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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.

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Induction of apoptosis selectively in melanoma cells by Ad-hTERT-E1a-Apoptin. (A) Flow cytometry analysis of A375 cells infected with the recombinant adenoviruses. (B) Fluorescence images of the adenovirus-infected A375 cells stained with Annexin V/PI. (C) Flow cytometry analysis of B16 cells infected with recombinant adenoviruses. (D) Fluorescence images of the adenovirus-infected B16 cells stained with Annexin V/PI. (E) Flow cytometry analysis of HEM cells infected with recombinant adenoviruses. (F) Fluorescence images of the adenovirus-infected HEM cells stained with Annexin V/PI. Representative images of three independent experiments at 100× magnification were used to show Annexin V binding. Infection with only Ad-CMV-E1a and Ad-CMV-E1A-Apoptin elevated the percentage of apoptotic normal HEM human epidermal melanocytes (E and F). However, all of the recombinant adenoviruses, except for Ad-mock, resulted in significant apoptosis in A375 (A and B) and B16 (C and D) melanoma cells. 1. Control; 2. Ad-mock; 3. Ad-CMV-Apoptin; 4. Ad-hTERT-Apoptin; 5. Ad-CMV-E1a; 6. Ad-hTERT-E1a; 7. Ad-CMV-E1a-Apoptin; 8. Ad-hTERT-E1a-Apoptin.
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Figure 3: Induction of apoptosis selectively in melanoma cells by Ad-hTERT-E1a-Apoptin. (A) Flow cytometry analysis of A375 cells infected with the recombinant adenoviruses. (B) Fluorescence images of the adenovirus-infected A375 cells stained with Annexin V/PI. (C) Flow cytometry analysis of B16 cells infected with recombinant adenoviruses. (D) Fluorescence images of the adenovirus-infected B16 cells stained with Annexin V/PI. (E) Flow cytometry analysis of HEM cells infected with recombinant adenoviruses. (F) Fluorescence images of the adenovirus-infected HEM cells stained with Annexin V/PI. Representative images of three independent experiments at 100× magnification were used to show Annexin V binding. Infection with only Ad-CMV-E1a and Ad-CMV-E1A-Apoptin elevated the percentage of apoptotic normal HEM human epidermal melanocytes (E and F). However, all of the recombinant adenoviruses, except for Ad-mock, resulted in significant apoptosis in A375 (A and B) and B16 (C and D) melanoma cells. 1. Control; 2. Ad-mock; 3. Ad-CMV-Apoptin; 4. Ad-hTERT-Apoptin; 5. Ad-CMV-E1a; 6. Ad-hTERT-E1a; 7. Ad-CMV-E1a-Apoptin; 8. Ad-hTERT-E1a-Apoptin.

Mentions: As shown in Figure 3, infection of all recombinant adenoviruses resulted in apoptosis of A375 and B16 cells, whereas in HEM cells, only infection with Ad-CMV-E1a and Ad-CMV-E1a-Apoptin induced apoptosis. In A375 and B16 cells, infection with the adenoviruses expressing Apoptin were observed predominantly in a late apoptotic stage; however, infection with Ad-CMV-E1a or Ad-hTERT-E1a were seen in mainly an early apoptotic stage. In HEM cells, because of the lost of the apoptotic inducing effect of Apoptin, Ad-CMV-E1a-Apoptin infected cells presented mainly in an early apoptotic stage similar to the Ad-CMV-E1a infected cells. These results indicated that Ad-hTERT-E1a-Apoptin induced apoptosis in melanoma cells specifically and induced apoptosis more rapidly than the control viruses.


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)

Induction of apoptosis selectively in melanoma cells by Ad-hTERT-E1a-Apoptin. (A) Flow cytometry analysis of A375 cells infected with the recombinant adenoviruses. (B) Fluorescence images of the adenovirus-infected A375 cells stained with Annexin V/PI. (C) Flow cytometry analysis of B16 cells infected with recombinant adenoviruses. (D) Fluorescence images of the adenovirus-infected B16 cells stained with Annexin V/PI. (E) Flow cytometry analysis of HEM cells infected with recombinant adenoviruses. (F) Fluorescence images of the adenovirus-infected HEM cells stained with Annexin V/PI. Representative images of three independent experiments at 100× magnification were used to show Annexin V binding. Infection with only Ad-CMV-E1a and Ad-CMV-E1A-Apoptin elevated the percentage of apoptotic normal HEM human epidermal melanocytes (E and F). However, all of the recombinant adenoviruses, except for Ad-mock, resulted in significant apoptosis in A375 (A and B) and B16 (C and D) melanoma cells. 1. Control; 2. Ad-mock; 3. Ad-CMV-Apoptin; 4. Ad-hTERT-Apoptin; 5. Ad-CMV-E1a; 6. Ad-hTERT-E1a; 7. Ad-CMV-E1a-Apoptin; 8. Ad-hTERT-E1a-Apoptin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Induction of apoptosis selectively in melanoma cells by Ad-hTERT-E1a-Apoptin. (A) Flow cytometry analysis of A375 cells infected with the recombinant adenoviruses. (B) Fluorescence images of the adenovirus-infected A375 cells stained with Annexin V/PI. (C) Flow cytometry analysis of B16 cells infected with recombinant adenoviruses. (D) Fluorescence images of the adenovirus-infected B16 cells stained with Annexin V/PI. (E) Flow cytometry analysis of HEM cells infected with recombinant adenoviruses. (F) Fluorescence images of the adenovirus-infected HEM cells stained with Annexin V/PI. Representative images of three independent experiments at 100× magnification were used to show Annexin V binding. Infection with only Ad-CMV-E1a and Ad-CMV-E1A-Apoptin elevated the percentage of apoptotic normal HEM human epidermal melanocytes (E and F). However, all of the recombinant adenoviruses, except for Ad-mock, resulted in significant apoptosis in A375 (A and B) and B16 (C and D) melanoma cells. 1. Control; 2. Ad-mock; 3. Ad-CMV-Apoptin; 4. Ad-hTERT-Apoptin; 5. Ad-CMV-E1a; 6. Ad-hTERT-E1a; 7. Ad-CMV-E1a-Apoptin; 8. Ad-hTERT-E1a-Apoptin.
Mentions: As shown in Figure 3, infection of all recombinant adenoviruses resulted in apoptosis of A375 and B16 cells, whereas in HEM cells, only infection with Ad-CMV-E1a and Ad-CMV-E1a-Apoptin induced apoptosis. In A375 and B16 cells, infection with the adenoviruses expressing Apoptin were observed predominantly in a late apoptotic stage; however, infection with Ad-CMV-E1a or Ad-hTERT-E1a were seen in mainly an early apoptotic stage. In HEM cells, because of the lost of the apoptotic inducing effect of Apoptin, Ad-CMV-E1a-Apoptin infected cells presented mainly in an early apoptotic stage similar to the Ad-CMV-E1a infected cells. These results indicated that Ad-hTERT-E1a-Apoptin induced apoptosis in melanoma cells specifically and induced apoptosis more rapidly than the control viruses.

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