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Prodrug-activating Gene Therapy with Rabbit Cytochrome P450 4B1/4-Ipomeanol or 2-Aminoanthracene System in Glioma Cells.

ABSTRACT: OBJECTIVE:We determined the cytotoxic properties of cytochrome P450 4B1 (CYP4B1) activated 4-ipomeanol (4-ipo) and 2-aminoanthracene (2-AA) in rat glioma to verify the CYP4B1/4-ipo or 2-AA system for prodrug-activating gene therapy. METHODS:The cyp4B1 cDNA was cloned into pcDNA3.1/Hygro from rabbit lung total RNA (pcDNA-cyp4B1). Lentiviral vector encoding firefly luciferase (fLuc) was infected into C6 (rat glioma), and the fLuc-expressing cell was selected (C6-L). After transfection with pcDNA-cyp4B1 vector into C6-L, the single clone expressing cyp4B1 gene was selected (C6-CL). Prodrug for various concentrations of 4-ipo or 2-AA was treated for 72 h and 96 h. The cell survival rate of C6-CL was determined using MTT assay and trypan-blue dye exclusion methods. RESULTS:By RT-PCR analysis, fLuc and CYP4B1 expression was detected in C6-CL, but not in C6. MTT assay and trypan-blue dye exclusion showed that IC50 of C6-CL was 0.3 mM and <0.01 mM after 4-ipo or 2-AA treatment at 96 h or 72 h exposure, respectively. Cell survivals of C6-CL were more rapidly reduced after treatment with 4-ipo or 2-AA than those of C6-L cells. The cell survival rate with MTT and trypan-blue dye exclusion assay was well correlated with fLuc activity in C6-CL cells. CONCLUSION:CYP4B1-based prodrug-activating gene therapy may have the potential to treat glioma and the cytotoxic effects of CYP4B1 enzyme activated 4-ipo or 2-AA in C6, and could be clearly determined by bioluminescent activity in C6-CL.

SUBMITTER: Jang SJ

PROVIDER: S-EPMC4042932 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Prodrug-activating Gene Therapy with Rabbit Cytochrome P450 4B1/4-Ipomeanol or 2-Aminoanthracene System in Glioma Cells.

Jang Su Jin SJ Kang Joo Hyun JH Lee Tae Sup TS Kim Sung Joo SJ Kim Kwang Il KI Lee Yong Jin YJ Cheon Gi Jeong GJ Choi Chang Woon CW Lim Sang Moo SM

Nuclear medicine and molecular imaging 20100807 3

<h4>Objective</h4>We determined the cytotoxic properties of cytochrome P450 4B1 (CYP4B1) activated 4-ipomeanol (4-ipo) and 2-aminoanthracene (2-AA) in rat glioma to verify the CYP4B1/4-ipo or 2-AA system for prodrug-activating gene therapy.<h4>Methods</h4>The cyp4B1 cDNA was cloned into pcDNA3.1/Hygro from rabbit lung total RNA (pcDNA-cyp4B1). Lentiviral vector encoding firefly luciferase (fLuc) was infected into C6 (rat glioma), and the fLuc-expressing cell was selected (C6-L). After transfecti ...[more]

PMID: 24899949

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Project description:Presently, we investigate the mechanisms whereby intratumoral expression of a cyclophosphamide-activating hepatic cytochrome P450 gene enhances therapeutic activity when cyclophosphamide is given on an every 6-day (metronomic) schedule. In P450-deficient 9L gliosarcomas grown in severe combined immunodeficient mice, metronomic cyclophosphamide substantially decreased tumor microvessel density and induced a approximately 70% loss of endothelial cells that began after the second cyclophosphamide treatment. These responses were accompanied by increased expression of the endogenous angiogenesis inhibitor thrombospondin-1 in tumor-associated host cells but by decreased expression in 9L tumor cells. These antiangiogenic responses preceded tumor regression and are likely key to the therapeutic activity of metronomic cyclophosphamide. Unexpectedly, 9L/2B11 tumors, grown from 9L cells infected with retrovirus encoding the cyclophosphamide-activating P450 2B11, exhibited antiangiogenic responses very similar to 9L tumors. This indicates that the tumor endothelial cell population is well exposed to liver-activated cyclophosphamide metabolites and that intratumoral P450 confers limited additional anti-endothelial cell bystander activity. In contrast, an increase in apoptosis, which preceded the antiangiogenic response, was substantially enhanced by intratumoral P450 2B11 expression. 9L/2B11 tumor regression was accompanied by an overall loss of tumor cellularity and by substantial enlargement of remaining P450-immunoreactive tumor cells as the number of P450-positive tumor cell decreased and the P450 protein content declined with cyclophosphamide treatment. We conclude that metronomic cyclophosphamide regresses P450-expressing tumors by two independent but complementary mechanisms: increased tumor cell killing via intratumoral P450-catalyzed prodrug activation, coupled with strong antiangiogenic activity, which is primarily associated with hepatic prodrug activation.

Dataset Information

Prodrug-activating Gene Therapy with Rabbit Cytochrome P450 4B1/4-Ipomeanol or 2-Aminoanthracene System in Glioma Cells.

Publications

Prodrug-activating Gene Therapy with Rabbit Cytochrome P450 4B1/4-Ipomeanol or 2-Aminoanthracene System in Glioma Cells.

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