Cellular Effect of Curcumin and Citral Combination on Breast Cancer Cells: Induction of Apoptosis and Cell Cycle Arrest

Abstract

Purpose: The unmanageable side effects caused by current chemotherapy regimens to treat cancer are an unresolved problem. Although many phytonutrients are useful as chemoprevention without side effects, their effects are slower and smaller than conventional chemotherapy. In the present work, we examined the cumulative effect of two phytonutrients, curcumin and citral, on breast cancer cell lines and compared their effect with the known chemotherapy regimen of cyclophosphamide, methotrexate, and 5-fluorouracil.

Methods: Using cultured breast cancer and normal epithelial cells, the cytotoxic and apoptotic effect of curcumin and citral was evaluated in vitro. The synergistic effect of curcumin and citral was calculated by a combination index study using the method by Chou and Talalay. Cell death pathways and mechanisms were analyzed by measuring intracellular reactive oxygen species (ROS) and apoptotic protein levels.

Results: Curcumin and citral caused dose and time dependent cell death and showed a synergistic effect at effective concentration EC50 and above concentrations in breast cancer cells without disturbing normal breast epithelial cells. With combination curcumin and citral treatment, apoptosis induction and cell cycle arrest at G0/G1 phase in breast cancer cells were observed. Curcumin and citral generated ROS and activated p53 and poly (ADP-ribose) polymerase-1 mediated apoptotic pathways.

Conclusion: The results of this study suggest that curcumin and citral in combination may be a useful therapeutic intervention for breast cancer.

Keywords: Apoptosis; Breast cancer cell line; Cell cycle checkpoints; Citral; Curcumin.

Figure 1. Survival of MCF 7 and MDA MB 231 cells after exposure to chemotherapeutic drugs. Dose dependent cell viability of MCF 7, MDA MB 231, and MCF 10A cells, after combination treatment with cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) was assessed by MTT assay. The concentration of each combination is shown below the graph. Data were plotted as percent viability (% control). At a zero concentration of drugs, % viability was considered 100%. The data represent the mean±standard deviation of one of the three similar experiments each performed in triplicate.

Figure 2. Clonogenic survival of breast cancer (MCF 7 and MDA MB 231) and breast epithelial (MCF 10A) cell lines treated with various concentrations of curcumin and citral for 24 hours. Data were plotted as percent viability (% control). At a zero concentration of curcumin and citral, the % viability was considered 100%. The data represent the mean±standard deviation of one of the three similar experiments each performed in triplicate.

Figure 3. Combined effect of curcumin (Cur) and citral (Cit) on the survival of MCF 7 and MDA MB 231 cells. (A) The concentration used of Cur1, Cur2, Cur3, and Cur4 was 10, 20, 40, and 80 µM, respectively. Whereas, the concentration used of Cit1, Cit2, Cit3, and Cit4 was 20, 40, 80, and 160 µM, respectively. The concentration of cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) was 3.5, 0.75, and 1.5 mM, respectively. Data were plotted as percent viability (% control). At a zero concentration of drugs, curcumin and citral, the % viability was considered 100%. The data represent the mean±standard deviation of one of the three similar experiments each performed in triplicate. (B) Combination Index (CI) of curcumin and citral. The graph shows the mean values of the combination index at the affected fractions of 25.0% (IC₂₅), 50.0% (IC₅₀), and 75.0% (IC₇₅), when curcumin and citral were used in combination in MCF 7 and MDA MB 231 cells. A CI value less than 1 indicates synergism, a CI not different from 1 indicates an addition, and a CI higher than 1 indicates antagonism. The data represent the mean±standard deviation of one of the three similar experiments each performed in triplicate.

Figure 4. Induction of apoptosis in MCF 7 cells by curcumin and citral treatment. (A) Annexin V-fluorescein isothiocyanate/propidium iodide (PI) dual staining of the MCF 7 cells exposed to cyclophosphamide, methotrexate, and 5-fluorouracil (CMF), curcumin or citral and to the combination of curcumin and citral. Graph represents the distribution of apoptotic cells based on annexin V/PI staining. The apoptotic cells are stained with annexin V (green fluorescence), and the necrotic cells are stained with PI (red fluorescence). The image showed the stained cells treated with curcuminn (40 µM) and citral (80 µM). Scale bar, 20 µm. (B) TUNEL assay. Apoptosis detection in MCF 7 cells treated with CMF, curcumin or citral and the combination of curcumin and citral for 24, 48, and 72 hours. Cells were observed under microscope and % apoptotic cells were counted out of total 100 cells. The image showed the stained cells treated with curcumin (40 µM) and citral (80 µM). Scale bar, 10 µm. The data represent the means of three independent experiments performed in triplicate, with standard deviations represented by vertical bars. ^*p< 0.05.

Figure 5. Measurement of DNA damage in MCF 7 cells caused by curcumin and citral treatment. (A) The DNA damage in MCF 7 cells exposed to CMF, curcumin or citral and the combination of curcumin and citral were assessed by COMET assay. A1, untreated MCF 10 A cells; A2, untreated MCF 7 cells; A3, CMF treated MCF 7 cells, and A4, curcumun (40 µM) and citral (80 µM) treated MCF 7 cells. (B) Graph showed the measure of COMET parameters. The data represent the means of three independent experiments performed in triplicate.

Figure 6. Distribution of MCF 7 cells in various phases of cell cycle; exposed to cyclophosphamide, methotrexate, and 5-fluorouracil (CMF), curcumin or citral and the combination of curcumin and citral; analyzed by flow cytometry. The data represent the means of three independent experiments performed in triplicate, with standard deviations represented by vertical bars. ^*p<0.05.

Figure 7. Intracellular reactive oxygen species (ROS) generation and total glutathione (GSH) content. (A) Effect of curcumin and citral on intracellular ROS. The graph shows the generation of ROS in MCF7 and MCF 10A cells, treated with cyclophosphamide, methotrexate, and 5-fluorouracil (CMF), curcumin or citral and the combination of curcumin and citral. The data represent the means of three independent experiments performed in triplicate, with standard deviations represented by vertical bars. (B) Effect of curcumin and citral on the total GSH content. The total GSH content of MCF 7 cells exposed to cyclophosphamide, methotrexate, and 5-fluorouracil (CMF), curcumin or citral and the combination of curcumin and citral was measured. Values in graph represent the total GSH content in µM. The data represent the means of three independent experiments performed in triplicate, with standard deviations represented by vertical bars. ^*p<0.05.

Figure 8. Measurement of levels of apoptotic proteins. The apoptotic protein levels in the MCF 7cells treated with cyclophosphamide, methotrexate, and 5-fluorouracil (CMF), curcumin or citral and the combination of curcumin and citral in MCF 7 and untreated MCF 10A cells were measured using the Sandwich Enzyme-Linked Immunosorbent Assay kit. Values of all protein represented their absorbance at 450 nm. The data represent the means of three independent experiments performed in triplicate, with standard deviations represented by vertical bars. ^*p<0.05.