Celecoxib é um antiinflamatório não esteroidal (AINE), inibidor seletivo da ciclooxigenase-2 (COX-2) usado em pesquisas recentes como agente anticarcinogênico. Os seus efeitos anti-neoplásicos dependem por um lado da sua capacidade de inibir a COX-2, mas por outro lado também age por mecanismos que independem da COX-2, em resumo o seu mecanismo de ação ainda não é completamente conhecido. O objetivo desta tese foi estudar os efeitos do celecoxib sobre as taxas de apoptose e os índices de proliferação celular de quatro linhagens celulares, Hn-6, Hn-19, Hn-30, Hn-31, de CECP e uma linhagem de queratinócitos mutada (HaCat), além de verificar se há correlação entre a expressão das proteínas COX-2, pAkt, ß-catenina, CD1 e NFKB e a inibição da proliferação celular. As células foram divididas em dois grupos: a, grupo controle; b, células cultivadas tratadas com celecoxib. A análise da expressão das proteínas pAkt, NFKB, ß-catenina, COX-2 e CD1 foi feita através da técnica de Western-blot. A indução de apoptose foi estudada com o Kit de Anexina. A proliferação celular foi monitorada através de curva de crescimento, com contagem celular na câmara de Neubauer e com o teste de viabilidade celular (Kit Cell Titer96) e a localização intracelular das proteínas foi avaliada por imunofluorescência. Os resultados mostraram significante aumento no índice celular de apoptose e diminuição da proliferação celular. Após o tratamento com celecoxib, a imunofluorescência mostrou que a proteína CD1 teve diminuição da expressão nuclear, a ß-catenina exibiu discreto aumento citoplasmático, o pAkt também passou a ser expresso no citoplasma da Hn6, enquanto as outras proteínas estudadas mantiveram o mesmo padrão de localização na célula. O western blot complementou os resultados da imunofluorescência indicando uma diminuição nos níveis de CD1. / Celecoxib, a cyclooxygenase-2 (COX-2) selective nonsteroidal anti-inflammatory drug, is a new anticarcinogenic agent. Its antitumor effects depend on the one hand on its COX-2-inhibiting potency, but on the other hand on COX-2-independent mechanisms, which until now have not been fully understood. The aim of this research was to study the effects of celecoxib in growth inhibition and apoptosis induction in four Head and Neck Squamous Cell Carcinoma (HNSCC) cell lines, HN6, HN19, HN30, HN31 and HaCat an immortalized keratinocyte cell line, and verify if there is a correlation between the growth inhibition and the expression of COX-2, pAkt, ß-catenin, CD1 and NFKB. The Western Blot was used to analyze the COX-2, pAkt, ß-catenin, CD1 and NFKB protein expression level. Apoptosis induction was studied with the Annexin V kit. The cell lines proliferation will be measured through a growth curve with the Neubauer chamber and MTS method (KitCell Titer96), the proteins intracellular site was assed by immunofluorescence technic. The same cell lines without any treatment were used as controls. Results showed a significant increase in apoptotic cells index, and growth inhibition in cell lines treated with celecoxib. The proteins localization was determined through immunofluorescence. In control group the CD1 was located mostly in nucleus, after treatment CD1 nuclear localization was reduced, it could also be noticed an increase in cytoplasmic expression of ß-catenin in all cell lines while pAkt cytoplasmic increase was present exclusevely in Hn6, the other proteins maintained their cellular localization,. The Western Blot results showed considerable reduction in CD1 levels with exception of Hn19 cell line.

Ziel dieser Studie war es, die Häufigkeit der Expression von EGFR, HER-2 sowie COX-2 im Zervixkarzinom zu eruieren. Dabei galt es herauszufinden, ob Unterschiede hinsichtlich des Nachweises dieser drei, möglicherweise therapeutisch relevanten Moleküle zwischen den primären, nicht vortherapierten und operierten Karzinomen und den multimodal vorbehandelten Rezidiven gab. In der vorliegenden retrospektiven Arbeit wurden 45 TMMR-operierte Primärtumoren und 28 LEER-operierte Rezidivtumoren der Universitätsfrauenklinik Leipzig (Triersches Institut) einbezogen und zusätzlich hinsichtlich der prognostischen Überlebensanalyse durch das Tumorstadium, Lymphknotenmetastasen und Rezidivauftreten sowie histologischer Charakteristika untersucht. Dazu wurden Tissue - Microarrays angefertigt mit anschließender immunhistochemischer Untersuchung dieser. Die Ergebnisse zeigten, dass die TMMR-Operation die Überlebensprognose signifikant verbessert, denn lediglich bei den LEER-therapierten Rezidivtumoren erlitten die Patientinnen sowohl Fernmetastasen als auch erneute Rezidive. Weder die Expression der drei untersuchten Moleküle noch die histopathologischen Parameter haben eine prognostische Relevanz. Es gibt keine signifikanten Zusammenhänge zwischen der Häufigkeit der Expression von EGFR, HER-2 sowie COX-2 und Primär-, bzw. Rezidivtumoren, sodass diese Moleküle keine Targets für eine individualisierte, zielgerichtete Therapie beim Zervixkarzinom darstellen.

L’inflammation est un phénomène affectant des millions de personnes à travers le monde. Il existe une grande variété de médiateurs inflammatoires impliqués dans différentes fonctions biologiques, dont la cyclooxygénase-2. Bien que de nombreux inhibiteurs sélectifs de la COX-2 aient été développés et commercialisés, ceux-ci présentent des effets secondaires dont la gravité a entraîné, dans certains cas, l'arrêt de leur commercialisation.De nos jours, les méthodes in silico sont de plus en plus employées dans les stratégies de découverte de nouvelles molécules à visée thérapeutique. Au cours de ce projet, nous nous sommes appuyés sur les modèles pharmacophoriques et les méthodes de docking afin de guider et de prioriser la synthèse de molécules de structures diverses et originales, susceptibles de présenter les meilleures affinités pour la cible étudiée. Ainsi, des prédictions réalisées avec le logiciel TOMOCOMD-CARDD combinées à des tests biologiques, ont permis d’identifier le cyclocoumarol comme une molécule potentiellement anti-inflammatoire. Dans le cadre de ces travaux nous nous sommes intéressés à la synthèse et l’étude d’analogues du cyclocoumarol en tant qu’inhibiteurs sélectifs de la COX-2. La pharmacomodulation autour du cyclocoumarol et la mise en place de stratégies de synthèse judicieuses ont permis d’obtenir une série d’analogues. Divers outils bioinformatiques ont été utilisés : le logiciel LigandScout a permis de construire des pharmacophores sélectifs de la COX-2 et les études de docking ont permis de comprendre les modes de liaisons des différents composés. Enfin, le logiciel SeeSAR, a permis de prédire l’affinité des molécules les plus susceptibles d’inhiber sélectivement la COX-2. Les tests biologiques ont confirmé leur activité inhibitrice envers la COX-2 avec une inhibition non significative vis-à-vis de COX-1. Parmi les molécules synthétisées, le 4-OMe cyclocoumarol a démontré une activité et une sélectivité très intéressantes, comparables au NS-398, un inhibiteur sélectif connu de la COX-2. A partir des résultats biologiques obtenus, un travail de phamacomodulation autour de ces dérivés du cyclocoumarol a été réalisé en utilisant des outils in silico dans le but de prédire l’affinité de nouveaux composés et de découvrir de nouveaux inhibiteurs sélectifs de la COX-2.Mots clés : cyclocoumarol, benzalacétones, warfarines, pharmacophores, docking, criblage virtuel, COX-2, repositionnement / Inflammation is a phenomenon affecting millions of people throughout the world. There is a broad range of inflammatory mediators implied in different biological functions including the cyclooxygenase-2. Although many selective inhibitors selective of COX-2 have been developed and marketed, they have displayed diverse side effects leading, in some cases, to their with drawal from the market. Nowadays, in silico methods are more and more used in the drug discovery process. In this project, we have used pharmacophoric models and docking methods to guide and prioritize the synthesis of molecules, presenting different and original structures, with enhanced affinity for the biological target. Thus, predictions realized with the TOMOCOMD-CARDD software together with biological tests enable to identify the cyclocoumarol as a potential anti-inflammatory molecule. As part of these works, the synthesis of and the study of cyclocoumarol analogues as selective inhibitors of COX-2 have been realized. Pharmacomodulation of cyclocoumarol and development of synthesis strategies led to a serie of cyclocoumarol analogues. Several bioinformatics tools have been used: selective COX-2 pharmacophores were elucidated using LigandScout and docking studies (Surflex) were conducted to understand the binding mode of different compounds. Finally, SeeSAR enabled to predict the affinity of the molecules the most susceptible to inhibit selectively COX-2. Biological tests confirmed their inhibitory activity against COX-2 and showed no significant inhibition for COX-1. Among the synthesized molecules, the 4-OMe cyclocoumarol has demonstrated an activity and a selectivity very interesting, similar to NS-398, a known selective COX-2 inhibitor.Based on the biological results obtained, a pharmacomodulation study of cyclocoumarol derivatives has been realized using in silico tools in order to predict the affinity of new compounds and to discover new selective inhibitors of COX-2.Keywords : cyclocoumarol, benzalacetones, warfarines, pharmacophores, docking, virtual screening, COX-2, repositioning

Herpes Zoster é uma doença causada pela reativação do vírus Varicela Zoster nos gânglios sensoriais, caracterizada pelo desenvolvimento de lesões na pele e dor. Não há modelos animais disponíveis para estudo da patofisiologia da doença. No entanto, um modelo murino que utiliza o HSV-1 tem sido usado para tal fim, visto que os animais desenvolvem lesões zosteriformes e desenvolvem hipersensibilidade na pata infectada. Não há dados na literatura acerca da resposta imune que se desenvolve nos gânglios da raiz dorsal destes animais. Logo, o objetivo deste trabalho foi o de avaliar células e mediadores inflamatórios presentes nos gânglios da raiz dorsal e sua relação com a hiperalgesia durante a infecção cutânea por HSV-1. Durante a fase aguda da infecção, os camundongos desenvolveram hiperalgesia nas patas ipsilaterais a partir do 3 dia pós-infecção, que perdurou até o 7 dia pós-infecção. A maior carga viral foi detectada nos gânglios L4, L5 e L6, os quais compõem o nervo ciático, que inerva a área infectada. O tratamento dos animais infectados com dexametasona ou fucoidina resultou na redução do comportamento de hiperalgesia, a partir do 5 dia pós-infecção, que corresponde ao período em que a migração de leucócitos passa a aumentar nos gânglios da raiz dorsal. Macrófagos, neutrófilos e linfócitos T CD4 foram detectados nos gânglios durante a infecção aguda. No entanto, linfócitos T CD8 estavam ausentes. A expressão do mRNA de TNF- e COX-2 estava aumentada nos gânglios, e o tratamento de animais infectados com drogas inibidoras de ambos resultou na redução da hiperalgesia. Os receptores do tipo Toll-like e da IL-1 não participam da geração da hipersensibilidade herpética. Após 50 dias da infecção, constatou-se que alguns animais apresentavam comportamento de hiperalgesia irreversível, semelhante à neuralgia pós-herpética humana (NPH). Não houve diferença significativa na incidência da NPH em animais de linhagens ou sexos diferentes. Ainda, o tratamento com drogas anticonvulsivantes e antidepressivas, mas não com morfina e anti-inflamatórios, resultou na redução transiente da hiperalgesia. Neste período, não há participação da inflamação na manutenção da hiperalgesia. A expressão de TNF- e COX-2 retorna aos níveis basais, e não são mais detectados neutrófilos e macrófagos. No entanto, a migração de linfócitos T CD4+ e CD8+ aos gânglios aumenta de maneira tempo-dependente. Durante a NPH, detectou-se uma intensa ativação das células satélites gliais, que contribuem para a manutenção da hiperalgesia pós-herpética. Nossos resultados demonstram que a manutenção hiperalgesia herpética é resultado da intensa resposta inflamatória que ocorre nos gânglios da raiz dorsal infectados, com aumento da produção de TNF- e COX-2, importantes mediadores para a hipersensibilidade. No entanto, durante a neuralgia pós-herpética, não há participação de células ou mediadores inflamatórios, mas de células da glia, as quais são importantes na manutenção da hiperalgesia. / Herpes Zoster is a disease caused by reactivation of varicella zoster virus in sensory ganglia, characterized by dermal rash and pain. There are no animal models available to study the pathophysiology of the disease. A murine model of HSV-1 infection on the hind paw skin has been used to study HZ, since mice develop HZ-like skin lesions and pain-related responses. There are no data available about the immune response in dorsal root ganglion (DRG) of these mice. Thus, the aim of this study was to evaluate cells and inflammatory mediators present in DRGs and its relationship with hiperalgesia during HSV-1 cutaneous infection. During the acute phase of infection, mice developed hyperalgesia in ipsilateral paws from 3 days post-infection, which persisted until 7 days post-infection. The highest viral load was detected in ganglia L4, L5 and L6. Treatment of infected mice with fucoidin or dexamethasone resulted in the reduction of hyperalgesic behavior, from the 5th post-infection day, which corresponds to the period in which leukocyte migration increase in the dorsal root ganglia. Macrophages, neutrophils and CD4 + T lymphocytes were detected in the ganglia during acute infection. However, CD8 + T lymphocytes were absent. The mRNA expression of TNF- and COX-2 was increased in dorsal root ganglia, and the treatment of infected mice with drugs that inhibits both mediators resulted in reduced hyperalgesia. The Toll-like receptors and IL-1 does not participate in the generation of herpetic hypersensitivity. After 50 days of infection, it was found that some animals presented irreversible hyperalgesic behavior, like human post-herpetic neuralgia (PHN). There was no significant difference in the incidence of PHN in animals of different genders or strains. Furthermore, treatment with anticonvulsant and antidepressant drugs, but not morphine and anti-inflammatory, resulted in transient reduction of hyperalgesia. In this period, there is no participation of inflammation in the hyperalgesia maintenance of. The expression of TNF- and COX-2 returns to baseline levels, and neutrophils and macrophages are no longer detected. However, the migration of CD4 + and CD8 + to ganglia increases in a time-dependent manner. During NPH, an intense activation of glial cells satellites was detected, that contributes to the maintenance of post-herpetic hyperalgesia. Our results demonstrate that herpetic hyperalgesia maintenance is a result of an intense inflammatory response that occurs in the infected dorsal root ganglia, with increased production of TNF- and COX-2. However, during post-herpetic neuralgia, there is involvement of glial cells, which are important in hyperalgesia maintenance.

A transformação de Aspergillus nidulans, com RNA de macrófagos de ratos, resultou na linhagem RT2, produtora de uma glicoproteína antiinflamatória. Nosso objetivo foi avaliar esta linhagem genenomicamente e caracterizar esta glicoproteína quanto à natureza bioquímica e sua atividade. Para tal, foi realizado RAPD e análise fenotípica desta linhagem. A Nandina foi purificada e submetida à espectrometria de massa para sequenciamento e identificação dos carboidratos. Testes da atividade antiinflamatória in vivo foram realizados em peritonite e edema de pata e inibição dos receptores de glicocorticóides. Os testes in vitro, sobre a produção das COXs e de PGE2, foram realizados em cultura de macrófagos. Os resultados mostraram que a linhagem RT2 é resultante da UT448, mas contém diferenças em seu genoma. A proteína purificada possui 40KDa. A espectrometria de massa caracterizou dois fragmentos da proteína e sua glicosilação. Os testes in vivo mostraram que a proteína inibe o edema e o influxo leucocitário e que esta atividade não é dependente de glicocorticóides, mas sim da inibição in vitro de COX-2, mas não de COX-1 e nem de PGE2. / Aspergillus nidulans transformation with rat macrophage RNA results on RT2 strain, producer of an antiinflammatory glycoprotein. Our objective was to evaluate this strain genomically and characterize biochemically and activity of its glycoprotein. To this, RAPD and fenotipical analysis were performed. The Nandin was purified and mass spectrometry analyzed to sequencing and carbohydrates analysis. Antiinflammatory activity testes in vivo in peritonitis and edema, and glucocorticoid receptors inhibition were performed. The in vitro testes, over expression and activity of COXs and PGE2, were performed in macrophage culture. The results show that RT2 strain came from UT448, but have genomics differences. The purified glycoprotein has been 40KDa. The mass spectrometry sequenced two protein fragments and showed that glycosylation. The in vivo testes showed that the glycoprotein has antiinflammatory activity inhibiting the edema and leukocyte influx. The RU38486 experiments evidenced that activity is not glucocorticoid receptors dependent, but in vivo inhibition of COX-2, but not COX-1 neither its product PGE2.

No / There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.

Cancer is a stem cell disease and populations of cancer stem cells (CSCs) are evident in many cancer types. CSCs exhibit similarities to normal embryonic and adult stem cells: they are able to self-renew and have the potential to give rise to a diverse array of differentiated progeny. CSCs are responsible for driving tumourigenesis and metastasis, and are inherently resistant to chemotherapy and radiotherapy. This cell population is enriched after treatment and, as a result of their tumourigenic capability, can re-populate tumour growth resulting in patient relapse, often with increased chemotherapeutic resistance. Increasing evidence supports that only by targeting this population of cells will a cure for cancer be possible. Hence, it is essential to identify pathways within CSC populations that can be targeted therapeutically. Cyclooxygenase-2 (COX-2) is an enzyme associated with inflammation and disease, and is upregulated in many cancers types. The COX-2 / prostaglandin E2 (PGE2) signalling pathway is associated with increased tumour growth, metastasis, immune evasion and overall worse patient prognosis. Recent evidence has identified that COX-2 is further upregulated in CSC populations isolated from cancer cell lines. Previously, we have shown that inhibition of COX-2 reduces CSC sphere-forming ability, a characteristic of stem cell self-renewal, suggesting a role for COX-2 in maintaining CSC populations. This work was carried out in both human and canine osteosarcoma cell lines with similar results. Cancer in dogs is a major health concern among an aging pet population. Many cancer types exhibit similarities between these species, suggesting that naturally occurring canine cancer may be a potential model for the human disease. The aim of this PhD project was to investigate the role of COX-2 in CSCs in a comparative cancer study. CSCs that express stem cell markers have been isolated from a panel of canine and human cancer cell lines including, mammary carcinoma and transitional cell carcinoma of the urinary bladder. CSCs over-express COX-2 compared to non-CSCs, therefore to determine the role of COX-2 in CSC biology the selective COX-2 inhibitor mavacoxib, a non-steroidal anti-inflammatory drug currently licenced for treating osteoarthritis in dogs, was utilised. Our results demonstrate that inhibiting COX-2 has a multifaceted impact on CSC biology, including reducing self-renewal capacity, clonogenicity, proliferation, migration, invasion and in vivo tumourigenicity. To confirm that mavacoxib is mediating these CSC-specific effects via inhibition of COX-2 rather than through unknown off-target effects, we generated canine specific-small interfering RNA to specifically reduce gene expression of COX-2. Our results confirm that mavacoxib exerts its anti-tumour effects via inhibition of COX-2. This project has highlighted a plethora of CSC-specific COX-2 effects, and to gain further insight we compared the global gene expression profiles of CSCs compared to non- CSCs isolated from a canine bladder carcinoma cell line. This data revealed that both mavacoxib and COX-2 specific siRNA target similar pathways within the two cell populations, confirming that mavacoxib exerts its effects in a COX-2 dependent manner. Interestingly, mavacoxib reduced the expression of a number of stemness related genes in the CSC population, including NOTCH and Wnt, suggesting that mavacoxib can inhibit CSC related pathways. Our overall results are comparable between canine and human cancer cell lines supporting the concept of naturally occurring tumours in dogs as models for the human disease. In conclusion, COX-2 plays an important role not only in maintaining CSC populations but also in their function, and targeting COX-2 in CSCs may provide therapeutic benefit.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world and Taiwan. The major factors involved in the molecular pathogenesis for the development of HCC had been explored in recent years. An extensive array of growth factors and their receptors had been identified and may act as positive and negative modulators in different stages of hepatocarcinogenesis. Current therapeutic approaches for HCC include surgical resection (include liver transplantation), trans-arterial embolization (TAE), alcohol injection, etc. However, the effect is limited due to most of the HCC patients present with advanced stages of the disease. Therefore, this underscores the need for the development of novel therapeutic strategies. It is pivotal to set up an orthotopic hepatoma model for the development of novel intervention strategies for HCC. Under the guidance of ultrasound, we are able to create hepatoma in the liver lobe of Sprague-Dawley (SD) rats by injection of Novikoff (N1-S1) hepatoma cells. In addition, sonographic technique was employed for the monitoring of tumor growth in this animal model in the following subprojects. The continuous, non-invasive measurement of orthotopic hepatoma development will be a valuable tool for the evaluation of effects of drugs for treatment of HCC. In Chapter 1, the study employed a relatively non-invasive approach to establish an orthotopic HCC model in immune-competent rats. This was done by ultrasound-guided implantation of cancer cells and the model was used to evaluate the therapeutic efficacy of short-term and low-dose epirubicin chemotherapy. Ultrasound-guided implantation of Novikoff hepatoma cells led to the formation of orthotopic HCC in 60.4% of the SD rats. Moreover, tumor sizes measured by ultrasound significantly correlated with those measured by calipers after sacrificing the animals (P < 0.00001). The rate of tumor induction by ultrasound-guided implantation was comparable to that of laparotomy (55/91, 60.4% vs. 39/52, 75%) and no significant difference in sizes of tumor was noted between the two groups. Moreover, there was a significant correlation in tumor size measurement by ultrasound and computerized tomography. In tumor-bearing rats, short-term and low-dose epirubicin chemotherapy caused a significant reduction in tumor growth, and was found to be associated with enhanced apoptosis and attenuated proliferation as well as a decrease in microvessel density in tumors. In chapter 2, we investigated the chemopreventive effects of celecoxib in the growth of orthotopic rat HCC and the possible signal pathways involved. The status of COX-2 expression in rat Novikoff HCC was consistent with that in human HCC. Both Western blot and PCR tests had proved that N1-S1 was a HCC model presenting with low COX-2 enzymes in tumor cells. Then, low doses of celecoxib was shown to effectively inhibited the proliferation and increased the apoptosis of N1-S1 cells in vitro, which were also safe to the normal hepatocytes. Moreover, chemoprevention by celecoxib inhibiting the HCC tumor growth was shown in rat orthotropic HCC model. Tumor incidence was not affected by the celecoxib prevention, but, tumor weight was found significantly suppressed by the drug. Possible mechanisms of chemoprevention by celecoxib seen in the animal model were thought to be related to the anti-angiogenic, anti-proliferative and anti-hCSC characters of the drug. In chapter 3, we tried to test the combined inhibitory effects of low doses of celecoxib and epirubicin on the growth of HCC. Combined low doses of epirubicin and celecoxib was effective in inhibiting the hepatic cancer stem cells, tumor angiogenesis, tumor cell proliferation, as well as promoting cancer apoptosis. These are compatible with the effects of the individual drugs on HCC growth shown in the previous two chapters. In general, combination therapy expressed more effectiveness in tumor suppression and less bone marrow suppression than the individual drugs used alone. Taken together, ultrasound-guided implantation of Novikoff hepatoma cells is an effective means of establishing orthotopic HCC in SD rats, which is suitable and convenient for therapeutic trial of anti-HCC treatment. In the current study, we had proved the efficacies of low doses of two drugs, epirubicin and celecoxib, acting individually, as well as the combined effects of them in treating HCC in this model.

Hepatic fibrosis represents a process of healing and scarring in response to chronic liver injury. Interleukin-10 (IL-10) is a cytokine that downregulates the proinflammatory response and has a modulatory effect on hepatic fibrogenesis. The aim of this study was to investigate whether IL-10 gene therapy possesses anti-hepatic fibrogenesis in mice. Liver fibrosis was induced by long-term thioacetamide administration in mice. Human IL-10 expression plasmid was delivered via electroporation after liver fibrosis established. IL-10 gene therapy reversed hepatic fibrosis and prevented cell apoptosis in a thioacetamide-treated liver. RT-PCR revealed IL-10 gene therapy could reduce liver transforming growth factor-£]1¡]TGF-£]1¡^, tumor necrosis factor-£\¡]TNF-£\¡^, collagen £\1, cell adhesion molecule, and tissue inhibitors of metalloproteinase¡]TIMPs¡^mRNA upregulation. Following gene transfer, the activation of £\-smooth muscle actin¡]£\-SMA¡^and cyclooxygenase-2¡]COX-2¡^were significantly attenuated. In brief, electroporative IL-10 gene therapy might be an effective therapeutic reagent for liver fibrosis with potential future clinical applications.

The purpose of this study was to assess the prognostic significance and clinical correlations of cyclooxgenase-2 expression (COX) in a cohort of patients treated with radiation (RT) for post-mastectomy chest wall relapse (PMCWR). Between 1975 and 1999, 113 patients were treated for isolated PMCWR. All patients were treated with biopsy and/or excision of the CWR followed by RT. Median follow-up was 10 years. All clinical data including demographics, pathology, staging, receptor status, HER-2/neu status, and adjuvant therapy were entered into a computerized database. Paraffin-embedded CWR specimens were retrieved from 42 patients, of which 38 were evaluated, created into a tissue microarray, stained by immunohistochemical methods for COX, and graded 0-3+. A score of 2-3+ was considered positive. Overall survival from original diagnosis for the entire cohort was 44% at 10 years. Survival rate after chest wall recurrence was 28% at 10 years. The distant metastasis-free survival rate after CWR was 40% at 10 years. Local-regional control of disease was achieved in 79% at 10 years after CWR. COX was considered positive in 13 of 38 cases. COX was inversely correlated with ER (p= .045) and PR (p = .028), and positively correlated with HER-2/neu (p =.003). COX was also associated with a shorter time to PMCWR. The distant metastasis-free rate for COX negative patients was 70% at 10 years, compared with 31% at 10 years for COX-2 positive patients (p = 0.029). COX positive had a poorer local-regional progression-free rate of 19% at 10 years, compared with 81% at 10 years for COX negative (p = 0.003). Outcome following RT for PMCWR is relatively poor. Positive COX correlated with other markers of poor outcome including a shorter time to local relapse, negative ER/PR and positive Her-2/neu status. Positive COX correlated with higher distant metastasis and lower local-regional control of disease. If confirmed with larger studies, these data have implications with respect to the concurrent use of COX-2 inhibitors and radiation for PMCWR.