人端粒酶逆转录酶启动子调控下活性半胱氨酸蛋白酶-3治疗人卵巢癌

The efficacy of autocatalytic casapse-3 driven by human telomerase reverse transcriptase promoter on human ovarian carcinoma

目的构建人端粒酶逆转录酶启动子(hTERTp)调控下的活性半胱氨酸蛋白酶-3(caspase-3)重组腺病毒载体并检测其对人卵巢癌的治疗作用。方法构建表达 hTERTp 调控下活性caspase-3的重组腺病毒载体 AdHT-rev-casp3;以 CMV 启动子调控下活性 caspase-3的重组腺病毒载体Ad-rev-casp3为对照,分别应用 Western 印迹、细胞计数试剂盒(CCK-8)法、流式细胞术和 TUNEL 检测AdHT-rev-casp3作用后卵巢癌细胞系 AO 及正常人脐静脉内皮细胞 HUVEC 中活性 caspae-3蛋白 p17和聚腺苷二磷酸-核糖多聚酶(PARP)裂解片段 p85的表达水平、细胞存活率和凋亡率;建立裸鼠人卵巢癌皮下及腹腔移植瘤模型,Western 印迹检测 AdHT-rev-casp3作用后裸鼠肿瘤及肝脏组织中活性caspase-3的表达情况,观测作用前后裸鼠生存率及肿瘤体积的变化以及裸鼠体内肝酶 ALT 和 AST 水平。结果 AO 细胞在 AdHT-rev-casp3(MOI=70)感染后明显表达活性 caspase-3蛋白 p17和 PARP 的裂解片段 p85蛋白,细胞存活率为55%,凋亡率为26%,而 HUVEC 在 AdHT-rev-casp3感染后无明显p17和 p85表达,细胞存活率和凋亡率与阴性对照组差异无统计学意义;AdHT-rev-casp3作用后的裸鼠肿瘤组织中明显表达活性 caspase-3,而肝脏组织中则无表达;AdHT-rev-casp3能明显延长荷瘤裸鼠的生存期[(177±12)d vs(106±11)d],抑瘤率为60%,其作用后裸鼠体内的肝酶水平无明显增高。结论 hTERTp 系统调控下 rev-caspase-3的重组腺病毒 AdHT-rev-casp3同时具有较强的致细胞凋亡能力和肿瘤靶向性,能明显抑制卵巢癌移植瘤的生长,延长荷瘤裸鼠的生存期,并明显降低 rev-caspase-3对肝脏的毒性作用。

Objective 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. Methods Recombinant adenovirus expressing autocatalytic caspase- 3 （rev-csapase-3） driven by hTERTp, AdHT-rev-casp3, was constructed. Ad-rev-casp3 expressing revcaspase-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-ribcse 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 mm^3 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. Results 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. Conclusion Recombinant adenovirus AdHT-rev-casp3 expressing revcaspase-3 driven by hTERTp effectively causes cell apoptosis targeting tumor, significantly suppresses tumor growth and prolongs the mouse survival duration, with mild liver toxicity.