Juan R. Mestre

Inhibition of cyclooxygenase-2 gene expression by p53.

Oncogenes enhance the expression of cyclooxygenase (Cox)-2, but interactions between tumor suppressor genes and Cox-2 have not been studied. In the present work, we have compared the levels of Cox-2 and the production of prostaglandin E2 in mouse embryo fibroblasts that do not express any p53 ((10)1) versus the same cell line ((10.1)Val5) engineered to overexpress wild-type (wt) p53 at 32 °C or mutant p53 at 39 °C. Cells expressing wt p53 showed about a 10-fold decrease in synthesis of prostaglandin E2 compared with those expressing mutant p53. Levels of Cox-2 protein and mRNA were markedly suppressed by wt p53 but not by mutant p53. Nuclear run-offs revealed decreased rates of Cox-2 transcription in cells expressing wt p53. The activity of the Cox-2 promoter was reduced by 85% in cells expressing wt p53 but was reduced only by 30% in cells expressing mutant p53 compared with cells null for p53. The effect of p53 on the suppression of Cox-2 promoter activity was localized to the first 40 base pairs 5′ from the transcription start site. Electrophoretic mobility shift assay revealed that p53 competed with TATA-binding protein for binding to mouseCox-2 or human Cox-2 promoter extending from −50 to +52 base pairs. The results of this study suggest that interactions between p53 and Cox-2 could be important for understanding why levels of Cox-2 are undetectable in normal cells and increased in many tumors.

Aspirin prevents tumors in a murine model of familial adenomatous polyposis

BACKGROUND Both human and murine studies suggest that anti-inflammatory drugs prevent intestinal neoplasia. The purpose of this study was to investigate the role of aspirin as a chemopreventive agent for colorectal cancer. METHODS We administered aspirin to the Min/+ mouse, an animal with a germline mutation in Apc, a gene that is essential for normal epithelial cell growth and differentiation. Apc mutation increases cytoplasmic beta-catenin, a regulatory protein associated with the cytoskeleton. Min/+ mice develop multiple intestinal adenomas and exhibit altered cell growth in the preneoplastic intestinal epithelium. RESULTS Aspirin decreased the rate of tumor formation in Min/+ mice by 44%. Aspirin also normalized enterocyte growth by increasing apoptosis and proliferation in the preneoplastic intestinal mucosa. Finally, aspirin produced a decrease in intracellular beta-catenin levels, suggesting that modulation of this protein is associated with tumor prevention. CONCLUSIONS These data confirm a role for aspirin in suppression of Apc-associated intestinal carcinogenesis.

Inhibition of Cyclooxygenase-2 Expression: An Approach to Preventing Head and Neck Cancer

ABSTRACT Cyclooxygenase (COX) catalyzes the formation of prostaglandins (PG) from arachidonic acid. A large body of evidence has accumulated to suggest that COX‐2, the inducible form of COX, is important in carcinogenesis. In this study, we determined whether (1) COX‐2 was overexpressed in squamous cell carcinoma of the head and neck (HNSCC) and whether (2) retinoids, a class of chemopreventive agents, blocked epidermal growth factor (EGF)‐mediated activation of COX‐2 expression. Levels of COX‐2 mRNA were determined in 15 cases of HNSCC and 10 cases of normal oral mucosa. Nearly a 100‐fold increase in amounts of COX‐2 mRNA was detected in HNSCC. By immunoblot analysis, COX‐2 protein was detected in 6 of 6 cases of HNSCC but was undetectable in normal mucosa. Because retinoids protect against oral cavity cancer, we investigated whether retinoids could suppress EGF‐mediated induction of COX‐2 in cultured oral squamous carcinoma cells. Treatment with EGF led to increased levels of COX‐2 mRNA, COX‐2 protein, and synthesis of PG. These effects were suppressed by a variety of retinoids. Based on the results of this study, it will be important to establish whether newly developed selective COX‐2 inhibitors are useful in preventing or treating HNSCC. Moreover, the anticancer properties of retinoids may be due, in part, to inhibition of COX‐2 expression. Combining a retinoid with a selective COX‐2 inhibitor may be more effective than either agent alone in preventing cancer of the upper aerodigestive tract.

Renal Cell Carcinoma Induces Prostaglandin E2 and T-Helper Type 2 Cytokine Production in Peripheral Blood Mononuclear Cells

AbstractBackground: Patients with renal cell carcinoma (RCC) do not develop an effective antitumor immune response, despite significant infiltration by lymphocytes. Tumor production of immunosuppressive factors may account for this failure. The object of this study was to investigate the production of immunosuppressive mediators, especially prostaglandin E2 (PGE2), by RCC. Methods: Peripheral blood mononuclear cells (PBMC) were cocultured with conditioned medium (CM) from human RCC cell lines in the presence or absence of NS-398, a selective cyclooxygenase 2 (COX-2) inhibitor. Supernatants were analyzed for levels of PGE2, interleukin (IL)-10, IL-6, IL-2, interferon-γ, and IL-12. The effects of RCC CM on PBMC proliferation were also examined. The expression of basal and stimulated COX-2 messenger RNA in the cell lines was assessed by reverse transcriptase-polymerase chain reaction. Results: RCC CM significantly increased PGE2 production by PBMC. T-helper type 2 (Th2) cytokine production was also significantly increased. Th1 cytokines were unchanged or decreased. RCC CM increased proliferation of PBMC. Coculture with NS-398 reduced PBMC PGE2 production to below control levels and significantly decreased IL-6 production and PBMC proliferation. NS-398 had no effect on cellular production of IL-10 or Th1 cytokines. Conclusions:Human RCC inhibits the host antitumor immune response by promoting PGE2 production and Th2 cytokines in PBMC. Selective inhibition of COX-2 may have a role in abrogating this effect.

Glucocorticoid pretreatment induces cytokine overexpression and nuclear factor-κB activation in macrophages

BACKGROUND Glucocorticoids are widely used in treating inflammatory diseases. The contribution of adrenal glucocorticoids to inflammatory regulation is unknown. Endogenous glucocorticoids, as distinct from synthetic analogues, not only suppress but also enhance immune functions. Elevated circulating cortisol levels are characteristic of injured patients. In a model of trauma, an early glucocorticoid surge occurs concomitantly with decreased cellular cytokine responses. Cytokine production elevated late after injury is associated with increased mortality. We hypothesized that this glucocorticoid surge mediates the later heightened macrophage responses. MATERIALS AND METHODS The murine macrophage like cells RAW 264.7 were incubated with corticosterone (35 ng/mL), or vehicle control, for 1 h, after which the cells were washed and corticosterone-free medium added. At 0, 3, 6, 12, and 24 h after removal of the corticosterone, the cells were stimulated with lipopolysaccharide (LPS) and interferon-gamma. Supernatant tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and nitrite levels were measured. In separate experiments the effect of pretreatment with corticosterone on TNF-alpha, IL-6, and nitrite mRNA expression as well as nuclear factor-kappaB and glucocorticoid receptor activity was determined. CD14 receptor expression was determined by flow cytometry. RESULTS Glucocorticoid pretreatment caused significantly increased RAW 264.7 cell production of nitrite, IL-6 and TNF-alpha. mRNA for these inflammatory mediators was induced 6 h after the corticosterone pretreatment, and was associated with activation of nuclear factor-kappaB in the presence of activated glucocorticoid receptor. Cell surface-expression of CD14 was likewise increased. CONCLUSIONS The results of this study demonstrate a novel role for glucocorticoids and provide a mechanism for the late upregulation in macrophage function after injury.

Overlapping CRE and E-box promoter elements can independently regulate COX-2 gene transcription in macrophages.

Macrophage cyclooxygenase‐2 (COX‐2) transcription is mediated through the collaboration of different promoter elements. Here, the role of an overlapping cyclic AMP responsive element (CRE)/E‐box was investigated. Nuclear proteins bound both the CRE and E‐box, which synergized with other promoter elements to induce COX‐2 transcription. Endotoxin induced binding of nuclear proteins to the CRE and E‐box and each element independently induced higher COX‐2 transcription levels than the overlapping CRE/E‐box. Transcription factors associated with the CRE binding complex included c‐Jun and CRE binding protein and with the E‐box binding complex USF‐1; their overexpression significantly induced COX‐2 transcription. Therefore, both CRE and E‐box promoter elements regulate COX‐2 transcription in macrophages.

Cyclooxygenase-2 Inhibition Improves Macrophage Function in Melanoma and Increases the Antineoplastic Activity of Interferon γ

AbstractBackground: Melanoma inhibits macrophage tumoricidal activity and increases the expression of cyclooxygenase-2 (COX-2). In this study, we sought to determine whether inhibition of COX-2 could restore macrophage function and hence maximize the antitumor activity of the immune stimulant interferon γ (IFNγ). Methods: Peritoneal macrophages were exposed to B16 melanoma-conditioned medium for 24 hours with or without the COX-2 inhibitor NS-398 and then were stimulated with lipopolysaccharide and IFNγ. Cytotoxic activity, nitrite production, and cytokine production by the stimulated macrophages were measured. In addition, B16 melanoma cells were implanted intradermally into mice treated with IFNγ (14,000 U on alternate days) alone or with a combination of IFNγ and a COX-2 inhibitor (NS-398 or nimesulide). Mice were assessed for tumor growth and survival. Results: Macrophage cytotoxicity and nitrite production were significantly suppressed by melanoma-conditioned medium (P < .01). This was prevented by 200 μM of NS-398 (P < .05). In vivo, combined treatment with IFNγ and a COX-2 inhibitor caused a significant inhibition of tumor growth (P < .01) and improved survival (P = .02) compared with controls. Conclusions: COX-2 inhibition reversed melanoma-induced suppression of macrophage function, and combined treatment of IFNγ plus a COX-2 inhibitor was maximally effective in reducing tumor growth and improving survival.

Decreased peritoneal macrophage NF-κB translocation to the nucleus in protein energy malnutrition: a role for the glucocorticoid response?

BACKGROUND & AIMS Protein energy malnutrition (PEM) induces immune suppression leading to increased morbidity and mortality. The mechanism(s) underlying PEM-mediated immune suppression remain unclear. Plasma glucocorticoid levels are elevated in PEM and it has been postulated that these increased levels may mediate macrophage (MØ) dysfunction in PEM. We have previously shown that nuclear factor kappa B (NF-kappaB) activation in response to LPS stimulation is diminished in peritoneal macrophages (PMØs) from PEM mice. We hypothesized that decreased NF-kappaB activation in lipopolysaccharide (LPS)-stimulated PMØs in PEM is mediated through increased circulating glucocorticoid levels. METHODS Mice were randomized to six groups of n = 15 each as follows: (1) control diet (24% casein) (C); (2) protein-free diet (PF); (3) mice with subcutaneously implanted corticosterone pellet on the control diet; (4) mice with subcutaneously implanted placebo pellet on the control diet; (5) adrenalectomized mice on the control diet; (6) adrenalectomized mice on the PF diet. Within each group, the mice were pair-fed for 7 days. At the end of the experimental time period, PMØs were harvested and NF-kappaB activation determined by electrophoretic mobility shift assay (EMSA). RESULTS Elevated circulating glucocorticoids diminished NF-kappaB activation but adrenalectomy failed to restore this diminution in a murine model of PEM. CONCLUSION NF-kappaB translocation to the nucleus in PEM is independent of elevated circulating glucocorticoid levels.

Cyclo-oxygenase inhibitor and interferon-γ act synergistically to reduce melanoma growth and improve survival in a murine model

ConclusionsIn a murine model of melanoma, combined treatment with interferon-γ plus a COX-2 inhibitor was maximally effective in reducing tumour growth and improving survival. This improvement in anti-tumour activity correlated with alterations in arginine metabolism within the tumour-associated macrophage in favour of increased nitric oxide production.

Melanoma-induced suppression of macrophage nitric oxide production: reversal by a cyclo-oxygenase 2 inhibitor

ConclusionsCOX-2 inhibition can reverse the immunosuppressive effects of melanoma on macrophage function by preventing the inhibition of nitric oxide synthesis. This indicates a potential role for COX-2 inhibitors with immunostimulatory therapy in melanoma.