Targeted Anti-Tumor Research of Mesenchymal Stem Cells Delivered Enzyme-Prodrug Gene CYP450.

Cytochrome P450(CYP450-CYP1A2 /CYP2B6/CYP2C9) was transfected into human bone marrow-derived mesenchymal stem cells (hBMSCs), and the targed anti-tumor effect of BMSC-CYP450 cooperated with enzyme-prodrug(Dacarbazine (DTIC)/Cyclophosphamide (CPA)) was measured to provide laboratory data base for gene directed enzyme prodrug targeted anti-tumor therapy (GDEPT) which used BMSC as vehicles.

We respectively cloned CYP1A2/CYP2B6/CYP2C9 cDNA from human liver and constructed recombinant adenovirus vectors(pAd5CMV-NpA-CYP1A2/ pAd5CMV-NpA-CYP2B6/pAd5CMV-NpA-CYP2C9) which titer was 1×10¹² pfu/mL. These hBMSCs were separated, cultured, purified, and detected by morphology, flow cytometry, osteogenic, adipogenic and chondrogenic induction, and RT-PCR(A surface marker for the identification of MSCs-the neural ganglioside GD2 gene). The tropism of BMSCs for cancer cells was detected by Transwell inserts technique. These recombinant vectors were transferred into BMSCs and A375/K562 cells, and the expression of EGFP and CYP1A2/CYP2B6/CYP2C9 was detected by fluorescence microscope, RT-PCR and Western blot respectively. Inverted microscope, MTT and Annexin V-FITC/PI detected the anti-tumor effect of CYP450 recombinant adenovirus vectors combined with chemotherapeutic prodrug DTIC/CPA in vitro. A human melanoma(A375) BALB/c nude mice model and a human myelocytic leukemia(K562) BALB/c nude mice model was constructed and detected by immuno-histochemistry analysis. The CYP1A2 gene tranfected BMSCs were injected into the A375 BALB/c nude mice model in combination with DTIC through caudal vein, while CYP2B6/CYP2C9 gene tranfected BMSCs were injected into K562 BALB/c nude mice model in combination with CPA in the same way. The measurement of tumors size, fluorescence microscope and TUNEL were used to detect anti-tumor effect of BMSCs-CYP1A2 cooperating with DTIC and BMSCs-CYP2B6/CYP2C9 with CPA in vivo.

We constructed the recombinant adenovirus vectors pAd5CMV-NpA-CYP1A2/pAd5CMV-NpA-CYP2B6/pAd5CMV-NpA-CYP2C9 and pAd5CMV-NpA-EGFP successfully. BMSCs was separated successfully, and it respectively showed that BMSCs can migrate through the polycarbonate filter toward K562 and A375 cells in the lower chamber in vitro. Fluorescence microscope detected the expression of EGFP, while both RT-PCR and Western blot detected high expression of CYP1A2/CYP2B6/CYP2C9 in gene-transfected group cells. Inverted microscope, MTT and Annexin V-FITC/PI confirmed that BMSCs transferred with CYP1A2/CYP2B6/CYP2C9 recombinant adenovirus vectors could activate DTIC/CPA and increase its anti-tumor effect(In the DTIC/CPA concentration(0.05 mmol/L/2.5 mmol/L) which BMSCs was relatively safe, the cell apoptosis was (38.38±2.27)% (P<0.01), (42.69±2.03)% (P<0.01) and (39. 51±1.94)% (P<0.01) in BMSCs-CYP1A2+A375 group, BMSCs-CYP2B6+K562 group and BMSCs-CYP2C9+K562 group respectively. ). A375 and K562 BALB/c nude mice model was constructed successfully. The sizes of the tumor in the nude mice treated with transfected BMSCs and DTIC/CPA were significantly smaller than control case and changed along with concentration(P< 0.01, P< 0.05).BMSCs was congregated to tumor site in fluorescence microscope. Apoptosis of tumor cells was conspicuously more in BMSCs-CYP1A2+A375/BMSCs-CYP2B6+K562/BMSCs-CYP2C9+K562 treatment group than in control group by TUNEL.

BMSCs had the tropism for cancer cells in vitro and vivo. DTIC can be catalyzed by CYP2E1/CYP1A2, while CPA by CYP2B6/CYP2C9 in vitro and vivo. BMSC-based enzyme prodrug system of CYP2E1/CYP1A2 and DTIC can induce A375 cells apoptosis, while BMSC-based enzyme prodrug system of CYP2B6/CYP2C9 and CPA can induce K562 cells apoptosis in vitro and targetedly in vivo.

No relevant conflicts of interest to declare.