Gene Therapy For Colorectal Cancer With Recombinant Adenovirus 5 Based On Loss Of The Insulin-like Growth Factor 2 Imprinting

Aim:To explore the feasibility of the recombinant adenovirus containing IGF2 imprinting system to gene-targeted therapy for human colorectal cancer (CRC) cell lines.Methods:(1) The H19 promoter, enhancer and CTCF fragments of human IGF2 imprinting were amplified by PCR and cloned into the plasmid pDC-312.(2) The E1A and EGFP fragments were amplified in TopK and pEGFP-C1 vector by PCR and then cloned into the recombinant plasmid with IGF2 Imprinting to construct a shuttle plasmid.(3) Both the shuttle plasmid pDC312-E1A and the skeleton plasmid Ad5 were transfected into HEK293 cell line by using lipo2000 after amplified and purified in Top10 competent cells, named Ad312-E1A and Ad312-EGFP.(4) HT-29 and HCT-8, SW480 and GES-1, showed loss of IGF2 imprinting (LOI) and maintenance of IGF2 imprinting (MO1), separately, were infected by Ad312-EGFP, and the expression of EGFP was detected.(5) Oncolytic adenovirus H101 was used as a positive control, four cell lines were infected by Ad312-E1A and H101 to detect mRNA and protein expression of E1A after 48 h. Meanwhile, anti-tumor effect of Ad312-E1A in vitro was verified by CCK-8 and flow cytometry.(6) The anti-tumor effect in vivo was assessed by xenografted models transfected with PBS, EGFP, H101 and Ad312-E1A, separately.Results:(1) The pDC312-E1A and Ad5 were successfully transfected into HEK293 cells, and EGFP expression was highest in 48 h. Cytopathic effect was changed as positive in 10 to 13 days, and the virus titer was appropriate (1×109 pfu/ml) by 3 rounds amplification of virus.(2) The recombinant adenovirus Ad312-EGFP was transfected into four cell lines, and the E1A was only expressed in IGF2 LOI cell lines (HT-29 and HCT-8) after 48 h.(3) Four cell lines were infected with the recombinant adenovirus Ad312-EGFP. After 48 h, mRNA and protein expression of E1A was only appeared in IGF2 LOI cells (HT-29 and HCT-8); The cell viability of HT-29 and HCT-8 transfected with 10 pfu/cell was decreased into (69.7±5.7)% and (65.2±7.1)%, separately, and cell apoptosis was increased into (33.4±4.1)% and (29.5±2.7)%, separately; Whereas, E1A was only expressed in HT-29 with p53 mutation after four cell lines infected with H101, the cell viability of HT-29 transfected with 10 pfu/cell was decreased into (59.4±2.4)%,and cell apoptosis was increased into (38.6±3.2)%.(4) Furthermore, the nude mice, carrying HT-29 xenografted tumors infected with PBS, EGFP, H101 and Ad312-E1A, were cured by muti-point injective method, indicating both H101 and Ad312-E1A significantly inhibited tumor growth and improved their prognoses.Conclusions:(1) We constructed successfully the recombinant adenoviruses Ad312-E1A, which carried E1A gene and IGF2 imprinting system.(2) We found that the Ad312-E1A could kill effectively the human colorectal cancer cell lines with IGF2 LOI in vivo and in vitro.(3) In the future, Ad312-E1A may be regarded as a novel way for CRC therapy, which provides a new strategy for CRC with gene-targeted therapy.