Helicobacter pylori disrupts gastric mucosal homeostasis by stimulating macrophages to secrete CCL3.

BACKGROUND: Helicobacter pylori (H. pylori) is the predominant etiological agent of gastritis and disrupts the integrity of the gastric mucosal barrier through various pathogenic mechanisms. After H. pylori invades the gastric mucosa, it interacts with immune cells in the lamina propria. Macrophages are central players in the inflammatory response, and H. pylori stimulates them to secrete a variety of inflammatory factors, leading to the chronic damage of the gastric mucosa. Therefore, the study aims to explore the mechanism of gastric mucosal injury caused by inflammatory factors secreted by macrophages, which may provide a new mechanism for the development of H. pylori-related gastritis. METHODS: The expression and secretion of CCL3 from H. pylori infected macrophages were detected by RT-qPCR, Western blot and ELISA. The effect of H. pylori-infected macrophage culture medium and CCL3 on gastric epithelial cells tight junctions were analyzed by Western blot, immunofluorescence and transepithelial electrical resistance. EdU and apoptotic flow cytometry assays were used to detect cell proliferation and apoptosis levels. Dual-luciferase reporter assays and chromatin immunoprecipitation assays were used to study CCL3 transcription factors. Finally, gastric mucosal tissue inflammation and CCL3 expression were analyzed by hematoxylin and eosin staining and immunohistochemistry. RESULTS: After H. pylori infection, CCL3 expressed and secreted from macrophages were increased. H. pylori-infected macrophage culture medium and CCL3 disrupted gastric epithelial cells tight junctions, while CCL3 neutralizing antibody and receptor inhibitor of CCL3 improved the disruption of tight junctions between cells. In addition, H. pylori-infected macrophage culture medium and CCL3 recombinant proteins stimulated P38 phosphorylation, and P38 phosphorylation inhibitor improved the disruption of tight junctions between cells. Besides, it was identified that STAT1 was a transcription factor of CCL3 and H. pylori stimulated macrophage to secret CCL3 through the JAK1-STAT1 pathway. Finally, after mice were injected with murine CCL3 recombinant protein, the gastric mucosal injury and inflammation were aggravated, and the phosphorylation level of P38 was increased. CONCLUSIONS: In summary, our findings demonstrate that H. pylori infection stimulates macrophages to secrete CCL3 via the JAK1-STAT1 pathway. Subsequently, CCL3 damages gastric epithelial tight junctions through the phosphorylation of P38. This may be a novel mechanism of gastric mucosal injury in H. pylori-associated gastritis.

ID1 expressing macrophages support cancer cell stemness and limit CD8+ T cell infiltration in colorectal cancer.

Elimination of cancer stem cells (CSCs) and reinvigoration of antitumor immunity remain unmet challenges for cancer therapy. Tumor-associated macrophages (TAMs) constitute the prominant population of immune cells in tumor tissues, contributing to the formation of CSC niches and a suppressive immune microenvironment. Here, we report that high expression of inhibitor of differentiation 1 (ID1) in TAMs correlates with poor outcome in patients with colorectal cancer (CRC). ID1 expressing macrophages maintain cancer stemness and impede CD8+ T cell infiltration. Mechanistically, ID1 interacts with STAT1 to induce its cytoplasmic distribution and inhibits STAT1-mediated SerpinB2 and CCL4 transcription, two secretory factors responsible for cancer stemness inhibition and CD8+ T cell recruitment. Reducing ID1 expression ameliorates CRC progression and enhances tumor sensitivity to immunotherapy and chemotherapy. Collectively, our study highlights the pivotal role of ID1 in controlling the protumor phenotype of TAMs and paves the way for therapeutic targeting of ID1 in CRC.

Expression and Significance of N-myc downstream regulated gene 2 in the process of Esophageal Squamous Cell Carcinogenesis.

It has been reported that the expression of tumor suppressor gene N-myc downstream-regulated gene 2 (NDRG2) was significantly reduced in human solid tumors, including esophageal squamous cell carcinoma (ESCC). This study aimed to explore whether the difference of NDRG2 expression exists in different stages of ESCC and provides a basis for the early diagnosis and prognosis of ESCC. Immunohistochemical staining was used to investigate the expression level of NDRG2 in samples from 91 patients with mild-to-moderate dysplasia, early ESCC, and advanced ESCC. The relationship between the expression of NDRG2 and clinicopathological characteristics of the patients was analyzed. The results showed that positive expression rates of NDRG2 in tissues adjacent to early ESCC (76.7%), or from mild-to-moderate dysplasia (74.1%), and early ESCC (83.3%) were significantly higher than in tissue from advanced ESCC (55.9%). The positive expression rate in advanced ESCC was significantly lower than in the other three tissue types (p < 0.05). There was a significant difference (p < 0.05) and correlation (Cramer's V = 0.351, p = 0.019, <0.05) between the expression of NDRG2 and the clinical stage in the 64 patients with ESCC. In conclusion, this study found that the expression of NDRG2 gradually decreased with the progression of esophageal lesions into advanced ESCC. This difference in positive expression rate was more obvious in male patients and patients under 60 years of age. Therefore, the detection of NDRG2 plays an important role in differentiating early ESCC from advanced ESCC.

TRIB3 reduces CD8+ T cell infiltration and induces immune evasion by repressing the STAT1-CXCL10 axis in colorectal cancer.

High CD8+ T cell infiltration in colorectal cancer (CRC) should suggest a favorable prognosis and a satisfactory response to immunotherapy; however, the vast majority of patients with CRC do not benefit from immunotherapy due to poor T cell infiltration. Therefore, a better understanding of the mechanisms for T cell exclusion from CRC tumors is needed. Tribbles homolog 3 (TRIB3) has been implicated as an oncoprotein, but its role in regulating antitumor immune responses has not been defined. Here, we demonstrated that TRIB3 inhibits CD8+ T cell infiltration in various CRC mouse models. We showed that TRIB3 was acetylated by acetyltransferase P300, which inhibited ubiquitination and subsequent proteasomal degradation of TRIB3. Ectopically expressed TRIB3 inhibited signal transducer and activator of transcription 1 (STAT1) activation and STAT1-mediated CXCL10 transcription by enhancing the epidermal growth factor receptor signaling pathway, causing a reduction in tumor-infiltrating T cells. Genetic ablation of Trib3 or pharmacological acceleration of TRIB3 degradation with a P300 inhibitor increased T cell recruitment and sensitized CRCs to immune checkpoint blockade therapy. These findings identified TRIB3 as a negative modulator of CD8+ T cell infiltration in CRCs, highlighting a potential therapeutic target for treating immunologically "cold" CRCs.

Rictor Activates Cav 1 Through the Akt Signaling Pathway to Inhibit the Apoptosis of Gastric Cancer Cells.

BACKGROUND: Rapamycin-insensitive companion of mammalian target of rapamycin (Rictor) protein is a core subunit of mammalian target of rapamycin complex 2, and is associated with cancer progression. However, the biological function of Rictor in cancer, particularly its clinical relevance in gastric cancer (GC) remains largely unknown. METHODS: Rictor expression and its association with clinicopathologic characteristics in GC were analyzed by immunohistochemistry. Effect of Rictor and Caveolin-1 (Cav 1) on GC cells apoptosis was evaluated via overexpression experiment in vitro. Mechanisms of Rictor and Cav 1 in GC were explored through overexpression and knockdown, by immunofluorescence and western blot analyses. RESULTS: Rictor was upregulated in GC, and mainly located in the cytoplasm of cancer cells. Moreover, higher Rictor levels were associated with worse prognosis. Rictor could inhibit GC cell apoptosis and promote cell growth in vitro. The results of immunofluorescence revealed that Cav 1 localized in GC cell membrane but did not co-localize with Rictor. Further, Rictor regulated apoptosis-related proteins, long non-coding RNAs and also activated cellular signaling, thereby positively regulating Cav 1 expression. This effect was attenuated by the Akt inhibitor ly294002. Cav 1 did not significantly affect the ability of Rictor to inhibit tumor cell apoptosis. CONCLUSIONS: Rictor is upregulated in GC and associated with worse prognosis. It inhibits tumor apoptosis and activates Cav 1 through the Akt signaling pathway to inhibit the apoptosis of GC cells. Rictor is, therefore, a promising prognostic biomarker and possible therapeutic target in GC patients.

Modulation of gut microbiota protects against viral respiratory tract infections: a systematic review of animal and clinical studies.

BACKGROUND: Earlier studies suggest that probiotics have protective effects in the prevention of respiratory tract infections (RTIs). Whether such benefits apply to RTIs of viral origin and mechanisms supporting the effect remain unclear. AIM: To determine the role of gut microbiota modulation on clinical and laboratory outcomes of viral RTIs. METHODS: We conducted a systematic review of articles published in Embase and MEDLINE through 20 April 2020 to identify studies reporting the effect of gut microbiota modulation on viral RTIs in clinical studies and animal models. The incidence of viral RTIs, clinical manifestations, viral load and immunological outcomes was evaluated. RESULTS: We included 58 studies (9 randomized controlled trials; 49 animal studies). Six of eight clinical trials consisting of 726 patients showed that probiotics administration was associated with a reduced risk of viral RTIs. Most commonly used probiotics were Lactobacillus followed by Bifidobacterium and Lactococcus. In animal models, treatment with probiotics before viral challenge had beneficial effects against influenza virus infection by improving infection-induced survival (20/22 studies), mitigating symptoms (21/21 studies) and decreasing viral load (23/25 studies). Probiotics and commensal gut microbiota exerted their beneficial effects through strengthening host immunity. CONCLUSION: Modulation of gut microbiota represents a promising approach against viral RTIs via host innate and adaptive immunity regulation. Further research should focus on next generation probiotics specific to viral types in prevention and treatment of emerging viral RTIs.

Challenge in the new era: Translational medicine in gastrointestinal endoscopy and early cancer.

Translational medicine is a new medical model that has emerged over the past 20 years and is dedicated to bridging the gap between basic and clinical research. At the same time, the diagnosis and treatment of digestive diseases, especially gastrointestinal endoscopy, have been rapidly developed. The emergence of new techniques for gastrointestinal endoscopy has changed the therapeutic spectrum of some diseases and brought huge benefits to patients. Targeted therapy has positively affected the individualized and precise treatment of patients with advanced gastrointestinal cancer. The construction of a standardized biobank provides a strong guarantee for clinicians to conduct translational medical research. Translational medicine has brought good development opportunities, but it also faces challenges. The training of translational medicine researchers and the transformation of educational models require sufficient attention for further development.

Inhibitory Role of Pentraxin-3 in Esophageal Squamous Cell Carcinoma.

BACKGROUND: Esophageal cancer is the sixth leading cause of cancer-related death worldwide. Pentraxin-3 (PTX3) is a member of the PTX superfamily. Here, we investigated the role of PTX3 in esophageal squamous cell carcinoma (ESCC). METHODS: The effect of PTX3 on ESCC cell proliferation, colony formation, apoptosis, migration, and invasion was investigated using cell viability assays, colony formation assays, flow cytometry, and migration and invasion assays. The effect of PTX3 on the tumorigenicity of ESCC in vivo was investigated with xenograft studies in nude mice. RESULTS: PTX3 overexpression in ESCC cells reduced cellular proliferation and colony formation (P < 0.05) and increased the rate of apoptosis (P < 0.05). PTX3 expression had no significant effect on the migratory or invasive potential of ESCC cells. In our mouse model of human ESCC, we achieved 100% successful tumor establishment. Compared with the control and empty vector-expressing groups, the PTX3-expressing group formed significantly smaller tumors (P < 0.05). CONCLUSIONS: This study indicates that PTX3 might play an inhibitory role in ESCC.

MiR-26a and miR-144 inhibit proliferation and metastasis of esophageal squamous cell cancer by inhibiting cyclooxygenase-2.

The altered expression of miRNAs is involved in carcinogenesis of esophageal squamous cell carcinoma (ESCC), but whether miRNAs regulate COX-2 expression in ESCC is not clear. To this end, the expression levels of miR-26a and miR-144 in ESCC clinical tissues and cell lines were investigated by qRT-PCR. COX-2 and PEG2 were quantified by western blot and ELISA. Decrease in miR-26a and miR-144 expression in ESCC was found by a comparison between 30 pairs of ESCC tumor and adjacent normal tissues as well as in 11 ESCC cell lines (P < 0.001). Co-transfection of miR-26a and miR-144 in ESCC cell lines more significantly suppressed cell proliferation, migration, and invasion than did either miR-26a or miR-144 alone (all P < 0.001), as shown by assays of CCK8, migration and invasion and flow cytometry. The inhibitory effect of these two miRNAs in vivo was also verified in nude mice xenograft models. COX-2 was confirmed as a target of miR-26a and miR-144. In conclusion, miR-26a and miR-144 expression is downregulated in ESCC. Co-expression of miR-26a and miR-144 in ESCC cells resulted in inhibition of proliferation and metastasis in vitro and in vivo, suggesting that targeting COX-2 may be the mechanism of these two miRNAs.

Cyclooxygenase-2, a Potential Therapeutic Target, Is Regulated by miR-101 in Esophageal Squamous Cell Carcinoma.

BACKGROUND & AIMS: Cyclooxygenase-2 (COX-2) is known to promote the carcinogenesis of esophageal squamous cell carcinoma (ESCC). There are no reports on whether microRNAs (miRNAs) regulate COX-2 expression in ESCC. This study investigated the effect of miR-101 on ESCC through modulating COX-2 expression in ESCC. METHODS: Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify miR-101 expression in ESCC clinical tissues and cell lines. The effects of miR-101 on ESCC progression were evaluated by cell counting kit-8 (CCK8), transwell migration and invasion assays, as well as by flow cytometry. The COX-2 and PEG2 levels were determined by western blot and enzyme-linked immunosorbent assays (ELISA). The luciferase reporter assay was used to verify COX-2 as a direct target of miR-101. The anti-tumor activity of miR-101 in vivo was investigated in a xenograft nude mouse model of ESCC. RESULTS: Downregulation of miR-101 was confirmed through comparison of 30 pairs of ESCC tumor and adjacent normal tissues (P < 0.001), as well as in 11 ESCC cell lines and a human immortalized esophageal cell line (P < 0.001). Transfection of miR-101 in ESCC cell lines significantly suppressed cell proliferation, migration, and invasion (all P < 0.001). The antitumor effect of miR-101 was verified in a xenograft model. Furthermore, COX-2 was shown to be a target of miR-101. CONCLUSIONS: Overexpression of miR-101 in ESCC inhibits proliferation and metastasis. Therefore, the miR-101/COX-2 pathway might be a therapeutic target in ESCC.

EGF Suppresses the Initiation and Drives the Reversion of TGF-ß1-induced Transition in Hepatic Oval Cells Showing the Plasticity of Progenitor Cells.

Transforming growth factor-ß1 (TGF-ß1) induces hepatic progenitors to tumor initiating cells through epithelial-mesenchymal transition (EMT), thus raising an important drawback for stem cell-based therapy. How to block and reverse TGF-ß1-induced transition is crucial for progenitors' clinical application and carcinogenic prevention. Rat adult hepatic progenitors, hepatic oval cells, experienced E-cadherin to N-cadherin switch and changed to α-smooth muscle actin (α-SMA) positive cells after TGF-ß1 incubation, indicating EMT. When TGF-ß1 plus EGF were co-administrated to these cells, EGF dose-dependently suppressed the cadherin switch and α-SMA expression. Interestingly, if EGF was applied to TGF-ß1-pretreated cells, the cells that have experienced EMT could return to their epithelial phenotype. Abruption of EGF receptor revealed that EGF exerted its blockage and reversal effects through phosphorylation of ERK1/2 and Akt. These findings suggest an important attribute of EGF on opposing and reversing TGF-ß1 effects, indicating the plasticity of hepatic progenitors.

Association of single nucleotide polymorphisms in the prostaglandin-endoperoxide synthase 2 (PTGS2) and phospholipase A2 group IIA (PLA2G2A) genes with susceptibility to esophageal squamous cell carcinoma.

BACKGROUND: The prostaglandin-endoperoxide synthase 2 (PTGS2) and phospholipase A2 group IIA (PLA2G2A) genes encode enzymes that are involved in arachidonic acid and prostaglandin biosynthesis. Dysregulation of both genes is associated with inflammation and carcinogenesis, including esophageal squamous cell carcinoma (ESCC). We therefore hypothesized that there is an association between single nucleotide polymorphisms (SNPs) in these genes and susceptibility to ESCC. METHODS: We performed a gene-wide tag SNP-based association study to examine the association of SNPs in PTGS2 and PLA2G2A with ESCC in 269 patients and 269 healthy controls from Taihangshan Mountain, Henan and Hebei Provinces, the rural area of China which has the highest incidence of esophageal cancer in the world. Thirteen tag SNPs in PLA2G2A and 4 functional SNPs in PTGS2 were selected and genotyped using a high-throughput Mass Array genotyping platform. RESULTS: We found a modest increased risk of ESCC in subjects with the PTGS2 rs12042763 AA genotype (OR=1.23; 95% CI, 1.00- 3.04) compared with genotype GG. For PLA2G2A, a decreased risk of ESCC was observed in subjects with the rs11677 CT (OR=0.51, 95%CI, 0.29-0.85) or TT genotype (OR=0.51, 95%CI, 0.17-0.96) or the T carriers (CT+TT) (OR=0.52, 95%CI, 0.31-0.85) when compared with the CC genotype. Also for PLA2G2A, rs2236771 C allele carriers were more frequent in the control group (P=0.02). Subjects with the GC (OR=0.55, 95%CI, 0.33-0.93) or CC genotype (OR=0.38, 95% CI, 0.16-0.94) or the C carriers (GC+CC) (OR=0.52, 95%CI, 0.32- 0.85) showed a negative association with ESCC susceptibility. CONCLUSIONS: Our results suggest that PTGS2 and PLA2G2A gene polymorphisms may modify the risk of ESCC development.

Serum peptide mapping in gastric precancerous lesion and cancer.

OBJECTIVE: To construct and verify a diagnostic model using proteomic analysis of serum samples for identifying gastric precancerous lesions and gastric cancer (GC). METHODS: The serum samples from 25 patients with gastric precancerous lesions (chronic atrophic gastritis with mild to moderate dysplasia), 25 GC patients and 25 healthy controls were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Spectral peaks with significant difference among the groups were identified and used as a diagnostic model for detecting gastric precancerous lesions and GC. The serum peptide map model was validated using an independent sample set including 15 healthy volunteers, 15 precancerous and 15 GC patients. RESULTS: The spectral peaks for the peptides with mass-to-charge (m/z) values of 1741 and 4210 were the most significantly different among the three groups. The sensitivity of this diagnostic model for detecting healthy controls, patients with gastric precancerous lesions and patients with GC was 80.0% (12/15), 66.7% (10/15) and 66.7% (10/15) respectively, while the specificity was 66.7% (20/30), 73.3% (22/30) and 73.3% (22/30), respectively. CONCLUSION: Our diagnostic model is useful for diagnosing gastric precancerous lesions and GC.

SULF1 inhibits proliferation and invasion of esophageal squamous cell carcinoma cells by decreasing heparin-binding growth factor signaling.

BACKGROUND: Heparin-binding growth factor signaling is involved in the pathogenesis and development of human cancers. It can be regulated by sulfation of cell-surface heparan sulfate proteoglycans (HSPG). SULF1 is a heparin-degrading endosulfatase which can modulate the sulfation of HSPGs. AIM: The purpose of this study was to elucidate the role of SULF1 in modulating proliferation and invasion of esophageal squamous cell carcinoma (ESCC) by decreasing heparin-binding growth factor signaling. METHODS: We restored SULF1 expression in the ESCC cell line KYSE150, and examined the effects of SULF1 expression on the proliferation and invasion of KYSE150 cells. In addition, we investigated the expression of SULF1 in human ESCC tissues and analyzed the correlation of SULF1 expression with clinicopathologic characteristics of ESCC. RESULTS: Our study shows that re-expression of SULF1 in ESCC cell line results in the downregulation of hepatocyte growth factor-mediated activation of MAPK pathways with a resultant decrease in cell invasiveness. Cell proliferation was also inhibited in SULF1-transfected KYSE150 cells. Immunohistochemical assays reveal that SULF1 is expressed in nearly half of the human ESCC tissues but not in normal esophageal epithelial cells. SULF1 expression in human ESCC tissues is negatively correlated with tumor size and tumor invasion. CONCLUSION: This study identified that SULF1 inhibits proliferation and invasion of ESCC by decreasing heparin-binding growth factor signaling and suggested that SULF1 plays an inhibiting role in the pathogenesis of ESCC.

Association between gastric cancer and -1993 polymorphism of TBX21 gene.

AIM: To investigate the association between the polymorphism of TBX21 gene and the risk of gastric cancer in a Chinese population. METHODS: The -1993 polymorphism located in TBX21 gene promoter region was identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The risk between TBX21 gene genotype and gastric cancer was determined by multivariate logistic regression analysis in 220 gastric cancer patients and 262 cancer-free controls matched by age, sex and ethnicity. RESULTS: Compared with the TBX21 -1993TT genotype, the -1993CC genotype exhibited a significantly elevated risk for gastric cancer [Odds ratio (OR) = 3.42, 95% confidence interval (CI): 1.41-8.31]. The relationship between the -1993 polymorphic genotype and the invasive status such as lymph node and distant metastasis was found among the gastric cancer patients (OR = 4.02, 95% CI: 1.87-8.66; OR = 7.02, 95% CI: 3.44-14.34, respectively). CONCLUSION: TBX21 -1993 polymorphism might contribute to the risk of gastric cancer, especially to the distant metastasis.

Promoter methylation regulates cigarette smoke-stimulated cyclooxygenase-2 expression in esophageal squamous cell carcinoma.

OBJECTIVE: Cigarette smoke extracts (CSE) could promote esophageal squamous cell carcinoma (ESCC) through upregulation of cyclooxygenase-2 (COX-2) expression. Promoter methylation mediates the transcriptional modulation of the COX-2 gene. The aim of the study was to explore whether COX-2 promoter methylation regulated COX-2 expression and functional activity in ESCC exposed to CSE. METHODS: The methylation status of COX-2 promoter in two human ESCC cell lines, EC109 and TE-1, was examined using bisulfite sequencing analysis. COX-2 mRNA and protein expression were detected by reverse-transcription polymerase chain reaction and Western blot. Prostaglandin E2 (PGE2) was examined by enzyme linked immunosorbent assay (ELISA). RESULTS: The promoter was hypermethylated in TE-1 which had a low level of COX-2 expression and was hypomethylated in EC109 with a relatively high level of COX-2 expression. Stimulation by cigarette smoke ethanol extract (EE) resulted in increased COX-2 expression in EC109, but not in TE-1. Treatment with 5-aza-2'-deoxycytidine (5-aza-DC) demethylated the promoter and upregulated COX-2 expression as well as PGE(2) production in TE-1, especially followed by EE stimulation. No significant effect was observed in EC109. CONCLUSION: These findings suggest that promoter methylation may be one of the mechanisms regulating COX-2 expression in ESCC in response to stimulation of CSE.

Epigenetic inactivation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma.

AIM: To investigate the expression and methylation status of the secreted frizzled-related protein 2 (SFRP2) in esophageal squamous cell carcinoma (ESCC) and explore its role in ESCC carcinogenesis. METHODS: Seven ESCC cell lines (KYSE 30, KYSE150, KYSE410, KYSE510, EC109, EC9706 and TE-1) and one immortalized human esophageal epithelial cell line (Het-1A), 20 ESCC tissue samples and 20 paired adjacent non-tumor esophageal epithelial tissues were analyzed in this study. Reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of SFRP2 in cell lines, primary ESCC tumor tissue, and paired adjacent normal tissue. Methylation status was evaluated by methylation-specific PCR and bisulfite sequencing. The correlation between expression and promoter methylation of the SFRP2 gene was confirmed with treatment of 5-aza-2'-deoxycytidine. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2-transfected cells and examined them with regard to cell proliferation, colony formation, apoptosis and cell cycle in vivo and in vitro. RESULTS: SFRP2 mRNA was expressed in the immortalized normal esophageal epithelial cell line but not in seven ESCC cell lines. By methylation-specific PCR, complete methylation was detected in three cell lines with silenced SFRP2 expression, and extensive methylation was observed in the other four ESCC cell lines. 5-aza-2'-deoxycytidine could restore the expression of SFRP2 mRNA in the three ESCC cell lines lacking SFRP2 expression. SFRP2 mRNA expression was obviously lower in primary ESCC tissue than in adjacent normal tissue (0.939 ± 0.398 vs 1.51 ± 0.399, P < 0.01). SFRP2 methylation was higher in tumor tissue than in paired normal tissue (95% vs 65%, P < 0.05). The DNA methylation status of the SFRP2 correlated inversely with the SFRP2 expression. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2 transfectants and control counterparts by introducing pcDNA3.1/v5 hisA -SFRP2 or pcDNA3.1/v5 hisA -empty vector into KYSE30 cells lacking SFRP2 expression. After transfection, the forced-expression of SFRP2 was confirmed by the RT-PCR. In comparison with the control groups, stably-expressed SFRP2 in KYSE 30 cells significantly reduced colony formation in vitro (47.17% ± 15.61% vs 17% ± 3.6%, P = 0.031) and tumor growth in nude mice (917.86 ± 249.35 mm(3)vs 337.23 ± 124.43 mm(3), P < 0.05). Using flow cytometry analysis, we found a significantly higher number of early apoptotic cells in SFRP2-transfected cells than in the control cells (P = 0.025). The mean cell number in the S and G2-M phases of the cell cycle was also significantly lower in SFRP2-transfected KYSE30 cells compared with mock transfected counterparts. CONCLUSION: Silencing of SFRP2 expression through promoter hypermethylation may be a factor in ESCC carcinogenesis through loss of its tumor-suppressive activity.

Aberrant methylation of the 3q25 tumor suppressor gene PTX3 in human esophageal squamous cell carcinoma.

AIM: To identify the novel methylation-silenced gene pentraxin 3 (PTX3) in esophageal squamous cell carcinoma (ESCC). METHODS: PTX3 mRNA expression was examined in six human ESCC cell lines, one human immortalized normal esophageal epithelial cell line, primary ESCC tumor tissue, and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction (RT-PCR). Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels. Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene. RESULTS: In the majority of ESCC cell lines, we found that PTX3 expression was down-regulated due to gene promoter hypermethylation, which was further confirmed by bisulphite genomic sequencing. Demethylation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines. Methylation was more common in tumor tissues (85%) than in adjacent nontumor tissues (25%) (P < 0 .01). CONCLUSION: PTX3 is down-regulated through promoter hypermethylation in ESCC, and could potentially serve as a biomarker of ESCC.

Dual inhibition of 5-LOX and COX-2 suppresses esophageal squamous cell carcinoma.

COX-2 and 5-LOX are up-regulated in ESCC. This study aims to determine the efficacy of COX-2 inhibitor, 5-LOX inhibitor and their combination on ESCC. Nimesulide can suppress cell growth and promote apoptosis, accompanied with a decrease of PGE(2) production. AA861 has the similar effect with a down-regulation of LTB(4). In animal experiment, the tumor volumes in drug-treated groups were significantly smaller with the lowest rates of Ki-67 positive cells. In conclusion, either COX-2 inhibitor or 5-LOX inhibitor can suppress ESCC. Dual inhibition of COX-2 and 5-LOX pathway may present a superior anticancer efficacy to either inhibition of COX-2 or 5-LOX alone.

Epigenetic inactivation of the SFRP1 gene in esophageal squamous cell carcinoma.

INTRODUCTION: The secreted frizzled-related protein 1 (SFRP1) gene, as a Wnt signaling modulator, is frequently inactivated by promoter methylation in many tumors including gastric cancer, breast cancer, oral squamous cell carcinoma, and esophageal adenocarcinoma. However, the role of SFRP1 in esophageal squamous cell carcinoma (ESCC) is not clear. In this study, we investigated the epigenetic inactivation of the SFRP1 gene in ESCC. METHODS: Nine ESCC cell lines, two immortalized human esophageal epithelial cell lines, twenty ESCC tissues, and paired adjacent nontumor tissues were analyzed in the study. Methylation-specific polymerase chain reaction (PCR), bisulfite sequencing, reverse-transcription PCR, immunohistochemistry, and chromatin immunoprecipitation assay were used to detect SFRP1 promoter methylation, expression of the SFRP1 gene, and histone modification in the SFRP1 promoter region. RESULTS: The SFRP1 promoter was found to be highly methylated in 95% (19/20) of the ESCC tissues and in nine ESCC cell lines, compared with 65% (13/20) of the paired nontumor tissues. Moreover, we confirmed that complete methylation of the SFRP1 gene promoter was correlated with its greatly reduced expression level. After individual treatment with 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA), the messenger RNA (mRNA) level of the SFRP1 gene was not obviously rescued in the EC9706 cell line. Combined incubation with DAC and TSA can, however, substantially increase the SFRP1 mRNA expression level in the EC9706 cell line. Chromatin immunoprecipitation assay showed that acetylated histone H3 and H4 were found in the SFRP1 promoter region. CONCLUSION: Promoter hypermethylation of SFRP1 is a frequent event in ESCC. Promoter methylation and histone acetylation may cooperatively regulate expression of the SFRP1 gene.