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The gas hydrogen sulphide (H2S) is normally produced in large amounts in the central nervous system during the metabolism of sulphur-containing aminoacids. H2S was recently shown to influence long-term potentiation in the rat hippocampus; this finding suggested that the gas may act as a neuromodulator in the brain. We therefore tested the effect of the gas on the release of corticotropin-releasing hormone (CRH) from rat hypothalamic explants. CRH immunoreactivity in the incubation media was taken as a marker of peptide release. We found that the addition of NaHS to incubation media was consistently associated with a concentration-dependent decrease in KCl-stimulated CRH release, whereas basal secretion was unaffected. Increased endogenous H2S production may be also obtained using an indirect precursor of H2S formation, S-adenosyl-L-methionine (SAMe). The latter mimicked the effects of NaHS, since it reduced potassium-stimulated CRH release. In vivo, SAMe showed no effect on hypothalamo-pituitary-adrenal (HPA) function under resting conditions, but inhibited stress-related glucocorticoid increase.

Immunocytochemical studies have revealed that 10 μM quercetin reduced the steady state levels of p21-ras proteins in both colon cancer cell lines and primary colorectal tumors. These findings were confirmed by Western blot and flow cytometric analysis showing that the inhibition of p21-ras expression by quercetin was time- and concentration-dependent. Twenty-four-hour treatment with 10 μM quercetin reduced p21-ras levels to about 50% of control values. Quercetin was similarly effective in inhibiting the expression of K-, H-, and N-ras proteins. Moreover, the effect of quercetin on ras oncogene expression was not dependent on the cell cycle position of colon cancer cells and appeared to be specific and not merely a consequence of overall inhibition of protein synthesis. Northern blot analysis revealed that quercetin produced in colon cancer cells an early (30 min) reduction of the steady state levels of K-, H-, and N-ras mRNAs. This reduction was also present after 6 hr of flavonoid treatment. These effects of quercetin suggest a possible chemopreventive role for this compound in colorectal carcinogenesis. Int. J. Cancer 85:438–445, 2000. ©2000 Wiley-Liss, Inc.

We investigated by immunocytochemistry the expression of the Ca(2+) binding protein S100A2 in 62 cases of laryngeal squamous-cell carcinoma (SCC). S100A2 was detected in 18/19 (95%) low-grade tumors and in 22/43 (51%) high-grade tumors, which were partially keratinizing. The remaining 21/43 (49%) high-grade tumors were non-keratinizing, anaplastic tumors and clearly S100A2-negative. In normal laryngeal squamous epithelium and in laryngeal SCC, S100A2 expression was strictly associated with that of cytokeratins 14 (P = 0.0002) and 17 (P = 0.0021), suggesting an association of S100A2 expression and cell commitment to squamous differentiation. A correlation was found between S100A2 tumor positivity and longer relapse-free (P = 0.0005) and overall (P = 0.0095) survival.

The effect of individual administration of low doses of highly purified eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (1 g/kg body weight) on the growth of Morris hepatocarcinoma 3924A transplanted in ACI/T rats was investigated. Both EPA and DHA inhibited growth of the hepatocarcinoma (50% reduction of tumor weight or volume at the 19th day after transplantation for both of the n-3 PUFA groups). EPA treatment reduced the percentage of proliferating tumor cells labeled with BUdR (10-fold), whereas DHA did not. Conversely, DHA supplementation induced a doubling of the number of cells undergoing apoptosis (labeled by TUNEL), whereas EPA treatment was much less effective. Analysis of changes in phospholipid fatty acids in tumor-cell membranes after both treatments with EPA and DHA showed a significant reduction in arachidonic-acid levels. EPA and docosapentaenoic acid (DPA), its elongation product, were increased in the phospholipids from EPA-treated animals. DHA and EPA, but not DPA, were increased in the DHA-treated group. It is concluded from the results of the present study that the anti-tumoral effect of EPA is related mainly to its inhibition of cell proliferation, whereas that of DHA corresponds with its induction of apoptosis. The alterations in fatty-acid composition induced by EPA or DHA appear to be factors underlying their differential actions on cell proliferation and apoptosis.

Eleven cases of meningiomas were investigated for the presence of estrogen and progesterone receptors. These tumors specifically bound estradiol. This binding activity almost exclusively resulted from the presence of high numbers of type II estrogen binding sites (EBS). Estrogen receptors were absent or present at low concentrations. Competition analysis showed that the flavonoid, quercetin, competed for tritiated estradiol binding to type II EBS; both rutin and hesperidin did not. In addition, 10 microM quercetin, unlike rutin or hesperidin, was effective in inhibiting in vitro bromodeoxyuridine incorporation by meningioma cells. Although the mechanism of the antiproliferative activity of quercetin remains to be clarified, it is possible that this flavonoid may regulate cell growth through a ligand interaction with type II EBS.

Cyclooxygenase (COX)-1 and -2 catalyze the formation of prostaglandins (PG). Given the role of COX and PG during intrathymic T cell development in the mouse, we investigated the expression and localization of these isozymes in the human thymus. mRNA and proteins correspondent to COX-1 and -2 were observed from whole thymus extracts. By immunohistochemistry, COX-2 was selectively localized in the medulla and it was predominant in a subset of stromal cells. By contrast, COX-1 was diffusely and exclusively present in the cortex, both in thymocytes at early stages of differentiation and in cytokeratin-positive epithelial cells, as demonstrated by double immunostaining and flow cytometry analysis. COX-2-positive cells in the medulla expressed cytokeratin and HLA-DR molecules, but they were negative for dendritic or macrophagic antigens. In addition, COX-2-positive cells expressed both the epidermal growth factor receptor and its ligand, the transforming growth factor-alpha. The inducible isoform of the PGE(2) synthase was also present in the same cells, while was absent from COX-1-expressing cells of the cortex. Finally, electron microscopy confirmed that COX-2 was mainly localized in the cytoplasm of cytokeratin-positive cells, along the rough endoplasmic reticulum. In conclusion, COX-2 and the inducible isoform of PGE(2) synthase appear to be constitutively and selectively present in medullary epithelial cells of the human thymus, whereas COX-1 is predominantly present in the thymic cortex, both in the stroma and in developing thymocytes.

n-3 Polyunsaturated fatty acids (PUFA) have been shown to inhibit chemical-induced carcinogenesis of colon in rats (Reddy, B.S., and Maruyama, H., Cancer Res. 46:3367-3370, 1986) and to normalize altered proliferative patterns of the colonic mucosa in human subjects at high risk for colon cancer (Anti, M., Gastroenterology 103, 883-891, 1992 and Gastroenterology 107, 1709-1715, 1994). To determine whether n-3 PUFA also influence the normal cellular turnover, we investigated the effects of dietary supplementation with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the two major n-3 PUFA, on cell proliferation, apoptosis, and crypt morphology in normal colonic mucosa. Moreover we analyzed some metabolic pathways such as fatty acid incorporation into membrane phospholipids and peroxisomal I]-oxidation involved in the effects of n-3 PUFA. Sixty male inbred ACI/T rats were randomly divided into three groups of 20 each, receiving oleic acid (control), EPA, or DHA (1 g/kg body wt/d) as ethyl esters by gastric gavage for 10 d. Cell proliferation and apoptosis were examined by bromodeoxyuridine and by in situ nick end labeling staining, respectively; fatty acid composition of phospholipid (PL) classes by thin-layer chromatography and gas-liquid chromatography and the activity of palmitoyl-CoA oxidase, the limiting enzyme of peroxisomal t-oxidation, were measured fluorimetrically by a coupled peroxidase reaction in mucosal homogenate. Both EPA and DHA inhibited cell proliferation and induced apoptosis in normal colonic mucosa of rats, but they did not modify crypt morphology and the total number of cells per crypt. EPA and DHA treatment decreased arachidonic acid content in phosphatidylethanolamine (PE) and increased EPA in all PL classes. After DHA treatment, DHA itself increased in phosphatidylcholine (PC) and PE but not in phosphatidylinositot (PI). As a consequence of the modifications in fatty acid composition, n-6/n-3 PUFA ratio decreased in total PL, in PC and PE fractions of both EPA and DHA groups. On the contrary, n-6/n-3 PUFA ratio did not change in the PI fraction. Unsaturation index was not modified in total PL as well as in PL classes. A significant increase in peroxisomal ~-oxidation (140% in EPA and 170% in DHA rats) was observed after both EPA and DHA supplementation. The increase in this metabolic pathway resulted in an overproduction of hydrogen peroxide. The changes in cell proliferation and apoptosis were related to changes in the composition of membrane PL and in fatty acid peroxisomal metabolism. The observation that arachidonic acid level is not affected in PC and PI suggests that eicosanoids are not involved in n-3 PUFA effects. Moreover, the induction of peroxisomal ~-oxidation with a consequent increase in hydrogen peroxide production suggests that a mild increase in reactive oxygen species could be implicated in the inhibition of cell proliferation and in the induction of apoptosis by n-3 PUFA. Our finding that EPA and DHA did not modify crypt morphology and the total number of cells per crypt, although they altered cell proliferation and apoptosis in normal colonic mucosa, suggests a possible use of these fatty acids as dietary chemopreventive agents.

The immunohistochemical distribution of S-100, a protein originally isolated from the brain, has been investigated at the light and electron microscopic levels in rat and man urinary systems. In both species the antigen essentially exhibited the same location, restricted, with different degrees of staining, to certain cells in the kidney, i.e. collecting tubules, thin limbs of Henle's loop and renal papillae.