Side Effects of Curcumin: Epigenetic and Antiproliferative Implications for Normal Dermal Fibroblast and Breast Cancer Cells

Abstract

Background: Curcumin is a yellow-orange pigment obtained from the plant Curcuma longa, which is known to exert beneficial effects in several diseases, including cancer. However, at high doses, it may produce toxic and carcinogenic effects in normal cells. In this context, we studied the effects of curcumin on normal human dermal fibroblast (HDF) cells and breast cancer cells (MCF7).

Methods: We used cellular viability and growth assays to evaluate the antiproliferative action of curcumin, analyzed the endogenous glutathione levels, conducted cell cycle, apoptosis, and necrosis analyses, and performed immunodetection of glutathionylated and acetylated H3 histones.

Results: We found that HDFs are more sensitive to curcumin treatment than MCF7 cells, resulting in pronounced arrest of cell cycle progression and higher levels of cellular death. In both cell types, the homeostasis of the redox cellular environment did not change after curcumin treatment; however, significant differences were observed in glutathione (GSH) levels and in S-glutathionylation of H3 histones.

Conclusion: Curcumin administration can potentially confer benefits, but high doses may be toxic. Thus, its use as a dietary supplement or in cancer therapies has a double edge.

Keywords: curcumin; glutathione; histone glutathionylation; human cancer cells; post-translational modifications (PTMs).

Figure 1

Effect of curcumin treatment on cell growth and cell viability in human dermal fibroblasts (HDFs) and breast cancer cells (MCF7). (A) Cell viability was determined by an MTT assay after exposure to an increasing concentration of curcumin for 24 h. (B) Cell growth represented as the fold increase in the number of viable cells over 24, 48, and 72 h for HDF and MCF7 cells exposed to 5, 10, and 20 μM of curcumin. The calculated doubling time index is shown in the table. The values in the figures are expressed as the mean ± SD of five independent experiments, each performed in triplicate. A significant difference in cell viability between the two lines was observed in the 20, 40, and 80 μM curcumin treatments. In both cell lines, significant differences in cell growth between the control cells and the curcumin-treated cells were observed (at 48 h and 72 h for all used doses and at 24 h for the HDF cell line in the 10 µM and 20 µM curcumin treatments). * p < 0.05 and ** p < 0.001.

Figure 2

Post-translational modifications (PTMs) of the H3 histone induced by curcumin in MCF7 and HDF cells. (A) Histone acetylation evaluated using an acetylation assay kit (Abcam) and analyzed and quantified by ELISA. The percentage of acetylation was calculated as described in the Materials and Methods section. The control for both cell lines was set as 100% histone acetylation. (B) Histone S-glutathionylation and total H3 evaluated by Western blotting with anti-glutathione (GSH) and anti-H3 antibodies. The histograms represent the densitometric analysis (n = 3). Lamin B1 has been used as a loading control. (C) Ratio of total H3 vs. glutathionylated H3. The histogram represents the values reported in the table, calculated by densitometric analysis. ** p < 0.001.

Figure 3

Effect of curcumin on glutathione levels in HDF and MCF7 cell lines. Quantification of total cellular GSH and GSSG of HDF (A) and MCF7 cells (B). Analysis was performed using a DTNB-glutathione reductase recycling assay and the amount of GSH and GSSG was normalized to protein content. (C) GSH:GSSG ratio. Values are expressed as the mean ± SD of six separate experiments.

Figure 4

Effect of curcumin on cell cycle progression in MCF7 and HDF cells. (A) Breast cancer cells (MCF-7) and (B) human dermal fibroblasts (HDFs) treated with 10 µM of curcumin for 24 h and stained with propidium iodide (PI). DNA content was analyzed by flow cytometry. Results are represented as patterns of cells stained with propidium iodide (PI) before and after curcumin treatment (left). The percentage of cell population at different phases (G1, S, and G2/M) of the cell cycle is shown by histograms (right). The images are representative of three separate experiments. * p < 0.05 and ** p < 0.001 versus an untreated control.

Figure 5

Curcumin-induced apoptosis. HDF and MCF7 cells were treated with 10 μM of curcumin for 24 h followed by washing, trypsinization, and incubation with Annexin V-FITC and propidium iodide (PI). Representative cytograms from three independent experiments are shown for (A) MCF7 and (B) HDF cells. (C) Apoptosis and necrosis quantified as a fold increase with respect to the control (untreated cells). Total apoptosis was calculated by considering early apoptosis (lower right) and late apoptosis (upper right). Data are the mean of at least three experiments, and error bars represent ± SD ** p ≤ 0.001 versus control.