Tomato powder impedes the development of azoxymethane-induced colorectal cancer in rats through suppression of COX-2 expression via NF-κB and regulating Nrf2/HO-1 pathway

Cancer is one of the leading causes of death worldwide. Since dietary factors have been connected to a reduced risk of a diversity of human cancers, in this study we investigated the effects of tomato powder (TP) on the development of azoxymethane (AOM)-induced colorectal cancer in Wistar rats, and possible mechanism(s) by which TP shows its chemopreventive activity. Here we show that TP added to feed at 5% rate decreases the rate of aberrant crypt foci (ACF) and reduces the development of adenocarcinoma and growth of AOM-induced colorectal cancer in rats. In addition, we demonstrate that TP supplementation shows its chemopreventive activities through inhibition of cyclooxygenase-2 (COX-2) expression via NF-κB pathway and promotion of apoptosis, as well as regulating Nrf2/HO-1 signaling pathway in colorectal tissue of AOM-treated rats. Our findings identify an intimate connection between dietary supplementation of TP and the decreased risk of colorectal cancer in rats, and suggest that consumption of TP would be a natural candidate for the prevention of colorectal cancer in men.     

Effects of quercetin and beta-carotene supplementation on azoxymethane-induced colon carcinogenesis and inflammatory responses in rats fed with high-fat diet rich in omega-6 fatty acids

Chronic inflammation in gastrointestinal tract has been suggested as a risk factor for tumor formation. The effect of dietary supplementation of quercetin or beta-carotene on colon carcinogenesis and inflammatory response in rats fed with high-fat diet rich in omega-6 fatty acids was assessed. Animals were exposed to two weekly subcutaneous injections of AOM (azoxymethane) at a single dose of 15 mg/kg body weight. A portion of rats from each group was sacrificed at 8 weeks after the last AOM treatment to determine ACF (aberrant crypt foci) formation. Colonic mucosa expression of iNOS (inducible nitric oxide) and COX-2 (cyclooxygenase-2) protein, and blood PGE2 (prostaglandin E2) level were measured. The remaining groups of animals were sacrificed at 33 weeks after the last AOM treatment to examine colon tumor formation. Rats on high-fat diet developed more aberrant crypt foci (P<0.05) compared with those of rats on regular diet. In the same vein, but in contrast to the effect seen with regular diet, the high-fat diet induced a significant up-regulation of iNOS expression. There was no significant change in the extent of COX-2 expression or in the PGE2 levels. Quercetin or beta-carotene supplementation reduced the number of ACF only in animals fed high-fat diet (p<0.05), however, no significant difference in tumor incidence was found. At week 33, the expression of iNOS was reduced by quercetin without a statistical significance, and COX-2 expression was slightly reduced in rats on beta-carotene supplementation. No change in PGE2 levels was observed. Whilst dietary antioxidants are considered as effective suppressors for precancerous lesion formation in colons exposed to high-risk diet, it is clear that elucidating the role of individual antioxidants in colon tumor formation coupled with an understanding of the molecular mechanisms involved would benefit colon cancer prevention strategies.     

Protein kinase CK2 promotes cancer cell viability via up-regulation of cyclooxygenase-2 expression and enhanced prostaglandin E2 production

Augmented expression of protein kinase CK2 is associated with hyperproliferation and resistance to apoptosis in cancer cells. Effects of CK2 are at least partially linked to signaling via the Wnt/β-catenin pathway, which is dramatically enhanced in colon cancer. Cyclooxygenase-2 (COX-2), a Wnt/β-catenin target gene, has been associated with enhanced cancer progression and metastasis. However, the possibility that a connection may exist between CK2 and COX-2 has not been explored previously. Here we investigated changes in COX-2 expression and activity upon CK2 modulation and evaluated how these changes affected cell viability. COX-2 expression and cell viability decreased upon selective inhibition of COX-2 with SC-791 or CK2 with 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), both in human colon (HT29-ATCC, HT29-US, DLD-1) and breast (ZR-75) cancer cells, as well as in human embryonic kidney (HEK-293T) cells. On the other hand, ectopic CK2α expression promoted up-regulation of COX-2 by activating the Wnt/β-catenin pathway in HEK-293T cells. Noteworthy, over-expression of either CK2α, β-catenin or COX-2, as well as supplementation of the medium with prostaglandin E2 (PGE2), all were individually sufficient to overcome limitations in cell viability triggered by CK2 inhibition either upon addition of DMAT or over-expression of a dominant negative CK2α variant. Altogether, these findings provide new insight to the role of CK2 in cancer by up-regulating COX-2 expression and thereby PGE2 production.     

Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in macrophage J774A.1

The present study focuses on the effect of various naturally occurring flavonoids (apigenin, galangin, morin, naringenin, quercetin, and silymarin) on nitric oxide (NO) and prostaglandin E2 (PGE2) production induced by lipopolysaccharide (LPS) in the macrophage cell line J774A.1. Moreover, we evaluated flavonoid modulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzyme expression by western blot analysis. Apigenin and quercetin (0.5-50 microM) were the most potent inhibitors of NO production and this effect was concentration-dependent and significant at 5 and 50 microM. These data were consistent with the modulation of iNOS enzyme expression. A similar pattern was observed considering the inhibitory effect of flavonoids on LPS-induced PGE2 release and COX-2 expression. Quercetin, galangin, apigenin, and naringenin markedly decreased PGE2 release and COX-2 expression in a concentration-dependent manner. This study suggests that inhibition of iNOS and COX-2 expression by flavonoids may be one of the mechanisms responsible for their anti-inflammatory effects.     

Potential misidentification of cyclooxygenase-2 by western blot analysis and prevention through the inclusion of appropriate controls

Cyclooxygenase (COX)-2 plays an important role in the development of cancer and has been recognized as a potential therapeutic target. Because nonsteroidal anti-inflammatory drugs (NSAIDs) are able to inhibit the activity of this enzyme, the potential efficacy of such drugs for purposes of cancer prevention or therapy is an area of intense research. Therefore, it is of critical importance to unequivocally determine the expression levels of COX-2 protein in tumor cells. In this regard, there are several conflicting reports in the literature where the same type of tumor cell lines were reported as COX-2 positive and as COX-2 negative. We found that during Western blot analysis of COX-2 positive and COX-2 negative cells, different antibodies to COX-2 protein are able to generate strong signals, which are false-positives and can be confused with COX-2. Thus, we believe that some of the conflicting reports on COX-2 expression in tumor cell lines could be the result of improper interpretation of the Western blot signals. Here, we present some of these pitfalls and suggest the inclusion of appropriate controls to unequivocally identify COX-2 protein levels.     

Silymarin is a selective estrogen receptor beta (ERbeta) agonist and has estrogenic effects in the metaphysis of the femur but no or antiestrogenic effects in the uterus of ovariectomized (ovx) rats

Silymarin is a widely used standardized mixture of flavonolignans and its major component Silybinin binds to cytosolic estrogen receptors. Here, we demonstrate that this binding is exclusive to the estrogen receptor beta (ERbeta). Treatment of ovariectomized (ovx) rats with silymarin or estradiol (E2) may allow differentiation of biological effects mediated by the ERalpha or ERbeta. E2 inhibited serum LH, cholesterol, LDL and HDL concentrations in the blood and increased gene expression of IGF1, HbEGF and C3 in the uterus, while silymarin was totally ineffective or antagonistic in altering these parameters. Both, E2 and silymarin inhibited expression of uterine ERbeta gene. Hence, in the pituitary, liver (where the lipoproteins are synthesized) and uterus E2 acts primarily via the ERalpha. Exclusive estrogenic effects of silymarin were observed in the metaphysis of the femur (MF), on osteoblast parameters (gene expression of IGF1, TGFbeta1, osteoprotegerin, collagen-1alpha1, osteocalcin (OC)) and on the osteoclast activity marker tartrate resistant acid phosphatase (TRAP) gene expression of adult ovx rats. Our RT-PCR method detects ERbeta gene expression in all organs including developing bones but not in the MF of adult ovx rats. We conclude therefore, that the effects of silymarin in this part of the bone cannot be exerted via the ERalpha because it does not bind to this receptor subtype. Despite the failure to detect ERbeta mRNA in the MF of our animals the possibility exists that ERbeta protein is present and may mediate the effects of silymarin. Another possibility may be that the effect of silymarin and therefore possibly also of E2 in the MF may be mediated via other possibly not yet identified receptors or via an ERbeta splice variant which is not detected by our PCR-method.     

Different expression of cyclooxygenase-2 (COX-2) in selected nonmelanocytic human cutaneous lesions

The aim of our study was to elucidate the possible involvement of COX-2 in the development and/or progression of nonmelanocytic skin lesions. To evaluate the usefulness of that enzyme as a potential molecular marker, we examined the intensity and spatial distribution of COX-2 expression in selected types of such tumors using the same immunohistochemical procedure as in our earlier studies of melanocytic cancers. We examined 20 benign epithelial lesions, 11 precancerous lesions, 21 basal cell carcinomas (BCC), 14 squamous cell carcinomas (SCC) and eight fibromas. The levels of COX-2 expression detected in benign lesions and in normal skin were comparable. Elevated expression of this protein may play a role in the development of SCC, as indicated by strong immunostaining both in SCCs and precancerous lesions. Significantly stronger staining in SCCs compared to BCCs may indicate a role of COX-2 in cancer malignancy and serve as an indicator useful for differential diagnostics of the two types of cancer. Strong staining in all skin layers of SCC may help in detecting cancer cells infiltrating surrounding skin layers.     

Regulation of COX-2 expression by miR-146a in lung cancer cells

Prostaglandins are a class of molecules that mediate cellular inflammatory responses and control cell growth. The oxidative conversion of arachidonic acid to prostaglandin H₂ is carried out by two isozymes of cyclooxygenase, COX-1 and COX-2. COX-1 is constitutively expressed, while COX-2 can be transiently induced by external stimuli, such as pro-inflammatory cytokines. Interestingly, COX-2 is overexpressed in numerous cancers, including lung cancer. MicroRNAs (miRNAs) are small RNA molecules that function to regulate gene expression. Previous studies have implicated an important role for miRNAs in human cancer. We demonstrate here that miR-146a expression levels are significantly lower in lung cancer cells as compared with normal lung cells. Conversely, lung cancer cells have higher levels of COX-2 protein and mRNA expression. Introduction of miR-146a can specifically ablate COX-2 protein and the biological activity of COX-2 as measured by prostaglandin production. The regulation of COX-2 by miR-146a is mediated through a single miRNA-binding site present in the 3′ UTR. Therefore, we propose that decreased miR-146a expression contributes to the up-regulation and overexpression of COX-2 in lung cancer cells. Since potential miRNA-mediated regulation is a functional consequence of alternative polyadenylation site choice, understanding the molecular mechanisms that regulate COX-2 mRNA alternative polyadenylation and miRNA targeting will give us key insights into how COX-2 expression is involved in the development of a metastatic condition.     

Effect of zinc supplementation on type 2 diabetes parameters and liver metallothionein expressions in Wistar rats

Zinc is a trace metal and acts as an active component of various enzymes. Zinc deficiency has been suggested to be associated with the development of diabetes. The present study investigated the role of zinc supplementation on prevention of diabetic conditions. A double-disease model mimicking hyperlipidemia and type 2 diabetes was created by applying high-fat diet and streptozotocin (STZ) to Wistar rats. We demonstrated that zinc supplementation improved symptoms of diabetes such as polydipsia and increased serum level of high-density lipoprotein cholesterol, indicating that zinc supplementation has a potential beneficial effect on diabetic conditions. The level of maldondialdehyde (MDA), an oxidative stress marker, was reduced in liver by zinc supplementation in high fat-fed rats with or without STZ injection. Meanwhile, we observed an increase in the expression of metallothioneins (MTs) in liver of rats treated with zinc. This suggests that the induction of MTs in liver, which has been shown to be important in scavenging free radicals, could be one of the underlying mechanisms of zinc supplementation on reducing MDA levels in liver. Finally, we found that zinc levels in liver were increased while there was no change in serum zinc levels, indicating that local zinc level might be a critical factor for the induction of MTs. Also, the level of MTs could potentially be an index of zinc bioavailability. Taken together, these results suggest that both zinc and MT could play an important role in balancing nutrition and metabolism to prevent diabetic development.     

Cyclooxygenase-2 (COX-2) in acute myocardial infarction: cellular expression and use of selective COX-2 inhibitor

Our previous work has shown strong expression of COX-2 in the myocardium of patients with end-stage ischemic heart failure. The purpose of this study was to determine the cellular expression of this enzyme in the setting of acute myocardial infarction (AMI) and determine the role of COX-2 in experimental animals using a selective COX-2 inhibitor. Experimental AMI was induced in rats by ligating the left coronary artery. Animals were either treated with a selective COX-2 inhibitor (5 mg x kg(-1) x day(-1)) or vehicle. Three days after ligation, cardiac function was assessed and infarct size was determined. Myocardial specimens were immunostained with antiserum to COX-2. Plasma concentration of prostanoids was measured by enzyme immunoassay. There was strong expression of COX-2 in the myocytes, endocardium, vascular endothelial cells, and macrophages in the infarcted zone of the myocardium. In contrast, little expression was seen in the myocardium of control rats. Animals treated with the COX-2 inhibitor showed a significant improvement in left ventricular (LV) end-diastolic pressure (P < 0.05) and LV systolic pressure (P < 0.01), and a reduction in infarct size (P < 0.05). Inhibition of COX-2 significantly decreased plasma concentration of thromboxane B2 (P < 0.05); however, it did not affect 6-keto-prostaglandin F1alpha. Induction of COX-2 during AMI appears to contribute to myocardial injury, and treatment with the specific inhibitor of the enzyme ameliorated the course of the disease.     

Antiproliferative effects of COX-2 inhibitor celecoxib on human breast cancer cell lines

The inducible COX-2 enzyme is over-expressed in human breast cancer and its over-expression generally correlates with angiogenesis, deregulation of apoptosis and worse prognosis. This observation may explain the beneficial effect of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors on breast cancer treatment. Here, we evaluated the antiproliferative activity of celecoxib, a selective COX-2 inhibitor, and its nitro-oxy derivative on human breast cancer cells characterized by low and high COX-2 expression, respectively. In ERα(+) MCF-7 cells celecoxib and its derivative induce a strong inhibition of cell growth, inhibition that is associated with the reduction of ERα expression and activation. These effects may be directly associated with ERK and Akt suppression and with PP2A and PTEN induction. In this cell line the drugs exert only weak effect on COX-2 level while they are able to reduce aromatase expression. On the contrary, in ERα(-) MDA-MB-231 cells, both drugs induce a marked inhibition of COX-2, inhibition that is associated with the reduction of aromatase expression and of cell proliferation. In both cell lines the effects of the drugs are associated with the suppression of cell invasion.     

Camptothecin-induced apoptosis in non-small cell lung cancer is independent of cyclooxygenase expression

Recent observations show a positive correlation between the expression of cyclooxygenase (COX), especially COX-2), and cancer development. Here we tested the hypothesis that expression of COX-2 could influence apoptosis in lung cancer cell lines. To address this question, we determined the effects of camptothecin-induced apoptosis on three lung cancer cell lines which over express COX-1 (CORL23), COX-2 (MOR-P) and neither isoform (H-460), and determine if these effects were prostaglandin mediated. We also compared the effects of non-selective and isoenzyme selective COX-2 inhibitors on camptothecin-induced apoptosis in these three cell lines. Camptothecin induced apoptosis in all three cell lines independently of COX-1 or COX-2 expression. Indomethacin, a non-selective COX inhibitor and NS398, a selective COX-2 inhibitor had no effect on camptothecin-induced apoptosis at concentrations that abolished prostaglandin production. In conclusion, these finding suggest that the COX pathway is not involved in camptothecin-induced apoptosis of non-small cell lung cancer cell lines.     

COX-2 is overexpressed in primary prostate cancer with metastatic potential and may predict survival. A comparison study between COX-2, TGF-β, IL-10 and Ki67

Background: The immune modulating molecules cyclooxygenase-2 (COX-2), transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) have regulatory roles in cancer progression. There are conflicting data regarding the roles of these molecules in prostate cancer. To elucidate the prognostic impact of these proteins and provide information on prognosis and treatment, we compared the expression of COX-2, TGF-β, and IL-10 in prostate cancer specimens with or without metastases. Ki67 was included as a measure of growth fraction of tumor cells. Methods: Digital video analysis images from tumor cell areas and tumor stromal areas were analyzed on formalin fixed, paraffin-embedded and immunohistochemical stained cancer specimens from 59 patients: 32 patients with metastases and 27 patients without clinical, biochemical, or radiological evidence of metastases within 10 years after diagnosis. The expression of COX-2 was scored as negative, weak, moderate, or strong. The expressions of TGF-β and IL-10 were assessed as proportions of moderately or strongly stained cells. Ki67 was detected as strong nuclear staining in proliferating cells. Results: In primary cancers in the metastatic group, COX-2, TGF-β and Ki67 were stronger expressed in epithelial tumor cell and tumor stromal areas compared with non-metastatic cancers (for all markers, p < 0.0001). High intensity of COX-2 staining in tumor areas was strongly associated with death from prostate cancer in univariate analyses (hazard ratio [HR] 95% CI, 4.0 (1.1–14.5)). In multivariate analyses, the risk estimate was strengthened but did not reach significance. No associations to death were found for the other markers. Conclusion: High expression of COX-2, TGF-β and Ki67 were in metastatic primary prostate carcinoma compared to non-metastatic cancers. High expression of COX-2 was associated to death from prostate carcinoma.     

The synergistic induction of cyclooxygenase-2 in lung fibroblasts by angiotensin II and pro-inflammatory cytokines

Although we have demonstrated that Angiotensin II (Ang II) signaling plays a role in colon and lung tumorigenesis, the precise mechanisms by which Ang II stimulates tumorigenesis remain unclear. The aim of this study was to investigate the synergistic induction of COX-2 by Ang II and pro-inflammatory cytokines in lung fibroblasts. We also compared the efficiencies of Ang II-dependent COX-2 induction in lung epithelial cells and stromal cells. Ang II induced COX-2 expression in lung fibroblasts in a dose-dependent manner (10(-9) to 10(-7) M) through the Ang II subtype 1 receptor (AT(1)). In addition, Ang II synergistically stimulated the induction of COX-2 by pro-inflammatory cytokines, IL-1beta, or TNF-alpha. Our results indicate that the pro-tumorigenic function of Ang II is attributable, in part, to its strong stimulatory effect of COX-2 expression in lung fibroblasts in which synergistic stimulation with pro-inflammatory cytokines was evident. It is also suggested that the AT(1) receptor in lung fibroblasts may be a rational target for chemoprevention of lung cancer.     

Viscum album exerts anti-inflammatory effect by selectively inhibiting cytokine-induced expression of cyclooxygenase-2

Viscum album (VA) preparations are extensively used as complementary therapy in cancer and are shown to exert anti-tumor activities which involve the cytotoxic properties, induction of apoptosis, inhibition of angiogenesis and several other immunomodulatory mechanisms. In addition to their application in cancer therapy, VA preparations have also been successfully utilized in the treatment of several inflammatory pathologies. Owing to the intricate association of inflammation and cancer and in view of the fact that several anti-tumor phytotherapeutics also exert a potent anti-inflammatory effect, we hypothesized that VA exerts an anti-inflammatory effect that is responsible for its therapeutic benefit. Since, inflammatory cytokine-induced cyclo-oxygenase-2 (COX-2) and prostaglandin E2 (PGE2) play a critical role in the pathogenesis of inflammatory diseases, we investigated the anti-inflammatory effect of VA on regulation of cyclo-oxygenase expression and PGE2 biosynthesis by using human lung adenocarcinoma cells (A549 cells) as a model. A549 cells were stimulated with IL-1β and treated with VA preparation (VA Qu Spez) for 18 hours. PGE2 was analysed in the culture supernatants by enzyme immunoassay. Expression of COX-2 and COX-1 proteins was analyzed by immunoblotting and the expression of COX-2 mRNA was assessed by semi-quantitative RT-PCR. We found that VA Qu Spez inhibit the secretion of IL-1β-induced PGE2 in a dose-dependent manner. Further, we also show that this inhibitory action was associated with a reduced expression of COX-2 without modulating the COX-1 expression. Together these results demonstrate a novel anti-inflammatory mechanism of action of VA preparations wherein VA exerts an anti-inflammatory effect by inhibiting cytokine-induced PGE2 via selective inhibition of COX-2.     

Cyclooxygenase-2 expression in macrophages: modulation by protein kinase C-alpha

Cyclooxygenase-2 (COX-2) is an inducible enzyme responsible for high levels of PG production during inflammation and immune responses. Previous studies with pharmacological inhibitors suggested a role for protein kinase C (PKC) in PG production possibly by regulating COX-2 expression. In this study, we addressed the role of PKC-alpha in the modulation of COX-2 expression and PGE2 synthesis by the overexpressing of a dominant-negative (DN) mutant of this isoenzyme in the mouse macrophage cell line RAW 264.7. We investigated the effect of various stimuli on COX-2 expression, namely, LPS, IFN-gamma, and the intracellular parasite Leishmania donovani. Whereas LPS-induced COX-2 mRNA and protein expression were down-regulated in DN PKC-alpha-overexpressing clones, IFN-gamma-induced COX-2 expression was up-regulated in DN PKC-alpha-overexpressing clones with respect to normal RAW 264.7 cells. Measurements of PGE2 levels revealed a strong correlation between PGE2 secretion and IFN-gamma-induced COX-2 mRNA and protein levels in DN PKC-alpha-overexpressing clones. Taken together, these results suggest a role for PKC-alpha in the modulation of LPS- and IFN-gamma-induced COX-2 expression, as well as in IFN-gamma-induced PGE2 secretion.