Efficient Non-viral Gene Therapy With Del1 Fragments in Mice
Oral cancer patients account for only a small percentage of all cancer patients. There are several types of oral cancer, but around 90% of these are squamous cell carcinomas. The major treatments for oral cancer include surgery, chemotherapy, and radiotherapy, and cancer gene therapy approaches are currently being developed.
Del1 is an extracellular matrix (ECM) protein produced both by blood vessel endothelium and by hypertrophic chondrocytes in developing embryos. The protein includes the following five recognizable protein domains: three epidermal growth factor repeats (referred to as E1, E2 and E3), and two discoidin domains (referred to as C1 and C2). The E3 domain has the ability to increase transfection efficiency and, at high concentrations, it induces apoptosis 1). In addition, the C1 domain is essential for the deposition of Del1 in the ECM, and the E3 domain enhances this C1-mediated deposition 2). We have reported that a fusion protein comprising the E3 and C1 domains of Del1 (E3C1) showed promising results in an in vitro gene therapy model. In the present study, the efficiency of E3C1 was evaluated in an in vivo study and the mechanisms of apoptosis by Del1 were analyzed in vitro.
Materials & Methods:
For the in vivostudy, cells of the human oral squamous cell carcinoma cell line SCCKN were injected into nude mice in order to generate explanted tumors. cDNAs encoding E3C1 domains of Del1 (E3C1) were inserted into pcDNA3D and injected into tumors every 7 days with a transfection reagent, jet-PEI. For the in vitro study, Cos-7 cells were treated with Del1 or its mutants. Immunocytochemistry was performed in order to examine focal adhesion. Cell signaling molecules were analyzed by Western blot analysis.
Results:
Mice that were treated with negative control vector died over the 42-day observation period. In contrast, increases in tumor volume after treatment with the E3C1-encoding vector were suppressed, and 50% of the treated mice survived. Specimens from explanted tumor treated with the E3C1-encoding vector showed apoptosis.
The presence of Del1 in the ECM inhibited adhesion of COS-7 cells to ECM at an early stage. Decreases in phosphorylation of Bad were observed in conjunction with apoptosis induced by E3.
Conclusions:
The present study suggested that the E3C1 fragment induced apoptosis by disrupting cell-ECM adhesion. This activity may be used for cancer gene therapy. Repeated local injection of DNA for E3C1 using a non-viral vector may be a useful approach for cancer patients.
Reffernce:
1) Hisataka Kitano, Shinichiro Kokubun, Chiaki Hidai. The Extracellular Matrix Protein Del1 Induces Apoptosis via its Epidermal Growth Factor Motif, Biochemical and Biophysical Research Communications (BBRC)., 393(4):757-761, 2010.
2) Chiaki Hidai, Hisataka Kitano, Shinichiro Kokubun. The Del1 deposition domain can immobilize 3α-hydroxysteroid dehydrogenase in the extracellular matrix without interfering with enzymatic activity, Bioprocess Biosyst Eng., 32(5): 569-573, 2009.