Stromal Protection in Combination with EGCG Lymphocytic Leukemia B Cells and May Overcome Their Curcumin Inhibits Prosurvival Pathways in Chronic

Purpose: Chronic lymphocytic leukemia (CLL) is incurable with current chemotherapy treatments. Curcumin (diferuloylmethane), an active ingredient in the spice turmeric, inhibits tumor metastasis, invasion, and angiogenesis in tumor cell lines.We evaluated the effects of curcumin on the viability of primary CLL B cells and its ability to overcome stromal mediated protection. Experimental Design: The in vitro effect of curcumin on primary CLL B cells was evaluated using fluorescence activated cell sorter analysis andWestern blotting. For some experiments, CLL B cells were cocultured with human stromal cells to evaluate the effects of curcumin on leukemia cells cultured in their microenvironment. Finally, the effect of curcumin in combination with the green tea extract epigallocatechin-3 gallate (EGCG) was evaluated. Results:Curcumin induced apoptosis in CLLB cells in a dose-dependent (5-20 Amol/L)manner and inhibited constitutively active prosurvival pathways, including signal transducers and activators of transcription 3 (STAT3), AKT, and nuclear factor nB. Moreover, curcumin suppressed expression of the anti-apoptotic proteinsMcl-1and X-linked inhibitor of apoptosis protein (XIAP), and up-regulated the pro-apoptotic protein BIM. Coculture of CLL B cells with stromal cells resulted in elevated levels of STAT3, increased expression of Mcl-1and XIAP, and decreased sensitivity to curcumin.Whencurcuminwas administered simultaneously with EGCG, antagonism was observed for most patient samples. In contrast, sequential administration of these agents led to substantial increases in CLL B-cell death and could overcome stromal protection. Conclusions: Curcumin treatment was able to overcome stromal protection of CLL B cells on in vitro testing and to synergize with EGCG when administered in a sequential fashion. Additional evaluation of curcumin as a potential therapeutic agent for treatment of CLL seems warranted. B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in North America and, with the possible exception of allogeneic stem cell transplant, is incurable with current treatments (1). CLL B cells are largely noncycling (2), and their accumulation is primarily secondary to decreased apoptosis rather than increased proliferation (3). Notably, the apoptotic resistance of CLL B cells to chemotherapeutic agents and monoclonal antibodies is, in part, related to increased levels of anti-apoptotic proteins Mcl-1, Bcl-2, and X-linked inhibitor of apoptosis protein (XIAP; refs. 1, 3–7). CLL B cells have also been found to have a constitutively active signal transducers and activators of transcription 3 (STAT3) pathway (8, 9), possess an autocrine vascular endothelial growth factor signaling loop (10), and express constitutively activated nuclear factor nB (NFnB; ref. 11) and phosphatidylinositol-3 kinase. Importantly, selective inhibition of phosphatidylinositol-3 kinase induces apoptosis of CLL B cells (12, 13). Previous work by our laboratory and others suggests that autocrine survival signals and interactions between the CLL B cell and its microenvironment influence apoptotic resistance and sensitivity to chemotherapeutic agents (8–10, 14). In the marrow, physical contact between stromal elements and leukemic cells promotes CLL B-cell survival (15–18), an effect mediated in part through integrins (e.g., very late antigen-4; ref. 18) on the surface of CLL B cells and their interaction with various ligands (e.g., vascular cell adhesion molecule-1, fibronectin, iC3b) expressed on marrow stromal cells (19–22). To add to the complexity of environmental/stromal protection, several other interactions (mediated by contact and soluble factors) between CLL B cells and their microenvironment have been shown to promote survival, proliferation, and upregulation of anti-apoptotic proteins (20, 22–25). There is now great interest in identifying pharmacological agents that are able to modulate these interactions that impact survival pathways in the hope of identifying potential novel therapies for treatment of CLL. Naturally occurring compounds are a potential source of agents that could modulate these Cancer Therapy: Preclinical Authors’Affiliation:Division of Hematology, Mayo Clinic, Rochester, Minnesota Received 6/19/08; revised 9/26/08; accepted10/12/08. Grant support: National Cancer Institute (NCI CA113408, NCI CA95241) CLL Global Research Foundation, CLL Topics, and Polyphenon E International commercial research support (T.D. Shanafelt). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints:Tait D. Shanafelt, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905. Phone: 507-284-1961; Fax: 507-266-9277; E-mail: shanafelt.tait@mayo.edu. F2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-1511 www.aacrjournals. org Clin Cancer Res 2009;15(4) February15, 2009 1250 American Association for Cancer Research Copyright © 2009 on September 26, 2011 clincancerres.aacrjournals. org Downloaded from DOI:10.1158/1078-0432.CCR-08-1511