Recombinant adenovirus encoding H-ras ribozyme induces apoptosis in laryngeal cancer cells through caspase- and mitochondria-dependent pathways.
Previously, we designed a ribozyme that targets the H-ras oncogene at the 12th codon mutation site (Chang et al., 1997). Ribozymes have antisense molecule and site-specific ribonuclease potential. In this study, an adenoviral vector was used to transduce the H-ras ribozyme into laryngeal cancer cells (HEp-2). This served to downregulate the H-ras gene expression in which this ribozyme performed antisense activity due to HEp-2 cells containing wild-type alleles in the 12th H-ras codon. Together, our data demonstrated that the recombinant adenovirus encoding H-ras ribozyme can be broadly regarded as a cytotoxic gene therapy in laryngeal cancer cells regardless of containing wild-type or mutant ras gene. In addition, the mechanism through which the H-ras ribozyme inhibited tumor growth was apoptosis and involved both caspase- and mitochondria-mediated pathways. The activators caspase-8 and -9 as well as the effector caspase-3 in the induction phase of apoptosis and the substrate PARP of caspase-3 in the execution phase were activated 48h following the H-ras ribozyme treatment. Mitochondrial events characterized by the production of superoxide anion and the release of cytochrome c started at 24h. Mitochondrial transmembrane potential loss occurred 48h after the ribozyme treatment. However, Bcl-2 delayed cytochrome c release to the cytosol, but it could not protect the apoptosis effect, suggesting that cytochrome c release from mitochondria may not play a role in H-ras ribozyme-induced apoptosis.
Wang
Chih-Hung
CH
Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, ROC.
Tsai
Li-Jen
LJ
Tsao
Yeou-Ping
YP
Hsieh
Jer-Tsong
JT
Chien
Wei-Wen
WW
Liao
Ching-Len
CL
Wang
Hsing-Won
HW
Liu
Hsiao-Sheng
HS
Chen
Show-Li
SL
D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000235:genetics* / Q000502:physiology; D020169:Caspases / Q000378:metabolism; D002455:Cell Division; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation; D019254:Genes, bcl-2; D011905:Genes, ras; D015316:Genetic Therapy; D006801:Humans; D007822:Laryngeal Neoplasms / Q000201:enzymology / Q000473:pathology / Q000628:therapy; D008928:Mitochondria / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics*; D014407:Tumor Cells, Cultured
D003574:Cytochrome c Group; D016337:RNA, Catalytic; D020169:Caspases