OBJECTIVETo investigate the expression of TMEMl6A in the colon of intractable functional constipation patients and its clinical implications.

METHODSA total of 30 patients with intractable chronic functional constipation were selected as trial group (full thickness tissue of sigmoid colon), 30 patients with colon cancer as control group (distant tissues at least 5 cm away from cancer). Tissues from two groups were collected in our hospital from February 2012 to June 2014 and confirmed by pathological diagnosis. Immunofluorescence staining, RT-PCR and Western blot were performed to detect the mRNA and protein expression of TMEM16A and c-kit in colon.

RESULTSTMEM16A and c-kit protein expressions were observed in similar sites of colon tissues in two groups. Expressions of TMEM16A and C-kit in colon tissues detected by immunofluorescence, RT-PCR, and Western blotting were significantly lower in trial group than those in control group (TMEM16A: mean A 1.84±0.25 vs. 3.65±0.32, P<0.05, gray intensity ratio 0.66±0.07 vs. 1.04±0.17, P<0.05, relative mRNA 0.41±0.05 vs. 1.00, P<0.05; c-kit: mean A 3.38±0.24 vs. 5.06±0.31, gray intensity ratio 0.64±0.06 vs. 0.98±0.09, relative mRNA 0.18±0.08 vs. 1.00, all P<0.05).

CONCLUSIONSTMEM16A expression in colon tissues of intractable functional constipation patients is significantly lower and may adjust the movement of colonic smooth muscle by regulating the c-kit expression. TMEMl6A may be used as a new candidate target for diagnosis and treatment of intractable functional constipation.

Anoctamin-1 ; Blotting, Western ; Chloride Channels ; Colon, Sigmoid ; Colonic Neoplasms ; Constipation ; Humans ; Muscle, Smooth ; Neoplasm Proteins ; Proteomics ; Proto-Oncogene Proteins c-kit ; RNA, Messenger

OBJECTIVETo investigate the expression of TMEM16A in gastric carcinoma and its clinical implications.

METHODSA total of 72 surgical specimens of gastric carcinoma were collected for examination of TMEM16A expression with immunohistochemical staining.

RESULTSTMEM16A expression was detected in the cytoplasm and cell membrane of the tumor cells. Of the 72 specimens of the tumor tissues, the total positivity rate of TMEM16A expression was 80.56% (58/72), significantly higher than the rate in the adjacent tissues (4.17%, 3/72, P<0.005).

CONCLUSIONAberrant expression of TMEM16A occurs in the majority of gastric carcinoma cases. TMEM16A can be used as a new candidate target for diagnosis and treatment of gastric carcinoma.

Adult ; Aged ; Anoctamin-1 ; Carcinoma ; metabolism ; pathology ; Chloride Channels ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Stomach Neoplasms ; metabolism ; pathology

OBJECTIVETo explore the effects of ANO1 overexpression on the proliferation, detachment, spreading, and migration of laryngocarcinoma Hep-2 cell line.

METHODSANO1-overexpressing Hep-2 cell line was selected as the assay group, and Hep-2 cell line with empty plasmid was selected as the control group. MTT assay was used to detect the proliferation abilities of Hep-2 cells in both two groups. Cell detachment assay and spreading assay were used to detect the detachment and spreading abilities of Hep-2 cells. Boyden chamber invasion assay, wound healing assay in vitro, and niflumic acid block chloride channel were used to detect the migration abilities of Hep-2 cells. All data were analyzed by SPSS 10.0 software package.

RESULTSCell proliferation assay by MTT showed that, compared with the control group, the optical density value of assay group was not significantly different (P=0.62). The results of cell detachment assay and cell spreading assay showed the cell detachment rates and cell spreading rates in assay group were significantly higher than those in control group (P<0.0001). The results of Boyden chamber invasion assay showed the percentages of cells migrating through the membrane in assay group were significantly higher than those in control group (P<0.0001). The results of in vitro wound healing experiments showed the wound area rate in assay group was significantly lower than that in control group (P<0.0001). The results of niflumic acid blocking chloride channel experiments showed the wound area rates in assay group were significantly higher than those in control group (P<0.0001).

CONCLUSIONANO1 overexpression does not remarkably alter the proliferation rate of cancer cells, but increases the migration, spreading, and detachment capacities of head and neck squamous cell carcinoma.

Anoctamin-1 ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Chloride Channels ; metabolism ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Neoplasm Proteins ; metabolism

OBJECTIVETo detect the effects of ANO1 overexpression on the biological behaviors of human laryngeal squamous cell carcinoma Hep-2 cells.

METHODSA Hep-2 cell line stably overexpressing ANO1 were examined with flow cytometry, soft agar assay, wound healing assay, siRNA experiments, and chloride channel block with DIDS to observe the effect of ANO1 overexpression on the growth, migration and invasion of the cells.

RESULTSFlow cytometry revealed a comparable cell percentage in G0/G1 phase between ANO1-overexpressing cells and the control cells (P>0.05). The two cells showed no significant difference in soft agar assay (P>0.05), but in wound healing experiments, ANO1-overexpressing cells showed significantly accelerated migration (P<0.05), whereas siRNA-mediated silencing of ANO1 significantly inhibited the cell migration (P<0.05). Treatment with DIDS resulted in an effective block of the ANO1 chloride channel activity and obviously decreased the migration speed of Hep-2 cells.

CONCLUSIONANO1 overexpression does not significantly affect the proliferation of cancer cells, but can enhance the migration ability of head and neck squamous cell carcinoma, suggesting the value of ANO1 as a new gene therapy target for head and neck squamous cell carcinoma.

Anoctamin-1 ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Chloride Channels ; metabolism ; Gene Silencing ; Head and Neck Neoplasms ; metabolism ; pathology ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Neoplasm Proteins ; metabolism ; RNA, Small Interfering

OBJECTIVETo identify the expression of DOG-1 in gastrointestinal stromal tumors (GIST) and to explore its potential association with clinicopathological features of GIST.

METHODSTwo tissue microarrays (TMA) were used for the study. Each TMA contained 80 tissue samples of GIST from 80 different patients, with each tumor represented by one core, and paraffin-embedded sections of 40 samples from normal gastrointestinal tissue were used as control. Immunohistochemistry staining (SABC method) was performed on TMA and paraffin-embedded sections to detected the expression of c-Kit (CD117) and DOG-1.

RESULTSImmunohistochemistry showed that in 80 GIST patients, 76 cases (95.0%) were DOG-1 positive and 67 cases (83.8%) were CD117 positive. The positive rate of DOG-1 was higher than that of CD117 (P<0.05). In 13 GIST samples of negative CD117, the positive rate of DOG-1 was 100%. Expressions of both DOG-1 and CD117 were negative in all the 40 samples of normal gastrointestinal tissue. The positive expression of DOG-1 and CD117 was not significantly different in spindle cell type (96.0% vs. 96.0%, P>0.05) and in mixed cell type (91.7% vs. 75.0%, P>0.05). While in the epithelioid cell type, the DOG-1 expression was higher than CD117 expression (94.1% vs. 52.9%, P<0.05). The positive expression of DOG-1 and CD117 was not associated with age, gender, location and risk stratification of the tumors (all P>0.05).

CONCLUSIONSDOG-1 expression is up-regulated in gastrointestinal stromal tumors, especially in epithelioid cell GIST, and may be used as a new marker in the diagnosis of GIST.

Adult ; Aged ; Anoctamin-1 ; Biomarkers, Tumor ; metabolism ; Chloride Channels ; metabolism ; Female ; Gastrointestinal Neoplasms ; metabolism ; Gastrointestinal Stromal Tumors ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Young Adult

OBJECTIVETo investigate the expression of DOG-1 in gastrointestinal stromal tumors (GIST) and its diagnostic application.

METHODSImmunohistochemical EnVision technique was used to assess the expression of DOG-1 in 84 cases of GIST in comparison with CD117 and CD34.

RESULTSAll 84 cases of GIST consisted of variable proportions of spindle and epithelioid tumor cells or just one type of the tumor cell. The expression rates of DOG-1, CD117 and CD34 were 91.3% (42/46), 95.7% (44/46) and 82.6% (38/46), in the group of very low and low risk GIST, and were 100% (38/38), 100% (38/38) and 78.9% (30/38), respectively, in the group of moderate and high risk GIST. True leiomyomas, schwannomas, fibromatosis and normal gastrointestinal mucoca did not express these markers. Moreover, the sensitivity and specificity of DOG-1 in the detection of GIST were similar to those of CD117, without statistical difference (P > 0.05) between the two markers. However, the sensitivity and specificity of DOG-1 detection of moderate and high risk GIST were significantly higher than those of CD34 (P < 0.01).

CONCLUSIONSDOG-1 is a novel marker of gastrointestinal stromal tumors. It has the sensitivity and specificity higher than CD34, especially in the detection of moderate and high risk GIST. Combined DOG-1 and CD117 immunohistochemistry will likely improve the diagnostic accuracy of GIST.

Adult ; Aged ; Anoctamin-1 ; Antigens, CD34 ; metabolism ; Biomarkers, Tumor ; analysis ; Chloride Channels ; Female ; Gastrointestinal Stromal Tumors ; diagnosis ; metabolism ; pathology ; Humans ; Male ; Membrane Proteins ; metabolism ; Middle Aged ; Neoplasm Proteins ; metabolism ; Proto-Oncogene Proteins c-kit ; metabolism ; Sensitivity and Specificity

The aim of the present study was to investigate whether the physiological features of Ano1 were affected by enhanced green fluorescent protein (EGFP) fusing at Ano1 C-terminal. The eukaryotic expression vectors of Ano1 and EGFP-Ano1 were constructed, and these plasmids were transfected into Fischer rat thyroid follicular epithelial (FRT) cells using liposome. The expression and location of Ano1 were examined by using inverted fluorescence microscope. The ability of Ano1 to transport iodide was detected by kinetics experiment of fluorescence quenching. The results showed that both Ano1 and EGFP-Ano1 were expressed on FRT cell membrane and could be activated by Ca(2+). There was no significant difference of the ability to transport iodide between Ano1 and EGFP-Ano1. These results suggest Ano1 and EGFP-Ano1 have similar physiological feature.
Animals ; Anoctamin-1 ; Cell Membrane ; physiology ; Chloride Channels ; metabolism ; Epithelial Cells ; physiology ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; Microscopy, Fluorescence ; Plasmids ; Rats ; Recombinant Fusion Proteins ; metabolism ; Thyroid Gland ; cytology ; Transfection

OBJECTIVETo study the immunophenotype and c-kit or platelet derived growth factor receptor alpha (PDGFRA) gene mutations in CD117-negative gastrointestinal stromal tumors (GISTs).

METHODSTen cases of GISTs with typical histologic features but no CD117 expression were retrieved from the archival of Department of Pathology, Peking Union Medical College Hospital, China. The cases were further evaluated for the presence of c-kit exons 9, 11, 13 and 17 mutations and PDGFRA exons 12 and 18 mutations. DNA was extracted from the paraffin-embedded tumor tissue. The PCR products were sequenced directly for the mutations. An immunohistochemical study for CD117, CD34, smooth muscle actin, desmin, S-100 protein, WT-1 and DOG-1 was also performed.

RESULTSEight of the 10 cases had the mutation tests completed. C-kit mutation in exon 9 was detected in only one case. Amongst the 10 cases studied, CD34 was expressed in 9 cases. Smooth muscle actin was focally positive in 2 cases. None of them expressed desmin or S-100 protein. DOG-1 and WT-1 were diffusely positive in 5 and 4 cases, respectively. In addition, DOG1 was diffusely but weakly positive in 1 case and focally expressed in 2 cases. Three cases were focally positive for WT-1.

CONCLUSIONPathologic diagnosis of CD117-negative GISTs can be facilitated with the application of a panel of immunohistochemical markers, including DOG-1 and WT-1.

Actins ; metabolism ; Adult ; Aged ; Anoctamin-1 ; Antigens, CD34 ; metabolism ; Chloride Channels ; Exons ; Female ; Gastrointestinal Stromal Tumors ; genetics ; metabolism ; pathology ; Humans ; Immunophenotyping ; Male ; Membrane Proteins ; metabolism ; Middle Aged ; Mutation ; Neoplasm Proteins ; metabolism ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; metabolism ; WT1 Proteins ; metabolism ; Young Adult

Anoctamin-1 ; Antineoplastic Agents ; administration & dosage ; Benzamides ; Biomarkers, Tumor ; metabolism ; Chloride Channels ; Drug Delivery Systems ; methods ; Gastrointestinal Neoplasms ; genetics ; metabolism ; pathology ; therapy ; Gastrointestinal Stromal Tumors ; genetics ; metabolism ; pathology ; therapy ; Humans ; Imatinib Mesylate ; Indoles ; administration & dosage ; Membrane Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Piperazines ; administration & dosage ; Proto-Oncogene Proteins c-kit ; metabolism ; Pyrimidines ; administration & dosage ; Pyrroles ; administration & dosage ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism