Suppression of metastasis by nuclear factor kB inhibitors in an in vivo lung metastasis model of chemically induced hepatocellular carcinoma

2 Daiyu-kai Institute of Medical Science, 64 Goura, Nishiazai, Azai-cho, Ichinomiya 491-0113; and 3 To evaluate the suppressive effects of nuclear factor kappa B (NF- κB) inhibitors on metastasis, three agents, pentoxifylline (PTX, 0.5% in diet), N-acetyl-L-cysteine (NAC, 0.5% in diet), and aspirin (ASP, 0.5% in diet) were applied in an in vivo highly metastatic rat hepatocellular carcinoma (HCC) model in F344 male rats. Ad- ministration of NF-κB inhibitors for 8 weeks after induction of highly metastatic HCC by sequential treatment with diethylnitro- samine and N-nitrosomorpholine did not cause any significant change in survival rate or body weight. The incidence of HCC was 100% at week 23, regardless of treatment with NF-κB inhibitors. PTX, NAC, and ASP did not exert any significant effect on the de- velopment or differentiation of HCCs, although PTX tended to de- crease the multiplicity of HCC. Although no lung metastasis was observed in the rats killed at the end of the period of carcinogen exposure, lung metastasis was found in 100% of animals in all the groups at the end of the experiment. Multiplicity of lung me- tastasis was significantly decreased by PTX and NAC, whereas ASP was without significant influence. The size of metastatic nod- ules was also significantly reduced in the PTX treatment group. Furthermore, the inhibitory κ-B (IκB) protein level, considered to be a marker for the degree of NF-κB transcription, was signifi- cantly suppressed by PTX. mRNA expression in HCC for vascular cell adhesion molecule-1 (VCAM-1), which is considered to play a key role in attachment of cancer cells to the endothelium, was significantly suppressed by PTX. Among the splicing variants of VEGF, VEGF-A120, VEGF-A144, VEGF-A164, and VEGF-A188, sup- pressed mRNA expression of VEGF-A188 appeared to be corre- lated with suppression of lung metastasis formation. In conclusion, the present study demonstrated that NF-κB inhibitors have the potential to inhibit lung metastasis from rat HCCs in vivo, and PTX is especially promising. Its mechanism of action may involve suppression of VCAM-1 and VEGF-A188 production. (Cancer Sci 2004: 95: 18-24) e have recently established an in vivo lung metastasis model in which hepatocellular carcinoma (HCC) in- duced by sequential treatment with two hepatocarcinogens, di- ethylnitrosamine (DEN) and N-nitrosomorpholine (NMOR), very frequently metastasizes to the lung. 1) This model has ad- vantages for investigation of the mechanisms of multistep me- tastasis by malignant tumors, and for the assessment of the efficacy of therapeutic treatments against metastasis in vivo. Our previous study demonstrated that aspirin (ASP) has the potential to inhibit lung metastasis by rat HCCs in vivo, al- though the observed suppressive effect was marginal.2) The mechanism appeared to involve a decrease in the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion mol- ecule-1 (VCAM-1), which play important roles in attachment of tumor cells to the vascular endothelium. 2)