2003 Volume 134 Issue 3 Pages 359-364
We investigated the mechanism of apoptosis induced by bafilomycin A1, an inhibitor of vacuolar H+-ATPase. Bafilomycin A1 significantly inhibited the growth of MXN-1 human gastric cancer cells. Bafilomycin A1 induced apoptosis as demonstrated by DNA ladder formation and the TUNEL method. We designed a flow cytometric assay to detect the alteration in lysosomal pH using a fluorescent probe, fluorescein isothiocyanate-conjugated dextran. This assay revealed that bafilomycin A1 dramatically increased lysosomal pH. However, bafilomycin A1 induced neither significant decrease in mitochondrial transmembrane potential nor the release of mitochondrial cytochrome c into the cytoplasm. Western blotting showed that cathepsin D, but not cathepsin L, was released into the cytoplasm. The activity of caspase-3 was significantly increased by bafilomycin A1. However, cathepsin D did not directly cleave procaspase-3. These findings suggest that bafilomycin A1-induced apoptosis in MKN-1 cells is mediated by other proteases released after lysosomal dysfunction followed by activation of caspase-3 in a cytochrome c-independent manner. The present study showed that flow cytometric analysis of lysosomal pH can be useful to evaluate lysosomal protease-mediated apoptosis.
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