title: [Construction of autocatalytic caspase-3 and its effects of inducing apoptosis in human ovarian carcinoma].
abstract: OBJECTIVE
  To construct the autocatalytic caspase-3 and investigate its apoptosis-inducing effect in ovarian cancer in vitro and in vivo.
  
  
  METHODS
  PCR recombination technique was used to construct autocatalytic caspase-3 which is named as rev-caspase-3, and Ad-Max system was used to prepare recombinant adenovirus containing rev-caspase-3, which is named as Ad-rev-casp3. Immunohistochemistry was used to detect active caspase-3 expression. Cell counting kit, flow cytometry and western blot were used to measure cell survival rate, apoptotic rate, cell cycle distribution and the expressions of p17, active subunit of caspase-3, and p85, the poly (adenosine diphosphate-ribose) polymerase (PARP) cleavage segment, respectively. Transmission electron microscope was used to detect cell ultrastructure, and real time PCR was used to detect apoptosis-related gene expression. Subcutaneous tumor models and abdominally spread tumor models of human ovarian carcinoma were established using AO cells in BALB/c nude mice. The mouse survival rates were measured for abdominally spread tumor models, and the volume of tumor nodules were determined for subcutaneous tumor models following the treatments of rev-caspase-3.
  
  
  RESULTS
  Active caspase-3 protein was significantly expressed, and the expression levels of active subunit of caspase-3, p17, and the PARP cleavage segment, p85, were significantly elevated in cells treated with rev-caspase-3. The decrease of cell survival rate and the increase of cell apoptotic rate were detected following Ad-rev-casp3 treatment. Treatments with Ad-rev-casp3 [multiplicity of infection (MOI) was 70] resulted in survival rate of 30.3% and apoptotic rate of 40.2%. There was a significant increase in cell number of S-phase (56.5%), while there was no significant apoptosis (3.4%) following treatments with Ad-rev-casp3 at a low dosage of MOI=10. Cells treated with rev-caspase-3 displayed significant apoptotic morphology. The levels of active caspase-3 gene expressions (9.44) significantly increased. Rev-caspase-3 treatment significantly prolonged survival, the mean survival duration was (213 +/- 16) days, and suppressed tumor growth (tumor growth suppression rate was 70%), when compared with treatment with phosphate buffered saline (PBS).
  
  
  CONCLUSIONS
  Recombinant adenovirus containing rev-caspase-3 can significantly induce apoptosis of ovarian carcinoma cells, suppress tumor growth and prolong the mouse survival duration.
authors.affiliation: Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Song
authors.forename: Keng
authors.initials: K
authors.lastname: Shen
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000235:genetics / Q000494:pharmacology / Q000627:therapeutic use*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects*; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D004195:Disease Models, Animal; D005260:Female; D005434:Flow Cytometry; D015316:Genetic Therapy; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms; D010051:Ovarian Neoplasms / Q000235:genetics / Q000628:therapy*; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3