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. Author manuscript; available in PMC: 2014 Jan 15.
Published in final edited form as: Clin Cancer Res. 2009 Feb 15;15(4):1250–1258. doi: 10.1158/1078-0432.CCR-08-1511

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

Fig. 5

(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.