E-cadherin mRNA Expression Research Articles

The effect of octamer-binding transcription factor 4 pseudogene 5 on bladder cancer cell line T24 migration and invasion

Objective To investigate octamer-binding transcription factor 4 pseudogene 5 (OCT4-pg5) expression in bladder cancer cell lines and its effect on T24 migration and invasion and the potential mechanism. Methods The real-time quantitative reverse transeriptase-polymerase chain reaction (RT-qPCR) was conducted to detect OCT4-pg5 expression in 5 kinds of bladder cancer cell lines (5637, EJ, BIU-87, T24, TCCSUP) and one normal Bladder epithelial cell line SV-HUC-1. Si-OCT4-pg5 vector and its control vector were transfected into T24 cells by Lipofectamine 2000, and OCT4-pg5 expression was detected by RT-qPCR. Next, wound healing assay and Transwell assay were used to examine the effects of OCT4-pg5 on T24 cell migration and invasion. Moreover, the possible mechanism by which OCT4-pg5 mediated T24 migration and invasion was explored using Western blotting and RT-qPCR. Results In comparison to SV-HUC-1, OCT4-pg5 levels were up-regulated in bladder cancer cell lines, especially in T24 cell (P<0.01). RT-qPCR results showed that OCT4-pg5 espression was obviously down-regulated after being translated with si-OCT4-pg5 vector. OCT4-pg5 down-expression in T24 cell could inhibit cell migration[ (28.50±4.08)% and (43.26±5.49)%, P<0.01] and invasion [ (104.50±6.95) and (140.50±8.90) cells, P<0.01]. In addition, down-regulation of OCT4-pg5 significantly reduced Vimentin (0.60±0.06) and β-catenin (0.70±0.08) mRNA expression, and improved E-cadherin mRNA expression (1.50±0.10) when compared with their contral groups (1.00±0.02, 1.00±0.04 and 1.00±0.05, respectively, P<0.01). Moreover, the same results were also obtained by western blotting. Conclusion OCT4-pg5 is overexpression in bladder cancer cells. And OCT4-pg5 down-expression in T24 cell could inhibit cell migration and invasion. OCT4-pg5 mediated T24 migration and invasion probably induced by regulating E-cadherin, Vimentin, β-catenin expression. Key words: Bladder cancer; Octamer-binding transcription factor 4 pseudogene 5; Migration; Invasion

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.

Garcinol Sensitizes NSCLC Cells to Standard Therapies by Regulating EMT-Modulating miRNAs.

Garcinol, a dietary factor obtained from Garcinia indica, modulates several key cellular signaling pathways as well as the expression of miRNAs. Acquired resistance to standard therapies, such as erlotinib and cisplatin, is a hallmark of non-small cell lung cancer (NSCLC) cells that often involves miRNA-regulated epithelial-to-mesenchymal transition (EMT). We used A549 cells that were exposed to transforming growth factor beta 1 (TGF-β1), resulting in A549M cells with mesenchymal and drug resistant phenotype, and report that garcinol sensitized resistant cells with mesenchymal phenotype to erlotinib as well as cisplatin with significant decrease in their IC50 values. It also potentiated the apoptosis-inducing activity of erlotinib in A549M and the endogenously mesenchymal H1299 NSCLC cells. Further, garcinol significantly upregulated several key EMT-regulating miRNAs, such as miR-200b, miR-205, miR-218, and let-7c. Antagonizing miRNAs, through anti-miRNA transfections, attenuated the EMT-modulating activity of garcinol, as determined by mRNA expression of EMT markers, E-cadherin, vimentin, and Zinc Finger E-Box Binding Homeobox 1 (ZEB1). This further led to repression of erlotinib as well as cisplatin sensitization, thus establishing the mechanistic role of miRNAs, particularly miR-200c and let-7c, in garcinol-mediated reversal of EMT and the resulting sensitization of NSCLC cells to standard therapies.

ANP/NPRA Inhibits Epithelial-Mesenchymal Transition of Airway by Targeting Smad3 in Asthma

Atrial natriuretic peptide (ANP) has a protective effect on allergic disorders of airway. It could inhibit transforming growth factor beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) of human airway cells. In this study, we investigated the role and mechanism of ANP in airway EMT with asthma. Asthma model was established with BALB/c mice using ovalbumin. Asthmatic mice were respectively treated with ANP, NPRA antagonist (A71915) or ANP+A71915. The protein expressions of TGF-β1, ANP, epithelial cadherin (E-Cadherin), α-smooth muscle actin (α-SMA), Smad3 and phosphorylated-Smad3 in lung tissues were detected by immunohistochemistry and Western blotting. The mRNA expressions of E-Cadherin, α-SMA, Smad3 in lung tissues were measured using real-time PCR. The levels of TGF-β1, IL-4 and IL-5 in bronchoalveolar lavage fluid, cyclic guanosine monophosphate (cGMP) in lung tissues, and ANP in serum were assessed by ELISA. Compared with controls, mRNA and protein expressions of E-Cadherin were decreased, whereas α-SMA were increased in asthma group, which could be reversed by ANP. cGMP level was higher in ANP group than asthma group, whereas p-Smad3 expression was lower. TGF-β1, IL-4 and IL-5 levels were higher in asthma group than controls, which could be reversed by ANP. The effect of ANP in asthma group could be inhibited by A71915. Therefore, in asthma, ANP/NPRA could inhibit airway EMT by targeting Smad3 via attenuating phosphorylation of Smad3. cGMP signaling may be involved into this process. This may provide a potential of ANP in treatment on refractory asthma with airway remodeling.

Yangyin Yiqi Mixture Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Rats through Inhibiting TGF-β1/Smad Pathway and Epithelial to Mesenchymal Transition.

Objective The aim of the current study was to investigate the protective effect of Yangyin Yiqi Mixture (YYYQ) on Bleomycin-induced pulmonary fibrosis in rats based on TGF-β1/Smad signal pathway and epithelial to mesenchymal transition (EMT). Methods 120 Wistar rats were randomly divided into six groups: control group, BLM group, BLM + Pred group, BLM+YYYQ-L group, BLM+YYYQ-M group, and BLM+YYYQ-H group. Rats were given an intratracheal instillation of 3 mg/kg BLM to establish the pulmonary fibrosis model and followed by different dosages of YYYQ (11, 22, 44g/kg, via intragastric gavage) or prednisone soluble (4.2mg/kg, via intragastric gavage) or water. After 14 days and 28 days, tissue sections were stained with hematoxylin-eosin and Masson's trichrome to observe histopathological changes. Protein levels of TGF-β1, CTGF, Interleukin 18, and hydroxyproline were detected by ELISA method, and mRNA expressions of TGF-β1, TβRI, TβRII, Smad3, Smad7, α-SMA, E-cadherin, laminin, and collagen I were detected by RT-PCR. Results TGF-β1, CTGF, Interleukin 18, and hydroxyproline levels and mRNA expression of TGF-β1, TβRI, TβRII, Smad3, α-SMA, laminin, and collagen I were significantly increased (p <0.01), while Smad7 and E-cadherin levels were significantly decreased in BLM group (p <0.01). YYYQ-M and YYYQ-H group had downregulated the TGF-β1, CTGF, hydroxyproline contents, and mRNA expression of TGF-β1, TβRI, TβRII, Smad3, α-SMA, laminin, and collagen I and upregulated mRNA levels of Smad7 and E-cadherin significantly (p <0.01 or p <0.05). The result from the present study, which was also supported by histological evidence, suggested that YYYQ-M group and YYYQ-H group exhibited better treatment effect on Bleomycin-induced pulmonary fibrotic rats when compared to that of BLM + Pred group (p <0.01). Meanwhile, the effect of YYYQ, in three different dosages, on the level of interleukin 18 was not significant. Conclusion These results showed that YYYQ has the potential of ameliorating the progression of pulmonary fibrosis, and the mechanism may be related to suppressing TGF-β1/Smad signal pathway and EMT in BLM-induced pulmonary fibrosis of rats.

E-Cadherin gene expression in oral cancer: Clinical and prospective data

BackgroundLow protein expression of E-cadherin in oral squamous cell carcinoma (OSCC) has been associated with clinical and histopathological traits such as metastases, recurrence, low survival and poor tumor differentiation, and it is considered a high-risk marker of malignancy. However, it is still unknown whether low expression of E-cadherin is also present at the mRNA level in OSCC cases. Objective: The aim of this study was to compare E-cadherin mRNA expression in OSCC patients and controls and to correlate the expression with clinical and prospective characteristics.Material and MethodsForty patients and 40 controls were enrolled. E-cadherin mRNA expression was evaluated by quantitative real-time polymerase chain reaction using TaqMan probes.ResultsE-cadherin mRNA expression was significantly decreased in OSCC patients compared to that of controls (p<0.001). Whereas no significant association between clinical parameters and E-cadherin expression levels was observed, we noted lower E-cadherin expression levels in patients with positive lymph node metastasis.ConclusionsE-cadherin mRNA expression was markedly diminished in OSCC, in agreement with previous results that examined E-cadherin expression at the protein level. E-cadherin is downregulated in the early clinical stages of OSCC, and its mRNA levels do not change significantly in the advanced stages, suggesting that there is limited usefulness of this parameter for predicting disease progression. Key words:Oral cancer, E-Cadherin, gene expression.

Effect of Withaferin A on epithelial-mesenchymal transition and apoptosis in cells of lung squamous cell carcinoma

Objective To study the regulation of Withaferin A on apoptosis-related proteins in lung squamous cell carcinoma and its effect on cell epithelial-mesenchymal transition. Methods Lung squamous cell carcinoma cell line SK-MES-1 were cultured in vitro. The SK-MES-1 cells were treated with the final mass concentration of 0 (control), 5, 10, 20, and 40 μg/ml for 28 h, and the general morphology and shedding of the cells were observed under phase contrast microscope; MTT assay was used for detection of cell viability; flow cytometry was used for detection of apoptosis; immunofluorescence and real-time fluorescent polymerase chain reaction (RT-PCR) was used for detection of apoptosis-related proteins and genes bcl-2 and bax, and expression of the epithelial marker E-cadherin and the interstitial marker Vimentin. Results Different concentrations (0, 5, 10, 20, 40 μg/ml) of Withaferin A inhibited the activity of SK-MES-1 cells with cell viability of 0.62±0.05, 0.42±0.04, and 0.33±0.06, 0.21±0.03, 0.17±0.04, respectively, which suggesting that this inhibition was related to the concentration of Withaferin A (F = 386.505, P = 0.005). After treatment with SK-MES-1 cells for 24 h at different concentrations (0, 5, 10, 20, 40 μg/ml) of Withaferin A, the apoptosis rates of each group were (0.180±0.011)%, (0.310±0.013)%, (0.500±0.021)%, (0.540±0.018)%, and (0.410±0.027)%, which suggesting that Withaferin A rarely caused apoptosis, mostly necrotic cells (F = 1 065.78, P = 0.124). In SK-MES-1 cells treated with different concentrations of Withaferin A for 24 h, the results of immunofluorescence showed that the expression of Vimentin was decreased in the experimental group at a concentration of 20 μg/ml compared with the control group, and the fluorescence intensity was lower than that of the control group, but the fluorescence intensity of E-cadherin was higher than that of the control group; the intensity did not change significantly in the experimental group with the other concentrations. While the expression levels of bax and bcl-2 proteins in the control group and the experimental group did not change significantly. RT-PCR results showed that the mRNA expression of E-cadherin (6.7±0.6 and 6.4±0.9) in the experimental group at a concentration of 20 and 40 μg/ml was significantly higher than that in the control group (4.2±1.0), and the difference was statistically significant (both P < 0.05), and the mRNA expression of Vimentin (4.7±0.5 and 4.7±0.5) was significantly lower than that in the control group (7.2±0.7), and the difference was statistically significant (both P < 0.05). Conclusion Withaferin A can inhibit the growth of lung squamous cell carcinoma cells and inhibit the process of epithelial-mesenchymal transition, but it has no obvious relationship with apoptosis and apoptosis-related proteins. Key words: Withaferin A; Squamous cell carcinoma, lung; Apoptosis; Epithelial-mesenchymal transition

Mechanism underlying the inhibitory effect of overexpression of tumor suppressor gene DKK3 on migration and invasion of human cutaneous malignant melanoma cells

Objective To investigate the expression of DKK3 in human cutaneous malignant melanoma cells and tissues, and to evaluate the effect of transfection with DKK3 gene on migration and invasion of a malignant melanoma cell line A375. Methods Western blot analysis was performed to measure the relative expression of DKK3 in human cutaneous melanoma cell lines HM, A375, WM451, SK-MEL-1, Hs-695T, MDA-MB-435s and WM35, as well as pigmented nevus tissues. Real-time fluorescence-based quantitative PCR (RT-PCR) was conducted to determine the mRNA expression of DKK3 in 58 melanoma tissues (including primary melanoma and metastatic melanoma) and 30 pigmented nevus tissues from Chongqing Traditional Chinese Medicine Hospital between August 2014 and June 2017. The pcDNA3.1 (+) -Flag-Vector (control group) and pcDNA3.1 (+) -Flag-DKK3 (transfection group) were transfected into A375 melanoma cells separately. RT-PCR and Western blot analysis were performed to verify the overexpression of DKK3, and to evaluate the effect of DKK3 overexpression on the expression of molecules related to the migration and invasion of melanoma cells. Cell scratch assay, Transwell migration and invasion assay were conducted to assess the effect of DKK3 on the migration and invasion of A375 cells. Statistical analysis was done by a two-sample t-test for comparisons between two groups, one-way analysis of variance (ANOVA) for intergroup comparison, and least significant difference (LSD) -t test for multiple comparisons with the SPSS 13.0 software. Results DKK3 protein was absent or lowly expressed in the human melanoma cell lines, but highly expressed in the pigmented nevus tissues. There were significant differences in the mRNA expression of DKK3 among the primary melanoma tissues (2-ΔΔCt:[0.325 ± 0.150] × 10-3) , metastatic melanoma tissues ([0.142 ± 0.210] × 10-3) and pigmented nevus tissues ([0.634 ± 0.120] × 10-3, F = 46.57, P < 0.05) . In addition, the mRNA expression of DKK3 was significantly lower in the metastatic melanoma tissues than in the primary melanoma tissues and pigmented nevus tissues (LSD-t = 2.48, 3.12, both P < 0.05) . After transfection with DKK3, cell scratch assay showed that the migration rate was significantly lower in the transfection group (22.11% ± 5.11%) than in the control group (54.36% ± 23.22%, t = 2.36, P < 0.001) . Transwell migration and invasion assay revealed that the number of A375 cells crossing the Transwell chamber was significantly lower in the transfection group (265 ± 33, 76 ± 18 respectively) than in the control group (429 ± 41, 135 ± 21 respectively; t = 1.24, 1.35 respectively, both P < 0.001) . After overexpression of DKK3 in the A375 cells in the transfection group, the mRNA and protein expression of E-cadherin were up-regulated, while the mRNA and protein expression of N-cadherin, vimentin, matrix metalloproteinase 2 (MMP2) , MMP7 and MMP11 were down-regulated compared with the control group. Conclusions The expression of DKK3 is down-regulated in the melanoma cell lines and tissues, and the migration and invasion of A375 cells are markedly inhibited by overexpression of DKK3. DKK3 may be a target for inhibiting the metastasis of cutaneous malignant melanoma. Key words: Melanoma; Genes, tumor suppressor; Cell movement; Invasion; Dickkopf-related protein 3; A375 cells

Low expression of PDK1 inhibits renal cell carcinoma cell proliferation, migration, invasion and epithelial mesenchymal transition through inhibition of the PI3K-PDK1-Akt pathway

As the most commonly occurring form of primary renal tumor, renal cell carcinoma (RCC) is a malignancy accompanied by a high mortality rate. 3-phosphoinositide-dependent protein kinase 1 (PDK1) has been established as a protein target and generated considerable interest in both the pharmaceutical and academia industry. The aim of the current study was to investigate the effect of si-PDK1 on the RCC cell apoptosis, proliferation, migration, invasion and epithelial mesenchymal transition (EMT) in connection with the PI3K-PDK1-Akt pathway. Microarray analysis from the GEO database was adopted to identify differentially expressed genes (DEGs) related to RCC, after which the positive expression of the PDK1 protein in tissue was determined accordingly. The optimal silencing si-RNA was subsequently selected and RCC cell lines 786-O and A498 were selected and transfected with either a si-PDK1 or activator of the PI3K-PDK1-Akt pathway for grouping purposes. The mRNA and protein expressions of PDK1, the PI3K-PDK1-Akt pathway-, EMT- and apoptosis-related genes were then evaluated. The effect of si-PDK1 on cell proliferation, apoptosis, invasion and migration was then analyzed. Through microarray analysis of GSE6344, GSE53757, GSE14762 and GSE781, PDK1 was examined. PDK1 was determined to be highly expressed in RCC tissues. Si-PDK1 exhibited marked reductions in relation to the mRNA and protein expression of PDK1, PI3K, AKT as well as Vimentin while elevated mRNA and protein expressions of E-cadherin were detected, which ultimately suggested that cell migration, proliferation and invasion had been inhibited coupled with enhanced levels of cell apoptosis. While a notable observation was made highlighting that the PI3K-PDK1-Akt pathway antagonized the effect of PDK1 silencing. Taken together, the key observations of this study provide evidence suggesting that high expressions of PDK1 are found in RCC, while highlighting that silencing PDK1 could inhibit RCC cell proliferation, migration, invasion and EMT by repressing the PI3K-PDK1-Akt pathway.

Anti-metastasis activity of curcumin against breast cancer via the inhibition of stem cell-like properties and EMT

BackgroundCurcumin is a polyphenolic compound with potent chemopreventive and anti-cancer efficacy. PurposeTo explore the potential anti-metastasis efficacy of curcumin in breast cancer stem-like cells (BCSCs), which are increasingly considered to be the origin of the recurrence and metastasis of breast cancer. MethodsA CCK8 assay was performed to evaluate cell viability, and a colony formation assay was conducted to determine cell proliferation in MCF-7 and MDA-MB-231 adherent cells. Transwell and wound healing assays were used to detect the effect of curcumin on cell migration and invasion in MDA-MB-231 cells. Mammospheres were cultured with serum free medium (SFM) for three generations and the BCSC surface marker CD44+CD24−/low subpopulation was measured by flow cytometry. Mammosphere formation and differentiation abilities were determined after cell treatment with curcumin. Then, a reverse transcription-quantitative polymerase chain reaction assay was conducted to detect the relative mRNA level of epithelial-mesenchymal transition (EMT) marker genes and western blot analysis was performed to determine the protein expression of stem cell genes in mammospheres treated with curcumin. ResultsCurcumin exhibited anti-proliferative and colony formation inhibiting activities in both the MCF-7 and MDA-MB-231 cell lines. It also suppressed the migration and invasion of MDA-MB-231 cells. The CD44+CD24−/low subpopulation was larger in mammospheres when MCF-7 and MDA-MB-231 adherent cells were cultured with SFM. Further studies revealed that curcumin inhibited mammosphere formation and differentiation abilities. Moreover, curcumin down-regulated the mRNA expression of Vimentin, Fibronectin, and β-catenin, and up-regulated E-cadherin mRNA expression levels. Western blot analysis demonstrated that curcumin decreased the protein expression of stem cell genes including Oct4, Nanog and Sox2. ConclusionThe results of the present study suggest that the inhibitor effects of curcumin on breast cancer cells may be related to resistance to cancer stem-like characters and the EMT process. These data indicate that curcumin could function as a type of anti-metastasis agent for breast cancer.

Effect of total triterpenoids of Chaenomeles speciosa on PPARγ/SIRT1/NF-κBp65 signaling pathway and intestinal mucosal barrier of ulcerative colitis induced by DSS in mice

To observe the effect of total triterpenoids of Chaenomeles speciosa on PPARγ/SIRT1/NF-κBp65 signaling pathway and intestinal mucosal barrier of ulcerative colitis induced by dextran sulfate sodium (DSS) in mice, C57BL/6 mice were randomly divided into normal group, model group, total triterpenoids of C. speciosa (50, 100 mg·kg⁻¹) groups and sulfasalazine (250 mg·kg⁻¹) group. The ulcerative colitis (UC) model was induced by orally administering 2.5% DSS to the experimental mice, and the corresponding drugs were given to each group 3 days before the administration with 2.5% DSS. The normal group and the model group were given the equal volume of 0.5% carboxymethyl cellulose sodium solution by gavage continuously for 10 days, q.d. The general conditions of the mice were observed on a daily basis, and the disease activity index (DAI) score was recorded. On the 10th day after the treatment, mice were put to death, the contents of TNF-α, IL-1β, IL-6, IFN-γ, IL-4 and IL-10 in the blood were detected, colon length was measured, colon mucosa damage index (CMDI) score was calculated, and MPO activity detection and histomorphology analysis were conducted. Real-time PCR was applied to detect the mRNA expressions of E-cadherin, occluding,MUC2 and TFF3; the protein expressions of SIRT1, IKKβ, p-IKKβ, IκBα, p-IκBα and cytosol and nucleus PPARγ, NF-κBp65 in intestinal tissue were detected by western blot. The results indicated that total triterpenoids of C. speciosa (50, 100 mg·kg⁻¹) could significantly improve the general conditions of UC mice, reduce the DAI, CMDI and histopathological scores, increase the colon length, reduce the colonic mucosa ulcers, erosion and inflammatory infiltration, restore the normal intestinal mucosal barrier function, reduce the contents of TNF-α, IL-1β, IL-6, IFN-γ, increase the contents of IL-4 and IL-10 in the blood, inhibit MPO activity in colon tissue, up-regulate the mRNA expressions of E-cadherin, occludin, MUC2 and TFF3 in colon tissue, down-regulate the protein expressions of cytosol PPARγ, tissue p-IKKβ, p-IκBα and nucleus NF-κBp65 in the colon tissue, decrease the p-IKKβ/IKKβ and p-IκBα/IκBα ratios, up-regulate the protein expressions of nucleus PPARγ, tissue SIRT1 and cytosol NF-κBp65 (P<0.05 or P<0.01, respectively), with a dose-effect relationship between the total triterpenoids of C. speciosa treated groups. These findings suggested that total triterpenoids of C. speciosa had a significantly therapeutic effect on UC mice induced by DSS, its mechanism might be related to the regulation of PPARγ/SIRT1/NF-κBp65 signaling pathway, the inhibition of pro-inflammatory factor formation and the up-regulation of protein expression of protective factors.

GLI-1 facilitates the EMT induced by TGF-β1 in gastric cancer.

To explore how GLI-1 affects the EMT induced by TGF-β1 in gastric cancer. Following 24 hours of culture of SGC-7901 cells in presence of TGF-β1, we observed the changes in morphology as well as mRNA and protein expressions of GLI-1, E-cadherin and Vimentin by RT-PCR and Western blot. Transwell assay was conducted to evaluate the changes in invasion ability of SGC-7901 cells. Then, SGC-7901 cells were co-treated with TGF-β1 and GANT 61, and changes of the above indexes were also detected using the corresponding methods. In presence of TGF-β1, EMT was initiated in SGC-7901 cells EMT with increased cell invasion ability, and the mRNA and protein expressions of E-cadherin were downregulated, while those of the GLI-1 and Vimentin were upregulated. Conversely, the co-treatment of TGF-β1 and GANT 61 suppressed the increased cell invasion ability induced only by TGF-β1, and the changes in mRNA and protein expressions of these factors were abolished. We found that GLI-1 facilitates the EMT induced by TGF-β1 in SGC-7901 cells, which may serve as a potential target in developing the clinical treatment of gastric cancer.

LncRNA NBR2 inhibits epithelial-mesenchymal transition by regulating Notch1 signaling in osteosarcoma cells.

Long noncoding RNAs (lncRNAs) have been identified to have increasingly important roles in tumorigenesis, and they may serve as novel biomarkers for cancer therapy. Recent studies have demonstrated that lncRNA NBR2 (neighbor of BRCA1 gene 2), a novel identified lncRNA, is decreased in several cancers; however, the role of NBR2 in the development of osteosarcoma has not been elucidated. In our study, we found that NBR2 expression was downregulated in osteosarcoma tissues, and osteosarcoma cases with lower NBR2 expression exhibited a shorter overall survival time compared with those with higher NBR2 expression. NBR2 overexpression inhibited osteosarcoma cell proliferation, invasion, and migration but did not increase apoptosis. Furthermore, RNA-binding protein immunoprecipitation assays confirmed that NBR2 directly binds to Notch1 protein. Furthermore, overexpression of Notch1 in NBR2-overexpressing osteosarcoma cells reversed the effects of NBR2 on cell proliferation, invasion, migration, and epithelial-mesenchymal transition. The in vivo results showed that NBR2 overexpression inhibited tumor growth in nude mice that were inoculated with osteosarcoma cells. NBR2 overexpression also suppressed the messenger RNA (mRNA) expression of Notch1, N-cadherin, and vimentin and increased the mRNA expression of E-cadherin in the tumor tissues. These data indicated that NBR2 served as a tumor suppressor gene in osteosarcoma and inhibited osteosarcoma cell proliferation, invasion, and migration. The current study provides a novel insight and treatment strategy for osteosarcoma.

Effects of PLK1 on proliferation, invasion and metastasis of gastric cancer cells through epithelial-mesenchymal transition.

Effects of polo-like kinase (PLK1) on proliferation, migration and invasion capacities of gastric cancer cells through epithelial-mesenchymal transition (EMT) were investigated. Small-interfering ribonucleic acid (siRNA) with targeted interference in PLK1 gene was designed and transfected into gastric cancer MGC-803 cells via Lipofectamine to inhibit the expression of PLK1 gene in MGC-803 cells. The proliferation of MGC-803 cells was detected via methyl thiazolyl tetrazolium (MTT) assay. The mRNA and protein expression of PLK1 and EMT-related marker (E-cadherin) was detected via real-time polymerase chain reaction and western blot analysis, respectively. The effects of interference in PLK1 gene on migration and invasion of MGC-803 cells were studied via wound healing assay and Transwell chamber assay, respectively. Results of MTT assay showed that compared with that in control group, the cell proliferation in PLK1 siRNA group was significantly inhibited (p<0.01). Compared with those in control group, the mRNA and protein expression of PLK1 in PLK1 siRNA group was significantly decreased (p<0.01), but the mRNA and protein expression of E-cadherin was obviously upregulated (p<0.01). Results of wound healing assay and invasion assay showed that the capacity of migration and invasion of MGC-803 cells in PLK1 siRNA group was significantly inhibited compared with those in control group (p<0.01). In conclusion, PLK1 enhances the proliferation, migration and invasion of gastric cancer MGC-803 cells through affecting EMT.

Polarized M2c macrophages have a promoting effect on the epithelial-to-mesenchymal transition of human renal tubular epithelial cells

This study planned to explore the effects of M2c macrophages on epithelial-to-mesenchymal transition (EMT) of human renal proximal tubular epithelial cells (HK-2). Human monocytic leukaemia cells were induced by TPA and IL-10 to differentiate M2c macrophages. Subsequently HK-2 cells were co-cultured with the M2c macrophages in Transwell chamber. After 48 h of co-culturing the HK-2 cells were detected in the mRNA and protein expression of E-cadherin, α-SMA and vimentin with RT-PCR, immunofluorescence and Western blot respectively. Besides, the migration ability of the HK-2 cells was estimated with Transwell migration assay. ANOVA was used to compare the difference between groups and Student's t-test to conduct multiple comparisons of two groups. P < 0.05 was considered statistically significant. The results showed that the mRNA and protein expression of α-SMA and vimentin of the HK-2 cells were increased but the E-cadherin decreased significantly after 48 h co-culturing with the M2c macrophages (P < 0.05 or P < 0.01). And the migration ability of HK-2 cells were also increased significantly (P < 0.05). It may be concluded that polarized M2c macrophages may have a promoting effect on the EMT of HK-2 cells.

Effects of osthole on the proliferation, invasion and migration of nasopharyngeal carcinoma cells CNE2

Objective To study the effects of osthole on the proliferation, invasion and migration of nasopharyngeal carcinoma cells CNE2, and to investigate the possible molecular mechanism involved in epithelial to mesenchymal transition (EMT) of CNE2. Methods CNE2 cells were cultured in vitro and were treated with 0, 20, 40 and 80 μg/ml osthole for 24 or 48 hours, and then methyl thiazolyl tetrazolium (MTT) assay and Transwell assay were used to explore their effects on the cell proliferation, invasion and migration while cells treated with 0 μg/ml osthole were used as the control group. Meanwhile, the mRNA and protein levels of markers of EMT (E-cadherin and vimentin ) and Wnt/β-catenin signaling (β-catenin and cyclin D1) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting respectively. Results After treatment for 24 and 48 hours, the inhibitory rates of treatment with various concentration of osthole (0, 20, 40, 80 μg/ml) were 0.00%±0.00%, 7.45%±0.87%, 14.12%±2.29%, 27.26%±0.43% and 0.00%±0.00%, 13.44%±0.84%, 29.03%±0.78%, 57.49%±1.70%, with significant differences (F=174.33, P<0.001; F=1 041.40, P<0.001), and the following contrast between each two groups met the statistical significance (all P<0.01). The migration cells per field of CNE2 cells treated with 0, 20, 40, 80 μg/ml osthole for 48 hours were 52.13±4.49, 29.00±4.49, 18.50±1.93, 13.75±2.77, which exhibited a significant difference (F=200.37, P<0.001), and the following contrast between each two groups met the statistical significance (all P<0.01). The invasion cells per field of CNE2 cells treated with 0, 20, 40, 80 μg/ml osthole for 48 hours were 46.63±2.87, 24.13±2.87, 16.75±5.29, 11.00±1.77 respectively, which exhibited a significant difference (F=131.92, P<0.001), and the following contrast between each two groups met the statistical significance (all P<0.01). Meanwhile, the relative mRNA and protein expressions of E-cadherin in 0, 20, 40 and 80 μg/ml osthole treated-cells (exposure for 48 hours) were 1.00±0.13, 2.61±0.03, 3.12±0.09, 3.60±0.06 (F=20.92, P<0.001) and 0.22±0.03, 0.35±0.01, 0.60±0.04, 0.82±0.03 (F=178.63, P<0.001) respectively, and the differences were statistically significant, and further pairwise comparison showed the differences were statistically significant (all P<0.05). Furthermore, the relative mRNA and protein levels of vimentin, β-catenin, cyclin D1 in 0, 20, 40 and 80 μg/ml osthole treatment for 48 hours were statistically significant difference (mRNA level of vimentin: 1.00±0.12, 0.68±0.03, 0.56±0.01, 0.40±0.09, F=9.48, P<0.010; mRNA level of β-catenin: 1.00±0.14, 0.78±0.04, 0.69±0.07, 0.46±0.12, F=4.84, P<0.050; mRNA level of cyclin D1: 1.00±0.09, 0.82±0.03, 0.58±0.09, 0.40±0.03, F=9.49, P<0.010; protein level of vimentin: 0.85±0.02, 0.74±0.01, 0.34±0.01, 0.27±0.01, F=610.58, P<0.001; protein level of β-catenin: 0.83±0.00, 0.44±0.02, 0.39±0.00, 0.23±0.03, F=985.74, P<0.001; protein level of cyclin D1: 0.86±0.02, 0.67±0.00, 0.35±0.01, 0.25±0.01, F=910.57, P<0.001), and further pairwise comparison showed the differences were statistically significant (all P<0.05). Conclusion Osthole can inhibit the proliferation, invasion and migration of CNE2 cells, which is related to the regulation of Wnt/β-catenin signal pathway and then suppressing of EMT. Key words: Osthole; Nasopharyngeal neoplasms; Cell proliferation; Neoplasm invasiveness; Cell movement

Honokiolinhibits the proliferation and invasion abilityof pancreatic cancer cells and the mechanism

Objective To observe the effect and mechanism of honokiol on the proliferation and invasion of SW1990cell. Methods SW1990 were treated with honokiol, Cell Counting Kit-8 (CCK-8) assay was used to detect the cell proliferation. cell apoptotic rate was detected by flow cytometry, cell invasion ability was evaluated by tumour invasion assay; and the mRNA and protein expression of E-cadherin and Wnt/β-cateninsignal were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Results Honokiol inhibited the growth of SW1990 cell in a time-dependent and concentration-dependent manner. With the increase of honokiol concentration, the late stage apoptosis of SW1990 were (2.10±0.44)%, (5.25±1.39)%, (25.75±1.00)%, (60.19±3.14)%, respectively (F=572.336, P=0.000). The number of invaded cells were (170.40±12.20), (95.20±6.98), (54.80±5.63), (21.40±4.93) cells, respectively (F=321.703, P=0.000). The mRNA and protein expression of β-catenin, Vimentin, B-cell lymphoma/leukemia (bcl)-xl, Survivin, c-Myc were down-regulated and bax, Caspase-3, E-cadherin were up-regulated in a concentration-dependent manner, which was detected by RT-PCR and Western blotting. Conclusion Honokiol inhibitedproliferation and invasion of SW1990 cell obviously, which may be through down-regulating the Wnt/β-catenin signalpathway. Key words: Honokiol; Pancreatic cancer; Proliferation; Apoptosis; Invasion

Antimetastatic Effects of Carbon-Ion Beams on Malignant Melanomas.

The goal of this work was to clarify the effect of carbon-ion beams on reduction of the metastatic potential of malignant melanoma using in vitro and in vivo techniques. We utilized a 290 MeV/u carbon beam with a 6-cm spread-out Bragg-peak (SOBP), 137Cs γ rays or 200 kVp X rays for irradiation, and in vitro murine melanoma B16/BL6 cells that were implanted into C57BL/6J mice. The metastatic abilities (migration, invasion and adhesion) were suppressed by carbon ion treatment at all doses that were tested, whereas invasion and migration tended to increase after X-ray irradiation at low dose. Biological effects of carbon ions increased with linear energy transfer (LET) for both cell killing and metastatic abilities, although the effects were more pronounced for migration and invasion. mRNA expression of E-cadherin was significantly downregulated with low-dose photon exposures, but increased with dose or LET. Expression of Mel-CAM and L1-CAM was upregulated after low-dose photon exposure, but decreased with dose, especially after carbon-ion treatment. Conversely, these molecules showed a reversal in expression changes, especially after low-dose photon exposure. Cell-cell adhesion may be an important contributor to the antimetastatic effect of carbon ion treatment. The number of lung metastases after local tumor irradiation significantly decreased with increased dose and LET, with carbon ions being more effective than γ rays. Integrating dose-response curves to examine the relationship between cell killing and lung metastasis clearly showed that carbon ions inhibit lung metastasis more efficiently than photons at the iso-effective level of cell killing. Thus, carbon ions were more effective than photon beams, not only at killing tumor cells, but also at inhibiting metastatic spread caused by tumor cells that survived irradiation.

Abstract 4019: The chronic intermittent hypoxia enhances cancer progression in lung cancer mouse model

Abstract Background: Obstructive sleep apnoea (OSA) is a very prevalent disorder charaterized by chronic intermittent hypoxia (CIH) and some reports suggested that OSA is related to increased incidence of cancer as well as cancer progression. The purpose of this study was to evaluate whether intermittent hypoxia with a time course similar to that found in OSA influence lung cancer development and progression in metastatic lung cancer animal model. Methods: C57/BL6 mice were divided into two groups; control condition (room air) or CIH. The mice of CIH group were exposed to IH for 2 weeks before and immediately for 2 weeks after tumour induction by tail vein injection of carcinoma cells. At day 14, the mice were sacrificed and measured cancer growth. Hypoxia inducible factor (HIF-1α), vascular endothelial growth factor (VEGF) and metastasis related matrix metalloproteinases (MMP) were measured by ELISA and gelatin zymography in lung tissue and serum, respectively. The mRNA expressions of epithelial mesenchymal transition markers were measured by real-time PCR in lung tissue. Results: The marked increase in tumor number and volume was found in CIH group (p &amp;lt; 0.05). The accumulation of total cell and neutrophils in BAL fluids and the activation of MMP -2 and -9 in serum were also increased by CIH. The level of HIF-1α in the nuclear fraction of pneumocytes and the expression of VEGF were increased significantly in CIH group compared to the control (p &amp;lt; 0.05). The mRNA expression of E-cadherin was decreased in CIH group compared to the control group while vimentin was increased. Conclusions: Our data shows that CIH is associated with enhanced proliferative and migratory properties of cancer. These results suggest evidence for the causal link between OSA and lung cancer progression. Key words: chronic intermittent hypoxia, obstructive sleep apnea, lung cancer Citation Format: SangHaak Lee, In Kyoung Kim, Hyeon Hui Kang, Hee Young Kwon, Hye Sun Kang, Chang Dong Yeo. The chronic intermittent hypoxia enhances cancer progression in lung cancer mouse model [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 4019.

Phytic acid improves intestinal mucosal barrier damage and reduces serum levels of proinflammatory cytokines in a 1,2-dimethylhydrazine-induced rat colorectal cancer model.

Phytic acid (PA) has been demonstrated to have a potent anticarcinogenic activity against colorectal cancer (CRC). Defects of the intestinal mucosal barrier and inflammation processes are involved in the development and progression of CRC. In the present study, we evaluated the effect of PA on the intestinal mucosal barrier and proinflammatory cytokines. After a 1-week acclimatisation period, sixty Wistar male rats were divided into the following five groups, with twelve rats per group: the control group (CG), model group (MG), low-PA-dose group (0·25 g/kg per d), middle-PA-dose group (0·5 g/kg per d), and high-PA-dose group (1 g/kg per d). 1,2-Dimethylhydrazine (DMH) at a dosage of 30 mg/kg of body weight was injected weekly to induce CRC for 18 weeks. We examined the expression of genes related to the intestinal mucosal barrier in the model. The results demonstrated that tumour incidence was decreased following PA treatment. The mRNA and protein expression of mucin 2 (MUC2), trefoil factor 3 (TFF3) and E-cadherin in the MG were significantly lower than those in the CG (P<0·05). The mRNA and protein expression of claudin-1 in the MG were significantly higher than those in the CG (P<0·05). PA elevated the mRNA and protein expression of MUC2, TFF3 and E-cadherin, and diminished the mRNA and protein expression of claudin-1. Furthermore, PA decreased serum levels of proinflammatory cytokines, which included TNF-α, IL-1β and IL-6. In conclusion, this study suggests that PA has favourable effects on the intestinal mucosal barrier and may reduce serum proinflammatory cytokine levels.