Nicola Maggiano

Cyclooxygenase-2 expression is induced during human megakaryopoiesis and characterizes newly formed platelets

Cyclooxygenase (COX)-1 or -2 and prostaglandin (PG) synthases catalyze the formation of various PGs and thromboxane (TX) A2. We have investigated the expression and activity of COX-1 and -2 during human megakaryocytopoiesis. We analyzed megakaryocytes from bone marrow biopsies and derived from thrombopoietin-treated CD34+ hemopoietic progenitor cells in culture. Platelets were obtained from healthy donors and patients with high platelet regeneration because of immune thrombocytopenia or peripheral blood stem cell transplantation. By immunocytochemistry, COX-1 was observed in CD34+ cells and in megakaryocytes at each stage of maturation, whereas COX-2 was induced after 6 days of culture, and remained detectable in mature megakaryocytes. CD34+ cells synthesized more PGE2 than TXB2 (214 ± 50 vs. 30 ± 10 pg/106 cells), whereas the reverse was true in mature megakaryocytes (TXB2 8,440 ± 2,500 vs. PGE2 906 ± 161 pg/106 cells). By immunostaining, COX-2 was observed in <10% of circulating platelets from healthy controls, whereas up to 60% of COX-2-positive platelets were found in patients. A selective COX-2 inhibitor reduced platelet production of both PGE2 and TXB2 to a significantly greater extent in patients than in healthy subjects. Finally, we found that COX-2 and the inducible PGE-synthase were coexpressed in mature megakaryocytes and in platelets. We conclude that both COX-isoforms contribute to prostanoid formation during human megakaryocytopoiesis and that COX-2-derived PGE2 and TXA2 may play an unrecognized role in inflammatory and hemostatic responses in clinical syndromes associated with high platelet turnover.

Dilated Intercellular Spaces of Esophageal Epithelium in Nonerosive Reflux Disease Patients with Physiological Esophageal Acid Exposure

OBJECTIVES:It has been demonstrated that dilation of intercellular spaces of esophageal epithelium is a marker of tissue injury in GERD patients with a pathological esophageal acid exposure time. To evaluate the relationship among ultrastructural changes, acid esophageal exposure, and GERD symptoms, intercellular space diameters have been assessed in nonerosive reflux disease (NERD) patients with/without abnormal acid exposure time.METHODS:Following a pharmacological wash-out, 20 NERD patients underwent upper endoscopy, esophageal manometry, and 24-h pH monitoring. Biopsies were taken at 5 cm above the lower esophageal sphincter and intercellular space diameters were measured on transmission electron microscopy photomicrographs. Seven asymptomatic controls underwent the same protocol.RESULTS:Acid exposure time was in the normal range in all controls and in 11 patients (NERD pH-negative); it was abnormal in 9 patients (NERD pH-positive). Mean intercellular space diameter in NERD pH-negative and in NERD pH-positive patients was three times greater than in controls (1.45 and 1.49 μm vs 0.45, p < 0.001). Mean values of maximum intercellular spaces in all NERD patients were greater, two-fold or more, than those in controls (p < 0.001). No difference in mean and maximal space diameters was observed between NERD pH-positive and pH-negative patients.CONCLUSIONS:Dilation of intercellular spaces is a feature of NERD patients, irrespective of esophageal acid exposure, and can be considered an objective, structural marker of GERD symptoms. Impaired esophageal mucosal resistance, even to small amounts of acid refluxate, plays a key role in the pathophysiology of NERD.

Dietary supplementation with eicosapentaenoic and docosahexaenoic acid inhibits growth of Morris hepatocarcinoma 3924A in rats: effects on proliferation and apoptosis

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. Int. J. Cancer 75:699–705, 1998.© 1998 Wiley‐Liss, Inc.

Quercetin inhibits p21‐RAS expression in human colon cancer cell lines and in primary colorectal tumors

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.

Cyclooxygenase-2 expression in endometrial carcinoma: correlation with clinicopathologic parameters and clinical outcome.

Cyclooxygenase‐2 (COX‐2) is overexpressed in endometrial hyperplasia and carcinoma, but no data have been reported until now about the expression of COX‐2 and its possible clinical significance in endometrial carcinoma. We investigated by immunohistochemistry the expression of COX‐2 in a single institutional series of primary untreated endometrial carcinoma patients. The relationship between COX‐2 expression and microsatellite instability (MI) status was also analyzed.

beta-carotene at high concentrations induces apoptosis by enhancing oxy-radical production in human adenocarcinoma cells.

This is the first report demonstrating a relationship between apoptosis induction and changes of intracellular redox potential in the growth-inhibitory effects of high concentrations of beta-carotene in a tumor cell line. beta-Carotene inhibited the growth of human WiDr colon adenocarcinoma cells in a dose- and time-dependent manner, induced apoptosis, and blocked Bcl-2 expression. These effects were accompanied by an enhanced production of intracellular reactive oxygen species (ROS). The addition of the antioxidant alpha-tocopherol blocked both the pro-oxidant and the growth-inhibitory effects of the carotenoid. These findings suggest that beta-carotene may act as an inductor of apoptosis by its pro-oxidant properties.

Lactobacillus acidophilus protects tight junctions from aspirin damage in HT-29 cells

Background/Aims: Non-steroidal anti-inflammatory drugs cause enterocyte damage inducing an increase of intestinal permeability. Tight junctions are the key structures in the permeability of the intestinal mucosa. ZO-1 is a tight junction associated protein considered a good marker of their integrity. It has been suggested that probiotics could play a protective role in the intestinal barrier function. We determined, in vitro, whether the heat-killed Lactobacillus acidophilus strain LB (LaLB) with its spent culture supernatant protects tight junctions of HT-29 cells from aspirin (ASA) damage. Methods: HT-29 cells were treated with ASA alone or ASA and LaLB with its spent culture supernatant together. Morphological alterations of tight junctions were evaluated by immunofluorescence using an anti-ZO-1 antibody. Moreover, a semiquantitative assay for ZO-1 was performed by Western blot. Results: Immunofluorescence analysis showed a fragmented and granulous ZO-1 staining, after ASA treatment. Using both ASA and LaLB with its spent culture supernatant together, we found a fine continuous linear web at cell-cell contacts similarly to control. Western blot revealed that ASA inhibited ZO-1 expression and LaLB with its spent culture supernatant counteracted this effect. Conclusions: This pilot study shows, for the first time, the protective effect of LaLB with its spent culture supernatant on tight junctions from ASA damage. These results suggest that probiotics could play a role in the prevention of ASA-induced alterations of intestinal permeability.

Prognostic significance of cyclooxygenase-2 in laryngeal squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) overexpression is an unfavorable prognostic marker in laryngeal squamous cell carcinoma (SCC). EGFR stimulates cyclooxygenase‐2 (COX‐2) expression in normal human keratinocytes and squamous carcinoma cells. Based on these observations a prognostic role of COX‐2 expression in laryngeal SCC can be hypothesized. Consequently, COX‐2 expression was studied in laryngeal SCC (median follow‐up = 47 months; range: 2–87 months) by quantitative immunohistochemistry (n = 61) and EGFR by binding assay (n = 51). Well‐differentiated regions of laryngeal SCC revealed strong COX‐2 immunostaining, whereas histologically normal areas neighboring tumor as well as poorly‐differentiated tumors were negative. Immunohistochemical results were confirmed by Western blot analyses. Coxs regression analysis showed that the combination of low levels of COX‐2 integrated density and high levels of EGFR covariates provided strong prediction, at 5‐year follow‐up, of both poor overall survival (χ2 = 12.905; p = 0.0016) and relapse‐free survival (χ2 = 9.209; p = 0.01). In vitro studies on CO‐K3 cell line, obtained from an EGFR positive, COX‐2 negative poorly‐differentiated laryngeal SCC, revealed that EGF stimulation failed to induce COX‐2 expression and PGE2 production suggesting a change in EGFR signaling pathway. These findings indicate that COX‐2 is overexpressed in less aggressive, low grade laryngeal SCC, whereas its expression is lost when tumors progress to a more malignant phenotype.

Cell proliferation’ differentiation’ and apoptosis are modified by n−3 polyunsaturated fatty acids in normal colonic mucosa

Supplementation with low doses of eicosapentaenoic (EPA) or docosahexaenoic (DHA) acid was used here to investigate changes in epithelial proliferation’ differentiation’ and apoptosis in normal rat colonic mucosa. ACI/T rats received by oral administration low doses of purified EPA or DHA ethyl esters (1g/kg body weight) and colonic mucosa was analyzed for cell proliferation’ differentiation’ and apoptosis. n−3 Polyunsaturated fatty acid incorporation into membrane phospholipids was investigated as reflections of fatty acid metabolism. Both EPA and DHA suppressed colonocyle proliferation and increased the numbers of differentiating and apoptotic cells without modification of the crypt morphology and the number of cells per crypt columns. A significant incorporation of the supplemented fatty acids into total phospholipids was observed. This enrichment was accompanied by a decreased content in arachidonic acid. The observation that EPA and DHA do not alter crypt morphology although they modify cell turnover in normal colonic mucosa suggests a possible use of these fatty acids as dietary chemopreventive agents.

Dietary alpha-linolenic acid reduces COX-2 expression and induces apoptosis of hepatoma cells.

Fatty acid synthetase (FAS) is overexpressed in various tumor tissues, and its inhibition and/or malonyl-CoA accumulation have been correlated to apoptosis of tumor cells. It is widely recognized that both ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) depress FAS expression in liver, although epidemiological and experimental reports attribute antitumor properties only to ω-3 PUFA. Therefore, we investigated whether lipogenic gene expression in tumor cells is differently regulated by ω-6 and ω-3 PUFAs. Morris hepatoma 3924A cells were implanted subcutaneously in the hind legs of ACI/T rats preconditioned with high-lipid diets enriched with linoleic acid or α-linolenic acid. Both-high lipid diets depressed the expression of FAS and acetyl-CoA carboxylase in tumor tissue, this effect correlating with a decrease in the mRNA level of their common sterol regulatory element binding protein-1 transcription factor. Hepatoma cells grown in rats on either diet did not accumulate malonyl-CoA. Apoptosis of hepatoma cells was induced by the α-linolenic acid-enriched diet but not by the linoleic acid-enriched diet. Therefore, in this experimental model, apoptosis is apparently independent of the inhibition of fatty acid synthesis and of malonyl-CoA cytotoxicity. Conversely, it was observed that apoptosis induced by the α-linolenic acid-enriched diet correlated with a decrease in arachidonate content in hepatoma cells and decreased cyclooxygenase-2 expression.