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

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.

Figure 1

Schematic of the recombinant adenoviruses and the adenovirus-mediated transgene expression. (A) Schematic diagram depicting the organization elements in the recombinant adenoviruses. Polyadenylation signal sequence is designated as pA. The indicated cells were infected with Ad-mock (control) or indicated recombinant viruses at a MOI of 100 for 48 h. Western blot analysis was performed to detect (B) Apoptin or E1a protein from A375 cells, (C) Apoptin or E1a expression in B16 cells, or (D) Apoptin or E1a expression in HEM cells. Infection of normal HEM human epidermal melanocytes (D) with Ad-CMV-E1a or Ad-CMV-E1a-Apoptin, but not Ad-hTERT-E1a or Ad-hTERT-E1a-Apoptin, resulted in production of E1a proteins, whereas in A375 (B) and B16 (C) melanoma cells, infection with all these replication-competent recombinant adenoviruses generated E1a proteins. In HEM cells (D), infection with Ad-CMV-E1a-Apoptin and Ad-CMV-Apoptin resulted in Apoptin production, whereas infection with Ad-hTERT-Apoptin or Ad-hTERT-E1a-Apoptin resulted in barely detectable of Apoptin production. In A375 (B) and B16 (C) cells, infection with Ad-CMV-Apoptin, Ad-hTERT-Apoptin, Ad-CMV-E1a-Apoptin, or Ad-hTERT-E1a-Apoptin generated significant Apoptin production. 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.

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.

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.

Figure 4

Ad-hTERT-E1A-Apoptin suppression of melanoma in the C57BL/6 mice model. (A) Tumor growth kinetics of mice that received intratumorally injections. (B) Survival curve of mice treated intratumorally. (C) Tumor growth kinetics of mice that received intravenously injections. (D) Survival curve of mice treated intravenously. The day that the first injection performed was considered as starting day 0. Data were represented as mean ± SD (A and C). Ad-CMV-E1a-Apoptin or Ad-hTERT-E1a-Apoptin significantly inhibited the growth of tumors in both introtumoral (A) and systemic (C) delivery groups. Although Ad-CMV-Apoptin, Ad-hTERT-Apoptin, Ad-CMV-E1a or Ad-hTERT-E1a had some inhibitory effect on tumors in both experimental groups, the antitumor effects of Ad-CMV-Apoptin and Ad-hTERT-Apoptin in systemic delivery group were marginal (A and C). Furthermore, increased mean survival was also observed in Ad-CMV-E1a-Apoptin or Ad-hTERT-E1a-Apoptin treated mice in comparison with saline, Ad-mock, Ad-CMV-Apoptin, Ad-hTERT-Apoptin, Ad-CMV-E1a or Ad-hTERT-E1a treated mice in the tumor models (B and D). 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.

Figure 5

Ad-hTERT-E1a-Apoptin reduction of pulmonary metastatic melanoma and resulting survival benefits. (A) Survival curve. (B) Representative photographs of lungs from control and treatment groups. The day that the first injection performed was considered as starting day 0. Saline or Ad-mock treated mice had the worst mean survival and Ad-CMV-E1a-Apoptin or Ad-hTERT-E1a-Apoptin treated mice had the most improved mean survival (A). Furthermore, both numbers and sizes of lung tumor nodules were reduced in mice treated with Ad-CMV-E1a-Apoptin or Ad-hTERT-E1a-Apoptin compared with those treated with saline, Ad-mock, Ad-CMV-Apoptin, Ad-hTERT-Apoptin, Ad-CMV-E1a or Ad-hTERT-E1a (B). 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.