Therapeutic Approaches in COVID-19 Patients: The Role of the Renin-Angiotensin System.

Two and a half years after COVID-19 was first reported in China, thousands of people are still dying from the disease every day around the world. The condition is forcing physicians to adopt new treatment strategies while emphasizing continuation of vaccination programs. The renin-angiotensin system plays an important role in the development and progression of COVID-19 patients. Nonetheless, administration of recombinant angiotensin-converting enzyme 2 has been proposed for the treatment of the disease. The catalytic activity of cellular ACE2 (cACE2) and soluble ACE2 (sACE2) prevents angiotensin II and Des-Arg-bradykinin from accumulating in the body. On the other hand, SARS-CoV-2 mainly enters cells via cACE2. Thus, inhibition of ACE2 can prevent viral entry and reduce viral replication in host cells. The benefits of bradykinin inhibitors (BKs) have been reported in some COVID-19 clinical trials. Furthermore, the effects of cyclooxygenase (COX) inhibitors on ACE2 cleavage and prevention of viral entry into host cells have been reported in COVID-19 patients. However, the administration of COX inhibitors can reduce innate immune responses and have the opposite effect. A few studies suggest benefits of low-dose radiation therapy (LDR) in treating acute respiratory distress syndrome in COVID-19 patients. Nonetheless, radiation therapy can stimulate inflammatory pathways, resulting in adverse effects on lung injury in these patients. Overall, progress is being made in treating COVID-19 patients, but questions remain about which drugs will work and when. This review summarizes studies on the effects of a recombinant ACE2, BK and COX inhibitor, and LDR in patients with COVID-19.

Targeting the Leukotriene Pathway for Colon Cancer Interception.

The role of chronic inflammation and arachidonic acid (AA) metabolism in tumor progression has been well characterized for variety of cancers, with compelling data for colon cancer. Several preclinical and clinical studies primarily focused on inhibiting the cyclooxygenase pathways using NSAIDs and aspirin for colon cancer prevention. However, emerging evidence clearly supports the pro-tumorigenic role of 5-lipoxygenase and its downstream leukotriene pathway within AA metabolism. As discussed in the current issue, targeting the leukotriene pathway by cysteinyl leukotriene receptor antagonist (LTRA) montelukast suppressed formation of aberrant crypt foci (ACF) and cell proliferation in colonic epithelium, suggesting the potential of LTRAs for colon cancer prevention. Although this is a short clinical chemoprevention trial to explore the effects of LTRAs against ACF development, it is a significant and timely study opening avenues to further explore the possibilities of using LTRAs in other inflammation-associated precancerous lesions as well. In this spotlight commentary, we highlight the implications of their data and the opportunities for developing LTRAs as potential candidates for colorectal cancer interception. See related article by Higurashi et al., p. 661.

Hinokitiol produces vasodilation in aortae from normal and angiotensin II- induced hypertensive rats via endothelial-dependent and independent pathways.

Hinokitiol is a natural bioactive compound with numerous pharmacological properties. Here, we aimed to examine hinokitiol's effects on vascular relaxation. Cumulative relaxation responses to hinokitiol were assessed in isolated aortae from normotensive and angiotensin II-induced hypertensive rats in the presence and absence of selective inhibitors. Hinokitiol produced vasodilation of phenylephrine preconstricted aortae using both normotensive and hypertensive rats. In normotensive rats, hinokitiol's vasodilation was reduced by endothelial denudation and nitric oxide synthase (NOS), guanylate cyclase, and cyclooxygenase inhibition. Also, hinokitiol vasodilation was attenuated by ß-receptors, adenylate cyclase, Ca2+-activated K+ channels and hyperpolarization inhibition. Moreover, hinokitiol exhibited a blocking activity on Ca2+ mobilization through voltage dependent Ca2+ channels (VDCC). However, its effect was not changed by muscarinic receptor and Sarc-K+ ATP channels blocking but was enhanced by blocking voltage-dependent K+ channels. However, in angiotensin II-induced hypertension, hinokitiol vasodilating activity was attenuated by NOS inhibition and it blocked Ca2+ mobilization through VDCC, while its vasodilation was partially attenuated by Sarc-K+ ATP channels blocking. However, the vasodilating effect of hinokitiol was not attenuated by either cyclooxygenase, ß-receptor, Ca2+-activated K+ channels, or voltage-dependent potassium channels inhibition, but was enhanced by blocking hyperpolarization. Hinokitiol's vasodilating effect in normotensive and hypertensive vessels is mediated through both endothelium-dependent and endothelium-independent mechanisms.

Occurrence of anti-Toxoplasma gondii antibodies in meat and dairy goat herds in Rio Grande do Norte, Brazil / Ocorrência de anticorpos anti-Toxoplasma gondii em rebanhos caprinos de corte e leiteiros do Rio Grande do Norte, Brasil

Toxoplasmosis is caused by Toxoplasma gondii, which is the main causative agent of abortion in small ruminants. Goats are among the animals that are most susceptible to this protozoon, and the disease that it causes leads to significant economic losses and has implications for public health, since presence of the parasite in products of goat origin is one of the main sources of human infection. Because of the significant economic impact, there is an urgent need to study the prevalence of T. gondii infection among goats in Sertão do Cabugi, which is the largest goat-producing region in Rio Grande do Norte. In the present study, the ELISA assay was used to test 244 serum samples from nine farms, located in four different municipalities in the Sertão do Cabugi region, which is an important goat-rearing region. The results showed that the prevalence of anti-T. gondii antibodies was 47.1% and that there was a significant association between positivity and the variables of age (≥ 34 months), location (Lajes, Angicos and Afonso Bezerra) and farm (all the farms). The avidity test was applied to all the 115 ELISA-positive samples to distinguish between acute and chronic infection. One hundred and three samples (89.6%) displayed high-avidity antibodies, thus indicating that most of the animals presented chronic infection, with a consequent great impact on the development of the goat production system and a risk to human health.

A toxoplasmose é causada pelo Toxoplasma gondii, principal agente causador de aborto em pequenos ruminantes. Os caprinos são uns dos animais mais suscetíveis a esse protozoário, levando a perdas econômicas significativas e implicações para a saúde pública, uma vez que a presença do parasito em produtos de origem caprina é uma das principais fontes de infecção humana. Devido ao impacto econômico significativo torna-se urgente estudar a prevalência da infecção, pelo T. gondii, entre caprinos do Sertão do Cabugi, a maior região produtora de caprinos no Rio Grande do Norte. O presente estudo utilizou o ELISA para testar 244 amostras de soro de 9 fazendas, situadas em 4 diferentes cidades na região do Sertão do Cabugi; uma importante região de criação de cabras. Os resultados mostraram uma prevalência de 47,1% para anticorpos anti- T. gondii e uma significativa associação entre a positividade e as variáveis idade (≥ 34 meses), localização (Lajes, Angicos e Afonso Bezerra e propriedade (todas as fazendas). O teste de avidez foi aplicado a todas as 115 amostras positivas pelo ELISA para discriminar entre infecção aguda e crônica. Cento e três amostras (89,6%) apresentaram anticorpos de alta avidez; indicando que a maioria dos animais estavam em infecção crônica, gerando um grande impacto sobre o desenvolvimento do sistema de produção em cabras e um risco para a saúde humana.

Stretch independent regulation of prostaglandin E(2) production within the isolated guinea-pig lamina propria.

OBJECTIVE: To use an isolated preparation of the guinea-pig bladder lamina propria (LP) to investigate the effects of adenosine tri-phosphate (ATP) and nitric oxide (NO) on the release of prostaglandin E(2) (PGE(2)). MATERIALS AND METHODS: The bladders of female guinea-pigs (200-400 g) were isolated and opened to expose the urothelial surface. The LP was dissected free of the underlying detrusor muscle and cut into strips from the dome to base. Strips were then incubated in Krebs buffer at 37 degrees C. Each tissue piece was then exposed to the stable ATP analogue, BzATP, and a NO donor, diethylamine-NONOate (DEANO), and the effect on PGE(2) output into the supernatant determined using the Parameter(TM) PGE(2) enzyme immunoassay kit (R & D Systems, Abingdon, UK). Experiments were repeated in the presence of purinergic receptor and cyclooxygenase (COX) enzymes, COX I and COX II, antagonists. The cellular location of COX I, COX II and neuronal NO synthase (nNOS) within the bladder LP was also determined by immunohistochemistry. RESULTS: PGE(2) production was significantly increased by BzATP. Antagonist studies showed the purinergic stimulation involved both P(2)X and P(2)Y receptors. The BzATP response was inhibited by the COX inhibitor indomethacin (COX I >COX II) but not by DUP 697 (COX II >COX I). Thus, BzATP stimulation occurs because of COX I stimulation. NO had no effect on PGE(2) production over the initial 10 min of an exposure. However, PGE(2) output was increased 100 min after exposure to the NO donor. In the presence of NO, the BzATP stimulation was abolished. Immunohistochemistry was used to confirm the location of COX I to the basal and inner intermediate urothelial layers and to cells within the diffuse layer of LP interstitial cells. In addition, nNOS was also located in the basal urothelial layers whilst COX II was found in the interstitial cell layers. CONCLUSIONS: There is complex interaction between ATP and NO to modulate PGE(2) release from the bladder LP in the un-stretched preparation. Such interactions suggest a complex interrelationship of signals derived from this region of the bladder wall. The importance of these interactions in relation to the physiology of the LP remains to be determined.

Cyclooxygenase-2-dependent prostaglandin (PG) E2 downregulates matrix metalloproteinase-3 production via EP2/EP4 subtypes of PGE2 receptors in human periodontal ligament cells stimulated with interleukin-1alpha.

BACKGROUND: Prostaglandin E2 (PGE2), which exerts its actions via EP receptors (EP1, EP2, EP3, and EP4), is a bioactive metabolite produced by cyclooxygenase (COX)-1 and/or COX-2 from arachidonic acid. In the present study, we investigated whether COX-2-derived PGE2 regulated matrix metalloproteinase (MMP)-3 production in human periodontal ligament (PDL) cells stimulated with interleukin (IL)-1alpha and which EP receptors were involved in PGE2 regulation of IL-1alpha-induced MMP-3 production. METHODS: Human PDL cells obtained from periodontally healthy subjects were stimulated with vehicle or IL-1alpha in the presence or absence of indomethacin (a COX-1/COX-2 inhibitor), NS-398 (a specific COX- 2 inhibitor), PGE2, EP receptor agonists, dibutyryl cAMP, and forskolin. PGE2 levels were assayed by enzyme-linked immunosorbent assay (ELISA). MMP-3 levels and caseinolytic activities were evaluated by ELISA and casein zymography, respectively. RESULTS: IL-1alpha enhanced both MMP-3 and PGE2 production. Indomethacin and NS-398 enhanced IL-1alpha-induced MMP-3 production in PDL cells, to the same extent, although both the agents completely inhibited IL-1alpha-induced PGE2 production. Exogenous PGE2 reduced IL-1alpha-induced MMP-3 production in a dose-dependent manner. Butaprost, a selective EP2 agonist, and ONO-AE1-329, a selective EP4 agonist, significantly inhibited IL-1alpha-induced MMP-3 production, although butaprost was less potent than ONO-AE-1-329. Dibutyryl cAMP, a cAMP analog, and forskolin, an adenylate cyclase activator, significantly inhibited IL-1alpha-stimulated MMP-3 production in PDL cells. CONCLUSIONS: These data suggest that COX-2-dependent PGE2 downregulates IL-1alpha-elicited MMP-3 production by cAMP-dependent pathways via EP2/EP4 receptors in human PDL cells. cAMP-elevating agents such as EP2/EP4 receptor activators may regulate the destruction of extracellular matrix components in periodontal tissue.

Local gene transduction of cyclooxygenase-1 increases blood flow in injured atherosclerotic rabbit arteries.

BACKGROUND: Cyclooxygenase-1 (COX-1) is the rate-limiting component in the synthesis of prostacyclin (PGI2), an important vasodilator and antithrombotic molecule. In balloon-injured, atherosclerosis-free porcine arteries, COX-1 gene transduction increases PGI2 production, induces durable vasodilation, and reduces thrombus formation. We tested the effectiveness of COX-1 local gene transduction for the prevention of postangioplasty restenosis in atherosclerotic arteries in a hypercholesterolemic rabbit model. METHODS AND RESULTS: We injured 1 carotid artery in 43 Watanabe heritable hyperlipidemic rabbits and performed local gene transduction using a viral vector containing the COX-1 gene (AdCOX-1, n=22) or no genes (Adnull, n=21). Three days later, AdCOX-1-treated arteries stimulated with arachidonic acid produced 100% more PGI2 (P<0.01), 400% more prostaglandin E2 (PGE2) (P<0.01), 400% more prostaglandin E1 (PGE1) (P<0.01), and 250% more cAMP (P<0.05) than Adnull-treated arteries. Twenty-eight days after treatment, Doppler sonography showed that blood flow velocity was preserved in AdCOX-1-treated arteries (ratio 0.92, injured compared with contralateral uninjured carotid artery) but reduced in Adnull-treated arteries (ratio 0.39), suggesting that AdCOX-1 prevented restenosis after injury. COX-1-transduced arteries also showed 80% greater lumen area 28 days after injury (P<0.01). CONCLUSIONS: The effectiveness of COX-1 in preventing restenosis and preserving normal blood flow 28 days after injury results from increased lumen area caused by durable vasodilation. COX-1 efficacy correlates with an early increase in the production of PGI2, PGE2, PGE1 (known to cause vasodilation), and cAMP. These results demonstrate for the first time that COX-1 gene transduction is an effective treatment for the prevention of postangioplasty restenosis of atherosclerotic arteries under clinically relevant conditions.

COX-2: a molecular target for colorectal cancer prevention.

Cyclooxygenase (COX), a key enzyme in the prostanoid biosynthetic pathway, has received considerable attention due to its role in human cancers. Observational and randomized controlled studies in many different population cohorts and settings have demonstrated protective effects of nonsteroidal anti-inflammatory drugs (NSAIDs; the inhibitors of COX activity) for colorectal cancers (CRCs). COX-2, the inducible isoform of cyclooxygenase, is overexpressed in early and advanced CRC tissues, which portends a poor prognosis. Experimental studies have thus identified important mechanisms and pathways by which COX-2 plays an important role in carcinogenesis. Selective COX-2 inhibitors have been approved for use as adjunctive therapy for patients with familial polyposis. The role of COX-2 inhibitors is currently being evaluated for use in wider populations.

Celecoxib inhibits prostate cancer growth: evidence of a cyclooxygenase-2-independent mechanism.

PURPOSE: Selective cyclooxygenase-2 (COX-2) inhibitors may suppress carcinogenesis by both COX-2-dependent and COX-2-independent mechanisms. The primary purpose of this study was to evaluate whether celecoxib or rofecoxib, two widely used selective COX-2 inhibitors, possess COX-2-independent antitumor activity. EXPERIMENTAL DESIGN: PC3 and LNCaP human prostate cancer cell lines were used to investigate the growth inhibitory effects of selective COX-2 inhibitors in vitro. To complement these studies, we evaluated the effect of celecoxib on the growth of PC3 xenografts. RESULTS: COX-1 but not COX-2 was detected in PC3 and LNCaP cells. Clinically achievable concentrations (2.5-5.0 micromol/L) of celecoxib inhibited the growth of both cell lines in vitro, whereas rofecoxib had no effect over the same concentration range. Celecoxib inhibited cell growth by inducing a G(1) cell cycle block and reducing DNA synthesis. Treatment with celecoxib also led to dose-dependent inhibition of PC3 xenograft growth without causing a reduction in intratumor prostaglandin E(2). Inhibition of tumor growth occurred at concentrations (2.37-5.70 micromol/L) of celecoxib in plasma that were comparable with the concentrations required to inhibit cell growth in vitro. The highest dose of celecoxib led to a 52% reduction in tumor volume and an approximately 50% decrease in both cell proliferation and microvessel density. Treatment with celecoxib caused a marked decrease in amounts of cyclin D1 both in vitro and in vivo. CONCLUSIONS: Two clinically available selective COX-2 inhibitors possess different COX-2-independent anticancer properties. The anticancer activity of celecoxib may reflect COX-2-independent in addition to COX-2-dependent effects.

[6]-Gingerol inhibits COX-2 expression by blocking the activation of p38 MAP kinase and NF-kappaB in phorbol ester-stimulated mouse skin.

[6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has a wide array of pharmacologic effects. The present study was aimed at unraveling the molecular mechanisms underlying previously reported antitumor promoting effects of [6]-gingerol in mouse skin in vivo. One of the well-recognized molecular targets for chemoprevention is cyclooxygenase-2 (COX-2) that is abnormally upregulated in many premalignant and malignant tissues and cells. In our present study, topical application of [6]-gingerol inhibited COX-2 expression in mouse skin stimulated with a prototype tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Since the transcription factor nuclear factor-kappaB (NF-kappaB) is known to regulate COX-2 induction, we attempted to determine the effect of [6]-gingerol on TPA-induced activation of NF-kappaB. Pretreatment with [6]-gingerol resulted in a decrease in both TPA-induced DNA binding and transcriptional activities of NF-kappaB through suppression of IkappaBalpha degradation and p65 nuclear translocation. Phosphorylation of both IkappaBalpha and p65 was substantially blocked by [6]-gingerol. In addition, [6]-gingerol inhibited TPA-stimulated interaction of phospho-p65-(Ser-536) with cAMP response element binding protein-binding protein, a transcriptional coactivator of NF-kappaB. Moreover, [6]-gingerol prevented TPA-induced phosphorylation and catalytic activity of p38 mitogen-activated protein (MAP) kinase that regulates COX-2 expression in mouse skin. The p38 MAP kinase inhibitor SB203580 attenuated NF-kappaB activation and subsequent COX-2 induction in TPA-treated mouse skin. Taken together, our data suggest that [6]-gingerol inhibits TPA-induced COX-2 expression in mouse skin in vivo by blocking the p38 MAP kinase-NF-kappaB signaling pathway.

Human hepatic and renal microsomes, cytochromes P450 1A1/2, NADPH:cytochrome P450 reductase and prostaglandin H synthase mediate the formation of aristolochic acid-DNA adducts found in patients with urothelial cancer.

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Understanding which human enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual's susceptibility to this plant carcinogen. Using the (32)P postlabeling assay, we examined the ability of microsomal samples from 8 human livers and from 1 human kidney to activate AAI, the major component of the plant extract AA, to metabolites forming adducts in DNA. Microsomes of both organs generated DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed from AAI by all human hepatic and renal microsomes. To define the role of human microsomal enzymes in the activation of AAI, we investigated the modulation of AAI-DNA adduct formation by cofactors and selective inhibitors of microsomal reductases, cytochrome P450 (CYP) enzymes, NADPH:CYP reductase and NADH:cytochrome b(5) reductase. We also determined whether the activities of CYP and NADPH:CYP reductase in different human hepatic microsomal samples correlated with the levels of AAI-DNA adducts formed by the same microsomal samples. On the basis of these studies, we attribute most of the activation of AAI in human hepatic microsomes to CYP1A2. In contrast to human hepatic microsomes, in human renal microsomes NADPH:CYP reductase is more effective in AAI activation. In addition, prostaglandin H synthase is another enzyme activating AAI in renal microsomes. The results demonstrate for the first time the potential of microsomal enzymes in human liver and kidney to activate AAI by nitroreduction.

Clinical implications of expression of cyclooxygenase-2 related to angiogenesis in uterine endometrial cancers.

BACKGROUND: Angiogenesis is essential for development, growth and advancement of solid tumors. Cyclooxygenase (cox)-2 is recognized as an angiogenic factor in various tumors. This prompted us to study the clinical implications of cox-2 expression and angiogenesis in uterine endometrial cancers. PATIENTS AND METHODS: Fifty patients underwent curative resection for uterine endometrial cancers. In uterine endometrial cancers, cox-2 levels were determined by enzyme immunoassay, and the localization and counts of microvessels were determined by immunohistochemistry. RESULTS: There was a significant correlation between microvessel counts and cox-2 levels in uterine endometrial cancers. Cox-2 localized in the cancer cells, but not in the stromal cells of uterine endometrial cancer tissues. Cox-2 levels decreased with the advancement. Furthermore, cox-2 levels significantly correlated with VEGF levels in uterine endometrial cancers. CONCLUSIONS: VEGF associated with cox-2 might work on angiogenesis at an early status in growth. Therefore, long-term administration of cox-2 inhibitors might be effective in the suppression of recurrent initiation of uterine endometrial cancers after curative resection.

Intestinal surgical resection disrupts electrical rhythmicity, neural responses, and interstitial cell networks.

BACKGROUND & AIMS: Surgical manipulations of the gastrointestinal (GI) tract, including intestinal resection and anastomosis, lead to motility disorders including a decrease in phasic and segmental contractions. The aims of the present investigation were to determine mechanisms underlying the loss of motility in a murine model of intestinal resection and to follow-up the recovery of intestinal motility after surgical manipulation. METHODS: Segments of ileum were removed from mice and the intestines were reconstructed. After surgery, the structure and activity of the ileal muscles, 0-5 cm oral and aboral to the site of resection, were examined at 5 and 24 hours with intracellular microelectrode recordings, isometric force measurements, Kit-like immunohistochemistry, and electron microscopy. RESULTS: Five hours after surgery there was loss of electrical slow waves and phasic contractions in muscles near the site of resection. This defect decreased as a function of distance above and below the resection. Tissues in the affected region were poorly responsive to carbachol and transmural nerve stimulation. Kit-like immunohistochemistry revealed disruption in interstitial cell of Cajal (ICC) networks at the level of the myenteric and deep muscular plexuses. Lesions in ICCs decreased with distance from the site of resection. Slow waves and mechanical activity recovered at the site of anastomosis 24 hours after surgery and recovered more rapidly when tissues were incubated in the inducible nitric oxide synthase (iNOS) inhibitor, L-N6 -(1-Iminoethyl) lysine hydrochloride (L-NIL). CONCLUSIONS: Loss of intestinal motility after surgery is associated with acute disruption of ICC networks, slow waves, and phasic contractions. This activity partially recovered within 24 hours after surgery.

Infection, fever, and exogenous and endogenous pyrogens: some concepts have changed.

For many years, it was thought that bacterial products caused fever via the intermediate production of a host-derived, fever-producing molecule, called endogenous pyrogen (EP). Bacterial products and other fever-producing substances were termed exogenous pyrogens. It was considered highly unlikely that exogenous pyrogens caused fever by acting directly on the hypothalamic thermoregulatory center since there were countless fever-producing microbial products, mostly large molecules, with no common physical structure. In vivo and in vitro, lipopolysaccharides (LPSs) and other microbial products induced EP, subsequently shown to be interleukin-1 (IL-1). The concept of the 'endogenous pyrogen' cause of fever gained considerable support when pure, recombinant IL-1 produced fever in humans and in animals at subnanomolar concentrations. Subsequently, recombinant tumor necrosis factor-alpha (TNF-alpha), IL-6 and other cytokines were also shown to cause fever and EPs are now termed pyrogenic cytokines. However, the concept was challenged when specific blockade of either IL-1 or TNF activity did not diminish the febrile response to LPS, to other microbial products or to natural infections in animals and in humans. During infection, fever could occur independently of IL-1 or TNF activity. The cytokine-like property of Toll-like receptor (TLR) signal transduction provides an explanation by which any microbial product can cause fever by engaging its specific TLR on the vascular network supplying the thermoregulatory center in the anterior hypothalamus. Since fever induced by IL-1, TNF-alpha, IL-6 or TLR ligands requires cyclooxygenase-2, production of prostaglandin E2 (PGE2) and activation of hypothalamic PGE2 receptors provides a unifying mechanism for fever by endogenous and exogenous pyrogens. Thus, fever is the result of either cytokine receptor or TLR triggering; in autoimmune diseases, fever is mostly cytokine mediated whereas both cytokine and TLR account for fever during infection.

NS398 reduces hypoxia-inducible factor (HIF)-1alpha and HIF-1 activity: multiple-level effects involving cyclooxygenase-2 dependent and independent mechanisms.

Tissue hypoxia is a common feature in solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a critical transcription factor that regulates the expression of genes encoding factors that influence tumor growth including vascular endothelial growth factor. Previous studies have demonstrated that post-transcriptional modification events are important for regulation of HIF-1alpha protein expression and HIF-1 transcriptional activity. Prostaglandin E2 (PGE2), a major end product of the cyclooxygenase-2 (COX-2) enzyme, induces the basal and hypoxia-induced nuclear relocalization of HIF-1alpha. This is suppressed by NS398, a COX-2 selective inhibitor. NS398 also inhibits hypoxia-induced angiogenesis, which may be mediated by the inhibition of HIF-1 function in a COX-2-dependent manner. Here, we show that NS398 reduces HIF-1alpha and HIF-1 transcriptional function in both COX-2 positive PC-3 cells and COX-2 negative HCT116 cells under normoxic and hypoxic conditions. On the one hand, NS398 decreases the expression of HIF-1alpha mRNA and reduces HIF-1alpha synthesis in a COX-2/PGE2 dependent way, which can be restored by addition of exogenous PGE2 that activates the phosphatidylinositol 3-kinase/AKT/p70s6k signaling pathway. On the other hand, NS398 accelerates HIF-1alpha degradation by moderately increasing ubiquitination and remarkably promoting the clearance of ubiquitylated protein, an effect most likely independent of COX-2/PGE2 since exogenous PGE2 fails to reverse it. Finally, NS398 decreases hypoxia-induced shifted form of HIF-1alpha and attenuates HIF-1 activation in greater extent under hypoxic than normoxic conditions. These data not only confirm the inhibitory effect of NS398 on HIF-1alpha and HIF-1 transcriptional activity but also demonstrate that such an effect occurs at multiple levels involving both COX-2 dependent and independent mechanisms.

Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition.

Nonsteroidal anti-inflammatory drugs (NSAIDs) represent one of the most highly utilized classes of pharmaceutical agents in medicine. All NSAIDs act through inhibiting prostaglandin synthesis, a catalytic activity possessed by two distinct cyclooxygenase (COX) isozymes encoded by separate genes. The discovery of COX-2 launched a new era in NSAID pharmacology, resulting in the synthesis, marketing, and widespread use of COX-2 selective drugs. These pharmaceutical agents have quickly become established as important therapeutic medications with potentially fewer side effects than traditional NSAIDs. Additionally, characterization of the two COX isozymes is allowing the discrimination of the roles each play in physiological processes such as homeostatic maintenance of the gastrointestinal tract, renal function, blood clotting, embryonic implantation, parturition, pain, and fever. Of particular importance has been the investigation of COX-1 and -2 isozymic functions in cancer, dysregulation of inflammation, and Alzheimer's disease. More recently, additional heterogeneity in COX-related proteins has been described, with the finding of variants of COX-1 and COX-2 enzymes. These variants may function in tissue-specific physiological and pathophysiological processes and may represent important new targets for drug therapy.

COX-2 and its inhibition as a molecular target in the prevention and treatment of lung cancer.

Lung cancer is the leading cause of cancer death in the USA. Conventional therapy using chemotherapy, radiation therapy, and a combination of the two, has yielded modest improvement in patient outcome. Dysfunction and dysregulation of many molecular processes and signaling pathways are involved in the development and growth of malignant lung tumors, and in conferring resistance to standard cancer treatments. Cyclooxygenase (COX)-2, an enzyme involved in prostaglandin production in pathologic states, is often overexpressed in premalignant and malignant lesions. Overexpression of COX-2 in lung cancer is associated with more aggressive biologic tumor behavior and adverse patient outcome. In preclinical studies, inhibition of this enzyme with selective COX-2 inhibitors enhances tumor response to radiation and chemotherapeutic agents. These findings quickly led to clinical studies. Phase I and II clinical trials of the combination of selective COX-2 inhibitors with radiotherapy, chemotherapy, or both in patients with lung cancer have been initiated and some preliminary results are available. In this review, the relationship between overexpression of COX-2 and lung cancer, the antitumor effect of selective COX-2 inhibitors, and the rationale for using selective COX-2 inhibitors combined with radiotherapy and chemotherapy, will be described. Current clinical protocols and preliminary findings will also be summarized.

Eicosanoid metabolism in squamous cell carcinoma cell lines derived from primary and metastatic head and neck cancer and its modulation by celecoxib.

Eicosanoid metabolism through cyclooxygenases (COXs) and lipoxygenases (LOXs) generates various lipids that play a role in squamous cell carcinogenesis. We used pairs of head and neck squamous cell carcinoma (HNSCC) cell lines derived from primary and metastatic tumors of the same patient to analyze eicosanoid metabolites by ESI-LC/MS/MS and COX/LOX expression by western immunoblotting. The effects of celecoxib on eicosanoid synthesis and HNSCC cell growth were examined. Prostaglandin E2 (PGE2) was the major metabolite in three of six cell lines. COX-2 was detected in three cell lines, which produced PGE2 (two from metastases). We found low expression of COX-1 at similar intensities for each pair of cell lines. 5-LOX was detected in all cells. Some expressed 12-LOX, 15-LOX-1, and 15-LOX-2, but there was no correlation between enzyme expression and endogenous product content. Exogenous arachidonic acid did not change the profile of eicosanoid biosynthesis. Low doses of celecoxib inhibited formation of PGE2 in UMSCC-14A cells by 84% as early as 6 hours. In contrast, 5-HETE, 12-HETE, and 15-HETE levels were increased by approximately 40-, 5- and 3-fold, respectively, with a decline to baseline levels within 24 hours. High dose celecoxib increased the 12-HETE level 2.3-fold after 3 days of incubation. Celecoxib inhibited growth of all HNSCC cell lines in a concentration-dependent manner regardless of their COX expression (IC50 values after 3 days; 33 to 62 microM). Our findings provide new informations about individual eicosanoids produced by HNSCC cells and their differential regulation by the selective COX-2 inhibitor celecoxib.

Molecular biology of sporadic gastric cancer: prognostic indicators and novel therapeutic approaches.

Both the availability of multiple treatment modalities and novel therapeutic targets make the correct prognostic stratification and the identification of truly predictive factors an issue of major debate in gastric cancer. Along with "classic" prognostic factors such as those related to the diffusion of the tumour at diagnosis (i.e., depth of gastric wall infiltration, locoregional lymph nodes or distant metastases) or those concerning the pathologic characteristics of the tumour, other, innovative, factors should be considered if a better definition of the characteristics of the tumour is to be given. These biological factors are often derived from the genetic process, which is thought to represent a crucial step to gastric cancer (DNA copy number changes, microsatellite instability, thymidilate synthase, E-cadherin, beta-catenin, mucin antigen, p53, c-erb B-2, COX-2, matrix metalloproteinases, VEGFR and EGFR). Some of those putative prognostic indicators can also be considered predictive of response to therapy as they are a molecular target either to chemotherapeutics (i.e., thymidilate synthase that is targeted by 5FU) or to a new class of antineoplastic molecules (i.e., c-erb B-2 targeted by trastuzumab, COX-2 by NSAIDs, matrix metalloproteinases, EGFR and VEGFR by specific inhibitors).