Song Yue, Shen Keng, Yu Jing-rong
Department of Gynecology and Obstetrics, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China.
Zhonghua Yi Xue Za Zhi. 2007 Nov 6;87(41):2919-24.
To construct recombinant adenoviral vector expressing autocatalysis caspase-3 driven by human telomerase reverse transcriptase promoter (hTERTp), and investigate its antitumor effect on ovarian cancer in vitro and in vivo.
Recombinant adenovirus expressing autocatalytic caspase-3 (rev-csapase-3) driven by hTERTp, AdHT-rev-casp3, was constructed. Ad-rev-casp3 expressing rev-caspase-3 driven by cytomegalovirus promoter (CMVp) was used as a positive control. hTERT positive human ovarian cancer cells of the line AO and hTERT-negative human umbilical venous endothelial cells (HUVECs) were cultured and transfected with AdHT-rev-casp3, Ad-rev-casp3, or Ad-EGFG expressing enhanced green fluorescent protein as control group. Western blotting, Cell Counting Kit (CCK-8), flow cytometry, and TUNEL were used to detect the expression of p17, active subunit of caspase-3, and p85, a poly ADP-ribose polymerase (PARP) cleavage fragment, and they were also used to measure the cell survival rate and apoptotic rate. Western blotting was used to detect the expression of active caspase-3 and its substrate PARP in the AO cells and HUVECs. Twenty nude BALB/c mice were inoculated subcutaneously with AO cells to establish subcutaneous tumor models, when the tumor grew to the volume of 150 mm3 the rats were divided into 4 equal groups to undergo intra-tumor injection of AdHT-rev-casp3, Ad-rev-casp3, Ad-EGFG, and phosphate-buffered saline (PBS) respectively, the survival rate tumor inhibition rate was observed, 72 days later the mice were killed with their livers and tumors taken out, and Western blotting was used to detect the expression of active caspase-3. Another 40 mice underwent intraperitoneal injection of AO cells to establish intraperitoneal transplanted tumor models, 21 days later the rats were divided into 4 equal groups to be injected intraperitoneally with AdHT-rev-casp3, Ad-rev-casp3, Ad-EGFG, or PBS, the survival rate was observed, and the blood levels of alanine transaminase (ALT) and aspartate transaminase (AST) were detected.
Following the administration of AdHT-rev-casp3, active caspase-3 protein was significantly expressed, and the levels of p17 and p85 expressions were significantly elevated in AO cells, while no expressions of p17 and p85 was observed in HUVEC. In contrast, both AO and HUVEC expressed high levels of p17 and p85 protein after administrations of Ad-rev-casp3. AdHT-rev-casp3 dose-dependently killed the hTERT positive AO cells, however, showed no killing effect on the hTERT-negative HUVEC cells; whereas Ad-rev-casp3 was cytotoxic independent of the hTERT status of the cells. The killing effect of Ad-rev-casp3 was stronger than that of AdHT-rev-casp3. Treated with AdHT-rev-cap3 the expression levels of the caspase-3 fragment p17 and PARP cleavage fragment p85 of the AO cells were significantly higher than those before the treatment, however, the expression levels of p17 and p85 were both weaker than those of the AO cells treated with Ad-rev-casp-3. Though treated with AdHT-rev-casp-3, there was still no remarkable expression of p17 and p85 in the HUVECs, however, rather high protein expression levels of p17 and p85 was shown. After treatment with AdHT-rev-casp3 remarkable expression of active caspase-3 was seen in the tumor collected from the mouse body, but not in the liver; however, high caspase-3 expression level was shown in both the liver and tumor after the treatment of Ad-rev-casp-3. 53 days after treatment the tumor suppression rate of the AdHT-rev-casp3 and ad-rev-casp-3 groups were 60% and 70% respectively, both significantly higher than that of the control group. The survival rates of the mice treated with AdHT-rev-casp3 and Ad-rev-casp-3 were both significantly longer than that of the PBS group; however the survival rate of the Ad-rev-casp-3 group was longer than that of the AdHT-rev-casp3 group. The serum ALT and AST levels were not significantly elevated in the AdHT-rev-casp3-treated mice, whereas 7-9-times that before treatment in the Ad-rev-casp3-treated mice.
Recombinant adenovirus AdHT-rev-casp3 expressing rev-caspase-3 driven by hTERTp effectively causes cell apoptosis targeting tumor, significantly suppresses tumor growth and prolongs the mouse survival duration, with mild liver toxicity.
构建由人端粒酶逆转录酶启动子（hTERTp）驱动的表达自催化型半胱天冬酶-3的重组腺病毒载体，并研究其对卵巢癌的体内外抗肿瘤作用。
构建由hTERTp驱动的表达自催化型半胱天冬酶-3（rev-csapase-3）的重组腺病毒AdHT-rev-casp3。以由巨细胞病毒启动子（CMVp）驱动的表达rev-caspase-3的Ad-rev-casp3作为阳性对照。培养hTERT阳性的人卵巢癌细胞系AO和hTERT阴性的人脐静脉内皮细胞（HUVECs），并分别用AdHT-rev-casp3、Ad-rev-casp3或表达增强型绿色荧光蛋白的Ad-EGFG作为对照组进行转染。采用蛋白质免疫印迹法、细胞计数试剂盒（CCK-8）、流式细胞术和TUNEL法检测半胱天冬酶-3活性亚基p17和多聚ADP核糖聚合酶（PARP）裂解片段p85的表达，同时检测细胞存活率和凋亡率。用蛋白质免疫印迹法检测AO细胞和HUVECs中活性半胱天冬酶-3及其底物PARP的表达。将20只裸BALB/c小鼠皮下接种AO细胞建立皮下肿瘤模型，待肿瘤长至150 mm3时，将大鼠分为4组，分别瘤内注射AdHT-rev-casp3、Ad-rev-casp3、Ad-EGFG和磷酸盐缓冲液（PBS），观察存活率和肿瘤抑制率，72天后处死小鼠，取出肝脏和肿瘤，用蛋白质免疫印迹法检测活性半胱天冬酶-3的表达。另取40只小鼠腹腔注射AO细胞建立腹腔移植瘤模型，21天后将大鼠分为4组，分别腹腔注射AdHT-rev-casp3、Ad-rev-casp3、Ad-EGFG或PBS，观察存活率，并检测血清丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）水平。
给予AdHT-rev-casp3后，AO细胞中活性半胱天冬酶-3蛋白显著表达，p17和p85表达水平显著升高，而HUVEC中未观察到p17和p85的表达。相比之下，给予Ad-rev-casp3后，AO和HUVEC中p17和p85蛋白均高表达。AdHT-rev-casp3剂量依赖性地杀伤hTERT阳性的AO细胞，但对hTERT阴性的HUVEC细胞无杀伤作用；而Ad-rev-casp3对细胞具有细胞毒性，与细胞的hTERT状态无关。Ad-rev-casp3的杀伤作用强于AdHT-rev-casp3。用AdHT-rev-cap3处理后，AO细胞中半胱天冬酶-3片段p17和PARP裂解片段p85的表达水平显著高于处理前，但p17和p85的表达水平均弱于用Ad-rev-casp-3处理的AO细胞。用AdHT-rev-casp-3处理后，HUVECs中仍未观察到p17和p85的显著表达，但p17和p85的蛋白表达水平较高。用AdHT-rev-casp3处理后，从小鼠体内采集的肿瘤中可见活性半胱天冬酶-3的显著表达，但肝脏中未观察到；而用Ad-rev-casp-3处理后，肝脏和肿瘤中均显示出高半胱天冬酶-3表达水平。处理53天后，AdHT-rev-casp3组和Ad-rev-casp-3组的肿瘤抑制率分别为60%和70%，均显著高于对照组。用AdHT-rev-casp3和Ad-rev-casp-3处理的小鼠存活率均显著长于PBS组；但Ad-rev-casp-3组的存活率长于AdHT-rev-casp3组。用AdHT-rev-casp3处理的小鼠血清ALT和AST水平未显著升高，而用Ad-rev-casp3处理的小鼠血清ALT和AST水平是处理前的7 - 9倍。
由hTERTp驱动的表达rev-caspase-3的重组腺病毒AdHT-rev-casp3能有效诱导肿瘤细胞凋亡，显著抑制肿瘤生长，延长小鼠存活时间，且肝毒性较轻。
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