Gene therapy using vector-mediated transfer of
prodrug activating genes is a promising treatment approach for malignant
tumors. As demonstrated recently, the novel
prodrug activating gene coding for rabbit
cytochrome P450 4B1 (
CYP4B1) is able to induce
tumor cell death at low micromolar concentrations in
glioblastoma cells
after treatment with the
prodrug 4-ipomeanol (4-IM) in vitro and in vivo. The rabbit
CYP4B1 converts this
prodrug and other furane analogs and aromatic
amines, such as
2-aminoanthracene, to highly toxic alkylating metabolites, whereas the human
isoenzyme exhibits only minimal enzymatic activity. In the present study, the
cDNA encoding rabbit
CYP4B1 was used for pharmacogene
therapy of
hepatocellular carcinoma (HCC). Cell clones derived from the human HCC cell lines Hep3B, HuH-7, and HepG2 and stably expressing the chimeric
protein CYP4B1-EGFP (the
CYP4B1 coding sequence fused to the
enhanced green fluorescent protein (EGFP) gene) were selected. HCC clones expressing EGFP served as controls. 4-IM rapidly induced
tumor cell death in CYP4B1-EGFP-expressing clones at low concentrations (a 50% lethal dose of between 0.5 and 2 microg/mL). No signs of toxicity were found in control cells expressing EGFP even at high
prodrug concentrations (20 microg/mL). Cell death occurred by apoptosis and was independent of functional p53. A pronounced direct bystander effect was observed in Hep3B cells, whereas bystander HepG2 and HuH-7 cells were highly resistant to toxic 4-IM metabolites. These results demonstrate that the
CYP4B1/4-1M system efficiently and rapidly induces cell death in HCC cells, and that a cell line-specific mechanism may exist that limits the extent of the bystander effect of this novel
prodrug activating system.