Abstract 58: 5-HT promotes hepatocellular carcinoma by influencing β-catenin
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Abstract 5-hydroxytryptamine (5-HT), a neurotransmitter and vasoactive factor, has been reported to promote proliferation of serum-deprived human hepatocellular carcinoma cells (HCC) but the detailed intracellular mechanism is still unknown. As Wnt/β-catenin signaling is highly dysregulated in a majority of HCC, this study explored the regulation of Wnt/β-catenin signaling by 5-HT. 5-HT promoted proliferation of serum-deprived HuH-7 and HepG2 cells and also increased total β-catenin levels, and active β-catenin protein levels compared to just control cells under serum free medium without 5-HT. Furthermore, 5-HT increased β-catenin levels in the presence of cycloheximide, a protein synthesis inhibitor, suggesting increased β-catenin levels due to inhibition of β-catenin degradation. Quantitative real-time polymerase chain reaction (qPCR) showed increased β-catenin downstream target genes, Axin1, cyclin D1, dickoppf-1 (DKK1) and glutamine synthetase (GS), in serum-deprived HCC cell lines treated with 5-HT. We next studied the expression of various 5-HT were receptors (5-HT1D, 5-HT2A, 5-HT2B, 5-HT5 and 5-HT7) by qPCR in 33 pairs of HCC tumors and corresponding adjacent non-tumor tissues. Receptors 5-HT1D (21/33, 64%), 5-HT2B (12/33, 36%) and 5-HT7 (15/33, 45%) were overexpressed in HCC tumour tissues whereas receptor 5-HT5 was reduced (30/33, 91%) in HCC tumour tissues. Receptor 5-HT2A did not show any significant statistical difference between HCC tumour and corresponding non-tumour tissues. We further investigated whether antagonists of the 5-HT receptors attenuated the activity of 5-HT both in vitro and in vivo and we narrowed our study to 5-HT7 antagonist as the expression of 5-HT7 was found to be significantly associated to liver histology and venous infiltration. Antagonist of receptor 5-HT7, SB258719, attenuated growth of serum-deprived HCC cell lines and primary tumor tissues in the presence of 5-HT. Furthermore, SB258719 reduced tumor growth in a xenograft mouse model accompanied by reduced β-catenin and increased GSK-3β levels as observed by immunohistochemical analysis. Conclusion: This study provides evidence of Wnt/β-catenin signaling activation in 5-HT induced proliferation of serum-deprived HCC cells. The proliferation is inhibited by 5-HT7 antagonist, SB258719, which may represent a potential therapeutic target for hepatocarcinogenesis. Citation Format: Sarwat Fatima, Shi Xiaoki, Lin Zesie, Chen Guo, John W. Ho, Nikki P. Lee, Xiang Bian Zhao. 5-HT promotes hepatocellular carcinoma by influencing β-catenin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 58. doi:10.1158/1538-7445.AM2015-58Keywords:
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Abstract Background Cartilage destruction is the main characteristic of osteoarthritis (OA), and osteopontin (OPN) is elevated in OA articular cartilage; however, the reason for the increased OPN level is not determined. In addition, Wnt/β-catenin signaling participates in the progression of OA. The aim of the present study was to evaluate whether canonical Wnt signaling could regulate the expression of OPN in human chondrocytes in vitro. Methods Human chondrocytes were cultured in vitro, and we first assayed the mRNA levels of OPN and β-catenin in chondrocytes. Next, we performed transient transfection of TCF 4 shRNA into chondrocytes to inhibit TCF 4 expression and explore changes in the OPN level. Then, the Wnt/β-catenin signaling inhibitor Dickkopf-1 (Dkk-1) was incubated with chondrocytes, and we assayed the changes in β-catenin and OPN. Results Our results showed that the expression of both β-catenin and OPN was increased in OA chondrocytes, but there were no correlations between β-catenin and OPN expression. TCF4 shRNA downregulated the expression of TCF 4 and OPN in chondrocytes, while after treatment with rDKK-1 at a concentration of 400 ng/ml for 24 h, the mRNA and protein expression of both β-catenin and OPN was significantly decreased in chondrocytes. Conclusions Elevated OPN expression might be regulated by the β-catenin/TCF-4 pathway, and the Wnt/β-catenin inhibitor DKK1 could inhibit the expression of β-catenin and OPN in OA chondrocytes.
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Elevated β-catenin levels in human colorectal cancer (CRC) cells lead to increased trans-activation of 'protumorigenic' β-catenin/T-cell factor (TCF) target genes such as cyclin D1. Therefore, possible targets for the anti-CRC activity of nonsteroidal anti-inflammatory drugs (NSAIDs) are β-catenin and catenin-related transcription (CRT). We tested the antiproliferative activity and the effects on levels of β-catenin and cyclin D1 protein, as well as CRT (measured using a synthetic β-catenin/TCF-reporter gene [TOPflash]), of a panel of NSAIDs (indomethacin, diclofenac, sulindac sulphide and sulphone, rofecoxib; range 10–600 μM) on SW480 human CRC cells in vitro. Following NSAID treatment, there was no consistent relationship between reduced cell proliferation, induction of apoptosis and changes in β-catenin protein levels or CRT. All the NSAIDs, except rofecoxib, decreased nuclear β-catenin content and cyclin D1 protein levels in parallel with their antiproliferative activity. However, cyclin D1 downregulation occurred prior to a decrease in total β-catenin protein levels and there was no correlation with changes in CRT, suggesting the existence of CRT-independent effects of NSAIDs on cyclin D1 expression. In summary, NSAIDs have differential effects on β-catenin protein and CRT, which are unlikely to fully explain their effects on cyclin D1 and their antiproliferative activity on human CRC cells in vitro.
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Abstract Background Cartilage destruction is the main characteristic of osteoarthritis (OA), and osteopontin (OPN) is elevated in OA articular cartilage; however, the reason for the increased OPN level is not determined. In addition, Wnt/β-catenin signaling participates in the progression of OA. The aim of the present study was to evaluate whether canonical Wnt signaling could regulate the expression of OPN in human chondrocytes in vitro. Methods Human chondrocytes were cultured in vitro, and we first assayed the mRNA levels of OPN and β-catenin in chondrocytes. Next, we performed transient transfection of TCF 4 shRNA into chondrocytes to inhibit TCF 4 expression and explore changes in the OPN level. Then, the Wnt/β-catenin signaling inhibitor Dickkopf-1 (Dkk-1) was incubated with chondrocytes, and we assayed the changes in β-catenin and OPN. Results Our results showed that the expression of both β-catenin and OPN was increased in OA chondrocytes, but there were no correlations between β-catenin and OPN expression. TCF4 shRNA downregulated the expression of TCF 4 and OPN in chondrocytes, while after treatment with rDKK-1 at a concentration of 400 ng/ml for 24 h, the mRNA and protein expression of both β-catenin and OPN was significantly decreased in chondrocytes. Conclusions Elevated OPN expression might be regulated by the β-catenin/TCF-4 pathway, and the Wnt/β-catenin inhibitor DKK1 could inhibit the expression of β-catenin and OPN in OA chondrocytes.
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To determine the relationship between expression pattern of E-cadherin, beta-catenin and cyclin D1, and clinicopathologic parameters in patients with head and neck squamous cell carcinomas (HNSCC).The authors evaluated the immunohistochemical expression pattern of E-cadherin, beta-catenin in relationship with cyclin D1 overexpression, degree of histologic differentiation, clinical stage, and nodal status in 146 head and neck squamous cell carcinomas (HNSCC). The authors also evaluated the expression of E-cadherin/beta-catenin complex, E-cadherin/cyclin D1, and beta-catenin/cyclin D1 double staining with confocal laser scanning microscope.Aberrant expressions in 78% of E-cadherin, 77% of beta-catenin, and 69% of cyclin D1 in the HNSCC were observed. There was correlation of aberrant expression of E-cadherin and nodal status. Cyclin D1 overexpression was also correlated to clinical stage and nodal status. Significant relation was observed between E-cadherin and beta-catenin expression patterns. Co-expression of E-cadherin and beta-catenin was significantly detected. However, there was no correlation of cyclin D1 overexpression with E-cadherin or beta-catenin expression patterns.These results suggest that aberrant expression of E-cadherin, E-cadherin/beta-catenin complex, and cyclin D1 may be involved in clinical stage and/or nodal status, and analysis of the pattern of E-cadherin, cyclin D1, and E-cadherin/beta-catenin complex may be good prognostic marker of HNSCC.
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Abstract Background: Cartilage destruction is the main characteristic of osteoarthritis (OA), and osteopontin (OPN) is elevated in OA articular cartilage; however, the reason for the increased OPN level is not determined. In addition, Wnt/β-catenin signaling participates in the progression of OA. The aim of the present study was to evaluate whether canonical Wnt signaling could regulate the expression of OPN in human chondrocytes in vitro.Methods: Human chondrocytes were cultured in vitro, and we first assayed the mRNA levels of OPN and β-catenin in chondrocytes. Next, we performed transient transfection of TCF 4 shRNA into chondrocytes to inhibit TCF 4 expression and explore changes in the OPN level. Then, the Wnt/β-catenin signaling inhibitor Dickkopf-1 (Dkk-1) was incubated with chondrocytes, and we assayed the changes in β-catenin and OPN.Results: Our results showed that the expression of both β-catenin and OPN was increased in OA chondrocytes, but there were no correlations between β-catenin and OPN expression. TCF4 shRNA downregulated the expression of TCF 4 and OPN in chondrocytes, while after treatment with rDKK-1 at a concentration of 400 ng/ml for 24 h, the mRNA and protein expression of both β-catenin and OPN was significantly decreased in chondrocytes.Conclusions: Elevated OPN expression might be regulated by the β-catenin/TCF-4 pathway, and the Wnt/β-catenin inhibitor DKK1 could inhibit the expression of β-catenin and OPN in OA chondrocytes.
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Background and purpose:Studies have shown that some molecular markers could serve as prognostic factors for nasopharynx carcinoma, but the predictive role of catenins and cyclin D1 remains uncertain for the disease. Our paper is to investigate the expression of catenins(α-,β- and γ-) and cyclin D1 in nasopharyngeal carcinoma as well as to analysis their relation to clinic factors and prognosis. Methods:We retrieved 38 paraffin-embedded specimens of nasopharynx carcinoma, immunohistochemistry was used to examine the expression of α-,β- and γ-catenin , cyclin D1 and tumor proliferation activity marker ki-67.Results:Reduced expression of α-,β- ,γ-catenin and cyclin D1 was observed in most of the tumors. Our preliminary study demonstrated that there was no significant correlation between their expression with T-stage, N-stage, clinical stage and primary tumor volume, as well as with ki-67 stain. In unviarance analysis, patients with reduced expression of β-catenin had poorer prognosis than those with high expression, 5 year overall survival and disease free survival rates of these two groups were 53.2%, 29.0% and 81.9%, 76.0%, respectively(P0.05). The significance of different expression of α-catenin was marginal(P=0.061) when analyzing its impact on disease free survival rate.Conclusions:There are abnormal expression of α-,β- ,γ-catenin and cyclin D1 in nasopharyngeal carcinoma. Our preliminary result shows that no significant correlation is found between their expression and tumor proliferation, clinical stage and metastasis. Reduced expression of β- catenin might be a negative prognostic factor in terms of survival rates. More cases should be further analyzed to clarify the issue.
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Indomethacin-induced G 1 arrest and apoptosis of human colorectal cancer (CRC) cells is associated with a dose-dependent decrease in β-catenin protein levels. β-catenin plays a pivotal role in the WNT signalling pathway and its expression is frequently dysregulated at early stages of colorectal carcinogenesis. The objective of this study was to investigate the effect of indomethacin on catenin expression and downstream WNT signalling events in human CRC cells. β-catenin, γ-catenin and T-cell facter (TCF) target gene (c yclin D1 , c- MYC and PPARδ ) expression was studied following indomethacin treatment of SW480 and HCT116 cells. C yclin D1 was used as a model TCF target gene for analysis of β-catenin–TCF-4 DNA binding and trans -activation. Indomethacin treatment was associated with a specific decrease in β-catenin (but not γ-catenin) expression. Resulting TCF target gene expression was gene specific ( cyclin D1 , decreased; c- MYC , increased; PPARδ , no significant change) . Cyclin D1 promoter analysis revealed that indomethacin disrupted formation of a β-catenin–TCF-4–DNA complex. Indomethacin-induced G 1 arrest and apoptosis is associated with specific β-catenin down-regulation in human CRC cells in vitro . Differential expression of TCF target genes following indomethacin treatment implies complex effects on multiple genes which play an important role in colorectal carcinogenesis.
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Background: β‐catenin gene mutations have been reported in vast majority of pilomatrixomas (PMXs). β‐catenin, a component of the adhesion molecules of the cytoskeleton, is degraded at the cytoplasm. Excess cytoplasmic β‐catenin enters into the nucleus and activates the transcription of several genes encoding c‐myc, cyclin D1 and others. Sublocation of β‐catenin has been demonstrated by immunohistochemistry. The aim of this study was to determine the role of β‐catenin‐related proteins in various benign trichogenic tumors. Methods: We investigated the expression of β‐catenin, E‐cadherin, c‐myc and cyclin D1 immunohistochemically, and the expression of these molecules were compared between two groups consisting of 12 PMXs and 12 other benign trichogenic tumors (OBTTs). Results: In PMX group, nuclear and/or cytoplasmic expression of β‐catenin was associated with a loss of membranous expression of E‐cadherin (p = 0.002). In OBTT group, a membranous expression of E‐cadherin and β‐catenin was observed, and there was a stronger nuclear immunoreactivity of cyclin D1 compared with PMX group (p = 0.006). Conclusions: In PMX, nuclear and/or cytoplasmic β‐catenin expression of tumoral cells is not related with β‐catenin‐related gene expressions (c‐myc or cyclin D1). The molecular behaviour of OBTTs is clearly different from that of PMXs in terms of to E‐cadherin and β‐catenin expression.
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