Chengshi Quan

Tight junction protein, claudin-6, downregulates the malignant phenotype of breast carcinoma.

Claudin-6 is a protein component of tight junctions and its expression has been found to be undetectable or at low levels in some human and rat breast cancer cells. Here we investigated the effect of claudin-6 upregulation on the malignant phenotype of human MCF-7 breast cancer cells. MCF-7 sublines with stable claudin-6 expression were established by transfection with a pcDNA3.1-claudin-6 expressing vector. The expression of claudin-6 on mRNA and protein levels was confirmed by reverse transcription-PCR, western blot and immunofluorescent assays. Then the effects of claudin-6 on cell proliferation and cell cycle were examined by 5-diphenyl terazolium-bromide assay and flow cytometry, respectively. Colony-forming assays were used to examine two-dimensional and three-dimensional colony forming ability. Invasive and migratory traits of claudin-6 expressing cells were determined by matrigel-based Boyden chamber invasion assay and monolayer wound-healing assay. The structure and function of tight junctions in both parental and claudin-6 expression MCF-7 cells were evaluated by measuring transepithelial electrical resistance. Immunofluorescent assays showed that transfected cells expressed claudin-6 on their membranes. Cells with high level expression of claudin-6 grew slowly and had a higher rate of death than control cells. Anchorage-independent growth, invasive and migratory traits were also substantially decreased in cells with claudin-6 expression; whereas the transepithelial electrical resistance was increased in the claudin-6 transfected cells. In conclusion, these results suggest that claudin-6 may function as a cancer suppressor; its downregulation may contribute to the malignant progression of certain types of breast cancers.

The expression patterns and correlations of claudin-6, methy-CpG binding protein 2, DNA methyltransferase 1, histone deacetylase 1, acetyl-histone H3 and acetyl-histone H4 and their clinicopathological significance in breast invasive ductal carcinomas

BackgroundClaudin-6 is a candidate tumor suppressor gene in breast cancer, and has been shown to be regulated by DNA methylation and histone modification in breast cancer lines. However, the expression of claudin-6 in breast invasive ductal carcinomas and correlation with clinical behavior or expression of other markers is unclear. We considered that the expression pattern of claudin-6 might be related to the expression of DNA methylation associated proteins (methyl-CpG binding protein 2 (MeCP2) and DNA methyltransferase 1 (DNMT1)) and histone modification associated proteins (histone deacetylase 1 (HDAC1), acetyl-histone H3 (H3Ac) and acetyl- histone H4 (H4Ac)).MethodsWe have investigated the expression of claudin-6, MeCP2, HDAC1, H3Ac and H4Ac in 100 breast invasive ductal carcinoma tissues and 22 mammary gland fibroadenoma tissues using immunohistochemistry.ResultsClaudin-6 protein expression was reduced in breast invasive ductal carcinomas (P < 0.001). In contrast, expression of MeCP2 (P < 0.001), DNMT1 (P = 0.001), HDAC1 (P < 0.001) and H3Ac (P = 0.004) expressions was increased. Claudin-6 expression was inversely correlated with lymph node metastasis (P = 0.021). Increased expression of HDAC1 was correlated with histological grade (P < 0.001), age (P = 0.004), clinical stage (P = 0.007) and lymph node metastasis (P = 0.001). H3Ac expression was associated with tumor size (P = 0.044) and clinical stage of cancers (P = 0.034). MeCP2, DNMT1 and H4Ac expression levels did not correlate with any of the tested clinicopathological parameters (P > 0.05). We identified a positive correlation between MeCP2 protein expression and H3Ac and H4Ac protein expression.ConclusionsOur results show that claudin-6 protein is significantly down-regulated in breast invasive ductal carcinomas and is an important correlate with lymphatic metastasis, but claudin-6 down-regulation was not correlated with upregulation of the methylation associated proteins (MeCP2, DNMT1) or histone modification associated proteins (HDAC1, H3Ac, H4Ac). Interestingly, the expression of MeCP2 was positively correlated with the expression of H3Ac and H3Ac protein expression was positively correlated with the expression of H4Ac in breast invasive ductal carcinomaVirtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4549669866581452

Clinicopathologic significance of claudin-6, occludin, and matrix metalloproteinases −2 expression in ovarian carcinoma

BackgroundTight junctions (TJs) are mainly composed of claudins, occludin, and tight junction adhesion molecules (JAM). The invasive and metastatic phenotype of highly invasive cancer cells has been related to abnormal structure and function of TJs, and with expression of activated matrix metalloproteinases (MMPs). The relevance of these mechanisms responsible for the invasion and metastasis of ovarian carcinoma is unclear. Similarly, it is not known if the expression of claudin-6, occludin and MMP2 is related with the clinical properties of these tumors.MethodsExpression of claudin-6, occludin, and MMP2 was detected in samples of human ovarian cancer tissues by immunohistochemistry and correlated with the clinical properties of the tumors.ResultsThe positive expression rates of claudin-6 and MMP-2 were higher in ovarian papillary serous carcinomas than n ovarian serous adenomas (P < 0.05). There were no differences in the expression of occludin (P > 0.05). The expression of claudin-6 and occludin in ovarian cancer was not correlated with patient age, pathological grade, clinical stage, and metastasis (P > 0.05). MMP-2 expression was enhanced with increased clinical stage and metastasis (P < 0.05), but was unrelated to patient age or tumor grade (P > 0.05). There were no apparent correlations between expression of claudin-6, occludin and MMP-2 in ovarian cancer tissue (P > 0.05).ConclusionsOur data suggest, for the first time, that the claudin-6 and MMP-2 are up-regulated in ovarian papillary serous carcinomas, MMP-2 expression was enhanced with increased clinical stage and metastasis. Claudin-6 and MMP-2 may play a positive role in the invasion and metastasis of ovarian cancer.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1775628454106511.

The distinct expression patterns of claudin-2, -6, and 11 between human gastric neoplasms and adjacent non-neoplastic tissues

BackgroundCancers have a multifactorial etiology a part of which is genetic. Recent data indicate that expression of the tight junction claudin proteins is involved in the etiology and progression of cancer.MethodsTo explore the correlations of the tight junction proteins claudin-2,-6, and −11 in the pathogenesis and clinical behavior of gastric cancer, 40 gastric cancer tissues and 28 samples of non-neoplastic tissues adjacent to the tumors were examined for expression of claudin-2,-6, and −11 by streptavidin-perosidase immunohistochemical staining method.ResultsThe positive expression rates of claudin-2 in gastric cancer tissues and adjacent tissues were 25% and 68% respectively (P < 0.001). The positive expression rates of claudin-6 in gastric cancer tissues and adjacent tissues were 55% and 79% respectively (P = 0.045 < 0.05). In contrast, the positive expression rates of claudin-11 in gastric cancer tissues and gastric cancer adjacent tissues were 80% and 46% (P = 0.004 < 0.01). Thus in our study, the expression of claudin-2, and claudin-6 was down regulated in gastric cancer tissue while the expression of claudin-11 was up regulated. Correlations between claudin expression and clinical behavior were not observed.ConclusionOur study provides the first evidence that claudin-2,-6, and −11 protein expression varies between human gastric cancers and adjacent non-neoplastic tissues.Virtual slidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/5470513569630744

Apoptosis signal-regulating kinase 1 is associated with the effect of claudin-6 in breast cancer

BackgroundPrevious studies have demonstrated that claudin-6 functions as a cancer suppressor in human MCF-7 breast cancer cells. The growth inhibitory effect could be attributed to inhibition of cell proliferation and induction of apoptosis. The purpose of the current study was to examine the involvement of apoptosis signal-regulating kinase 1 (ASK1) in the anticancer effect of claudin-6.MethodsImmunohistochemical analysis was performed to evaluate the ASK1 protein expression and the correlation between ASK1, claudin-6 and clinicopathological features in 85 samples of breast invasive ductal carcinomas (IDC). Western blotting and RT-PCR was carried out to examine the expression of ASK1 and claudin-6 in MCF-7 cell clones transfected with claudin-6.ResultsImmunohistochemical analysis showed that ASK1 expression was significantly related with that of claudin-6 in breast invasive ductal carcinomas (P < 0.05). In addition, a positive correlation between ASK1 and C-erb B 2 protein expression was identified (P < 0.05). Western blotting and RT-PCR consistently revealed that the level of ASK1 protein and mRNA was upregulated in MCF-7 cell clones transfected with claudin-6.ConclusionsOur data suggests, for the first time, that the ASK1 signal may play a positive role in the inhibitory effect of claudin-6 in breast cancer.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1200314318763661

Gene silencing of claudin‑6 enhances cell proliferation and migration accompanied with increased MMP‑2 activity via p38 MAPK signaling pathway in human breast epithelium cell line HBL‑100

Disruption or loss of tight junction structure and function is associated with tumor growth, invasion and metastasis in tumors of human epithelial origin. Since claudin is the most important backbone protein of tight junctions, the downregulation or loss of claudin expression is hypothesized to be important for tumor development and metastasis. In the current study, RNA interference (RNAi) was used to knock down the expression of claudin‑6 to investigate the effect of claudin‑6 downregulation on the malignant phenotype in the human breast epithelium cell line HBL‑100. The junctional function was investigated by measuring the transepithelial electrical resistance across the confluent epithelial cell layer. Manual cell counting and wound healing assays were performed to examine cell proliferation and migration. Changes in matrix metalloproteinase‑2 (MMP‑2) expression and activity were examined by reverse transcription polymerase chain reaction (RT‑PCR) and gelatin zymography. The expression of p38 mitogen‑activated protein kinases (MAPKs) and phosphorylated p38 MAPK were measured by western blot analysis. Claudin‑6 knockdown resulted in significantly lower transepithelial electrical resistance (P<0.001), higher growth rate (P<0.001) and migratory ability (P<0.001) accompanied with an increased MMP‑2 expression and activity (P<0.001). Furthermore, a decreased expression of phosphorylated p38 MAPK (P<0.001) was detected in HBL‑100 cells. These observations support the hypothesis that a decreased expression of claudin‑6 contributes to the malignant progression of human breast cancer.

Tight junction protein claudin-6 inhibits growth and induces the apoptosis of cervical carcinoma cells in vitro and in vivo.

Claudin-6, a member of claudin family integral membrane proteins, has recently been reported to be a tumor suppressor for breast cancer. However, whether it plays a role in other types of cancer remains unclear. In the present study, we showed that the expression of claudin-6 is down-regulated in cervical carcinoma tissues as revealed by immunohistochemistry. Through over-expressing claudin-6 in HeLa and C33A cervical carcinoma cells, we found that claudin-6 is localized at plasma membrane and it increases transepithelial electrical resistance of the cells. Gain of claudin-6 expression suppresses cell proliferation, colony formation in vitro, and tumor growth in vivo. The effects are accompanied and potentially caused by promotion of tumor cell apoptosis. Taken together, these results suggest that claudin-6 may function as a tumor suppressor and loss of claudin-6 contributes to enhanced tumorigenic properties of cervical carcinoma cells.

Transdifferentiation of Human Hair Follicle Mesenchymal Stem Cells into Red Blood Cells by OCT4

Shortage of red blood cells (RBCs, erythrocytes) can have potentially life-threatening consequences for rare or unusual blood type patients with massive blood loss resulting from various conditions. Erythrocytes have been derived from human pluripotent stem cells (PSCs), but the risk of potential tumorigenicity cannot be ignored, and a majority of these cells produced from PSCs express embryonic ε- and fetal γ-globins with little or no adult β-globin and remain nucleated. Here we report a method to generate erythrocytes from human hair follicle mesenchymal stem cells (hHFMSCs) by enforcing OCT4 gene expression and cytokine stimulation. Cells generated from hHFMSCs expressed mainly the adult β-globin chain with minimum level of the fetal γ-globin chain. Furthermore, these cells also underwent multiple maturation events and formed enucleated erythrocytes with a biconcave disc shape. Gene expression analyses showed that OCT4 regulated the expression of genes associated with both pluripotency and erythroid development during hHFMSC transdifferentiation toward erythroid cells. These findings show that mature erythrocytes can be generated from adult somatic cells, which may serve as an alternative source of RBCs for potential autologous transfusion.

The effects of shRNA-mediated gene silencing of transcription factor SNAI1 on the biological phenotypes of breast cancer cell line MCF-7

To research the effects of silencing transcription factor SNAI1 on the in vitro biological phenotypes of breast cancer cell line MCF-7, based on the gene sequence of SNAI1, we linked shRNA with the green fluorescent protein-expressing eukaryotic expression vector pGCsilencer™ U6/Neo/GFP, and transfected it into MCF-7 cells. The SNAI1 gene-silencing effect was authenticated by RT-PCR and immunofluorescence. We then examined the effect of gene silencing on the expression of epithelial and mesenchymal markers and on their biological phenotypes of the target cells. Finally, we explained that SNAI1 was bound to E-cadherin in MCF-7 cells by ChIP. Silencing SNAI1 upregulated the expression of epithelial markers claudin-4, claudin-7, and E-cadherin, while expression of the mesenchymal marker matrix metalloproteinase-2 was downregulated. The capacity for proliferation, migration, and invasion was diminished. SNAI1 binds to the E-cadherin gene promoter and inhibits its transcription. We can conclude that silencing gene SNAI1 inhibits expression of properties that are associated with the malignant phenotype of MCF-7 cells and reverses the epithelial–mesenchymal transition process by regulating relevant target gene E-cadherin.

CLDN6 enhances chemoresistance to ADM via AF-6/ERKs pathway in TNBC cell line MDAMB231

Claudin-6 (CLDN6), a critical tight junction protein acting as a tumor suppressor in breast cancer, is also considered to be a stem cell marker. Triple-negative breast cancer (TNBC) is a subtype of claudin-low and stem cell-like breast cancer which is chemoresistant to multiple anti-cancer drugs. The aim of our study was to determine whether CLDN6 plays a role in chemoresistance of TNBC. We found that overexpression of CLDN6 in TNBC cell line MDAMB231 significantly inhibited cell growth, migration, and invasion. The expression of CLDN6 increased the IC50 of adriamycin (ADM) and promoted the clonogenic survival. CLDN6 inhibited ADM-induced apoptosis and senescence in MDAMB231 cells. However, P-gp, a resistance-related protein highly associated with chemoresistance, was downregulated by CLDN6 overexpression in MDAMB231 cells. Epithelial mesenchymal transition (EMT) marker E-cadherin was increased, and vimentin was decreased by CLDN6. In addition, stem cell markers OCT4, SOX2, and Nanog were dramatically increased. CLDN6 colocalized and interacted with AF-6. Overexpression of CLDN6 increased the expression of afadin (AF-6) and hampered the activation of ERK signaling. PMA, a specific ERK activator, reversed the expression of EMT and stem cell markers, and decreased chemoresistance of MDAMB231 cells to ADM with a decreased IC50 and an increased apoptosis resulting from CLDN6. Together, we conclude that CLDN6 enhances the chemoresistance to ADM via activating the AF-6/ERK signaling pathway and up-regulating cancer stem cell characters in MDAMB231 cells.