Induction of apoptotic DNA fragmentation mediated by mitochondrial pathway with caspase-3-dependent BID cleavage in human gastric cancer cells by a new nitroxyl spin-labeled derivative of podophyllotoxin.

Abstract

4-[4''-(2'', 2'', 6'', 6''-tetramethyl-l''-piperidinyloxy) amino]-4'-demethyl-epipodophyllotoxin (GP7) is a new semi-synthesized nitroxyl spin-labeled derivative of podophyllotoxin with anti-leukemic and anti-osteosarcoma effects. The purpose of the present study is to investigate the anti-gastric cancer (GC) effects of GP7 and the possible involvement of caspase pathway in GP7-induced apoptotic DNA fragmentation in human GC cells. Effects of GP7 on the proliferation of human GC cell lines MKN28, AGS, BGC-823 and HGC-27 in different degrees of differentiation and normal human gastric epithelial cell line GES-1 were studied by MTT assay and compared with the effects of etoposide. Effects of GP7 on cell viability and heat shock protein 90 expression of BGC-823 and HGC-27 cells were analyzed by trypan blue exclusion test and western blotting, respectively. Effects of GP7 on apoptotic DNA fragmentation and caspase pathway of BGC-823 and HGC-27 cells were detected by agarose gel electrophoresis, colorimetric assay and western blotting. Caspase-3 inhibitor was used to manipulate the activity of caspase-3. GP7 inhibited concentration- and time-dependently the proliferation of human GC cells, and the inhibitory effect of GP7 on the proliferation of BGC-823 or HGC-27 cells was 1.15- or 1.21-fold higher than that of etoposide. GP7 downregulated heat shock protein 90, improved the anti-GC effects of adriamycin, cisplatin, 5-fluorouracil and their combinations, induced apoptotic DNA fragmentation, activations of caspase-9 and -3 but not -8, cytochrome-c release and BID cleavage in BGC-823 and HGC-27 cells. Caspase-3 inhibitor abrogated GP7-induced BID cleavage, decreased cytochrome-c release, caspase-9 and -3 activities and apoptotic DNA fragmentation but increased cell viability in BGC-823 and HGC-27 cells. Our findings indicate that GP7 is a promising anti-GC derivative of podophyllotoxin, and GP7-induced apoptosis in human GC cells may be mediated by mitochondrial pathway with caspase-3-dependent BID cleavage.