The anti-adipogenic effects of (-)epigallocatechin gallate are dependent on the WNT/β-catenin pathway

(-)Epigallocatechin gallate (EGCG) is the most abundant catechin in green tea and reportedly has anti-obesity and anti-adipogenic effects. In this study, we determined that the up-regulation of the WNT/β-catenin pathway is the anti-adipogenic mechanisms of EGCG in 3T3-L1 cells. EGCG treatment down-regulates the expression of major genes involved in the adipogenesis pathway including peroxisome proliferator-activated receptor (PPAR)γ, CCAAT/enhancer binding protein (C/EBP)α, fatty acid binding protein (FABP)4 and fatty acid synthase (FASN), while up-regulating the nuclear level of β-catenin. Knockdown of β-catenin using small interfering (si) RNA attenuated the inhibitory effects of EGCG on intracellular lipid accumulation. β-catenin siRNA transfection also recovered terminal adipocyte markers such as FABP4, FASN, lipoprotein lipase and adiponectin, which were down-regulated by EGCG. The DNA binding activities as well as the expression levels of PPARγ and C/EBPα, which were down-regulated by EGCG, were significantly restored by β-catenin siRNA transfection. In addition, we found that EGCG efficiently up-regulates the WNT/β-catenin pathway. Among the members of the WNT/β-catenin pathway, the expressions of low density lipoprotein receptor-related protein (LRP)5, LRP6, disheveled (DVL)2 and DVL3 were significantly up-regulated, while AXIN expression was down-regulated by EGCG, and the phosphorylation of glycogen synthase kinase 3β was increased. These results suggest that EGCG activates the WNT/β-catenin pathway, resulting in the up-regulation of β-catenin, which down-regulates the major genes of the adipogenesis pathway. Taken together, our findings clearly show that the anti-adipogenic effects of EGCG are, at least partially, dependent on the WNT/β-catenin pathway.     

Suppression of nasopharyngeal carcinoma cell by targeting β-catenin signaling pathway

Aim: To investigate the role of β-catenin in pathogenesis of nasopharyngeal carcinoma (NPC). Methods: Cellular proliferation, apoptosis, matrix penetration assay, and western blotting were employed to determine cell biological changes in NPC cell lines transfected with β-catenin siRNA. Immunohistochemistry staining was used to detect β-catenin and Ki-67 expression in NPC tissue. Results: β-Catenin was upregulated in NPC cell lines and tissues compared with chronic nasopharyngitis tissue. β-Catenin knockdown dramatically inhibited cellular growth, migration and invasion, but induced apoptosis of NPC cells. Further study showed that downstream genes of β-catenin signaling pathway including cyclin D1, c-Myc, MMP2 and MMP9 expression were suppressed in NPC cell lines transfected with β-catenin siRNA. Conclusion: Targeting β-catenin signaling pathway may be a noval strategy for NPC therapy.     

Identification of a novel crosstalk between casein kinase 2α and NPM-ALK in ALK-positive anaplastic large cell lymphoma

It was previously reported that β-catenin contributes to the tumorigenesis of ALK-positive anaplastic large cell lymphoma (ALK⁺ALCL), and the oncogenic effects of β-catenin in these tumors are promoted by NPM-ALK, an abnormal fusion protein characteristic of ALK⁺ALCL. In this study, we hypothesized that NPM-ALK promotes the oncogenic activity of β-catenin via its functional interactions with the Wnt canonical pathway (WCP). To test this hypothesis, we examined if NPM-ALK modulates the gene expression of various members in the WCP. Using a Wnt pathway-specific oligonucleotide array and Western blots, we found that the expression of casein kinase 2α (CK2α) was substantially downregulated in ALK⁺ALCL cells in response to siRNA knockdown of NPM-ALK. CK2α is biologically important in ALK⁺ALCL, as its inhibition using 4,5,6,7-tetrabromobenzotriazole or siRNA resulted in a significant decrease in cell growth and a substantial decrease in the β-catenin protein level. Furthermore, CK2α co-immunoprecipitated with NPM-ALK and regulated its level of serine phosphorylation, a feature previously shown to correlate with the oncogenic potential of this fusion protein. To conclude, this study has revealed a novel crosstalk between NPM-ALK and CK2α, and our data supports the model that these two molecules work synergistically to promote the tumorigenicity of these lymphomas.     

Absolute β-catenin concentrations in Wnt pathway-stimulated and non-stimulated cells

The intracellular level of the proto-oncoprotein β-catenin is a parameter for the activity of the Wnt pathway, which has been linked to carcinogenesis. The paper introduces a novel sandwich-based ELISA for the determination of the β-catenin concentration in lysates from cells or tissues. The advantages of the method were proven by determining β-catenin levels in cell lines and in cells after activation of the Wnt pathway. Analysis revealed high β-catenin concentrations in the cell lines HeLa, KB, HT1080, MCF-7, U-87 and U-373, which had not been described before. β-Catenin concentrations were compared in HEK293 and C57MG cells after activation of the Wnt pathway. The β-catenin concentrations increased by different factors depending on whether the Wnt pathway was activated by incubation with LiCl or with Wnt-3a-conditioned medium. This finding indicated that the β-catenin level depends on the way and level of Wnt pathway activation. The quantitative analysis of β-catenin in colorectal tumours revealed high β-catenin levels in tumours with truncating mutations in the APC gene.     

Overexpression of fatty acid synthase in Middle Eastern epithelial ovarian carcinoma activates AKT and Its inhibition potentiates cisplatin-induced apoptosis

Fatty acid synthase (FASN), the enzyme responsible for de novo synthesis of fatty acids, has been shown to be deregulated in several cancers, including epithelial ovarian carcinoma (EOC). In this study, we investigated the function of the FASN signaling pathway in a large series of Middle Eastern EOC patient samples, a panel of cell lines and nude mouse model. Using immunohistochemistry, we detected overexpression of FASN in 75.5% (114/151) of the tumor samples. Overexpression of FASN was associated significantly with tumor proliferative marker Ki-67 (P = 0.0009), activated AKT (P = 0.0117) and XIAP (P = 0.0046). Treatment of EOC cell lines with C-75, a selective inhibitor of FASN, caused inhibition of EOC cell viability via induction of apoptosis. Inhibition of FASN by C-75 led apoptosis via the mitochondrial pathway. FASN inhibition caused downregulation of activated AKT and its downstream targets. In addition, inhibition by FASN siRNA caused downregulation of FASN and activation of caspases, suggesting the role of FASN in C-75 mediated apoptosis. Furthermore, treatment of EOC cells with subtoxic doses of C-75 augmented the effect of cisplatin-mediated induction of apoptosis. Finally, treatment of EOC cell line xenografts with a combination of C-75 and cisplatin resulted in growth inhibition of tumors in nude mice through downregulation of FASN and activation of caspases. Altogether, our results show overexpression of FASN in Middle Eastern EOC, suggesting that FASN may be a potential therapeutic target in a subset of EOC, alone or in combination with other conventional chemotherapeutic agents.     

下调脂肪酸合成酶表达对膀胱癌UMUC3细胞增殖、迁移及侵袭能力的影响*

目的: 研究脂肪酸合成酶(FASN)表达对膀胱癌UMUC3细胞增殖、迁移、侵袭的影响,探讨其内在可能机制。方法:免疫组化法检测30例膀胱癌和15例正常膀胱组织FASN蛋白的表达;用脂质体2000分别转染FASN siRNA和无义siRNA至UMUC3细胞,筛选、鉴定siFASN和siControl稳定的细胞,siFASN组细胞设为实验组,siControl组设为对照组;采用蛋白印迹法(Western blot)和实时荧光定量PCR(RT-PCR)法分别检测siFASN组和siControl组细胞FASN蛋白及mRNA的表达,MTT法检测siFASN组和siControl组细胞增殖情况,划痕试验、Transwell试验分别检测siFASN组和siControl组细胞迁移、侵袭能力。结果:FASN蛋白在膀胱癌组织中过表达,且与病理分期、分级密切相关(P＜0.05)。与siControl组相比,siFASN组细胞FASN mRNA及蛋白表达下调(P＜0.05),细胞增殖活力明显下降(P＜0.05),迁移能力明显下降(P＜0.05),穿膜细胞数量明显减少(P＜0.05)。结论:FASN过表达在膀胱癌发生、发展中发挥重要作用,下调FASN表达能抑制膀胱癌细胞的增殖、迁移、侵袭能力,抑制FASN表达有望成为一种新的膀胱癌治疗方法。     

Dishevelled proteins and CYLD reciprocally regulate each other in CML cell lines

Dishevelled (Dvl) proteins are activated by Wnt pathway stimulation and have crucial roles in the regulation of β-catenin destruction complex. CYLD is a tumor suppressor and a deubiquitination enzyme. CYLD negatively regulates the Wnt/β-catenin signaling pathway by deubiquitinating Dvl proteins. Loss of function and mutations of CYLD were linked to different types of solid tumors. Loss of function in CYLD is associated with Dvl hyper ubiquitination, resulting in the transmission of Wnt signaling to downstream effectors. β-catenin upregulation is observed during disease progression in chronic myeloid leukemia (CML). Deregulated Dvl signaling may be a reason for β-catenin activation in CML; and CYLD may contribute to Dvl deregulation. First, we evaluated mRNA expression in three CML cell lines and mRNA expression of the CYLD gene was found to be present in all (K562, MEG01, KU812). Unlike solid tumors sequencing revealed no mutations in the coding sequences of the CYLD gene. DVL genes were silenced by using a pool of siRNA oligonucleotides and gene expression differences in CYLD was determined by RT-PCR and western blot. CYLD protein expression decreased after Dvl silencing. An opposite approach of overexpressing Dvl proteins resulted in upregulated CYLD expression. While previous reports have described CYLD as a regulator of DVL proteins; our data suggests the presence of a more complicated reciprocal regulatory mechanism in CML cell lines.     

Knockdown of β-catenin controls both apoptotic and autophagic cell death through LKB1/AMPK signaling in head and neck squamous cell carcinoma cell lines

The Wnt/β-catenin pathway regulates the viability and radiosensitivity of head and neck squamous cancer cells (HNSCC). Increased β-catenin predisposes HNSCC patients to poor prognosis and survival. This study was conducted to determine the mechanism by which β-catenin regulates the viability of HNSCC. AMC-HN-3, -HN-8, UM-SCC-38, and -SCC-47 cells, which were established from human head and neck cancer specimens, and underwent cell death following β-catenin silencing. β-Catenin silencing significantly induced G1 arrest and increased the expression of Bax and active caspase-3, which demonstrates the sequential activation of apoptotic cascades following treatment of HNSCC with targeted siRNA. Intriguingly, β-catenin silencing also induced autophagy. Here, we confirm that the number of autophagic vacuoles and the expression of type II light chain 3 were increased in cells that were treated with β-catenin siRNA. These cell death modes are most likely due to the activation of LKB1-dependent AMPK following β-catenin silencing. The activated LKB1/AMPK pathway in AMC-HN-3 cells caused G1 arrest by phosphorylating p53 and suppressing mTOR signaling. In addition, treating AMC-HN-3 cells with LKB1 siRNA preserved cell viability against β-catenin silencing-induced cytotoxicity. Taken together, these results imply that following β-catenin silencing, HNSCC undergo both apoptotic and autophagic cell death that are under the control of LKB1/AMPK. To the best of our knowledge, these results suggest for the first time that novel crosstalk between β-catenin and the LKB1/AMPK pathway regulates the viability of HNSCC. This study thus presents new insights into our understanding of the cellular and molecular mechanisms involved in β-catenin silencing-induced cell death.     

L1-mediated colon cancer cell metastasis does not require changes in EMT and cancer stem cell markers

Aberrant activation of Wnt/β-catenin signaling is common in most sporadic and inherited colorectal cancer (CRC) cells leading to elevated β-catenin/TCF transactivation. We previously identified the neural cell adhesion molecule L1 as a target gene of β-catenin/TCF in CRC cells. Forced expression of L1 confers increased cell motility, invasion, and tumorigenesis, and the induction of human CRC cell metastasis to the liver. In human CRC tissue, L1 is exclusively localized at the invasive front of such tumors in a subpopulation of cells displaying nuclear β-catenin. We determined whether L1 expression confers metastatic capacities by inducing an epithelial to mesenchymal transition (EMT) and whether L1 cosegregates with cancer stem cell (CSC) markers. We found that changes in L1 levels do not affect the organization or expression of E-cadherin in cell lines, or in invading CRC tissue cells, and no changes in other epithelial or mesenchymal markers were detected after L1 transfection. The introduction of major EMT regulators (Slug and Twist) into CRC cell lines reduced the levels of E-cadherin and induced fibronectin and vimentin, but unlike L1, Slug and Twist expression was insufficient for conferring metastasis. In CRC cells L1 did not specifically cosegregate with CSC markers including CD133, CD44, and EpCAM. L1-mediated metastasis required NF-κB signaling in cells harboring either high or low levels of endogenous E-cadherin. The results suggest that L1-mediated metastasis of CRC cells does not require changes in EMT and CSC markers and operates by activating NF-κβ signaling.     

Neural cell adhesion molecule potentiates the growth of murine melanoma via β-catenin signaling by association with fibroblast growth factor receptor and glycogen synthase kinase-3β

The neural cell adhesion molecule (NCAM) was recently shown to be involved in the progression of various tumors with diverse effects. We previously demonstrated that NCAM potentiates the cellular invasion and metastasis of melanoma. Here we further report that the growth of melanoma is obviously retarded when the expression of NCAM is silenced. We found that the proliferation of murine B16F0 melanoma cells, their colony formation on soft agar, and growth of transplanted melanoma in vivo are clearly inhibited by the introduction of NCAM siRNA. Interestingly, change of NCAM expression level is shown to regulate the activity of Wnt signaling molecule, β-catenin, markedly. This novel machinery requires the function of FGF receptor and glycogen synthase kinase-3β but is independent of the Wnt receptors, MAPK-Erk and PI3K/Akt pathways. In addition, NCAM is found to form a functional complex with β-catenin, FGF receptor, and glycogen synthase kinase-3β. Moreover, up-regulation of NCAM140 and NCAM180 appears more potent than NCAM120 in activation of β-catenin, suggesting that the intracellular domain of NCAM is required for facilitating the β-catenin signaling. Furthermore, the melanoma cells also exhibit distinct differentiation phenotypes with the NCAM silencing. Our findings reveal a novel regulatory role of NCAM in the progression of melanoma that might serve as a new therapeutic target for the treatment of melanoma.