Suppression of Wnt/β-catenin signaling inhibits prostate cancer cell proliferation

Although mounting evidence has demonstrated an important role of Wnt/β-catenin signaling in the development and progression of cancer, the therapeutic potential of small molecules that target this pathway for prostate cancer remains largely unknown. We reported herein that the highly invasive androgen-independent PC-3 and DU145 human prostate cancer cells exhibited higher levels of Wnt/β-catenin signaling than the androgen-dependent LNCaP prostate cancer cells and non-cancerous PZ-HPV-7 and PWR-1E prostate cells, and that exogenous Wnt3A treatment exaggerated the difference of the Wnt/β-catenin signaling levels among these prostate cells. Furthermore, we demonstrated that the non-steroidal anti-inflammatory drug, sulindac sulfide, the cyclooxygenase-2 (COX-2) selective inhibitor, celecoxib, and the nitric oxide-donating aspirin derivative, NO-ASA, blocked Wnt/β-catenin signaling in PC-3 and DU145 cells. These effects occurred at concentrations comparable to those required to inhibit cell proliferation, indicating that the inhibitory effect of these drugs on prostate cancer cell proliferation may involve the suppression of Wnt/β-catenin signaling. Finally, we showed that a novel small molecule inhibitor of Wnt/β-catenin signaling, PKF118- 310, inhibited Wnt/β-catenin signaling and proliferation in prostate cancer cells within the same concentration range. Together, these results suggest that small molecules that inhibit Wnt/β-catenin signaling have therapeutic potential for the prevention or treatment of prostate cancer.     

Role of Wnt/β-catenin signaling in drug resistance of pancreatic cancer

Pancreatic cancer is characterized by its intrinsic resistance to cytotoxic agents. But the underlying molecular mechanism is unclear. Studies demonstrate that angiogenesis, presence of highly resistant cancer stem cells (CSCs), dysregulation of cell cycle and apoptosis are main aspects of mechanisms of pancreatic cancer chemoresistance. Interestingly, recent investigations of Wnt/β-catenin signaling suggest roles for the signaling in these four aspects and the pathogenesis of pancreatic cancer. Conceivably, the dysregulation of Wnt/β-catenin signaling pathway is involved in pancreatic cancer chemoresistance. Though researchers have proven it in some other cancer types, however, there is no direct evidence for this reasoning in pancreatic cancer. Designing effective experiment setups to define the function and mechanism of Wnt/β-catenin signaling in pancreatic cancer chemoresistance and subsequently targeting the signaling to improve the sensitivity of chemotherapy in pancreatic cancer require a full understanding of the molecular mechanisms of Wnt/β-catenin signaling pathway in angiogenesis, maintaining of highly resistant CSCs, regulation of cell cycle and apoptosis in pancreatic cancer.     

SFRP1通过抑制Wnt/β-catenin通路促进心肌细胞增殖的分子机制

目的 探讨SFRP1是否通过抑制Wnt/β-catenin通路促进心肌细胞增殖。方法 选取c57BL/6雄性小鼠18只,出生后1 d、7 d及28 d的c57BL/6小鼠各6只分为3组,分离和提取心肌组织,检测心肌组织中SFRP1的mRNA及蛋白表达水平。利用出生后1 d的乳鼠心脏培养小鼠原代心肌细胞,予SFRP1-shRNA慢病毒转染细胞,免疫荧光检测心肌细胞增殖标志物Ki67和PH3表达变化,Western blot检测胞浆β-catenin蛋白表达变化。结果 SFRP1 mRNA和蛋白水平在小鼠出生后逐渐下降(P<0.05)。转染SFRP1-shRNA慢病毒后,SFRP1蛋白表达水平均下降(P<0.05),其中shRNA-2干扰效果最好。与空载组相比,shRNA组细胞Ki67和PH3表达下降,同时胞浆β-catenin蛋白表达水平升高(P<0.05)。结论 SFRP1通过抑制Wnt/β-catenin信号通路促进心肌细胞增殖。     

Inhibition of Wnt/β-catenin signaling mediates ursolic acid-induced apoptosis in PC-3 prostate cancer cells

BackgroundUrsolic acid, a pentacyclic triterpenoid, is known to exert antitumor activity in breast, lung, liver and colon cancers. Nonetheless, the underlying mechanism of ursolic acid in prostate cancer cells still remains unclear. To investigate the antitumor mechanism, the apoptotic mechanism of ursolic acid via Wnt/β-catenin signaling was examined in PC-3 prostate cancer cells.MethodsCytotoxicity assay, flow cytometry, immunofluorescence assay and western blotting were performed.ResultsUrsolic acid showed cytotoxicity against PC-3, LNCaP and DU145 prostate cancer cells with IC₅₀ of 35 μM, 47 μM and 80 μM, respectively. Also, ursolic acid significantly increased the number of ethidium homodimer stained cells and apoptotic bodies, and dose-dependently enhanced the sub-G1 apoptotic accumulation in PC-3 cells. Consistently, western blotting revealed that ursolic acid effectively cleaved poly (ADP-ribose) polymerase (PARP), activated caspase-9 and -3, suppressed the expression of survival proteins such as Bcl-X_L, Bcl-2 and Mcl-1, and upregulated the expression of Bax in PC-3 cells. Interestingly, ursolic acid suppressed the expression of Wnt5α/β and β-catenin, and enhanced the phosphorylation of glycogen synthase kinase 3 β (GSK3β). Furthermore, the GSK3β inhibitor SB216763 or Wnt3a-conditioned medium (Wnt3a-CM) reversed the cleavages of caspase-3 and PARP induced by ursolic acid in PC-3 cells.ConclusionsOur findings suggest that ursolic acid induces apoptosis via inhibition of the Wnt5/β-catenin pathway and activation of caspase in PC-3 prostate cancer cells. These results support scientific evidence that medicinal plants containing ursolic acid can be applied to cancer prevention and treatment as a complement and alternative medicine (CAM) agent.     

GSK-3β suppresses the proliferation of rat hepatic oval cells through modulating Wnt/β-catenin signaling pathway

Aim:

Glycogen synthase kinase 3β (GSK-3β) plays a crucial role in hepatic biology, including liver development, regeneration, proliferation and carcinogenesis. In this study we investigated the role of GSK-3β in regulation of growth of hepatic oval cells in vitro and in liver regeneration in partially hepatectomized rats.

Methods:

WB-F344 cells, the rat hepatic stem-like epithelial cells, were used as representative of oval cells. Cell viability was examined using a WST-8 assay. The cells were transfected with a recombinant lentivirus expressing siRNA against GSK-3β (GSK-3βRNAiLV) or a lentivirus that overexpressed GSK-3β (GC-GSK-3βLV). Adult rats underwent partial (70%) hepatectomy, and liver weight and femur length were measured at d 7 after the surgery. The expression of GSK-3β, phospho-Ser⁹-GSK-3β, β-catenin and cyclin D1 was examined with immunoblotting assays or immunohistochemistry.

Results:

Treatment of WB-F344 cells with the GSK-3β inhibitor SB216763 (5 and 10 μmol/L) dose-dependently increased the levels of phospho-Ser⁹-GSK-3β, but not the levels of total GSK-3β, and promoted the cell proliferation. Knockout of GSK-3β with GSK-3βRNAiLV increased the cell proliferation, whereas overexpression of GSK-3β with GC-GSK-3βLV decreased the proliferation. Both SB216763 and GSK-3βRNAiLV significantly increased the levels of β-catenin and cyclin D1 in the cells, whereas GSK-3β overexpression decreased their levels. In rats with a partial hepatectomy, administration of SB216763 (2 mg/kg, ip) significantly increased the number of oval cells, the levels of phospho-Ser⁹-GSK-3β, β-catenin and cyclin D1 in liver, as well as the ratio of liver weight to femur length at d 7 after the surgery.

Conclusion:

GSK-3β suppresses the proliferation of hepatic oval cells by modulating the Wnt/β-catenin signaling pathway.     

阿魏酸对脂多糖诱导的OA软骨细胞Wnt/β-catenin信号通路的影响

目的探讨阿魏酸(Ferulic acid,FA)对脂多糖(LPS)诱导的OA软骨细胞Wnt/β-catenin信号通路的影响。方法于4月龄新西兰大白兔膝软骨提取软骨细胞,体外培养后分为4组,空白对照组、OA模型组、OA+FA低剂量组和OA+FA高剂量组,将2μg/ml的LPS加入培养基中培养软骨细胞,构建OA软骨细胞模型,对照组加入等体积的细胞培养液,选取合适的阿魏酸浓度,确定低剂量组和高剂量组,低剂量组和高剂量组则分别加入5μmol/ml、20μmol/ml的阿魏酸进行共培养,甲苯胺蓝染色观察正常软骨细胞形态,qRT-PCR法检测4组软骨细胞Ⅱ型胶原基因的表达(COL2)、软骨细胞基质金属蛋白(MMP-13)基因的表达,CCK-8法检测软骨细胞增殖情况,Western blot检测Wnt2、GSK-3β、β-catenin、COL2、MMP-13、Bcl-2、Bax蛋白的表达。结果甲苯胺蓝染色法鉴定软骨细胞,软骨细胞在显微镜下呈现形态不规则、梭形透光度良好的多层细胞,细胞核红染,呈球形;qRT-PCR和Western blot结果显示OA模型组COL2基因和蛋白的表达量显著低于空白组...     

MicroRNA-147b suppresses the proliferation and invasion of non-small-cell lung cancer cells through downregulation of Wnt/β-catenin signalling via targeting of RPS15A

Deregulation of microRNAs (miRNAs) leads to malignant growth and aggressive invasion during cancer occurrence and progression. miR-147b has emerged as one of the cancer-related miRNAs that are dysregulated in multiple cancers. Yet, the relevance of miR-147b in non-small-cell lung cancer (NSCLC) remains unclear. In the present study, we aimed to report the biological function and signalling pathways mediated by miR-147b in NSCLC. Our results demonstrate that miR-147b expression is significantly downregulated in NSCLC tissues and cell lines. Overexpression of miR-147b decreased the proliferative ability, colony-forming capability, and invasive potential of NSCLC cells. Notably, our study identified ribosomal protein S15A (RPS15A), an oncogene in NSCLC, as a target gene of miR-147b. Our results showed that miR-147b negatively modulates RPS15A expression in NSCLC cells. An inverse correlation between miR-147b and RPS15A was evidenced in NSCLC specimens. Moreover, miR-147b overexpression downregulated the activation of Wnt/β-catenin signalling via targeting of RPS15A. Overexpression of RPS15A partially reversed the miR-147b-mediated antitumour effect in NSCLC cells. Collectively, these findings reveal that miR-147b restricts the proliferation and invasion of NSCLC cells by inhibiting RPS15A-induced Wnt/β-catenin signalling and suggest that the miR-147b/RPS15A/Wnt/β-catenin axis is an important regulatory mechanism for malignant progression of NSCLC.     

Tetrandrine inhibits Wnt/β-catenin signaling and suppresses tumor growth of human colorectal cancer

As one of the most common malignancies, colon cancer is initiated by abnormal activation of the Wnt/β-catenin pathway. Although the treatment options have increased for some patients, overall progress has been modest. Thus, there is a great need to develop new treatments. We have found that bisbenzylisoquinoline alkaloid tetrandrine (TET) exhibits anticancer activity. TET is used as a calcium channel blocker to treat hypertensive and arrhythmic conditions in Chinese medicine. Here, we investigate the molecular basis underlying TET's anticancer activity. We compare TET with six chemotherapy drugs in eight cancer lines and find that TET exhibits comparable anticancer activities with camptothecin, vincristine, paclitaxel, and doxorubicin, and better than that of 5-fluorouracil (5-FU) and carboplatin. TET IC?? is ≤5 μM in most of the tested cancer lines. TET exhibits synergistic anticancer activity with 5-FU and reduces migration and invasion capabilities of HCT116 cells. Furthermore, TET induces apoptosis and inhibits xenograft tumor growth of colon cancer. TET treatment leads to a decrease in β-catenin protein level in xenograft tumors, which is confirmed by T-cell factor/lymphocyte enhancer factor and c-Myc reporter assays. It is noteworthy that HCT116 cells with allelic oncogenic β-catenin deleted are less sensitive to TET-mediated inhibition of proliferation, viability, and xenograft tumor growth. Thus, our findings strongly suggest that the anticancer effect of TET in colon cancer may be at least in part mediated by targeting β-catenin activity. Therefore, TET may be used alone or in combination as an effective anticancer agent.     

AGGF1通过激活Wnt/β-catenin信号通路促进结直肠癌发生发展

目的 研究G补缀FHA域血管新生因子1（angiogenic factor with G-patch and FHA domain 1,AGGF1）调控Wnt/β-catenin信号通路对结直肠癌细胞（colorectal cancer,CRC）增殖、侵袭和上皮间质转化（epithelial mesenchymal transition,EMT）的可能机制。方法 通过裸鼠荷瘤免疫组化法检测AGGF1在正常组织和癌组织中的表达。qRT-PCR和Western blotting检测NCM460、SW620、HT29、HCT116、SW480中AGGF1的mRNA和蛋白表达水平。于HCT116细胞中过表达AGGF1,分为Vector组和AGGF1组;于SW620细胞中敲减AGGF1,分为shControl组和shAGGF1组。CCK-8法和克隆形成试验测定细胞活性和细胞克隆数目。划痕试验和Transwell试验检测细胞迁移和侵袭情况。免疫荧光检测细胞E-cadherin、N-cadherin的表达。Western blotting检测细胞E-cadherin、N-cadherin、AGGF1、β-catenin、糖原合酶激酶-3β （glycogen synthase kinase-3β,GSK-3β）、p-GSK-3β蛋白表达水平。结果 AGGF1蛋白表达在CRC组织中明显增高。AGGF1 mRNA和蛋白表达水平在HCT116细胞中最低,在SW620细胞中最高。CCK-8法、克隆形成试验、划痕试验和Transwell试验结果显示,在HCT116细胞中,与Vector组比较,AGGF1组48,72,96 h细胞活性、克隆细胞数目、相对迁移距离、侵袭细胞数显著增加（P<0.05或P<0.01）;而在SW620细胞中,与shControl组比较,shAGGF1组结果相反（P<0.05或P<0.01）。免疫荧光和Western blotting检测结果显示,与Vector组比较,AGGF1组E-cadherin荧光表达和蛋白表达水平降低（P<0.01）,N-cadherin增强（P<0.05或P<0.01）,同时上调AGGF1、β-catenin、p-GSK-3β/GSK-3β蛋白表达（P<0.01）;与shControl组比较,shAGGF1组E-cadherin荧光表达和蛋白表达水平显著升高（P<0.01）,N-cadherin显著降低（P<0.05或P<0.01）,同时下调AGGF1、β-catenin、p-GSK-3β/GSK-3β蛋白表达（P<0.05或P<0.01）。结论 AGGF1可通过激活Wnt/β-catenin信号通路异常表达,上调β-catenin、p-GSK-3β、N-cadherin表达,下调E-cadherin表达,促进CRC细胞增殖、迁移、侵袭和EMT,从而促进CRC发生发展。     

MicroRNA-520e targets AEG-1 to suppress the proliferation and invasion of colorectal cancer cells through Wnt/GSK-3β/β-catenin signalling

MicroRNA-520e (miR-520e) is increasingly being recognized as a cancer-related miRNA in multiple cancer types; however, little is known about its role in colorectal cancer. In this study, we determined the specific role of miR-520e in colorectal cancer. Expression of miR-520e was lower in colorectal cancer tissues compared to normal tissues. Overexpression of miR-520e significantly decreased the proliferation, colony formation and invasion of colorectal cancer cells, while inhibition of miR-520e exhibited the opposite effect. Moreover, miR-520e was found to target the 3′-untranslated region of astrocyte elevated gene-1 (AEG-1) and inhibit AEG-1 expression in colorectal cancer cells. An inverse correlation between miR-520e and AEG-1 expression was confirmed in colorectal cancer tissues. Notably, miR-520e suppressed the phosphorylation of glycogen synthase kinase-3β and decreased the expression of β-catenin, leading to inactivation of Wnt/β-catenin signalling in colorectal cells. A rescue assay confirmed that miR-520e regulates cell proliferation, invasion and Wnt/β-catenin signalling through targeting AEG-1. Taken together, these results indicate that miR-520e plays a critical role in regulating colorectal cancer cell proliferation and invasion by inhibiting Wnt/β-catenin signalling via AEG-1. Our study highlights the importance of the miR-520e/AEG-1/Wnt/β-catenin signalling axis in colorectal cancer, thus targeting miR-520e may represent an effective therapeutic strategy.     

Ophiopogonin D suppresses TGF-β1-mediated metastatic behavior of MDA-MB-231 breast carcinoma cells via regulating ITGB1/FAK/Src/AKT/β-catenin/MMP-9 signaling axis

Ophiopogonin D, a steroidal glycoside extracted from the Traditional Chinese Medicine Ophiopogon japonicus, shows anti-tumor property in several lines of cancers; however, its effect on triple-negative breast cancer (TNBC) has not been investigated. In this study, the anti-metastatic effect of Ophiopogonin D in TNBC cells as well as the underlying mechanism in such process was explored. Ophiopogonin D dose-dependently decreased cell proliferation of MDA-MB-231 cells. Meanwhile, Ophiopogonin D significantly inhibited TGF-β1-induced metastatic behavior of MDA-MB-231 cells, including EMT, anoikis resistance as well as migration and invasion, via suppressing MMP-9 activity. Mechanically, Ophiopogonin D achieved its effect through efficiently abolishing ITGB1 expression, thus reducing the phosphorylation of FAK, Src and AKT, as well as upregulating nuclear β-catenin. ITGB1 overexpression partly recovered Ophiopogonin D's inhibitory effect on metastatic behavior via activating MMP-9. These results demonstrated that Ophiopogonin D could suppress TGF-β1-mediated metastatic behavior of MDA-MB-231 cells by regulating ITGB1/FAK/Src/AKT/β-catenin/MMP-9 signaling axis, which might provide new insight for the control of TNBC metastasis.     

GSK3β/β-catenin signaling is correlated with the differentiation of glioma cells induced by wogonin

Malignant gliomas are the most common and most aggressive primary brain tumor, and for which differentiation therapy has emerged as a promising candidate strategy. In this study, we used in vitro and in vivo assays to examine the differentiation effects of wogonin, a major active constituent of Scutellaria baicalensis, on glioma C6 and U251 cells. We found that wogonin can suppress cell proliferation and induce G0/G1 arrest under a concentration-dependent manner. Wogonin also triggered significant reduction in the G1 cell-cycle regulatory proteins cyclin D1, cyclin-dependent kinase 2 and 4 along with overexpression of cell-cycle inhibitory proteins p27. Immunofluorescence and western blot analysis indicated that wogonin increased the expression of lineage-specific differentiation marker glial fibrillary acidic protein (GFAP). In mechanisms, we verified that wogonin significantly diminished the phosphorylated level of protein kinase B (AKT), and maintenance of low β-catenin expression level was dependent on glycogen synthase kinase 3β (GSK3β) activation at Ser9. Blocking GSK3β/β-catenin pathway was required for wogonin-induced proliferation inhibition and terminal differentiation by using canonical activator lithium chloride (LiCl) and inhibitor dickkopf-1 (Dkk1). Moreover, intravenous administration of wogonin delayed the growth of C6 glioma in the intracranial tumor model. These findings provide the evidence and mechanistic support for wogonin-based differentiation therapies for malignant glioblastoma. Furthermore, inhibition of GSK3β/β-catenin pathway may be a key and requisite factor in glioma differentiation.     

MicroRNA-367-3p overexpression represses the proliferation and invasion of cervical cancer cells through downregulation of SPAG5-mediated Wnt/β-catenin signalling

MicroRNA-367-3p (miR-367-3p) has been previously reported as a cancer-related miRNA that is dysregulated in various cancer types and functions either as an oncogenic or as tumour suppressive miRNA. However, whether miR-367-3p is dysregulated in cervical cancer and, further, whether it contributes to the development and progression of the disease remains unknown. Here, our results demonstrated that miR-367-3p expression was markedly decreased in both cervical cancer tissues and cell lines compared with corresponding controls. In vitro experiments revealed that miR-367-3p overexpression repressed the proliferation and invasion of cervical cancer cells. Notably, sperm-associated antigen 5 (SPAG5) was identified as a target gene of miR-367-3p. Moreover, decreased expression of miR-367-3p was correlated with high expression of SPAG5 in cervical cancer tissue specimens. SPAG5 inhibition or miR-367-3p overexpression significantly downregulated Wnt/β-catenin signalling in cervical cancer cells. However, the antitumour effect mediated by miR-367-3p overexpression was partially reversed by SPAG5 overexpression. Overall, these findings demonstrate that miR-367-3p overexpression restricts the proliferation and invasion of cervical cancer cells through targeting SPAG5 to downregulate Wnt/β-catenin signalling, suggesting a mechanism for the tumour suppressive function of miR-367-3p in cervical cancer. Our study highlights the involvement of miR-367-3p/SPAG5/Wnt/β-catenin signalling axis in regulating the malignant progression of cervical cancer.     

Wnt1/β-catenin/DKK1信号通路与儿童原发性肾病综合征糖皮质激素反应异质性关系研究

目的观察不同糖皮质激素(GC)反应原发性肾病综合征(PNS)患儿外周血浆、单个核细胞中GC治疗前后Wnt1/β-catenin/DKK1信号通路表达的变化,探讨该信号通路在儿童PNS发病中的可能作用及与GC反应的关系。方法选取初发PNS患儿48例作为PNS组,按照GC治疗的反应分为2组:GC敏感型肾病综合征组(SSNS组n=29);GC耐药型肾病综合征组(SRNS组n=19);另选取健康体检儿15名作为对照组。PNS患儿分两个时间点抽取外周血标本:①病初未用GC时;②足量GC治疗6周时。采用双抗体夹心酶联免疫吸附试验法(ELISA)、实时荧光定量PCR检测(qPCR)检测Wnt1、β-catenin、DKK1的表达水平。结果 GC治疗前,SSNS组及SRNS组β-catenin表达水平高于对照组;SRNS组β-catenin表达水平高于SSNS组;GC治疗6周时,SSNS组及对照组β-catenin表达水平低于SRNS组;SSNS组β-catenin表达水平高于对照组;SSNS组及SRNS组β-catenin表达水平低于治疗前。结论 PNS患儿存在β-catenin表达上调,提示β-catenin蛋白可能参与了PNS发病,不同GC反应PNS患儿β-catenin高表达可能与PNS患儿GC耐药密切相关,PNS患儿β-catenin升高可作为GC耐药的标志之一。GC可能影响β-catenin表达水平。不同GC反应PNS患儿Wnt1和DKK1表达差异无统计学意义,提示Wnt1、DKK1蛋白与患儿PNS发生及GC耐药无相关性。     

The CRD of Frizzled 7 exhibits chondroprotective effects in osteoarthritis via inhibition of the canonical Wnt3a/β-catenin signaling pathway

Osteoarthritis (OA) is a chronic inflammatory joint disease without effective drugs. Frizzled 7 (FzD7) binds its ligand Wnt3a through an extracellular cysteine-rich domain (CRD) to transduce the canonical Wnt/β-catenin signaling pathway, which has been strongly implicated in OA pathogenesis. Effects of recombinant protein of FzD7 CRD on Wnt/β-catenin signaling and chondral destruction was evaluated in this study. Firstly, increased protein levels of FzD7, Wnt3a and β-catenin were detected in human OA cartilage implying that the canonical Wnt/β-catenin signaling mediated by Wnt3a and FzD7 executes an essential role in OA. Then we showed that FzD7 CRD antagonized the Wnt3a/β-catenin signaling pathway in a dose-dependent manner by binding Wnt3a. In addition, FzD7 CRD increased the expression of glycosaminoglycans (GAGs), Collagen II, aggrecan and reduced the expression of matrix metalloproteinase (MMP)-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) in Wnt3a-stimulated human chondrocytes. Furthermore, a single intra-articular injection of the FzD7 CRD was efficacious in destabilization of the medial meniscus (DMM) mouse OA model, significantly improving Osteoarthritis Research Society International (OARSI) histology scores compared to mice treated with PBS. The results indicate that the FzD7 CRD exhibits chondroprotective effects by binding Wnt3a to suppress the Wnt3a/β-catenin signaling. Targeting the FzD7 CRD may be a novel therapy for the treatment of OA.