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Nicotinamide mononucleotide adenylyltransferase2 overexpression enhances colorectal cancer cell-kill by Tiazofurin

Cancer Gene Therapy volume 20, pages 403–412 (2013)Cite this article

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Abstract

Colorectal cancer cells exhibit limited cytotoxicity towards Tiazofurin, a pro-drug metabolized by cytosolic nicotinamide mononucleotide adenylyltransferase2 (NMNAT2) to thiazole-4-carboxamide adenine dinucleotide, a potent inhibitor of inosine 5′-monophosphate dehydrogenase required for cellular guanylate synthesis. We tested the hypothesis that colorectal cancer cells that exhibit low levels of NMNAT2 and are refractory to Tiazofurin can be rendered sensitive to Tiazofurin by overexpressing NMNAT2. Transfection of hNMNAT2 resulted in a six- and threefold cytoplasmic overexpression in Caco2 and HT29 cell lines correlating with Tiazofurin-induced enhanced cell-kill. Folate receptors expressed on the cell surface of 30–50% colorectal carcinomas were exploited for cellular targeting with Tiazofurin encapsulated in folate-tethered nanoparticles. Our results indicated that in wild-type colorectal cancer cells, free Tiazofurin-induced EC50 cell-kill was 1500–2000 μM, which was reduced to 66–156 μM in hNMNAT2-overexpressed cells treated with Tiazofurin encapsulated in non-targeted nanoparticles. This efficacy was improved threefold by encapsulating Tiazofurin in folate-tethered nanoparticles to obtain an EC50 cell-kill of 22–59 μM, an equivalent of 100–300 mg m−2 (one-tenth of the approved dose of Tiazofurin in humans), which will result in minimal toxicity leading to cancer cell-kill. This proof-of-principle study suggests that resistance of colorectal cancer cell-kill to Tiazofurin can be overcome by sequentially overexpressing hNMNAT2 and then facilitating the uptake of Tiazofurin by folate-tethered nanoparticles, which enter cells via folate receptors.

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Abbreviations

FR:

folate receptors-α

IMPDH:

inosine 5′-monophosphate dehydrogenase

NMNAT:

nicotinamide 5′-mononucleotide adenylyltransferase

TAD:

thiazole-4-carboxamide adenine dinucleotide

TRMP:

tiazofurin 5′-monophosphate

TR:

Tiazofurin (2-ß-D-ribofuranosylthiazole-4-carboxamide).

References

  1. André T, Boni C, Mounedji-Boudiaf L, Navarro M, Tabernero J, Hickish T et al. Multicenter International Study of Oxaliplatin/5-Fluorouracil/Leucovorin in the Adjuvant Treatment of Colon Cancer (MOSAIC) Investigators. N Engl J Med 2004; 350: 2343–2351.

    Article  Google Scholar 

  2. Wu C, Goldberg RM . Colorectal cancer in 2012: revisiting landmark trials and identifying new therapies. Nat Rev Clin Oncol 2013; 10: 71–72.

    Article  CAS  Google Scholar 

  3. de Gramont A, Van Cutsem E, Schmoll HJ, Tabernero J, Clarke S, Moore MJ et al. Bevacizumab plus oxaliplatin-based chemotherapy as adjuvant treatment for colon cancer (AVANT): a phase 3 randomised controlled trial. Lancet Oncol 2012; 13: 1255–1233.

    Article  Google Scholar 

  4. Arkenau HT, Arnold D, Cassidy J, Diaz-Rubio E, Douillard JY, Hochster H et al. Efficacy of oxaliplatin plus capecitabine or infusional fluorouracil/leucovorin in patients with metastatic colorectal cancer: a pooled analysis of randomized trials. J Clin Oncol 2008; 26: 5910–5917.

    Article  CAS  Google Scholar 

  5. Emanuelli M, Carnevali F, Saccucci F, Pierella F, Amici A, Raffaelli N et al. Molecular cloning, chromosomal localization, tissue mRNA levels, bacterial expression, and enzymatic properties of human NMN adenylyltransferase. J Biol Chem 2001; 276: 406–412.

    Article  CAS  Google Scholar 

  6. Schweiger M, Hennig K, Lerner F, Niere M, Hirsch-Kauffmann M, Specht T et al. Characterization of recombinant human nicotinamide mononucleotide adenylyltransferase (NMNAT), a nuclear enzyme essential for NAD synthesis. FEBS Lett 2001; 492: 95–100.

    Article  CAS  Google Scholar 

  7. Raffaelli N, Sorci L, Amici A, Emanuelli M, Mazzola F, Magni G . Identification of a novel human nicotinamide mononucleotide adenylyltransferase. Biochem Biophys Res Commun 2002; 297: 835–840.

    Article  CAS  Google Scholar 

  8. Zhang X, Kurnasov OV, Karthikeyan S, Grishin NV, Osterman AL, Zhang H . Structural characterization of a human cytosolic NMN/NaMN adenylyltransferase and implication in human NAD biosynthesis. J Biol Chem 2003; 278: 13503–13511.

    Article  CAS  Google Scholar 

  9. Yalowitz JA, Xiao S, Biju MP, Antony AC, Cummings OW, Deeg MA et al. Characterization of human brain nicotinamide 5′-mononucleotide adenylyltransferase-2 and expression in human pancreas. Biochem J 2004; 377: 317–326.

    Article  CAS  Google Scholar 

  10. Berger F, Lau C, Dahlmann M, Ziegler M . Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms. J Biol Chem 2005; 280: 36334–36341.

    Article  CAS  Google Scholar 

  11. Cooney DA, Jayaram HN, Gebeyehu G, Betts CR, Kelley JA, Marquez VE et al. The conversion of 2-ß-D-ribofuranosylthiazole-4-carboxamide to an analog of NAD with potent IMP dehydrogenase-inhibitory properties. Biochem Pharmacol 1982; 31: 2133–2136.

    Article  CAS  Google Scholar 

  12. Jayaram HN, Smith AL, Glazer RI, Johns DG, Cooney DA . Studies on the mechanism of action of 2-beta-D-ribofuranosylthiazole-4-carboxamide (NSC 286193)-II. Relationship between dose level and biochemical effects in P388 leukemia in vivo. Biochem Pharmacol 1982; 31: 3839–3845.

    Article  CAS  Google Scholar 

  13. Jayaram HN . Biochemical mechanisms of resistance to tiazofurin. Adv Enzyme Regul 1985; 24: 67–89.

    Article  CAS  Google Scholar 

  14. Jayaram HN, Zhen W, Gharehbaghi K . Biochemical consequences of resistance to tiazofurin in human myelogenous leukemic K562 cells. Cancer Res 1993; 53: 2344–2348.

    CAS  PubMed  Google Scholar 

  15. Tricot GJ, Jayaram HN, Lapis E, Natsumeda Y, Nichols CR, Kneebone P et al. Biochemically directed therapy of leukemia with tiazofurin, a selective blocker of inosine 5'- phosphate dehydrogenase activity. Cancer Res 1989; 49: 3696–3701.

    CAS  PubMed  Google Scholar 

  16. Wright DG, Boosalis MS, Waraska K, Oshry LJ, Weintraub LR, Vosburgh E . Tiazofurin effects on IMP-dehydrogenase activity and expression in the leukemia cells of patients with CML blast crisis. Anticancer Res 1996; 16: 3349–3351.

    CAS  PubMed  Google Scholar 

  17. Grifantini M, Tiazofurine ICN . Pharmaceuticals. Curr Opin Investig Drugs 2000; 1: 257–262.

    CAS  PubMed  Google Scholar 

  18. Maroun JA, Eisenhauer E, Cripps C, Maksymiuk A . Phase II study of tiazofurin in colorectal cancer: A National Cancer Institute of Canada study. Cancer Treat Rep 1987; 71: 1297–1298.

    CAS  PubMed  Google Scholar 

  19. Jayaram HN, Lapis E, Tricot G, Kneebone P, Paulik E, Zhen W et al. Clinical pharmaco kinetic study of tiazofurin administered as a 1-hour infusion. Int J Cancer 1992; 51: 182–188.

    Article  CAS  Google Scholar 

  20. Zhen W, Jayaram HN, Weber G . Antitumor activity of tiazofurin in human colon carcinoma HT-29. Cancer Invest 1992; 10: 505–511.

    Article  CAS  Google Scholar 

  21. Lapis KY, Bocsi J, Tovari J, Bartha I, Timar J, Raso E . Antiinvasive effects of Tiazofurin on liver-metastatic human colon carcinoma xenografts. Anticancer Res 1996; 16: 3323–3331.

    CAS  PubMed  Google Scholar 

  22. Ahluwalia GS, Jayaram HN, Plowman JP, Cooney DA, Johns DG . Studies on the mechanism of action of 2-ß-D-ribofuranosylthiazole-4-carboxamide-V. Factors governing the response of murine tumors to tiazofurin. Biochem Pharmacol 1984; 33: 1195–1203.

    Article  CAS  Google Scholar 

  23. Lee RJ, Low PS . Folate-mediated tumor cell targeting of liposome-entrapped doxorubicin in vitro. Biochim Biophys Acta 1995; 1233: 134–144.

    Article  Google Scholar 

  24. Zhang Y, Guo L, Roeske RW, Antony AC, Jayaram HN . Pteroyl-γ-glutamate-cysteine synthesis and its application in folate receptor-mediated cancer cell targeting using folate-tethered liposomes. Anal Biochem 2004; 332: 168–177.

    Article  CAS  Google Scholar 

  25. Xiang G, Wu J, Lu Y, Liu Z, Lee RJ . Synthesis and evaluation of novel ligand for folate mediated targeting liposomes. Int J Pharm 2008; 356: 29–36.

    Article  CAS  Google Scholar 

  26. Suzuki R, Takizawa T, Kuwata Y, Mutoh M, Ishiguro N, Utoguchi N et al. Effective anti-tumor activity of oxaliplatin encapsulated in transferrin-PEG-liposomes. Int J Pharm 2008; 346: 143–150.

    Article  CAS  Google Scholar 

  27. Kalli KR, Oberg AL, Keeney GL, Christianson TJ, Low PS, Knutson KL et al. Folate receptor alpha as a tumor target in epithelial ovarian cancer. Gynecol Oncol 2008; 108: 619–626.

    Article  CAS  Google Scholar 

  28. Shia J, Klimstra DS, Nitzkorski JR, Low PS, Gonen M, Landmann R et al. Immuno histochemical expression of folate receptor alpha in colorectal carcinoma: patterns and biological significance. Hum Pathol 2008; 39: 498–505.

    Article  CAS  Google Scholar 

  29. D’Angelica M, Ammori J, Gonen M, Klimstra DS, Low PS, Murphy L et al. Folate receptor-α expression in resectable hepatic colorectal cancer metastases: patterns and significance. Modern Pathol 2011; 24: 1221–1228.

    Article  Google Scholar 

  30. Xiao S, Tang YS, Khan RA, Zhang Y, Kusumanchi P, Stabler SP et al. Influence of physiologic folate deficiency on human papillomavirus type 16 (HPV16)-harboring human keratinocytes in vitro and in vivo. J Biol Chem 2012; 287: 12559–12577.

    Article  CAS  Google Scholar 

  31. Antony A, Tang YS, Khan RA, Biju MP, Xiao X, Li QJ et al. Translational upregulation of folate receptors is mediated by homocysteine via RNA-heterogeneous nuclear ribonucleoprotein E1 interactions. J Clin Invest 2004; 113: 285–301.

    Article  CAS  Google Scholar 

  32. Egan RW . Hydrophile-lipophile balance and critical micelle concentration as key factors influencing surfactant disruption of mitochondrial membranes. J Biol Chem 1976; 251: 4442–4447.

    CAS  PubMed  Google Scholar 

  33. Gharehbaghi K, Sreenath A, Hao Z, Paull KD, Szekers T, Cooney DA et al. Comparison of biochemical parameters of benzamide riboside, a new inhibitor of IMP dehydrogenase, with tiazofurin and selenazofurin. Biochem Pharmacol 1994; 48: 1413–1419.

    Article  CAS  Google Scholar 

  34. Nagai M, Natsumeda Y, Weber G . Proliferation-linked regulation of type II IMP dehydrogenase gene in human normal lymphocytes and HL-60 leukemic cells. Cancer Res 1992; 52: 258–261.

    CAS  PubMed  Google Scholar 

  35. Franchetti P, Cappellacci L, Pasqualini M, Petrelli R, Jayaprakasan V, Jayaram HN et al. Synthesis, conformational analysis, and biological activity of new analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) as IMP dehydrogenase inhibitors. Bioorg Med Chem 2005; 13: 2045–2053.

    Article  CAS  Google Scholar 

  36. Ahluwalia GS, Cooney DA, Marquez VE, Jayaram HN, Johns DG . Studies on the mechanism of action of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide). VI. Biochemical and pharmacological studies on the degradation of thiazole-4-carboxamide adenine dinucleotide (TAD). Biochem Pharmacol 1986; 35: 3783–3790.

    Article  CAS  Google Scholar 

  37. Pillwein K, Schuchter K, Ressmann G, Gharehbaghi K, Knoflach A, Cermak B et al. Cytotoxicity, differentiating activity and metabolism of tiazofurin in human neuroblastoma cells. Int J Cancer 1993; 55: 92–95.

    Article  CAS  Google Scholar 

  38. Gabizon AA . Pegylated liposomal doxorubicin: metamorphosis of an old drug into a new form of chemotherapy. Cancer Invest 2001; 19: 424–436.

    Article  CAS  Google Scholar 

  39. Low PS, Antony AC . Folate receptor-targeted drugs for cancer and inflammatory diseases. Adv Drug Deliv Rev 2004; 56: 1055–1058.

    Article  CAS  Google Scholar 

  40. Anderson RM, Bitterman KJ, Wood JG, Medvedik O, Cohen H, Lin SS et al. Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels. Biol Chem 2002; 277: 18881–18890.

    Article  CAS  Google Scholar 

  41. Guo X, Eans TRJ, Sumanth S, Armesilla AL, Darling JL, Schatzlein A et al. In vitro evaluation of cancer-specific NF-kB-CEA enhancer-promoter system for 5-fluorouracil prodrug gene therapy in colon cancer cells. Br J Cancer 2007; 97: 745–754.

    Article  CAS  Google Scholar 

  42. Wang Y, Canine BF, Hatefi A . HSV-TK/GCV cancer suicide gene therapy by a designed recombinant multifunctional vector. Nanomedicine 2011; 7: 193–200.

    Article  CAS  Google Scholar 

  43. Zhang J, Wei F, Wang H, Li H, Qiu W, Ren P et al. A novel oncolytic adenovirus expressing Escherichia coli cytosine deaminase exhibits potent antitumor effect on human solid tumors. Cancer Biother Radiopharm 2010; 25: 487–495.

    Article  CAS  Google Scholar 

  44. Sun XL, Jayaram HN, Gharehbaghi K, Li QJ, Xiao X, Antony AC . Modulation of the cytotoxicity of 3′-azido-3′-deoxythymidine and methotrexate after transduction of folate receptor cDNA into human cervical carcinoma: Identification of a correlation between folate receptor expression and thymidine kinase activity. Cancer Res 1999; 59: 940–946.

    CAS  PubMed  Google Scholar 

  45. Sega EL, Low PS . Tumor detection using folate receptor-targeted imaging agents. Cancer Metastasis Re 2008; 27: 655–664.

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the United States Veterans Affairs Merit Review Award to Hiremagalur N Jayaram, PhD.

Author contributions

HNJ and ACA contributed to conception and design of the research, acquision of data, analysis and interpretation of data, drafting of the manuscript. PK, YZ, MBJ, NHJ, RAK and YT contributed to the acquisition of data, analysis and interpretation of data and final approval of the manuscript.

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Authors and Affiliations

  1. Research Service, Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, IN, USA

    P Kusumanchi, M B Jani, R A Khan, A C Antony & H N Jayaram

  2. Department of Medicine (Hematology–Oncology), Indiana University School of Medicine, Indianapolis, IN, USA

    P Kusumanchi, Y Zhang, N H Jayaram, R A Khan, Y Tang & A C Antony

  3. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA

    H N Jayaram

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Correspondence to H N Jayaram.

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Competing interests

A provisional patent application based on this work was filed by the technology transfer program of the United States Department of Veteran Affairs, Washington, DC for Drs Hiremagalur N Jayaram, Asok C Antony and Praveen Kusumanchi. The remaining authors, Drs Yonghua Zhang, Mehul B Jani, Nagesh H Jayaram, Rehana A Khan and Yingsheng Tang declare no conflict of interest.

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Kusumanchi, P., Zhang, Y., Jani, M. et al. Nicotinamide mononucleotide adenylyltransferase2 overexpression enhances colorectal cancer cell-kill by Tiazofurin. Cancer Gene Ther 20, 403–412 (2013). https://doi.org/10.1038/cgt.2013.33

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