Curcumin, the principle component of the spice turmeric, has been used as an anti-inflammatory medication in India and China for centuries. Recent studies, predominantly using actively dividing cell lines, have suggested that this compound could be used as a chemopreventative or therapeutic agent for epithelial tumors. As curcumin has been reported to inhibit the NIK/IKK complex, an activity that would be expected to induce apoptosis in B cell malignancies, we sought to determine whether curcumin induces apoptosis in vitro in primary chronic lymphocytic leukemia (B-CLL) cells. Primary leukemic cells were incubated with varying dosages of curcumin, followed by assessment for apoptosis. The role of PPARg or NF-kB signaling in curcumin-induced apoptosis was examined by cotreatment with a PPARg antagonist or EMSA of nuclear NFkB complexes. We also examined whether a clinically achievable concentration of curcumin (1 mM) would augment the apoptotic effects of fludarabine, dexamethasone, vincristine or the PDE4 inhibitor rolipram. In B-CLL cells from 14 patients, curcumin-induced apoptosis with a mean EC 50 of 5.5 mM. In contrast, the EC 50 for whole mononuclear cells from a healthy donor was 21.8 mM. In a 48 hr wash-out time course, curcumin-induced apoptosis was time-dependent, with a substantial reduction in apoptosis observed when curcumin was removed after 5 hr. Curcumin treatment reduced basal nuclear NF-kB levels and 1 mM curcumin augmented both vinca alkaloid and PDE4 inhibitor-induced apoptosis in B-CLL cells. Our studies suggest that curcumin may augment the efficacy of established or experimental therapies for B-CLL. Am. J. Hematol. 82:23-30, 2007. V V C 2006 Wiley-Liss, Inc.

CD40, a 48-kDa transmembrane protein belonging to the tumor necrosis factor receptor superfamily, is expressed on B lymphocytes as well as dendritic cells, macrophages, epithelial cells, and hematopoietic progenitor cells (1). Its ligand (CD40L) 2 is found on activated T lymphocytes; interaction between the receptor and ligand induces B cell activation and proliferation, Ig secretion, memory cell formation, and isotype switching (2). The critical role that CD40 plays in B cell function is made evident by the absence of germinal centers and secondary immune responses in CD40-deficient mice and the association of mutations in CD40 and CD40L with the human disease X-linked hyper-IgM syndrome (3). In addition, CD40 signaling is also involved in neoplastic cell proliferation. The interaction between CD40 and its ligand can inhibit apoptosis of normal and transformed B cells induced by engagement of the B cell receptor (BCR), serum deprivation, or treatment with a chemotherapeutic agent (4). For example, triggering of CD40 by the anti-CD40 monoclonal antibody G28-5 inhibits apoptosis induced by the chemotherapeutic agent fludarabine in B cells from patients with chronic lymphocytic leukemia (CLL) (5). A variety of second messengers are activated after treatment of B cells with CD40L, including NF-B (6). In WEHI 231 murine B lymphoma cells, a model often used to study NF-B and its effects on B cell survival, CD40L stimulation following BCR engagement rescues cells from apoptosis (7). BCR engagement of WEHI 231 cells normally leads to an initial transient increase in p50/c-Rel NF-B factor activity at 1 h, which is followed by a rapid decline in levels (8), and then to apoptosis (9). Studies from our laboratories have shown that CD40L-mediated rescue of WEHI 231 B cells is due in part to maintenance of NF-B factor binding (10), although the nature of the subunits responsible was not determined.The mammalian NF-B family members are p65 (RelA), RelB, c-Rel, p105/p50, and p100/p52. These subunits contain a 300-amino acid long region termed the Rel homology domain, which is involved in subunit dimerization and binding to DNA (11). For the most part, the different members can form heteroand homodimers, which vary significantly in their transactivation potential (11, 12). As we first showed by antisense and ectopic c-Rel expression studies in splenic B lymphocytes and WEHI 231 B cells (9), NF-B transcription factors play critical roles in proliferation control and B cell survival (13, 14). NF-B, which regulates the c-myc, and c-myb genes (8,15,16), has also been implicated in promoting neoplastic transformation (13,14). In most non-B cells, NF-B is sequestered in the cytoplasm bound to specific inhibitory proteins (I B (inhibitor of B protein)) of which I B-␣ is the prototype. In B cells, NF-B activity is constitutive, but can be further induced or modulated (17)(18)(19). Activation of classical NF-B (p50/p65) through the canonical pathway is mediated via the I B kinase complex, con-* This work was supported by National Instit...

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Type 4 cyclic AMP (cAMP) phosphodiesterase (PDE4) inhibitors, a class of compounds in clinical development that activate cAMP-mediated signaling by inhibiting cAMP catabolism, offer a feasible means by which to potentiate glucocorticoid-mediated apoptosis in lymphoid malignancies such as B-cell chronic lymphocytic leukemia (B-CLL). In this study, we show that PDE4 inhibitors up-regulate glucocorticoid receptor (GRa) transcript levels in B-CLL cells but not T-CLL cells or Sezary cells or normal circulating T cells, B cells, monocytes, or neutrophils. Because GRa transcript half-life does not vary in CLL cells treated with the prototypic PDE4 inhibitor rolipram, the 4-fold increase in GRa mRNA levels observed within 4 h of rolipram treatment seems to result from an increase in GRa transcription. Rolipram treatment increases levels of transcripts derived from the1A3 promoter to a greater extent than the 1B promoter.Treatment of B-CLL cells with two other PDE4 inhibitors currently in clinical development also augments GR transcript levels and glucocorticoid-mediated apoptosis. Washout studies show that simultaneous treatment with both drug classes irreversibly augments apoptosis over the same time frame that GR upregulation occurs. Although treatment of B-CLL cells with glucocorticoids reduces basal GRa transcript levels in a dose-related manner, cotreatment with rolipram maintained GRa transcript levels above baseline. Our results suggest that as a result of their unusual sensitivity to PDE4 inhibitor^mediated up-regulation of GRa expression, treatment of B-CLL patients with combined PDE4 inhibitor/glucocorticoid therapy may be of therapeutic benefit in this disease.Glucocorticoids are an important component of standard therapy for several lymphoid malignancies, including multiple myeloma, acute lymphoblastic leukemia, and diffuse large B-cell lymphoma. As early studies in patients with B-cell chronic lymphocytic leukemia (B-CLL) showed that addition of prednisone to chlorambucil augmented response rate but not median survival, glucocorticoids are not generally a standard component of initial therapy for patients with B-CLL (1, 2). Nonetheless, two studies of high-dose glucocorticoid therapy have suggested that glucocorticoids can be of clinical benefit to a subset of patients with treatment-refractory B-CLL (3, 4).Despite frequent responses to glucocorticoid treatment, monotherapy with glucocorticoids is not curative in any lymphoid malignancy, but the mechanisms underlying clinical glucocorticoid resistance remain controversial. Structural alterations in the glucocorticoid receptor (GR) are commonly identified in lymphoid cell lines that have been selected for glucocorticoid resistance by prolonged culture in dexamethasone, but comparable alterations in primary malignant lymphoid cells have been only infrequently reported (5 -9). A detailed analysis of treated B-CLL patients failed to identify abnormalities in either the DNA-or steroid-binding domains of leukemic GRs (10). Nonstructural modifications of ...