Construction of autocatalytic caspase-3 and its effects of inducing apoptosis in human ovarian carcinoma
- VernacularTitle:活性半胱氨酸天冬氨酸蛋白酶3分子的构建及其致卵巢上皮性癌细胞凋亡的实验研究
- Author:
Yue SONG
;
Keng SHEN
;
- Publication Type:Journal Article
- Keywords:
Ovarian neoplasms;
Caspase;
Apoptosis
- From:
Chinese Journal of Obstetrics and Gynecology
2000;0(12):-
- CountryChina
- Language:Chinese
-
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-easp3.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 plT,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 ultrastrueture,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).Conclusion Recombinant adenovirus containing rev-caspase-3 can significantly induce apoptosis of ovarian carcinoma cells,suppress tumor growth and prolong the mouse survival duration.
