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1.  Measuring telomere length for the early detection of precursor lesions of esophageal squamous cell carcinoma 
BMC Cancer  2013;13:578.
Esophageal cancer is the sixth leading cause of cancer death worldwide; current early detection screening tests are inadequate. Esophageal balloon cytology successfully retrieves exfoliated and scraped superficial esophageal epithelial cells, but cytologic reading of these cells has poor sensitivity and specificity for detecting esophageal squamous dysplasia (ESD), the precursor lesion of esophageal squamous cell carcinoma (ESCC). Measuring telomere length, a marker for chromosomal instability, may improve the utility of balloon cytology for detecting ESD and early ESCC.
We examined balloon cytology specimens from 89 asymptomatic cases of ESD (37 low-grade and 52 high-grade) and 92 age- and sex-matched normal controls from an esophageal cancer early detection screening study. All subjects also underwent endoscopy and biopsy, and ESD was diagnosed histopathologically. DNA was extracted from the balloon cytology cells, and telomere length was measured by quantitative PCR. A receiver operating characteristic (ROC) curve was plotted for telomere length as a diagnostic marker for high-grade dysplasia.
Telomere lengths were comparable among the low- and high-grade dysplasia cases and controls, with means of 0.96, 0.96, and 0.92, respectively. The area under the ROC curve was 0.55 for telomere length as a diagnostic marker for high-grade dysplasia. Further adjustment for subject characteristics, including sex, age, smoking, drinking, hypertension, and body mass index did not improve the use of telomere length as a marker for ESD.
Telomere length of esophageal balloon cytology cells was not associated with ESCC precursor lesions. Therefore, telomere length shows little promise as an early detection marker for ESCC in esophageal balloon samples.
PMCID: PMC3882883  PMID: 24308314
Esophageal squamous cell carcinoma; Esophageal squamous dysplasia; Early detection; Screening; Balloon cytology; Telomeres
2.  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 mm3 vs 337.23 ± 124.43 mm3, 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.
PMCID: PMC3280398  PMID: 22363119
Esophageal squamous cell carcinoma; Secreted frizzled-related protein 2; Methylation; Tumor suppressor gene; Wnt signaling pathway
3.  Detection of Esophageal Squamous Cell Carcinoma by Cathepsin B Activity in Nude Mice 
PLoS ONE  2014;9(3):e92351.
Background and Objective
Despite great progress in treatment, the prognosis for patients with esophageal squamous cell carcinoma (ESCC) remains poor, highlighting the importance of early detection. Although upper endoscopy can be used for the screening of esophagus, it has limited sensitivity for early stage disease. Thus, development of new diagnosis approach to improve diagnostic capabilities for early detection of ESCC is an important need. The aim of this study was to assess the feasibility of using cathepsin B (CB) as a novel imaging target for the detection of human ESCC by near-infrared optical imaging in nude mice.
Initially, we examined specimens from normal human esophageal tissue, intraepithelial neoplasia lesions, tumor in situ, ESCC and two cell lines including one human ESCC cell line (Eca-109) and one normal human esophageal epithelial cell line (HET-1A) for CB expression by immunohistochemistry and western blot, respectively. Next, the ability of a novel CB activatable near-infrared fluorescence (NIRF) probe detecting CB activity presented in Eca-109 cells was confirmed by immunocytochemistry. We also performed in vivo imaging of tumor bearing mice injected with the CB probe and ex vivo imaging of resected tumor xenografts and visceral organs using a living imaging system. Finally, the sources of fluorescence signals in tumor tissue and CB expression in visceral organs were identified by histology.
CB was absent in normal human esophageal mucosa, but it was overexpressed in ESCC and its precursor lesions. The novel probe for CB activity specifically detected ESCC xenografts in vivo and in vitro.
CB was highly upregulated in human ESCC and its precursor lesions. The elevated CB expression in ESCC allowed in vivo and in vitro detection of ESCC xenografts in nude mice. Our results support the usefulness of CB activity as a potential imaging target for the detection of human ESCC.
PMCID: PMC3950293  PMID: 24618814
4.  Establishment of using serum YKL-40 and SCCA in combination for the diagnosis of patients with esophageal squamous cell carcinoma 
BMC Cancer  2014;14:490.
Elevated serum YKL-40 levels have been observed in various cancers. We evaluated the diagnostic performance of serum YKL-40 alone or in combination with the CEA, CYFRA21-1 and SCCA tumor markers for patients with esophageal squamous cell carcinoma (ESCC).
YKL-40 was detected in ESCC cell lines and tissues by real-time RT-PCR, Western blotting and ELISA. YKL-40 protein expression was determined in 20 ESCC tumor tissues using immunohistochemistry. Serum YKL-40 was measured by ELISA in 126 healthy donors, 59 patients with benign esophageal diseases and 150 patients with ESCC. Serum CEA, CYFRA21-1 and SCCA were determined by electrochemiluminescence.
YKL-40 mRNA and protein were observed in ESCC cancer cell lines, tissues and cell culture media, respectively. YKL-40 expression was observed in 17 of 20 ESCC samples (85%). Serum YKL-40 concentration was significantly elevated in patients with ESCC (Range: 6.95-502.10 ng/ml) compared with patients with benign diseases (Range: 1.21-429.30 ng/ml; P = 0.038) and healthy controls (Range: 2.56-132.26 ng/ml; P < 0.001). ROC curves demonstrated that serum YKL-40 has a sensitivity of 72.70%, a specificity of 84.13% and an AUC of 0.874 for the diagnosis of ESCC, which was superior to CEA (Sen: 8.00%; Spe: 96.80%, AUC = 0.652), CYFRA21-1 (Sen: 40.00%; Spe: 92.06%, AUC = 0.746) and SCCA (Sen: 32.67%; Spe: 94.44%, AUC = 0.789). The YKL-40 and SCCA combination was better for diagnosing ESCC (Sen: 82.00%, Spe: 79.37%, PPV: 82.55 and NPV: 78.74; AUC = 0.917) than the YKL-40 and CEA combination (Sen: 74.00%, Spe: 83.20%, PPV: 84.09 and NPV: 72.73; AUC = 0.877), the YKL-40 and CYFRA21-1 combination (Sen: 82.00%, Spe: 77.78%, PPV: 81.46% and NPV: 78.40%; AUC = 0.897) or the CEA, CYFRA21-1 and SCCA combination (Sen: 56.67%, Spe: 84.80%, PPV: 81.73 and NPV: 61.99; AUC = 0.831). Associations between serum YKL-40 levels and the clinic characteristics of ESCC were not significant, with the exception of age (p = 0.001).
ESCC tumor cells and tissues express YKL-40. Serum YKL-40 may be a potential biomarker for ESCC. Serum YKL-40 in combination with SCCA significantly increases the sensitivity of detecting ESCC.
PMCID: PMC4094903  PMID: 25001061
YKL-40; Esophageal cancer; ESCC
5.  Over-Expression of CDC25B and LAMC2 mRNA and Protein in Esophageal Squamous Cell Carcinomas and Pre-Malignant Lesions in Subjects from a High-Risk Population in China 
Molecular events associated with the initiation and progression of esophageal squamous cell carcinoma (ESCC) remain poorly understood, but likely hold the key to effective early detection approaches for this almost invariably fatal cancer. CDC25B and LAMC2 are two promising early detection candidates emerging from new molecular studies of ESCC. To further elucidate the role of these two genes in esophageal carcinogenesis, we performed a series of studies to: (i) confirm RNA over-expression; (ii) establish the prevalence of protein over-expression; (iii) relate protein over-expression to survival; and (iv) explore their potential as early detection biomarkers. Results of these studies indicated that CDC25B mRNA was over-expressed (≥2-fold over-expression in tumor compared to normal) in 64% of the 73 ESCC cases evaluated, while LAMC2 mRNA was over-expressed in 89% of cases. CDC25B protein expression was categorized as positive in 59% (144/243) of ESCC cases on a tumor tissue microarray, and non-negative LAMC2 patterns of protein expression were observed in 82% (225/275) of cases. Multivariate-adjusted proportional hazard regression models showed no association between CDC25B protein expression score and risk of death (Hazard Ratio [HR] for each unit increase in expression score = 1.00, P=0.90), however, several of the LAMC2 protein expression patterns strongly predicted survival. Using the cytoplasmic pattern as the reference (the pattern with the lowest mortality), cases with a diffuse pattern had a 254% increased risk of death (HR=3.52, P=0.007), cases with no LAMC2 expression had a 169% increased risk of death (HR=2.69, P=0.009), and cases with a peripheral pattern had a 130% greater risk of death (HR=2.30, P=0.02). CDC25B protein expression scores in subjects with esophageal biopsies diagnosed as normal (n=35), dysplastic (n=23), or ESCC (n=32) increased significantly with morphologic progression. For LAMC2, all normal and dysplastic patients had a continuous pattern of protein expression, while all ESCCs showed alternative, non-continuous patterns. This series of studies showed that both CDC25B and LAMC2 over-express RNA and protein in a significant majority of ESCC cases. The strong relation of LAMC2 pattern of protein expression to survival suggests a role in prognosis, while CDC25B’s association with morphologic progression indicates a potential role as an early detection marker.
PMCID: PMC2729558  PMID: 18559558
esophageal cancer; quantitative RT-PCR; tissue microarray; survival; early detection; CDC25B; LAMC2
6.  DUSP6, a tumor suppressor, is involved in differentiation and apoptosis in esophageal squamous cell carcinoma 
Oncology Letters  2013;6(6):1624-1630.
Dual-specificity phosphatase 6 (DUSP6), a specific negative feedback regulator of phosphorylated extracellular signal-regulated kinase, was found to play an important role in numerous types of solid tumors as a tumor suppressor. In this study, 64.2% (61/95) of esophageal squamous cell carcinoma (ESCC) specimens studied exhibited reduced DUSP6 protein expression, compared with 91% (81/89) of normal esophageal specimens that displayed moderate or strong DUSP6 protein expression in tissue microarray analysis. In total, 36.8% (7/19) of the tumor biopsies displayed at least two-fold downregulation of DUSP6 compared with their paired normal counterparts, by qPCR. Significant loss of DUSP6 was observed in EC9706 and KYSE150 ESCC cell lines by immunoblotting assay. Low DUSP6 protein expression was significantly associated with pathological grade in ESCC by immunohistochemistry (P<0.05). Treatment with 5-aza-2′-deoxycytidine restored DUSP6 expression in the two ESCC cell lines, and the expression varied according to the drug concentration. Methylation-specific PCR analysis showed methylation-specific products in the two ESCC cell lines. We observed significant differences in the early and total apoptotic proportion between the control and experimental groups of the two ESCC cell lines and their transfectants (P<0.001) by annexin/propidium iodide assay. The presence of cleaved PARP product, a marker of caspase-mediated apoptosis, expressed in the two pCMV-DUSP6 transfectants in marked contrast to the parental and pCMV-transfected EC9706 and KYSE150 cells, was observed by immunoblotting. Overall, our results support the role of DUSP6 as a novel candidate tumor suppressor gene in ESCC, which may be a potential prognostic marker for ESCC.
PMCID: PMC3834198  PMID: 24260056
dual-specificity phosphatase 6; differentiation; apoptosis; methylation; esophageal squamous cell carcinoma
7.  None-endoscopic Screening for Esophageal Squamous Cell Carcinoma- A Review 
Esophageal cancer (EC) is the eighth most common cancer and sixth most frequent cause of cancer mortality worldwide. Esophageal squamous cell carcinoma (ESCC) is the most common type of EC. ESCC develops by progression from premalignant lesions, which are called esophageal squamous dysplasia (ESD). Prevention is the most effective strategy for controlling this disease. Generally, two methods may be defined for ESCC prevention. The aim of the first preventive method is to prevent the initiation of ESD by avoiding the known risk factors, or primary prevention. Secondary prevention focuses on detection of the disease in its early curable stage, thus preventing its progression into advanced stages. Endoscopy with iodine staining and biopsy is the diagnostic choice for ESD. However it is invasive and expensive, and not accepted by asymptomatic ESD cases. Therefore, it is necessary to find a non-endoscopic screening method. Despite the large number of studies conducted worldwide, no approved method has been developed for ESCC screening. Regarding the multi-factorial nature of ESCC, it is proposed that the use of a combination of various criteria, such as cytological examination, risk factors, genetic alteration, and molecular markers may result in the development of a comprehensive and effective ESCC screening program.
PMCID: PMC4017690  PMID: 24829644
Esophageal squamous cell carcinoma; Screening; Non-endoscopic; Review
8.  Combining proteomics, serum biomarkers and bioinformatics to discriminate between esophageal squamous cell carcinoma and pre-cancerous lesion*  
Objective: Biomarker assay is a noninvasive method for the early detection of esophageal squamous cell carcinoma (ESCC). Searching for new biomarkers with high specificity and sensitivity is very important for the early detection of ESCC. Serum surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS) is a high throughput technology for identifying cancer biomarkers using drops of sera. Methods: In this study, 185 serum samples were taken from ESCC patients in a high incidence area and screened by SELDI. A support vector machine (SVM) algorithm was adopted to analyze the samples. Results: The SVM patterns successfully distinguished ESCC from pre-cancerous lesions (PCLs). Also, types of PCL, including dysplasia (DYS) and basal cell hyperplasia (BCH), and healthy controls (HC) were distinguished with an accuracy of 95.2% (DYS), 96.6% (BCH), and 93.8% (HC), respectively. A marker of 25.1 kDa was identified in the ESCC patterns whose peak intensity was observed to increase significantly during the development of esophageal carcinogenesis, and to decrease obviously after surgery. Conclusions: We selected five ESCC biomarkers to form a diagnostic pattern which can discriminate among the different stages of esophageal carcinogenesis. This pattern can significantly improve the detection of ESCC.
PMCID: PMC3520450  PMID: 23225851
Biomarker; Esophageal squamous cell carcinoma; Surface-enhanced laser desorption/ionization; Support vector machine
9.  Hedgehog signaling activation in the development of squamous cell carcinoma and adenocarcinoma of esophagus 
Hedgehog (Hh) signaling is frequently activated in human cancer, including esophageal cancer. Most esophageal cancers are diagnosed in the advanced stages, therefore, identifying the very alterations that drive esophageal carcinogenesis may help designing novel strategies to diagnose and treat the disease. Analysis of Hh signaling in precancerous lesions is a critical first step in determining the significance of this pathway for carcinogenesis. Here we report our data on Hh target gene expression in 174 human esophageal specimens [28 esophageal adenocarcinomas (EAC), 19 Barrett’s esophagus, 103 cases of esophageal squamous cell carcinoma (ESCC), and 24 of squamous dysplastic lesions], and in two rat models of esophageal cancer. We found that 96% of human EAC express Hh target genes. We showed that PTCH1 expression is the most reliable biomarker. In contrast to EAC, only 38% of ESCC express Hh target genes. We found activation of Hh signaling in precancerous lesions of ESCCs and EACs in different degrees (21% and 58% respectively). Expression of Hh target genes is frequently detected in severe squamous dysplasia/ carcinoma in situ (p=0.04) and Barrett’s esophagus (p=0.01). Unlike EAC, sonic hedgehog (Shh) expression was rare in ESCCs. Consistent with the human specimen data, we found a high percentage of Hh signaling activation in precancerous lesions in rat models. These data indicate that Hh signaling activation is an early molecular event in the development of esophageal cancer, particularly EAC.
PMCID: PMC3325770  PMID: 22509480
Esophageal adenocarcinoma (EAC); esophageal squamous cell carcinoma (ESCC); hedgehog (Hh); patched-1 (PTCH1 for humans and Ptch1 for animals); Gli2; sFRP-1; human homologue of hedgehog-interaction protein (HHIP); rat model; Barrett’s esophagus (BE)
10.  Loss of disabled-2 expression is an early event in esophageal squamous tumorigenesis 
AIM: Disabled-2 (DAB2) is a candidate tumor-suppressor gene identified in ovarian cancer that negatively influences mitogenic signal transduction of growth factors and blocks ras activity. In a recent study, we observed down-regulation of DAB2 transcripts in ESCCs using cDNA microarrays. In the present study, we aimed to determine the clinical significance of loss of DAB2 protein in esophageal tumorigenesis, hypothesizing that DAB2 promoter hypermethylation-mediated gene silencing may account for loss of the protein.
METHODS: DAB2 expression was analyzed by immunohistochemistry in 50 primary esophageal squamous cell carcinomas (ESCCs), 30 distinct hyperplasia, 15 dysplasia and 10 non-malignant esophageal tissues. To determine whether promoter hypermethylation contributes to loss of DAB2 expression in ESCCs, methylation status of DAB2 promoter was analyzed in DAB2 immuno-negative tumors using methylation-specific PCR.
RESULTS: Loss of DAB2 protein was observed in 5/30 (17%) hyperplasia, 10/15 (67%) dysplasia and 34/50 (68%) ESCCs. Significant loss of DAB2 protein was observed from esophageal normal mucosa to hyperplasia, dysplasia and invasive cancer (Ptrend < 0.001). Promoter hypermethylation of DAB2 was observed in 2 of 10 (20%) DAB2 immuno-negative ESCCs.
CONCLUSION: Loss of DAB2 protein expression occurs in early pre-neoplastic stages of development of esophageal cancer and is sustained down the tumorigenic pathway. Infrequent DAB2 promoter methylation in ESCCs suggests that epigenetic gene silencing is only one of the mechanisms causing loss of DAB2 expression in ESCCs.
PMCID: PMC4124415  PMID: 17009406
Disabled-2; DOC-2; Esophageal cancer; Promoter hypermethylation; Dysplasia
11.  Expression of p14ARF, p15INK4b, p16INK4a and skp2 increases during esophageal squamous cell cancer progression 
Esophageal carcinoma is the sixth most common cause of cancer-related mortality in the world. Senescence and apoptosis are assumed to be two main mechanisms that inhibit age-related carcinogenesis. p14ARF, p15INK4b and p16INK4a, which are known to induce senescence by regulating G1 cell cycle arrest, have been identified as senescence markers. However, the mechanism by which senescence and apoptosis causes neoplasia in esophageal squamous cell carcinoma (ESCC) has not been identified. In this study, 20 cases of normal esophageal tissues, 11 cases of esophageal intraepithelial dysplasia (EID) and 60 cases of ESCC were obtained and pathologically diagnosed. Immunohistochemical staining was performed to assess the expression of p14ARF, p15INK4b, p16INK4a, skp2, bcl-2 and ki-67. The senescence markers p14ARF and p16INK4a were found to be expressed in 15 and 10% of the normal tissues, 82 and 73% of the EID cases and 100 and 88% of the ESCC cases, respectively. The expression of p15INK4b was low in normal tissues, while 92% of the ESCC specimens were diffusely and markedly stained, involving the basal, middle and upper portion of the epithelium. The nuclear expression markers ki-67 and skp2 were highly expressed in ESCC tissues (100 and 72%, respectively). bcl-2 was expressed weakly in normal tissues (10%) and demonstrated various staining patterns in carcinoma specimens (strong in 60%, negative in 40%). MI was 0.09% in normal tissues and 0.95% in the ESCC specimens. Apart from the increased proliferation in esophageal carcinogenesis, as indicated in the ki-67 and skp2 indices, there was an increased expression of senescence-associated molecular markers in the ESCC specimens, which indicates that the senescence pathway may be activated and become a part of cancer development. Of greatest interest to us was that, when compared with clinical information, the expression of the senescence markers was markedly high in the poorly differentiated specimens with lymph node metastasis, indicating that senescence markers may have diagnostic potential in clinical settings.
PMCID: PMC3438583  PMID: 22970012
senescence; apoptosis; carcinogenesis; esophageal squamous cell carcinoma
12.  Hypermethylation of multiple tumor-related genes associated with DMNT3b upregulation served as a biomarker for early diagnosis of esophageal squamous cell carcinoma 
Epigenetics  2011;6(3):307-316.
This study was designed to determine the significance of DNA methyltransferases (DNMTs) in DNA hypermethylation in esophageal squamous cell carcinoma (ESCC) and to identify DNA methylation markers in serum for the early diagnosis of ESCC. A promoter methylation profile of 12 tumor-related genes was assessed using methylation-specific PCR in ESCC and paired non-tumor tissue samples from 47 patients. Expression levels of DNMTs were examined by real-time reverse transcription-PCR and immunohistochemistry. Using MethyLight, the methylation status of five genes was analyzed in serum samples from 45 patients and 15 healthy individuals. A total of 46 (97.9%) of 47 ESCC samples showed methylation in at least one of the examined genes, and methylation was most frequent for RAR-β (46.8%), DAPK (46.8%), p16 (44.7%) and CDH1 (42.6%). Methylation of RASSF1A was significantly correlated with the poorly differentiated tumors and the early pathologic tumor classification (p = 0.035 and p = 0.046, respectively). Tumoral DNMT3b mRNA upregulation was significantly correlated with hypermethylation of multiple tumor-related genes (p = 0.021). In addition, hypermethylation of cell-free serum DNA was common in ESCC patients and diagnostic accuracy was increased when methylation of multiple genes (RAR-β, DAPK, CDH1, p16 and RASSF1A) were analyzed in combination (ROC AUC 0.911, 82.2% sensitivity and 100% specificity). The present study suggests that hypermethylation of multiple tumor-related genes may be involved in the pathogenesis of ESCC and mediated by the increase of DNMT3b expression. A cluster of multiple methylated genes in serum DNA has the potential as a novel biomarker for ESCC diagnosis.
PMCID: PMC3092679  PMID: 21150312
hypermethylation; esophageal squamous cell carcinoma; DNA methyltransferase 3b; serum; biomarker; diagnosis
13.  Study on RIZ1 gene promoter methylation status in human esophageal squamous cell carcinoma 
AIM: To investigate the promoter region methylation status of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) in the human esophageal squamous cell carcinoma (ESCC) cell lines and tissues and verify the relationship between methylation of RIZ1 and oncogenesis, tumor progression and metastasis etc of ESCC.
METHODS: Methylation-specific polymerase chain reaction (MSP) was used to investigate the promoter region methylation status of RIZ1 in 6 ESCC cell lines. One cell line where RIZ1 promoter region methylation was detected was selected for the next study, where the cell line was treated with 5-aza-CdR. Real-time polymerase chain reaction was used to investigate its influence on the transcription of RIZ1. Experiments using frozen pathological specimens from 47 ESCC patients were performed using the same MSP methodology.
RESULTS: Promoter methylation of RIZ1 gene was detected in TE13, CaEs17 and EC109 cell lines and the cell line TE13 was chosen for further study. The expression of RIZ1 mRNA in TE-13 was up-regulated after treatment with 5-aza-CdR. The rate of methylation in carcinomas tissues was significantly higher than those in matched neighboring normal and distal ending normal tissue, and the deviation of data was statistically significant (χ2 = 24.136, P < 0.01). Analysis of the gender, age familial history, tumour deviation, tumour saturation, lymph gland displacement and clinical staging of 47 samples from ESCC patients showed that the fluctuation of data was not statistically significant.
CONCLUSION: Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression in human ESCC. RIZ1 is considered to be a potential tumor suppressor gene and may be a biological parameter for testing early stage human ESCC.
PMCID: PMC3280405  PMID: 22363126
Retinoblastoma protein-interacting zinc finger gene 1; Tumor suppressor genes; Esophageal squamous cell carcinoma; Promoter methylation; Methylation-specific polymerase chain reaction
14.  Increased matrix metalloproteinase activation in esophageal squamous cell carcinoma 
Esophageal squamous cell carcinomas (ESCC) are usually asymptomatic and go undetected until they are incurable. Cytological screening is one strategy to detect ESCC at an early stage and has shown promise in previous studies, although improvement in sensitivity and specificity are needed. Proteases modulate cancer progression by facilitating tumor invasion and metastasis. In the current study, matrix metalloproteinases (MMPs) were studied in a search for new early detection markers for ESCC.
Protein expression levels of MMPs were measured using zymography in 24 cases of paired normal esophagus and ESCC, and in the tumor-associated stroma and tumor epithelium in one sample after laser capture microdissection (LCM). MMP-3 and MMP-10 transcripts in both the epithelium and stroma in five cases were further analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).
Gelatin zymography showed bands corresponding in size to MMP-2, MMP-3, MMP-9, and MMP-10 enzymes in each of the 24 cancer cases. MMP levels tended to be higher in tumors than paired normal tissue; however, only the 45 kDa band that corresponds to the activated form of MMP-3 and MMP-10 was strongly expressed in all 24 tumors with little or no expression in the paired normal foci. LCM-based analysis showed the 45 kDA band to be present in both the stromal and epithelial components of the tumor microenvironment, and that MMP-3 and MMP-10 mRNA levels were higher in tumors than paired normal tissues for each compartment.
Increased levels of MMPs occur in ESCC suggesting their up-regulation is important in esophageal tumorigenesis. The up-regulated gene products have the potential to serve as early detection markers in the clinic.
PMCID: PMC2958908  PMID: 20920372
15.  Temporal evolution in caveolin 1 methylation levels during human esophageal carcinogenesis 
BMC Cancer  2014;14:345.
Esophageal cancer ranks eighth among frequent cancers worldwide. Our aim was to investigate whether and at which neoplastic stage promoter hypermethylation of CAV1 is involved in human esophageal carcinogenesis.
Using real-time quantitative methylation-specific PCR (qMSP), we examined CAV1 promoter hypermethylation in 260 human esophageal tissue specimens. Real-time RT-PCR and qMSP were also performed on OE33 esophageal cancer cells before and after treatment with the demethylating agent, 5-aza-2’-deoxycytidine (5-Aza-dC).
CAV1 hypermethylation showed highly discriminative ROC curve profiles, clearly distinguishing esophageal adenocarcinomas (EAC) and esophageal squamous cell carcinomas (ESCC) from normal esophagus (NE) (EAC vs. NE, AUROC = 0.839 and p < 0.0001; ESCC vs. NE, AUROC = 0.920 and p < 0.0001). Both CAV1 methylation frequency and normalized methylation value (NMV) were significantly higher in Barrett’s metaplasia (BE), low-grade and high-grade dysplasia occurring in BE (D), EAC, and ESCC than in NE (all p < 0.01, respectively). Meanwhile, among 41 cases with matched NE and EAC or ESCC, CAV1 NMVs in EAC and ESCC (mean = 0.273) were significantly higher than in corresponding NE (mean = 0.146; p < 0.01, Student’s paired t-test). Treatment of OE33 EAC cells with 5-Aza-dC reduced CAV1 methylation and increased CAV1 mRNA expression.
CAV1 promoter hypermethylation is a frequent event in human esophageal carcinomas and is associated with early neoplastic progression in Barrett’s esophagus.
PMCID: PMC4035847  PMID: 24885118
CAV1; Hypermethylation; EAC; ESCC
16.  Prognostic CpG Methylation Biomarkers Identified by Methylation Array in Esophageal Squamous Cell Carcinoma Patients 
Background: Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with poor prognosis. We aimed to identify a panel of CpG methylation biomarkers for prognosis prediction of ESCC patients.
Methods: Illumina's GoldenGate methylation array, supervised principal components, Kaplan-Meier survival analyses and Cox regression model were conducted on dissected tumor tissues from a training cohort of 40 ESCC patients to identify potential CpG methylation biomarkers. Pyrosequencing quantitative methylation assay were performed to validate prognostic CpG methylation biomarkers in 61 ESCC patients. The correlation between DNA methylation and RNA expression of a validated marker, SOX17, was examined in a validation cohort of 61 ESCC patients.
Results: We identified a panel of nine CpG methylation probes located at promoter or exon1 region of eight genes including DDIT3, FES, FLT3, NTRK3, SEPT5, SEPT9, SOX1, and SOX17, for prognosis prediction in ESCC patients. Risk score calculated using the eight-gene panel statistically predicted poor outcome for patients with high risk score. These eight-gene also showed a significantly higher methylation level in tumor tissues than their corresponding normal samples in all patients analyzed. In addition, we also detected an inverse correlation between CpG hypermethylation and the mRNA expression level of SOX17 gene in ESCC patients, indicating that DNA hypermethylation was responsible for decreased expression of SOX17.
Conclusions: This study established a proof-of-concept CpG methylation biomarker panel for ESCC prognosis that can be further validated by multiple cohort studies. Functional characterization of the eight prognostic methylation genes in our biomarker panel could help to dissect the mechanism of ESCC tumorigenesis.
PMCID: PMC4057483  PMID: 24936140
esophageal squamous cell carcinoma; CpG methylation; DNA methylation array; pyrosequencing; prognosis.
17.  Inactivation of miR-34a by aberrant CpG methylation in Kazakh patients with esophageal carcinoma 
Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with dismal prognosis and high incidence and mortality in Kazakh population. MiR-34a, a direct p53 target gene, possesses tumor-suppressive properties as they mediate apoptosis, cell cycle arrest, and senescence. The reduced expression of miR-34a by methylation in various cancers has been reported.
To determine whether aberrant miR-34a methylation occurs in esophageal cancer, the DNA methylation of 23 CpGs sites in the miR-34a promoter was quantitatively analyzed in relation to the translation initiation site by MALDI -TOF mass spectrometry in 59 ESCC tissues and 34 normal tissues from the Kazakh population. Real-time PCR was used to detect the inhibition of miR-34a expression levels and to evaluate their association with methylation.
We found that miR-34a is more frequently methylated in ESCC (0.133 ± 0.040) than in controls (0.066 ± 0.045, P < 0.01). A nearly two-fold increase in miR-34a expression for the hypomethylated promoter was found in normal esophageal tissues than ESCC with hypermethylation (P <0.0001), pointing to a negative relationship between miR-34a CpG sites methylation and expression(r = −0.594, P = 0.042). The hypermethylation of miR-34a CpG_8.9 was associated with the advanced UICC stage III/IV of the esophageal cancers, and the hypermethylation of CpG_8.9 and CpG_5 of miR-34a was significantly correlated with lymph node metastasis.
Our findings suggest that miR-34a is involved in the etiology of ESCC and that hypermethylated miR-34a is a potential biomarker for ESCC diagnosis and prognosis. Moreover, targeting miR-34a methylation by demethylating agents may offer a novel strategy for anticancer therapy of ESCC.
PMCID: PMC3931274  PMID: 24528540
MiR-34a; Esophageal squamous cell carcinoma; Kazakh; Methylation
18.  Contribution of nestin positive esophageal squamous cancer cells on malignant proliferation, apoptosis, and poor prognosis 
The stem cell-associated intermediate filament nestin has recently been linked with neoplastic transformation, but the specific mechanism by which nestin positive tumor cells leads to malignant invasion and metastasis behaviors of esophageal squamous cell carcinoma (ESCC) remains unclear.
To obtain insight into the biological role of nestin in ESCC, we explored the association of the nestin phenotype with malignant proliferation and apoptosis in esophageal squamous cancer cells. Nestin expression was determined in ESCC specimens and cell lines, and correlated with clinicopathological properties, including clinical prognosis and proliferative markers. The association of the nestin phenotype with apoptotic indicators was also analyzed.
Nestin was expressed in ESCC specimens and cell lines. ESCC patients with nestin-positive tumors had significantly shorter median survival and progression-free survival times than those with nestin-negative tumors. Positive staining for the proliferation markers Ki67 and PCNA (proliferating cell nuclear antigen) was detected in 56.9% and 60.2% of ESCC specimens, respectively, and was strongly correlated with the nestin phenotype. Notably, expression of cyclin dependent kinase-5 (CDK5) and P35 was detected in 53.8% and 48.4% of ESCC specimens, respectively, and was strongly associated with the nestin phenotype.
Our data demonstrated nestin expression in ESCC specimens and cell lines, and revealed a strong association of the nestin phenotype with poor prognosis in ESCC patients. Furthermore, we showed that nestin positive ESCC cells played an important role in the malignant proliferation and apoptosis.
PMCID: PMC4071021  PMID: 24966803
Esophagus; Cancer; Esophageal squamous cell carcinoma; Nestin; Intermediate filament; Proliferation; Apoptosis
19.  Overexpression of DNA damage-induced 45 α gene contributes to esophageal squamous cell cancer by promoter hypomethylation 
Environmental factors-induced dysfunction of esophageal squamous epithelium, including genomic DNA impairment and apoptosis, play an important role in the pathogenesis of esophageal squamous cell cancer. DNA damage-induced 45α (GADD45α) has been found promoting DNA repair and removing methylation marker, Therefore, in this study we will investigate whether GADD45α expression is induced and its mechanism in esophageal squamous cell cancer.
Two human esophageal squamous cell lines (ESCC), ECA109 and KYSE510 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). Lipofectamine 2000 was used to transfect cells. mRNA level of GADD45α was measured by reverse transcription-quantitive PCR (RT-qPCR), protein level of GADD45α was detected by western blot and Immunohistochemistry. Global DNA methylation of tissue sample was measured using the Methylamp Global DNA Methylation Quantification Ultra kit (Epigentek Group) and promoter methylation was measured by bisulfite sequencing.
GADD45a mRNA and protein levels were increased significantly in tumor tissue than that in adjacent normal tissue. Hypomethylation of global genomic DNA and GADD45α promoter were found in ESCC. The cell sensitivity to Cisplatin DDP was decreased significantly in Eca109 and Kyse510 cells, in which GADD45α expression was down-regulated by RNA interference (RNAi). In addition, silence of GADD45a expression in ESCC cells inhibited proliferation and promoted apoptosis.
Overexpression of GADD45α gene is due to DNA hypomethylation in ESCC. GADD45α may be a protective factor in DDP chemotherapy for esophageal squamous cell carcinoma.
PMCID: PMC3364148  PMID: 22313682
Esophageal squamous cell cancer; GADD45α; DNA methylation; DNA damage
20.  Identification of a DNA Methylome Profile of Esophageal Squamous Cell Carcinoma and Potential Plasma Epigenetic Biomarkers for Early Diagnosis 
PLoS ONE  2014;9(7):e103162.
DNA methylation is a critical epigenetic mechanism involved in key cellular processes. Its deregulation has been linked to many human cancers including esophageal squamous cell carcinoma (ESCC). This study was designed to explore the whole methylation status of ESCC and to identify potential plasma biomarkers for early diagnosis. We used Infinium Methylation 450k array to analyze ESCC tissues (n = 4), paired normal surrounding tissues (n = 4) and normal mucosa from healthy individuals (n = 4), and combined these with gene expression data from the GEO database. One hundred and sixty eight genes had differentially methylated CpG sites in their promoter region and a gene expression pattern inverse to the direction of change in DNA methylation. These genes were involved in several cancer-related pathways. Three genes were validated in additional 42 ESCC tissues and paired normal surrounding tissues. The methylation frequency of EPB41L3, GPX3, and COL14A1 were higher in tumor tissues than in normal surrounding tissues (P<0.017). The higher methylation frequency of EPB41l3 was correlated with large tumor size (P = 0.044) and advanced pT tumor stage (P = 0.001). The higher methylation frequency of GPX3 and COL14A1 were correlated with advanced pN tumor stage (P = 0.001 and P<0.001). The methylation of EPB41L3, GPX3, and COL14A1 genes were only found in ESCC patients' plasma, but not in normal individuals upon testing 42 ESCC patients and 50 healthy individuals. Diagnostic sensitivity was increased when methylation of any of the 3 genes were counted (64.3% sensitivity and 100% specificity). These differentially methylated genes in plasma may be used as biomarkers for early diagnosis of ESCC.
PMCID: PMC4106874  PMID: 25050929
21.  Gene amplification of EGFR, HER2, FGFR2 and MET in esophageal squamous cell carcinoma 
International Journal of Oncology  2013;42(4):1151-1158.
Molecular targeted therapy is expected to be a promising therapeutic approach for the treatment of esophageal squamous cell carcinoma (ESCC); however, the gene amplification status of molecular targeted genes in ESCC remains largely unclear. The gene amplification of EGFR, HER2, FGFR2 and MET was examined using a real-time PCR-based copy number assay of 245 ESCC surgical specimens of formalin-fixed, paraffin-embedded samples. Fluorescence in situ hybridization (FISH) and comparative genomic hybridization analyses verified the results of the copy number assay. EGFR mutation was detected using the Scorpions-ARMS method. The EGFR status and drug sensitivity to an EGFR tyrosine kinase inhibitor was then evaluated in vitro. Gene amplification of EGFR and HER2 was observed in 7% (16/244) and 11% (27/245) of the ESCC specimens. A multivariate analysis revealed that HER2 amplification was a significant predictor of a poor prognosis in patients with stage III post-operative ESCC. The L861Q type of EGFR mutation with hypersensitivity to EGFR tyrosine kinase inhibitor was found in one of the eight ESCC cell lines and one del745 type of EGFR mutation was identified in 107 clinical samples. In addition, we demonstrated for the first time that FGFR2 amplification was observed in 4% (8/196) of the ESCC specimens. MET amplification was observed in 1% (2/196). In conclusion, the frequent gene amplification of EGFR, HER2 and FGFR2 and the presence of active EGFR mutations were observed in ESCC specimens. Our results strongly encourage the development of molecular targeted therapy for ESCC.
PMCID: PMC3622677  PMID: 23426935
EGFR; HER2; FGFR2; MET; esophageal squamous cell carcinoma
22.  Genome-Wide Gene Expression Profile Analyses Identify CTTN as a Potential Prognostic Marker in Esophageal Cancer 
PLoS ONE  2014;9(2):e88918.
Esophageal squamous cell carcinoma (ESCC) is one of the most common fatal malignances of the digestive tract. Its prognosis is poor mainly due to the lack of reliable markers for early detection and prognostic prediction. Here we aim to identify the molecules involved in ESCC carcinogenesis and those as potential markers for prognosis and as new molecular therapeutic targets.
We performed genome-wide gene expression profile analyses of 10 primary ESCCs and their adjacent normal tissues by cDNA microarrays representing 47,000 transcripts and variants. Candidate genes were then validated by semi quantitative reverse transcription-PCR (RT-PCR), tissue microarrays (TMAs) and immunohistochemistry (IHC) staining.
Using an arbitrary cutoff line of signal log ratio of ≥1.5 or ≤−1.5, we observed 549 up-regulated genes and 766 down-regulated genes in ESCCs compared with normal esophageal tissues. The functions of 302 differentially expressed genes were associated with cell metabolism, cell adhesion and immune response. Several candidate deregulated genes including four overexpressed (CTTN, DMRT2, MCM10 and SCYA26) and two underexpressed (HMGCS2 and SORBS2) were subsequently verified, which can be served as biomarkers for ESCC. Moreover, overexpression of cortactin (CTTN) was observed in 126/198 (63.6%) of ESCC cases and was significantly associated with lymph node metastasis (P = 0.000), pathologic stage (P = 0.000) and poor survival (P<0.001) of ESCC patients. Furthermore, a significant correlation between CTTN overexpression and shorter disease-specific survival rate was found in different subgroups of ESCC patient stratified by the pathologic stage (P<0.05).
Our data provide valuable information for establishing molecules as candidates for prognostic and/or as therapeutic targets.
PMCID: PMC3925182  PMID: 24551190
23.  Predicting esophageal squamous cell carcinoma and squamous dysplasia: risk modeling in a high risk area in Iran 
Archives of Iranian Medicine  2012;15(1):18-21.
Identifying people at higher risk of having squamous dysplasia, the precursor lesion for esophageal squamous cell carcinoma (ESCC), would allow targeted endoscopic screening.
We used multivariate logistic regression models to predict ESCC and dysplasia as outcomes. The ESCC model was based on data from the Golestan Case-Control Study (total n=871; cases=300), and the dysplasia model was based on data from a cohort of subjects from a GI clinic in Northeast Iran (total n=724; cases=26). In each of these analyses, we fit a model including all risk factors known in this region to be associated with ESCC. Individual risks were calculated using the linear combination of estimated regression coefficients and individual-specific values for covariates. We used cross-validation to determine the area under the curve (AUC) and to find the optimal cut points for each of the models.
The model had an area under the curve of 0.77 (95% CI: 0.74–0.80) to predict ESCC with 74% sensitivity and 70.4% specificity for the optimum cut point. The area under the curve was 0.71 (95% CI: 0.64–0.79) for dysplasia diagnosis, and the classification table optimized at 61.5% sensitivity and 69.5% specificity. In this population, the positive and negative predictive values for diagnosis of dysplasia were 6.8% and 97.8%, respectively.
Our models were able to discriminate between ESCC cases and controls in about 77%, and between individuals with and without squamous dysplasia in about 70% of the cases. Using risk factors to predict individual risk of ESCC or squamous dysplasia still has limited application in clinical practice, but such models may be suitable for selecting high risk individuals in research studies, or increasing the pretest probability for other screening strategies.
PMCID: PMC3294378  PMID: 22208438
24.  Elevated Maspin Expression Is Associated with Better Overall Survival in Esophageal Squamous Cell Carcinoma (ESCC) 
PLoS ONE  2013;8(5):e63581.
Tumor suppressor maspin is a differentially regulated gene in the progression of many types of cancer. While the biological function of maspin in blocking tumor invasion and metastasis is consistent with the loss of maspin expression at the late stage of tumor progression, the differential expression and the biological significance of maspin in early stage of tumor progression appear to be complex and remain to be elucidated. In the current study, we examined the expression of maspin in 84 esophageal squamous cell carcinoma (ESCC) cases (stages I–III) and 55 non-tumor adjacent esophageal tissue specimens by immunohistochemical (IHC) staining. The correlation of maspin with clinicopathological parameters was analyzed. Compared to normal esophageal squamous tissue where 80% (47/55) of the cases expressed maspin at a low to moderate level, all ESCC specimens (100% (84/84)) were positive for maspin expression at a moderate to high level. ESCC with low or moderate maspin expression had significantly shorter postoperative survival rates compared to those that had high maspin expression (p<0.001). Since the correlation of maspin with ESCC histology and the correlation of maspin with ESCC prognosis seem to be at odds, we further investigated the biological function of maspin in ESCC using the established ESCC cell lines. The expression of maspin in five human esophageal squamous cancer cell lines (T12, E450, KYSE150, EC109, and KYSE510) was examined by the Western blot. ESCC cell line KYSE510 that did not express maspin and was stably transfected by maspin cDNA or an empty vector. The resulting transfected cells were characterized in vitro. Maspin expression significantly inhibited cell proliferation, motility and matrigel invasion. Taken together, our data suggest that the transient up-regulation of maspin in the early development of ESCC may be a defense mechanism against further transition towards more malignant phenotypes, ultimately slowing down ESCC tumor progression.
PMCID: PMC3661574  PMID: 23717449
25.  Loss of heterozygosity analysis of microsatellites on multiple chromosome regions in dysplasia and squamous cell carcinoma of the esophagus 
The objective of this study was to characterize the molecular events in the carcinogenesis of esophageal squamous cell carcinoma (ESCC) and to identify biomarkers for early detection of the disease. Matched precancerous and cancerous tissues resected from 34 esophageal cancer patients from Chongqing, southern China, were compared to evaluate the extent of loss of heterozygosity (LOH). Sixteen microsatellite markers on chromosome regions 3p, 4p, 5q, 8p, 9p, 9q, 11p, 13q and 17p were used for PCR-based LOH analysis. The overall frequency of LOH at the 16 microsatellite loci was significantly increased as the pathological status of the resection specimens changed from low-grade dysplasia (LGD) to high-grade dysplasia (HGD) and SCC (P<0.001). A total of 8 markers showed LOH in the LGD samples. In addition, heterozygosity was regained at 4 loci in the SCC samples of 4 patients, respectively, in comparison to the results for these loci in the HGD samples. The overall rate of LOH increased significantly with the deterioration of the lesions, indicating that tumorigenesis of the esophageal squamous epithelia is a progressive process involving accumulative changes in LOH. The 8 loci showing allelic loss in the LGD samples may be involved in the early-stage tumorigenesis of ESCC, and LOH analysis at these loci may help improve the early detection of this disease. Regain of heterozygosity found in certain patients suggests the possibility of genetic heterogeneity in the tumori-genesis of esophageal cancer.
PMCID: PMC3440705  PMID: 22977611
esophageal squamous cell carcinoma; microsatellite; loss of heterozygosity

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