359-64.
 (4) Martinez-Zaguilan, R., et al. (1993), Am J Physiol 265, C1015-29.