Cytotoxic effects of curcumin in human retinal pigment epithelial cells

Abstract

Background: Curcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE) cells in vitro.

Methodology/principal findings: Cellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM) and delayed apoptosis (above 1 µM). The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein.

Conclusion: It is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (∼10 µM) has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as concomitant therapy of cancer or in the treatment of eye diseases, retinal function should be monitored carefully.

Figure 1. Curcumin inhibits expression and secretion of VEGF.

A. Concentration-dependent effect of curcumin on the gene expression of VEGF-A. The mRNA level was determined by real-time RT-PCR after stimulation of the cells with curcumin for 2 and 6 h, respectively. B. Concentration-dependent effect of curcumin on the secretion of VEGF from RPE cells. The effects were determined in the absence (control) and presence of PDGF (10 ng/ml) and CoCl₂ (150 µM), respectively, for 6 h. The level of VEGF-A₁₆₅ in the cultured media was determined by ELISA. The concentration of curcumin (in µM) is given in the bars. The vehicle control was made with ethanol (0.2%). Data are means ± SEM of 3–6 independent experiments carried out in triplicate using cells from different donors. Significant difference vs. untreated control: *P<0.05; **P<0.01. Significant difference vs. PDGF and CoCl₂, respectively: ^• P<0.05; ^•• P<0.01.

Figure 2. Effects of curcumin on the gene expression of bFGF (A) and HGF (B).

The mRNA levels were determined by real-time RT-PCR after stimulation of the cells with curcumin for 2 and 6 h, respectively. C. Effect of curcumin on the secretion of bFGF from RPE cells. The level of bFGF in the cultured media was determined by ELISA after stimulation of the cells with curcumin for 6 and 24 h, respectively. The concentration of curcumin (in µM) is given in the bars. The vehicle control was made with ethanol (0.2%). Data are means ± SEM of 4–5 independent experiments carried out in triplicate using cells from different donors. Significant difference vs. untreated control: *P<0.05; **P<0.01.

Figure 3. Concentration-dependent effects of curcumin on the proliferation (A, B) and chemotaxis (C) of RPE cells.

The rate of BrdU incorporation was measured after a 24 h (A) and 96 h (B) incubation with the agents, respectively. The concentration of curcumin (in µM) is given in the bars. The effects were determined in the absence (control) and presence of PDGF (10 ng/ml). The vehicle control was made with ethanol (0.2%). Data are means ± SEM of 4–8 independent experiments carried out in triplicate using cells from different donors, and are expressed in percent of untreated control (100%). Significant difference vs. untreated control: *P<0.05; **P<0.01; ***P<0.001. Significant difference vs. PDGF control: ^• P<0.05; ^•• P<0.01.

Figure 4. Concentration-dependent effects of curcumin (A) and triamcinolone acetonide (B) on the viability of RPE cells.

The percentage of viable cells was evaluated 24 h after addition of the test substances to the culture medium. Data are means ± SEM of 5–7 independent experiments carried out in triplicate using cells from different donors, and are expressed in percent of untreated control (100%). Significant difference vs. control: *P<0.05; **P<0.01.

Figure 5. Concentration- and time-dependent effect of curcumin on the rate of internucleosomal DNA fragmentation of RPE cells.

The rate of DNA fragmentation was determined in the cell-free culture supernatants (reflecting cellular necrosis) and cytoplasmic lysates of the cells (reflecting apoptosis) after 6 (A) and 24 h (B) of cell culturing. The concentration of curcumin (in µM) is given in the bars. The vehicle control was made with ethanol (0.2%). Data are means ± SEM of 4–8 independent experiments carried out in triplicate using cells from different donors, and are expressed in percent of untreated control (100%). Significant difference vs. untreated control: *P<0.05; **P<0.01.

Figure 6. Mechanisms of curcumin-induced RPE cell apoptosis/necrosis.

The rate of DNA fragmentation in the cultured media was determined after a 6 h stimulation of the cultures with curcumin (50 µM). The curcumin-induced DNA fragmentation was inhibited in the presence of the following agents: the caspase-3 inhibitor Ac-DEVD-CHO (DEVD; 100 µM), the calpain inhibitor PD150606 (PD; 100 µM), the cell-permeable calcium chelator BAPTA-AM (100 µM), the reducing agent dithiothreitol (DTT; 3 mM), and cyclosporin A (CsA; 1 µM), respectively. The curcumin-induced DNA fragmentation was not inhibited by the caspase-8 inhibitor Ac-IETD-CHO (IETD; 100 µM) and a neutralizing anti-TNFα antibody (20 µg/ml), respectively. Data are means ± SEM of 3–5 independent experiments carried out in triplicate using cells from different donors. Significant difference vs. untreated control: *P<0.05. Significant difference vs. curcumin control: ^• P<0.05.

Figure 7. Effects of curcumin on the gene expression of caspase-3 (A) and Smac (B).

The mRNA levels were determined by real-time RT-PCR after stimulation of the cells with curcumin for 2, 6, and 24 h, respectively. The concentrations of curcumin (in µM) are given in the bars. The vehicle control was made with ethanol (Eth; 0.2%). Data are means ± SEM of 4 independent experiments carried out in triplicate using cells from different donors. Significant difference vs. untreated control: *P<0.05.

Figure 8. Curcumin induces phosphorylation of p38 MAPK and decreases phosphorylation of Akt in RPE cells.

A. Dose-dependence of the curcumin effect on the phosphorylation level of p38 MAPK. As control, PDGF (10 ng/ml) was tested. B. The inhibitor of p38 MAPK activation, SB203580 (SB; 10 µM), decreased the curcumin-induced phosphorylation of p38 MAPK. Curcumin was tested at 50 and 100 µM. C. Dose-dependence of the curcumin effect on the phosphorylation level of Akt. The cultures were stimulated with the agents for 15 min. The amounts of total proteins are shown above; the amounts of phosphorylated proteins are shown below. Similar results were obtained in 3 independent experiments using cells from different donors.

Figure 9. Effects of curcumin on the protein level (A, B) and mRNA expression (C) of HSP70 in RPE cells.

The effect on the protein level was determined by Western blotting after a 6 h (A) and a 24 h (B) stimulation of the cells with curcumin. Similar results were obtained in 3 independent experiments using cells from different donors. In A and B, untreated cells were used as control; the positive control was made by incubation of the cells at 42°C for 1 h followed by incubation at 37°C for 3 h. The vehicle control was made with ethanol (Eth; 0.2%). The gene expression level (C) was determined with real-time RT-PCR after stimulation of the cells with curcumin for 2, 6, and 24 h, respectively. Data are means ± SEM of 3–4 independent experiments carried out in triplicate using cells from different donors. Significant difference vs. untreated control: *P<0.05.