Curcumin enhances the cytogenotoxic effect of etoposide in leukemia cells through induction of reactive oxygen species

Figures & data

Figure 1 Influence of curcumin on cytotoxicity induced by etoposide in HL-60 cells, primary CD34⁺ cells, and granulocytes.

Notes: (A) Cells were incubated with different concentrations of curcumin and/or etoposide for 24 hours, stained using propidium iodide, and analyzed by flow cytometry. Percentage of dead HL-60 cells. (B) Cooperation pattern of curcumin (7, 10, and 20 μM) and etoposide (3, 5, and 10 μM) combinations in induction of cytotoxicity in HL-60 cells. (C) Percentage of dead CD34⁺ cells after incubation with curcumin and/or etoposide. (D) Percentage of dead granulocytes after incubation with investigated compounds. *P<0.05; **P<0.01; ***P<0.001 vs control; ^##P<0.01; ^###P<0.001 vs etoposide. The experiments were repeated three times. Data are presented as mean ± standard error of the mean.
Abbreviations: CD, cluster of differentiation; CI, combination index; Cur, curcumin; Eto, etoposide.

Figure 2 Influence of curcumin on phosphorylation of H2AX and percentage of sub-G1 fraction induced by etoposide.

Notes: (A) Cells were treated with curcumin and/or etoposide for 4 hours. Intensity of fluorescence of γ-H2AX was measured by flow cytometry. (B) Representative contour plots of histone γ-H2AX vs DNA staining of HL-60 cells. (C) Percentage of sub-G1 fraction of cell cycle was evaluated by flow cytometry. (D) Representative histograms of cell cycle distribution and sub-G1 fraction of HL-60 cells. *P<0.05; ***P<0.001 vs control; ^#P<0.05; ^##P<0.01; ^###P<0.001 vs etoposide. The experiments were repeated three times. Data are presented as mean ± standard error of the mean.
Abbreviations: CD, cluster of differentiation; Cur, curcumin; Eto, etoposide; PE-A, phycoerythrin-area.

Figure 3 Pro-oxidant activity of curcumin and etoposide in HL-60 cells.

Notes: (A) HL-60 cells were incubated with curcumin and/or etoposide for 24 hours. GSH content was detected spectrophotometrically. (B) ROS content in cells was detected using CM-H₂DCFDA staining and analyzed by flow cytometry. (C) Representative histograms of CM-H₂DCFDA-derived fluorescence. (D) Cells were preincubated for 1 hour with NAC, followed by incubation with curcumin and/or etoposide for 24 hours. Cytotoxicity was detected using PI staining and analyzed by flow cytometry. (E) Cells were preincubated for 1 hour with NAC followed by incubation with curcumin and/or etoposide for 24 hours. Apoptotic cells were detected using Annexin-V/PI staining and analyzed by flow cytometry. *P<0.05; **P<0.01; ***P<0.001 vs appropriate control; ^#P<0.05; ^###P<0.001 vs etoposide. Data are presented as mean ± standard error of the mean.
Abbreviations: APC, allophycocyanin; Cur, curcumin; Eto, etoposide; GSH, glutathione; NAC, N-acetyl-l-cysteine; CM-H₂DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; PI, propidium iodide; ROS, reactive oxygen species.

Figure 4 Leukemia progression and effect of etoposide and/or curcumin on body and spleen weights of BNML rats.

Notes: (A) Scheme of experiment. (B) Body weight of rats before and 23 days after inoculation of leukemic cells. (C) Weight of spleen in rats before and 23 days after inoculation of leukemic cells. (D) Representative pictures of healthy (day 0) and leukemic (day 23) spleen. (E) Effect of curcumin (100 or 200 mg/kg) and/or etoposide (50 mg/kg) on weight of spleen in rats 23 days after inoculation of leukemic cells. Each bar represents mean ± SEM. N=8. *P<0.05; ***P<0.001 vs day 0. ^###P<0.001 vs etoposide alone.
Abbreviations: BNML, Brown Norway myeloid leukemia; Cur, curcumin; Eto, etoposide; SEM, standard error of the mean; Cont, control.

Figure 5 Effect of etoposide and/or curcumin on leukemic cells and healthy B-cells in the spleens of BNML rats.

Notes: BNML rats were treated with curcumin (100 or 200 mg/kg) and/or etoposide (50 mg/kg). Spleens were excised on day 23 after inoculation of leukemic cells. (A) Detection of BNML cells in the rat spleen by flow cytometry. (B) Percentage of leukemic cells in the spleen. (C) Percentage of healthy B-cells in the spleen. (D) Apoptosis rate in leukemic cells. (E) Apoptosis rate in B-cells. Flow cytometry phenotyping and staining with Annexin-V/7AAD. Each bar represents mean ± SEM. *P<0.05; **P<0.01; ***P<0.001 vs day 0. ^#P<0.05; ^##P<0.01 vs etoposide alone.
Abbreviations: 7AAD, 7-amino-actinomycin D; BNML, Brown Norway myeloid leukemic cells; CD, cluster of differentiation; Cont, control; Cur, curcumin; Eto, etoposide; FITC, fluorescein isothiocyanate; GR, granulocytes; lymph, lymphocytes; SEM, standard error of the mean; SSC, side scatter; FSC, forward scatter; RM124-RPE, RM124-R-phycoerythrin.

Figure 6 Effect of etoposide and/or curcumin on expression of survivin in leukemic cells and healthy B-cells isolated from the spleens of BNML rats and in HL-60 cells. Notes: BNML rats were treated with curcumin (100 mg/kg or 200 mg/kg) and/or etoposide (50 mg/kg). Spleens were excised on day 23 after inoculation of leukemic cells. HL-60 cells were incubated with different concentrations of curcumin and/or etoposide for 24 hours. (A) Expression of survivin in BNML cells. (B) Expression of survivin in B-cells. (C) Expression of surviving in HL-60 cells by quantitative RT-PCR. EF2 served as a housekeeping gene. Each bar represents mean ± SEM. The analysis of survivin expression was repeated three times.

Abbreviations: BNML, Brown Norway myeloid leukemia; Cur, curcumin; EF, elongation factor; Eto, etoposide; RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean.