Cytotoxic activity of curcumin towards CCRF-CEM leukemia cells and its effect on DNA damage

Abstract

The cytotoxic activity of curcumin towards CCRF-CEM human T-cell leukemia cells was measured by the MTT assay. Tumor cells were more sensitive to the cytotoxic activity of curcumin or curcumin-Cu (II)compared to normal cells, and the IC(50) of curcumin towards CCRF-CEM cells was 8.68 microM, and that of curcumin-Cu (II) was 8.14 microM. The cell cycle distribution of curcumin-treated CCRF-CEM cells was analyzed by flow cytometry. DNA damage induced by oxidants such as curcumin-Cu (II) ions is considered as one of the main causes of cell inactivation. Therefore, we analyzed the effect of curcumin on DNA damage by agarose gel electrophoresis and atomic force microscopy (AFM). Gel electrophoresis analyses showed that curcumin or Cu (II) alone failed to cause DNA damage in pBR322 plasmid DNA as compared with the normal plasmid. However, DNA plasmids were mostly damaged after treatment with curcumin of different concentrations in the presence of Cu (II). Two forms were observed by means of AFM: closed circular plasmids and linear plasmids. DNA damage induced by a combination of curcumin and Cu (II) was also found by agarose gel electrophoresis, which was applied as control method to verify the results obtained by AFM.

Figure 1

Structure of curcumin.

Figure 2

Proposed mechanism for curcumin–Cu (II) induced DNA damage.

Figure 3

Effects of curcumin and curcumin-Cu (II) on the viability of CCRF-CEM and Vero cells as determined by the MTT assay. A: Treatment with curcumin; B: Treatment with curcumin-Cu (II). The values for each concentration tested represent the average (mean ± SD) from eight replicate wells and are representative of three separate experiments.

Figure 4

Cell cycles distribution of CCRF-CEM cells after treatment with different concentrations of curcumin for 48 h. Flow cytometric analysis of CCRF-CEM cell cycle distribution, (A) Untreated control cells; (B) Cells treated with 15 μM curcumin; (C) The results were analyzed by Mod Fit LT 3.0. Data are presented as mean ± SD (n = 3). **p < 0.01, *p < 0.05; p value compared with the control group (0 μM).

Figure 5

Agarose gel electrophoretic patterns of plasmid DNA treated with curcumin in the presence of Cu (II) ions (0.2 mM). pBR322 plasmid DNA (0.5 μg) was incubated for 1 h at 37 ºC in the presence of the following additives: lane 1, no addition (DNA control); lane 2, 0.2 mM curcumin without copper; lane 3, copper without curcumin; lanes 4–8, copper plus curcumin at different concentrations, 0.01, 0.025, 0.05, 0.1, and 0.2 mM, respectively.

Figure 6

Untreated pBR332 DNA plasmid. A: Two-dimensional representation. Size = 2 µm × 2 µm; B: Three-dimensional representation. Size = 400 nm × 400 nm × 400 nm.

Figure 7

AFM imaging of pBR332 DNA plasmids treated with curcumin and/or Cu (II). A: Plasmid 0.05 µg plus 0.02 mM Cu (II) for 2 h; B: Plasmid 0.05 µg plus 0.02 mM curcumin for 2 h; C: Plasmid 0.05 µg plus 0.02 mM Cu (II) plus 0.02 mM curcumin for 2 h.