Anti-Inflammatory Curcumin Inhibits Activation-Induced Cytosine Deaminase (AID) within Cycling Normal and CLL B Lymphocytes.

Abstract 4606

Curcumin is a natural phenolic compound within the spice, Curcuma longa. It has noted anti-inflammatory effects, in large part due to its potent suppressive effect on the NF-kB signaling pathway. AID is a NF-kB-regulated enzyme, essential for B cell Ig class switch recombination and somatic hypermutation and recently shown to promote oncogenic transformation within both B cell and non-B cell lineages. This study has examined the effect of curcumin on the division-linked upregulation of AID protein and mRNA within several human B cell populations: in vitro-activated normal and CLL B lymphocytes and the AID-positive, pre-germinal center B cell line, CL-01. CFSE-labeled, IgM+ human B2 cells isolated from spleen/tonsil were pre-activated for 4–5 days with stimuli likely encountered in sites of inflammation, i.e. limiting surrogate C3dg-coated antigen (anti-IgM: anti-CD21: dextran) + IL-4 + BAFF. Peripheral blood B-CLL cells were activated with TLR-9 ligand, ODN-2006, + IL-15. Curcumin at doses from 6 to 50 μ M, and parallel DMSO vehicle controls, were pulsed into dividing B cell cultures (day 3, 4, or 5 of activation), and AID mRNA and protein assessed after 1 to 2 days. In experiments with CL-01 B cells, the kinetics of curcumin-induced AID suppression was further analyzed. Messenger RNA was monitored by both quantitative and qualitative RT-PCR; AID protein was assessed by two-color flow cytometry of CFSE-labeled cells and immunoblotting. The above experiments revealed that curcumin can significantly down-regulate AID mRNA and protein, in dose dependent fashion within each of the above B cell populations. Following a 16h pulse of curcumin (25 μ M), AID mRNA within CL-01 cells was inhibited by 60% (p=0.001), and accompanied by ~ 60% decrease in AID protein. Within cultures of replicating normal human B lymphocytes, a similar pulse of curcumin reduced total culture AID mRNA by an average of 70% in 3 experiments. AID protein in blasts representing 3–4 divisions was reduced by 79%, and in those representing 1–2 divisions by 58%, within a representative experiment. AID mRNA was evident within all in vitro-activated B-CLL clones tested (total = 6 clones at the time of submission). This was significantly reduced by a 15–24 hr pulse with curcumin (20-25 μ M): 42% inhibition (p=0.02)). The inhibitory effects of curcumin were evident in both IgHV mutated and unmutated clones. Within stimulated B-CLL assessed for AID protein (4 total clones, of which 2 were positive), a 20 hr pulse of curcumin at 40 μ M and 20 μ M reduced AID expression in one clone by 80% and 40%, respectively. In the other clone, a maximal tested dose of 20 μ M curcumin reduced AID protein by only 12%. Suppression of AID mRNA in the CL-01 cell line was noted as soon as 3 hours following exposure to curcumin and was preceded by inhibition of NF-kB activation, both baseline and BAFF-induced. The latter was determined through monitoring intracellular levels of phospho-p65^-Ser(529) – an active phosphorylated form of NF-kB RelA. Taken together, these findings suggest that NF-kB- and AID-suppressing curcumin may be useful in reducing the risk of malignant transformation and B-CLL progression into more malignant subclones, as well as treating B cell autoimmune diseases driven by pathogenic, somatically-mutated IgG autoantibodies.