Littérature scientifique sur le sujet « Trefoil factor 1 »

Trefoil factors are small peptides found in several mammalian tissues including gut, respiratory tract and brain. Their physiological function is not well understood. Among them, trefoil factor 3 (intestinal trefoil factor) is known to be cytoprotective in the gut. However, the molecular mechanism and secondary mediators of trefoil factor 3 action are not known. In the present study, we examined whether the cyclooxygenase pathway is involved in trefoil factor 3 action. We showed that trefoil factor 3 significantly induces the production of prostaglandin E(2) and prostaglandin I(2) in IEC-18 cells (an intestinal epithelial cell line) in a dose dependent manner. Western blot and immunohistochemistry revealed that trefoil factor 3 (2.5 microM) up-regulates the expression of cyclooxygenase-2 but not cyclooxygenase-1 in IEC-18 cells. Treating cells with trefoil factor 3 (10 microM) significantly attenuated reactive oxygen species-induced IEC-18 cell injury. This effect is blocked by NS-398 (10 microM), a selective cyclooxygenase-2 inhibitor. Moreover, we demonstrated that exogenously administered carbacyclin (1 microM, a stable analogue of prostaglandin I(2)) and/or prostaglandin E(2) (1 microM) caused a significant reduction of reactive oxygen species-induced cell injury, mimicking the effect of trefoil factor 3. In summary, our results indicate that trefoil factor 3 activates cyclooxygenase-2 in intestinal epithelium to produce prostaglandin I(2) and prostaglandin E(2), which function as survival factors and mediate the cytoprotective action of trefoil factor 3 against oxidant injury.

1. The availability of recombinant epidermal growth factor provides a potentially exciting development for the treatment of gastrointestinal ulceration. However, because of its potent mitogenic activity, there is a need for strategies which reduce the dose required. Intestinal trefoil factor stimulates mucosal healing without increasing proliferation. Studies were undertaken to examine the biological effects of rat intestinal trefoil factor and/or human epidermal growth factor upon gastrointestinal epithelial cell functions pertinent to mucosal protection, using two wounding models. 2. The study of epithelial restitution in vitro demonstrated a marked synergistic effect on the rate of migration of the wound edge when intestinal trefoil factor was used in combination with epidermal growth factor. There was no increased cellular proliferation due to the addition of intestinal trefoil factor to the cells when given alone, or to the stimulatory effect of cells treated with epidermal growth factor. In the rat model of gastric ulceration, the presence of both epidermal growth factor and intestinal trefoil factor protected against the development of indomethacin-induced gastric lesions. 3. We conclude that combination therapy of epidermal growth factor with intestinal trefoil factor could provide a more potent, safer approach to the treatment of human gastrointestinal ulceration.

Trefoil factor 3 (intestinal trefoil factor) is a cytoprotective factor in the gut. Herein we compared the effect of trefoil factor 3 with tumor necrosis factor-α on 1) activation of NF-κB in intestinal epithelial cells; 2) expression of Twist protein (a molecule essential for downregulation of nuclear factor-κB activity in vivo); and 3) production of interleukin-8. We showed that Twist protein is constitutively expressed in intestinal epithelial cells. Tumor necrosis factor-α induced persistent degradation of Twist protein in intestinal epithelial cells via a signaling pathway linked to proteasome, which was associated with prolonged activation of NF-κB. In contrast to tumor necrosis factor, trefoil factor 3 triggered transient activation of NF-κB and prolonged upregulation of Twist protein in intestinal epithelial cells via an ERK kinase-mediated pathway. Unlike tumor necrosis factor-α, transient activation of NF-κB by trefoil factor 3 is not associated with induction of IL-8 in cells. To examine the role of Twist protein in intestinal epithelial cells, we silenced the Twist expression by siRNA. Our data showed that trefoil factor 3 induced interleukin-8 production after silencing Twist in intestinal epithelial cells. Together, these observations indicated that 1) trefoil factor 3 triggers a diverse signal from tumor necrosis factor-α on the activation of NF-κB and its associated molecules in intestinal epithelial cells; and 2) trefoil factor 3-induced Twist protein plays an important role in the modulation of inflammatory cytokine production in intestinal epithelial cells.

Abstract The functional role of autocrine trefoil factor-1 (TFF1) in mammary carcinoma has not been previously elucidated. Herein, we demonstrate that forced expression of TFF1 in mammary carcinoma cells resulted in increased total cell number as a consequence of increased cell proliferation and survival. Forced expression of TFF1 enhanced anchorage-independent growth and promoted scattered cell morphology with increased cell migration and invasion. Moreover, forced expression of TFF1 increased tumor size in xenograft models. Conversely, RNA interference-mediated depletion of TFF1 in mammary carcinoma cells significantly reduced anchorage-independent growth and migration. Furthermore, neutralization of secreted TFF1 protein by polyclonal antibody decreased mammary carcinoma cell viability in vitro and resulted in regression of mammary carcinoma xenografts. We have therefore demonstrated that TFF1 possesses oncogenic functions in mammary carcinoma cells. Functional antagonism of TFF1 can therefore be considered as a novel therapeutic strategy for mammary carcinoma.

11022 Background: Patients with breast cancer frequently develop bone metastases, which are responsible for high morbidity and reduced quality of life. The early identification of patients with a high probability of relapsing in this site could be used to select candidates for tailored therapy with bone-specific drugs such as bisphosphonates or RANK-L inhibitors. We aimed to identify a pattern of tissue markers in primary breast cancer that could predict bone metastatization. Methods: Expression of different markers was retrospectively analyzed in frozen breast cancer tissue samples from 90 patients comprising 30 cases with no evidence of disease (NEDP), 30 with bone metastases (BMP) and 30 with visceral metastases (VMP). Eight transcripts were analyzed by Quantitative Real time PCR: trefoil factor 1(TFF1), bone sialoprotein (IBSP), heparanase (HPSE), secreted protein acidic and rich in cysteine (SPARC), connective tissue growth factoe (CTGF), B2 microglobulin (B2M) and receptor activator of Nf-kB (RANK). Immunohistochemistry of TFF1 was performed on a part of the case series. Results: Marker expression analysis in the 3 different subgroups showed at least twofold higher median values of all markers in NEDP and VMP subgroups than in BMP. In particular, TFF1, B2M and CXCR4 levels showed statistically significant values. Median TFF1 value in BMP patients was 430.64 compared to 115.83 and 32.79 in VMP and NEDP, respectively (p=0.0043). Considering markers as dichotomous variables, TFF1 expression in BMP reached 59% compared to 21% and 23 % in NEDP and VMP, respectively (p=0.0022). Univariate analysis confirmed that TFF1 predicted the relapse and also the site of relapse. Immunohistochemistry data on TFF1 revealed that this protein was expressed only by cancer cells. Furthermore, the accuracy of the marker did not change at RNA or protein level, thus excluding a post transcriptional control of the RNA. Conclusions: In this preliminary study we identified a gene expression pattern in primary breast cancer that can identify patients destined to relapse to the bone. In particular, TFF1 would seem to be a suitable marker for bone metastatization and a possible target for the development of new drugs.

2009 - 2010
The maintenance of gastrointestinal tissue integrity is physiologically essential in the presence of the persistent harassment of microbial flora and injurious agents. Even though the repair of the gastric epithelium may be modulated by several factors, the epithelial continuity also depends on a family of small peptides called trefoil factors (TFFs). The trefoil factors family comprises the gastric peptides pS2/TFF1, the spasmolytic peptide (SP)/TFF2 and the intestinal trefoil factor (ITF)/TFF3; they are characterized by a three looped domain, the “trefoil domain”, stabilized by three disulphide bridges. TFF1 and TFF3 also have a seventh cysteine that allows the formation of omo- and/or hetero-dimers. On the other hand TFF2 presents only a monomeric form, containing two trefoil domains in the same polypeptide chain. TFFs are small protease-resistant proteins that are abundantly produced by mucus-secreting cells of the gastrointestinal tract onto the mucosal surface. TFFs are essential in the protection of the mucosal epithelia against a wide range of biological threats, thus contributing to the mucosal repair. The signaling events that mediate the cellular responses elicited by TFFs are only partially understood. Moreover there are convincing evidence that TFFs do play an important role in tumorigenesis, even though their specific roles in cancer are still unclear. TFF1 expression is strongly induced after mucosal injury and it has been proposed that TFF1 functions as a gastric tumor suppressor gene. Several studies confirm that TFF1 expression is frequently lost in gastric cancer because of deletions, mutations or methylation of the TFF1 gene. Infection by Helicobacter pylori, a class 1 carcinogen according to WHO classification, is thought to promote stomach carcinogenesis through induction of aberrant DNA methylation. Samples from infected patients show lower expression of TFF1. Recent studies have also shown that there is a direct relationship between Helicobacter pylori and the dimeric form of the protein. In fact, it was demonstrated that the core oligosaccharide portion of H. pylori lipopolysaccharide (RF-LPS) is able to bind to TFF1. It also seems that the loss of TFF1 is an important event in shaping the NF-kB-mediated inflammatory response during the progression to gastric tumorigenesis, being TFF1 a negative regulator of NF-kB signalling. It is thus emerging a clear correlation between loss of TFF1, the development of inflammatory disease and the neoplastic process. Recent analyses made by our research group allowed us to point out the up-regulation of TFF1 gene expression in rats fed on copper deficient diets, and allowed us to find out the unexpected ability of TFF1 to bind copper ions. The presence of a cysteine surrounded by several negatively charged residues in the carboxy-terminus of the protein suggested the presence of a copper-binding site. Afterwards, it was shown that Cys58 and at least three Glu surrounding residues are essential to efficiently bind copper. Moreover, the incubation of the native peptide with copper salts increases the fraction of peptide omodimers produced by inter-molecular oxidation of Cys58 and disulphide bond formation. The Ph.D. research project was aimed at characterising the structure-function relationship of the TFF1-Cu complex. Briefly, we studied the influence of copper on known TFF1 biological activities and on its gene regulation, then we investigated its involvement in the TFF1 mediated mechanisms of Helicobacter pylori virulence and infection. A preliminary Real Time PCR quantitative analysis showed that copper deficiency positively modulates tff1 expression in an adenocarcinoma cell line (AGS), thus confirming our previous data obtained in vivo in copper deficient rat intestine. In order to map possible copper responsive elements in the proximal promoter sequence, we analysed the expression of a reporter gene (Luciferase) driven by deletion constructs of the tff1 gene promoter. AGS cells transfected with the deletion constructs allowed us to identify the upstream 5’ gene sequence -583/-435 as a promoter region sensitive to the changes of copper concentration. In fact, copper chelation treatments with bathocuproine disulfonate (BCS) were able to stimulate an increase of the promoter activity of the corresponding deletion construct. Following the sequence analysis (Transfac software) we focused our attention on a putative SP1 binding site identified in this region, whose binding ability was then confirmed by electrophoretic mobility shift assay (-561/-552). In agreemente with our in vitro results, it was also observed that copper favours the native TFF1 dimer formation in the culture medium of MCF-7 and HT29-MTX cells (a mucus-secreting clone obtained from the HT29 colon cancer cell line), thus confirming a possible role of the metal in the balance between the monomeric and the dimeric forms To evaluate the effect of copper-TFF1 interaction on the well known motogenic activity of the protein, we performed wound healing assays on an inducible clone of gastric cancer cells (AGS) able to overexpress the peptide (AGS-AC1). As expected, the overexpression of TFF1 stimulates an appreciable increase of cell migration, and copper chelation (BCS) undo the benefits of the increased peptide level. Our previous results showed that copper treatments decreased the amount of secreted protein in culture medium. Further experiments demonstrated that induced AGS-AC1 cells are able to store intracellularly higher amount of copper if compared to uninduced AGS-AC1 cells. This evidence suggests that TFF1 levels may also play a role also in the uptake/traffic of copper in this in vitro model. Finally, we studied the combined influence of TFF1 and copper in Helicobacter pylori infections. Our results demonstrate that Cu-TFF1 complex promotes H. pylori colonization of AGS cells. In fact, AGS-AC1 cells overexpressing TFF1 are more efficiently colonised by H. pylori wild-type (str. P12) if compared to uninduced cells. The presence of copper in a duplicate experiment further increases the colonization, as well as copper chelation by bathocuproine disulfonate (BCS) reduces the observed effect. The same result was obtained with H. pylori str. P12Δ479, an isogenic mutant expressing a truncated LPS core still able to bind to TFF1. On the other hand, H. pylori P12Δ1191, unable to bind TFF1, is not affected by copper levels in the culture medium. Parallel experiments were carried out on mucus secreting HT29-E12 goblet cells, to compare and/or confirm the results obtained in AGS-AC1. The results show that also in HT29-E12 cells the H. pylori colonization follows a similar trend, increasing when incubated in the presence of Cu and decreasing after BCS treatment. The present work contributed interesting results in the field of the biochemistry of the epithelia, in the wake of the research in progress in our laboratory aimed at studying the biological activities of the newly identified metalloprotein Cu-TFF1, whose properties are still poorly characterized. On the basis of the previous structural pieces of evidence we observed that the protein level and the balance of its oligomeric forms can be affected and regulated by copper ions. In turn, this delicate equilibrium is able to affect the integrity and the rheological properties of the epithelial barrier, thus representing a fine tuner, or an Achille’s heel, through which pathogenic microrganisms and deregulated proliferation of neoplastic cells may take advantage for their invasiveness. The role of copper in the TFFs biochemistry represents a new finding in the puzzling and versatile functions of this interesting peptide family, whose thorough comprehension still reserves many questions and surprises. [edited by author]
IX n.s.

Asthma is an incapacitating disease of the respiratory system, which causes extensive morbidity and mortality worldwide. Asthma affects more than 300 million people globally(Masoli et al. 2004). In Australia, it affects 10.2% of the population (Masoli et al. 2004) and causes 60,000 people to be hospitalised annually. Health care expenditure due to asthma in Australia was $606 million in 2004–2005. There are four primary biological factors that function in the initiation and exacerbation of asthma. Airway inflammation is important as it is often the first response to an airway insult, initiating the three other components: bronchoconstriction, mucus hyper-secretion and hyper-reactivity. The mediators involved in asthma are still not well understood, and current anti-inflammatory corticosteroid treatments are not effective with all asthmatics. As there is currently no cure for asthma, and airway inflammation is the primary component of the disease, it is important that we understand and investigate the mediators of airway inflammation to look for a potential cure and to produce better therapeutics to treat the inflammation. Trefoil factors (TFFs) and secretoglobins (SCGBs) are small secreted proteins involved in the mediation of inflammation and epithelial restitution. TFFs are pro-inflammatory and SCGBs anti-inflammatory by nature. The hypothesis of this study is that in response to induced acute airway inflammation, the expression of TFF1 and TFF3 will increase and expression of SCGB1A1 and SCGB3A2 will decrease in non-asthmatics (N-A), asthmatics medicating with bronchodilators (A-BD) and asthmatics medicating with corticosteroids (A-ST). When comparing the three groups, we expect to see higher expression of the TFFs in the A-BD group compared to the N-A and A-ST groups, indicating that inflammation is mediated by TFFs in asthma and that corticosteroid medication controls their expression as part of the control of inflammation. We expect to see the opposite with SCGBs, with a greater decrease in the A-BD group compared to the other two groups, suggesting that the A-BD group has the least anti-inflammatory activity in response to inflammatory insult. Epigenetic modification plays a role in the regulation of genes that initiate disease states such as inflammatory conditions and cancers. Histone acetylation is one such modification, which involves the acetylation of histones in chromatin by histone acetyltransferases (HATs). This increases the transcription of genes involved with inflammation or enrols histone deacetylases (HDACs) to down-regulate the transcription of inflammatory genes. These HATs and HDACs work in a homeostatic fashion; however, in the event of inflammation, increased HAT activity can stimulate further inflammation, which is believed to be the mechanism involved in some inflammatory diseases. This study hypothesises that in response to inflammation, the expression of HDACs (HDAC1-5) will decrease and the expression of HATs (NCOA1-3, HAT-1 and CREBBP) will increase in all groups. When comparing the expression between the groups, it was expected that a greater decrease in HDACs and a greater increase in HATs will be seen in the A-BD group compared to the other two groups. This would identify histone acetylation as a mechanism involved in the inflammatory condition of asthma and indicate that corticosteroids may treat the inflammation in asthma at least in part by controlling histone acetylation. The aim of the project was to compare the expression of inflammatory genes TFF1, TFF3, SCGB1A1 and SCGB3A2, as well as to compare the gene expression of HDAC1-5, NCOA1-3, HAT-1 and CREBBP within and between N-A (n=15), A-BD (n=15) and A-ST (n=15) groups in response to inflammation. This was performed by collecting airway cells and proteins by sputum induction in three sessions. The sessions were coordinated into an initial baseline collection (SI-1), followed by a second session at least one week later (SI-2) and a third session, six hours after SI-2 to collect a sample containing the resultant acute inflammation caused in SI-2 (SI-3). Analysis of the SI-1 and SI-2 samples in all three groups had high amounts of variability between samples. The samples were taken at least one weak apart and the environmental stimuli on each participant outside of the testing sessions could not be controlled. For this reason, the SI-1 samples were not used for analysis; instead SI-2 and SI-3 samples were compared as they were same-day collections, reducing the probability of differences being due to anything other than the sputum induction. The gene expressions of the TFFs, SCGBs, HDACs and HATs were analysed using real-time PCR. Western blot analysis was performed to analyse the protein concentrations of the TFFs and SCGBs in secreted fractions of the sputum collection. Both the secreted and intracellular protein fractions collected from the sputum inductions for pre- and post-inflammation (SI-2, SI-3) samples of the N-A and A-BD groups were analysed using a proteomic method called iTRAQ. This allowed the comparison of the change in protein expression as a result of airway inflammation in each group. This technique was used as a discovery method to identify novel proteins that are modulated by induced acute airway inflammation. Any proteins of interest would then be further validated and used for future research. Inflammation was achieved in the SI-3 samples of the N-A group with a 21% unit increase in % neutrophils compared to SI-2 (p=0.01). The N-A group had a marked 5.5-fold decrease in HDAC1 gene expression in SI-3 compared to SI-2 (p=0.03). No differences were seen in any of the TFFs, SCGBs or any of the rest of the HDACs and HATs. Western blot data did not display any significant changes in the protein levels of the TFFs and SCGBs analysed. However, non-significant analysis of the data displayed increases in TFF1 and TFF3, and decreases in SCGB1A1 and SCGB3A2 for the majority of SI-3 samples compared to SI-2. The A-BD group also presented a marked increase in neutrophils in the SI-3 samples compared to SI-2 (27% unit increase, p=0.04). The A-BD group had a significant increase in TFF3 and SCGB1A1 gene expression concomitant with induced acute airway inflammation. A 7.3-fold increase in TFF3 (p=0.05) in SI-3 indicated that TFF3 is linked to inflammation in asthmatics. A 2.8-fold increase in SCGB1A1 (p=0.03) indicated that this gene is also up-regulated, suggesting that this SCGB is expressed to try to combat induced acute airway inflammation. No significant changes were seen in any of the other genes analysed. Western blot data did not display any significant changes in the protein levels of the TFFs and SCGBs analysed. However, non-significant analysis of the data displayed an increase in TFF1 and TFF3, and a decrease in SCGB1A1 and SCGB3A2 in SI-3, similar to that seen in the N-A group. The A-ST group was different from the A-BD group, characterised by the use of inhaled corticosteroid medication to treat asthma symptoms. Inhaled corticosteroids are known to treat asthma symptoms through the control of inflammation. Therefore, it was expected that corticosteroid medication would also control the expression of TFFs, SCGBs, HATs and HDACs. Gene expression results only identified a 7.6-fold decrease in HDAC2 expression in SI-3 (p=0.001), which is proposed to be due to the up-regulation of HDAC2 protein that is known to be a function of corticosteroid use. Western blot data did not display any significant changes in the protein levels of the TFFs and SCGBs analysed. The gene expression in SI-2 and SI-3 in each group was compared. When comparing the A-BD group to the N-A group, a 9-fold increase in TFF3 (p=0.008) and a 34-fold increase in SCGB1A1 (p=0.03) were seen in the SI-3 samples. Comparisons of the A-ST group to the N-A group had an increased expression in SI-2 samples for HDAC5 (3.6-fold, p=0.04), NCOA2 (8.5-fold, p=0.04), NCOA3 (17-fold, p=0.01), HAT-1 (36-fold, p=0.003) and CREBBP (13-fold, p=0.001). The SI-3 samples in the A-ST group compared to the N-A group had increased expression for HDAC1 (6.4-fold, p=0.04), HDAC5 (5.2-fold, p=0.008), NCOA2 (9.6-fold, p=0.03), NCOA3 (16-fold, p=0.06), HAT-1 (41-fold, p<0.001) and CREBBP (31-fold, p=0.001). Comparisons of the A-ST group to the A-BD group had SI-2 increases in HDAC1 (3.8-fold, p=0.03), NCOA3 (4.5-fold, p=0.03), HAT-1 (5.3-fold, p=0.01) and CREBBP (23-fold, p=0.001), while SI-3 comparisons saw a decrease in HDAC2 (41-fold, p=0.008) and increases in HAT-1 (4.3-fold, p=0.003) and CREBBP (40-fold, p=0.001). Results showed that TFF3 and SCGB1A1 expression is higher in asthmatics than non-asthmatics and that histone acetylation is more active in the A-ST group than either the N-A or A-BD group, which suggests that histone acetylation activity may be positively correlated with asthma severity. The iTRAQ proteomic analysis of the secreted protein samples identified the SCGB1A1 protein and found it to be decreased in both the N-A and A-BD groups post-inflammation, but significantly so only in the A-BD group. Although no significant results were obtained from the western blot data, both groups displayed a decrease in SCGB1A1 concentration in SI-3 samples, suggesting a correlation with the proteomic data. Only 31 peptides were identified from the secreted samples. The intracellular iTRAQ analysis successfully identified 664 peptides, eight of which had differential expression in association with induced acute airway inflammation. Significant increases were seen in the A-BD group in SI-3 compared to SI-2 than in the N-A group in chloride intracellular channel protein 1, keratin-19, eosinophil cationic protein, calnexin, peroxiredoxin-5, and ATP-synthase delta subunit, while decreases were seen in cystatin-A and mucin-5AC. The iTRAQ analysis was only a discovery measure and further validation must be performed. In summary, the expression of TFFs and SCGBs differed between non-asthmatics and asthmatics. It is clear that TFF3 is active in the airway inflammation associated with asthma as indicated by an increase associated with inflammation in the A-BD group compared to the N-A group. Results for HDAC and HAT genes showed high HAT expression in the A-ST group compared to the N-A and A-BD groups, suggesting that histone acetyltransferases may be responsible for the characteristic unregulated inflammatory symptoms of asthmatics taking corticosteroids. Interestingly, corticosteroid medication did not seem to silence the expression of the analysed HAT genes, which indicates that corticosteroids may not control inflammation by direct regulation of HATs, but instead by competition, most probably with HDAC2 protein. As a discovery tool, iTRAQ is a potent method to both identify and compare the concentration of proteins between samples. The method is a powerful first step into the identification of novel proteins that are regulated in response to different treatments.

Abstract

The trefoil factor family of secreted proteins contains three members; trefoil factor 1 or TFF1, trefoil factor 2 or TFF2, and trefoil factor 3 or TFF3. These three proteins share a conserved 42-43 amino acid domain containing 6 cysteine residues resulting in three disulfide bonds that holds the protein in a characteristic three-loop or "trefoil structure" known as the P domain. TFF1 is primarily localized to the stomach and secreted by the gastric mucosa while TFF2 and TFF3 are primarily localized to the colon and duodenum and secreted by the goblet cells. All three of these proteins play a protective role in the gastrointestinal tract where they are normally localized and have been identified as possible tumor suppressors, however, these proteins are also upregulated in cancer within tissues where they are not normally expressed including the breast, pancreas, prostate, and liver. The mechanisms by which two of these factors, TFF1 and TFF3, promote tumorigenesis remain largely undefined. In this dissertation we will attempt to elucidate these mechanisms as well as the regulation of these two proteins in both pancreatic and prostate cancer. Many of the underlying genetic and molecular mechanisms involved in the development of both pancreatic and prostate cancer remain largely unknown and as a result, therapeutic and diagnostic tools for treating these diseases are not as effective as they could be. By deciphering the role of TFF1 and TFF3 in these cancers, they could potentially serve as new therapeutic targets or biomarkers for treating both diseases.

Chapter 2 of this dissertation will examine the functional role of TFF1 promoting tumorigenesis in pancreatic and prostate cancer. We will show that TFF1 expression is critical for the viability of both pancreatic and prostate cancer cells and that reduction of TFF1 expression in these cells results in decreased tumorigenicity when implanted in immunocompromised mice. It will also be demonstrated that TFF1's function in promoting tumorigenicity is its ability to assist tumor cells overcome the tumor suppressive barrier of senescence. Thirdly, we show that the form of senescence that TFF1 assists in allowing the cells overcome is oncogene-induced senescence (OIS). Lastly, a cell cycle array identifies the potential downstream target p21CIP, a cyclin-dependent kinase inhibitor and OIS marker, whose expression is induced by loss of TFF1 expression.

In Chapter 3 of this work, we examine the role of another trefoil factor family member, TFF3, and its role in promoting prostate tumorigenesis. Just as with TFF1, it appears that TFF3 3 expression is critical for prostate cancer cell viability and tumorigenicity using the same experimental techniques used in Chapter 2. Using a genetically defined model of prostate cancer, a PI3-kinase-dependent regulatory mechanism of TFF3 emerges in this prostate cancer context. Using this system we begin to see a divergence in both regulation and function of TFF1 and TFF3 in prostate cancer. Finally, a mouse model expressing TFF3 was developed to monitor the histopthological changes associated with expression of this protein. Initial characterization of this model suggests a hyperplastic phenotype coinciding with TFF3 expression in the prostate.

The two studies in this dissertation establish a role of TFF1 and TFF3 in both prostate and pancreatic tumorigenesis and demonstrate that ablation of expression of both proteins is a potent inhibitor of tumorigenesis. With this knowledge, it is possible that TFF1 and TFF3 may become a potential therapeutic target or diagnostic marker for better treatment of prostate and pancreatic cancer.

Dissertation

碩士
國立中興大學
食品暨應用生物科技學系所
99
Digestive system damages are common problems in people who lived in a stressful life. Mucosal defense in gastrointestinal tract includes local gastric mucosal defense mechanisms and neurohormonal regulation. The surface epithelial cells secrete mucus, bicarbonate and generate prostaglandins, heat shock proteins, trefoil factor peptides (TFFs), and cathelicidins to defense or regulate the mucosal damages. The TFFs mediate mucosal repair by stimulating cell migration, inhibiting apoptosis and inflammation, and promote the barrier function of mucus. In this study, attempts of expressing recombinant human trefoil factor 1 (TFF1) by well known Escherichia coli and GRAS Lactococcus lactis were proceeded. In the first part of this study, a novel recombinant human TFF1 gene was designed according to the preferred codons of L. lactis. Then the recombinant human TFF1-expressing plasmids were constructed. The recombinant human TFF1-expressed plasmids for L. lactis including constitutive pNZDSASm-sacBATFF1, and nisin-inducible pNZNS-sacBATFF1 and pNZNUB-TFF1. Isopropyl β-D-1-thiogalactopyranoside (IPTG)-indicible pET-sacBATFF1 and pET-TFF1 are the recombinant human TFF1-expressed plasmids of E. coli. Owing to the unstable expression of L. lactis system, the optimized recombinant human TFF1 was purified from fermented E. coli BL21(DE3) transformants and the purified recombinant human TFF1 was identified by native-PAGE, Western blot, and peptide fingerprinting. The bioactivity of recombinant human TFF1 was analyzed by wound healing assay of C2BBe1 cell (clone of Caco-2 cell). Results showed that the recombinant human TFF1 exhibited reclosing up wound of C2BBe1 cell monolayer and could be improved is increased by adding few FBS. The recombinant human TFF1, treated with modified gastrointestinal pH, pH 2.4 and pH 7.0 buffer showed that the recombinant human TFF1 is more active after pH 2.4 treatment. Results suggest that the acidic environment in stomach might be benefitial to active conformation of recombinant human TFF1.

碩士
國立中興大學
食品暨應用生物科技學系所
101
Gastrointestinal dameges are caused by irregular eating habits and life stress, finally result in tumor ulceration. Therefore gastrointestinal diseases have become modern civilized illness. Human gastrointestinal tract epithelial tissue repair their damages,by secreting HCO3-, heat shock proteins, trefoil factor, and antimicrobial peptides. Among these repairing factors trefoil factor can stimulate cell migration, by inhibit cell suicide and inflammation repair gastrointestinal epithelium. Apart from gastrointestinal dameges allergic diseases is also a civilized illness. When the body was invaded by bacteria or foreign objects, the immune system release histamine and cause inflammation. The acute inflammation of the immune system for pathogen invasion caused symptoms, not only destruct pathogens but also make normal cells destructive. Interleukin -10 (interleukin 10, IL-10) can adjust chronic, acute inflammation inhibiting cytokines such as IL-1, IL-2, and tumor necrosis factor production TFN, and interleukin -10 is classified as anti-inflammatory cytokines. In this study, the recombinant human trefoil factor 1(TFF1) were expressed by GRAS grade Lactococcus lactis. In the first part of this study, the induction nisin concentration of MNICE system (pNZNS-SacBATFF1) was re-established, therefore accomplish more stable rTFF1 extracellular expression. Another acid-inducible system(MPHI)(pNZAUS-SacBATFF1)was constructed, and confirmed the expression. The two systems fermented by using fed batch in a 5L fermenter .MNICE obtain 418.2 μg / L and MPHI available 441.7μg / L of recombinant human trefoil factor 1. The purified recombinant human trefoil factor 1 from two systems were identified by homodimer determination, native –PAGE, MALDI-TOF MS. The bioactivity of recombinant human trefoil factor 1 from two systems were analyzed by wound healing assay of AGS cells. Results showed that the recombinant human TFF1 from two systems exhibited wound healing capacities of AGS cell. The recombinant human TFF1, treated with pH 2.4 buffer which showed better active wound healing capacities. This results suggest that the acidic environment in stomach might be benefitial to active conformation of recombinant human TFF1. In another part of this study a novel recombinant human IL-10 gene was designed according to the preferred codons of L. lactis. Then the recombinant human IL-10-expressing plasmids was constructed(pNZDSASm-sacBAIL-10). The rIL-10 was not secreted extracellular but was detected as pre-IL-10 by western blot analysis. In conclusion the MpHI system fermented by using fed batch can obtain high purity and biological activity trefoil factor I. In the near future can be applied to healthcare industry.