Cyclooxygenase-2 Promoter Research Articles

Abstract 122: Activation of Nuclear Factor Erythroid 2-related Factor 2 (Nrf2) Enhances Cyclooxygenase 2 Expression via Promoter Antioxidant Response Element in Preglomerular Vascular Smooth Muscle Cells (PGVSMCs)

Nrf2 is a key cytoprotective transcription factor driving many antioxidant and anti-inflammatory genes expression via binding to antioxidant response elements (AREs) in promoters of targeting genes. However, it was reported that Nrf2 also increased expression of pro-inflammatory and pro-oxidative genes IL-6 and NOX4 through the same mechanism. We detected 2 putative ARE consensus sequences within 1500 nucleotides (nts) located in the genomic region upstream of the transcription start site in rat cyclooxygenase 2 (COX2) gene. Therefore, we hypothesized that activation of Nrf2-dependent AREs upregulates COX2 expression in rat PGVSMCs. Incubation of the cells for 24 hours with Nrf2 activator, tert-butylhydroquinone (tBHQ), did not significantly alter Nrf2 expression but dose-dependently increased COX2 gene and protein in a dose response manner (5-20μM). Twenty μM of tBHQ treatment enhanced expression of COX2 gene and protein by 10.7±2.2 and 9.2±3.4 fold (both p<0.01, N=3), COX2 activity (from COX2-based product of fluorescent resorufin) by 229.9±28.8% (p<0.005, N=6) but reactive oxygen species (ROS) generated in the cells (from oxidized dihydroethidium-based fluorescent intensity) were reduced by 35.1±10.2% (p<0.01, N=4). tBHQ also significantly promoted Nrf2 nuclear translocation by 86±7.4% (p<0.05, N=3) and potentiated Nrf2 activity by 387.5±19.2% (p<0.005, N=4) determined with cell fraction immunoblotting and Nrf2-ARE binding-based enzyme-linked immunosorbent assay. Nrf2 expression was dramatically knocked down with markedly decreased COX2 expression and activity in the cells transfected with Nrf2 specific siRNA and treated with tBHQ compared to those transfected with the scramble control siRNA and treated with tBHQ. Chromatin immunoprecipitation (CHIP)-based PCR analysis revealed an enhanced recruitment of Nrf2 to the endogenous COX2 promoter spanning the ARE (-417 to -426 nts) after 24 hour-tBHQ treatment but knockdown of Nrf2 gene with siRNA significantly diminished the tBHQ-mediated role, suggesting that there is a functional ARE located in COX2 promoter. In conclusion, these results show a novel role of Nrf2 in inducing COX2 expression through binding to promoter ARE in the absence of increased ROS in rat PGVSMCs.

Evaluation of anti-acne properties of phloretin in vitro and in vivo.

This study aimed to investigate the anti-acne properties of phloretin in vitro and in vivo. Anti-microbial activity against Propionibacterium acnes (P. acnes), Propionibacterium granulosum (P. granulosum) and Staphylococcus epidermidis (S. epidermidis) were observed by the minimum inhibitory concentration (MIC) and disc diffusion methods. The anti-inflammatory effects were studied in HaCaT cells based on P. acnes-induced inflammatory mediators, including PGE2 and COX-2, examined through enzyme-linked immunosorbent assay (ELISA) and luciferase reporter gene assay. Thirty healthy subjects with whiteheads participated in the clinical study. Comedo counting, and the amount of sebum and porphyrin were measured before treatment and following 4 consecutive weeks of treatment with phloretin. Phloretin showed anti-microbial activities against P. acnes, P. granulosum, S. epidermidis with the MIC of 0.5, 0.5 and 0.25 mg mL(-1) , respectively. P. acnes-induced activation of the COX-2 promoter was markedly attenuated by phloretin treatment. Consistent with these results, inhibition of PGE2 production was also observed. In 1-month, placebo-controlled trials, phloretin showed clinically and statistically significant reduction of comedo counts and sebum output level. Compared to before treatment, whiteheads, blackheads, papules, sebum output level and amount of sebum and porphyrin were significantly decreased at 4 weeks in the test group. This study revealed that phloretin inhibits the growth of P. acnes, P. granulosum, and S. epidermidis. In addition, we demonstrated that phloretin attenuates COX-2 and PGE2 expression during the P. acnes-induced upregulation of inflammatory signalling. Clinical studies further suggested that treatment with formulations containing phloretin confers anti-acne benefits. Based on these results, we suggest that phloretin may be introduced as a possible acne-mitigating agent.

Protein Phosphatase 2A in Lipopolysaccharide-Induced Cyclooxygenase-2 Expression in Murine Lymphatic Endothelial Cells.

The lymphatic endothelium plays an important role in the maintenance of tissue fluid homeostasis. It also participates in the pathogenesis of several inflammatory diseases. However, little is known about the underlying mechanisms by which lymphatic endothelial cell responds to inflammatory stimuli. In this study, we explored the mechanisms by which lipopolysaccharide (LPS) induces cyclooxygenase (COX)-2 expression in murine lymphatic endothelial cells (SV-LECs). LPS caused increases in cox-2 mRNA and protein levels, as well as in COX-2 promoter luciferase activity in SV-LECs. These actions were associated with protein phosphatase 2A (PP2A), apoptosis signal-regulating kinase 1 (ASK1), JNK1/2 and p38MAPK activation, and NF-κB subunit p65 and C/EBPβ phosphorylation. PP2A-ASK1 signaling blockade reduced LPS-induced JNK1/2, p38MAPK, p65 and C/EBPβ phosphorylation. Transfection with PP2A siRNA reduced LPS’s effects on p65 and C/EBPβ binding to the COX-2 promoter region. Transfected with the NF-κB or C/EBPβ site deletion of COX-2 reporter construct also abrogated LPS’s enhancing effect on COX-2 promoter luciferase activity in SV-LECs. Taken together, the induction of COX-2 in SV-LECs exposed to LPS may involve PP2A-ASK1-JNK and/or p38MAPK-NF-κB and/or C/EBPβ cascade.

Dysregulated CRTC1 activity is a novel component of PGE2 signaling that contributes to colon cancer growth.

First identified as a dedicated CREB (cAMP response element-binding protein) co-activator, CRTC1 (CREB-regulated transcription co-activator 1) has been widely implicated in various neuronal functions because of its predominant expression in the brain. However, recent evidences converge to indicate that CRTC1 is aberrantly activated in an expanding number of adult malignancies. In this study, we provide strong evidences of enhanced CRTC1 protein content and transcriptional activity in mouse models of sporadic (APC(min/+) mice) or colitis-associated colon cancer azoxymethane/dextran sulfate sodium (AOM/DSS-treated mice), and in human colorectal tumors specimens compared with adjacent normal mucosa. Among signals that could trigger CRTC1 activation during colonic carcinogenesis, we demonstrate that treatment with cyclooxygenase 2 (COX2) inhibitors reduced nuclear CRTC1 active form levels in colonic tumors of APC(min/+) or AOM/DSS mice. In accordance, prostaglandins E2 (PGE2) exposure to human colon cancer cell lines promoted CRTC1 dephosphorylation and parallel nuclear translocation, resulting in enhanced CRTC1 transcriptional activity, through EP1 and EP2 receptors signaling and consecutive calcineurin and protein kinase A activation. In vitro CRTC1 loss of function in colon cancer cell lines was associated with reduced viability and cell division rate as well as enhanced chemotherapy-induced apoptosis on PGE2 treatment. Conversely, CRTC1 stable overexpression significantly increased colonic xenografts tumor growth, therefore demonstrating the role of CRTC1 signaling in colon cancer progression. Identification of the transcriptional program triggered by enhanced CRTC1 expression during colonic carcinogenesis, revealed some notable pro-tumorigenic CRTC1 target genes including NR4A2, COX2, amphiregulin (AREG) and IL-6. Finally, we demonstrate that COX2, AREG and IL-6 promoter activities triggered by CRTC1 are dependent on functional AP1 and CREB transcriptional partners. Overall, our study establishes CRTC1 as new mediator of PGE2 signaling, unravels the importance of its dysregulation in colon cancer and strengthens its use as a bona fide cancer marker.

Magnolin inhibits cell migration and invasion by targeting the ERKs/RSK2 signaling pathway.

BackgroundMagnolin is a natural compound abundantly found in Magnolia flos, which has been traditionally used in oriental medicine to treat headaches, nasal congestion and anti-inflammatory reactions. Our recent results have demonstrated that magnolin targets the active pockets of ERK1 and ERK2, which are important signaling molecules in cancer cell metastasis. The aim of this study is to evaluate the effects of magnolin on cell migration and to further explore the molecular mechanisms involved.MethodsMagnolin-mediated signaling inhibition was confirmed by Western blotting using RSK2+/+ and RSK2−/− MEFs, A549 and NCI-H1975 lung cancer cells, and by NF-κB and Cox-2 promoter luciferase reporter assays. Inhibition of cell migration by magnolin was examined by wound healing and/or Boyden Chamber assays using JB6 Cl41 and A549 human lung cancer cells. The molecular mechanisms involved in cell migration and epithelial-to-mesenchymal transition were determined by zymography, Western blotting, real-time PCR and immunocytofluorescence.ResultsMagnolin inhibited NF-κB transactivation activity by suppressing the ERKs/RSK2 signaling pathway. Moreover, magnolin abrogated the increase in EGF-induced COX-2 protein levels and wound healing. In human lung cancer cells such as A549 and NCI-H1975, which harbor constitutive active Ras and EGFR mutants, respectively, magnolin suppressed wound healing and cell invasion as seen by a Boyden chamber assay. In addition, it was observed that magnolin inhibited MMP-2 and −9 gene expression and activity. The knockdown or knockout of RSK2 in A549 lung cancer cells or MEFs revealed that magnolin targeting ERKs/RSK2 signaling suppressed epithelial-to-mesenchymal transition by modulating EMT marker proteins such as N-cadherin, E-cadherin, Snail, Vimentin and MMPs.ConclusionsThese results demonstrate that magnolin inhibits cell migration and invasion by targeting the ERKs/RSK2 signaling pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1580-7) contains supplementary material, which is available to authorized users.

Abstract 1019: hnRNPA2/B1 upregulates COX-2 expression and tumor growth and predicts poor prognosis in human lung cancers

Abstract Constitutive high expression of COX-2 can promote tumor development and has been regarded as a hallmarker for cancers. However, the excise regulation mechanism of COX-2 and its role in lung tumoirgenesis remains unknown. In this study, we discovered and identified hnRNPA2/B1 as a novel regulator of COX-2 promoter in non-small cell lung cancer (NSCLC) cells by a DNA probe-biotin-streptavidin-agarose pulldown and proteomic technique. We showed that hnRNPA2/B1 specifically bound to COX-2 core promoter region, activated COX-2 promoter activity and up-regulated COX-2 expression. Knockdown of hnRNPA2/B1 expression by siRNA significantly suppressed tumor cell growth in NSCLC cell lines in vitro and in a animal model in vivo by inhibiting COX-2 expression. In contrast, overexpression of hnRNPA2/B1 promoted COX-2 expression and tumor cell growth in NSCLC cells. Micro-tissue array and immunohistochemical assay showed that hnRNPA2/B1 expression correlated with COX-2 expression and high expression of hnRNPA2/B1 and COX-2 was associated with worse prognosis in patients with NSCLCs. Mechanistically, hnRNPA2/B1 functions as a regulator of COX-2 to control tumor growth through its interaction with p300, a transcriptional co-activator, in NSCLC cells. Immunoprecipitation and confocal immunofluorescence microscope analysis confirmed the direct interaction of hnRNPA2/B1 with p300 proteins. Overexpression of p300, but not its histon acetyltransferase domain deletion mutation, enhanced the acetylation of hnRNPA2/B1, facilitated the binding of hnRNPA2/B1 to COX-2 promoter, thereby promoted COX-2 expression and tumor cell growth. Therefore, these results indicate that hnRNPA2/B1 acts as an activator to regulate COX-2 expression and tumor growth through the interaction with p300 in NSCLC cells and suggest that the hnRNPA2/B1/COX-2 signaling is a potential therapeutic target for lung cancers. Note: This abstract was not presented at the meeting. Citation Format: Jingshu Wang, Yang Xuan, Wei Guo, Guangwei Du, Wuguo Deng. hnRNPA2/B1 upregulates COX-2 expression and tumor growth and predicts poor prognosis in human lung cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1019. doi:10.1158/1538-7445.AM2015-1019

Abstract 1816: Impact of 5FU and radiation on replication of interferon-expressing adenovirus

Abstract Pancreatic Cancer is a disease for which there are very limited efficacious treatments, resulting in a 5-year survival rate for affected patients around only 6%. A recombinant oncolytic adenovirus (OAd-IFN) developed by our group to target pancreatic cancer utilizes enhanced oncolysis with an overexpression of the adenoviral death protein, enhanced infectivity with a genetically modified fiber, conditional replication targeted to pancreatic cancer using the Cox-2 promoter, and an expression of human interferonαto enhance the therapeutic effects of chemotherapy and radiation through the antitumor effects of the cytokine. Our previous work analyzing the therapeutic abilities of IFN expressing vectors in human pancreatic cancer xenografts in immonudeficient nude mice and in syngeneic hamster pancreatic cancer nodules in immunocompetent Syrian hamsters, showed a more evident antitumor effect of OAd-IFN in the latter models. This suggests the success of OAd-IFN as an immune system stimulator, making it a promising candidate for combination therapy approaches. The goal of this study is to investigate the possibility for use of OAd-IFN as a therapy for pancreatic cancer in conjunction with 5FU and radiation. Specifically, the adenoviral replicative ability was measured within pancreatic cancer Mia-Paca 2 cells under various conditions of 5FU and radiation exposure. Replication was analyzed by a correlation to the increase in amount of viral DNA extracted from treated cells at two time points post infection. In two preliminary trials, cells were infected with the virus two days prior to receiving 5FU and radiation, and another group of cells was treated with 5FU before viral infection. Based on the initial data collected, radiation seemed to have no effect on viral replication. 5FU treatment prior to viral infection resulted in an increase in replication at low dose, and a decrease in replication with high dose, however, this effect was only seen at the first time point 3 days after infection. Similarly, 5FU treatment post infection showed a minor dose dependent inhibition of viral replication at high doses, with little to no effect observed in the low dose. Combining viral infection with 5FU appears to have an impact on the replication of OAd-IFN in vitro; however, further trials need to be done to explore the effect of various timing among viral infection and therapy treatments as well as different therapy concentrations and viral titers to further analyze the potential combination therapies. Citation Format: Jordan Sell. Impact of 5FU and radiation on replication of interferon-expressing adenovirus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1816. doi:10.1158/1538-7445.AM2015-1816

Abstract 2714: Effects of endosulfan on COX-2 expression via NADPH oxidase and the ROS, MAPK, and Akt pathways

Abstract Endosulfan is correlated with endocrine disruption, reproductive, and immune dysfunctions. Recently, endosulfan was shown to have an effect on inflammatory pathways, but its influence on cyclooxygenase-2(COX-2) expression is unclear. This study investigated the effects of COX-2 and molecular mechanisms by endosulfan in murine macrophage RAW 264.7 cells. Endosulfan significantly induced COX-2 protein and mRNA levels, as well as COX-2 promoter-driven luciferase activity and the production of prostaglandin E2, a major COX-2 metabolite. Transfection experiments with several human COX-2 promoter constructs revealed that endosulfan activated NF-KB, C/EBP, AP-1, and CREB. Moreover, Akt and mitogen-activated protein kinases (MAPK) were significantly activated by endosulfan. Moreover, endosulfan increased production of the ROS and the ROS-producing NAPDH-oxidase (NOX) family oxidases, NOX2, and NOX3. These results demonstrate that endosulfan induces COX-2 expression via NADPH oxidase, ROS, and Akt/MAPK pathways. These findings provide further insight into the signal transduction pathways involved in the inflammatory effects of endosulfan. Citation Format: Eun Hee Han, Sang Mi Jun, Young Ho Chung. Effects of endosulfan on COX-2 expression via NADPH oxidase and the ROS, MAPK, and Akt pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2714. doi:10.1158/1538-7445.AM2015-2714

HCV NS5A Up-Regulates COX-2 Expression via IL-8-Mediated Activation of the ERK/JNK MAPK Pathway.

Chronic hepatitis C virus (HCV) infection leads to intrahepatic inflammation and liver cell injury, which are considered a risk factor for virus-associated hepatitis, cirrhosis, and hepatocellular carcinoma worldwide. Inflammatory cytokines are critical components of the immune system and influence cellular signaling, and genetic imbalances. In this study, we found that cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) were significantly induced by HCV infection and HCV NS5A expression, and induction of COX-2 correlated with HCV-induced IL-8 production. We also found that the ERK and JNK signaling pathways were involved in the regulation of IL-8-mediated COX-2 induction in response to HCV infection. Using a promoter-linked reporter assay, we identified that the C/EBP regulatory element within the COX-2 promoter was the dominant factor responsible for the induction of COX-2 by HCV. Silencing C/EBP attenuated HCV-induced COX-2 expression. Our results revealed that HCV-induced inflammation promotes viral replication, providing new insights into the involvement of IL-8-mediated COX-2 induction in HCV replication.

Mucin 1 Regulates Cox-2 Gene in Pancreatic Cancer.

Eighty percent of pancreatic ductal adenocarcinomas (PDAs) overexpress mucin 1 (MUC1), a transmembrane mucin glycoprotein. MUC1(high) PDA patients also express high levels of cyclooxygenase 2 (COX-2) and show poor prognosis. The cytoplasmic tail of MUC1 (MUC1-CT) partakes in oncogenic signaling, resulting in accelerated cancer progression. Our aim was to understand the regulation of Cox-2 expression by MUC1. Levels of COX-2 and MUC1 were determined in MUC1(-/-), MUC1(low), and MUC1(high) PDA cells and tumors using reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry. Proliferative and invasive potential was assessed using MTT and Boyden chamber assays. Chromatin immunoprecipitation was performed to evaluate binding of MUC1-CT to the promoter of COX-2 gene. Significantly higher levels of COX-2 mRNA and protein were detected in MUC1(high) versus MUC1(low/null) cells, which were recapitulated in vivo. In addition, deletion of MUC1 gene and transient knockdown of MUC1 led to decreased COX-2 level. Also, MUC1-CT associated with the COX-2 promoter at ∼1000 base pairs upstream of the transcription start site, the same gene locus where nuclear factor κB p65 associates with the COX-2 promoter. Data supports a novel regulation of COX-2 gene by MUC1 in PDA, the intervention of which may lead to a better therapeutic targeting in PDA patients.

COX-2 gene promoter DNA methylation status in eutopic and ectopic endometrium of Egyptian women with endometriosis

The pathophysiology of COX-2 expression in endometriosis is a matter of debate. The aim was to investigate the role of DNA methylation of the NF-IL6 site within the promoter of COX-2 gene in the pathogenesis of endometriosis. The endometrial tissues (ectopic and eutopic) were collected from 60 women with endometriosis and 30 women without endometriosis (control group). The methylation status of COX-2 was examined by methylation-specific PCR. Quantitative real-time PCR (RT-PCR) was performed to measure COX-2 mRNA levels in endometrial tissues. We found significantly higher levels of COX-2 in ectopic endometriotic tissue compared with eutopic tissue. Also, we found that the frequencies of methylation status of the NF-IL6 site within the COX-2 promoter in the eutopic and ectopic endometrial tissues of endometriosis groups were significantly decreased in comparison to controls (P=0.002, P=0.000 respectively). Our study demonstrated that DNA hypomethylation of the NF-IL6 site within the promoter of COX-2 gene could be a key mechanism for its elevated expression in the eutopic and ectopic tissues of endometriosis.

Inhibition of PKC-Induced COX-2 and IL-8 Expression in Human Breast Cancer Cells by Glucosamine.

Breast cancer is a common cancer leading to many deaths among females. Cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) are two highly expressed inflammatory mediators to be induced by the protein kinase C (PKC) signaling via various inflammatory stimuli and both contribute significantly to cancer metastasis/progression. Glucosamine has been shown to act as an anti-inflammation molecule. The aim of this study was to clarify the role and acting mechanism of glucosamine during the PKC-regulation of COX-2/IL-8 expression and the associated impact on breast cancer. In MCF-7 breast cancer cells, glucosamine effectively suppresses the PKC induction of COX-2 and IL-8 promoter activity, mRNA and protein levels, as well as the production of prostaglandin E(2) (PGE(2)) and IL-8. Glucosamine is able to promote COX-2 protein degradation in a calpain-dependent manner and IL-8 protein degradation in calpain-dependent and proteasome-dependent manners. The MAPK and NF-κB pathways are involved in PKC-induced COX-2 expression, but only the NF-κB pathway is involved in PKC-induced IL-8 expression. Glucosamine attenuates PKC-mediated IκBα phosphorylation, nuclear NF-κB translocation, and NF-κB reporter activation. Both PGE(2) and IL-8 promote cell proliferation and IL-8 induces cell migration; thus, glucosamine appears to suppress PKC-induced cell proliferation and migration. Furthermore, glucosamine significantly inhibits the growth of breast cancer xenografts and this is accompanied by a reduction in COX-2 and IL-8 expression. In conclusion, glucosamine seems to attenuate the inflammatory response in vitro and in vivo and this occurs, at least in part by targeting to the NF-κB signaling pathway, resulting in an inhibition of breast cancer cell growth.

Histone acetyltransferase p300 is induced by p38MAPK after photodynamic therapy: the therapeutic response is increased by the p300HAT inhibitor anacardic acid

Oxidative stress mediated by photodynamic therapy (PDT) mediates the tumoricidal effect, but has also been shown to induce the expression of prosurvival molecules, such as cyclooxygenase-2 (COX-2), which is involved in tumor recurrences after PDT. However, the molecular mechanism is still not fully understood. In this study, we found that activated p38MAPK could significantly up-regulate the activity and expression of histone acetyltransferase p300 (p300HAT) in A375 and C26 cells treated with ALA-and chlorin e6 (Ce6)-mediated photodynamic treatment. A colony-formation assay showed that PDT-induced cytotoxicity was dramatically elevated in the presence of the p300HAT inhibitor anacardic acid (AA). Further studies showed that increased p300HAT acetylates histone H3 and NF-κB p65 subunit to up-regulate the COX-2 expression, which was reduced by AA or p300HAT shRNA. Using chromatin immunoprecipitation analysis, we found that the augmented acetylation of histone H3 and NF-κB increases their binding to the COX-2 promoter region. These in vitro findings were further verified in mice bearing murine C26 and human A375 tumors treated with liposomal Ce6 mediated PDT. Meanwhile, the combination of PDT and AA resulted in greater tumor regression in BALB/c mice bearing C26 tumors, compared with PDT only or combined with COX-2 inhibitor. Finally, we demonstrated that suppression of the PDT-induced p300HAT activity also resulted in the decreased expression of survivin, restoring caspase-3 activity and sensitizing PDT-treated cells from autophagy to apoptosis due to the Becline-1 cleavage. This study demonstrates for the first time the molecular mechanisms involved in histone modification induced by PDT-mediated oxidative stress, suggesting that HAT inhibitors may provide a novel therapeutic approach for improving PDT response.

436. Oncolytic Adenoviruses Targeted to Esophageal Adenocarcinoma

In the United States and other Western countries, the incidence of esophageal adenocarcinoma (EAC) has increased in recent years. Given the poor overall survival of these patients, the need for novel therapeutics has never been greater. Our research group has designed conditionally replicative oncolytic adenoviruses (CRAd) which use the cyclooxygenase-2 (Cox2) promoter to control the tissue specificity of viral replication. EAC, along with many other human gastrointestinal malignancies, has a high expression of Cox2. We hypothesize that our vectors will selectively replicate within EAC cells, thereby minimizing potential deleterious effects on normal tissues.A chimeric 5/3 fiber was employed to maximize infectivity. Viral replication was under control of the Cox2 promoter. In addition, the E3 region was modified to allow for expression of either a luciferase or interferon alpha (IFN) transgene. The vectors were thoroughly tested in vitro using both crystal violet and MTS assays to assess cell viability following viral infection in multiple EAC cell lines. Furthermore, the in vitro cytocidal effect was also characterized when the Cox2-IFN vector was combined with cisplatin and radiation. Additionally, a nude mouse model was used to test the in vivo antitumor effect. Finally, the vectors were tested ex vivo using tissue samples derived from resected human EAC specimens. A Krumdiek tissue slicer was used to prepare the samples, which were then infected with virus and subsequently analyzed.Systemic IFN has demonstrated some success in the treatment of EAC, but its use is limited by systemic side effects. Our vectors provide a way of delivering IFN locally to the tumor. Here, the Cox2-IFN virus demonstrated a strong cytocidal effect at early time points in all tested cell lines. In fact, it caused significantly more cell death than an otherwise identical vector expressing luciferase (p<0.05), thereby demonstrating the profound effect of IFN expression. Additionally, there was a dose-dependent increase in cytocidal effect when the Cox2-IFN virus was combined with cisplatin and radiation. In vivo, that same tumor-specific IFN-expressing vector demonstrated a profound anti-tumor effect when compared to the groups treated with a wild type virus and a saline control. Replication dependent reporter expression (luciferase) was measured to assess the viral replication in the patient-derived tissue slices. Both the Cox2-controlled vector and the vector lacking a promoter showed strong replication in the EAC tissue slices as expected. Importantly, in the normal esophagus samples, there was significantly less replication in the Cox2-controlled group (p<0.05). Viral copy number (using the E4 primer) was also used to quantify viral replication. Here, a similar trend was observed as there was virtually no replication of the Cox2 virus within the normal tissue samples.We have demonstrated that a CRAd driven by the Cox2 promoter has a strong oncolytic effect both in vivo and in vitro. Moreover, our Cox2-controlled vector has demonstrated cancer-specific replication. We believe that our novel vector possesses both therapeutic efficacy and specificity, which makes it an ideal candidate for clinical translation.

435. Effects of Chemotherapy and Radiation on Replication of Interferon-Expressing Adenovirus in Pancreatic Cancer Cells

Pancreatic Cancer is a disease for which there are very limited efficacious treatments, resulting in a 5-year survival rate for affected patients around only 6%. Our group has developed a recombinant oncolytic adenovirus (OAd-IFN) aimed for use as an additional treatment for this disease. Manipulations in the viral genome include enhanced oncolysis with an overexpression of the adenoviral death protein, enhanced infectivity with a genetically modified fiber that has replaced the serotype 3 Ad fiber with that of a serotype 5 Ad, conditional replication targeted to pancreatic cancer cells by linking replication with the Cox-2 promoter, and an expression of the gene for human interferon α within the E3 region to enhance the therapeutic effects of chemotherapy and radiation through the antitumor effects of the cytokine.Our previous work analyzing the therapeutic abilities of IFN expressing vectors in human pancreatic cancer xenografts in immonudeficient nude mice and in syngeneic hamster pancreatic cancer nodules in immunocompetent Syrian hamsters, showed a more evident antitumor effect of OAd-IFN in the latter models. This suggests the success of OAd-IFN as an immune system stimulator, making it a promising candidate for combination therapy approaches. In vitro studies in hamster pancreatic cancer cell lines showed significantly improved killing effects when OAd-IFN was added to conventional chemotherapy and radiation treatments, as well as greater killing ability with triple therapies using OAd-IFN versus an OAd without IFN. Moreover, killing effects became more pronounced over time, showing that the oncolytic impact of the IFN depends on replication of the virus to express the IFN gene.The goal of this study is to investigate the effects of chemotherapy and radiation on the replication of our OAd-IFN, since understanding these interactions is necessary to evaluate the possibility of using this virus as part of a combined therapeutic regimen. Specifically, the adenoviral replicative ability was measured within pancreatic cancer Mia-Paca 2 cells under various conditions of 5FU and radiation exposure. Replication was analyzed by quantifying viral DNA extracted from treated cells at two time points post infection. In preliminary trials, cells were infected with the virus either two days prior to receiving 5FU and radiation or one day post 5FU treatment. Based on the initial data collected, radiation seemed to have no effect on viral replication. 5FU treatment prior to viral infection resulted in an increase in replication at low dose, and a decrease in replication at high dose, however, this effect was only seen at the first time point. Similarly, 5FU treatment post infection showed minor inhibition of viral replication at high doses, with little to no effect observed in the low dose. Further trials including a simultaneous treatment with 5FU and virus are being done to better explore these observed effects, and future trials will be conducted exploring for similar patterns using Gemcitabine and Cisplatin in place of 5FU.

Suppressor of cytokine signaling 1 is a positive regulator of TGF-β-induced prostaglandin production in human follicular dendritic cell-like cells.

PGs are emerging as important immune modulators. Since our report on the expression of PG synthases in human follicular dendritic cells, we investigated the potential immunoregulatory function of PGs and their production mechanisms. In this study, we explored the intracellular signaling molecules mediating TGF-β-induced cyclooxygenase (COX)-2 augmentation in follicular dendritic cell-like cells. TGF-β triggered phosphorylation of Smad3 and ERK, which were essential for the increase in COX-2 protein. Interestingly, depletion of suppressor of cytokine signaling 1 (SOCS1) resulted in an almost complete inhibition of Smad3 phosphorylation and COX-2 induction. Nuclear translocation of Smad3 was inhibited in SOCS1-depleted cells. SOCS1 knockdown also downregulated TGF-β-stimulated Snail expression and its binding to the Cox-2 promoter. In contrast, overexpression of SOCS1 gave rise to a significant increase in Snail and COX-2 proteins. SOCS1 was reported to be a negative regulator of cytokine signaling by various investigators. However, our current data suggest that SOCS1 promotes TGF-β-induced COX-2 expression and PG production by facilitating Smad3 phosphorylation and Snail binding to the Cox-2 promoter. The complete understanding of the biological function of SOCS1 might be obtained via extensive studies with diverse cell types.

A reporter mouse for optical imaging of inflammation in mdx muscles.

BackgroundDuchenne muscular dystrophy (DMD) is due to mutations in the gene coding for human DMD; DMD is characterized by progressive muscle degeneration, inflammation, fat accumulation, and fibrosis. The mdx mouse model of DMD lacks dystrophin protein and undergoes a predictable disease course. While this model has been a valuable resource for pre-clinical studies aiming to test therapeutic compounds, its utility is compromised by a lack of reliable biochemical tools to quantifiably assay muscle disease. Additionally, there are few non-invasive assays available to researchers for measuring early indicators of disease progression in mdx mice.MethodsMdx mice were crossed to knock-in mice expressing luciferase from the Cox2 promoter. These reporter mice (Cox2FLuc/+DMD−/−) were created to serve as a tool for researchers to evaluate muscle inflammation. Luciferase expression was assayed by immunohistochemistry to insure that it correlated with muscle lesions. The luciferase signal was quantified by optical imaging and luciferase assays to verify that the signal correlated with muscle damage. As proof of principle, Cox2FLuc/+DMD−/− mice were also treated with prednisolone to validate that a reduction in luciferase signal correlated with prednisone treatment.ResultsIn this investigation, a novel reporter mouse (Cox2FLuc/+DMD−/− mice) was created and validated for non-invasive quantification of muscle inflammation in vivo. In this dystrophic mouse, luciferase is expressed from cyclooxygenase 2 (Cox2) expressing cells and bioluminescence is detected by optical imaging. Bioluminescence is significantly enhanced in damaged muscle of exercised Cox2FLuc/+DMD−/− mice compared to non-exercised Cox2FLuc/+DMD+/+ mice. Moreover, the Cox2 bioluminescent signal is reduced in Cox2FLuc/+DMD−/− mice in response to a course of steroid treatment. Reduction in bioluminescence is detectable prior to measurable therapy-elicited improvements in muscle strength, as assessed by traditional means. Biochemical assay of luciferase provides a second means to quantify muscle inflammation.ConclusionsThe Cox2FLuc/+DMD−/− mouse is a novel tool to evaluate the therapeutic benefits of drugs intended to target inflammatory aspects of dystrophic pathology. This mouse model will be a useful adjunct to traditional outcome measures in assessing potential therapeutic compounds.

C-Src/Pyk2/EGFR/PI3K/Akt/CREB-activated pathway contributes to human cardiomyocyte hypertrophy: Role of COX-2 induction

Thrombin and COX-2 regulating cardiac hypertrophy are via various signaling cascades. Several transcriptional factors including CREB involve in COX-2 expression. However, the interplay among thrombin, CREB, and COX-2 in primary human neonatal ventricular cardiomyocytes remains unclear. In this study, thrombin-induced COX-2 promoter activity, mRNA and protein expression, and PGE2 synthesis were attenuated by pretreatment with the inhibitors of c-Src (PP1), Pyk2 (PF431396), EGFR (AG1478), PI3K/Akt (LY294002/SH-5), and p300 (GR343), or transfection with siRNAs of c-Src, Pyk2, EGFR, p110, Akt, CREB, and p300. Moreover, thrombin-stimulated phosphorylation of c-Src, Pyk2, EGFR, Akt, CREB and p300 was attenuated by their respective inhibitors. These results indicate that thrombin-induced COX-2 expression is mediated through PAR-1/c-Src/Pyk2/EGFR/PI3K/Akt linking to CREB and p300 cascades. Functionally, thrombin-induced hypertrophy and ANF/BNP release were, at least in part, mediated through a PAR-1/COX-2-dependent pathway. We uncover the importance of COX-2 regarding human cardiomyocyte hypertrophy that will provide a therapeutic intervention in cardiovascular diseases.

Human Cardiac Hypertrophy Induced by Thrombin/COX‐2 is Mediated Through a c‐Src/Pyk2/EGFR/CREB‐dependent Pathway

Thrombin and cyclooxygenase-2 (COX-2) are elevated, and regulate several inflammatory situations via modulating complex signaling cascades, which participates in many cardiovascular diseases. It has been presented that thrombin induces COX-2 expression through a PAR (protease- activated receptor)-1-dependent manner. The aim of this study was to identify whether thrombin-induced COX-2 expression and prostaglandin E2 (PGE2) release was mediated via a c-Src-dependent transactivation of epidermal growth factor receptor (EGFR) pathway in primary human cardiomyocytes. First, the data showed that thrombin-induced COX-2 promoter activity, mRNA and protein expression, and PGE2 synthesis was prohibited by pretreatment with the pharmacological inhibitors of c-Src, Pyk2, EGFR, PI3K/Akt, and p300, or transfection with siRNAs of c-Src, Pyk2, EGFR, p110, Akt, CREB, and p300. Next, thrombin stimulated activation of phosphorylation of c-Src, Pyk2, EGFR, Akt, MAPKs, and then in turn activated CREB and p300, all of which were repressed by their respective inhibitors. Thrombin-induced COX-2 expression and PGE2 generation is mediated through PAR-1/c-Src/Pyk2-mediated EGFR/PI3K/Akt/MAPKs linking to CREB and p300 cascades. Functionally, thrombin magnified cell hypertrophy and atrial natriuretic factor (ANF) release via these signaling components. Understanding the mechanisms of COX-2 expression by thrombin/PAR-1 system provides a rational therapeutic intervention regarding cardiovascular diseases.

Cyclooxygenase-2 and microRNA-155 expression are elevated in asthmatic airway smooth muscle cells.

Cyclooxygenase-2 (COX-2) expression and PGE2 secretion from human airway smooth muscle cells (hASMCs) may contribute to β2-adrenoceptor hyporesponsiveness, a clinical feature observed in some patients with asthma. hASMCs from patients with asthma exhibit elevated expression of cytokine-responsive genes, and in some instances this is attributable to an altered histone code and/or microRNA expression. We hypothesized that COX-2 expression and PGE2 secretion might be elevated in asthmatic hASMCs in response to proinflammatory signals in part due to altered histone acetylation and/or microRNA expression. hASMCs obtained from nonasthmatic and asthmatic human subjects were treated with cytomix (IL-1β, TNF-α, and IFN-γ). A greater elevation of COX-2 mRNA, COX-2 protein, and PGE2 secretion was observed in the asthmatic cells. We investigated histone H3/H4-acetylation, transcription factor binding, mRNA stability, p38 mitogen-activated protein kinase signaling, and microRNA (miR)-155 expression as potential mechanisms responsible for the differential elevation of COX-2 expression. We found that histone H3/H4-acetylation and transcription factor binding to the COX-2 promoter were similar in both groups, and histone H3/H4-acetylation did not increase after cytomix treatment. Cytomix treatment elevated NF-κB and RNA polymerase II binding to similar levels in both groups. COX-2 mRNA stability was increased in asthmatic cells. MiR-155 expression was higher in cytomix-treated asthmatic cells, and we show it enhances COX-2 expression and PGE2 secretion in asthmatic and nonasthmatic hASMCs. Thus, miR-155 expression positively correlates with COX-2 expression in the asthmatic hASMCs and may contribute to the elevated expression observed in these cells. These findings may explain, at least in part, β2-adrenoceptor hyporesponsiveness in patients with asthma.