Vacuolar H+-ATPase inhibitor induces apoptosis via lysosomal dysfunction in the human gastric cancer cell line MKN-1.
We investigated the mechanism of apoptosis induced by bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPase. Bafilomycin A(1) significantly inhibited the growth of MKN-1 human gastric cancer cells. Bafilomycin A(1) 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 A(1) dramatically increased lysosomal pH. However, bafilomycin A(1) 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 A(1). However, cathepsin D did not directly cleave procaspase-3. These findings suggest that bafilomycin A(1)-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.
Nakashima
Shigehito
S
Third Department of Internal Medicine, Mie University School of Medicine, Tsu, Mie, 514-8507.
Hiraku
Yusuke
Y
Tada-Oikawa
Saeko
S
Hishita
Terutoshi
T
Gabazza
Esteban C
EC
Tamaki
Shigenori
S
Imoto
Ichiro
I
Adachi
Yukihiko
Y
Kawanishi
Shosuke
S
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002402:Cathepsin D / Q000378:metabolism; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020287:In Situ Nick-End Labeling; D008247:Lysosomes / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors
D004791:Enzyme Inhibitors; D018942:Macrolides; C040929:bafilomycin A1; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002402:Cathepsin D; D006180:Proton-Translocating ATPases