Induction of antiproliferative effect by diosgenin through activation of p53,release of apoptosis-inducing factor （AIF） and modulation of caspase-3 activity in different human cancer cells

Previously, we demonstrated that a plant steroid, diosgenin, altered cell cycle distribution and induced apoptosis in the human osteosarcoma 1547 cell line. The objective of this study was to investigate if the antiproliferative effect of diosgenin was similar for different human cancer cell lines such as laryngocarcinoma HEp-2 and melanoma M4Beu cells. Moreover, this work essentially focused on the mitochondrial pathway. We found that diosgenin had an important and similar antiproliferative effect on different types of cancer cells. In addition, our new results show that diosgenin-induced apoptosis is caspase-3 dependent with a fall of mitochondrial membrane potential, nuclear localization of AIF and poly (ADP-ribose) polymerase cleavage. Diosgenin treatment also induces p53 activation and cell cycle arrest in the different cell lines studied.     

Inhibition of cyclooxygenase activity reduces rotavirus infection at a postbinding step

Elevated levels of prostaglandins (PGs), products of cyclooxygenases (COXs), are found in the plasma and stool of rotavirus-infected children. We sought to determine the role of COXs, PGs, and the signal transduction pathways involved in rotavirus infection to elucidate possible new targets for antiviral therapy. Human intestinal Caco-2 cells were infected with human rotavirus Wa or simian rotavirus SA-11. COX-2 mRNA expression and secreted PGE2 levels were determined at different time points postinfection, and the effect of COX inhibitors on rotavirus infection was studied by an immunofluorescence assay (IFA). To reveal the signal transduction pathways involved, the effect of MEK, protein kinase A (PKA), p38 mitogen-activated protein kinase (MAPK), and NF-kappaB inhibitors on rotavirus infection was analyzed. In infected Caco-2 cells, increased COX-2 mRNA expression and secreted PGE2 levels were detected. Indomethacin (inhibiting both COX-1 and COX-2) and specific COX-1 and COX-2 inhibitors reduced rotavirus infection by 85 and 50%, respectively, as measured by an IFA. Indomethacin reduced virus infection at a postbinding step early in the infection cycle, inhibiting virus protein synthesis. Indomethacin did not seem to affect viral RNA synthesis. Inhibitors of MEK, PKA, p38 MAPK, and NF-kappaB decreased rotavirus infection by at least 40%. PGE2 counteracted the effect of the COX and PKA inhibitors but not of the MEK, p38 MAPK, and NF-kappaB inhibitors. Conclusively, COXs and PGE2 are important mediators of rotavirus infection at a postbinding step. The ERK1/2 pathway mediated by PKA is involved in COX induction by rotavirus infection. MAPK and NF-kappaB pathways are involved in rotavirus infection but in a PGE2-independent manner. This report offers new perspectives in the search for therapeutic agents in treatment of severe rotavirus-mediated diarrhea in children.     

Diosgenin,a plant steroid,induces apoptosis in human rheumatoid arthritis synoviocytes with cyclooxygenase-2 overexpression

In the present study, we have shown for the first time that a plant steroid, diosgenin, causes an inhibition of the growth of fibroblast-like synoviocytes from human rheumatoid arthritis, with apoptosis induction associated with cyclooxygenase-2 (COX-2) up-regulation. Celecoxib, a selective COX-2 inhibitor, provoked a large decrease in diosgenin-induced apoptosis even in the presence of exogenous prostaglandin E₂, whereas interleukin-1β, a COX-2 inducer, strongly increased diosgenin-induced apoptosis of these synoviocytes. These findings suggest that the proapoptotic effect of diosgenin is associated with overexpression of COX-2 correlated with overproduction of endogenous prostaglandin E₂. We also observed a loss of mitochondrial membrane potential, caspase-3 activation, and DNA fragmentation after diosgenin treatment.     

Nutritionally essential fatty acids and biologically indispensable cyclooxygenases

The study of cyclooxygenases (COXs), targets of aspirin and related drugs, is rooted in the discovery of essential fatty acids (EFAs). There are two COXs that convert EFAs, primarily arachidonic acid, to prostaglandins. Each COX is involved with distinct biologies. COX-1 expression is constitutive while COX-2 is inducible. The two COXs might have evolved partly to permit prostaglandin formation at different tissue sites. However, COX-2 is sometimes induced in cells already expressing COX-1, and in these instances, COX-2 functions while COX-1 is latent. This can occur because of unique biochemical properties of COX-2 that enable cells to form prostaglandins when arachidonic acid comprises a small fraction of available fatty acids and the concentrations of peroxides that are necessary for COX to function are low.     

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.     

Vitamin C suppresses proliferation of the human melanoma cell SK-MEL-2 through the inhibition of cyclooxygenase-2 (COX-2) expression and the modulation of insulin-like growth factor II (IGF-II) production

Vitamin C plays a crucial role in the suppression of proliferation of several types of cancer. Over-expression of cyclooxygenase (COX)-2 and type I insulin-like growth factor (IGF) receptor are important for proliferation and protection from apoptosis in malignancies. However, its specific mechanisms, especially the interaction between COX-2 expression and IGF-I axis mediated by vitamin C, remain yet to be clarified. Therefore, we investigated the effects of vitamin C on the proliferation of melanoma cells via the modulation of COX-2 expression and IGF-I axis. As a result, we found that 1.0 mM vitamin C inhibits the proliferation of SK-MEL-2 without induction of apoptosis. At that moment, IGF-II production was decreased, followed by the inhibition of COX-2 activity. IGF-IR expression was also down-regulated by vitamin C treatment. It coincided with the result from the inhibition of COX-2 by NS-398 and COX-2 siRNA. In addition, the decreased IGF-IR expression by vitamin C was restored by the treatment of recombinant prostaglandin E2. Finally, we determined whether the signal pathway would be involved in vitamin C-induced IGF-II and IGF-IR down-regulation. When the cells were exposed to SB203580, a specific inhibitor of p38 MAPK, COX-2 expression was dramatically recovered. In addition, phosphorylated p38 MAPK was increased after vitamin C treatment. Taken together, vitamin C suppresses proliferation of the human melanoma cell line SK-MEL2 via the down-regulation of IGF-II production and IGF-IR expression, which is followed by the activation of p38 MAPK and the inhibition of COX-2 expression.     

Structure-function relationship for saponin effects on cell cycle arrest and apoptosis in the human 1547 osteosarcoma cells: a molecular modelling approach of natural molecules structurally close to diosgenin

In this paper, eight natural molecules structurally close to diosgenin (five saponins: diosgenin, hecogenin, tigogenin, sarsasapogenin, smilagenin; two steroidal alkaloids: solasodine, solanidine; one sterol: stigmasterol) have been tested for their biological activities on human 1547 osteosarcoma cells. Differences in activity were studied in term of proliferation rate, cell cycle distribution and apoptosis induction. By using molecular modelling, two structural characteristics were calculated: spatial conformation and electron transfer capacity. The second property has been investigated by the HOMO repartition and the corresponding energy. Correlation between the experimental and the theoretical data permit us to highlight the importance of the hetero-sugar moiety and the 5,6-double bond in the biological activity (apoptosis and cell cycle arrest) on the human 1547 cell line. The importance of conformation at C-5 and C-25 carbon atoms was also discussed.     

COX-2 inhibitor, NS398, enhances Fas-mediated apoptosis via modulation of the PTEN-Akt pathway in human gastric carcinoma cell lines

A variety of human cancer cells are resistant to Fas ligand and anti-Fas antibody induced apoptosis. Previously, we reported that human gastric carcinoma cell lines were resistant to the anti-Fas antibody, CH-11, without interferon-gamma pretreatment in vitro. Cyclooxygenase (COX)-2 is known to be expressed in many human malignancies, and is correlated with tumor progression and resistance to apoptosis. This study examined whether NS398, a COX-2 inhibitor, inhibited cell proliferation and increased Fas-mediated apoptosis in human gastric carcinoma cell lines. Treatment of NS398 inhibited cell proliferation in MKN-45, which expressed the highest level of COX-2 among seven human gastric carcinoma cell lines, in a dose- and time-dependent manner, in contrast to less prominent effects in KATO-III, which expresses no COX-2. Although the treatment of CH-11 induced apoptosis in both cells, the simultaneous treatment of NS398 and CH-11 remarkably induced apoptosis, as confirmed by Hoechst 33258 staining and the terminal deoxynucleotidyl transferase- mediated dUTP-digoxigenin nick-end labeling (TUNEL) method in MKN-45. Flow cytometric analysis also revealed the increased pre-G1 fraction by the simultaneous treatment. The treatment of NS398 induced upregulation of Bad and PTEN, and downregulation of phosphorylated Akt (Thr308). These findings suggest that COX-2 might inhibit Fas-mediated apoptosis in human gastric carcinoma cell lines, especially MKN-45, by modulating PTEN and Akt.     

COX-2 mediates hepatitis B virus X protein abrogation of p53-induced apoptosis

The oncogenic hepatitis B virus X protein (HBx) and cyclooxygenase (COX)-2 are highly co-expressed in chronic hepatitis, cirrhosis and well-differentiated hepatocellular carcinoma (HCC). Although HBx is shown to activate COX-2, the functional consequences of this interaction in hepatocarcinogenesis remain unknown. Using an engineered hepatoma cell system in which the expression of wild-type p53 can be chemically modulated, we show here that COX-2 mediates HBx actions in opposing p53. Enforced expression of HBx sequestrates p53 in the cytoplasm and significantly abolishes p53-induced apoptosis. The anti-apoptotic Mcl-1 protein is suppressed by p53 but reactivated by HBx. The abrogation of apoptosis is completely reversed by specific COX-2 inhibition, suggesting that HBx blocks p53-induced apoptosis via activation of COX-2/PGE₂ pathway. We further show that COX-2 inhibition blocks HBx reactivation of Mcl-1, linking this protein to the anti-apoptotic function of COX-2. These results demonstrate that COX-2 is an important survival factor mediating the oncogenic actions of HBx. Over-expression of HBx and COX-2 may provide a selective clonal advantage for preneoplastic or neoplastic hepatocytes and contribute to the initiation and progression of HCC.     

Coupling between cyclooxygenases and terminal prostanoid synthases

Biosynthesis of prostanoids is regulated by three sequential enzymatic steps, namely phospholipase A2, cyclooxygenase (COX), and terminal prostanoid synthase. Recent evidence suggests that lineage-specific terminal prostanoid synthases, including prostaglandin (PG) E2, PGD2, PGF2alpha, PGI2, and thromboxane synthases, show distinct functional coupling with upstream COX isozymes, COX-1 and COX-2. This can account, at least in part, for segregated utilization of the two COX isozymes in distinct phases of PG-biosynthetic responses. In terms of their localization and COX preference, terminal prostanoid synthases are classified into three categories: (i) the perinuclear enzymes that prefer COX-2, (ii) the cytosolic enzyme that prefers COX-1, and (iii) the translocating enzyme that utilizes both COXs depending on the stimulus. Additionally, altered supply of arachidonic acid by phospholipase A2s significantly affects the efficiency of COX-terminal prostanoid synthase coupling. In this review, we summarize our recent understanding of the coupling profiles between the two COXs and various terminal prostanoid synthases.     

Signaling pathways regulating aromatase and cyclooxygenases in normal and malignant breast cells

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C(19) androgens to C(18) estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin PGE(2) increases intracellular cAMP levels and stimulates estrogen biosynthesis, and our recent studies have shown a strong linear association between CYP19 expression and the sum of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression in breast cancer specimens. Knowledge of the signaling pathways that regulate the expression and enzyme activity of aromatase and cyclooxygenases (COXs) in stromal and epithelial breast cells will aid in understanding the interrelationships of these two enzyme systems and potentially identify novel targets for regulation. The effects of epidermal growth factor (EGF), transforming growth factor-beta (TGFbeta), and tetradecanoyl phorbol acetate (TPA) on aromatase and COXs were studied in primary cultures of normal human adipose stromal cells and in cell cultures of normal immortalized human breast epithelial cells MCF-10F, estrogen-responsive human breast cancer cells MCF-7, and estrogen-unresponsive human breast cancer cells MDA-MB-231. Levels of the constitutive COX isozyme, COX-1, were not altered by the various treatments in the cell systems studied. In breast adenocarcinoma cells, EGF and TGFbeta did not alter COX-2 levels at 24h, while TPA induced COX-2 levels by 75% in MDA-MB-231 cells. EGF and TPA in MCF-7 cells significantly increased aromatase activity while TGFbeta did not. In contrast to MCF-7 cells, TGFbeta and TPA significantly increased activity in MDA-MB-231 cells, while only a modest increase with EGF was observed. Untreated normal adipose stromal cells exhibited high basal levels of COX-1 but low to undetectable levels of COX-2. A dramatic induction of COX-2 was observed in the adipose stromal cells by EGF, TGFbeta, and TPA. Aromatase enzyme activity in normal adipose stromal cells was significantly increased by EGF, TGFbeta and TPA after 24h of treatment. In summary, the results of this investigation on the effects of several paracrine and/or autocrine signaling pathways in the regulation of expression of aromatase, COX-1, and COX-2 in breast cells has identified more complex relationships. Overall, elevated levels of these factors in the breast cancer tissue microenvironment can result in increased aromatase activity (and subsequent increased estrogen biosynthesis) via autocrine mechanisms in breast epithelial cells and via paracrine mechanisms in breast stromal cells. Furthermore, increased secretion of prostaglandins such as PGE(2) from constitutive COX-1 and inducible COX-2 isozymes present in epithelial and stromal cell compartments will result in both autocrine and paracrine actions to increase aromatase expression in the tissues.     

Cyclooxygenase-2 modulates cellular growth and promotes tumorigenesis

Cyclooxygenase (COX)-2 and the prostaglandins resulting from its enzymatic activity have been shown to play a role in modulating cell growth and development of human neoplasia. Evidence includes a direct relationship between COX-2 expression and cancer incidence in humans and animal models, increased tumorigenesis after genetic manipulation of COX-2, and significant anti-tumor properties of non-steroidal anti-inflammatory drugs in animal models and in some human cancers. Recent data showed that COX-2 and the derived prostaglandins are involved in control of cellular growth, apoptosis, and signal through a group of nuclear receptors named peroxisome proliferator-activated receptors (PPARs). In this article we will review some of the findings suggesting that COX-2 is involved in multiple cellular mechanisms that lead to tumorigenesis.     

shRNA-targeted Cyclooxygenase (COX)-2 inhibits proliferation, reduces invasion and enhances chemosensitivity in laryngeal carcinoma cells

Cyclooxygenase-2 (COX-2), one isoform of cyclooxygenase proinflammatary enzymes, is a causal factor for tumor development, invasion, metastasis, and chemoresistance. It is frequently overexpressed in a variety of human malignancies, including laryngeal carcinoma. To investigate its possibility as a therapeutic target for the treatment of laryngeal carcinoma, we employed RNA interference technology to downregulate endogenous gene COX-2 expression in laryngeal carcinoma cells and analyzed its phenotypical changes. Results showed that shRNA-mediated downregulation of COX-2 expression in human laryngeal carcinoma cells significantly inhibited cell proliferation and colony formation in vitro and reduced the potential of tumorigenicity in vivo. The specific downregulation led to cell arrest in the G(0)/G(1) phase of cell cycle and final apoptosis induction. The increased apoptosis was associated with the ratios of Bcl-2 or Bcl-xL/Bax. In the present study, we also observed that the downregulation of COX-2 could obviously enhanced the cytotoxic effect of Taxanes both in vitro and in vivo. All these results suggest that knockdown of COX-2 expression can lead to potent antitumor activity and chemosensitizing activity to taxanes in human laryngeal carcinomas.     

Cyclooxygenase (COX)-2 and COX-1 potentiate beta-amyloid peptide generation through mechanisms that involve gamma-secretase activity

In previous studies we found that overexpression of the inducible form of cyclooxygenase, COX-2, in the brain exacerbated beta-amyloid (A beta) neuropathology in a transgenic mouse model of Alzheimer's disease. To explore the mechanism through which COX may influence A beta amyloidosis, we used an adenoviral gene transfer system to study the effects of human (h)COX-1 and hCOX-2 isoform expression on A beta peptide generation. We found that expression of hCOXs in human amyloid precursor protein (APP)-overexpressing (Chinese hamster ovary (CHO)-APPswe) cells or human neuroglioma (H4-APP751) cells resulting in 10-25 nM prostaglandin (PG)-E2 concentration in the conditioned medium coincided with an approximately 1.8-fold elevation of A beta-(1-40) and A beta-(1-42) peptide generation and an approximately 1.8-fold induction of the C-terminal fragment (CTF)-gamma cleavage product of the APP, an index of gamma-secretase activity. Treatment of APP-overexpressing cells with the non-selective COX inhibitor ibuprofen (1 microM, 48 h) or with the specific gamma-secretase inhibitor L-685,458 significantly attenuated hCOX-1- and hCOX-2-mediated induction of A beta peptide generation and CTF-gamma cleavage product formation. Based on this evidence, we next tested the hypothesis that COX expression might promote A beta peptide generation via a PG-E2-mediated mechanism. We found that exposure of CHO-APPswe or human embryonic kidney (HEK-APPswe) cells to PG-E2 (11-deoxy-PG-E2) at a concentration (10 nM) within the range of PG-E2 found in hCOX-expressing cells similarly promoted (approximately 1.8-fold) the generation of the CTF-gamma cleavage product of APP and commensurate A beta-(1-40) and A beta-(1-42) peptide elevation. The study suggests that expression of COXs may influence A beta peptide generation through mechanisms that involve PG-E2-mediated potentiation of gamma-secretase activity, further supporting a role for COX-2 and COX-1 in Alzheimer's disease neuropathology.     

Actin cytoskeletal architecture regulates nitric oxide-induced apoptosis,dedifferentiation, and cyclooxygenase-2 expression in articular chondrocytes via mitogen-activated protein kinase and protein kinase C pathways

Nitric oxide (NO) in articular chondrocytes regulates differentiation, survival, and inflammatory responses by modulating ERK-1 and -2, p38 kinase, and protein kinase C (PKC) alpha and zeta. In this study, we investigated the effects of the actin cytoskeletal architecture on NO-induced dedifferentiation, apoptosis, cyclooxygenase (COX)-2 expression, and prostaglandin E2 production in articular chondrocytes, with a focus on ERK-1/-2, p38 kinase, and PKC signaling. Disruption of the actin cytoskeleton by cytochalasin D (CD) inhibited NO-induced apoptosis, dedifferentiation, COX-2 expression, and prostaglandin E2 production in chondrocytes cultured on plastic or during cartilage explants culture. CD treatment did not affect ERK-1/-2 activation but blocked the signaling events necessary for NO-induced dedifferentiation, apoptosis, and COX-2 expression such as activation of p38 kinase and inhibition of PKCalpha and -zeta. CD also suppressed activation of downstream signaling of p38 kinase and PKC, such as NF-kappaB activation, p53 accumulation, and caspase-3 activation, which are necessary for NO-induced apoptosis. NO production in articular chondrocytes caused down-regulation of phosphatidylinositol (PI) 3-kinase and Akt activities. The down-regulation of PI 3-kinase and Akt was blocked by CD treatment, and the CD effects on apoptosis, p38 kinase, and PKCalpha and -zeta were abolished by the inhibition of PI 3-kinase with LY294002. Our results collectively indicate that the actin cytoskeleton mediates NO-induced regulatory effects in chondrocytes by modulating down-regulation of PI 3-kinase and Akt, activation of p38 kinase, and inhibition of PKCalpha and -zeta     

Coupling between cyclooxygenase, terminal prostanoid synthase, and phospholipase A2.

We have recently shown that two distinct prostaglandin (PG) E(2) synthases show preferential functional coupling with upstream cyclooxygenase (COX)-1 and COX-2 in PGE(2) biosynthesis. To investigate whether other lineage-specific PG synthases also show preferential coupling with either COX isozyme, we introduced these enzymes alone or in combination into 293 cells to reconstitute their functional interrelationship. As did the membrane-bound PGE(2) synthase, the perinuclear enzymes thromboxane synthase and PGI(2) synthase generated their respective products via COX-2 in preference to COX-1 in both the -induced immediate and interleukin-1-induced delayed responses. Hematopoietic PGD(2) synthase preferentially used COX-1 and COX-2 in the -induced immediate and interleukin-1-induced delayed PGD(2)-biosynthetic responses, respectively. This enzyme underwent stimulus-dependent translocation from the cytosol to perinuclear compartments, where COX-1 or COX-2 exists. COX selectivity of these lineage-specific PG synthases was also significantly affected by the concentrations of arachidonate, which was added exogenously to the cells or supplied endogenously by the action of cytosolic or secretory phospholipase A(2). Collectively, the efficiency of coupling between COXs and specific PG synthases may be crucially influenced by their spatial and temporal compartmentalization and by the amount of arachidonate supplied by PLA(2)s at a moment when PG production takes place.