We observed that N-nitrosomorpholine (NMOR) given after a multi-carcinogenic treatment induces liver carcinomas with 56% lung metastasis. An additional treatment with diethylnitrosamine (DEN) with NMOR further enhanced the incidence of hepatocellular carcinoma (HCC) with lung metastasis. We have further revised the duration of NMOR treatment to establish an animal model with a simple experimental protocol and an appropriate experimental duration to facilitate investigation exploring the mechanisms of HCC metastasis and development of anti-metastatic therapeutics. We observed that DEN exposure followed by a 16-week treatment with NMOR to be a most efficient protocol for the induction of HCC metastasizing to the lung. In this review, we will discuss about the usefulness of animal models for induction of highly metastatic HCC and the assessment of the efficacy of anti-metastatic therapeutics. Additionally, we will also discuss use of these models in analysis of individual steps in the metastatic process by using non-steroidal anti-inflammatory drugs, aspirin and indomethacin, two nuclear factor kappa B (NF-κB) inhibitors, pentoxifylline and N-acetyl-L-cysteine.
 A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward and D. Forman, “Global Cancer Statistics,” CA: A Cancer Journal for Clinicians, Vol. 61, No. 2, 2011, pp. 69-90. doi:caac.20107[pii]10.3322/caac.20107
 D. B. Walsh, S. Downing, R. Nauta and M. N. Gomes, “Metastatic Cancer. A Relative Contraindication to Vena cava Filter Placement,” Cancer, Vol. 59, No. 1, 1987, pp. 161-163. doi:10.1002/1097-0142(19870101)59:1<161::AID-CNCR2820590131>3.0.CO;2-A
 J. E. Talmadge and I. J. Fidler, “AACR Centennial Series: The Biology of Cancer Metastasis: Historical Perspective,” Cancer Research, Vol. 70, No. 14, 2010, pp. 5649-5669. doi:10.1158/0008-5472.CAN-10-1040
 K. Morikawa, S. M. Walker, J. M. Jessup and I. J. Fidler, “In Vivo Selection of Highly Metastatic Cells from Surgical Specimens of Different Primary Human Colon Carcinomas Implanted into Nude Mice,” Cancer Research, Vol. 48, 1988, pp. 1943-1948.
 X. B. Li, J. Hamada, N. Takeichi, M. Hosokawa and H. Kobayashi, “Timing of UFT Administration Combined with Surgical Operation in Pulmonary Metastasis of Rat Mammary Carcinoma,” Japanese Journal of Cancer and Chemotherapy, Vol. 16, No. 7, 1989, pp. 2381-2385.
 T. Masui, H. Nakanishi, K. Inada, T. Imai, Y. Mizoguchi, H. Yada, M. Futakuchi, T. Shirai and M. Tatematsu, “Highly Metastatic Hepatocellular Carcinomas Induced in Male F344 Rats Treated with N-Nitrosomorpholine in Combination with Other Hepatocarcinogens Show a High Incidence of p53 Gene Mutations Along with Altered mRNA Expression of Tumor-Related Genes,” Cancer Letters, Vol. 112, No. 1, 1997, pp. 33-45. doi:10.1016/S0304-3835(96)04543-0
 M. Futakuchi, M. Hirose, T. Ogiso, K. Kato, M. Sano, K. Ogawa and T. Shirai, “Establishment of an in Vivo Highly Metastatic Rat Hepatocellular Carcinoma Model,” Japanese Journal of Cancer and Chemotherapy, Vol. 90, No. 11, 1999, pp. 1196-1202. doi:10.1111/j.1349-7006.1999.tb00695.x
 H. Enzmann, H. Zerban, S. A. Kopp, E. Loser and P. Bannach, “Effects of Low Doses of N-Nitrosomorpholine on the Development of Early Stages of Hepatocarcinogenesis,” Carcinogenesis, Vol. 16, No. 7, 1995, pp. 1513-1518. doi:10.1093/carcin/16.7.1513
 M. Volm, H. Zerban, J. Mattern and T. Efferth, “Overexpression of P-Glycoprotein in Rat Hepatocellular Carcinomas Induced with N-Nitrosomorpholine,” Carcinogenesis, Vol. 11, No. 1, 1990, pp. 169-172. doi:10.1093/carcin/11.1.169
 E. Weber and P. Bannasch, “Dose and Time Dependence of the Cellular Phenotype in Rat Hepatic Preneoplasia and Neoplasia Induced by Continuous Oral Exposure to N-Nitrosomorpholine,” Carcinogenesis, Vol. 15, No. 6, 1994, pp. 1235-1242. doi:10.1093/carcin/15.6.1235
 R. Umbas, J. A. Schalken, T. W. Aalders, B. S. Carter, H. F. Karthaus, H. E. Schaafsma, F. M. Debruyne and W. B. Isaacs, “Expression of the Cellular Adhesion Molecule E-Cadherin Is Reduced or Absent in High-Grade Prostate Cancer,” Cancer Research, Vol. 52, No. 18, 1992, pp. 5104-5109.
 P. Navarro, M. Gomez, A. Pizarro, C. Gamallo, M. Quintanilla and A. Cano, “A Role for the E-Cadherin cell-Cell Adhesion Molecule during Tumor Progression of Mouse Epidermal Carcinogenesis,” The Journal of Cell Biology, Vol. 115, No. 2, 1991, pp. 517-533. doi:10.1083/jcb.115.2.517
 S. Wakatsuki, R. Watanabe, K. Saito, T. Saito, A. Katagiri, S. Sato and Y. Tomita, “Loss of Human E-Cadherin (ECD) Correlated with Invasiveness of Transitional Cell Cancer in the Renal Pelvis, Ureter and Urinary Bladder,” Cancer Letters, Vol. 103, No. 1, 1996, pp. 11-17. doi:10.1016/0304-3835(96)04194-8
 H. Yoshino, M. Futakuchi, Y. M. Cho, K. Ogawa, F. Takeshita, N. Imai, S. Tamano and T. Shirai, “Modification of an in Vivo Lung Metastasis Model of Hepatocellular Carcinoma by Low Dose N-Nitrosomorpholine and Diethylnitrosamine,” Clinical and Experimental Metastasis, Vol. 22, No. 5, 2005, pp. 441-447. doi:10.1007/s10585-005-2807-9
 S. Ito, H. Nakanishi, T. Hirai, T. Kato, Y. Kodera, Z. Feng, Y. Kasai, K. Ito, S. Akiyama, A. Nakao and M. Tatematsu, “Quantitative Detection of CEA Expressing Free Tumor Cells in the Peripheral Blood of Colorectal Cancer Patients during Surgery with Real-Time RT-PCR on a LightCycler,” Cancer Letters, Vol. 183, No. 2, 2002, pp. 195-203. doi:10.1016/S0304-3835(02)00157-X
 M. Matsumura, Y. Niwa, Y. Hikiba, K. Okano, N. Kato, S. Shiina, Y. Shiratori and M. Omata, “Sensitive Assay for Detection of Hepatocellular Carcinoma Associated Gene Transcription (Alpha-Fetoprotein mRNA) in Blood,” Biochemical and Biophysical Research Communications, Vol. 207, No. 2, 1995, pp. 813-818. doi:10.1006/bbrc.1995.1259
 Y. Miyajima, K. Horibe, M. Fukuda, K. Matsumoto, S. Numata, H. Mori and K. Kato, “Sequential Detection of Tumor Cells in the Peripheral Blood and Bone Marrow of Patients with Stage IV Neuroblastoma by the Reverse Transcription-Polymerase Chain Reaction for Tyrosine Hydroxylase mRNA,” Cancer, Vol. 77, No. 6, 1996, pp. 1214-1219. doi:10.1002/(SICI)1097-0142(19960315)77:6<1214::AID-CNCR31>3.0.CO;2-2
 K. J. Luzzi, I. C. MacDonald, E. E. Schmidt, N. Kerkvliet, V. L. Morris, A. F. Chambers and A. C. Groom, “Multistep Nature of Metastatic Inefficiency: Dormancy of Solitary Cells after Successful Extravasation and Limited Survival of Early Micrometastases,” American Journal of Pathology, Vol. 153, No. 3, 1998, pp. 865-873. doi:10.1016/S0002-9440(10)65628-3
 M. Futakuchi, K. Ogawa, M. Sano, S. Tamano, F. Takeshita and T. Shirai, “Suppression of Lung Metastasis by Aspirin but Not Indomethacin in an in Vivo Model of Chemically Induced Hepatocellular Carcinoma,” Japanese Journal of Cancer Research, Vol. 93, No. 10, 2002, pp. 1175-1181. doi:10.1111/j.1349-7006.2002.tb01220.x
 N. Murashige, M. Kami and M. Ikeda, “Aspirin, COX-2, and the Risk of Colorectal Cancer,” The New England Journal of Medicine, Vol. 357, 2007, pp. 824-825; Author Reply pp. 824-825. doi:10.1056/NEJMc071797
 Y. J. Zhang, Y. J. Bao, Q. Dai, W. Y. Yang, P. Cheng, L. M. Zhu, B. J. Wang and F. H. Jiang, “mTOR Signaling Is Involved in Indomethacin and Nimesulide Suppression of Colorectal Cancer Cell Growth via a COX-2 Independent Pathway,” Annals of Surgical Oncology, Vol. 18, No. 2, 2011, pp. 580-588. doi:10.1245/s10434-010-1268-9
 M. Tsujii, S. Kawano and R. N. DuBois, “Cyclooxygenase-2 Expression in Human Colon Cancer Cells Increases Metastatic Potential,” Proceedings of the National Academy of Sciences of the United States, Vol. 94, No. 7, 1997, pp. 3336-3340. doi:10.1073/pnas.94.7.3336
 M. Farooqui, Y. Li, T. Rogers, T. Poonawala, R.J. Griffin, C.W. Song, K. Gupta, “COX-2 Inhibitor Celecoxib Prevents Chronic Morphine-Induced Promotion of Angiogenesis, Tumour Growth, Metastasis and Mortality, without Compromising Analgesia,” British Journal of Cancer, Vol. 97, No. 11, 2007, pp. 1523-1531. doi:10.1038/sj.bjc.6604057
 B. Singh, J. A. Berry, A. Shoher, G. D. Ayers, C. Wei and A. Lucci, “COX-2 Involvement in Breast Cancer Metastasis to Bone,” Oncogene, Vol. 26, No. 26, 2007, pp. 3789-3796. doi:10.1038/sj.onc.1210154
 Q. Chen and J. Massague, “Molecular Pathways: Vcam-1 as a Potential Therapeutic Target in Metastasis,” Clinical Cancer Research, Vol. 18, No. 20, 2012, pp. 5520-5525. doi:10.1158/1078-0432.CCR-11-2904
 J. P. Johnson, B. G. Stade, B. Holzmann, W. Schwable and G. Riethmuller, “De Novo Expression of Intercellular-Adhesion Molecule 1 in Melanoma Correlates with Increased Risk of Metastasis,” Proceedings of the National Academy of Sciences of the United States A, Vol. 86, No. 2, 1989, pp. 641-644. doi:10.1073/pnas.86.2.641
 C. Lawson, M. Ainsworth, M. Yacoub and M. Rose, “Ligation of ICAM-1 on Endothelial Cells Leads to Expression of VCAM-1 via a Nuclear Factor-KappaB-Independent Mechanism,” The Journal of Immunology, Vol. 162, No. 5, 1999, pp. 2990-2996.
 D. Zapolska-Downar and M. Naruszewicz, “Propionate Reduces the Cytokine-Induced VCAM-1 and ICAM-1 Expression by Inhibiting Nuclear Factor-Kappa B (NF-KappaB) Activation,” Journal of physiology and pharmacology, Vol. 60, No. 2, 2009, pp. 123-131.
 C. Weber, W. Erl, A. Pietsch and P. C. Weber, “Aspirin Inhibits Nuclear Factor-Kappa B Mobilization and Monocyte Adhesion in Stimulated Human Endothelial Cells,” Circulation, Vol. 91, No. 7, 1995, pp. 1914-1917. doi:10.1161/01.CIR.91.7.1914
 D. K. Biswas, C. M. Ahlers, B. J. Dezube and A. B. Pardee, “Pentoxifylline and Other Protein Kinase C Inhibitors Down-Regulate HIV-LTR NF-Kappa B Induced Gene Expression,” Molecular Medicine, Vol. 1, No. 1, 1994, pp. 31-43.
 M. Roederer, P. A. Raju, F. J. Staal and L. A. Herzenberg, “N-Acetylcysteine Inhibits Latent HIV Expression in Chronically Infected Cells,” AIDS Research and Human Retroviruses, Vol. 7, No. 6, 1991, pp. 563-567. doi:10.1089/aid.1991.7.563
 V. P. Sant, M. S. Nagarsenker, S. G. Rao and R. P. Gude, “Enhancement of Anti-Metastatic Activity of Pentoxifylline by Encapsulation in Conventional Liposomes and Sterically Stabilized Liposomes in Murine Experimental B16F10 Melanoma Model,” Journal of Pharmacy and Pharmacology, Vol. 52, No. 12, 2000, pp. 1461-1466. doi:10.1211/0022357001777667
 P. Redondo, E. Bandres, T. Solano, I. Okroujnov and J. Garcia-Foncillas, “Vascular Endothelial Growth Factor (VEGF) and Melanoma. N-Acetylcysteine Downregulates VEGF Production in Vitro,” Cytokine, Vol. 12, No. 4, 2000, pp. 374-378. doi:10.1006/cyto.1999.0566
 T. Cai, G. Fassina, M. Morini, M. G. Aluigi, L. Masiello, G. Fontanini, F. D’Agostini, S. De Flora, D. M. Noonan and A. Albini, “N-Acetylcysteine Inhibits Endothelial Cell Invasion and Angiogenesis,” Laboratory Investigation, Vol. 79, No. 9, 1999, pp. 1151-1159.
 S. Kawakami, Y. Kageyama, Y. Fujii, K. Kihara and H. Oshima, “Inhibitory Effect of N-Acetylcysteine on Invasion and MMP-9 Production of T24 Human Bladder Cancer cells,” Anticancer Research, Vol. 21, No. 1A, 2001, pp. 213-219.
 M. K. Jones, H. Wang, B. M. Peskar, E. Levin, R. M. Itani, I. J. Sarfeh and A. S. Tarnawski, “Inhibition of Angiogenesis by Nonsteroidal Anti-Inflammatory Drugs: Insight into Mechanisms and Implications for Cancer Growth and Ulcer Healing,” Nature Medicine, Vol. 5, No. 12, 1999, pp. 1418-1423. doi:10.1038/70995
 S. Abiru, K. Nakao, T. Ichikawa, K. Migita, M. Shigeno, M. Sakamoto, H. Ishikawa, K. Hamasaki, K. Nakata and K. Eguchi, “Aspirin and NS-398 Inhibit Hepatocyte Growth Factor-Induced Invasiveness of Human Hepatoma Cells,” Hepatology, Vol. 35, No. 5, 2002, pp. 1117-1124. doi:10.1053/jhep.2002.32676
 M. Futakuchi, K. Ogawa, S. Tamano, S. Takahashi and T. Shirai, “Suppression of Metastasis by Nuclear Factor Kappab Inhibitors in an in Vivo Lung Metastasis Model of Chemically Induced Hepatocellular Carcinoma,” Cancer Science, Vol. 95, No. 1, 2004, pp. 18-24. doi:10.1111/j.1349-7006.2004.tb03165.x
 V. Blank, P. Kourilsky and A. Israel, “NF-Kappa B and Related Proteins: Rel/Dorsal Homologies Meet Ankyrin-Like Repeats,” Trends in Biochemical Sciences, Vol. 17, No. 4, 1992, pp. 135-140. doi:10.1016/0968-0004(92)90321-Y
 G. Dong, Z. Chen, T. Kato and C. Van Waes, “The Host Environment Promotes the Constitutive Activation of Nuclear Factor-KappaB and Proinflammatory Cytokine Expression during Metastatic Tumor Progression of Murine Squamous Cell Carcinoma,” Cancer Research, Vol. 59, 14, 1999, pp. 3495-3504.
 S. Huang, C. A. Pettaway, H. Uehara, C. D. Bucana and I. J. Fidler, “Blockade of NF-KappaB Activity in Human Prostate Cancer Cells Is Associated with Suppression of Angiogenesis, Invasion, and Metastasis,” Oncogene, Vol. 20, No. 31, 2001, v4188-4197. doi:10.1038/sj.onc.1204535
 H. M. Ko, K. H. Seo, S. J. Han, K. Y. Ahn, I. H. Choi, G. Y. Koh, H. K. Lee, M. S. Ra and S. Y. Im, “Nuclear Factor KappaB Dependency of Platelet-Activating Factor-Induced Angiogenesis,” Cancer Research, Vol. 62, No. 6, 2002, pp. 1809-1814.