treatment with curcumin, disc cell cultures were either kept untreated, treated with 5 ng ml IL 1B alone or co treated with 20 uM curcumin for 60 min. Nuclear e tracts were prepared by washing trypsin harvested cells with 10 mM HEPES, containing 1. 5 mM MgCl2, 10 mM KCl, 1 mM PMSF, 5 mM DTT and 0. 1% protease inhibitors. Then, cells were lysed with 0. 1% NP 40 for 5 min, centrifuged for 5 min at 10000 rpm and supernatants were discarded. Nuclear pellets were washed with 0. 1% NP 40 and lysed for 20 min with 20 mM HEPES, containing 1. 5 mM MgCl2, 420 mM NaCl, 25% glycerol, 1 mM PMSF and 5 mM DTT as well as protease inhibitors. After cen trifugation, protein content was measured by Bradford assay. Nuclear e tracts of untreated, IL 1B treated and IL 1B curcumin treated cells were separated on a SDS polyacrylamid gel and transferred to a PVDF membrane.

The membrane was incubated with a p65 antibody followed by incubation with an appropriate HRP secondary antibody GSK-3 before analyzing chemiluminescence. PARP was used as a loading control. The assay was performed on cells from three independent biopsies. Transcription factor assay for NF ��B In order to detect specific NF ��B DNA binding activity in nuclear e tracts, the NF ��B Transcription Factor Assay was used according to the protocol provided by the manufacturer. Briefly, a specific double stranded DNA sequence containing the NF ��B response element was immobilized to the wells of a 96 well plate. Nuclear e tracts were prepared as described above and added to the coated wells.

NF ��B contained in the added nuclear e tract bound specifically to the NF ��B response element and was detected by addition of the provided specific primary antibody direc ted against NF ��B. A secondary antibody conju gated with HRP was added, a colorimetric readout at 655 nm was performed and data was quantified as indi cated in the protocol. The assay was performed on cells from two independent biopsies. Western blot for MAP kinases Whole cell e tracts of untreated, IL 1B treated and IL 1B curcumin treated cells were prepared after 15 min to investigate whether curcu min acts on typical MAP kinases. Protein content was measured by Bradford assay and immunoblotting of whole cell e tracts was performed as described for p65, but membranes were incubated with antibodies recognizing either unphosphorylated or phosphorylated p38, ERK or JNK before adding an HRP labeled rabbit secondary antibody and analyzing chemiluminescence.

Tubulin was used as a loading con trol. The assay was performed on samples from five in dependent e periments. Statistical analysis All quantitative data was statistically analyzed using a Mann Whitney U test on the SPSS statistics software and differences were consid ered statistically significant at p 0. 05. Results Cytoto icity of curcuma e tracts and curcumin Cytoto icity of curcuma e tracts and curcumin was determined after 6, 18 and 30 hours using the MTT assay. For the curcuma DMSO e tract, cell viability w