Annual Report 1998-1999

32. Effect of Curcumin on the Production of Nitric Oxide by Cultured Rat Mammary Gland

Nitric oxide (NO) is a unique biological messenger molecule that is synthesized from L-arginine by nitric oxide synthase (NOS). NO regulates blood pressure, smooth muscle relaxation, neuronal signaling, immune reactions, and aspects of development, cell motility and differentiation. NOS has at least three distinct isoforms, including the neuronal/brain (nNOS, bNOS or NOS1), inducible (iNOS or NOS2), and endothelial (eNOS or NOS3) types, and these are known to distribute across a wide spectrum of cell types and tissues. In our previous report we showed that three isoforms of NOS are present in the rat mammary gland, and we suggested that these NOS isoforms may correlate with mammary gland development and regulatory functions. Meanwhile, curcumin, the yellow pigment and a major component of turmeric which is a commonly used spice, has been shown to possess anti-carcinogenic and anti-inflammatory activities and inhibitory activity towards reactive oxygen-generating enzymes. We reported that curcumin had a preventive activity towards the promotion of radiation-induced mammary tumorigenesis based on results from an animal model system established in our laboratory. Now, we have examined the effect of curcumin on NO-generation by the rat mammary gland in culture to elucidate the effectiveness and usefulness of curcumin in the pathophysiology of the mammary gland.

Isolated mammary glands from female Wistar-MS rats were diced into approximately 3-mm cubes and each cube was cultured in the well of a 24-multiwell plate containing 2 ml of 5% fetal calf serum/Dulbecco's Modified Eagle Medium in the presence or absence of bacterial lipopolysaccharide (LPS, 0.5 g/ml) for 2 days in a mixture of 5% CO₂/95% air at 37°C. Curcumin (~100 M) was added at the same time to the LPS treated cultures. The nitrite concentrations in conditioned media were determined immediately after the termination of the culture with Griess reagent mixture. Tissue homogenates from the incubated mammary glands were also prepared for Western blot analyses of NOS isoforms. NO scavenging activity of curcumin was examined by incubation with NO donor, N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC 12) in a reaction mixture held at 37°C for 2 h. At the end of incubation, the reaction mixture was placed on ice and collected for determination of the nitrite concentration.

The amount of NO produced spontaneously by mammary glands in culture was relatively minute for the 2-day culture period, whereas the NO concentration in the conditioned media was significantly increased (almost 20-fold over the control) by the addition of LPS to the culture system. This enhancement of NO production by the mammary gland with LPS was reduced to 76% and to 59% by addition of 30 M and 100 M curcumin, respectively, to the culture (Fig. 14.). The iNOS (122 kDa) and eNOS (152 kDa) isoforms were detected in the mammary gland extracts at the terminus of the organ culture. The quantity of immunoreactive iNOS was apparently increased in the extract treated with LPS, while, the eNOS expression was clearly diminished in the corresponding tissue extract. Curcumin (100 M) obviously suppressed the iNOS expression in the mammary glands cultured with LPS, and recovery of the decline in eNOS expression was conversely observed. Incubation of NOC 12 resulted in linear dose-dependent NO production, and the presence of curcumin reduced the NO concentration in the mixture (Fig. 15.). These results indicate that curcumin has an inhibitory activity for iNOS induction by LPS in the mammary gland and a scavenging activity for NO radical, and they suggest that the therapeutic properties of curcumin regarding inflammation, cancer and other pathological conditions of mammary glands might be explained, at least partly, by its ability to inhibit iNOS expression and to scavenge NO.

Fig.14. Inhibition of NO production in LPS-stimulated mammary glands by curcumin.
A piece of the mammary gland was cultured, and at the end of culture the conditioned media were collected for the detection of nitrite (NO₂) concentration.
*: Significant difference from LPS alone control, p < 0.001.

Fig.15. Production of nitric oxide from NOC 12 and NO scavenging by curcumin.
NOC 12 in PBS was mixed with curcumin dissolved in alcohol and incubated at 37°C for 2 h. The nitrite (NO₂) concentration was determined with Griess reagent mixture. Values represent mean ± SE obtained from two independent experiments. Each experiment contained two tubes per replicate.
* : Significant difference from control (NOC 12 alone), p < 0.001.
§ : Significant difference from control (NOC 12 alone), p < 0.01.
# : Significant difference from control (NOC 12 alone), p < 0.05.