Protein Expression Levels Of E-cadherin Research Articles

Costunolide induces apoptosis and inhibits migration and invasion in H1299 lung cancer cells.

Costunolide being a sesquiterpene lactone, is known to have anticancer properties. The present study investigated the anticancer effects of costunolide against the H1299 human non-small-cell lung cancer (NSCLC) cell line. Inhibition of cell viability by costunolide was assessed via a MTT assay. Furthermore, the apoptotic rate was detected using Annexin V/propidium iodide labeling. A colony forming cell assay was performed to investigate the antiproliferative effects of costunolide. Wound healing and Transwell assays were performed to determine the inhibitory effects of costunolide on migration and invasion, respectively. Western blot analysis was undertaken to determine protein expression, and reverse transcription-quantitative PCR was performed to assess mRNA expression levels. The results demonstrated that costunolide inhibited the viability of H1299 cells, with a half maximal inhibitory concentration value of 23.93±1.67 µM and induced cellular apoptosis in a dose-dependent manner. Furthermore, the colony formation, migrative and invasive abilities of the H1299 cells were inhibited in a dose- or time-dependent manner. The protein expression levels of E-cadherin increased and those of N-cadherin decreased following treatment with costunolide, which suggested that costunolide inhibited epithelial-to-mesenchymal transition. The mRNA levels of B-Raf, E-cadherin, N-cadherin, integrins α2 and β1, as well as matrix metalloproteinases 2 were also found to be regulated costunolide. These findings indicate the potential of costunolide in the treatment of NSCLC.

Annexin A13 predicts poor prognosis for lung adenocarcinoma patients and accelerates the proliferation and migration of lung adenocarcinoma cells by modulating epithelial-mesenchymal transition.

This study aimed to investigate the role of ANXA13 in lung adenocarcinoma (LUAD) growth, migration, and the underlying mechanisms. Firstly, in the TCGA dataset for LUAD, ANXA13 is found to be highly expressed in patients with LUAD and high expression of ANXA13 predicted poor outcomes in LUAD patients. Consistently, the data of qRT-PCR showed that the expression of ANXA13 was higher in LUAD cell lines (Calu-3, LTEP-a-2, and NCI-H1395) than that in normal lung cell line BEAS2B. Then, we performed gain- and loss of function of ANXA13 in NCI-H1395 and Calu-3 cells, respectively. The results displayed that deficiency of ANXA13 suppresses cell proliferation, invasion, and migration in Calu-3 cells and overexpression of ANXA13 augments cell proliferation, invasion, and migration in NCI-H1395 cells. Finally, it was found that silencing of ANXA13 obviously raised the protein expression levels of E-cadherin and reduced the protein levels of N-cadherin, Vimentin, and Snail in Calu-3 cells whereas overexpression of ANXA13 obviously receded the protein expression levels of E-cadherin and enhanced the protein levels of N-cadherin, Vimentin, and Snail in NCI-H1395 cells. This study analyzed the biological effects of ANXA13 in LUAD cells, indicating that ANXA13 could regard as a therapeutic target for LUAD.

Effect of Silencing FMOD on Proliferation and Migration of H322 Cells and Its Mechanism

To investigate the regulation of fibromodulin (FMOD) on proliferation, adhesion and migration of non-small cell lung cancer cell line H322, and discuss its action mechanism. H322 cells were randomly divided into control group, small interfering RNA (siRNA) silencing FMOD ( FMOD siRNA) group and control siRNA (Con siRNA) group. FMOD siRNA and Con siRNA were transfected into H322 cells. The cell viability of each group was detected by CCK-8 method. The adhesion ability of cells was detected by fluorescein diacetate (FDA) fluorescent staining. The cell migration ability was detected by Transwell method. Real time-PCR was used to detect the mRNA expressions of Cyclin D1, intercellular adhesion molecule -1 （ICAM-1）, E-cadherin, FMOD, transforming growth factor-β (TGF-β), Smad2, Smad3, Smad4 and Smad7 in cells. The protein expressions of Cyclin D1, ICAM-1, E-cadherin, FMOD, TGF-β1, Smad2, Smad3, Smad4 and Smad7 were detected by Western blot. Compared with the Con siRNA group, the cell viability, cell adhesion and migration ability of the FMOD siRNA group were decreased, and the difference was statistically significant ( P<0.01). There was no significant difference between the control group and the Con siRNA group. Real time-PCR and Western blot results showed that the mRNA and protein expression levels of Cyclin D1, ICAM-1, TGF-β1, Smad2, Smad3 and Smad4 were decreased in FMOD siRNA group, compared with Con siRNA group, while the mRNA and protein expression levels of E-cadherin and Smad7 are elevated. Silencing of the FMOD gene significantly reduces the proliferation, adhesion and migration of H322 cells, which may be conducted by inhibiting the TGF-β/Smad signaling pathway.

The potential influence of long non-coding RNA PRKG1-AS1 on oral squamous cell carcinoma: A comprehensive study based on bioinformatics and in vitro validation.

Oral squamous cell carcinoma (OSCC) is one of the most frequent malignancies in oral cancer. Herein, we aimed to investigate the influence of lncRNA protein kinase cGMP-dependent type I-Antisense RNA 1 (PRKG1-AS1) in OSCC progression. Basing on the data acquired from TCGA database, the expression and prognostic value of PRKG1-AS1 in OSCC patients were assessed. The expression of PRKG1-AS1 in OSCC cells was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by Cell Counting Kit-8 (CCK8) and colony-forming assays. Transwell assay was employed to test cell invasion and migration. The protein expression of epithelial-mesenchymal transition (EMT)-related markers was detected by Western blotting. The consequences displayed that PRKG1-AS1 was highly expressed in OSCC tissues and high expression of PRKG1-AS1 predicted poor outcomes. The expression of PRKG1-AS1 was higher in CAL27, SCC-9, and SCC-4 than that in normal human oral keratinocytes (NHOK). The results of biological experiments showed that deficiency of PRKG1-AS1 suppressed cell growth, invasion, and migration in CAL27 cells, and over-expression of PRKG1-AS1 accelerated cell growth, invasion, and migration in SCC-4 cells. Finally, silencing of PRKG1-AS1 obviously facilitated the protein expression levels of E-cadherin and reduced levels of N-cadherin, Vimentin, and Snail in CAL27 cells whereas over-expression of PRKG1-AS1 led to opposite results in SCC-4 cells. These outcomes indicated that PRKG1-AS1 functioned as a facilitator in OSCC cell growth, migration, and invasion, which all might be achieved by regulating EMT.

LncRNA SNHG7 contributes to cell proliferation, invasion and prognosis of cervical cancer.

Long non-coding RNAs (lncRNAs) have been identified as oncogenes or tumor suppressor genes in the development of various cancers. However, the function of lncRNA SNHG7 in cervical cancer remains unclear. Therefore, the aim of this study was to investigate the potential role of lncRNA SNHG7 in cervical cancer and to explore the possible underlying mechanism. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of lncRNA SNHG7 in 60 cervical cancer tissues and adjacent normal tissues. Cell Counting Kit-8 (CCK-8) and transwell assays were used to study the effects of lncRNA SNHG7 on the proliferation and invasion of cervical cancer cells, respectively. Furthermore, Western blot was used to detect the expression levels of proteins in the epithelial-mesenchymal transition (EMT) process. LncRNA SNHG7 expression increased significantly in cervical cancer tissues. The inhibition of lncRNA SNHG7 expression markedly inhibited cervical cell proliferation and invasion. Meanwhile, the inhibition of lncRNA SNHG7 resulted in the increased protein expression level of E-cadherin, and decreased protein expressions of N-cadherin and Vimentin. In addition, the survival time of patients with high expression of lncRNA SNHG7 was remarkably shortened. LncRNA SNHG7 contributes to cell proliferation and invasion of cervical cancer. Moreover, SNHG7 has emerged as an independent and significant factor associated with poor survival of cervical cancer patients.

Incremental load training improves renal fibrosis by regulating the TGF‑β1/TAK1/MKK3/p38MAPK signaling pathway and inducing the activation of autophagy in aged mice.

Recent studies have confirmed that kidney tissue fibrosis is closely linked to the natural aging of organs. One of its major characteristics is the reduction of autophagic activity. However, to date, few studies have assessed whether incremental load training is able to improve the occurrence of renal fibrosis caused by natural aging and the underlying mechanisms. In the present study involving male C57/BL mice, an elderly exercise group (OY group) was subjected to progressive load-increasing rotary-bar training (5 days/week, lasting for 6 weeks), with an elderly control group (OC group) and a young control group (YC group) used as controls. Renal fibrosis and autophagy-associated indicators were assessed by Masson's staining, reverse transcription-quantitative PCR analysis, western blotting, immunofluorescence and transmission electron microscopy. The results suggested that collagen deposition in the basal part of the renal tubular epithelium and glomeruli in the OY group was significantly lower than that in the OC group. In the OC group, the protein expression levels of E-cadherin, Beclin 1 and light chain 3 were significantly decreased, and increases in α-smooth muscle actin-positive signals were observed in the glomerular matrix and renal capsule wall. Furthermore, the expression of transforming growth factor (TGF)-β1 and its downstream signaling molecules TGF-β-activated kinase 1 (TAK1), mitogen-activated protein kinase (MAPK) kinase (MKK3) and p38MAPK were downregulated following training. The present study confirmed that incremental load training may improve renal fibrosis in aged mice by regulating the TGF-β1/TAK1/MMK3/p38MAPK signaling pathway and inducing the activation of autophagy to reduce the synthesis of extracellular matrix and delay the epithelial-mesenchymal transition. The present study provides a novel experimental basis for the intervention of incremental load training to prevent senile renal fibrosis.

Epithelial-to-Mesenchymal Transition (EMT) and Signal Transducers and Activators of Transcription (STAT3) Are a Novel Therapeutic Strategy for Hepatocellular Carcinoma (HCC) Prognosis

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide and is the second most common cancer in Egypt. Poor diagnosis of HCC is correlated with vascular invasion and metastasis. Epithelial-to-mesenchymal transition (EMT) is the main step in the tumor invasion process whereby epithelial cells lose cell polarity with each other and then undergo a dramatic remodeling of the cytoskeleton. Also, EMT plays a pivotal role in metastasis when epithelial cell layers lose cell-cell contacts in tumor progression due to the loss of E-cadherin and increasing the ability of the spread into surrounding tissues. Signal transducers and activators of transcription (STAT) play a different cellular function as signal transducers in the cytoplasm and transcription activators in the nucleus. STAT3 gene plays a crucial role to affect EMT in cancer progression by promoting cell proliferation and survival through its function as a transcription factor of the tumor. The aim of our study is to investigate the gene expression of STAT3 in HCC (HepG-2) cell lines and the expression of cell differentiation and proliferation markers. Western blotting was used to examine the protein expression of STAT3, E-cadherin, and β-catenin signaling. Our results showed that STAT3 expression levels were detected, as well as a significant increase in the expression of proliferation marker as β-catenin and a significant decrease of differentiation marker as E-cadherin protein expression levels in HepG-2 cell lines. Conclusion Our finding provides novel evidence for using a molecular gene therapy as STAT3, which showed an effect on EMT that plays a pivotal role in the prognosis of HCC. The loss of E-cadherin expression is a hallmark of EMT, because β-catenin is associated with the cytoplasmic domain of E-cadherin. We have emphasized the significant role of EMT and STAT3 in HCC progression, which could be a potential application as a novel therapeutic strategy for HCC treatment.

Role of HMGN1 in epithelial-mesenchymal transition of kidney tubules in diabetic nephropathy mice

Objective This study aimed to investigate the renal expression change of high mobility of nucleosome binding protein 1 (HMGN1) in epithelia-mesenchymal transition (EMT) process, and to study the effect of HMGN1 on renal fibrosis in the diabetic nephropathy mice model. Methods 20 C57BL/6 mice were randomly divided into control group, model group, benazepril group, and insulin group. After 8 weeks of drug intervention, blood, urine and kidney tissue samples were taken from mice. The routine physiological and biochemical indexes were detected. Renal structure and fibrosis were detected by HE and Sirius red staining, respectively. Immunohistochemistry and in situ hybridization were used to investigate the protein and mRNA expression levels of HMGN1, CD68, F4/80, α-smooth muscle actin (α-SMA), and E-cadherin in renal tissue. Results Blood glucose, renal index, and urinary albumin to creatinine ratio (UACR) were significantly higher in the model group than those in the normal group. In the model group, HE staining showed glomerular hypertrophy and interstitial inflammatory cell infiltration, and Sirius red showed collagen deposition in the renal tissue. Compared with normal group, HMGN1, CD68, F4/80 positive cell counts and α-SMA protein expression were all increased, while E-cadherin protein expression was downregulated in the model group (all P<0.05). The above indexes were not improved significantly in the benazepril group. And after intervention of insulin, the expression levels of CD68 positive cell count and α-SMA protein were decreased and the expression levels of E-cadherin protein were increased compared with the model group (all P<0.05). The correlation analysis showed that the level of HMGN1 was correlated with CD68, F4/80, α-SMA, E-cadherin and collagen protein, while CD68 and f4/80 were correlated with α-SMA, collagen protein and blood glucose, respectively (all P<0.05). Conclusion HMGN1 is involved in the progression of diabetic nephropathy fibrosis, and its underlying mechanism might be related to the macrophage-mediated EMT process. Key words: High-mobility group nucleosome-binding protein 1; Epithelial-mesenchymal transition; Diabetic nephropathies; Renal fibrosis

Oroxylin A Suppresses the Cell Proliferation, Migration, and EMT via NF-κB Signaling Pathway in Human Breast Cancer Cells.

Oroxylin A is a natural extract and has been reported to have a remarkable anticancer function. However, the mechanism of its anticancer activity remains not quite clear. In this study, we examined the inhibiting effects of Oroxylin A on breast cancer cell proliferation, migration, and epithelial-mesenchymal transition (EMT) and its possible molecular mechanism. The cytoactive and inflammatory factors were analyzed via Cell Counting Kit-8 assay and ELISA assay, respectively. Flow cytometry and western blotting were used to assess the cell proliferation. In addition, a wound healing assay and transwell assay were used to detect cell invasion and migration. qRT-PCR and western blot were employed to determine the effect of Oroxylin A on the EMT formation. Moreover, expression level of protein related to NF-κB signaling pathway was determined by western blot. The results revealed that Oroxylin A attenuated the cytoactivity of MDA-MB-231 cells in a dose- and a time-dependent manner. Moreover, cell proliferation, invasion, and migration of breast cancer cells were inhibited by Oroxylin A compared to the control. The mRNA and protein expression levels of E-cadherin were remarkably increased while N-cadherin and Vimentin remarkably decreased. Besides, Oroxylin A suppressed the expression of inflammatory factors and NF-κB activation. Furthermore, we also found that supplement of TNF-α reversed the effects of Oroxylin A on the cell proliferation, invasion, migration, and EMT in breast cancer cells. Taken together, our results suggested that Oroxylin A inhibited the cell proliferation, invasion, migration, and EMT through inactivating NF-κB signaling pathway in human breast cancer cells. These findings strongly suggest that Oroxylin A could be a therapeutic potential candidate for the treatment of breast cancer.

Long non-coding RNA H19 promotes the proliferation and invasion of lung cancer cells and regulates the expression of E-cadherin, N-cadherin, and vimentin.

BackgroundThis study aimed to explore the effect of long non-coding RNA (LncRNA) H19 on the proliferation and invasion of lung carcinoma cells A549, and to determine its molecular targets.MethodsA549 cells were with either LncRNA H19 or LncRNA H19 shRNA, and the expression levels of LncRNA H19 were evaluated by quantitative real-time PCR (RT-PCR). We measured cell proliferation using the CCK-8 assay, cell counting assays, and colony formation assay in response to shLncRNA H19-2. Cell migration and invasion were assessed by wound healing assay and Transwell assay, respectively. The mRNA and protein expression levels of E-cadherin, N-cadherin, and vimentin were determined by RT-PCR and western blot, respectively.ResultsThe three LncRNA H19 shRNAs used in our study significantly reduced the expression levels of LncRNA H19 in A549 cells (P<0.05). Moreover, LncRNA H19 shRNA 2 (shLncRNA-2) was the most potent inhibitor of LncRNA H19 expression, and was selected for further experimentation. Transfection with shLncRNA H19-2 significantly decreased the proliferation, migration, and invasion of A549 cells, while overexpression of LncRNA H19 had the opposite effect in these cells (P<0.05). In response to shLncRNA H19-2, the expression levels of E-cadherin were notably elevated (P<0.05), while the expression levels of N-cadherin and vimentin were decreased (P<0.05). In contrast, overexpression of LncRNA H19 induced the expression of E-cadherin, and blocked the expression of N-cadherin, and vimentin (P<0.05).ConclusionOur results suggest that LncRNA H19 mediates the proliferation and invasion of lung cancer cells via upregulation of N-cadherin and vimentin, and downregulation of E-cadherin.

3,3′-Diindolylmethane Inhibits TNF-α- and TGF-β-Induced Epithelial–Mesenchymal Transition in Breast Cancer Cells

Epithelial–mesenchymal transition (EMT) is the initial event required by cancer cells. Thus, inhibition of the EMT process could have potential benefits for preventing the spread of cancers. The phytochemicals have been reported to have inhibitory activity against the EMT process in breast cancers, but the mechanism behind this effect has not been fully elucidated.3,3′-Diindolylmethane (DIM) is a major indole derived from bioactive compounds in cruciferous vegetables. In this study, we examined the effects of DIM cotreatment together with TNF-α/TGF-β on the EMT process as well as the mechanisms underlying its effects on human breast cancer cells. DIM significantly enhanced the mRNA and protein expression of E-cadherin and occludin in MCF-7 cells. The protein expression levels of E-cadherin and occludin in MCF-7 cells were significantly decreased after TNF-α/TGF-β treatment alone, but these effects were reversed by the DIM co-treatment. Furthermore, DIM with TNF-α/TGF-β co-treatment attenuated the phosphorylation of Smad2/3 and ERK1/2 proteins. DIM significantly inhibited the TNF-α/TGF-β-induced migration of breast cancer cells. Taken together, the results indicated that DIM effectively suppressed EMT processes through the inhibition of TNF-α/TGF-β-associated signaling pathways in breast cancer cells. Thus, DIM may be a novel preventive and/or therapeutic approach for the treatment of breast cancers.

Effect of asiatic acid on epithelial-mesenchymal transition of human alveolar epithelium A549 cells induced by TGF-β1.

Asiatic acid (AA) is a pentacyclic triterpenoid isolated from Centella asiatica (L.) Urban that possesses significant antitumor activities. In the present study, the mechanism of AA on transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) was investigated in the lung cancer cell line A549. To do so, cell morphological alterations were observed and recorded at different time periods. Cells treated with TGF-β1 were spindle-shaped and characterized as stromal cells, whereas AA-treated cells exhibited epithelial cell characteristics and increased intercellular adhesion. The MTT assay demonstrated that the high concentration of AA inhibited the viability of A549 cells treated with TGF-β. In addition, the wound healing and Transwell assays revealed that AA inhibited TGF-β1-induced invasion and migration of A549 cells. Furthermore, AA treatment increased the mRNA and protein expression levels of E-cadherin, and decreased the expression levels of snail family transcriptional repressor (Snail), N-cadherin, vimentin and β-catenin in TGF-β1-treated A549 cells. In conclusion, these results suggested that AA may inhibit TGF-β1-induced EMT in lung cancer through increased expression of E-cadherin, and inhibition of Snail, N-cadherin and vimentin expression.

Expression of lysine-specific demethylase 1 in hepatocellular carcinoma tissues and its effect on cell proliferation and invasion

Objective To investigate the expression of lysine-specific demethylase 1 (LSD1) in hepatocellular carcinoma tissues, and to explore the effects of down-regulation of LSD1 gene expression on proliferation and invasion of human hepatoma HepG2 cells. Methods A total of 91 cases of hepatocellular carcinoma undergoing elective surgery in our hospital and Henan Cancer Hospital were selected. Immunohistochemistry was used to detect the expression of LSD1 protein in hepatocellular carcinoma tissues and adjacent tissues. Human hepatoma HepG2 cells were cultured and divided into LSD1 interference sequence group, negative control group and blank group. Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect the expression of LSD1 gene in cells. Methyl thiazol tetrazolium (MTT) assay was used to detect cell proliferation. Transwell chamber was used to detect cell invasion. Western blotting was used to detect the expression of LSD1, Snail, E-cadherin and N-cadherin proteins in cells. Results The positive expression rate of LSD1 protein in hepatocellular carcinoma tissues was 81.32%, which was significantly higher than that in adjacent tissues (35.16%, P<0.01). The expression of LSD1 protein in hepatocellular carcinoma tissues was related to the degree of differentiation, TNM staging and vascular invasion (P<0.05). The relative expression level of LSD1 mRNA in LSD1 interference sequence group was 1.19±0.10, which was significantly lower than that in the negative control group and blank group (2.29±0.14 and 2.34±0.11, P<0.05). The absorbance (A) values at 24, 48, 72 and 96 h in the LSD1 interference sequence group were 0.23±0.05, 0.32±0.08, 0.38±0.08 and 0.52±0.03, respectively, which were significantly lower than those in the negative control group (0.38±0.08, 0.49±0.05, 0.67±0.04 and 0.77±0.10) and blank group (0.33±0.03, 0.52±0.09, 0.70±0.08 and 0.78±0.07, P<0.05). The number of invasive cells in the LSD1 interference sequence group was (86.3±3.9), which was significantly less than that in the negative control group (110.6±9.4) and the blank group (117.7±7.0, P<0.05). The relative expression levels of LSD1, Snail, and N-cadherin proteins in the LSD1 interference sequence group were (0.25±0.03, 0.30±0.02 and 0.32±0.03), which were significantly lower than those in the negative control group (0.77±0.06, 0.76±0.10 and 0.67±0.04) and blank group (0.84±0.14, 0.74±0.07 and 0.69±0.06, P<0.05), and the relative expression level of E-cadherin protein (0.71±0.05) was significantly higher than in the negative control group (0.37±0.02) and blank group (0.34±0.04, P<0.05). Conclusion LSD1 protein was highly expressed in hepatocellular carcinoma tissues, and might play an important role in the proliferation and invasion of human hepatoma HepG2 cells through epithelial-mesenchymal transition. Key words: Carcinoma, hepatocellular; Lysine-specific demethylase 1; Proliferation; Invasion; Epithelial-mesenchymal transition

Effect of endostatin on proliferation, invasion and epithelial-mesenchymal transition of basal cell carcinoma cell A431.

To investigate the effect of endostatin on the proliferation, invasion and epithelial-mesenchymal transition (EMT) of human basal cell carcinoma (BCC) cells (A431). CCK-8 assay and transwell chamber assay were performed to detect cell proliferation and invasion abilities, respectively. Western blot was performed for the detection of the expressions of EMT-related proteins levels. The therapeutic effect of endostatin on tumor formation was tested using a mouse xenograft model. After endostatin treatment, transwell assay showed that the number of invasive cells in the observation group and control group were (38.25±8.13) and (98.25±9.14), respectively; the relative expression level of E-cadherin protein in the observation group was (0.34±0.03), which was significantly higher than that in the control group (0.14±0.01); the relative expression levels of N-cadherin protein in the observation group was (0.18±0.05), which was significantly lower than that in the control group (0.43±0.03), (all p<0.05). The expression levels of Vimentin and Fibronectin proteins were significantly lower, while the expression levels of α-smooth muscle Actin (α-SMA) were significantly higher in the observation group than those in the control group. Treatment with endostatin significantly inhibited tumor growth in the mouse xenograft model. Therefore, endostatin can inhibit the proliferation, invasion and EMT in BCC.

Transforming Growth Factor β1 (TGF-β1)-Stimulated Integrin-Linked Kinase (ILK) Regulates Migration and Epithelial-Mesenchymal Transition (EMT) of Human Lens Epithelial Cells via Nuclear Factor κB (NF-κB).

BackgroundIn view of the high incidence of posterior capsule opacification (PCO) and the effects of TGF-β signaling on the epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs), our study aimed to explore the mechanism of the function of TGF-β signaling in LECs EMT.Material/MethodsHuman lens epithelial cells (HLEC-h3) were treated with TGF-β, ILK siRNA, ILK inhibitor, and NF-κB inhibitor to study the effects of TGF-β, ILK, and NF-κB on cell migration and EMT. Cell migration assay was used to measure cell migration ability. Western blot was performed to detect the expression of ILK, E-cadherin, and α-SMA at the protein level. QRT-PCR was used to detect the expression of ILK at the mRNA level.ResultsCompared with control cells, TGF-β treatment increased the expression level of ILK HLEC-h3, promoted migration of HLEC-h3 cells, increased the expression level of E-cadherin protein, and decreased the expression level of α-SMA protein. However, treatment with ILK siRNA, ILK inhibitor, and NF-κB inhibitor reversed the effects of TGF-β on HLEC-h3 cells.ConclusionsTGF-β-stimulated ILK regulates the migration and EMT of human LECs via NF-κB.

Pectolinarigenin inhibits non‑small cell lung cancer progression by regulating the PTEN/PI3K/AKT signaling pathway.

Lung cancer is the principal cause of cancer-associated mortality. Pectolinarigenin (Pec) reportedly has effective antitumor activity against certain cancer types. Phosphatase and tensin homolog (PTEN) is a well-known tumor suppressor and serves a vital role in cancer progression. However, the effect of Pec on non-small cell lung cancer (NSCLC) cell proliferation and metastasis, and the underlying mechanism, has not yet been elucidated. In the present study, it was demonstrated that Pec inhibited the proliferation of A549 and Calu-3 cells in dose- and time-dependent manners. The apoptosis rate significantly increased with increasing doses of Pec. Apoptosis-associated protein expression was additionally altered by Pec exposure. Pec was able to suppress the metastasis of NSCLC cells; it upregulated the mRNA and protein expression levels of E-cadherin, and downregulated the mRNA and protein expression levels of vimentin. Additionally, Pec was able to activate PTEN and subsequently downregulate the PI3K/protein kinase B (AKT) signaling pathway. In summary, Pec was able to inhibit cell proliferation, promote apoptosis and suppress metastasis in NSCLC cells through the PTEN/PI3K/AKT signaling pathway, indicating that Pec is a potential agent for NSCLC therapy.

Polyphyllin I inhibits growth and invasion of cisplatin-resistant gastric cancer cells by partially inhibiting CIP2A/PP2A/Akt signaling axis

The aberrant expression of cancerous inhibitor of protein phosphatase 2A (CIP2A) indicates poor prognosis and promotes EMT and metastasis. EMT, a crucial cellular process that occurs during cancer progression and metastasis, has been reported to promote drug resistance in several previous studies. Consequently, ongoing research has been focused on exploring therapeutic options for preventing EMT to delay or reverse drug resistance. Polyphyllin I (PPI) is a natural component extracted from Paris polyphylla that displays anti-cancer properties. In the present study, we investigated whether PPI can be used in the cisplatin (DDP)-resistant human gastric cancer cell line SGC7901/DDP. The results demonstrated that PPI treatment significantly inhibited cell proliferation, invasion and EMT. TGF-β1 is known to promote EMT-induced metastasis in numerous tumor types. PPI inhibited the invasion of TGFβ1-induced SGC7901/DDP cells in vitro. PPI also increased the mRNA and protein expression levels of E-cadherin but decreased the expression levels of vimentin. Further examination of the mechanism revealed that the CIP2A/PP2A/Akt pathway is partially involved in this regulation of EMT-related biomarkers and invasion. Furthermore, xenograft tests also confirmed the antitumor effects of PPI in vivo. We propose that PPI could be developed as a candidate drug for treating cancer invasion and migration.

Abstract 3158: DLL3 regulates migration and invasion of small cell lung cancer

Abstract Background: Delta-like protein 3 (DLL3) is a ligand of Notch signaling, which is reported to be a tumor suppressor in small cell lung cancer (SCLC). Previous studies suggest that DLL3 might be associated with neuroendocrine tumorigenesis through inhibition of Notch signaling unlike other activating ligands. Moreover, DLL3 was highly expressed in SCLC, but not in normal lung tissue. However, little is known about function of DLL3 in SCLC. In this study, we examine the effect of DLL3 in tumorigenesis of SCLC. Methods: The mRNA expression levels and proteins of DLL3, Notch receptors (Notch1, Notch2, Notch3 and Notch4), Hes1 and EMT markers (E-cadherin, Snail and Vimentin) were examined using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot in 9 SCLC cell lines (SBC-3, SBC-5, MS-1, RERF-LC-MA, H69, H82, H209, H529 and H1688). We used small interfering RNA (siRNA) to down-regulate the expression of DLL3 in SCLC cell lines. Anchorage-dependent and anchorage-independent cell growth was measured by MTT assay and migration and invasion were assessed by transwell assay. Results: The mRNA of DLL3 was expressed in all of 9 SCLC cell lines. The expression of DLL3 was especially higher in H82, H69, H209, H529 and H1688 cells. Notch1 protein was expressed in SBC-3, SBC-5, MS-1 and H82. Based on DLL3 expression analysis data, we used H82 and H69 in the following experiments. The suppression of DLL3 by siRNA resulted in the moderate inhibition of cell growth of H82 cells in both anchorage-dependent and anchorage-independent cell proliferation. The depletion of DLL3 prevented migration and invasion in the two cell lines. The expressions of Notch1 and Notch target gene, Hes1 were downregulated by DLL3 knockdown in both SCLC cells. Because Notch pathway was reported to regulate EMT, we evaluated the EMT markers when DLL3 was inhibited in SCLC cells. Snail was downregulated in DLL3 knockdown SCLC cells, while protein expression levels of E-cadherin and Vimentin were not changed in these DLL3 knockdown cells compared to control cells. Conclusions: DLL3 promotes the migration and invasion in SCLC cells by modulating Notch1 and Snail. Citation Format: Megumi Furuta, Jun Konishi Sakakibara, Tetsuaki Shoji, Yuta Takashima, Hajime Kikuchi, Eiki Kikuchi, Junko Kikuchi, Ichiro Kinoshita, Hirotoshi Dosaka Akita, Masaharu Nishimura. DLL3 regulates migration and invasion of small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3158.

Abstract 1396: Pathway-directed DNA demethylation as an epithelial to mesenchymal targeting strategy

Abstract It has been proposed that by silencing tumor suppressor genes, DNA methylation plays an important function in cancer initiation and progression. DNA hypomethylating agents are being developed as anti-cancer agents. However, this approach causes unrestricted hypomethylation, which could potentially activate tumor-promoting mechanisms. Therefore a strategy capable of inducing hypomethylation of selected genes is needed. Towards this goal, we designed a strategy to induce demethylation of a coordinated gene program by using Ten-eleven translocation (TET1) dioxygenase, which converts 5 -methylcytosine to 5-hydroxymethylcytosine. We constructed a fusion gene TET1-DBD containing the catalytic domain of TET1 and the DNA binding domain of Snail, the transcription factor that regulates epithelial-to-mesenchymal transition (EMT). We hypothesized that this fusion gene will bind to promoters targeted by Snail, induce hypomethylation, reactivate gene expression, and block EMT. To test this hypothesis, we used TET1-DBD and measured induction of EMT in an ovarian (SKOV3) and breast cancer (MCF10A) model driven by TGF-β. Genome-wide DNA methylation was assessed by Reduced Representation Bisulfite Sequencing (RRBS) to measure the downstream targets of TET1-DBD. TET1-DBD increased mRNA and protein expression levels of E-cadherin in transfected cells and prevented TGF-β induced expression of fibronectin and vimentin in MCF10A and SKOV3 cells. RRBS analysis demonstrated hypomethylation of genes targeted by Snail-DBD. Our results suggest that TET1-DBD selectively binds to and hypomethylates promoters of genes targeted by Snail, resulting in inhibition of the EMT program. We propose a novel approach to induce selective promoter hypomethylation and to block key oncogenic processes by using TET1 fused to a transcription factor “carrier”. Citation Format: HAO HUANG, Qing Yu, Maooj Kandpal, Ramana V. Davuluri, Daniela Matei. Pathway-directed DNA demethylation as an epithelial to mesenchymal targeting strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1396.

MicroRNA-206 regulates the epithelial-mesenchymal transition and inhibits the invasion and metastasis of prostate cancer cells by targeting Annexin A2.

The present study investigated the molecular mechanism by which microRNA-206 (miR-206) targets Annexin A2 (ANXA2) expression and inhibits the invasion and metastasis of prostatic cancer cells through regulation of the epithelial-mesenchymal transition (EMT). Using bioinformatics analysis, miR-206 was identified as the most promising candidate miRNA that targeted ANXA2. Prostate tissue specimens from 60 patients with prostate cancer, 30 patients with metastatic prostate cancer and 20 patients with benign prostatic hyperplasia (BPH) were examined for ANXA2 protein expression by immunohistochemistry and western blotting and for miR-206 expression by reverse transcription-quantitative polymerase chain reaction. Additionally, human prostate cancer PC-3 cells were transfected with miR-206 mimics, miR-206 inhibitors or a negative control sequence, and expression of ANXA2, E-cadherin and N-cadherin was detected by western blotting. Transwell assays were performed to determine the effect of altered miR-206 expression on the invasive behavior of PC-3 cells. Bioinformatics analysis predicted complementary binding between miR-206 and ANXA2 mRNA. ANXA2 protein expression was detected in a significantly higher proportion of BPH tissues (95%, 19/20) when compared with prostate cancer tissues (51.7%, 31/60; P<0.05). Similarly, ANXA2 was expressed in a significantly higher proportion of metastatic prostate cancer samples than that of prostate cancer samples (P<0.05). Expression of miR-206 was higher than that of ANXA2 in prostate cancer samples, but lower in BPH samples. Inhibition of miR-206 expression in PC-3 cells upregulated ANXA2 and E-cadherin protein expression levels, downregulated N-cadherin and vimentin, and promoted cell invasion in vitro. These data suggested that binding between miRNA-206 and ANXA2 mRNA may regulate EMT signaling, thereby suppressing the invasion and metastasis of prostatic cancer cells.