3.1 Construction of expression vector of pIRES2-TAT-apoptin-EGFP, pIRES2 -apoptin-EGFP and pIPRES2-TAT-EGFP
Apoptin, derived from chicken anemia virus, can successfully induce apoptosis in a wide range of human tumor cell lines. In contrast, normal cells are not affected when coexisting with apoptin protein. Thus, Apoptin is regarded as a potential anti-tumor gene drug to be used by transfection into tumor cells or fused with some CPPs such as TAT a transcriptional regulator protein of HIV-1.
In the present study, expression vectors of pIRES2-TAT-Apoptin-EGFP, pIRES2- Apoptin-EGFP and pIPRES2-TAT-EGFP were constructed and transfected into esophageal carcinoma cell lines. The construction progress was mentioned (Fig.1). DNA sequencing results showed that the recombinant plasmids were in accordance with the results as designed before.
3.2 Expression and subcellular localization of Apoptin and TAT-Apoptin
After transfection, Eca109 cells stained green fluorescence, indicating successful transfection of the recombinant vectors and expression within the cells. Following incubation with Rabbit anti-etag monoclonal antibody and the secondary goat anti-Rabbit antibody labeled with TRITC, Eca109 cells gave out red, which means the expression of Apoptin-etag and TAT-Apoptin-etag. As shown in Figure 2, Eca109 cells transfected with pIRES2-Apoptin-Etag-EGFP emitted both green and red, suggesting expression of both Apoptin and TAT-Apoptin and they were mainly located in the nucleus.
3.3 Inhibition effects of TAT-Apoptin and Apoptin on eca109 and te-1 cells
Eca109 and Te-1 cells were plated into 96-cell plates, and the DNA (ug): lipofectamine 2000 (ul) ratio was maintained at 1:1.5. As shown in Figure. 3A, the transfection efficiency was approximately 70%-80%. The corresponding MTT assay results were shown in Figure. 3B. After 24 or 48 hours of transfection, the viability of eca109 and te-1 cells treated with pIRES2-Apoptin-etag-EGFP and pIRES2-TAT-apoptin-etag-EGFP was significantly decreased compared to the cells treated with pIRES2-TAT-etag-EGFP, pIRES2-EGFP vector, and lipofectamine 2000 alone (p<0.05). The viability of cells treated with pIRES2-TAT-Apoptin-etag-EGFP was not different from that of cells treated with pIRES2-Apoptin-etag-EGFP (p>0.05) after 24h. However, after 48 hours, the viability of cells treated with pIRES2-TAT-Apoptin-etag-EGFP was significantly decreased compared to cells treated with pIRES2-Apoptin-etag-EGFP (p<0.05).
Fig.2 t-a-e-p: pIRES2-TAT-apoptin-etag- EGFP; a-e-p: pIRES2-apoptin-etag- EGFP; t-e-p: pIRES2-TAT--etag- EGFP; v-p: pIRES2-EGFP; lipo: lipofectamine2000 only.
3.4 Cell cycle distribution and apoptosis after transfection.
As shown in Fig. 4 and Fig. 5, both pIRES2-apoptin-etag-EGFP and pIRES2- TAT-Apoptin-etag-EGFP can induce G0/G1 in Eca109 and Te-1 cell lines after transfection. As shown in Fig. 4, Te-1 and Eca109 cells treated with pIRES2-Apoptin-etag-EGFP and pIRES2- TAT-Apoptin-etag-EGFP resulted in a significant increase in G0/G1 arrest, up to more than 50%, but there were no significant differences between the pIRES2-Apoptin-etag-EGFP and pIRES2- TAT-Apoptin-etag-EGFP groups.
As shown in Fig. 7, after transfection, both pIRES2-Apoptin-etag-EGFP and pIRES2- TAT-Apoptin-etag-EGFP can induce apoptosis in Eca109 cells. There were also no obvious differences between the two groups in apoptosis. This shows that both Apoptin and TAT-Apoptin both can induce apoptosis of esophageal tumor cells in vitro.