Vacuolar H+-ATPase inhibitor induces apoptosis via lysosomal dysfunction in the human gastric cancer cell line MKN-1
Vacuolar H+-ATPase inhibitor induces apoptosis via lysosomal dysfunction in the human gastric cancer cell line MKN-1
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DOI：
10.1093/jb/mvg153
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发表时间：
2003-09-01
影响因子：
2.7
通讯作者：
Kawanishi, S
中科院分区：
文献类型：
--
作者：
Nakashima, S;Hiraku, Y;Kawanishi, S
我们研究了空泡H+-ATP酶抑制剂巴弗洛霉素A（1）诱导细胞凋亡的机制。巴弗洛霉素A（1）对人胃癌MKN-1细胞生长有明显的抑制作用。DNA梯状条带形成和TUNEL法显示巴弗洛霉素A（1）诱导细胞凋亡。我们设计了一种流式细胞术检测溶酶体pH值的变化，使用荧光探针，荧光素异硫氰酸酯结合葡聚糖。该试验显示，巴弗洛霉素A（1）显著增加溶酶体pH值。然而，巴弗洛霉素A（1）既不诱导线粒体跨膜电位显著降低，也不诱导线粒体细胞色素c释放到细胞质中。Western blotting显示，组织蛋白酶D，而不是组织蛋白酶L，被释放到细胞质中。巴弗洛霉素A可显著增加caspase-3的活性（1）。然而，组织蛋白酶D并不直接切割半胱氨酸天冬氨酸蛋白酶原-3。这些发现表明，巴弗洛霉素A（1）诱导的MKN-1细胞凋亡是由溶酶体功能障碍后释放的其他蛋白酶介导的，随后以细胞色素c非依赖性方式激活caspase-3。目前的研究表明，溶酶体pH值的流式细胞仪分析可以用来评估溶酶体蛋白酶介导的细胞凋亡。
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.