Hui Ying Zhang

Gastroesophageal Reflux Might Cause Esophagitis Through a Cytokine-Mediated Mechanism Rather Than Caustic Acid Injury

BACKGROUND & AIMS Reflux esophagitis is believed to be caused by the caustic effects of refluxed gastric acid on esophageal epithelial cells. However, caustic chemical injuries develop rapidly whereas esophagitis might not appear until weeks after the induction of reflux in animal models. We studied early histologic events in the development of reflux esophagitis in a rat model and performed in vitro experiments to determine whether exposure to acidified bile salts causes esophageal epithelial cells to secrete chemokines that might contribute to inflammation. METHODS At various time points after esophagoduodenostomy, the rat esophagus was removed and inflammatory changes were analyzed by histologic analyses. Human esophageal squamous cell lines were exposed to acidified bile salts to evaluate their effects on cytokine production and immune-cell migration. RESULTS Reflux esophagitis started at postoperative day 3 with lymphocytic infiltration of the submucosa that progressed to the epithelial surface-these findings contradicted those expected from a caustic chemical injury. Basal cell and papillary hyperplasia preceded the development of surface erosions. Exposure of squamous cells to acidified bile salts significantly increased the secretion of interleukin-8 and interleukin-1beta; conditioned media from these cells caused significant increases in the migration rates of T cells and neutrophils. CONCLUSIONS These findings support, but do not prove, an alternative concept for the development of reflux esophagitis in which refluxed gastric juice does not directly damage the esophagus, but rather stimulates esophageal epithelial cells to secrete chemokines that mediate damage of esophageal tissue.

Acid and Bile Salt–Induced CDX2 Expression Differs in Esophageal Squamous Cells From Patients With and Without Barrett's Esophagus

BACKGROUND & AIMS It is not clear why only a minority of patients with gastroesophageal reflux disease (GERD) develop Barretts esophagus. We hypothesized that differences among individuals in molecular pathways activated when esophageal squamous epithelium is exposed to reflux underlie the development of Barretts metaplasia. METHODS We used esophageal squamous cell lines from patients who had GERD with Barretts esophagus (normal esophageal squamous [NES]-B3T and NES-B10T) and without Barretts esophagus (NES-G2T and NES-G4T) to study effects of acid and bile salts on expression of the CDX2 gene. Bay 11-705, Ad5 inhibitor kappaB(IkappaB)alpha-SR, and site-directed mutagenesis were used to explore effects of nuclear factor-kappaB (NF-kappaB) inhibition on CDX2 promoter activity; DNA binding of the NF-kappaB subunits p50 and p65 was assessed by chromatin immune-precipitation. RESULTS Acid and bile salts increased CDX2 messenger RNA (mRNA), protein, and promoter activity in NES-B3T and NES-B10T cells, but not in NES-G2T or NES-G4T cells. Inhibition of NF-kappaB abolished the increase in CDX2 promoter activity. Increased CDX2 promoter activity was associated with nuclear translocation of p50, which bound to the promoter. We found CDX2 mRNA in 7 of 10 esophageal squamous biopsy specimens from patients with Barretts esophagus, but in only 1 of 10 such specimens from patients who had GERD without Barretts esophagus. CONCLUSIONS Acid and bile salts induce CDX2 mRNA and protein expression in esophageal squamous cells from patients with Barretts esophagus, but not from GERD patients without Barretts esophagus. We speculate that these differences in acid- and bile salt-induced activation of molecular pathways may underlie the development of Barretts metaplasia.

Esophageal adenocarcinoma arising in Barrett esophagus

The major risk factors for esophageal adenocarcinoma are gastroesophageal reflux disease (GERD) and Barrett esophagus, a squamous-to-columnar cell metaplasia that predisposes to malignancy. Adenocarcinomas in Barrett esophagus are thought to arise through a sequence of growth-promoting, genetic alterations that accumulate until the cells have acquired the physiologic hallmarks of cancer proposed by Hanahan and Weinberg. Moreover, GERD and Barrett esophagus are associated with chronic esophagitis, and inflammation is a well known risk factor for cancer formation. The cell that gives rise to Barrett metaplasia is not known. It has been proposed that the metaplasia may arise from a change in the differentiation pattern of stem cells that either reside in the esophagus or are recruited to the esophagus from the bone marrow. Alternatively, it is possible that Barrett metaplasia develops through the conversion of one differentiated cell type into another. Regardless of the cell of origin, Barrett metaplasia ultimately must be sustained by stem cells, which might be identified by intestinal stem cell markers. An emerging concept in tumor biology is that cancer stem cells are responsible for sustaining tumor growth. If Barrett cancers develop from Barrett stem cells, then a therapy targeted at those stem cells might prevent esophageal adenocarcinoma. This report reviews the risk factors for Barrett esophagus and esophageal adenocarcinoma, the mechanisms by which genetic alterations might contribute to carcinogenesis in Barrett esophagus, and the role of stem cells in the development of Barrett metaplasia and adenocarcinoma.

Acid has antiproliferative effects in nonneoplastic Barrett's epithelial cells.

OBJECTIVES:For patients with Barretts esophagus, physicians commonly prescribe antisecretory medications in dosages above those required to heal reflux esophagitis because acid has been shown to have proproliferative and antiapoptotic effects on Barretts cancer cells and on Barretts mucosal explants. For a number of reasons, these model systems may not be ideal for determining the effects of acid on benign Barretts epithelial cells, however. We studied the effects of acid on proliferation and apoptosis in a nonneoplastic, telomerase-immortalized Barretts epithelial cell line.METHODS:Barretts cells were treated with two 3-minute exposures to acidic media. Cell growth was determined using cell counts, proliferation was studied by flow cytometry, cell viability was determined by trypan blue staining, and apoptosis was assessed by TUNEL and Annexin V. The expression levels of p53 and p21 were determined by Western blotting. p53 siRNA was used to study the effect of p53 inhibition on total cell numbers after acid exposure.RESULTS:Acid exposure significantly decreased total cell numbers at 24 h without affecting either cell viability or apoptosis. Acid exposure resulted in cell cycle prolongation that was associated with greater expression of p53, but not p21. The acid-induced decrease in total cell numbers was abolished by p53 RNAi.CONCLUSIONS:Acid exposure has p53-mediated, antiproliferative effects in nonneoplastic Barretts epithelial cells. These findings contradict the results of prior in vitro and ex vivo studies. We speculate that the prescription of antisecretory medications in dosages beyond those required to heal gastroesophageal reflux disease (GERD) symptoms and endoscopic signs could be detrimental. Controlled, prospective clinical trials are needed to determine the optimal level of acid suppression for patients with Barretts esophagus.

Cancer-related inflammation and Barrett's carcinogenesis: interleukin-6 and STAT3 mediate apoptotic resistance in transformed Barrett's cells

Cancer-related inflammation recently has been proposed as a major physiological hallmark of malignancy. Some genetic alterations known to promote cellular proliferation and induce malignant transformation also may participate in an intrinsic inflammatory pathway that produces a cancer-promoting inflammatory microenvironment. Little is known about this intrinsic inflammatory pathway in Barretts esophagus. We have used a series of nontransformed and transformed human Barretts epithelial cell lines developed in our laboratory to explore the potential contribution of interleukin (IL)-6 and signal transducer and activator of transcription (STAT3) (key molecules in the intrinsic inflammatory pathway) to Barretts carcinogenesis. We determined IL-6 mRNA expression and protein secretion and protein expression of activated phospho-STAT3 and its downstream target myeloid cell leukemia (mcl)-1 (Mcl-1). We used an IL-6 blocking antibody and two JAK kinase inhibitors (AG490 and JAK inhibitor I) to assess whether STAT3 activation is IL-6 dependent. We also used small interfering RNAs (siRNAs) to STAT3 and Mcl-1 to assess effects of STAT3 pathway inhibition on apoptosis. Phospho-STAT3 was expressed only by transformed Barretts cells, which also exhibited higher levels of IL-6 mRNA and of IL-6 and Mcl-1 proteins than nontransformed Barretts cells. STAT3 phosphorylation could be blocked by IL-6 blocking antibody and by AG490 and JAK inhibitor I. In transformed Barretts cells, rates of apoptosis following exposure to deoxycholic acid were significantly increased by transfection with siRNAs for STAT3 and Mcl-1. We conclude that activation of the IL-6/STAT3 pathway in transformed Barretts epithelial cells enables them to resist apoptosis. These findings demonstrate a possible contribution of the intrinsic inflammatory pathway to carcinogenesis in Barretts esophagus.

Unlike Esophageal Squamous Cells, Barrett's Epithelial Cells Resist Apoptosis by Activating the Nuclear Factor-κB Pathway

Apoptosis is an important mechanism for maintaining tissue homeostasis and for preventing the proliferation of cells with mutations that could result in malignancy. Barretts epithelium has been reported to be more resistant to apoptosis than normal esophageal squamous epithelium. We have explored the contribution of the nuclear factor-kappaB (NF-kappaB) pathway to apoptotic resistance in non-neoplastic, telomerase-immortalized esophageal squamous (NES) and Barretts (BAR-T) epithelial cell lines. We exposed these cells to UV-B irradiation in doses known to cause DNA damage and to induce apoptosis in normal cells, and studied apoptosis as well as the expression of phospho-H2AX, NF-kappaB, Bcl-2, XIAP, cIAP-1, and survivin proteins. We also used Bay 11-7085 and siRNAs to NF-kappaB and Bcl-2 to assess the effects of NF-kappaB and Bcl2 inhibition on apoptosis. UV-B irradiation at low doses (50 and 100 J/m(2)) caused DNA damage in both NES and BAR-T cells but significantly increased apoptosis only in NES cells. UV-B irradiation caused a decrease in the levels of NF-kappaB, Bcl-2, cIAP-1, XIAP, and survivin in NES cells but increased the levels of those proteins in BAR-T cells. The resistance of BAR-T cells to apoptosis induced by low-dose UV-B irradiation was abolished by Bay 11-7085 and by siRNA for NF-kappaB and was decreased significantly by siRNA for Bcl-2. We conclude that the ability of Barretts epithelial cells to activate the NF-kappaB pathway when they have sustained DNA damage allows them to resist apoptosis. This capacity to avoid apoptosis despite genotoxic damage may underlie the persistence and malignant predisposition of Barretts metaplasia.

Differences in activity and phosphorylation of MAPK enzymes in esophageal squamous cells of GERD patients with and without Barrett's esophagus

We hypothesized that, in esophageal squamous epithelial cells, there are differences among individuals in the signal transduction pathways activated by acid reflux that might underlie the development of Barretts esophagus. To explore that hypothesis, we immortalized nonneoplastic, esophageal squamous cells from patients with gastroesophageal reflux disease (GERD) with (NES-B3T) and without (NES-G2T) Barretts esophagus and used those cells to study acid effects on MAPK proteins. During endoscopy in patients with GERD with and without Barretts esophagus, we took biopsy specimens from the distal squamous esophagus to study MAPK proteins before and after esophageal perfusion with 0.1 N HCl. We used immunoblotting and Western blotting to study MEK1/2 phosphorylation at two activating sites (serines 217/221), MEK1 phosphorylation at an inhibitory site (threonine 286), and MEK1/2 activity. After acid exposure, both cell lines exhibited increased MEK1/2 phosphorylation at the activating sites; the NES-B3T cells had higher levels of MEK1 phosphorylation at the inhibitory site, however, and only the NES-G2T cells showed an acid-induced increase in MEK1/2 activity. Similarly, in the squamous epithelium of patients with GERD with and without Barretts esophagus, acid perfusion increased MEK1/2 phosphorylation at the activating sites in both patient groups; the Barretts patients had higher levels of MEK1 phosphorylation at the inhibitory site, however, and only the patients without Barretts demonstrated an acid-induced increase in ERK1/2 phosphorylation. In esophageal squamous cell lines and biopsies from patients with GERD with and without Barretts esophagus, we have found differences in MAPK pathways activated by acid exposure. We speculate that these differences might underlie the development of Barretts metaplasia.

Targeting the intrinsic inflammatory pathway: honokiol exerts proapoptotic effects through STAT3 inhibition in transformed Barrett's cells.

One way to link chronic inflammation with cancer is through the intrinsic inflammatory pathway, in which genetic alterations that induce malignant transformation also produce a cancer-promoting, inflammatory microenvironment. Signal transducer and activator of transcription 3 (STAT3) contributes to the intrinsic inflammatory pathway in Barretts esophagus. In human tumors, honokiol (a polyphenol in herbal teas) has growth-inhibitory and proapoptotic effects associated with suppressed activation of STAT3. We used human Barretts epithelial and esophageal adenocarcinoma cell lines to determine effects of honokiol on cell number, necrosis, apoptosis, and anchorage-independent growth and to explore STAT3s role in those effects. We determined Ras activity and expression of phosphorylated ERK1/2, phosphorylated Akt, and phosphorylated STAT3 in the presence or absence of honokiol. Cells were infected with constitutively active Stat3-C to assess effects of honokiol-induced STAT3 inhibition on apoptosis. Honokiol decreased cell number and increased necrosis and apoptosis in transformed Barretts cells, but not in nontransformed cells. In adenocarcinoma cells, honokiol also increased necrosis and apoptosis and decreased anchorage-independent growth. Within 30 min of honokiol treatment, transformed Barretts cells decreased expression of phosphorylated STAT3; decreases in Ras activity and phosphorylated ERK1/2 expression were detected at 24 h. Infection with Stat3-C significantly reduced apoptosis after honokiol treatment. Honokiol causes necrosis and apoptosis in transformed Barretts and esophageal adenocarcinoma cells, but not in nontransformed Barretts cells, and the proapoptotic effects of honokiol are mediated by its inhibition of STAT3 signaling. These findings suggest a potential role for targeting the intrinsic inflammatory pathways as a therapeutic strategy to prevent Barretts carcinogenesis.

Malignant transformation of non-neoplastic Barrett's epithelial cells through well-defined genetic manipulations.

Background Human Barretts cancer cell lines have numerous, poorly-characterized genetic abnormalities and, consequently, those lines have limited utility as models for studying the early molecular events in carcinogenesis. Cell lines with well-defined genetic lesions that recapitulate various stages of neoplastic progression in Barretts esophagus would be most useful for such studies. Methodology/Principal Findings To develop such model cell lines, we started with telomerase-immortalized, non-neoplastic Barretts epithelial (BAR-T) cells, which are spontaneously deficient in p16, and proceeded to knock down p53 using RNAi, to activate Ras by introducing oncogenic H-RasG12V, or both. BAR-T cells infected with either p53 RNAi or oncogenic H-RasG12V alone maintained cell-to-cell contact inhibition and did not exhibit anchorage-independent growth in soft agar. In contrast, the combination of p53 RNAi knockdown with expression of oncogenic H-RasG12V transformed the p16-deficient BAR-T cells, as evidenced by their loss of contact inhibition, by their formation of colonies in soft agar, and by their generation of tumors in immunodeficient mice. Conclusions/Significance Through these experiments, we have generated a number of transformed and non-transformed cell lines with well-characterized genetic abnormalities recapitulating various stages of carcinogenesis in Barretts esophagus. These lines should be useful models for the study of carcinogenesis in Barretts esophagus, and for testing the efficacy of chemopreventive and chemotherapeutic agents.

W1966 Unlike Deoxycholic Acid, Ursodeoxycholic Acid Does Not Cause DNA Damage or Activate the NF-κB Pathway in Barrett's Metaplasia In Vitro and In Vivo

namely Hi-maize 958 and Hi-maize 260. Probiotic; control diet plus lyophilized cultures (1x10^11 cfu/g) of Bifidobacterium lactis at 1% by weight. Synbiotic; to each of the RS diets was added Bifidobacterium lactis at 1%. After 4 weeks on these nutritionally balanced diets, colonic neoplasms were induced by 2 weekly injections of azoxymethane (15 mg/kg B.W). Colons were resected after 30 weeks of feeding for evaluation of neoplasm formation, short chain fatty acids (SCFA), and epithelial biology. Results: Rats fed the synbiotic combination of RS with Bifidobacterium lactis significantly lowered the incidence and multiplicity of colonic neoplasms (P<0.01, using two-way factorial Poissonmodel) by over 50% compared to the Control group. There was a trend for protection by either RS alone (P=0.07) while no protection against colorectal cancer was seen in the group supplemented with only Bifidobacterium lactis. Fermentation events (SCFA, pH) were altered by the inclusion of RS into the diet while the inclusion of Bifidobacterium lactis into the diet had no significant effect on the fermentation parameters. Conclusion(s): The synbiotic combination of resistant starch and Bifidobacterium lactis significantly protects against the development of colorectal cancer in the rat-azoxymethane model to a degree not seen by the components. This suggests matching of prebiotic to probiotic might be important to maximise health benefit. (1) Le Leu, RK. et al.. Synbiotic combination of resistant starch and bifidobacterium lactis can facilitate apoptotic deletion of carcinogen-damaged cells in the rat colon. (2005) J. Nutr. 135 996-1001