D-Aminoacid oxidase (DAO) was isolated from fresh porcine kidney; its cytotoxic potential was studied under in vitro and in vivo conditions. The isolated DAO was complexed with Fe<sub>2</sub>O<sub>3</sub> nanoparticles and its potential as an oxidation therapeutic agent was analysed. The ability of the complex in eliciting H<sub>2</sub>O<sub>2</sub> mediated cytotoxicity was studied on Dalton’s lymphoma ascites cells (DLA). The induction of apoptosis in DLA cells by Fe<sub>2</sub>O<sub>3</sub>-DAO complex was studied by morphological examination and alkaline single cell gel electrophoresis (comet assay). The antitumor activity of the complex was investigated by oral administration of the complex and the substrate D-alanine to tumor bearing Swiss albino mice and by targeting the complex to the tumor site, using an externally applied magnetic field. Fe<sub>2</sub>O<sub>3</sub>-DAO along with D-alanine showed remarkable cytotoxicity in a substrate concentration-dependent manner. Both morphological examination and comet assay revealed that Fe<sub>2</sub>O<sub>3</sub>-DAO/D-alanine induced apoptosis. Oral administration of Fe<sub>2</sub>O<sub>3</sub>-DAO and D-alanine along with magnetic targeting significantly suppressed tumor growth in mice. The present report provides the first evidence for the promising application of enzyme bound nanoparticles for targeted oxidation therapy.
K. B. Bechman and B. N. Ames, “Oxidative Decay of DNA,” The Journal of Biological Chemistry, Vol. 272, No. 32, 1997, pp. 633-636.
O. Ben-Yoseph and B. D. Ross, “Oxidation Therapy: The Use of a Reactive Oxygen Species-Generating Enzyme System for Tumor Treatment,” British Journal of Cancer, Vol. 70, 1994, pp. 1131-1135. doi:10.1038/bjc.1994.460
L. D. Stegman, H. Zheng, E. R. Neal, O. Ben-Yoseph, L. Pollegioni, M. Pilone and B. D. Ross, “Induction of Cytotoxic Oxidative Stress by D-Alanine in Brain Tumor Cells Expressing Rhodotorula gracilis: A Cancer Gene Therapy Strategy,” Human Gene Therapy, Vol. 9, No. 2, 1998, pp. 185-193. doi:10.1089/hum.1998.9.2-185
B. S. Berlett and E. R. Stadtman, “Protein Oxidation in Aging, Disease, and Oxidative Stress,” The Journal of Biological Chemistry, Vol. 272, No. 33, 1997, pp. 20313-20316. doi:10.1074/jbc.272.33.20313
B. Halliwell and J. M. Gutteridge, “Free Radicals, Lipid Peroxidation, and Cell Damage,” The Lancet, Vol. 324, No. 8411, 1984, p. 1095. doi:10.1016/S0140-6736(84)91530-7
T. Matsura, M. Kai, Y. Fujii and K. Yamada, “Hydrogen Peroxide-Induced Apoptosis in HL-60 Cells Requires Caspase-3 Activation,” Free Radical Research, Vol. 30, No. 1, 1999, pp. 73-83. doi:10.1080/10715769900300081
H. Yamakawa, Y. Ito, T. Naganawa, Y. Banno, S. Nakashima, S. Yoshimura, M. Sawada, Y. Nishimura, Y. Nozawa and N. Sakai, “Activation of Caspase 9 and 3 during H 2 O 2 -Induced Apoptosis of PC12 Cells Independent of Ceramide Formation,” Neurological Research, Vol. 22, No. 6, 2000, pp. 556-564.
H. N. Green and J. W. Westrop, “Hydrogen Peroxide and Tumor Therapy,” Nature, Vol. 181, 1958, pp. 128-129. doi:10.1038/181128a0
K. Sugiura, “Effect of Hydrogen Peroxide on Transplanted Rat and Mouse Tumors,” Nature, Vol. 182, 1958, pp. 1310-1311. doi:10.1038/1821310a0
J. Fang, T. Sawa, T. Akaike and H. Maeda, “Tumor-Targeted Delivery of Polyethylene Glycol-Conjugated for Antitumor Therapy via Enzymatic Generation of Hydrogen Peroxide,” Cancer Research, Vol. 62, No. 11, 2002, pp. 3138-3143.
H. Maeda, L.W. Seymour and Y. Miyamoto, “Conjugates of Anticancer Agents and Polymers: Advantages of Mac-Romolecular Therapeutics in Vivo,” Bioconjugate Chemisry, Vol. 3, No. 5, 1992, pp. 351-362.
J. Fang, D. Deng, H. Nakamura, T. Akuta, H. Qin, A. K. Iyer, K. Greish and H. Maeda, “Oxystress Inducing Anti-tumor Therapeutics via Tumor-Targeted Delivery of PEG-Conjugated D-Amino Acid Oxidase,” International Journal of Cancer, Vol. 122, No. 5, 2008, pp. 1135-1144. doi:10.1002/ijc.22982
O. D. Jayakumar, R. Ganguly, A. K. Tyagi, D.K. Chandrasekharan and C. K. K. Nair, “Water Dispersible Fe3O4 Nanoparticles Carrying Doxorubicin for Cancer Therapy,” Journal of Nanoscience and Nanotechnology, Vol. 9, 2009, pp. 6344-6348. doi:10.1166/jnn.2009.1369
L. W. Seymour, Y. Miyamoto, H. Maeda, M. Brereton, J. Strohalm, K. Ulbrich and R. Duncan, “Influence of Mo-Lecular Weight on Passive Tumor Accumulation of a Soluble Macromolecular Drug Carrier,” European Journal of Cancer, Vol. 31A, No. 5, 1995, pp. 766-770. doi:10.1016/0959-8049(94)00514-6
L. W. Seymour, S. P. Olliff, C. J. Poole, P. G. De Takats, R, Orme, D. R. Ferry, H. Maeda, T. Konno and D. J. Kerr, “A Novel Dosage Approach for Evaluation of SMANCS [Poly-(strene-co-maleyl-half-n-butylate)-neocarzinostatin] in the Treatment of Primary Hepatocellular Carcinoma,” International Journal of Oncology, Vol. 12, No. 6, 1998, pp. 1217-1223.
J. Grabis, G. Heidemane and D. Rasmane, “Preparation of Fe 3 3O 4 and γ-Fe 2 O 3 Nanoparticles by Liquid and Gas Phase Processes,” Materials Science (medziagotyra), Vol. 14, 2008, pp. 292-295.
J. D. Choi, “The Nature of N-5 Reactivity in Flavins and Inhibitor Complexes,” 1978. http://etd.lib.ttu.edu/theses/available/etd-04102009-1295015070716/unrestricted/31295015070716.pdf
T. Watanabe, Y. Motomura and T. Suga, “A New Colori-Metric Determination of and Urate Oxidase Activity,” Analytical Biochemistry, Vol. 86, No. 1, 1978, pp. 310-315. doi:10.1016/0003-2697(78)90347-0
D. K. Chandrasekharan, V. T. Kagiya and C. K. K. Nair, “Radiation Protection by 6-Palmitoyl Ascorbic Acid-2-Glucoside: Studies on DNA Damage in Vitro, ex Vivo, in Vivo and Oxidative Stress in Vivo,” Journal of Radiation Research, Vol. 50, No. 3, 2009, pp. 203-212. doi:10.1269/jrr.08090
H. Cerda, H. Delincee, H. Haine and H. Rupp, “The DNA ‘Comet Assay’ as a Rapid Screening Technique to Control Irradiated Food,” Mutation Research, Vol. 375, No. 2, 1997, p. 167. doi:10.1016/S0027-5107(97)00012-2
K. Konca, A. Lankoff, A. Banasik, H. Lisowka, T. Kuszewski, S. Gozdz, Z. Koza and A. Wojcik, “Across Platform Public Domain PC Image Analysis Programme for the Comet Assay,” Mutation Research, Vol. 534, No. 1-2, 2003, pp. 15-20.
D. Sandeep and C. K. K. Nair, “Protection of DNA and Membrane from γ-Radiation Induced Damage by the Extract of Acorus calamus Linn.: An in Vitro Study,” Environmental Toxicology and Pharmacology, Vol. 29, No. 3, 2010, pp. 302-307. doi:10.1016/j.etap.2010.02.006
G. G. Nair and C. K. K. Nair, “Amelioration of γ-Radiation Induced Genomic Insult and Oxidative Stress in Whole Body Irradiated Swiss Albino Mice by Sesamol,” International Journal of Low Radiation, Vol. 8, No. 1, 2011, pp. 20-34.
S. Wada, T. V. Khoa, Y. Kobayashi, T. Funayama, K. Yamamoto, M. Natsuhor and N. Ito, “Detection of Radiation-Induced Apoptosis Using the Comet Assay,” Journal of Veterinary Medical Science, Vol. 65, No. 11, 2003, pp. 1161-1166. doi:10.1292/jvms.65.1161
R. C. Wilkins, D. Wilkinson, H. P. Maharaj, P. V. Bellier, M. B. Cybulski and J. R. McLean, “Differential Apoptotic Response to Ionizing Radiation in Subpopulations of Human White Blood Cells,” Mutation Research, Vol. 513, No. 1-2, 2002, pp. 27-36.
T. Sawa, J. Wu, T. Akaike and H. Maeda, “Tumor-Targeting Chemotherapy by a Xanthine Oxidase-Polymer Conjugate That Generates Oxygen-free Radicals in Tumor Tissue,” Cancer Research, Vol. 60, 2000, pp. 666-671.