Construction of CYP2E1 recombinant adenovirus vector and its anti-tumor effects

Abstract

To construct the human cytochrome P-450 CYP2E1 (CYP2E1) recombinant adenovirus vector and detect its anti-tumor effects in combination with chemotherapeutic drugs so as to provide rationales for gene directed enzyme prodrug therapy (GDEPT). CYP2E1 cDNA was cloned from human liver and a recombinant adenovirus vector constructed at a titer of 1 × 10(12) pfu/ml. Human bone marrow-derived mesenchymal stem cells (BMSCs) were separated, cultured, purified and detected. The tropism of BMSCs for cancer cells was detected by Transwell technique. These recombinant vectors were transferred into BMSCs and A375 cells and the expressions of EGFP and CYP2E1 detected by fluorescence microscope, RT-PCR and Western blot respectively. Inverted microscope, MTT and Annexin V-FITC/PI were employed to detect the anti-tumor effect of CYP2E1 recombinant adenovirus vectors in combination with chemotherapeutic prodrug dacarbazine (DTIC). The recombinant adenovirus vectors pAd5CMV-NpA-CYP2E1 and pAd5CMV-NpA-EGFP were constructed successfully. BMSCs were successfully separated and they could migrate in vitro through a polycarbonate filter toward K562 and A375 cells in the lower chamber. Fluorescence microscope was used to detect the expression of EGFP while both RT-PCR and Western blot revealed a high expression of CYP2E1 in gene-transfected group cells. The dead cell counts of co-culture group of gene-transfected BMSCs and wild type A375 cells were significantly higher than those of the control under inverted microscope. The results of MTT showed that the growth inhibition of gene-transfected group cells was increased with DTIC concentration in a concentration dependent manner. IC(50) of group BMSCs-CYP2E1 + A375 was (0.17 ± 0.13) mmol/L, and that of the control group was (0.65 ± 0.20) mmol/L (P < 0.01). The DTIC concentration at which BMSCs were relatively safe might be selected at 0.05 mmol/L. Annexin V-FITC/PI confirmed that the apoptosis rate of BMSCs-CYP2E1 + A375 group was significantly higher than that of the control group after a 48-hour treatment of 0.05 mmol/L DTIC (26.8 ± 2.0 vs 8.7 ± 1.3, P < 0.01). With an in vitro tropism for cancer cells, BMSCs transferred with CYP2E1 recombinant adenovirus vectors have anti-tumor effects probably synergistically with chemotherapeutic drugs.