Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IκB kinase and NFκB activation in macrophages

Nitric oxide (NO) plays an important: role in inflammation and in the multiple stages of carcinogenesis. In this study, we investigated the inhibitory effects of curcumin and its metabolites, tetrahydrocurcumin, hexahydrocurcumin, and octahydrocurcumin, on the induction of NO synthase (NOS) in RAW 264.7 cells activated with lipopolysaccharide (LPS). Western blotting and northern blotting analyses demonstrated that curcumin strongly reduced 130-kDa protein and 4.5-kb mRNA levels of iNOS in LPS-activated macrophages compared with its metabolites, tetrahydrocurcumin, hexahydrocurcumin, and octahydrocurcumin. Moreover, electrophoretic mobility shift assay (EMSA) experiments indicated that curcumin blocked the LPS-induced binding of nuclear factor-kappaB (NF kappaB), a transcription factor necessary far iNOS induction to its P-32-labeled double-stranded oligonucleotide probe. The inhibition of NF kappaB activation occurred through the prevention of inhibitor kappaB (I kappaB) degradation. Transient transfection experiments also showed that curcumin inhibited NF kappaB-dependent transcriptional activity. Curcumin blocked the disappearance of inhibitory kappaB alpha (I kappaB alpha) and p65 from the cytosolic fraction, and inhibited the phosphorylation of I kappaB alpha. Furthermore, we showed that curcumin could inhibit the I kappaB kinase 1 (IKK1) and I kappaB kinase 2 (IKK2) activities induced by EPS, but tetrahydrocurcumin, hexahydrocurcumin, and octahydrocurcumin were less active. These results suggest that curcumin may exert its anti-inflammatory and anti-carcinogenic properties by suppressing the activation of NF kappaB through inhibition of IKK activity. (C) 2000 Elsevier Science Inc.