Human Trefoil Factor Research Articles

JAK/STAT3 Pathway in Human Intestinal Epithelial Cells During Trefoil Factor 3(TFF3) Mediated Cell Migration

Objective:Trefoil factor family is expressed in several tissues of the body and provides gastric and intestinal protection and healing. This research aims to indicate the mechanism involved in its function.Methods:The intestinal epithelial cells were pretreated with JAK inhibitor AG490 or the concentration of 60ug/ml human recombinant trefoil factor, while the levels of phospho-STAT3, E-cadherin and N-cadherin were detected by Western Blotting. The levels of Matrix Metalloproteinases, Ecadherin and N-cadherin were evaluated by quantitative real time PCR. The cell migration was assessed by the transwell assay and the scratch assay. The immunofluorescence method was performed to detect the reduction of molecular E-cadherin.Results:hTFF3 activates the JAK/STAT3 pathway in HT-29 cells. The effect of JAK/STAT3 pathway mechanism on cell migration promoted by hTFF3. TFF3 promoting cell migration is associated with increased gene transcription of MMPs. hTFF3 alters E-cadherin expression. hTFF3 activates the expression of N-cadherin and down-regulates E-cadherin expression in HT-29 Cells.Conclusion:We have shown that TFF3 activated the JAK/STAT3 pathway. TFF3 increased the level of Matrix Metalloproteinases and N-cadherin, decreased that of E-cadherin, while AG490 had the opposite effect. TFF3 accelerated cell migration and the AG490 relieved the migrating rate to control the levels. TFF3 activated JAK/STAT3 pathway which was associated with intestinal epithelial cell migration.

Chemical synthesis of human trefoil factor 1 (TFF1) and its homodimer provides novel insights into their mechanisms of action

TFF1 is a key peptide for gastrointestinal protection and repair. Its molecular mechanism of action remains poorly understood with synthetic intractability a recognised bottleneck. Here we describe the synthesis of TFF1 and its homodimer and their interactions with mucins and Helicobacter pylori. Synthetic access to TFF1 is an important milestone for probe and therapeutic development.

Correction: Chemical synthesis of human trefoil factor 1 (TFF1) and its homodimer provides novel insights into their mechanisms of action.

Correction for 'Chemical synthesis of human trefoil factor 1 (TFF1) and its homodimer provides novel insights into their mechanisms of action' by Nayara Braga Emidio et al., Chem. Commun., 2020, DOI: 10.1039/D0CC02321C.

1130P - Severe oral mucositis (SOM) mitigation by genetically modified lactococcus lactis bacteria (LLB) producing human trefoil factor 1 (hTFF1; AG013) in patients being treated with concomitant chemoradiation (CRT) for oral and oropharyngeal cancers (OCOPC)

BackgroundSOM is a devasting consequence of CRT. hTFF1 is a naturally occurring protein that can protect the mucosa. LLB were genetically modified (GM) to produce hTFF1, formulated as an oral rinse and attenuated SOM in patients receiving chemotherapy. The GM LLB lack all necessary components for survival and multiplication. The objective of this ongoing Phase 2 trial is to assess the safety and efficacy of AG013 as a SOM intervention. MethodsThis is a double-blind, randomized, placebo-controlled trial recruiting ∼200 patients with OCOPC at 48 sites (US and Europe). Patients (PTS) receive cumulative RT (cumRT) between 50Gy – 72Gy, 2.0-2.2Gy QD + QW/Q3W cisplatin. At least two mucosal sites at risk of SOM receive minimal cumRT of 50Gy. PTS are randomized 1:1 to receive placebo or AG013 [LLB strain sAGX0085 engineered to secrete hTFF1 (2x1011 CFU/15mL tid)] starting on CRT day 1 and continuing 2 weeks post-CRT. Beginning on CRT day 1 and continuing Q2W until resolution, OM is assessed by trained assessors and scores assigned centrally. The primary and secondary efficacy endpoints are SOM duration and incidence (WHO criteria). AEs are described by NCI-CTCv4. A DSMB performed a safety analysis following accrual of the first 24 PTS. Tumor response to CRT is evaluated for 1-year post CRT. Results71 PTS have been randomized across 48 study sites. Complete OM data are available for 42 PTS for whom blinded evaluation (active and placebo) demonstrated an overall SOM incidence of 52%. SOM was noted at 81 of 547 visits (14.8%). 25 PTS have stopped active treatment; 2 for non-compliance, 5 for AEs, and 18 lost to follow-up or unwilling or unable to conform to the protocol. Unexpected SAEs were noted in 9 PTS. No study drug-associated cases of bacteremia or sepsis were seen. DSMB review after the first 24 PTS concluded that there were no contraindications to study continuance. ConclusionsObservations based on blinded data suggest that AG013 offers a safe, well-tolerated, and potentially efficacious platform to deliver an effective protein intervention for SOM mitigation. Clinical trial identificationAG013-ODOM-201. Legal entity responsible for the studyOragenics, Inc. FundingOragenics, Inc. DisclosureS. Sonis: Advisory / Consultancy, consultant: Oragenics. All other authors have declared no conflicts of interest.

Production of Monoclonal Antibodies against Human Trefoil Factor 3 and Development of a Modified-Sandwich ELISA for Detection of Trefoil Factor 3 Homodimer in Saliva

BackgroundHuman trefoil factor (TFF) peptides consist of three members: TFF1, TFF2 and TFF3. TFF3 is the most abundant TFF peptide in saliva. TFF3 homodimer was suggested to be involved in apoptosis inhibition and malignancy. Determination of TFF3 homodimer expression profiles in saliva may lead to new information about oral biology and diseases. The objective of this study was to generate monoclonal antibodies (mAbs) against TFF3 and apply the produced mAbs for the establishment of ELISA for quantification of dimeric TFF3 in saliva.ResultsWith our modified hybridoma technique, three hybridoma clones producing anti-TFF3 mAbs having IgG isotype were generated. The mAbs were specific for TFF3 with no cross-reactivity to other TFFs. Using the generated mAbs, a modified-sandwich ELISA with high sensitivity for the quantification of dimeric TFF3 in saliva was developed. Using this ELISA, the amount of dimeric TFF3 in saliva could be measured.ConclusionsA modified-sandwich ELISA for the quantification of TFF3 dimeric form was established. The established ELISA will be a valuable tool for facilitating the investigation of the physiological roles and the diagnostic values of TFF3 in oral diseases. The concept of this modified-sandwich ELISA may be applied for the determination of other homodimeric peptides of interest.

Human Trefoil Factor 3 induces the transcription of its own promoter through STAT3

Human trefoil factor 3 (hTFF3) is a small peptide of potential therapeutic value. The mechanisms underlying the transcriptional regulation of hTFF3 remain unclear. The purpose of this study was to identify the core functional elements for the self-induction action of hTFF3 and transcription factors. First, truncated promoters were constructed to identify the functional regions of the hTFF3 promoter. Next, point mutation, chromatin immunoprecipitation, RNA interference, and gene overexpression experiments were performed to analyze the transcriptional binding sites responsible for the self-induced transcription of hTFF3. Our results revealed the −1450 bp to −1400 bp fragment of the hTFF3 promoter was the functional region for the self-induction action of hTFF3. Bioinformatics analysis confirmed that a STAT3 binding site is present in the −1417 bp to −1409 bp region. Subsequently, site-directed mutagenesis analysis determined that this STAT3 binding site was critical for the self-induction effect of hTFF3. ChIP experiments confirmed that STAT3 binds to the hTFF3 promoter. STAT3 overexpression and knockdown experiments revealed that STAT3 enhanced the self-induction effect and the expression of hTFF3. This study confirmed that hTFF3 exhibits self-induction action, and that STAT3 is the key transcription factor to maintain the function of self-induction.

Abstract 1233: Impairment of TFF3 induces cellular apoptosis in prostate cancer cell lines and overexpression of TFF3 may be a prognostic biomarker in human prostate cancers

Abstract Background: Human trefoil factor 3(TFF3)/ Intestinal trefoil factor(ITF) is originally known well as an essential regulator for epithelial restitution in gastrointestinal tract and also suggested as a potential cancer marker in serum. Furthermore, the aberrant overexpression of TFF3 has been reported in various human cancers, majorly colorectal cancers, suggesting that TFF3 may play a critical role in tumor growth. However, its molecular signaling pathway remains unclear. Objective: To clarify whether TFF3 is related to survival/anti-apoptotic cell signaling pathways, and to determine that tff3 could be a molecular marker in prostate cancers. Design, Setting, and Participants: TFF3 gene was silenced by using siRNA in prostate cancer two kinds of cell lines, LNCap and PC3, following investigation of genes related to apoptosis and cell migration. The expression of TFF3 was analyzed by immunohistochemistry in 109 prostate cancer and 106 normal prostate tissues. Results and Limitations: TFF3 silenced prostate cancer cell lines exhibited increased annexin V binding and the activation of caspase-9 and caspase-3 which are crucial for programmed cell death, consistent with an increased rate of apoptosis, reduced rate of proliferation and migration. In human prostate cancer tissue microarray, TFF3 was positive in 54.7% (49.1%, 2+; 5.6%, 1+) of cancer cases (59/108) and 2.8% (1.9%, 2+; 0.9%, 1+) of morphologically normal tissue cases (3/106), P<0.0001. Conclusions: The results suggest that TFF3 expression seems play a role in promoting cell survival and be a potential useful marker in prostate cancers. In these contexts, TFF3 in human prostate cancers deserves further investigation to exploit as a cancer marker and anticancer drug. Citation Format: So-Youn Kim, Ji-Ying Liu, Yeun-Jun Chung. Impairment of TFF3 induces cellular apoptosis in prostate cancer cell lines and overexpression of TFF3 may be a prognostic biomarker in human prostate 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 1233. doi:10.1158/1538-7445.AM2015-1233

Functional expression of recombinant human trefoil factor 1 by Escherichia coli and Brevibacillus choshinensis.

BackgroundTrefoil factor 1 (TFF1) mediates mucosal repair and belongs to a highly conserved trefoil factor family proteins which are secreted by epithelial cells in the stomach or colon mucous membrane. TFF1 forms a homodimer via a disulphide linkage that affects wound healing activity. Previous recombinant expressions of TFF1 were too low yield for industrial application. This study aims to improve the expression level of bioactive recombinant TFF1 (rTFF1) and facilitate application potency.MethodsThe rTFF1 gene rtff1 was synthesized, expressed by Escherichia coli and secreted by Brevibacillus choshinensis. The rTFF1s were purified. The polymeric patterns and wound healing capacities of purified rTFF1s were checked.ResultsIn Escherichia coli, 21.08 mg/L rTFF1 was stably expressed as monomer, dimer and oligomer in soluble fraction. In Brevebacillus choshinensis, the rTFF1 was secreted extracellularly at high level (35.73 mg/L) and formed monomer, dimer and oligomer forms. Both proteins from different sources were purified by Ni-NTA chromatography and exhibited the wound healing activities. The rTFF1 produced by B. choshinensis had better wound healing capability than the rTFF1 produced by E. coli. After pH 2.4 buffer treatments, the purified rTFF1 formed more oligomeric forms as well as better wound healing capability. Glycosylation assay and LC-MS/MS spectrometry experiments showed that the rTFF1 produced by B. choshinensis was unexpectedly glycosylated at N-terminal Ser residue. The glycosylation may contribute to the better wound healing capacity.ConclusionsThis study provides a potent tool of rTFF1 production to be applied in gastric damage protection and wound healing. The protein sources from B. choshinensis were more efficient than rTFF1 produced by E. coli.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0149-5) contains supplementary material, which is available to authorized users.

Serum human trefoil factor 3 is a biomarker for mucosal healing in ulcerative colitis patients with minimal disease activity.

The goals of treating ulcerative colitis (UC) have shifted from clinical remission to mucosal healing. Non-invasive biomarkers are required to assess mucosal healing as endoscopic assessment is inconvenient for patients. Enhanced expression of trefoil factor 3 (TFF3, a mucin-associated peptide) is observed after injury of the gastrointestinal tract. The present study was designed to evaluate TFF3 as a biomarker of mucosal healing in patients with UC. This cross-sectional study included consecutive patients with UC (18-65 years old, disease duration >3 months, either left-sided colitis or pancolitis) who had a Simple Clinical Colitis Activity Index (SCCAI) <6. Colonoscopy was done to assess the presence or absence of mucosal healing (defined using the Baron score) in all patients. Serum level of TFF3 was assessed in all patients and 20 healthy controls. Seventy-four patients were included [mean age 37.2±10.9 years, 47 males, median disease duration 4.8 years (IQR 3-8.3), median SCCAI = 0] in the study. Forty-three patients had mucosal healing (Baron score 0 or 1) and 31 did not (Baron score 2 or 3). Median TFF3 level in patients without mucosal healing was significantly higher than that in patients with mucosal healing [1.5 (IQR 1.2-1.9) vs 1.1 (IQR 0.8-1.3) ng/ml, p = 0.01] and healthy controls [0.85 (IQR 0.7-1.2) ng/ml, p < 0.001]. A serum TFF3 level of <1.27 ng/ml (as determined by the receiver operating characteristic curve; area under the curve 0.73) had sensitivity, specificity, positive predictive value and negative predictive value of 70, 68, 75 and 62%, respectively, for identifying patients with mucosal healing. Serum TFF3 can potentially be used as a biomarker to assess mucosal healing in UC patients.

Effect of adenovirus mediated human intestinal trefoil factor gene on intestinal epithelial cells

Objective To construct a recombinant adenovirus vector of human intestinal trefoil factor(hITF)and observe its effect on human intestinal epithelial cells(Caco-2). Methods A recombinant adenovirus vector containing hITF gene was constructed and infected Caco- 2 cells. The expression of hITF mRNA and protein were detected by reverse transcriotion-polymerase chain reaction(RT-PCR), Western blotting and immunofluorescence. Then, wounds were established in confluent monolayers of Caco-2 cells infected with recombinant adenovirus vector, and the width of the cells migration was measured in a blind manner. Results The recombinant adenovirus vector Ad-hITF was constructed and the infection efficiency was about 100%. RT- PCR and Western blotting assay demonstrated that hITF was transcribed and expressed successfully. hITF was proved to distribute in cytoplasm by immunofluorescence assay. In addition, Ad-hITF enhanced migration activity of HT-29 cells obviously. Conclusion Recombinant adenovirus vector containing hITF gene was successfully constructed and its effect was confirmed in promoting cells migration. This research lay foundations for the further application of hITF gene therapy. Key words: Intestinal trefoil factor; Adenovirus; Intestinal epithelial cells; Migration

TFF3 and HER2 expression and their correlation with survival in gastric cancer.

The molecular biomarkers human epidermal growth factor receptor-2 (HER2) and trefoil factor 3 (TFF3) are reported to play important roles in the pathogenesis of gastric cancer (GC). In this study, we investigated the clinicopathological and prognostic significance of TFF3 and HER2 expression in GC and explored the correlation between these two biomarkers. Ninety-two patients who were diagnosed with GC were enrolled. TFF3 and HER2 expression was determined on tumor tissues. The results showed that TFF3 and HER2 were positively expressed in 42.7 and 10.9% of the cases, respectively. There were significantly higher rates of TFF3 positivity in patients with deep invasive tumors and advanced stage ones. Patients with negative TFF3 staining survived longer than those with the presence of TFF3, with 5-year overall survival (OS) rates of 57.1 ± 7.1 and 39.5 ± 7.5%, respectively (P = 0.033). However, HER2 positivity was not significantly associated with OS (P = 0.262). Multivariate analysis demonstrated TFF3 expression to be an independent indicator for short-term survival, with a hazard ratio of 2.327 (95% confidence interval (CI), 1.202-4.507, P = 0.012). There was a trend that the expression of TFF3 was more frequent in HER2 negative tumors than in HER2 positive ones (positive rates: 16.3 vs. 4.7%, P = 0.098). Patients with HER2-negative/TFF3-negative GC presented higher OS than those with other phenotypes (P = 0.009). This study suggests that TFF3 is an independent indicator for survival in GC, while HER2 is not associated with the outcome. Patients with HER2-negative/TFF3-negative GC have the best outcome.

Human Trefoil Factor 2 Is a Lectin That Binds α-GlcNAc-capped Mucin Glycans with Antibiotic Activity against Helicobacter pylori

Helicobacter pylori infection is the major cause of gastric cancer and remains an important health care challenge. The trefoil factor peptides are a family of small highly conserved proteins that are claimed to play essential roles in cytoprotection and epithelial repair within the gastrointestinal tract. H. pylori colocalizes with MUC5AC at the gastric surface epithelium, but not with MUC6 secreted in concert with TFF2 by deep gastric glands. Both components of the gastric gland secretome associate non-covalently and show increased expression upon H. pylori infection. Although blood group active O-glycans of the Lewis-type form the basis of H. pylori adhesion to the surface mucin layer and to epithelial cells, α1,4-GlcNAc-capped O-glycans on gastric mucins were proposed to inhibit H. pylori growth as a natural antibiotic. We show here that the gastric glycoform of TFF2 is a calcium-independent lectin, which binds with high specificity to O-linked α1,4-GlcNAc-capped hexasaccharides on human and porcine stomach mucin. The structural assignments of two hexasaccharide isomers and the binding active glycotope were based on mass spectrometry, linkage analysis, (1)H nuclear magnetic resonance spectroscopy, glycan inhibition, and lectin competition of TFF2-mucin binding. Neoglycolipids derived from the C3/C6-linked branches of the two isomers revealed highly specific TFF2 binding to the 6-linked trisaccharide in GlcNAcα1-4Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-3)GalNAc-ol(Structure 1). Supposedly, lectin TFF2 is involved in protection of gastric epithelia via a functional relationship to defense against H. pylori launched by antibiotic α1,4-GlcNAc-capped mucin glycans. Lectin-carbohydrate interaction may have also an impact on more general functional aspects of TFF members by mediating their binding to cell signaling receptors.

Trefoil Factor Family Peptide 3 (TFF3) is Upregulated Under Experimental Conditions Similar to Dry Eye Disease and Supports Corneal Wound Healing Effects In Vitro

To elucidate the role of trefoil family peptide (TFF) 3 at the ocular surface under conditions similar to dry eye disease (DED) and in tears of patients suffering from DED. Trefoil family peptide 3 levels in tear samples from non-Sjögren's DED patients with moderate dry eye were analyzed by ELISA and compared with tears from healthy volunteers. A human corneal epithelial (HCE) cell line was treated with proinflammatory cytokines IL-1β and TNF-α, hyperosmolar medium, or scratching for up to 24 hours. Trefoil family peptide 3 gene expression and protein biosynthesis were analyzed by RT-PCR, immunofluorescence, and ELISA. Migration and proliferation of HCE cells under recombinant (r) human (h) trefoil factor family peptide 3 (TFF3) stimulation were investigated by scratching and bromodeoxyuridine (BrdU) proliferation assays. Tears of patients suffering from DED contained significantly higher TFF3 levels than tears from healthy volunteers. Stimulation of HCE cells with proinflammatory cytokines, culture under hyperosmolar conditions, or scratching resulted, with the exception of hyperosmolar conditions, in an increase in TFF3 expression and elevated secretion level of TFF3. Cell proliferation decreased and cell migration increased after 24-hours stimulation with rhTFF3. These results suggest that inflammatory factors or ocular surface damage as they occur in DED, lead to an increase of TFF3 tear film concentration, whereas hyperosmolarity does not. Our data underline a potential role for TFF3 as a candidate therapeutic for the ocular surface damage observed in DED.

Cloning and characterization of the human trefoil factor 3 gene promoter.

Human trefoil factor 3 (hTFF3) is a small-molecule peptide with potential medicinal value. Its main pharmacological function is to alleviate gastrointestinal mucosal injuries caused by various factors and promote the repair of damaged mucosa. However, how its transcription is regulated is not yet known. The aim of this study was to clone the hTFF3 gene promoter region, identify the core promoter and any transcription factors that bind to the promoter, and begin to clarify the regulation of its expression. The 5′ flanking sequence of the hTFF3 gene was cloned from human whole blood genomic DNA by PCR. Truncated promoter fragments with different were cloned and inserted into the pGL3-Basic vector to determine the position of the core hTFF3 promoter. Transcription element maintaining basic transcriptional activity was assessed by mutation techniques. Protein-DNA interactions were analyzed by chromatin immunoprecipitation (ChIP). RNA interference and gene over-expression were performed to assay the effect of transcription factor on the hTFF3 expression. The results showed that approximately 1,826 bp of the fragment upstream of hTFF3 was successfully amplified, and its core promoter region was determined to be from −300 bp to −280 bp through analysis of truncated mutants. Mutation analysis confirmed that the sequence required to maintain basic transcriptional activity was accurately positioned from −300 bp to −296 bp. Bioinformatic analysis indicated that this area contained a Sp1 binding site. Sp1 binding to the hTFF3 promoter was confirmed by ChIP experiments. Sp1 over-expression and interference experiments showed that Sp1 enhanced the transcriptional activity of the hTFF3 promoter and increased hTFF3 expression. This study demonstrated that Sp1 plays an important role in maintaining the transcription of hTFF3.

Expression and localization of trefoil factor family genes in rat submandibular glands

The trefoil factor (TFF) family, which comprises TFF1, TFF2 and TFF3, plays an essential role in epithelial regeneration within the gastrointestinal tract. All three TFFs are present in human saliva; TFF3 is the predominant trefoil peptide. Little is known about the expression and tissue distribution of TFFs in rats, which are commonly used as a model system for human studies. We investigated the localization of the TFF genes that encode secretory peptides in rat submandibular glands (SMG). All three TFFs were expressed in rat SMG, although their location varied. Substantial amounts of TFF1 were detected only in the cytoplasm of epithelial cells in the SMG granular convoluted tubules (GCT), while TFF2 and TFF3 were widely distributed in the cytoplasm of epithelial cells of intercalated ducts (ID), striated ducts (SD) and interlobular ducts (ILD). The three TFFs also were detected especially in the lumens of the SD and ILD. Semi-quantitative RT-PCR and in situ hybridization experiments confirmed TFF1, TFF2 and TFF3 mRNA expressions in the SMG. Greater expression of TFF peptides and mRNA was observed in male rats than in females. The broad expression of TFFs in rat SMG cells and lumens suggests that TFFs function in this organ by their secretion into the duct lumens. We also found differences in TFF expression profiles between rat and human SMG; therefore, caution should be exercised when using rats as a model for human TFF studies.

Recombinant Human Trefoil Factor 3 Ameliorates Bowel Injury: Its Anti-Inflammatory Effect on Experimental Necrotizing Enterocolitis

Aim. Recombinant human trefoil factor 3 (intestinal trefoil factor) has been suggested to be partially protective against necrotizing enterocolitis (NEC), but the mechanisms of this protection have not been defined. We investigated whether the protective effects of rhTFF3 are the result of an anti-inflammatory response. Methods. The rats were killed on day 4, the distal ileum was harvested for morphological studies and immunohistochemistry for NF-κB (p65), and the amounts of IL-1β, IL-6, and IL-10 in the intestinal tissue were measured using commercial ELISA assay kits. Results. In the neonatal NEC, IL-1β, IL-6, and IL-10 were significantly higher than in normal group. In normal group, IL-1β and IL-6 were significantly decreased, and the amount of IL-10 was markedly increased compared with NEC group. In the NEC model, immunohistochemical staining for NF-κB (p65) was demonstrated to be of a strong brown color and was distributed in the intestinal epithelium. Treatment with rhTFF3 significantly decreased the immunoreactivity of NF-κB (p65) in the NEC model. Conclusions. Intestinal inflammation was ameliorated after rhTFF3 was injected. rhTFF3 may protect against the intestinal injury of the neonatal rat NEC model by suppression of the inflammatory response.

Phase 1b, multicenter, single blinded, placebo‐controlled, sequential dose escalation study to assess the safety and tolerability of topically applied AG013 in subjects with locally advanced head and neck cancer receiving induction chemotherapy

Oral mucositis (OM) is a significant toxicity of induction chemotherapy for locally advanced head and neck cancer (LAHNC). The safety and tolerability of AG013, an oral rinse containing recombinant Lactococcus lactis secreting mucosal protectant human trefoil factor 1 (hTFF1), was evaluated in a phase 1b study in LAHNC subjects who received induction with cisplatin, 5-fluorouracil, with or without docetaxel. Preliminary efficacy data were also obtained. A total of 25 of 52 LAHNC subjects who were followed during induction cycle 1 developed ulcerative oral mucositis (UOM; World Health Organization grade > 2) and were randomized to AG013:placebo (5:2 ratio) for cycle 2. Dosing schedules of 1, 3, or 6 times daily were evaluated (2 × 10(11) , 6 × 10(11) , and 1.2 × 10(12) colony forming units per day, respectively). OM was evaluated daily from cycle 2, day 1 through 14, using World Health Organization criteria. Pharmacokinetic assessment was also conducted. AG013 bacteria were not detected in blood. Oral live AG013 bacterial and hTFF1 levels in saliva and oral mucosa were equivalent among treatment groups. The most frequently occurring adverse events were nausea, oral pain, fatigue, diarrhea, and mucosal inflammation. Only 12% (3 of 25 adverse events), mainly nausea, were attributed to the investigational medicinal product: AG013 or placebo. Efficacy analysis showed a 35% reduction in percentage of days with UOM in AG013-subjects versus placebo. All placebo subjects experienced ≥ 2 days of UOM, whereas 29% of AG013 subjects had UOM for 0 or 1 day. AG013 use resulted in fewer unscheduled office and emergency room visits. No differences were noted in mouth and throat soreness, opioid use, or gastrostomy tube placement. AG013 was safe and well tolerated. Preliminary efficacy data support further study.

Selective Estrogen Receptor Modulators Resveratrol, Genistein, Tamoxifen and Pterostilbene down‐regulate the Expression of Estrogen‐Regulated Trefoil Factor 1 (TFF1) Gene in MCF‐7 Breast Cancer Cells

The human trefoil factor 1 (TFF1) is a small cysteine‐rich secretory protein expressed in the breast tumor in response to estrogen. The selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene provide an effective treatment for estrogen receptor (ER) positive breast cancers. Our studies indicate that these SERMs at pharmacological concentrations modulate the expression of TFF1. The MCF‐7 breast cancer cells were treated with increasing concentrations of estrogen and SERMs such as resveratrol, genistein, tamoxifen and pterostilbene, and its effects were studied using RNA and protein analyses. The cell viability studies using estrogen showed increased cell growth even at low concentrations whereas the SERMs showed a concentration dependent cell growth arrest and death by apoptosis. Reverse Transcription‐Polymerase Chain Reaction (RT‐PCR) and Northern blot analyses indicated decreased levels of transcription of TFF1 mRNA. Western blot analysis showed a corresponding decrease in the expression of the protein. The above results indicate the potential use of these SERMs for the down‐regulation of TFF1 expression and consequently the regression of ER positive breast tumors. This work was supported by science research funds from Arnold and Marie Schwartz College of Pharmacy, Long Island University, NY.

Phase Ib, multicenter, single-blinded, placebo-controlled, sequential dose-escalation study to assess the safety and tolerability of topically applied AG013 in subjects with locally advanced head and neck cancer (LAHNC) receiving induction chemotherapy (ICT).

9024 Background: Oral mucositis (OM) is a significant toxicity of ICT in LAHNC. AG013, a mouth rinse composed of a recombinant Lactococcus lactis (sAGX0085) engineered to secrete human Trefoil Factor 1 (hTFF1) and deficient in the gene encoding thymidylate synthase, was investigated in patients receiving ICT (cisplatin, 5-fluorouracil +/-docetaxel:PF+/-T). TFFs have wound-healing properties and are protective of mucosal tissues. Methods: To evaluate the safety, tolerability, PK profile and efficacy of AG013 in attenuating ICT-associated OM, 52 patients (pts) with newly diagnosed LAHNC receiving ICT (24TPF, 1PF) were screened in ICT cycle (Cy) 1 for symptomatic OM (SOM). 25 pts who developed SOM in Cy1 were enrolled into 3 successive groups of at least 7 pts each in the active phase and randomly assigned at the Baseline visit (d1 of ICT Cy2) in a 5:2 ratio to receive AG013 or placebo during ICT Cy2. Dose was escalated in successive groups from 1 x/day (qd) to 3 x/day (tid) and to 6 x/day, resulting in doses of 2 x 1011, 6 x 1011, and 1.2 x 1012 colony-forming units (CFU)/day respectively. Pts were assessed daily from Cy 2 d1 to d14 for OM using WHO criteria. Safety endpoints and PK (mucosal, salivary and blood samples) were assessed. Results: AG013-sAGX0085 could not be detected in blood. Recovered oral live bacterial levels were high immediately after dosing, decreased by 90 minutes post dose, and undetectable at study end. No differences in hTFF1 serum levels were seen. Pts in the placebo group had ulcerative OM (UOM) on nearly 60% of the days vs. 35-40% days in the AG013 group. 29 % pts who received AG013 had 0 or 1 day of UOM vs. at least 2 days of UOM in the placebo group. Pts who received AG013 on any dosing schedule had a lower % of days with OM, fewer unplanned office and emergency room visits compared to pts who received placebo. No differences were noted in mouth and throat soreness, opioid use, or gastrostomy tube placement. Conclusions: Topical administration of AG013 appears safe, well tolerated and effective in reducing the severity and course of OM in patients receiving ICT for HNC.

Bacterial expression and purification of biologically active human TFF2

Human trefoil factor 2 (hTFF2) is considered as one of the most important initiators of mucosal healing in the gastrointestinal tract by promoting cell migration and suppressing apoptosis. However, it is hard to obtain hTFF2 from human tissue and many recombinant hTFF2 produced in vitro exist as fusion proteins. The purpose of the present study was to produce native hTFF2 while maintaining its biological activities. The open reading frame of hTFF2 was inserted into a pET-32a(+) expression vector, and hTFF2-TRX fusion protein was successfully expressed in Escherichia coli and purified by Nickel-nitrilotriacetic acid affinity chromatography and reverse-phase HPLC steps. The recombinant fusion protein (purity>95%) was cleaved by Factor Xa at 23 Degrees Celsius to release hTFF2. After removal of Factor Xa and undigested fusion proteins, hTFF2 was purified and identified by SDS-PAGE and Western blotting. The yield of recombinant hTFF2 was about 5 mg/L. The recombinant hTFF2 could promote IEC-6 cells migration and in vitro wound healing via the activation of ERK1/2. Recombinant hTFF2 could also inhibit apoptosis of HCT-116 cells induced by 50 μmol/L ceramide. In summary, our results showed that the recombinant hTFF2 was expressed in E. coli and successfully purified after cleavage with the fusion partner with high yield while maintaining its biological activities. Recombinant hTFF2 might be useful for investigating the molecular mechanism of hTFF2 and development of hTFF2-related drugs.