Limits...
Potent anti-tumor effects of a dual specific oncolytic adenovirus expressing apoptin in vitro and in vivo.

Li X, Liu Y, Wen Z, Li C, Lu H, Tian M, Jin K, Sun L, Gao P, Yang E, Xu X, Kan S, Wang Z, Wang Y, Jin N - Mol. Cancer (2010)

Bottom Line: When treated with Ad-hTERT-E1a-Apoptin, the subcutaneous primary tumor volume reduction was not only observed in intratumoral injection group but in systemic delivery mice.In the lung metastasis model, Ad-hTERT-E1a-Apoptin effectively suppressed pulmonary metastatic lesions.Furthermore, treatment of primary and metastatic models with Ad-hTERT-E1a-Apoptin increased mice survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetic Engineering Laboratory of PLA, Academy of Military Medical Sciences of PLA, Changchun, China.

ABSTRACT

Background: Oncolytic virotherapy is an attractive drug platform of cancer gene therapy, but efficacy and specificity are important prerequisites for success of such strategies. Previous studies determined that Apoptin is a p53 independent, bcl-2 insensitive apoptotic protein with the ability to specifically induce apoptosis in tumor cells. Here, we generated a conditional replication-competent adenovirus (CRCA), designated Ad-hTERT-E1a-Apoptin, and investigated the effectiveness of the CRCA a gene therapy agent for further clinical trials.

Results: The observation that infection with Ad-hTERT-E1a-Apoptin significantly inhibited growth of the melanoma cells, protecting normal human epidermal melanocytes from growth inhibition confirmed cancer cell selective adenoviral replication, growth inhibition, and apoptosis induction of this therapeutic approach. The in vivo assays performed by using C57BL/6 mice containing established primary or metastatic tumors expanded the in vitro studies. When treated with Ad-hTERT-E1a-Apoptin, the subcutaneous primary tumor volume reduction was not only observed in intratumoral injection group but in systemic delivery mice. In the lung metastasis model, Ad-hTERT-E1a-Apoptin effectively suppressed pulmonary metastatic lesions. Furthermore, treatment of primary and metastatic models with Ad-hTERT-E1a-Apoptin increased mice survival.

Conclusions: These data further reinforce the previously research showing that an adenovirus expressing Apoptin is more effective and advocate the potential applications of Ad-hTERT-E1a-Apoptin in the treatment of neoplastic diseases in future clinical trials.

Show MeSH

Related in: MedlinePlus

Assessment of the selective inhibition effect of Ad-hTERT-E1a-Apoptin on melanoma cells. Effects of the different MOIs and infection times on (A), A375 cell viability, (B) B16 cell viability, and (C), on HEM cell viability. Cells were seeded in 96-well plates (1 × 104 cells/well) one day before cells were infected with various concentrations (1 MOI, 10 MOI, and 100 MOI) of the indicated adenoviruses. Tumor viability was measured every day over a 4 days period by MTT colorimetric assay and all measurements were performed in triplicate. Data are presented as mean ± SD. In normal HEM human epidermal melanocytes (C), infection with Ad-CMV-E1a or Ad-CMV-E1a-Apoptin, but not Ad-CMV-Apoptin, Ad-hTERT-Apoptin, Ad-hTERT-E1a, or Ad-hTERT-E1a-Apoptin, induced growth inhibition. In contrast, in A375 (A) and B16 (B) melanoma cells, Ad-hTERT-E1a-Apoptin, Ad-hTERT-E1a-Apoptin, Ad-CMV-E1a, and Ad-hTERT-E1a infection resulted in significant growth inhibition. 1. Ad-mock; 2. Ad-CMV-Apoptin; 3. Ad-hTERT-Apoptin; 4. Ad-CMV-E1a; 5. Ad-hTERT-E1a; 6. Ad-CMV-E1a-Apoptin; 7. Ad-hTERT-E1a-Apoptin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2818692&req=5

Figure 2: Assessment of the selective inhibition effect of Ad-hTERT-E1a-Apoptin on melanoma cells. Effects of the different MOIs and infection times on (A), A375 cell viability, (B) B16 cell viability, and (C), on HEM cell viability. Cells were seeded in 96-well plates (1 × 104 cells/well) one day before cells were infected with various concentrations (1 MOI, 10 MOI, and 100 MOI) of the indicated adenoviruses. Tumor viability was measured every day over a 4 days period by MTT colorimetric assay and all measurements were performed in triplicate. Data are presented as mean ± SD. In normal HEM human epidermal melanocytes (C), infection with Ad-CMV-E1a or Ad-CMV-E1a-Apoptin, but not Ad-CMV-Apoptin, Ad-hTERT-Apoptin, Ad-hTERT-E1a, or Ad-hTERT-E1a-Apoptin, induced growth inhibition. In contrast, in A375 (A) and B16 (B) melanoma cells, Ad-hTERT-E1a-Apoptin, Ad-hTERT-E1a-Apoptin, Ad-CMV-E1a, and Ad-hTERT-E1a infection resulted in significant growth inhibition. 1. Ad-mock; 2. Ad-CMV-Apoptin; 3. Ad-hTERT-Apoptin; 4. Ad-CMV-E1a; 5. Ad-hTERT-E1a; 6. Ad-CMV-E1a-Apoptin; 7. Ad-hTERT-E1a-Apoptin.

Mentions: As expected, with longer infection times, growth of A375 or B16 cells infected with Ad-CMV-E1a, Ad-hTERT-E1a, Ad-CMV-E1a-Apoptin and Ad-hTERT-E1a-Apoptin were inhibited. However, cells infected with replication-incompetent adenoviruses (Ad-CMV-Apoptin, Ad-hTERT-Apoptin and Ad-mock) gradually resumed growth after 2 d. In contrast, the growth of HEM cells infected only with Ad-CMV-E1a or Ad-CMV-E1a-Apoptin, in which viral replication is controlled by the CMV promoter, were suppressed. As shown in Figure 2, the cell viabilities were dependent on the MOI of the recombinant adenoviruses to some extent. In A375 and B16 cells, infection with Ad-CMV-E1a or Ad-hTERT-E1a at an MOI of 10 or 100 induced cell growth inhibition of 30%-40% and 60%-70% after 4 d, respectively. When infected with 1 MOI or 10 MOI of Ad-CMV-E1a-Apoptin or Ad-hTERT-E1a-Apoptin, cell growth was inhibited by 10%-20% and 50%-60% after 4 d, respectively, and the growth of cells infected with 100 MOI was almost blocked (70%-80%). However, in HEM cells, the dose-dependent inhibition was only observed in the Ad-CMV-E1a and Ad-CMV-E1a-Apoptin experimental groups. It is notable that, in A375 and B16 cells, the replication-competent adenoviruses were much more potent than the replication-incompetent adenoviruses in cancer cell suppression. Furthermore, the growth inhibition of 10 MOI Ad-CMV-E1a-Apoptin or Ad-hTERT-E1a-Apoptin infected cells were similar to that of 100 MOI ofAd-CMV-E1a or Ad-hTERT-E1a, indicating that the adenoviruses with the dual cancer-specific genes were more effective than the normal replication-incompetent adenoviruses in inhibiting cell growth. That is, Ad-hTERT-E1a-Apoptin could replicate specifically in melanoma cells (A375 and B16) and restrict the cell growth selectively, and exhibited higher tumor-specific killing activity than the control viruses. The interaction of infection time and MOI was complex and synergistic, and cell viability showed an apparent dependent relationship on both factors.


Potent anti-tumor effects of a dual specific oncolytic adenovirus expressing apoptin in vitro and in vivo.

Li X, Liu Y, Wen Z, Li C, Lu H, Tian M, Jin K, Sun L, Gao P, Yang E, Xu X, Kan S, Wang Z, Wang Y, Jin N - Mol. Cancer (2010)

Assessment of the selective inhibition effect of Ad-hTERT-E1a-Apoptin on melanoma cells. Effects of the different MOIs and infection times on (A), A375 cell viability, (B) B16 cell viability, and (C), on HEM cell viability. Cells were seeded in 96-well plates (1 × 104 cells/well) one day before cells were infected with various concentrations (1 MOI, 10 MOI, and 100 MOI) of the indicated adenoviruses. Tumor viability was measured every day over a 4 days period by MTT colorimetric assay and all measurements were performed in triplicate. Data are presented as mean ± SD. In normal HEM human epidermal melanocytes (C), infection with Ad-CMV-E1a or Ad-CMV-E1a-Apoptin, but not Ad-CMV-Apoptin, Ad-hTERT-Apoptin, Ad-hTERT-E1a, or Ad-hTERT-E1a-Apoptin, induced growth inhibition. In contrast, in A375 (A) and B16 (B) melanoma cells, Ad-hTERT-E1a-Apoptin, Ad-hTERT-E1a-Apoptin, Ad-CMV-E1a, and Ad-hTERT-E1a infection resulted in significant growth inhibition. 1. Ad-mock; 2. Ad-CMV-Apoptin; 3. Ad-hTERT-Apoptin; 4. Ad-CMV-E1a; 5. Ad-hTERT-E1a; 6. Ad-CMV-E1a-Apoptin; 7. Ad-hTERT-E1a-Apoptin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2818692&req=5

Figure 2: Assessment of the selective inhibition effect of Ad-hTERT-E1a-Apoptin on melanoma cells. Effects of the different MOIs and infection times on (A), A375 cell viability, (B) B16 cell viability, and (C), on HEM cell viability. Cells were seeded in 96-well plates (1 × 104 cells/well) one day before cells were infected with various concentrations (1 MOI, 10 MOI, and 100 MOI) of the indicated adenoviruses. Tumor viability was measured every day over a 4 days period by MTT colorimetric assay and all measurements were performed in triplicate. Data are presented as mean ± SD. In normal HEM human epidermal melanocytes (C), infection with Ad-CMV-E1a or Ad-CMV-E1a-Apoptin, but not Ad-CMV-Apoptin, Ad-hTERT-Apoptin, Ad-hTERT-E1a, or Ad-hTERT-E1a-Apoptin, induced growth inhibition. In contrast, in A375 (A) and B16 (B) melanoma cells, Ad-hTERT-E1a-Apoptin, Ad-hTERT-E1a-Apoptin, Ad-CMV-E1a, and Ad-hTERT-E1a infection resulted in significant growth inhibition. 1. Ad-mock; 2. Ad-CMV-Apoptin; 3. Ad-hTERT-Apoptin; 4. Ad-CMV-E1a; 5. Ad-hTERT-E1a; 6. Ad-CMV-E1a-Apoptin; 7. Ad-hTERT-E1a-Apoptin.
Mentions: As expected, with longer infection times, growth of A375 or B16 cells infected with Ad-CMV-E1a, Ad-hTERT-E1a, Ad-CMV-E1a-Apoptin and Ad-hTERT-E1a-Apoptin were inhibited. However, cells infected with replication-incompetent adenoviruses (Ad-CMV-Apoptin, Ad-hTERT-Apoptin and Ad-mock) gradually resumed growth after 2 d. In contrast, the growth of HEM cells infected only with Ad-CMV-E1a or Ad-CMV-E1a-Apoptin, in which viral replication is controlled by the CMV promoter, were suppressed. As shown in Figure 2, the cell viabilities were dependent on the MOI of the recombinant adenoviruses to some extent. In A375 and B16 cells, infection with Ad-CMV-E1a or Ad-hTERT-E1a at an MOI of 10 or 100 induced cell growth inhibition of 30%-40% and 60%-70% after 4 d, respectively. When infected with 1 MOI or 10 MOI of Ad-CMV-E1a-Apoptin or Ad-hTERT-E1a-Apoptin, cell growth was inhibited by 10%-20% and 50%-60% after 4 d, respectively, and the growth of cells infected with 100 MOI was almost blocked (70%-80%). However, in HEM cells, the dose-dependent inhibition was only observed in the Ad-CMV-E1a and Ad-CMV-E1a-Apoptin experimental groups. It is notable that, in A375 and B16 cells, the replication-competent adenoviruses were much more potent than the replication-incompetent adenoviruses in cancer cell suppression. Furthermore, the growth inhibition of 10 MOI Ad-CMV-E1a-Apoptin or Ad-hTERT-E1a-Apoptin infected cells were similar to that of 100 MOI ofAd-CMV-E1a or Ad-hTERT-E1a, indicating that the adenoviruses with the dual cancer-specific genes were more effective than the normal replication-incompetent adenoviruses in inhibiting cell growth. That is, Ad-hTERT-E1a-Apoptin could replicate specifically in melanoma cells (A375 and B16) and restrict the cell growth selectively, and exhibited higher tumor-specific killing activity than the control viruses. The interaction of infection time and MOI was complex and synergistic, and cell viability showed an apparent dependent relationship on both factors.

Bottom Line: When treated with Ad-hTERT-E1a-Apoptin, the subcutaneous primary tumor volume reduction was not only observed in intratumoral injection group but in systemic delivery mice.In the lung metastasis model, Ad-hTERT-E1a-Apoptin effectively suppressed pulmonary metastatic lesions.Furthermore, treatment of primary and metastatic models with Ad-hTERT-E1a-Apoptin increased mice survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetic Engineering Laboratory of PLA, Academy of Military Medical Sciences of PLA, Changchun, China.

ABSTRACT

Background: Oncolytic virotherapy is an attractive drug platform of cancer gene therapy, but efficacy and specificity are important prerequisites for success of such strategies. Previous studies determined that Apoptin is a p53 independent, bcl-2 insensitive apoptotic protein with the ability to specifically induce apoptosis in tumor cells. Here, we generated a conditional replication-competent adenovirus (CRCA), designated Ad-hTERT-E1a-Apoptin, and investigated the effectiveness of the CRCA a gene therapy agent for further clinical trials.

Results: The observation that infection with Ad-hTERT-E1a-Apoptin significantly inhibited growth of the melanoma cells, protecting normal human epidermal melanocytes from growth inhibition confirmed cancer cell selective adenoviral replication, growth inhibition, and apoptosis induction of this therapeutic approach. The in vivo assays performed by using C57BL/6 mice containing established primary or metastatic tumors expanded the in vitro studies. When treated with Ad-hTERT-E1a-Apoptin, the subcutaneous primary tumor volume reduction was not only observed in intratumoral injection group but in systemic delivery mice. In the lung metastasis model, Ad-hTERT-E1a-Apoptin effectively suppressed pulmonary metastatic lesions. Furthermore, treatment of primary and metastatic models with Ad-hTERT-E1a-Apoptin increased mice survival.

Conclusions: These data further reinforce the previously research showing that an adenovirus expressing Apoptin is more effective and advocate the potential applications of Ad-hTERT-E1a-Apoptin in the treatment of neoplastic diseases in future clinical trials.

Show MeSH
Related in: MedlinePlus