Abstract
Preclinical and clinical studies of CYP gene-directed enzyme prodrug therapy have been focused on anticancer prodrugs activated by CYP2B enzymes, which have low endogenous expression in human liver; however, the gene therapeutic potential of CYP3A enzymes, which are highly expressed in human liver, remains unknown. This study investigated methoxymorpholinyl doxorubicin (MMDX; nemorubicin), a novel CYP3A-activated anticancer prodrug. Retroviral transfer of CYP3A4 increased 9L gliosarcoma cell chemosensitivity to MMDX 120-fold (IC50=0.2 nM in 9L/3A4 cells). In CHO cells, overexpression of P450 reductase in combination with CYP3A4 enhanced chemosensitivity to MMDX, and to ifosfamide, another CYP3A4 prodrug, 11- to 23-fold compared with CYP3A4 expression alone. CYP3A4 expression and MMDX chemosensitivity were increased in human lung (A549) and brain (U251) tumor cells infected with replication-defective adenovirus encoding CYP3A4. Coinfection with Onyx-017, a replication-conditional adenovirus that coamplifies and coreplicates the Adeno-3A4 virus, led to large increases in CYP3A4 RNA but only modest increases in CYP3A4 protein and activity. MMDX induced remarkable growth delay of 9L/3A4 tumors, but not the P450-deficient parental 9L tumors, in immunodeficient mice administered low-dose MMDX either intravenous or by direct intratumoral (i.t.) injection (60 μg kg−1, every 7 days × 3). Notably, the i.t. route was substantially less toxic to the mouse host. No antitumor activity was observed with intraperitoneal MMDX treatment, suggesting a substantial hepatic first pass effect, with activated MMDX metabolites formed in the liver having poor access to the tumor site. These studies demonstrate that human CYP3A4 has strong potential for MMDX prodrug-activation therapy and suggest that endogenous tumor cell expression of CYP3A4, and not hepatic CYP3A4 activity, is a key determinant of responsiveness to MMDX therapy in cancer patients in vivo.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Abbreviations
- CYP or P450:
-
cytochrome P450
- DMEM:
-
Dulbecco's modified Eagle's medium
- FBS:
-
fetal bovine serum
- GDEPT:
-
gene-directed enzyme prodrug therapy
- IFA:
-
ifosfamide
- MMDX:
-
methoxymorpholinyl doxorubin, also known as nemorubicin
- MOI:
-
multiplicity of infection
References
Michael M, Doherty MM . Tumoral drug metabolism: overview and its implications for cancer therapy. J Clin Oncol 2005; 23: 205–229.
Moolten FL . Drug sensitivity (‘suicide’) genes for selective cancer chemotherapy. Cancer Gene Ther 1994; 1: 279–287.
Roy P, Waxman DJ . Activation of oxazaphosphorines by cytochrome P450: application to gene-directed enzyme prodrug therapy for cancer. Toxicol In Vitro 2006; 20: 176–186.
Chen L, Yu LJ, Waxman DJ . Potentiation of cytochrome P450/cyclophosphamide-based cancer gene therapy by coexpression of the P450reductase gene. Cancer Res 1997; 57: 4830–4837.
Chase M, Chung RY, Chiocca EA . An oncolytic viral mutant that delivers the CYP2B1 transgene and augments cyclophosphamide chemotherapy. Nat Biotechnol 1998; 16: 444–448.
Jounaidi Y, Chen CS, Veal GJ, Waxman DJ . Enhanced antitumor activity of P450prodrug-based gene therapy using the low Km cyclophosphamide 4-hydroxylase P4502B11. Mol Cancer Ther 2006; 5: 541–555.
Jounaidi Y, Hecht JE, Waxman DJ . Retroviral transfer of human cytochrome P450genes for oxazaphosphorine-based cancer gene therapy. Cancer Res 1998; 58: 4391–4401.
Tyminski E, Leroy S, Terada K, Finkelstein DM, Hyatt JL, Danks MK et al. Brain tumor oncolysis with replication-conditional herpes simplex virus type 1 expressing the prodrug-activating genes, CYP2B1 and secreted human intestinal carboxylesterase, in combination with cyclophosphamide and irinotecan. Cancer Res 2005; 65: 6850–6857.
Niculescu-Duvaz I, Springer CJ . Introduction to the background, principles, and state of the art in suicide gene therapy. Mol Biotechnol 2005; 30: 71–88.
Zhang Y, Parker WB, Sorscher EJ, Ealick SE . PNP anticancer gene therapy. Curr Top Med Chem 2005; 5: 1259–1274.
Searle PF, Chen MJ, Hu L, Race PR, Lovering AL, Grove JI et al. Nitroreductase: a prodrug-activating enzyme for cancer gene therapy. Clin Exp Pharmacol Physiol 2004; 31: 811–816.
Trask TW, Trask RP, Aguilar-Cordova E, Shine HD, Wyde PR, Goodman JC et al. Phase I study of adenoviral delivery of the HSV-tk gene and ganciclovir administration in patients with current malignant brain tumors. Mol Ther 2000; 1: 195–203.
Cunningham C, Nemunaitis J . A phase I trial of genetically modified Salmonella typhimurium expressing cytosine deaminase (TAPET-CD, VNP20029) administered by intratumoral injection in combination with 5-fluorocytosine for patients with advanced or metastatic cancer. Protocol no: CL-017. Version: April 9, 2001. Hum Gene Ther 2001; 12: 1594–1596.
Freytag SO, Stricker H, Peabody J, Pegg J, Paielli D, Movsas B et al. Five-year follow-up of trial of replication-competent adenovirus-mediated suicide gene therapy for treatment of prostate cancer. Mol Ther 2007; 15: 636–642.
Braybrooke JP, Slade A, Deplanque G, Harrop R, Madhusudan S, Forster MD et al. Phase I study of MetXia-P450gene therapy and oral cyclophosphamide for patients with advanced breast cancer or melanoma. Clin Cancer Res 2005; 11: 1512–1520.
Chen L, Waxman DJ . Intratumoral activation and enhanced chemotherapeutic effect of oxazaphosphorines following cytochrome P-450 gene transfer: development of a combined chemotherapy/cancer gene therapy strategy. Cancer Res 1995; 55: 581–589.
Samel S, Keese M, Lux A, Jesnowski R, Prosst R, Saller R et al. Peritoneal cancer treatment with CYP2B1 transfected, microencapsulated cells and ifosfamide. Cancer Gene Ther 2006; 13: 65–73.
Jounaidi Y, Waxman DJ . Combination of the bioreductive drug tirapazamine with the chemotherapeutic prodrug cyclophosphamide for P450/P450-reductase-based cancer gene therapy. Cancer Res 2000; 60: 3761–3769.
McCarthy HO, Yakkundi A, McErlane V, Hughes CM, Keilty G, Murray M et al. Bioreductive GDEPT using cytochrome P4503A4 in combination with AQ4N. Cancer Gene Ther 2003; 10: 40–48.
Ghielmini M, Colli E, Bosshard G, Pennella G, Geroni C, Torri V et al. Hematotoxicity on human bone marrow- and umbilical cord blood-derived progenitor cells and in vitro therapeutic index of methoxymorpholinyldoxorubicin and its metabolites. Cancer Chemother Pharmacol 1998; 42: 235–240.
Kuhl JS, Duran GE, Chao NJ, Sikic BI . Effects of the methoxymorpholino derivative of doxorubicin and its bioactivated form versus doxorubicin on human leukemia and lymphoma cell lines and normal bone marrow. Cancer Chemother Pharmacol 1993; 33: 10–16.
Lau DH, Duran GE, Lewis AD, Sikic BI . Metabolic conversion of methoxymorpholinyl doxorubicin: from a DNA strand breaker to a DNA cross-linker. Br J Cancer 1994; 70: 79–84.
Lewis AD, Lau DH, Duran GE, Wolf CR, Sikic BI . Role of cytochrome P-450 from the human CYP3A gene family in the potentiation of morpholino doxorubicin by human liver microsomes. Cancer Res 1992; 52: 4379–4384.
Baldwin A, Huang Z, Jounaidi Y, Waxman DJ . Identification of novel enzyme–prodrug combinations for use in cytochrome P450-based gene therapy for cancer. Arch Biochem Biophys 2003; 409: 197–206.
Quintieri L, Geroni C, Fantin M, Battaglia R, Rosato A, Speed W et al. Formation and antitumor activity of PNU-159682, a major metabolite of nemorubicin in human liver microsomes. Clin Cancer Res 2005; 11: 1608–1617.
Alvino E, Gilberti S, Cantagallo D, Massoud R, Gatteschi A, Tentori L et al. In vitro antitumor activity of 3′-desamino-3′(2-methoxy-4-morpholinyl) doxorubicin on human melanoma cells sensitive or resistant to triazene compounds. Cancer Chemother Pharmacol 1997; 40: 180–184.
Lu H, Waxman DJ . Antitumor activity of methoxymorpholinyl doxorubicin: potentiation by cytochrome P4503A metabolism. Mol Pharmacol 2005; 67: 212–219.
Quintieri L, Rosato A, Napoli E, Sola F, Geroni C, Floreani M et al. In vivo antitumor activity and host toxicity of methoxymorpholinyl doxorubicin: role of cytochrome P4503A. Cancer Res 2000; 60: 3232–3238.
Ding S, Yao D, Burchell B, Wolf CR, Friedberg T . High levels of recombinant CYP3A4 expression in Chinese hamster ovary cells are modulated by coexpressed human P450reductase and hemin supplementation. Arch Biochem Biophys 1997; 348: 403–410.
Kozak M . Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 1986; 44: 283–292.
Finer MH, Dull TJ, Qin L, Farson D, Roberts MR . kat: a high-efficiency retroviral transduction system for primary human T lymphocytes. Blood 1994; 83: 43–50.
Chen CS, Jounaidi Y, Waxman DJ . Enantioselective metabolism and cytotoxicity of R-ifosfamide and S-ifosfamide by tumor cell-expressed cytochromes P450. Drug Metab Dispos 2005; 33: 1261–1267.
Miwa GT, West SB, Lu AY . Studies on the rate-limiting enzyme component in the microsomal monooxygenase system. Incorporation of purified NADPH-cytochrome c reductase and cytochrome P-450 into rat liver microsomes. J Biol Chem 1978; 253: 1921–1929.
Jounaidi Y, Waxman DJ . Use of replication-conditional adenovirus as a helper system to enhance delivery of P450prodrug-activation genes for cancer therapy. Cancer Res 2004; 64: 292–303.
Kivisto KT, Kroemer HK, Eichelbaum M . The role of human cytochrome P450enzymes in the metabolism of anticancer agents: implications for drug interactions. Br J Clin Pharmacol 1995; 40: 523–530.
Haaz MC, Rivory L, Riche C, Vernillet L, Robert J . Metabolism of irinotecan (CPT-11) by human hepatic microsomes: participation of cytochrome P-450 3A and drug interactions. Cancer Res 1998; 58: 468–472.
Yao D, Ding S, Burchell B, Wolf CR, Friedberg T . Detoxication of vinca alkaloids by human P450CYP3A4-mediated metabolism: implications for the development of drug resistance. J Pharmacol Exp Ther 2000; 294: 387–395.
Chen L, Waxman DJ . Cytochrome P450gene-directed enzyme prodrug therapy (GDEPT) for cancer. Curr Pharm Des 2002; 8: 1405–1416.
Salmons B, Lohr M, Gunzburg WH . Treatment of inoperable pancreatic carcinoma using a cell-based local chemotherapy: results of a phase I/II clinical trial. J Gastroenterol 2003; 38 (Suppl 15): 78–84.
Martinez C, Garcia-Martin E, Pizarro RM, Garcia-Gamito FJ, Agundez JA . Expression of paclitaxel-inactivating CYP3A activity in human colorectal cancer: implications for drug therapy. Br J Cancer 2002; 87: 681–686.
Schmidt R, Baumann F, Knupfer H, Brauckhoff M, Horn LC, Schönfelder M et al. CYP3A4, CYP2C9 and CYP2B6 expression and ifosfamide turnover in breast cancer tissue microsomes. Br J Cancer 2004; 90: 911–916.
Kivisto KT, Fritz P, Linder A, Friedel G, Beaune P, Kroemer HK . Immunohistochemical localization of cytochrome P4503A in human pulmonary carcinomas and normal bronchial tissue. Histochem Cell Biol 1995; 103: 25–29.
Murray GI, McFadyen MC, Mitchell RT, Cheung YL, Kerr AC, Melvin WT . Cytochrome P450CYP3A in human renal cell cancer. Br J Cancer 1999; 79: 1836–1842.
Murray GI, Taylor VE, McKay JA, Weaver RJ, Ewen SW, Melvin WT et al. Expression of xenobiotic metabolizing enzymes in tumours of the urinary bladder. Int J Exp Pathol 1995; 76: 271–276.
Engels FK, Ten Tije AJ, Baker SD, Lee CK, Loos WJ, Vulto AG et al. Effect of cytochrome P4503A4 inhibition on the pharmacokinetics of docetaxel. Clin Pharmacol Ther 2004; 75: 448–454.
Crespi CL, Penman BW, Hu M . Development of Caco-2 cells expressing high levels of cDNA-derived cytochrome P4503A4. Pharm Res 1996; 13: 1635–1641.
Yoshitomi S, Ikemoto K, Takahashi J, Miki H, Namba M, Asahi S . Establishment of the transformants expressing human cytochrome P450subtypes in HepG2, and their applications on drug metabolism and toxicology. Toxicol In Vitro 2001; 15: 245–256.
Lengler J, Omann M, Duvier D, Holzmüller H, Gregor W, Salmons B et al. Cytochrome P450reductase dependent inhibition of cytochrome P4502B1 activity: implications for gene directed enzyme prodrug therapy. Biochem Pharmacol 2006; 72: 893–901.
Kaminsky LS, Guengerich FP . Cytochrome P-450 isozyme/isozyme functional interactions and NADPH–cytochrome P-450 reductase concentrations as factors in microsomal metabolism of warfarin. Eur J Biochem 1985; 149: 479–489.
Shimada T, Mernaugh RL, Guengerich FP . Interactions of mammalian cytochrome P450, NADPH-cytochrome P450reductase, and cytochrome b(5) enzymes. Arch Biochem Biophys 2005; 435: 207–216.
Vasey PA, Bissett D, Strolin-Benedetti M, Poggesi I, Breda M, Adams L et al. Phase I clinical and pharmacokinetic study of 3′-deamino-3′-(2-methoxy-4-morpholinyl)doxorubicin (FCE 23762). Cancer Res 1995; 55: 2090–2096.
Zangar RC, Bollinger N, Verma S, Karin NJ, Lu Y . The nuclear factor-kappa B pathway regulates cytochrome P4503A4 protein stability. Mol Pharmacol 2008; 73: 1652–1658.
Chen CS, Jounaidi Y, Su T, Waxman DJ . Enhancement of intratumoral cyclophosphamide pharmacokinetics and antitumor activity in a P4502B11-based cancer gene therapy model. Cancer Gene Ther 2007; 14: 935–944.
Ichikawa T, Petros WP, Ludeman SM, Fangmeier J, Hochberg FH, Colvin OM et al. Intraneoplastic polymer-based delivery of cyclophosphamide for intratumoral bioconversion by a replicating oncolytic viral vector. Cancer Res 2001; 61: 864–868.
Duvillard C, Polycarpe E, Romanet P, Chauffert B . [Intratumoral chemotherapy: experimental data and applications to head and neck tumors]. Ann Otolaryngol Chir Cervicofac 2007; 124: 53–60.
Celikoglu F, Celikoglu SI, Goldberg EP . Bronchoscopic intratumoral chemotherapy of lung cancer. Lung Cancer 2008; 61: 1–12.
Almond BA, Hadba AR, Freeman ST, Cuevas BJ, York AM, Detrisac CJ et al. Efficacy of mitoxantrone-loaded albumin microspheres for intratumoral chemotherapy of breast cancer. J Control Release 2003; 91: 147–155.
Acknowledgements
We thank Jie Ma for assistance with intravenous injections and Dr Thomas Friedberg (University of Dundee) for providing CYP3A4 cDNA and CHO cells expressing CYP3A4. This study was supported in part by NIH grant CA49248 (to DJW).
Author information
Authors and Affiliations
Corresponding author
Additional information
Supplementary Information accompanies the paper on Cancer Gene Therapy website (http://www.nature.com/cgt)
Supplementary information
Rights and permissions
About this article
Cite this article
Lu, H., Chen, CS. & Waxman, D. Potentiation of methoxymorpholinyl doxorubicin antitumor activity by P450 3A4 gene transfer. Cancer Gene Ther 16, 393–404 (2009). https://doi.org/10.1038/cgt.2008.93
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/cgt.2008.93
Keywords
This article is cited by
-
Effects of traditional herbal formulae on human CYP450 isozymes
Chinese Journal of Integrative Medicine (2017)
-
EET signaling in cancer
Cancer and Metastasis Reviews (2011)
-
Adenoviral delivery of pan-caspase inhibitor p35 enhances bystander killing by P450 gene-directed enzyme prodrug therapy using cyclophosphamide+
BMC Cancer (2010)
-
Gef gene therapy enhances the therapeutic efficacy of doxorubicin to combat growth of MCF-7 breast cancer cells
Cancer Chemotherapy and Pharmacology (2010)
-
Cytochrome P450-derived eicosanoids: the neglected pathway in cancer
Cancer and Metastasis Reviews (2010)