18. Modulation by Curcumin of NF-kB Activation in Cultured Rat Mammary Gland

Makoto Onoda and Hiroshi Inano

Keywords: curcumin, NF-kB, IkB, mammary gland, nitric oxide, nitric oxide synthase

We found in a previous report that curcumin, a yellow pigment and a major component of turmeric, had the ability to inhibit induction of inducible nitric oxide synthase (iNOS) by lipopolysaccharide (LPS) in the rat mammary gland and to scavenge NO radicals. However, the NO-generation by iNOS within the mammary gland was not disturbed by the presence of curcumin after the pre-stimulation with LPS. It is, therefore, likely that curcumin does not affect the post-translational processes of iNOS de novo synthesis. In this context, we undertook an investigation to elucidate whether curcumin influenced the expression and translocation of NF -kB, the transcription factor that regulates iNOS gene transcription, and IkB, an inhibitory element of NF-kB, by using rat mammary gland tissue culture system.

Female Wistar-MS (8-week-old) rats were primed by implantation with pellets of 17-estradiol (0.5 mg/3-week-release type) and progesterone (35 mg/3-week-release type). After 3 weeks of priming, the rats were sacrificed by carbon dioxide asphyxiation and the inguinal mammary glands were excised asceptically for organ culture. The isolated mammary glands were diced into approximately 3 mm cubes, and each cube cultured in the well of 24-multiwell plates containing 2 ml of 5% fetal calf serum (FCS)/Dulbecco's Modified Eagle's Medium (DMEM) supplemented with antibiotics and antimycotic in a mixture of 5% CO₂/95% air at 37^oC for 2 days. The medium was then replaced with 5% FCS/DMEM in the presence or absence of LPS (0.1 or 0.5g/ml), and the culture was maintained for another 30 or 60 min. Curcumin (100-fold concentration) dissolved or suspended in absolute ethanol was added at the same time to the LPS treated cultures. At the end of the culture, the cultured mammary glands were collected and further processed for the preparation of mammary gland homogenates. The mammary glands cultured in various conditions were minced and homogenized in ice-chilled 5 mM Tris-HCl buffer (pH 7.5) containing 0.25 M sucrose, 5 mM EGTA and inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 mM sodium vanadate, 10 g/ml aprotinin, 5 g/ml leupeptin). The homogenates were then centrifuged at 600 xg for 10 min at 4^oC, and cytoplasmic (supernatant) and nuclear (precipitate) fractions were separately reconstituted in reducing sample buffer and loaded into the mini-gel system for SDS-PAGE. Subsequently, the separated proteins were electrotransferred to a nitrocellulose membrane. The membrane was reacted overnight with either anti-NF-kB p65 or anti-IkB body. NF-kB p65 and IkB were detected with 2nd antibody conjugated with alkaline phosphatase. The immunoreactivity was, then, visualized with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). The molecular weight of the immunoreactive bands was estimated from plots of molecular weight vs. relative mobility of rainbow marker standard proteins which were run simultaneously with the sample proteins.

The amount of NF-kB in the cytoplasmic fraction did not pronouncedly change after 30 and 60 min incubations with LPS (0.1 and 0.5 g/ml), however, that of the nuclear fractions was obviously increased in the mammary gland by LPS-stimulation (Fig. 10). In addition, IkB content slightly, but clearly, declined in cytoplasmic fractions of LPS-treated mammary gland. These indicate the degradation of IkB, NF-kB activation and the translocation of this transcription factor from cytoplasm to nucleus in the mammary gland in a short term after the stimulation by LPS.

On the other hand, the addition of curcumin (100M) with LPS (0.1 g/ml) to the culture clearly inhibited the translocation to the nucleus of NF-kB and recovered the reduction of IkBin the cytoplasmic fraction (Fig. 10), while, curcumin at the same dose did not show any inhibitory effect on the NF-kB translocation and IkB degradation in the mammary gland treated with a higher dose (0.5 g/ml) of LPS.

This may indicate a limited potential for curcumin on the iNOS inhibition under the current experimental conditions because of less solubility of curcumin in the in vitro culture system. In any event, NO produced harmfully within the mammary gland during acute and chronic pathophysiological conditions may perturb the regulation of normal mammary gland development and its function. Results shown here support the idea that curcumin possesses the ability to reduce induction of iNOS by LPS in the mammary gland.

The results also suggest that a part of the inhibitory activity of curcumin on iNOS induction in mammary gland may be due to inhibition of the NF-kB activation cascade following IkB degradation. Figure Legend.

Fig.10.

Immunoblot analyses of NF-kBp65 and IkB in nuclear and cytoplasmic fractions, respectively, obtained from cultured rat mammary glands. Each fraction (10 g/lane) was loaded onto the SDS-PAGE mini-gel system and the immunoreactive substances were visualized by Western blot analysis with an alkaline phosphatase-conjugated secondary antibody. Lane 1, non-treatment control; Lane 2, control + LPS (0.1 g/ml); Lane 3, control + LPS + curcumin (100 M).

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