title: Sequential combination therapy with flavopiridol and autocatalytic caspase-3 driven by amplified hTERT promoter synergistically suppresses human ovarian carcinoma growth in vitro and in mice.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Obstetrics and Gynecology, ShengJing Hospital, China Medical University, No. 36, Sanhao street, Heping District, Shen yang, 110004, China. ssyue@sohu.com.
authors.forename: Yue
authors.initials: Y
authors.lastname: Song
authors.affiliation: Department of Obstetrics and Gynecology, ShengJing Hospital, China Medical University, No. 36, Sanhao street, Heping District, Shen yang, 110004, China. ykxinxing@163.com.
authors.forename: Xing
authors.initials: X
authors.lastname: Xin
authors.affiliation: Department of Obstetrics and Gynecology, ShengJing Hospital, China Medical University, No. 36, Sanhao street, Heping District, Shen yang, 110004, China. yyt0706@163.com.
authors.forename: Xingyue
authors.initials: X
authors.lastname: Zhai
authors.affiliation: Department of Obstetrics and Gynecology, ShengJing Hospital, China Medical University, No. 36, Sanhao street, Heping District, Shen yang, 110004, China. xiazj@sj-hospital.org.
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Xia
authors.affiliation: 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. shenkeng@vip.sina.cn.
authors.forename: Keng
authors.initials: K
authors.lastname: Shen
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage*; D017209:Apoptosis; D053148:Caspase 3 / Q000235:genetics* / Q000378:metabolism; D002470:Cell Survival; D003131:Combined Modality Therapy; D005260:Female; D005419:Flavonoids / Q000008:administration & dosage*; D015316:Genetic Therapy; D006801:Humans; D007274:Injections, Intraperitoneal; D008819:Mice, Nude; D010051:Ovarian Neoplasms / Q000201:enzymology / Q000473:pathology / Q000628:therapy*; D010880:Piperidines / Q000008:administration & dosage*; D019098:Telomerase / Q000235:genetics*; D047368:Tumor Burden; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D005419:Flavonoids; D010880:Piperidines; C077990:alvocidib; D019098:Telomerase; C505407:CASP3 protein, human; D053148:Caspase 3