In vitro and in vivo effect of 5-FC combined gene therapy with TNF-α and CD suicide gene on human laryngeal carcinoma cell line Hep-2

Abstract

This study was aimed to investigate the effect of combined cancer gene therapy with exogenous tumor necrosis factor-alpha (TNF-α) and cytosine deaminase (CD) suicide gene on laryngeal carcinoma cell line Hep-2 in vitro and in vivo. Transfection of the recombinant eukaryotic vectors of pcDNA3.1 (+) containing TNF-α and/or CD into Hep-2 cells resulted in expression of TNF-α and/or CD gene in vitro. The significant increase in apoptotic Hep-2 cells and decrease of Hep-2 cell proliferation were observed using 5-FC treatment combined with TNF-a expression by CD/5-FC suicide system. Moreover, bystander effect was also observed in the TNF-α and CD gene co-expression group. Laryngeal squamous cell carcinoma (LSCC) mice model was established by using BALB/c mice which different transfected Hep-2 cells with pcDNA3.1 (+) containing TNF-α and/or CD were applied subcutaneously. So these mice are divided into four groups, namely, (1)Hep-2/TIC group; (2)Hep-2/CD group; (3)Hep-2/TNF-α group; (4)Hep-2/0 group. At day 29 after cell inoculation, volume of grafted tumor had significant difference between each two of them (P<0.05). These results showed that the products of combined CD and TNF-α genes inhibited the growth of transplanted LSCC in mice model. So by our observed parameters and many others results, we hypothesized that 5-FC combined gene therapy with TNF-αand CD suicide gene should be an effective treatment on Laryngeal carcinoma.

Figure 1. Electrophoresis analysis was performed on the RT-PCR-amplified products of TNF-α gene and revealed 700 bp amplified segments (Fig. 1 A).

Whereas the amplified products of CD gene showed specific band between 1.2 kb and 1.5 kb, which was consistent with target gene in terms of gene length (Fig. 1 B) (M means DNA Marker and 1 mans PCR-amplified products of TNF-α gene in Fig. 1A; M means DNA Marker and 1 mans PCR-amplified products of CD gene in Fig. 1B).

Figure 2. When cleavaging pcDNA3.1 (+)-CD, agarose gel electrophoresis analysis on cleavaged products revealed a band between 1.2 kb and 1.5 kb, which was consistent with target band (Fig. 2 A).

When cleavaging pcDNA3.1(+)-CD -TNF-α, electrophoresis showed protein band at around 700 bp (Fig. 2 B). By cleavaging pcDNA 3.1(+)-TNF-α-IRES, respectively, TNF-α, IRES and CD were obtained (Fig. 2 C). M means DNA Marker and 1 mans pcDNA3.1(+)-CD/Xho Ι+Apa Ι in Fig. 2 A; M means DNA Marker and 1 mans pcDNA3.1(+)-CD -TNF-α/EcoR Ι+Not Ιin Fig. 2 B; 1 mans pcDNA 3.1(+)-TNF-α-IRES-CD/EcoRΙ+NotΙ, 2 means pcDNA 3.1(+)-TNF-α-IRES-CD/XhoΙ+NotΙ and 3 means pcDNA 3.1(+)-TNF-α-IRES-CD/XhoΙ+ApaΙ in Fig. 2 C.

Figure 3. For Hep-2/0 cells which were not transfected with TNF-α gene, no noticeable TNF-α mRNA was seen (Fig. 3 A, band 1).

While in transfected Hep-2/TIC and Hep-2/TNF-α cells, electrophoresis on RT-PCR products revealed specific bands of TNF-α mRNA (Fig. 3B, band 2; Fig. 3C, band 1.). In Hep-2/0 cells which were not transfected with CD gene, no mRNA of CD gene was detected (Fig. 3C, band 2). While in transfected Hep-2/TIC and Hep-2/CD cells, electrophoresis on RT-PCR products revealed specific bands of mRNA of CD gene (Fig. 3B, band 1; Fig. 3C, band 2).

Figure 4. Line graph of survival rates in cells treated by 5-FC at varied concentrations for 4 days.

Figure 5. By-stander effects in varied transfected cells.

Figure 6. Apoptotic rates in cells treated by 5-FC for 4 days.

Figure 7. Concentrations of extracellular TNF-a protein in varied cells culture.

Figure 8. Influence of in vivo combined treatment of CD and TNF-a genes on the growth of grafted laryngeal carcinoma.

The left picture shows the measured tumor tissues (A: Hep-2/TIC; B: Hep-2/CD; C: Hep-2/Hep-2/TNF-α; D: Hep-2/0). The right picture shows the growth curve of grafted laryngeal carcinoma.

Figure 9. Pathological changes in the sections of varied grafted tumor tissue (Hep-2/TIC group: large amount of necrotic tumor tissue, with considerable lymphocyte infiltration and rare tumor cell division; Hep-2/CD group: considerable amount of necrotic tissue, with a few infiltrating lymphocytes; Hep-2/TNF-α group: large amount of necrotic tissue, with extremely rare infiltrating lymphocytes; Hep-2/0 group: tumor tissue of lowly differentiated squamous cell carcinoma, with frequent giant cells and dividing tumor cells, no significant tumor necrosis or lymphocyte infiltration was seen).