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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.

Song Y, Xin X, Zhai X, Xia Z, Shen K - J Ovarian Res (2014)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, ShengJing Hospital, China Medical University, No. 36, Sanhao street, Heping District, Shen yang, 110004, China. ssyue@sohu.com.

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.

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Related in: MedlinePlus

In vivoantitumor activity. A: Subcutaneous tumors derived from OVCAR3 cells were treated as shown. Tumor volumes were monitored over time (days) after inoculation of tumor cells. Values represent the mean of 5 mice per group. B: 1:PBS, 2: flavopiridol (10 mg/kg), 3: AdHTVP2G5-rev-casp3 (7 × 108 TCID50/mouse), 4: AdHTVP2G5-rev-casp3(7 × 108 TCID50/mouse) + flavopiridol(5 mg/kg). Survival curves and mean survival durations for animals bearing abdominally spread OVCAR3 tumors. The animals received three treatments using PBS, flavopiridol, AdHTVP2G5-rev-casp3 or flavopiridol + AdHTVP2G5-rev-casp3 as described in the text. Survival was then monitored: the survival duration in animals receiving treatment using AdHTVP2G5-rev-casp3, flavopiridol or flavopiridol + AdHTVP2G5-rev-casp3 were significantly differently from those in the mice that received treatment using controls (P < 0.01). In addition, synergisms of AdHTVP2G5-rev-casp3 and flavopiridol were observed, and significantly prolonged survival can be achieved using combined AdHTVP2-G5-rev-casp3 and flavopiridol therapy without an increase in doses(P < 0.05).
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Fig4: In vivoantitumor activity. A: Subcutaneous tumors derived from OVCAR3 cells were treated as shown. Tumor volumes were monitored over time (days) after inoculation of tumor cells. Values represent the mean of 5 mice per group. B: 1:PBS, 2: flavopiridol (10 mg/kg), 3: AdHTVP2G5-rev-casp3 (7 × 108 TCID50/mouse), 4: AdHTVP2G5-rev-casp3(7 × 108 TCID50/mouse) + flavopiridol(5 mg/kg). Survival curves and mean survival durations for animals bearing abdominally spread OVCAR3 tumors. The animals received three treatments using PBS, flavopiridol, AdHTVP2G5-rev-casp3 or flavopiridol + AdHTVP2G5-rev-casp3 as described in the text. Survival was then monitored: the survival duration in animals receiving treatment using AdHTVP2G5-rev-casp3, flavopiridol or flavopiridol + AdHTVP2G5-rev-casp3 were significantly differently from those in the mice that received treatment using controls (P < 0.01). In addition, synergisms of AdHTVP2G5-rev-casp3 and flavopiridol were observed, and significantly prolonged survival can be achieved using combined AdHTVP2-G5-rev-casp3 and flavopiridol therapy without an increase in doses(P < 0.05).

Mentions: To evaluate the in vivo efficacy of the sequential combination treatment of AdHTVP2G5-rev-casp3 with flavopiridol, we established mouse xenograft model by inoculating OVCAR3 cells subcutaneously in the nude mice and treated the mice with AdHTVP2G5-rev-casp3 and/or flavopiridol. Immunohistochemistry has confirmed the OVCAR3 xenograft. Fifty-three days after treatment, the tumor volume was 1458 ± 326, 1317 ± 295 and 487 ± 63 mm3 in mice bearing OVCAR3 xenograft treated with AdHTVP2G5-rev-casp3, flavopiridol and the sequential combination of AdHTVP2G5-rev-casp3 and flavopiridol, respectively. All treatments showed significant suppression of tumor growth at the end point of the study, with the tumor growth suppression rate of 33.6%, 40.1% and 77.8% for AdHTVP2G5-rev-casp3, flavopiridol and their combination, respectively (P < 0.05), as shown in Figure 4a. Moreover, we observed the synergism against xenograft growth between AdHTVP2G5-rev-casp3 and flavopiridol in vivo.Figure 4


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.

Song Y, Xin X, Zhai X, Xia Z, Shen K - J Ovarian Res (2014)

In vivoantitumor activity. A: Subcutaneous tumors derived from OVCAR3 cells were treated as shown. Tumor volumes were monitored over time (days) after inoculation of tumor cells. Values represent the mean of 5 mice per group. B: 1:PBS, 2: flavopiridol (10 mg/kg), 3: AdHTVP2G5-rev-casp3 (7 × 108 TCID50/mouse), 4: AdHTVP2G5-rev-casp3(7 × 108 TCID50/mouse) + flavopiridol(5 mg/kg). Survival curves and mean survival durations for animals bearing abdominally spread OVCAR3 tumors. The animals received three treatments using PBS, flavopiridol, AdHTVP2G5-rev-casp3 or flavopiridol + AdHTVP2G5-rev-casp3 as described in the text. Survival was then monitored: the survival duration in animals receiving treatment using AdHTVP2G5-rev-casp3, flavopiridol or flavopiridol + AdHTVP2G5-rev-casp3 were significantly differently from those in the mice that received treatment using controls (P < 0.01). In addition, synergisms of AdHTVP2G5-rev-casp3 and flavopiridol were observed, and significantly prolonged survival can be achieved using combined AdHTVP2-G5-rev-casp3 and flavopiridol therapy without an increase in doses(P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4302516&req=5

Fig4: In vivoantitumor activity. A: Subcutaneous tumors derived from OVCAR3 cells were treated as shown. Tumor volumes were monitored over time (days) after inoculation of tumor cells. Values represent the mean of 5 mice per group. B: 1:PBS, 2: flavopiridol (10 mg/kg), 3: AdHTVP2G5-rev-casp3 (7 × 108 TCID50/mouse), 4: AdHTVP2G5-rev-casp3(7 × 108 TCID50/mouse) + flavopiridol(5 mg/kg). Survival curves and mean survival durations for animals bearing abdominally spread OVCAR3 tumors. The animals received three treatments using PBS, flavopiridol, AdHTVP2G5-rev-casp3 or flavopiridol + AdHTVP2G5-rev-casp3 as described in the text. Survival was then monitored: the survival duration in animals receiving treatment using AdHTVP2G5-rev-casp3, flavopiridol or flavopiridol + AdHTVP2G5-rev-casp3 were significantly differently from those in the mice that received treatment using controls (P < 0.01). In addition, synergisms of AdHTVP2G5-rev-casp3 and flavopiridol were observed, and significantly prolonged survival can be achieved using combined AdHTVP2-G5-rev-casp3 and flavopiridol therapy without an increase in doses(P < 0.05).
Mentions: To evaluate the in vivo efficacy of the sequential combination treatment of AdHTVP2G5-rev-casp3 with flavopiridol, we established mouse xenograft model by inoculating OVCAR3 cells subcutaneously in the nude mice and treated the mice with AdHTVP2G5-rev-casp3 and/or flavopiridol. Immunohistochemistry has confirmed the OVCAR3 xenograft. Fifty-three days after treatment, the tumor volume was 1458 ± 326, 1317 ± 295 and 487 ± 63 mm3 in mice bearing OVCAR3 xenograft treated with AdHTVP2G5-rev-casp3, flavopiridol and the sequential combination of AdHTVP2G5-rev-casp3 and flavopiridol, respectively. All treatments showed significant suppression of tumor growth at the end point of the study, with the tumor growth suppression rate of 33.6%, 40.1% and 77.8% for AdHTVP2G5-rev-casp3, flavopiridol and their combination, respectively (P < 0.05), as shown in Figure 4a. Moreover, we observed the synergism against xenograft growth between AdHTVP2G5-rev-casp3 and flavopiridol in vivo.Figure 4

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, ShengJing Hospital, China Medical University, No. 36, Sanhao street, Heping District, Shen yang, 110004, China. ssyue@sohu.com.

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.

Show MeSH
Related in: MedlinePlus