Effects of hypoxia and limited diffusion in tumor cell microenvironment on bystander effect of P450 prodrug therapy.

Cytochrome P450 (CYP) enzyme 2B1 metabolizes the anticancer prodrug cyclophosphamide (CPA) to 4-hydroxy-CPA, which decomposes to the cytotoxic metabolites acrolein and phosphoramide mustard. We have evaluated the bystander cytotoxicity of CPA in combination with CYP2B1 gene-directed enzyme prodrug therapy using a cell culture-based agarose overlay technique. This method mimics the tumor microenvironment by limiting the diffusion of metabolites and by reducing the oxygen concentration to levels similar to those found in solid tumors. Under these conditions, the CYP activity of CYP2B1-expressing tumor cells was decreased by 80% compared to standard aerobic conditions. Despite this decrease in metabolic activity, a potent bystander effect was observed, resulting in up to 90% killing by CPA of a tumor cell population comprised of only approximately 20% CYP-expressing tumor cells. Similarly, transient transfection of a small fraction ( approximately 14%) of a human hepatoma Huh7 cell population with a CYP2B1 expression plasmid followed by short-term treatment with CPA (5 h) led to an eradication of 95% of the cells. No such bystander effect was observed without the agarose overlay. These findings suggest that the agarose overlay technique is very useful as an in vitro test system for investigation of the bystander effect of CYP/CPA and other enzyme/prodrug combinations under conditions that mimic the hypoxic conditions present in solid tumors in vivo.