Curcumin inhibits prosurvival pathways in chronic lymphocytic leukemia B cells and may overcome their stromal protection in combination with EGCG

Abstract

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 and Western 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 CLL B cells in a dose-dependent (5-20 micromol/L) manner and inhibited constitutively active prosurvival pathways, including signal transducers and activators of transcription 3 (STAT3), AKT, and nuclear factor kappaB. Moreover, curcumin suppressed expression of the anti-apoptotic proteins Mcl-1 and 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-1 and XIAP, and decreased sensitivity to curcumin. When curcumin was 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.

Fig. 1. Curcumin induces apoptosis in primary CLL B-cells in a dose-dependent manner

(A) Primary PBMC (≥90% CD5⁺/CD19⁺ lymphocytes) isolated from CLL patients (n=18) were treated with increasing doses of curcumin for 24 hours. Cells were harvested, stained with annexin/PI and analyzed by flow cytometry for the induction of cell death. Similarly, PBMC from normal individuals (n=5) were treated with curcumin and induction of cell death in CD19⁺ B-lymphocytes was analyzed by flow cytometry (annexin/PI positivity). Mean values were plotted with standard error bars. (B) Curcumin induced cell death involves PARP cleavage. Lysates from CLL B-cells (n=6, P=patient) treated with curcumin or DMSO were analyzed for PARP cleavage by Western blot. Curcumin-treated cells displayed cleavage of the native PARP (116 kDa) into its signature 85 kDa polypeptide fragment.

Fig. 2. Curcumin inhibits pro-survival signaling pathways active in CLL B-cells

Curcumin-treated CLL B-cells were analyzed by Western blot using phospho-specific antibodies to assess the phosphorylation profile of the pro-survival signaling molecules known to be constitutively elevated in CLL including: (A) STAT3 (n=4), (B) AKT (n=7) and (C) IκBα (n =7). Curcumin treatment decreased phosphorylation levels of STAT3, AKT, and IκBα in primary CLL B-cells. Total STAT3, AKT and IκBα were used as loading controls for the respective experiments. Representative figures of, at least, ten CLL B patients’ samples.

Fig. 3. Curcumin modulates the expression of certain pro- and anti-apoptotic proteins in CLL B-cells

Lysates of CLL B-cells isolated from various patients as indicated treated with curcumin were analyzed to assess the effect of curcumin on the anti-apoptotic proteins XIAP (n=3), Mcl-1 (n=6), and Bcl-2 (n=6) as well as the pro-apoptotic protein BIM (n=6) using specific antibodies. Actin was used as the loading control. Curcumin treatment of CLL B-cells suppressed the expression of XIAP (A) and Mcl-1 (B, top row) but, not Bcl-2 (B, middle row). Curcumin treatment of CLL B-cells also resulted in up-regulation of the pro-apoptotic protein BIM expression (C, top row). AKT is the upstream negative regulator of BIM expression. Inhibition of AKT-phosphorylation by curcumin (C, bottom row) is also shown.

Fig. 4. Coculture of CLL B-cells with HS-5 modulates STAT3 activation and apoptosis-regulatory proteins

(A) Primary CLL B-cells cocultured with HS-5 human stromal cells in transwells for 48 hours were harvested and the cell lysates (n=6) were analyzed for enhancement of STAT3 activation by western blot using a specific antibody to phosphorylated STAT3 (Ser 727). Total STAT3 level was also analyzed by stripping the membrane using a specific antibody. Actin was used as the loading control. Up-regulation of total STAT3 and enhancement of STAT3 activation from the basal level was observed when CLL B-cells were cocultured with stromal cells. (B & C) CLL B-cell lysates described in (A) were also analyzed for the expression of Mcl-1, XIAP and Bcl-2 by Western blot. Co-culture with stromal cells resulted in increased expression of Mcl-1 (B) and XIAP (C top row) in CLL B-cells. Bcl-2 expression remained unaltered (C middle row). (D) Modulation of curcumin induced apoptosis in CLL B cells when cocultured with stromal cells. Primary CLL B-cells (n=9) were cultured alone or together with HS-5 human stromal cells in either transwells or direct cell contact for 24 hours. Cells were then treated with the indicated doses of curcumin for 24 hours. Cells were harvested, stained with CD19-APC and annexin-FITC/PI by flow cytometric analysis. Viability of CD19⁺ lymphocytes was assessed and is represented by mean values with standard error bars. Higher dose curcumin (20 μM) appeared to overcome the effects of stromal protection.

Fig. 5. Effect of combination treatment with curcumin and EGCG on primary CLL B-cell survival

(A) Primary CLL B-cells (n=10) were treated with increasing doses of curcumin or EGCG alone or in combination using a constant ratio (1:10). After 24 hours of treatment, viability was assessed using annexin/PI staining. The mean value at each dose level is represented in the figure along with the standard error. (B) Combination index values at effective dose (ED) 50 (50% cell death) and ED90 (90% cell death) for curcumin and EGCG (constant ratio 1:10) for CLL B-cells from 10 patients were calculated using Calcusyn software. Values less than 1 imply synergy; values equal to 1 imply an additive effect; and values greater than 1 imply antagonism. As shown, the simultaneous administration of curcumin and EGCG led to antagonism in the majority of patients tested. (C) Sequential treatment is superior to concurrent therapy. Based on the results in Panel B suggesting that although both agents have single-agent activity they are antagonistic in the majority of patients when administered simultaneously, we next evaluated the effect of sequential administration. CLL B-cells were cultured (n=5) with sub-lethal doses of curcumin (C, 10 μM), EGCG (E, 100 μM) or both drugs together (C+E) for 24 hours. Cells were then washed and cultured for another 24 hours in either media alone or with the second agent (C then E; E then C) for an additional 24 hours using the same doses. Cells were harvested and apoptosis assessed using annexin/PI staining as analyzed by flow cytometry. The results show sequential administration (C then E; E then C) was dramatically superior to simultaneous administration (C+E) and that the E then C sequence appeared superior to the reverse. (D) Sequential treatment of CLL B cells with curcumin/EGCG overcomes stromal protection. CLL B-cells (n=5) were treated with curcumin (C), at 10 and 15 μM), EGCG (E) at 100 μM or together (C10+E and C15+E) either cocultured in direct contact with HS-5 stromal cells or cultured alone for 24 hours. Cells were then washed and cultured for another 24 hours in either media alone or with the second agent (C10 then E; C15 then E; E then C10 and E then C15) for an additional 24 hours. Cells were harvested and induction of apoptosis was assessed using annexin/PI staining as analyzed by flow cytometry. The results show sequential administration (E then C) was dramatically superior to simultaneous administration (C+E) or the reverse sequence and that this approach can overcome stromal protection. Results are presented as mean values with standard error bars.