Song Yue, Xin Xing, Zhai Xingyue, Xia Zhijun, Shen Keng
Department of Obstetrics and Gynecology, ShengJing Hospital, China Medical University, No. 36, Sanhao street, Heping District, Shen yang, 110004, China.
Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, No. 1, Shuai fuyuan Hutong, Dongcheng District, Beijing, China.
J Ovarian Res. 2014 Dec 21;7:121. doi: 10.1186/s13048-014-0121-3.
Induction of cell apoptosis and regulation of cell cycle are very attractive for treatments of tumors including ovarian carcinoma. Flavopiridol is a potent small molecular cyclin-dependent kinase(cdk) inhibitor, but its antitumor efficacy is not satisfied yet. Caspase-3 play a major role in the transduction of apoptotic signals and the execution of apoptosis in mammalian cells. We have successfully constructed the recombinant adenovirues AdHTVP2G5-rev-casp3 containing autocatalytic caspase-3 (rev-caspase-3) driven by amplified hTERT promoter system (TSTA-hTERTp). In this study, we applied it with flavopiridol to investigate their antitumor effect on ovarian cancer in vitro and in vivo.
Cell viabilities were determined using Cell Counting Kit 8 and flow cytometry. RT-PCR and immunoblotting assays were used to detect cellular apoptotic activities. Tumor growth and survival of mice bearing tumors were studied.
Flavopiridol or AdHTVP2G5-rev-casp3 at low dosage alone was mildly cytotoxic in vitro with a viability rate of 86.5 ± 4.7% for 300 nM flavopiridol and 88.9 ± 5.4% for AdHTVP2G5-rev-casp3 (MOI 20). By contrast, significant synergism of their sequential combination was observed, and the treatment of AdHTVP2G5-rev-casp3 (MOI 20) infection for 72 h, followed by flavopiridol (300 nM) for 48 h, can result in the most synergistic cell death, with cell survival rate and apoptotic rate of 11.6% and 69.7%, respectively. The sequential combination showed synergistic tumor suppression rate of 77.8%, which was significantly higher than that of AdHTVP2G5-rev-casp3 (33.6%) or flavopiridol (40.1%) alone. The mean survival of mice treated with the combination was 286 ± 8 d, which was synergistically longer than that of mice treated with AdHTVP2G5-rev-casp3 (141 ± 14d), flavopiridol (134 ± 10 d) or controls (106 ± 11 d) (P < 0.01).
The sequential combination of rev-caspase-3 and flavopiridol result in significant synergistic cell killing effects, significant tumor growth suppression and extended survival of mice bearing OVCAR3 cells. The combination should be further explored as a potential clinically useful regimen against ovarian cancer.
诱导细胞凋亡和调控细胞周期对包括卵巢癌在内的肿瘤治疗具有很大吸引力。黄酮哌啶醇是一种有效的小分子细胞周期蛋白依赖性激酶（cdk）抑制剂，但其抗肿瘤疗效尚不尽人意。半胱天冬酶-3在哺乳动物细胞凋亡信号转导和凋亡执行过程中起主要作用。我们成功构建了由扩增的人端粒酶逆转录酶启动子系统（TSTA-hTERTp）驱动的含自催化半胱天冬酶-3（rev-半胱天冬酶-3）的重组腺病毒AdHTVP2G5-rev-casp3。在本研究中，我们将其与黄酮哌啶醇联合应用，以研究它们在体外和体内对卵巢癌的抗肿瘤作用。
使用细胞计数试剂盒8和流式细胞术测定细胞活力。采用RT-PCR和免疫印迹分析检测细胞凋亡活性。研究荷瘤小鼠的肿瘤生长和存活情况。
单独使用低剂量的黄酮哌啶醇或AdHTVP2G5-rev-casp3在体外具有轻度细胞毒性，300 nM黄酮哌啶醇的存活率为86.5±4.7%，AdHTVP2G5-rev-casp3（感染复数为20）的存活率为88.9±5.4%。相比之下，观察到它们序贯联合具有显著的协同作用，AdHTVP2G5-rev-casp3（感染复数为20）感染72小时，随后用黄酮哌啶醇（300 nM）处理48小时，可导致最强的协同细胞死亡，细胞存活率和凋亡率分别为11.6%和69.7%。序贯联合显示协同肿瘤抑制率为77.8%，显著高于单独使用AdHTVP2G5-rev-casp3（33.6%）或黄酮哌啶醇（40.1%）。联合治疗的小鼠平均生存期为286±8天，协同地长于接受AdHTVP2G5-rev-casp3（141±14天）、黄酮哌啶醇（134±10天）或对照（106±11天）治疗的小鼠（P<0.01）。
rev-半胱天冬酶-3与黄酮哌啶醇的序贯联合产生显著的协同细胞杀伤作用、显著的肿瘤生长抑制作用，并延长了荷OVCAR3细胞小鼠的生存期。该联合方案应作为一种潜在的治疗卵巢癌的临床有效方案进一步探索。
