Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of I kappa B kinase and NF kappa B activation in macrophages

Nitric oxide (NO) plays an important: role in inflammation and in the multi ple stages of carcinogenesis. In this study, we investigated the inhibitory effects of curcumin and its metabolites, tetrahydrocurcumin, hexahydrocurc umin, and octahydrocurcumin, on the induction of NO synthase (NOS) in RAW 2 64.7 cells activated with lipopolysaccharide (LPS). Western blotting and no rthern blotting analyses demonstrated that curcumin strongly reduced 130-kD a protein and 4.5-kb mRNA levels of iNOS in LPS-activated macrophages compa red with its metabolites, tetrahydrocurcumin, hexahydrocurcumin, and octahy drocurcumin. Moreover, electrophoretic mobility shift assay (EMSA) experime nts indicated that curcumin blocked the LPS-induced binding of nuclear fact or-kappaB (NF kappaB), a transcription factor necessary far iNOS induction to its P-32-labeled double-stranded oligonucleotide probe. The inhibition o f 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 p 65 from the cytosolic fraction, and inhibited the phosphorylation of I kapp aB alpha. Furthermore, we showed that curcumin could inhibit the I kappaB k inase 1 (IKK1) and I kappaB kinase 2 (IKK2) activities induced by EPS, but tetrahydrocurcumin, hexahydrocurcumin, and octahydrocurcumin were less acti ve. These results suggest that curcumin may exert its anti-inflammatory and anti-carcinogenic properties by suppressing the activation of NF kappaB th rough inhibition of IKK activity. (C) 2000 Elsevier Science Inc.