The Anticancer Effect of Cytochrome P450 2E1 Gene Which Was Mediated by Recombination Adenovirus in Mesenchymal Stem Cells.

Dacarbazine (DTIC) can inhibit purine nucleotide synthesis after activated by P4502E1(CYP2E1) in liver and can be used for the treatment of malignant melanoma and Hodgkins lymphoma.

Objective: To explore the anticancer effect of the combination of CYP2E1 and DTIC.

Methods:

1. A full length cDNA fragment encoding the human CYP2E1 was cloned and was transfected into bone marrow mesenchymal stem cells (BMSCs) through recombinant adenovirus vector.

2. The recombinant adenovirus vectors pAd5CMV-CYP2E1 and pAd5CMV-EGFP were constructed and transfected into the packing cell lines HEK293 cells by liposome encapsulation method.

3. The expression of CYP2E1 gene was detected by Real-time PCR and Western Blot and contrasted to the cells which were transfected into the recombinant adenovirus including EGFP.

4. In vitro, gene transfected BMSCs were co-cultured with DTIC, and then the supernatant was added into the medium of human malignant melanoma M14 cells. Fluorescence microscopy, DNA Ladder, flowcytometry, electrophoresis were employed to tests the apoptosis, and MTT method was applied to detecting the IC50.

5. In the animal experiment, the CYP2E1 gene tranfected BMSCs were injected in combination with DTIC directly into the malignant melanomas on nude mice or through caudal vein, and the injected BMSCs transfected pAd5CMV-EGFP and DTIC in the same manner into nude control mice. The inhibiting effect of new planted tumors of experimental group was monitored through Immuno-histochemistry analysis and the shape and size of tumors were measured.

6. CYP2E1-EGFP gene tranfected BMSCs were injected into nude mice through caudal vein and tracked by fluorescence.

Results:

1. CYP2E1 gene(1482bp) was cloned successfully and it was conformed by DNA sequencing; Endonuclease and PCR analyses showed that recombinant adenovirus vector had been constructed and high titer recombinant adenovirosome(1×10¹²pfu/ml) had been packaged successfully.

2. BMSCs had been isolated and flowcytometry showed their phenotype were CD44(+),CD105(+), CD34(−)and CD45(−).

3. The mRNA and protein quantities of the group of CYP2E1 gene tranfected BMSCs were significantly higher than the control group (P<0.05, n=24).

4. In vitro, immunity fluorescence microscope, DNA Ladder, and flowcytometry all showed the supernatant of co-culture of CYP2E1 gene tranfected BMSCs and DTIC) significantly decreased the mortality of M14 cells compared with pAd5CMV-EGFP-BMSCs control group (P < 0. 05, n=24).

5. In vivo, the sizes of the tumor in the nude mice treated with transfected BMSCs and DTIC were significantly smaller than control case and changed along with concentration. (P< 0.05, n=20). No new tumor was found in the nude mouse inoculated with M14 cells after the injection of CYP2E1 transfected BMSCs and DTIC.

Conclusion: Recombination adenovirus vector was able to express CYP2E1 in BMSCs. Gene CYP2E1 transfected BMSCs and DTIC showed anticancer activity both in vitro and vivo.