Effects of curcumin on the proliferation and mineralization of human osteoblast-like cells: implications of nitric oxide

Abstract

Curcumin (diferuloylmethane) is found in the rhizomes of the turmeric plant (Curcuma longa L.) and has been used for centuries as a dietary spice and as a traditional Indian medicine used to treat different conditions. At the cellular level, curcumin modulates important molecular targets: transcription factors, enzymes, cell cycle proteins, cytokines, receptors and cell surface adhesion molecules. Because many of the curcumin targets mentioned above participate in the regulation of bone remodeling, curcumin may affect the skeletal system. Nitric oxide (NO) is a gaseous molecule generated from L-arginine during the catalization of nitric oxide synthase (NOS), and it plays crucial roles in catalization and in the nervous, cardiovascular and immune systems. Human osteoblasts have been shown to express NOS isoforms, and the exact mechanism(s) by which NO regulates bone formation remain unclear. Curcumin has been widely described to inhibit inducible nitric oxide synthase expression and nitric oxide production, at least in part via direct interference in NF-κB activation. In the present study, after exposure of human osteoblast-like cells (MG-63), we have observed that curcumin abrogated inducible NOS expression and decreased NO levels, inhibiting also cell prolifieration. This effect was prevented by the NO donor sodium nitroprusside. Under osteogenic conditions, curcumin also decreased the level of mineralization. Our results indicate that NO plays a role in the osteoblastic profile of MG-63 cells.

Figure 1

(a) Concentration-dependent effect of curcumin on MG-63 cells viability. Cells were incubated with various concentrations of curcumin for 24 h, after which cell viability was measured using MTT. Bars represent mean values, error bars represent SEM: n.s. (no significant difference compared to control cells), ** significant difference compared to control cells (p < 0.001). (b) Effect of curcumin (10 μM); L-NG-Nitroarginine Methyl Ester (L-NAME) (1 mM) and Sodium nitroprusside (SNP) (0.2 mM) at the indicated combinations after 24 h of exposure. Bars represent mean values, n.s. (no significant difference compared to control cells), * significant difference compared to control cells (p < 0.05), ** significant difference compared to control cells (p < 0.001).

Figure 2

(a) Nitrite production by MG-63 cells at 12 and 24 h. Nitrite production was expressed as percentage of control (unstimulated cells). ** (p < 0.001), n.s. (not significant) vs. control cells in both cases. Bars represent mean values; error bars represent SEM. (b) Temporal changes in the expression of iNOS mRNA after curcumin (10 μM) exposure after 6, 12 and 24 h. Dots represent mean values; error bars represent SEM. ** Significant difference compared to control cells (p < 0.001).

Figure 3

(a) Effect of curcumin (10 μM) and L-NAME (1 mM) on mineralized nodules in MG-63 cells. Cells were untreated or treated with osteogenic medium, osteogenic medium + curcumin, osteogenic medium + LNAME or osteogenic medium + curcumin and L-NAME for 21 days, and, then, mineralization was determined by AR-S staining. (b) Results are expressed as percentage of control. The data are expressed as mean ± SEM of three determinants. n.s. (no significant difference compared to control cells), * significant difference compared to control cells (p < 0.05), ** significant difference compared to control cells (p < 0.001).

Figure 4

(a) Time course of alterations in COLI mRNA and OCN mRNA. (b) Concentration after the 7, 15 and 21 day of exposure to 10 μM curcumin + osteogenic medium or osteogenic medium alone. Total cellular RNA was extracted from MG-63 cells as described in Methods. COLI and OCN mRNA amount was determined by performing RT-PCR. Data represent the mean ± SEM for a minimum of three separate experiments. ** Significant difference compared to day 15 cells (p < 0.01). Not significant differences were found between curcumin stimulated cells and controls.

Figure 5

(a) The ALP activity of MG-63 cells at 0.5, 1, 1.5, 2, 3, 7, 9, 12, 15 and 21 post seeding in the presence of either osteogenic medium or osteogenic medium + curcumin 10 μM. ** (p < 0.001 vs. day 0.5 in both conditions). Significant differences (p < 0.001) were found from day 9 of culture to the end of the experiment between the experimental conditions. (b) Time course of alterations in ALP mRNA concentration after the 3, 7, 15 and 21 day of exposure to 10 μM curcumin + osteogenic medium, osteogenic medium or culture medium (unstimulated). Total cellular RNA was extracted from MG-63 cells as described in Methods. ALP mRNA amount was determined by performing RT-PCR. Data represent the mean ± SEM for a minimum of three separate experiments. * Significant difference compared to unstimulated cells (p < 0.05). Not significant differences were found between curcumin stimulated cells and osteogenic medium.