E-cadherin mRNA Research Articles

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.

The hippo pathway effector Yes‐associated protein promotes epithelial proliferation and remodeling in chronic rhinosinusitis with nasal polyps

Hippo-Yes-associated protein (YAP) pathway plays an important role in epithelial cell proliferation and development. However, its possible role in chronic rhinosinusitis with nasal polyps (CRSwNP) remains unknown. We aim to investigate it on nasal epithelial proliferation and remodeling in CRSwNP. The expressions of hippo pathway components as well as Ki-67 and E-cadherin in the sinonasal mucosa and nasal epithelial cells were analyzed in 14 controls, 14 eosinophilic CRSwNP, and 14 noneosinophilic CRSwNP. Nasal epithelial cells from 6 controls, 6 eosinophilic CRSwNP, and 6 noneosinophilic CRSwNP were cultured and treated with lipopolysaccharide (LPS), Poly(I:C), or a selective YAP inhibitor verteporfin (VP). The hippo pathway components MST1, LATS1/2, YAP, and TEAD1 were increased in both eosinophilic and noneosinophilic CRSwNP, particularly in nasal epithelial cells, along with upregulation of Ki-67 and downregulation of E-cadherin. The mRNA levels of YAP positively correlated with the Ki-67 mRNA levels, and negatively associated with the E-cadherin mRNA levels in polyp tissues and epithelial cells from nasal polyps (NPECs). LPS and Poly(I:C) upregulated the YAP expression in nasal epithelial cells accompanied by increased TEAD1 and Ki-67 expression. Conversely, YAP inhibition by VP decreased TEAD1 and Ki-67 expression in NPECs. Hippo pathway components are abnormally upregulated in NPECs, and its effector YAP promotes nasal epithelial cells proliferation and remodeling in CRSwNP. It provides a rationale to explore inhibition of YAP as a novel therapeutic strategy for reducing the epithelial proliferation and remodeling in CRSwNP.

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.

MiR-200b attenuates IL-6 production through IKK\u03b2 and ZEB1 in human gingival fibroblasts

ObjectiveMicroRNAs (miRNAs) play important roles in biological processes such as cell differentiation, development, infection, immune response, inflammation and tumorigenesis. We previously reported that the expression of miR-200b was significantly increased in inflamed gingiva compared with non-inflamed gingiva. To elucidate the roles of miR-200b in the inflamed gingiva, we have analyzed the effects of miR-200b on the expression of IL-6 in human gingival fibroblasts (HGF).Materials and methodsTotal RNA and protein were extracted from HGF after stimulation by interleukin-1β (IL-1β; 1 ng/ml) or tumor necrosis factor-α (TNF-α; 10 ng/ml) and transfected with miR-200b expression plasmid or miR-200b inhibitor. IL-6, IL-1β, inhibitor of nuclear factor kappa-B kinaseβ (IKKβ), Zinc-finger E-box-binding homeobox 1 (ZEB1) and E-cadherin mRNA and protein levels were analyzed by real-time PCR and Western blot.ResultsIL-1β and TNF-α increased IL-6 mRNA and protein levels, and they were significantly suppressed by miR-200b overexpression, whereas they were further increased by miR-200b inhibitor in HGF. IKKβ and ZEB1 which are target genes of miR-200b negatively regulate E-cadherin. MiR-200b suppressed the expression of IKKβ and ZEB1 and increased E-cadherin mRNA and protein levels in HGF.ConclusionsThese results suggest that miR-200b attenuates inflammatory response via IKKβ and ZEB1 in periodontal tissue.

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.

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.

Up-regulation of miR-210 induced by a hypoxic microenvironment promotes breast cancer stem cells metastasis, proliferation, and self-renewal by targeting E-cadherin.

Breast cancer stem cells (BCSCs), a small subset of breast cancer cells with stem cell-like properties, are essential in tumor formation, metastasis, resistance to anticancer therapies, and cancer recurrence. MicroRNAs (miRNAs) are involved in tumorigenicity by regulating specific oncogenes and tumor-suppressor genes, and their roles in BCSCs are becoming apparent. A novel, 3-dimensional (3D), semisolid culture system was established to culture MCF-7 spheroid cells with high percentage of BCSCs. The differences in miRNA expression among the MCF-7 parental cells, BCSC-enriched MCF-7 spheroid cells, and CD44+/CD24- MCF-7 cells were evaluated by miRNA microarray, and the high expression of miR-210 in MCF-7 spheroid cells and CD44+/CD24- MCF-7 cells was verified by quantitative RT-PCR. MCF-7 cells were cultured in a hypoxic chamber to detect the effect of hypoxia on miR-210 expression and the stemness of the cells. The 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide (MTT), transwell, and sphere-formation assays were performed to detect the proliferation, migration, and self-renewal ability of miR-210-overexpressed MCF-7 cells and MCF-7 spheroid cells with miR-210 knocked down. The target of miR-210 was validated with a dual-luciferase reporter assay and Western blotting. In vivo xenograft assay and metastasis assay were performed to study the effects of miR-210 targeting E-cadherin on BCSCs growth and lung metastasis, and the tumors were assessed by immunohistochemistry and immunofluorescence. We developed a novel 3D, semisolid culture system to culture MCF-7 spheroid cells, which are enriched in BCSCs, and found, by performing miRNAs expression profiling, miR-210 was up-regulated in those cells compared with MCF-7 parental cells. High miR-210 expression was also detected in CD44+/CD24- MCF-7 cells and human CD44+/CD24- breast cancer cells, which was demonstrated to be partially due to the hypoxic microenvironment around BCSCs in MCF-7 spheroids or solid tumors. Ectopic expression of miR-210 in MCF-7 cells promoted their migration, invasion, proliferation, and self-renewal in both in vitro and in vivo studies. We further reported that miR-210 suppressed E-cadherin expression by targeting the open reading frame region of E-cadherin mRNA and by up-regulation of E-cadherin transcription repressor, Snail. Accordingly, E-cadherin overexpression compromises the migration, invasion, proliferation, and self-renewal ability of miR-210-overexpressed MCF-7 both in vitro and in vivo. These findings reveal a novel regulatory pathway centered on hypoxia-mediated miR-210 targeting of E-cadherin, which contributes to the properties and breast tumorigenesis of BCSCs.-Tang, T., Yang, Z., Zhu, Q., Wu, Y., Sun, K., Alahdal, M., Zhang, Y., Xing, Y., Shen, Y., Xia, T., Xi, T., Pan, Y., Jin, L. Up-regulation of miR-210 induced by a hypoxic microenvironment promotes breast cancer stem cells metastasis, proliferation, and self-renewal by targeting E-cadherin.

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.

Simultaneous Fluorescence Visualization of Epithelial-Mesenchymal Transition and Apoptosis Processes in Tumor Cells for Evaluating the Impact of Epithelial-Mesenchymal Transition on Drug Efficacy.

The epithelial-mesenchymal transition (EMT) process plays a pivotal role in acquiring invasive and metastatic properties and has been recognized as a crucial driver of epithelial-derived tumor malignancies. It is necessary to determine the role of EMT in promoting or suppressing carcinoma progression through investigating the relationship between EMT and apoptosis. We designed a multicolor fluorescent nanoprobe for simultaneously imaging the epithelial biomarker E-cadherin mRNA, the mesenchymal marker vimentin mRNA, and the apoptotic marker caspase-3. EMT and apoptosis progresses could be visually detected, which were used to study the effect of EMT on apoptosis and further assess the influence of EMT on drug efficacy in different cancer cells. We believe the designed nanoprobe can offer a new strategy for visualizing EMT and apoptosis in tumor cells and will be a promising tool to investigate the efficiency of drugs targeting EMT-related therapies in living cells.

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.

Liquid biopsy biomarkers of renal interstitial fibrosis based on urinary exosome

Renal interstitial fibrosis (RIF) is difficult to diagnose. This paper explored liquid biopsy markers in urinary exosomes derived from RIF patients. Urine samples from 32 patients with various degrees of RIF and 20 non-RIF patients were collected. The size and morphology of urinary exosomes isolated by polyethylene glycol were observed with electron microscopy. Protein biomakers of exosomes were analyzed by Western blot. qRT-PCR was used to detect the levels of biomarkers (miR-29c, miR-21, E-cadherin, and vimentin) of epithelial mesenchymal transition in urinary exosomes. The diagnostic value was detected with ROC curves. Results displayed successfully isolated urinary exosomes. The examined miRNAs and mRNAs were checked from all urinary exosomes samples, except for two cases of RIF which lacked E-cadherin mRNA. RNA levels were different in patients with diverse degrees of RIF. Urinary miR-29c was decreased with the progress of fibrosis. Levels of E-cadherin mRNA were first decreased and then increased. The contents of miR-21 and vimentin mRNA were also depended on the degrees of RIF. ROC curve analysis showed that the area under the curve (AUC) of miR-29c was 0.8621, statistically significant compared with the non-RIF group (P < 0.05). The miR-29c level within the urinary exosomes is a promising marker for the diagnosis of RIF.

Troglitazone inhibits the migration and invasion of PC-3 human prostate cancer cells by upregulating E-cadherin and glutathione peroxidase 3.

Troglitazone (TGZ) is a synthetic peroxisome proliferator-activated receptor γ (PPARγ) ligand that exhibits potential antitumor effects on a number of cancer subtypes, including prostate cancer. However, little is known about the effect of TGZ on metastasis in prostate cancer. The aim of the present study was to determine the inhibitory effect and mechanism underlying TGZ on cell growth, migration and invasion using the prostate cancer PC-3 cell line. Cellular migration and invasion were evaluated by performing a wound healing assay and Matrigel assay, respectively. The expression levels of mRNA and protein were determined by reverse transcription-quantitative polymerase chain reaction and western blotting. The results demonstrated that TGZ dose-dependently inhibited cell migration and invasion of PC-3 cells. The present study also revealed that TGZ increased the mRNA and protein levels of E-cadherin and glutathione peroxidase 3 (GPx3) in human prostate cancer PC-3 cells. In addition, GW9662, a PPARγ antagonist, attenuated the increased mRNA and protein levels of E-cadherin and GPx3, suggesting that the PPARγ-dependent signaling pathway was involved. Taken together, these results suggested that the anti-migration and anti-invasion effect of TGZ on PC-3 prostate cancer cells is, at least in part, mediated via upregulation of E-cadherin and GPx3. The present study also concluded that PPARγ may be used as a potential remedial target for the prevention and treatment of prostate cancer cell invasion and metastasis.

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

C-X-C Motif Ligand 1 (CXCL1) from melanoma cells down-regulates the invasion of their metastatic melanoma cells.

The surgical resection of a primary melanoma is sometimes followed by the immediate development of distant metastases, suggesting that the primary melanoma might control the metastatic process. We hypothesized that a paracrine factor(s) from primary melanoma cells might regulate the progression of metastasizing melanoma cells. Here we attempted to identify the factor(s) from primary melanoma cells that regulate the invasion ability of metastatic melanoma cells. We used two mouse melanoma cell lines, B16 and B16/BL6, that latter of which is a subline of B16 melanoma and shows high metastatic potential to lung. We investigated the interaction between the parent B16 cells and daughter B16/BL6 cells by invasion assay, cell morphology, cytokine array, RT-PCR, and gelatin-zymography. The conditioned medium (CM) from B16 significantly (p=0.02) inhibited the invasion ability of B16/BL6 cells. The morphology of the B16/BL6 cells was changed from bipolar shape to a multipolar shape following the addition of the CM from B16. The B16 cells produced high levels of C-X-C motif ligand 1 (CXCL1), CXCL10, and M-CSF compared to the B16/BL6 cells. CXCL1 significantly (p=0.01) decreased the invasion ability of B16/BL6 cells, but CXCL10 and M-CSF did not. The invasion-inhibitory activity of the CM from B16 was significantly (p=0.046) suppressed following the addition of a neutralizing anti-CXCL1 antibody. The CM of B16 and CXCL1 increased the E-cadherin mRNA level and decreased MMP2 activity of B16/BL6 cells. These findings suggested that primary melanoma cells might down-regulate the invasion activity of metastatic melanoma cells through CXCL1 signaling.

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 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.

Abstract 4476: Role of MAP-Kinase suppressor in IBD

Abstract BACKGROUND &amp; AIMS: Endogenous suppressor of Mitogen Activated Kinase Pathway (MAPK) are considered cancer suppressors. In this regard , Sprouty (SPRY) proteins that are endogenous suppressor of receptor tyrosine kinase signaling are reported to be tumor suppressors in various cancers, whereas we discovered that SPRY2 augments Epithelial Mesenchymal Transition (EMT) in colorectal cancer (CRC) [Oncogene, 2016 Jun 16; 35(24): 3151-62]. Significance of SPRY proteins in Inflammatory Bowel Disease (IBD) is not clearly understood. MATERIALS &amp; METHODS: Biopsies from Crohn's Colitis patients were assessed for SPRY, Beta-catenin, Occludin and ZO1 expression. Tight junction profiler was employed to assess expression of integrin proteins in CaCO2 colon cancer cell line. SPRY was suppressed in colon cancer cells by siRNA and CRISPR/Cas9 technology and changes were assessed by immunoblotting, real time PCR, phase-contrast and confocal microscopy. Spry1 and Spry2 floxed mice were utilized to dissect SPRY functions in mesenchymal cells. RESULTS: SPRY, E-cadherin, Beta-catenin and Occludin mRNA transcripts were significantly different in IBD patients (p&amp;lt;0.05). No significant changes in ZO1 mRNA transcripts were noted. Suppression of SPRY significantly decreased expression of several integrins in CaCO2 cells (p&amp;lt;0.05) . Treatment of CaCO2 cells with Dextran Sodium Sulfate (DSS) significantly altered SPRY, Beta-catenin, Occludin and ZO1 expressions that was reversed by SPRY suppression. Spry1-/- and Spry2-/- double mutant murine embryonic fibroblasts (MEFs) demonstrated restoration of several epithelial markers which protected cells from DSS-induced changes. CONCLUSIONS: SPRY expression in colonic epithelium regulates IBD. SPRY has prognostic value in IBD. Citation Format: Sharad Khare, Qiong Zhang. Role of MAP-Kinase suppressor in IBD [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 4476.

Abstract 4507: C-X-C motif ligand 1 from melanoma cells downregulates the invasion of their metastatic melanoma cells

Abstract [Introduction]The prognosis of patients with malignant melanoma is extremely poor because of the frequent metastasis. We have occasionally experienced the clinical cases that malignant melanoma patients rapidly developed distant metastases after the surgical resection of primary cancer lesion. These cases suggested that a primary tumor may control a metastatic tumor. We hypothesized that primary melanoma cells might produce a factor(s) which regulate the progression of metastatic melanoma cells. We reported that the conditioned medium from B16 cells significantly (p=0.02) suppressed the invasion ability of B16/BL6 cells, which is a subline of B16 melanoma and shows high metastatic potential to lung at the previous meeting. Then we investigated the growth and invasion interaction between primary malignant melanoma cells and high metastatic melanoma cells at the molecular level. [Materials and methods]We used two mouse melanoma cell lines, B16 and B16/BL6, that latter of which is a subline of B16 melanoma and shows high metastatic potential to lung. We investigated the interaction between the parent B16 cells and daughter B16/BL6 cells by invasion assay, cell morphology, cytokine array, RT-PCR, and gelatin-zymography. [Results]The conditioned medium (CM) from B16 cells significantly (p=0.02) inhibited the invasion ability of B16/BL6 cells. The morphology of the B16/BL6 cells was changed by the CM from bipolar in B16 cells to a multipolar shape. The B16 cells produced high levels of C-X-C motif ligand 1 (CXCL1), CXCL10, and M-CSF compared to the B16/BL6 cells. CXCL1 significantly (p=0.01) decreased the invasion ability of B16/BL6 cells, but CXCL10 and M-CSF did not. The invasion-inhibitory activity of the CM from B16 cells was significantly (p=0.046) suppressed following the addition of a neutralizing anti-CXCL1 antibody. The CM of B16 and CXCL1 increased the E-cadherin mRNA level and MMP2 activity of B16/BL6 cells. [Conclusion]Primary melanoma cells might down-regulate the invasion activity of metastatic melanoma cells through CXCL1 signaling. Citation Format: Takaharu Hatano, Masakazu Yashiro, Hisashi Motomura, Heishiro Fujikawa, Shusaku Maeda. C-X-C motif ligand 1 from melanoma cells downregulates the invasion of their metastatic melanoma cells [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 4507.