Rabbit cytochrome P450 4B1: A novel prodrug activating gene for pharmacogene therapy of hepatocellular carcinoma

Abstract

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.

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

“…2I). In contrast, 2-AA 20, a previously proposed pro-drug for r-P422 as the suicide gene (Rainov et al, 1998;Mohr et al, 2000;Jang et al, 2010), induced specific cytotoxicity in r-P422, h-P427 and also h-P + 12 expressing cells albeit the pro-drug activation rate seemed to be the highest with the r-P422 enzyme (8 ± 1.1% viable cells after 48 h at 290 µM; Fig. 2J).…”

“…We and others have demonstrated the efficiency of the CYP4B1/4-IPO suicide gene system in human cells, either with the rabbit isoform r-P422 (Rainov et al, 1998;Mohr et al, 2000) or with the reengineered human isoform h-P + 12 Wiek et al, 2015;Roellecke et al, 2016). Since an endogenous substrate for any of the CYP4B1 isoforms is unknown and in order to better understand potential endogenous functions and also to find new cytotoxic pro-drugs, we created a library of potential substrates shown in Fig.…”

Mechanisms of cell death induced by suicide genes encoding purine nucleoside phosphorylase and thymidine kinase in human hepatocellular carcinoma cells in vitro

“…Tumor therapy based on the expression of suicide genes has become an attractive therapeutic strategy for human malignancies such as hepatocellular carcinoma (HCC), [1][2][3][4][5][6] which is a malignant tumor with increasing incidence. 7,8 HCC is highly resistant to chemotherapeutic agents and radiotherapy, 9 which may be caused by molecular changes occurring during oncogenesis such as overexpression of the multidrug resistance gene 10 and loss or mutations of tumor suppressor genes.…”

Gene therapy of hepatocarcinoma: a long way from the concept to the therapeutical impact

“…The possibilities to use the rabbit cytochrome p450 4B1, which induces cell death following the treatment of transduced cells with 4-ipomenaol, or the use of E. coli purine nucleoside phosphorylase gene, which converts purine analogs into toxic metabolites, are now under investigation and show some interest as an alternative to the HSV-TK/GCV strategy considering they also exhibit a strong bystander effect. 70,127 The comparison between the purine nucleoside phosphorylase (PNP) strategy and the HSV-TK/GCV showed the interest of using the PNP/fludarabine combination, which exhibits a stronger killing effect than HSV-TK/GCV in HCC cells line with a mutated p53 genotype. 128 From the immunomodulation to the generation of the antitumoral response Viral or nonviral vector-mediated transfer of genes coding for immunomodulatory molecules or Tumor Associated Antigens, TAA, may generate or reactivate the immune response against tumor cells.…”