title: 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: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Gastroenterology, The Second Clinical Medical College, Jinan University, Shenzhen 518020, China.
authors.forename: Yu-Jie
authors.initials: YJ
authors.lastname: Yang
authors.affiliation: Department of Histology and Embryology, School of Basic Medicine, Lanzhou University, Key Laboratory of Pre-clinical Study for New Drugs of Gansu Province, Lanzhou 730000, China. Electronic address: qishn@lzu.edu.cn.
authors.forename: She-Ning
authors.initials: SN
authors.lastname: Qi
authors.affiliation: Department of Gastroenterology, The Second Clinical Medical College, Jinan University, Shenzhen 518020, China.
authors.forename: Rui-Yue
authors.initials: RY
authors.lastname: Shi
authors.affiliation: Department of Gastroenterology, The Second Clinical Medical College, Jinan University, Shenzhen 518020, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Yao
authors.affiliation: Department of Gastroenterology, The Second Clinical Medical College, Jinan University, Shenzhen 518020, China. Electronic address: wangls168@163.com.
authors.forename: Li-Sheng
authors.initials: LS
authors.lastname: Wang
authors.affiliation: Department of Gastrointestinal Oncology Surgery, Tumor Hospital of Gansu Province, Lanzhou 730050, China.
authors.forename: Hu-Qin
authors.initials: HQ
authors.lastname: Yuan
authors.affiliation: Reproductive Hospital, The First Clinical Medical College, Lanzhou University, Lanzhou 730000, China.
authors.forename: Yuan-Xue
authors.initials: YX
authors.lastname: Jing
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects*; D005047:Etoposide / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D011034:Podophyllotoxin / Q000031:analogs & derivatives / Q000494:pharmacology*; D013113:Spin Labels; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000378:metabolism
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D013113:Spin Labels; D005047:Etoposide; D045304:Cytochromes c; D053148:Caspase 3; D053453:Caspase 9; D011034:Podophyllotoxin; D005472:Fluorouracil; C072088:4'-demethylepipodophyllotoxin
keywords: Apoptosis; BID; Gastric cancer; Podophyllotoxin; Proliferation