A novel Bifidobacterium infantis-mediated TK/GCV suicide gene therapy system exhibits antitumor activity in a rat model of bladder cancer

Abstract

Bladder cancer is the ninth most common malignancy in the world. Successful clinical management remains a challenge. In order To search for novel targeted and efficacious treatment, we sought to investigate anti-tumor activity of BI-TK suicide gene therapy system in a rat model of bladder tumors. We first constructed and tested an anaerobic Bifidobacterium infantis-mediated thymidine kinase (BI-TK) suicide gene therapy system. To test the in vivo efficacy of this system, we established a rat model of bladder tumors, which was induced by N-methyl-nitrosourea perfusion. Bifidobacterium infantis containing the HSV-TK (i.e., BI-TK) were constructed by transformation of recombinant plasmid pGEX - TK. The engineered BI-TK was injected into tumor-bearing rats via tail vein, followed by intraperitoneal injection of ganciclovir (GCV). Using the rat model of bladder tumors, we found that bladder tumor burdens were significantly lower in the rats treated with BI-TK/GCV group than that treated with normal saline control group (p <0.05). While various degrees of apoptosis of the tumor cells were detected in all groups using in situ TUNEL assay, apoptosis was mostly notable in the BI-TK/GCV treatment group. Immunohistochemical staining further demonstrated that the BI-TK/GCV treatment group had the highest level of caspase3 protein expression than that of the empty plasmid group and normal saline group (p < 0.05). Thus, our results demonstrate that the Bifidobacterium infantis-mediated TK/GCV suicide gene therapy system can effectively inhibit rat bladder tumor growth, possibly through increasing caspase 3 expression and inducing apoptosis.

Figure 1

Construction and verification of Bifidobacterium infantis-mediated TK tumor-targeting suicide gene therapy system. Plasmid DNA was purified from anaerobic culture, digested with restriction enzymes, and resolved on 1% agarose gel. The expected 6.0 kb fragment of pGEX-TK is indicated by arrows.

Figure 2

Histologic evaluation of the MNU-induced rat bladder cancer. MNU-induced bladder tumor samples were retrieved and subjected to paraffin-embedded sectioning and H & E staining. (A) Normal saline group, (B) Bifutobacterium infantis with empty plasmid group, and (C) Bifutobacterium infantis-PGEX-TK group. Representative samples are shown. Magnification, 100×.

Figure 3

Apoptosis analysis of BI-TK/GCV treated rat bladder cancer. The TUNEL assay was carried out as described in Methods. Cells with positive staining were randomly counted in 10 high-power fields and the apoptosis index was calculated (mean SD). (A) Normal saline group (6.88 ± 1.40), (B) Bifutobacterium infantis with empty plasmid group (16.01 ± 3.48), and (C) Bifutobacterium infantis-PGEX-TK group (41.72 ± 4.27). There is statistically significant difference between each groups (p < 0.05). Representative samples are shown. Magnification, 100×.

Figure 4

Immunohistochemical staining of Caspase 3 expression in BI-TK/GCV treated rat bladder cancer. The percentage of positive caspase 3 staining was 6.88 ± 1.40% for the normal saline group(A), 16.01 ± 3.48% for the BI-pGEX-5X-1 group(B), and 41.72 ± 4.27% for the BI-TK group(C), respectively. The differences between each group were statistically significant (p <0.05). ,100×.