Cadherin Switch Research Articles

Electromagnetic field as a possible inhibitor of tumor invasion by declining E-cadherin/N-cadherin switching in triple negative breast cancer

ABSTRACT Breast cancer has been recognized as the most common cancer affecting women. Extremely low-frequency electromagnetic field (ELF-EMF) exposure can influence cellular activities such as cell-cell junctions and metastasis. However, more research is required to determine these fields’ underlying mechanisms of action. Since cadherin switching is an important process during EMT (epithelial-mesenchymal transition), in this study, cadherin switching was regarded as one of the probable mechanisms of the effect of ELF-EMFs on metastasis suppression. For five days, breast cells received a 1 Hz, 100mT ELF-EMF (2 h/day). Cell invasion and migration were assessed in vitro by the Scratch wound healing assay and Transwell culture chambers. The expression of E- and N-cadherin was assessed using real-time PCR, western blotting, and Immunocytochemistry. ELF-EMF dramatically reduced the migration and invasion of MDA-MB 231 malignant cells compared to sham exposure, according to the results of the scratch test and the Transwell invasion test. The mRNA and protein expression levels of E-cadherin showed an increase, while the N-cadherin expression was found with a decrease, in MDA-MB231 cells receiving 1 Hz EMF compared to sham exposure. E-cadherin’s mRNA and protein expression levels were enhanced in MCF10A cells receiving 1 Hz EMF compared to sham exposure. ELF-EMF can be used as a method for the multifaceted treatments of invasive breast cancer.

Tumoral Malignancy Decreases Coupled with Higher ROS and Lipid Peroxidation in HCT116 Colon Cancer Cells upon Loss of PRDX6.

Peroxiredoxin 6 (PRDX6) is an atypical member of the peroxiredoxin family that presents not only peroxidase but also phospholipase A2 and lysophosphatidylcholine acyl transferase activities able to act on lipid hydroperoxides of cell membranes. It has been associated with the proliferation and invasive capacity of different tumoral cells including colorectal cancer cells, although the effect of its removal in these cells has not been yet studied. Here, using CRISPR/Cas9 technology, we constructed an HCT116 colorectal cancer cell line knockout for PRDX6 to study whether the mechanisms described for other cancer cells in terms of proliferation, migration, and invasiveness also apply in this tumoral cell line. HCT116 cells lacking PRDX6 showed increased ROS and lipid peroxidation, a decrease in the antioxidant response regulator NRF2, mitochondrial dysfunction, and increased sensitivity to ferroptosis. All these alterations lead to a decrease in proliferation, migration, and invasiveness in these cells. Furthermore, the reduced migratory and invasive capacity of HCT116 cancer cells is consistent with the observed cadherin switch and decrease in pro-invasive proteins such as MMPs. Therefore, the mechanism behind the effects of loss of PRDX6 in HCT116 cells could differ from that in HepG2 cells which is coherent with the fact that the correlation of PRDX6 expression with patient survival is different in hepatocellular carcinomas. Nonetheless, our results point to this protein as a good therapeutic target also for colorectal cancer.

Clinical Implications of EMT in HNSCC: A Review of the Factors and Pathways at Play

_Background: _Epithelial-mesenchymal transition (EMT) is a biological process where epithelial cells acquire mesenchymal traits. Epithelial cells are characterized by tight cell-cell adhesions and apical-basal polarity, whereas mesenchymal cells are generally elongated in appearance with loose cell-cell interactions, allowing increased cell migration. Many studies have been done on EMT pathways in oral cavity carcinoma, but there are few studies about the possible clinical implications. _Aims_ _&_ _Objectives:_ This systematic review was carried out to find the clinical implications of EMT in HNSCC and bring together the molecular, genetic, and epigenetic pathways found to be acting on the cadherin switch. _Materials & Methods: _An extensive search for relevant papers was made on PubMed, Medline, and Google Search. Only good-quality studies pertaining to epithelial–mesenchymal transition in oral cavity cancers in humans were selected. Furthermore, all selected papers were assessed for their clinical relevance. _Results: EMT has role in prognostication, diagnostic dilemma, margin assessment, mandibular preservation, making OSCC more prone to systemic therapies and planning risk reduction in strategies. _The cadherin switch is regulated by transcription factors like Snail, SLUG, ZEB1, and ZEB2. It is also acted on by epigenetic modifiers. These transcription factors are regulated by multiple pathways like Wnt/β–catenin, PI3K/AKT, MAPK, etc. These pathways are in turn regulated by molecular and external agents like NNK from tobacco smoke, hypoxia, the DDB2 gene, reactive oxygen species (ROS), and melatonin.

Oncogenic Role of SATB2 In Vitro: Regulator of Pluripotency, Self-Renewal, and Epithelial-Mesenchymal Transition in Prostate Cancer.

Special AT-rich sequence binding protein-2 (SATB2) is a nuclear matrix protein that binds to nuclear attachment regions and is involved in chromatin remodeling and transcription regulation. In stem cells, it regulates the expression of genes required for maintaining pluripotency and self-renewal and epithelial-mesenchymal transition (EMT). In this study, we examined the oncogenic role of SATB2 in prostate cancer and assessed whether overexpression of SATB2 in human normal prostate epithelial cells (PrECs) induces properties of cancer stem cells (CSCs). The results demonstrate that SATB2 is highly expressed in prostate cancer cell lines and CSCs, but not in PrECs. Overexpression of SATB2 in PrECs induces cellular transformation which was evident by the formation of colonies in soft agar and spheroids in suspension. Overexpression of SATB2 in PrECs also resulted in induction of stem cell markers (CD44 and CD133), pluripotency-maintaining transcription factors (cMYC, OCT4, SOX2, KLF4, and NANOG), CADHERIN switch, and EMT-related transcription factors. Chromatin immunoprecipitation assay demonstrated that SATB2 can directly bind to promoters of BCL-2, BSP, NANOG, MYC, XIAP, KLF4, and HOXA2, suggesting SATB2 is capable of directly regulating pluripotency/self-renewal, cell survival, and proliferation. Since prostate CSCs play a crucial role in cancer initiation, progression, and metastasis, we also examined the effects of SATB2 knockdown on stemness. SATB2 knockdown in prostate CSCs inhibited spheroid formation, cell viability, colony formation, cell motility, migration, and invasion compared to their scrambled control groups. SATB2 knockdown in CSCs also upregulated the expression of E-CADHERIN and inhibited the expression of N-CADHERIN, SNAIL, SLUG, and ZEB1. The expression of SATB2 was significantly higher in prostate adenocarcinoma compared to normal tissues. Overall, our data suggest that SATB2 acts as an oncogenic factor where it is capable of inducing malignant changes in PrECs by inducing CSC characteristics.

EMT Dynamics in Lymph Node Metastasis of Oral Squamous Cell Carcinoma.

Epithelial-mesenchymal transition (EMT) enables tumor cell invasion and metastasis. Many studies have demonstrated the critical role of EMT in lymph node metastasis in oral squamous cell carcinoma (OSCC). During EMT, epithelial cancer cells lose intercellular adhesion and apical-basal polarity and acquire mesenchymal properties such as motility and invasiveness. A significant feature of EMT is cadherin switching, involving the downregulation of E-cadherin and upregulation of N-cadherin. The TGF-β/SMAD pathway can also induce EMT. We aimed to evaluate EMT markers as predictors of lymph node metastasis in OSCC. We performed genetic profiling of 159 primary OSCCs from TCGA and analyzed the expression of EMT markers, including cadherin switch genes (CDH1, CDH2), and TGF-β/SMAD pathway genes. Samples were divided into advanced (stage III-IV) and early (stage I-II) stage groups. Differential expression analysis was performed, as well as an independent validation study containing fresh OSCC samples. TGF-β/SMAD pathway genes such as SMAD6 were upregulated in advanced stage tumors. N-cadherin and SNAIL2 were overexpressed in node-positive tumors. Keratins were downregulated in these groups. Our findings demonstrate that EMT marker expression correlates with lymph node metastasis in OSCC. Developing therapies targeting regulators such as N-cadherin may prevent metastasis and improve outcomes.

Cholangiocarcinoma Malignant Traits Are Promoted by Schwann Cells through TGFβ Signaling in a Model of Perineural Invasion.

The term cholangiocarcinoma (CCA) defines a class of epithelial malignancies originating from bile ducts. Although it has been demonstrated that CCA patients with perineural invasion (PNI) have a worse prognosis, the biological features of this phenomenon are yet unclear. Our data show that in human intrahepatic CCA specimens with documented PNI, nerve-infiltrating CCA cells display positivity of the epithelial marker cytokeratin 7, lower with respect to the rest of the tumor mass. In an in vitro 3D model, CCA cells move towards a peripheral nerve explant allowing contact with Schwann cells (SCs) emerging from the nerve. Here, we show that SCs produce soluble factors that favor the migration, invasion, survival and proliferation of CCA cells in vitro. This effect is accompanied by a cadherin switch, suggestive of an epithelial-mesenchymal transition. The influence of SCs in promoting the ability of CCA cells to migrate and invade the extracellular matrix is hampered by a specific TGFβ receptor 1 (TGFBR1) antagonist. Differential proteomic data indicate that the exposure of CCA cells to SC secreted factors induces the upregulation of key oncogenes and the concomitant downregulation of some tumor suppressors. Taken together, these data concur in identifying SCs as possible promoters of a more aggressive CCA phenotype, ascribing a central role to TGFβ signaling in regulating this process.

YAP1 controls the N-cadherin-mediated tumor-stroma interaction in melanoma progression

The hallmark of epithelial-to-mesenchymal transition (EMT) is the switch from epithelial cadherin (E-cadherin) to neural cadherin (N-cadherin), allowing melanoma cells to form a homotypic N-cadherin-mediated adhesion with stromal fibroblasts. However, how cadherin switching is initiated, maintained, and regulated in melanoma remains elusive. Here, we report a novel mechanism underlying cadherin switching in melanoma cells that is regulated by stromal Yes-associated protein 1 (YAP1) signaling. The progression of a BRAF-mutant mouse melanoma was suppressed in vivo upon YAP1 ablation in cancer-associated fibroblasts (CAFs). On the contrary, overexpressing YAP1 in CAFs accelerated melanoma development. By RNA-Seq, N-cadherin was identified as a major downstream effector of YAP1 signaling in CAFs. YAP1 silencing reduced N-cadherin expression in CAFs, leading to the downregulation of N-cadherin in neighboring melanoma cells. N-cadherin ablation inhibited the PI3K-AKT signaling pathway in melanoma cells and melanoma cell proliferation. The findings suggest that YAP1 depletion in CAFs induces the downregulation of p-AKT signaling in melanoma cells through the N-cadherin-mediated interaction between melanoma cells and CAFs. The data underscore an important role of CAFs in regulating N-cadherin-mediated adhesion and signaling in melanoma and highlight that disentangling cadherin-mediated cell-cell interactions can potentially disrupt tumor-stroma interactions and reverse the tumor cell invasive phenotype.

Immunohistochemical assessment of the potential behavior of glandular odontogenic cyst and inflammatory periodontal cyst using E-cadherin and N-cadherin

BackgroundThis study is done to evaluate the immunohistochemical expression of E-cadherin and N-cadherin in glandular odontogenic cysts (GOC), inflammatory periodontal (IPDL) cysts, and dental follicles (DF), and if there is a correlation with their biological behavior GOC, IPDL cysts, and DF cases were collected and investigated by immunohistochemistry. The area percent of immunoexpressed markers was calculated by SPSS version 25.ResultsThe statistical analysis revealed a clear, statistically significant difference between the studied groups regarding the area percentage of the two immune-expression markers (p value = 0.000). The highest area percentage of the mean value of E-cadherin was recorded in IPDL cysts group with the most negligible value recorded in the group of GOC. On the other hand, the highest area percentage of the mean value of N-cadherin was documented in the group of GOC with the least recorded value was noted in the group of DF.ConclusionWe noted that the cadherin switch mechanism in the epithelial lining of odontogenic cysts is a critical step in the epithelial mesenchymal transition process which may associates with clinical behavior and may also impact the mode of treatment.

Cadherin switching in oral squamous cell carcinoma: A clinicopathological study.

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide as it represents the sixth most common cancer. Numerous molecular mechanisms have been explained to regulate OSCC progression, including epithelial-mesenchymal transition (EMT). Cadherin switching is the pivotal process that controls EMT in which E-cadherin reduces while N-cadherin elevates. This work aimed to clarify the role of cadherin switching in OSCC. Thirty paraffin-embedded tissue blocks of OSCC including six cases with lymph node metastasis were subjected to immunohistochemical staining using antibodies against E&N-cadherins. Cell cultures were performed using OSCC cell lines (SCC-15/SCC-25) from the human tongue. F-12K medium (Kaighn's Modification of Ham's F12 Medium) was added as EMT inducing media. E&N-cadherin mRNA gene expression levels were detected by real time-polymerase chain reaction (RT-PCR). Cadherin switching through N-cadherin elevation and E-cadherin reduction was evaluated at the histopathologic level in primary and metastatic OSCC as well as at the genetic level within OSCC cell culture. Cadherin switching showed a significant correlation between E&N-cadherins at different histopathological grades of OSCC and in metastatic OSCC. Moreover, the level of mRNA gene expression of E&N-cadherins in human 15 SCC and 25 SCC cell lines with EMT-inducing media exhibited a significant correlation. Cadherin switching is a crucial event in the EMT process. It may be used as a significant tool in the study of OSCC progression. Cadherin switching plays a significant role in the invasion and metastasis of OSCC.

Loss of PRDX6 Aborts Proliferative and Migratory Signaling in Hepatocarcinoma Cell Lines.

Peroxiredoxin 6 (PRDX6), the only mammalian 1-Cys member of the peroxiredoxin family, has peroxidase, phospholipase A2 (PLA2), and lysophosphatidylcholine (LPC) acyltransferase (LPCAT) activities. It has been associated with tumor progression and cancer metastasis, but the mechanisms involved are not clear. We constructed an SNU475 hepatocarcinoma cell line knockout for PRDX6 to study the processes of migration and invasiveness in these mesenchymal cells. They showed lipid peroxidation but inhibition of the NRF2 transcriptional regulator, mitochondrial dysfunction, metabolic reprogramming, an altered cytoskeleton, down-regulation of PCNA, and a diminished growth rate. LPC regulatory action was inhibited, indicating that loss of both the peroxidase and PLA2 activities of PRDX6 are involved. Upstream regulators MYC, ATF4, HNF4A, and HNF4G were activated. Despite AKT activation and GSK3β inhibition, the prosurvival pathway and the SNAI1-induced EMT program were aborted in the absence of PRDX6, as indicated by diminished migration and invasiveness, down-regulation of bottom-line markers of the EMT program, MMP2, cytoskeletal proteins, and triggering of the "cadherin switch". These changes point to a role for PRDX6 in tumor development and metastasis, so it can be considered a candidate for antitumoral therapies.

Prognostic impact of the loss of E-cadherin and de novo expression of N-cadherin at the invasive front of primary and recurrent oral squamous cell carcinoma.

The epithelial-mesenchymal transition (EMT) is a biological mechanism in multiple pathophysiological diseases. Related alterations in cadherin expression play a crucial role in carcinogenesis, progression, angiogenesis, and immune response. EMT cells exhibit a transition from an epithelial to a mesenchymal phenotype (cadherin-switch). This process is characterized by the de novo development of N-cadherin (N-CAD), which replaces E-cadherin (E-CAD) and signifies an increased migratory capacity and malignant transformation. The cadherin switch is a hallmark of EMT and has been studied in various cancer entities. We predicted that the cadherin switch in the primary and recurrent oral squamous cell carcinoma (re-OSCC) tissues is an inherent characteristic of the tumor, affects the biologic behavior, and further reflects the post-recurrence survival outcome of these patients. Survival outcome was analyzed by calculating the post-recurrence survival of the high-risk group and correlating the standardized h-score-based IHC expression of both cadherin types with the clinical follow-up. 94 patients with re-OSCC were observed within the cohort. Tissue samples from both primary and recurring tumors were collected. There was a significant association between loss of E-CAD expression and both oral cancer-specific and overall survival, (HR=2.72, CI:1.50-4.95, p=0.001) and (HR=3.84, CI:1.93-7.63, p=0.001), respectively, for expression loss higher than 60%. There was no statistically significant correlation between N-CAD de novo expression and Overall, oral cancer-specific and disease-free post-recurrence survival. The current study clearly shows that cadherin-switch, identified as E-CAD loss and N-CAD de novo expression in the invasion front of a re-OSCC, appears to be an inherent histological hallmark that does not change from primary manifestation to recurrence within the same tumor, regardless of the form of adjuvant therapy used for the primary tumor. The loss of E-CAD expression in re-OSCC is an independent risk factor for poor survival, and may be used to stratify therapy and de/escalate the multimodal treatment.

An in vitro culture platform for studying the effect of collective cell migration on spatial self-organization within induced pluripotent stem cell colonies

BackgroundHuman induced pluripotent stem cells (hiPSCs) provide an in vitro system to identify the impact of cell behavior on the earliest stages of cell fate specification during human development. Here, we developed an hiPSC-based model to study the effect of collective cell migration in meso–endodermal lineage segregation and cell fate decisions through the control of space confinement using a detachable ring culture system.ResultsThe actomyosin organization of cells at the edge of undifferentiated colonies formed in a ring barrier differed from that of the cells in the center of the colony. In addition, even in the absence of exogenous supplements, ectoderm, mesoderm, endoderm, and extraembryonic cells differentiated following the induction of collective cell migration at the colony edge by removing the ring-barrier. However, when collective cell migration was inhibited by blocking E-cadherin function, this fate decision within an hiPSC colony was altered to an ectodermal fate. Furthermore, the induction of collective cell migration at the colony edge using an endodermal induction media enhanced endodermal differentiation efficiency in association with cadherin switching, which is involved in the epithelial-mesenchymal transition.ConclusionsOur findings suggest that collective cell migration can be an effective way to drive the segregation of mesoderm and endoderm lineages, and cell fate decisions of hiPSCs.

SPRED2: A Novel Regulator of Epithelial-Mesenchymal Transition and Stemness in Hepatocellular Carcinoma Cells.

The downregulation of SPRED2, a negative regulator of the ERK1/2 pathway, was previously detected in human cancers; however, the biological consequence remains unknown. Here, we investigated the effects of SPRED2 loss on hepatocellular carcinoma (HCC) cell function. Human HCC cell lines, expressing various levels of SPRED2 and SPRED2 knockdown, increased ERK1/2 activation. SPRED2-knockout (KO)-HepG2 cells displayed an elongated spindle shape with increased cell migration/invasion and cadherin switching, with features of epithelial-mesenchymal transition (EMT). SPRED2-KO cells demonstrated a higher ability to form spheres and colonies, expressed higher levels of stemness markers and were more resistant to cisplatin. Interestingly, SPRED2-KO cells also expressed higher levels of the stem cell surface markers CD44 and CD90. When CD44+CD90+ and CD44-CD90- populations from WT cells were analyzed, a lower level of SPRED2 and higher levels of stem cell markers were detected in CD44+CD90+ cells. Further, endogenous SPRED2 expression decreased when WT cells were cultured in 3D, but was restored in 2D culture. Finally, the levels of SPRED2 in clinical HCC tissues were significantly lower than those in adjacent non-HCC tissues and were negatively associated with progression-free survival. Thus, the downregulation of SPRED2 in HCC promotes EMT and stemness through the activation of the ERK1/2 pathway, and leads to more malignant phenotypes.

Endothelial-to-mesenchymal transition: a precursor to pulmonary arterial remodelling in patients with idiopathic pulmonary fibrosis.

We have previously reported arterial remodelling in patients with idiopathic pulmonary fibrosis (IPF) and suggested that endothelial-to-mesenchymal transition (EndMT) might be central to these changes. This study aims to provide evidence for active EndMT in IPF patients. Lung resections from 13 patients with IPF and 15 normal controls (NCs) were immunostained for EndMT biomarkers: vascular endothelial cadherin (VE-cadherin), neural cadherin (N-cadherin), S100A4 and vimentin. Pulmonary arteries were analysed for EndMT markers by using computer- and microscope-assisted image analysis software Image ProPlus7.0. All the analysis was done with observer blinded to subject and diagnosis. Increased expression of mesenchymal markers N-cadherin (p<0.0001), vimentin (p<0.0001) and S100A4 (p<0.05) was noted with downregulation of junctional endothelial VE-cadherin (p<0.01) in the intimal layer of the arteries from patients with IPF compared to NCs. Cadherin switch was observed in IPF patients, showing increase in endothelial N-cadherin and decrease in VE-cadherin (p<0.01). There was also VE-cadherin shift from junctions to cytoplasm (p<0.01), effecting endothelial cell integrity in patients with IPF. In IPF, individual mesenchymal markers vimentin and N-cadherin negatively correlated with diffusing capacity of the lungs for carbon monoxide (r'= -0.63, p=0.03 and r'= -0.66, p=0.01). Further, N-cadherin positively correlated with arterial thickness (r'=0.58, p=0.03). This is the first study to demonstrate active EndMT in size-based classified pulmonary arteries from IPF patients and potential role in driving remodelling changes. The mesenchymal markers had a negative impact on the diffusing capacity of the lungs for carbon monoxide. This work also informs early origins of pulmonary hypertension in patients with IPF.

Differential expression of Cadherins switch and Caveolin-2 during stages of oral carcinogenesis.

Oral squamous cell carcinoma (OSCC) accounts for 90% of oral malignancies, which may be preceded by oral potentially malignant disorders (OPMDs). Cancer progression involves the downregulation of epithelial markers (E-cadherin) and the upregulation of mesenchymal markers (N-cadherin), which together characterise the epithelial-mesenchymal transition (EMT). Furthermore, caveolin can act on cell adhesion and migration events that regulate the expression of the E-cadherin/α-β-catenin complex, thus favouring aggressive biological behaviour. This study aimed to analyse the immunoexpression of E-cadherin, N-cadherin and caveolin-2 at different stages of oral carcinogenesis to identify reliable biomarkers to predict malignant potential. Expressions of E-cadherin and N-cadherin in 14 normal oral mucosae (NOM), 14 OPMD and 33 OSCC specimens were evaluated using immunohistochemistry. Clinicopathological parameters were also assessed. E-cadherin immunoexpression was significantly reduced during the progression of oral carcinogenesis (P = 0.0018). N-cadherin immunoexpression did not show any statistical differences between these groups. However, a representative number of N-cadherin-positive OSCC cases did not express E-cadherin. The expression of caveolin-2 increased significantly with the progression of the disease, from NOM to OSCC (P value: 0.0028). These findings indicate that cadherin switch and caveolin-2 immunoexpression may be regulatory events in oral carcinogenesis.

A prospective immunohistochemical study using E-cadherin and Ncadherin to depict cadherin switch in oral squamous cell carcinoma.

A prospective immunohistochemical study using E-cadherin and Ncadherin to depict cadherin switch in oral squamous cell carcinoma.

Hedgehog regulation of epithelial cell state and morphogenesis in the larynx.

The larynx enables speech while regulating swallowing and respiration. Larynx function hinges on the laryngeal epithelium which originates as part of the anterior foregut and undergoes extensive remodeling to separate from the esophagus and form vocal folds that interface with the adjacent trachea. Here we find that sonic hedgehog (SHH) is essential for epithelial integrity in the mouse larynx as well as the anterior foregut. During larynx-esophageal separation, low Shh expression marks specific domains of actively remodeling epithelium that undergo an epithelial-to-mesenchymal transition (EMT) characterized by the induction of N-Cadherin and movement of cells out of the epithelial layer. Consistent with a role for SHH signaling in regulating this process, Shh mutants undergo an abnormal EMT throughout the anterior foregut and larynx, marked by a cadherin switch, movement out of the epithelial layer and cell death. Unexpectedly, Shh mutant epithelial cells are replaced by a new population of FOXA2-negative cells that likely derive from adjacent pouch tissues and form a rudimentary epithelium. These findings have important implications for interpreting the etiology of HH-dependent birth defects within the foregut. We propose that SHH signaling has a default role in maintaining epithelial identity throughout the anterior foregut and that regionalized reductions in SHH trigger epithelial remodeling.

LncRNA TUG1 promotes the migration and invasion in type I endometrial carcinoma cells by regulating E–N cadherin switch

ObjectiveAccumulating evidence has demonstrated that lncRNA Taurine-upregulated gene 1 (TUG1) plays an important role in regulation of cell morphology, migration, proliferation and apoptosis. Our aim was to evaluate the oncogenic role of TUG1 in type I Endometrial Carcinoma (EC) and explore the precise mechanism of TUG1 involved in tumor progression. Materials and methodsThe GSE17025 data set was used to analyze the correlation of TUG1 expression with type I EC patients’ prognosis. Furthermore, TUG1 expression profiles were measured by qRT-PCR from carcinoma tissues and adjacent nonneoplastic tissues (NNT) of 105 type I EC patients. The regulation of epithelial–mesenchymal transition (EMT) related molecules, p-AKT and AKT by TUG1 knockdown was investigated using Western blot analysis; meanwhile, the oncogenic roles of TUG1 were evaluated using cell viability and transwell migration/invasion assay in Hec-1-A and Ishikawa cell lines. ResultsFirstly, we observed a significant association between higher TUG1 expression and lower survival rate in type I EC patients using the GSE17025 data set. A significant elevation of TUG1 levels was confirmed in type I EC tissues compared with NNT in the 105 type I EC patients, and high expression of TUG1 was associated with lymph vascular space invasion (LVSI) and lymph node metastasis (LNM). Subsequently, TUG1 knockdown could remarkably inhibit the Hec-1-A and Ishikawa cell invasion and migration in the functional experiment. Furthermore, our results showed that the protein levels of E-cadherin increased and N-cadherin decreased significantly, while β-catenin and Vimentin were not significantly altered upon TUG1 silencing in both Hec-1-A and Ishikawa cells. Finally, we found the p-AKT and AKT protein levels, and the rate of p-AKT/t-AKT has a tendency to be down-regulate in Hec-1-A cells, while the AKT pathway was not change significantly in Ishikawa cells after TUG1 knockdown. ConclusionCollectively, our data reveal that TUG1 might be regarded as an oncogenic molecule that promotes type I EC cells metastasis leading to tumor progression, at least partially, by regulating E–N cadherin switch and the AKT pathway.

Establishment and Preliminary Analysis of Lung Cancer Cell Line A549 with Stable MAP4 K4 Knockdown

To analyze the effect of knocking down MAP4 K4 expression on the proliferation and migration of cancer cells, and to explore its underlining molecular mechanisms. A stable knockdown MAP4 K4 cell line was constructed and the subcellular localization of the cells was determined with immunofluorescence, cell proliferation assay and cell migration assay. In addition, the effects of down-regulated MAP4 K4 expression were analyzed by examining the difference between the proliferation and migration of cancer cells in the knockdown group and those of the control group. MAP4 K4 was localized in focal adhesion and cell edges in A549 cells. Stable knockdown of MAP4 K4 expression induced cancer cells to grow in clusters and arrested the progression of the cell cycle and cell migration. Further analysis found that knocking down MAP4 K4 expression in A549 cells induced the accumulation of epithelial cell marker E-cadherin, and subsequently, the down-regulation of N-cadherin, a mesenchymal cell marker, thereby disrupting the "cadherin switch" and the epithelial-mesenchymal conversion. Then, the control group and the knockdown group both received the combined treatment of cisplatin at a final concentration of 5 μmol/L and paclitaxel at a final concentration of 20 nmol/L. The stably knocked down MAP4 K4 expressing cells showed significantly enhanced toxicity of chemotherapeutic drugs to cancer cells. The study shows that MAP4 K4 regulates the malignant phenotypes of cancer cells and chemoresistance by regulating "cadherin switch" to promote epithelial-mesenchymal transition in A549 cells.

Antitumor activity of irinotecan with ellagic acid in C6 glioma cells.

SUMMARYOBJECTIVE:Irinotecan-based combination chemotherapies in malignant gliomas need to be examined. The aim of this study was to investigate the synergetic effect of ellagic acid, a natural polyphenolic antioxidant compound, with irinotecan, an inhibitor of topoisomerase I enzyme, on the growth, cadherin switch, and angiogenic processes of a glioma cell line.METHODS:A combination of 100 μM ellagic acid and 100 μM irinotecan was applied to rat C6 glioma cells for 24th, 48th, and 72nd h. The cell proliferation was evaluated by 5-bromo-2′-deoxyuridine immunocytochemistry. The expression levels of vascular endothelial growth factor, E-cadherin, and N-cadherin were measured using real-time polymerase chain reaction and their immunoreactivities using immunocytochemistry.RESULTS:The treatment of irinotecan with combining ellagic acid enhanced antitumor activity and the synergistic effect of these reduced the cell proliferation of C6 glioma by inhibiting the cadherin switch and promoting the antiangiogenic processes.CONCLUSIONS:Further research is required to prove a negative relationship between C6 glial cell proliferation and irinotecan with ellagic acid application. Our preliminary data suggest that even with the extremely short-term application, irinotecan with ellagic acid may affect glioma cells at the level of gene and protein expression.