Expression Of Dvl-1 Research Articles

Curcumin promotes apoptosis of liver cancer cells by down-regulating WNT/β-catenin

Curcumin (C21H20O6) is considered to exert anticancer potential. However, the relationship between curcumin and β-catenin has not been fully clarified and its regulatory role in human liver cancer has not yet been confirmed. In this study, human liver cancer cells were exposed to curcumin and the proliferation of HepG2 cells was detected by MTT method along with analysis of cell cycle and apoptosis, as well as Wnt/β-catenin signaling proteins expression. Curcumin shows a time-dependent and dose-dependent effect on HepG2 cell proliferation. The IC50 at 24 h, 48 h and 72 h were 12.8±0.67) μmol/L, 8.8±0.43 μmol/L and 4.6±0.42 μmol/L, respectively. Curcumin could dose-dependently (5, 10 and 20 μmol/L) arrest HepG2 cells in the G2/M phase (p < 0.05) and increase apoptosis (p < 0.05). In addition, curcumin down-regulated total, cytoplasmic and nuclear β-catenin proteins in HepG2 cells, indicating inhibited Wnt/β-catenin signaling. Curcumin reduced the expression of Dvl-2, Dvl-3, GSK-3β (p-ser9), C-myc, and Survivin, and increased the expression of GSK-3 (p-tyr216) and Axin-2 without affecting total GSK-3β levels. Curcumin is able to inhibit liver cancer cell activities possibly through inhibition of Wnt/β-catenin signaling.

Lipid nanoparticles containing PlncRNA-1 promote apoptosis of liver cancer cells by down-regulating Wnt/beta-catenin signaling

PlncRNA-1 has an anti-cancer property. Liposome nanoparticles (LNPs) are used as carriers of targeted drugs and contribute to tumor-targeted drug delivery. However, the interaction between LNPs containing PlncRNA-1 and β-catenin, a key component of Wnt signaling pathway, and its role in liver cancer have not yet been confirmed. Human liver cancer cells were exposed to LNPs containing PlncRNA-1 at different concentrations followed by analysis of cell proliferation, cell cycle distribution and apoptosis. Wnt/β-catenin pathway-related gene expression was determined. LNPs containing PlncRNA-1 dose-dependently suppressed the proliferation of liver cancer HepG2 cells with IC50 at 24 h, 48 h and 72 h of 12.8±0.67 μmol/L, 8.8±0.43 μmol/L and 4.6±0.42 μmol/L, respectively. PlncRNA-1-loaded LNPs (5, 10, and 20 μmol/L) dose-dependently arrested HepG2 cells in G2/M phase and induced apoptosis (p > 0.05). Administration of LNPs carrying PlncRNA-1 decreased the expression of overall, cytoplasmic, and nuclear β-catenin, thereby suppressing Wnt/β-catenin pathway. Protein expression levels of GSK-3 (p-tyr216) and Axin-2 were increased, while protein expression levels of Dvl-2, Dvl-3, GSK-3β, c-myc and survivin were significantly decreased. Invasion and metastasis of malignant tumors is an important factor. Inhibiting invasion of cancer cells effectively might improve the prognosis of cancer patients. LNPs carrying PlncRNA-1 promoted cell cycle transition and cell apoptosis by decreasing the expression of Dvl-2, Dvl-3, intracellular β-catenin, cytoplasmic β-catenin, and total β-catenin protein, which may relate to the inhibition of Wnt/β-catenin pathway.

Dishevelled segment polarity protein 2 promotes gastric cancer progression through Wnt/β-catenin pathway

Dishevelled family proteins (DVL1–3), key scaffold proteins, act on canonical and non-canonical Wnt/β-catenin signaling pathway. DVL has been implicated in various tumor progression. However, its role and underlying mechanisms in gastric cancer (GC) remain unclear. The aim of this study was to investigate the role of DVL in GC development using cell lines and 209 GC specimens. We analyzed three orthologs of DVL in GC tissues and paired adjacent non-tumor tissues, and only DVL2 is highly expressed in GC tissues. We also analyzed clinicopathological data on DVL2 expression in gastric cancer specimens. In immunohistochemistry, DVL2 expression was up-regulated in GC tissues compared with paired adjacent non-tumor tissues (153/209, 73.2%). DVL2 expression level was significantly correlated with many clinicopathological parameters such as T stage (P < 0.001) and N stage (P < 0.001). Survival analysis showed that the overall survival (OS) of patients with high expression of DVL2 was significantly shorter than those with low expression. Multivariate Cox regression analysis revealed that DVL2 expression was an important and independent prognostic factor for gastric cancer patients (P = 0.011, HR=1.78, 95%CI (1.14–2.79). Depletion of endogenous DVL2 using short hairpin RNA (shRNA) inhibited GC cell proliferation, migration, and invasion. The abnormal activation of Wnt/β-catenin signaling pathway is mainly achieved through the abnormal expression of DVL2. DVL2 is highly expressed in gastric cancer tissues, which may be a new independent risk factor for the prognosis of gastric cancer patients. In gastric cancer, DVL2 overexpression plays a crucial role in the occurrence and development of gastric cancer, so it may become a new, effective and complementary therapeutic target for gastric cancer.

Network Pharmacology and Molecular Docking Analysis on Molecular Targets and Mechanisms of Fei Jin Sheng Formula in the Treatment of Lung Cancer.

Fei Jin Sheng Formula (FJSF) is widely used in clinical treatment of lung cancer. But the underlying active ingredients and mechanisms are unclear. To investigate the active components and functional mechanisms of FJSF in treating lung cancer using a network pharmacology approach and molecular docking combined with vitro experiments Methods: Based on the TCMSP and related literature, the chemical components of related herbs in FJSF were collected. The active components of FJSF were screened by ADME parameters, and the targets were predicted by the Swiss Target Prediction database. The "drug-active ingredient-target" network was constructed by Cytoscape. Disease-related targets of lung cancer were acquired from GeneCards, OMIM, and TTD databases. Then drug-disease intersection target genes were obtained through the Venn tool. GO analysis and KEGG pathway enrichment analysis were performed via the Metascape database. Cytoscape was used to construct a PPI network and perform topological analysis. Kaplan-Meier Plotter was used to analyze the relationship between DVL2 and the prognosis of lung cancer patients. xCell method was used to estimate the relationship between DVL2 and immune cell infiltration in lung cancer. Molecular docking was performed by AutoDockTools-1.5.6. The results were verified by experiments in vitro. FJSF contained 272 active ingredients and 52 potential targets for lung cancer. GO enrichment analysis is mainly related to cell migration and movement, lipid metabolism, and protein kinase activity. KEGG pathway enrichment analysis mainly involves PI3K-Akt, TNF, HIF-1, and other pathways. Molecular docking shows that the compound Xambioona, quercetin and methyl palmitate in FJSF has a strong binding ability with NTRK1, APC, and DVL2. Analysis of the data in UCSC to analyze the expression of DVL2 in lung cancer shows that DVL2 was overexpressed in lung adenocarcinoma tissues. Kaplan-Meier analysis shows that the higher DVL2 expression in lung cancer patients was associated with poorer overall survival and poorer survival in stage I patients. It was negatively correlated with the infiltration of various immune cells in the lung cancer microenvironment. Vitro Experiment showed that Methyl Palmitate (MP) can inhibit the proliferation, migration, and invasion of lung cancer cells, and its mechanism of action may be to downregulate the expression of DVL2. FJSF may play a role in inhibiting the occurrence and development of lung cancer by downregulating the expression of DVL2 in A549 cells through its active ingredient Methyl Palmitate. These results provide scientific evidence for further investigations into the role of FJSF and Methyl Palmitate in the treatment of lung cancer.

CircMMD reduction following tumor treating fields inhibits glioblastoma progression through FUBP1/FIR/DVL1 and miR-15b-5p/FZD6 signaling

BackgroundTumor treating fields (TTF) is the latest treatment for GBM. Circular RNA (circRNA) has been demonstrated to play critical roles in tumorigenesis. However, the molecular mechanism of TTF remained largely unknown and the role of circRNA in TTF was not reported. The aim of this study was to elucidate the role and mechanism of circMMD in TTF treatment of GBM.MethodsDivergent primer was designed to verify the existence of circMMD in GBM cells. The prognostic role of circMMD was explored in glioma specimens. The knockdown and overexpressed plasmids were used to evaluate the effect of circMMD on GBM cell proliferation and TTF efficacy. RNA pull-down and RNA immunoprecipitation were performed to identify binding proteins of circMMD. Subcutaneous and intracranial tumor models were established to validate findings in vivo.ResultsThe expression of circMMD was elevated in GBM and its high expression indicated poor prognoses. TTF intervention could reduce circMMD synthesis, which suppressed GBM proliferation and increased TTF-mediated apoptosis. The reduction of circMMD promoted the interaction between FUBP1 and FIR, which decreased DVL1 transcription. Meanwhile, decreased circMMD would promote the activity of miR-15b-5p to degrade FZD6. Finally, the diminished expression of DVL1 and FZD6 expression suppressed the activation of Wnt/β-catenin pathway.ConclusionsOur study revealed a novel mechanism of TTF that TTF-mediated reduction of circMMD could inhibit Wnt/β-catenin pathway to suppress GBM proliferation.

Dishevelled 2 regulates cancer cell proliferation and T cell mediated immunity in HER2-positive breast cancer

BackgroundDishevelled paralogs (DVL1, 2, 3) are key mediators of Wnt pathway playing a role in constitutive oncogenic signaling influencing the tumor microenvironment. While previous studies showed correlation of β-catenin with T cell gene expression, little is known about the role of DVL2 in modulating tumor immunity. This study aimed to uncover the novel interaction between DVL2 and HER2-positive (HER2+) breast cancer (BC) in regulating tumor immunity and disease progression.MethodsDVL2 loss of function studies were performed with or without a clinically approved HER2 inhibitor, Neratinib in two different HER2+ BC cell lines. We analyzed RNA (RT-qPCR) and protein (western blot) expression of classic Wnt markers and performed cell proliferation and cell cycle analyses by live cell imaging and flow cytometry, respectively. A pilot study in 24 HER2+ BC patients was performed to dissect the role of DVL2 in tumor immunity. Retrospective chart review on patient records and banked tissue histology were performed. Data were analyzed in SPSS (version 25) and GraphPad Prism (version 7) at a significance p < 0.05.ResultsDVL2 regulates the transcription of immune modulatory genes involved in antigen presentation and T cell maintenance. DVL2 loss of function down regulated mRNA expression of Wnt target genes involved in cell proliferation, migration, invasion in HER2+ BC cell lines (±Neratinib). Similarly, live cell proliferation and cell cycle analyses reveal that DVL2 knockdown (±Neratinib) resulted in reduced proliferation, higher growth arrest (G1), limited mitosis (G2/M) compared to non-targeted control in one of the two cell lines used. Analyses on patient tissues who received neoadjuvant chemotherapy (n = 14) further demonstrate that higher DVL2 expression at baseline biopsy pose a significant negative correlation with % CD8α levels (r = − 0.67, p < 0.05) while have a positive correlation with NLR (r = 0.58, p < 0.05), where high NLR denotes worse cancer prognosis. These results from our pilot study reveal interesting roles of DVL2 proteins in regulating tumor immune microenvironment and clinical predictors of survival in HER2+ BC.ConclusionOur study demonstrates potential immune regulatory role of DVL2 proteins in HER2+ BC. More in-depth mechanistic studies of DVL paralogs and their influence on anti-tumor immunity may provide insight into DVLs as potential therapeutic targets benefiting BC patients.

Suppression of Wnt/β-catenin Signaling in PDAC via METTL16-mediated N6-methyladenosine Modification of DVL2.

Pancreatic cancer is a formidable cause of cancer-related deaths worldwide and has witnessed a more than twofold increase in incidence over the last 25 years. The most frequently occurring form of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), accounting for the majority of pancreatic cancer cases. N6-methyladenosine (m6A), the most abundant transcript modification, has been implicated in the pathogenesis of numerous human cancers, including pancreatic cancer. Despite this, the functional role of methyltransferase-like 16 (METTL16), a critical m6A methyltransferase, in PDAC remains elusive. In this study, we demonstrate that METTL16 expression is significantly diminished in PDAC, rendering it a promising prognostic indicator. Strikingly, both in vitro and in vivo assays revealed accelerated metastasis and invasion of PDAC cells upon METTL16 knockdown, while overexpression of METTL16 exerted an opposite effect. Mechanistically, METTL16 regulates DVL2 expression by suppressing its translation via m6A modification, thereby regulating Wnt/β-catenin signaling., Our results unveil the downregulation of METTL16 as a concomitant increase in DVL2 levels via m6A modification promoting the progression of PDAC. Thus, we propose METTL16 as a novel therapeutic candidate for targeted PDAC treatment.

Effect of miR-34a on the Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells (BMSCs) in Hyperlipidemia Rats

This study intends to explore miR-34a’s effect on BMSCs osteogenic differentiation of hyperlipidemia. 20 SD rats were equally assigned into control group (normal diet) and high-fat group (research diet). Cells were transfected with miR-34a mimic or negative control followed by meausring miR-34a, Dvl2, PPAR-γ, ALP, Runx2, and sp7, and ALP activity. The number of mineralized nodules, adipocytes, miR-34a and PPAR-γ expression in high-fat group were significantly increased and Dvl2, ALP, Runx2, and sp7 mRNA showed opposite profiles. Meanwhile, Runx2, ALP protein, cytoplasm and nuclear blue-black particles, Dvl2 protein and mRNA in miR-34a mimic group were significantly downregulated (P &lt; 0.05). Additionally, the luciferase activity of wild-type plasmid+miR-34a mimic group was significantly lower than mutant group, indicating that miR-34a targets Dvl2. In conclusion, miR-34a inhibits the osteogenic differentiation of hyperlipidemia BMSCs by inhibiting the expression of Dvl2, Runx2 and ALP activity, indicating that it might be target in the hyperlipidemia treatment.

CYP2E1 plays a suppressive role in hepatocellular carcinoma by regulating Wnt/Dvl2/β-catenin signaling

ObjectiveKnowledge of the role of CYP2E1 in hepatocarcinogenesis is largely based on epidemiological and animal studies, with a primary focus on the role of CYP2E1 in metabolic activation of procarcinogens. Few studies have directly assessed the effects of CYP2E1 on HCC malignant phenotypes.MethodsThe expression of CYP2E1 in HCC tissues was determined by qRT-PCR, western blotting and immunohistochemistry. Overexpression of CYP2E1 in HCC cell was achieved by lentivirus transfection. The function of CYP2E1 were detected by CCK-8, wound healing, transwell assays, xenograft models and pulmonary metastasis model. TOP/FOPFlash reporter assay, western blotting, functional rescue experiments, Co-immunoprecipitation and reactive oxygen species detection were conducted to reveal the underlying mechanism of the tumor suppressive role of CYP2E1.ResultsCYP2E1 expression is down-regulated in HCC tissues, and this downregulation was associated with large tumor diameter, vascular invasion, poor differentiation, and shortened patient survival time. Ectopic expression of CYP2E1 inhibits the proliferation, invasion and migration and epithelial-to-mesenchymal transition of HCC cells in vitro, and inhibits tumor formation and lung metastasis in nude mice. Mechanistic investigations show that CYP2E1 overexpression significantly inhibited Wnt/β-catenin signaling activity and decreased Dvl2 expression in HCC cells. An increase in Dvl2 expression restored the malignant phenotype of HCC cells. Notably, CYP2E1 promoted the ubiquitin-mediated degradation of Dvl2 by strengthening the interaction between Dvl2 and the E3 ubiquitin ligase KLHL12 in CYP2E1-stable HCC cells. CYP2E1-induced ROS accumulation was a critical upstream event in the Wnt/β-Catenin pathway in CYP2E1-overexpressing HCC cells.ConclusionsThese results provide novel insight into the role of CYP2E1 in HCC and the tumor suppressor role of CYP2E1 can be attributed to its ability to manipulate Wnt/Dvl2/β-catenin pathway via inducing ROS accumulation, which provides a potential target for the prevention and treatment of HCC.Graphical

Abstract P5-10-04: Exploring novel connections between dishevelled (DVL) proteins and HER2-positive breast cancer-road to translation

Abstract Aberrant Wnt signaling contributes to every stage of tumorigenesis from immunosuppression in the tumor microenvironment (TME) and reduced tumor infiltrating lymphocytes (TIL) activity in the tumor niche to modulating several cancer hallmarks such as unrestrained growth, increased neoplastic cell survival, invasion, and metastasis. The overactive Wnt pathway is critical for tumor progression and hence a therapeutic target in multiple clinical trials. Dishevelled (DVL)s are the major scaffold proteins which relay signals in both canonical and non-canonical Wnt pathways and help maintain constitutive oncogenic signaling. Though DVL proteins are significant drivers of oncogenic Wnt signaling, little is known about their role in modulating tumor immunity. Previously we demonstrated that nuclear localization of DVL proteins is associated with transcription of several cancer-associated genes as well promotion of in vivo tumor growth. Moreover, our preliminary studies revealed significant positive association between high DVL expression and poor patient survival in different aggressive subtypes of breast cancer including HER2-positive breast cancer. Therefore, we hypothesize that (i) DVL depletion will reduce expression of classic Wnt markers involved in cancer cell proliferation and invasion in HER2-overexpressed in vitro models of breast cancer and (ii) clinically, high DVL protein expression will modulate markers of tumor immunity such as TIL score, levels of cytotoxic (CD8α) T-cells, neutrophil/platelet to lymphocyte ratio (NLR, PLR), eventually resulting in poor survival outcomes in HER2-positive breast cancer.To test our hypothesis, we performed DVL2 loss of function studies in two different HER2-overexpressed breast cancer cell lines such as SKBR3 (ER/PR-, HER2+) and BT474 (ER/PR+, HER2+) via lentiviral shRNA transduction and siRNA transfection. We analyzed RNA (RTqPCR) and protein (western blot) expression of classic Wnt markers and performed cell proliferation and cell cycle analyses by live cell imaging and flow cytometry, respectively. Additionally, we performed a pilot study including 10 HER2-positive breast cancer patients to dissect the role of DVL2 in tumor immunity. Retrospective chart review on patient records and banked tissue histology were performed to investigate the markers of tumor immunity and DVL proteins. Data were analyzed in SPSS (version 25) and GraphPad Prism (version 7) at a significance p&amp;lt;0.05.Initially, we identify higher DVL2 protein levels in tissues from HER2-positive breast cancer compared to non-cancer and other subtypes of breast cancer. Next, DVL2 depletion (NTC vs. sh- or siDVL2) results in reduced mRNA expression of Wnt target genes such as CYCLIN D1, OCT4, MMP7, VEGFA classically involved in cell proliferation, stem cell renewal, migration, and invasion respectively in both SKBR3 and BT474 cells. Similarly, live cell proliferation and cell cycle analyses reveal that DVL2 knockdown resulted in reduced proliferation, higher growth arrest (G1), limited mitosis (G2/M) compared to non-targeted control. Analyses on patient tissues further demonstrate that higher DVL2 expression pose a weak negative correlation with % TIL score (r=-0.17, NS) and % CD8α levels (r=-0.32, NS) while have a positive correlation with NLR (r=0.65, p=0.05), where high NLR denotes worse cancer prognosis. Though not statistically significant, these results from our pilot study reveal crucial roles of DVL2 proteins in regulating immunity and survival of HER2-positive breast cancer patients. Our study is noteworthy since it demonstrates possible immune regulatory role of DVL proteins in HER2-positive breast cancer which in turn, will aid in designing future clinical trials and in-depth mechanistic studies benefitting patients of different subtypes of breast cancer. Citation Format: Fahmida Rasha, Mingxiao V Yang, Isabel Castro-Piedras, Kathryn L. Furr, Annie Snitman, Sonia Y. Khan, Luis Brandi, Hafiz Khan, Nusrat Jahan, Michael W. Melkus, Kevin Pruitt, Rakhshanda Layeequr Rahman. Exploring novel connections between dishevelled (DVL) proteins and HER2-positive breast cancer-road to translation [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-04.

Dvl2 facilitates the coordination of NF-κB and Wnt signaling to promote colitis-associated colorectal progression.

Colitis‐associated colorectal cancer (CAC) arises due to prolonged inflammation and has distinct molecular events compared with sporadic colorectal cancer (CRC). Although inflammatory NF‐κB signaling was activated by pro‐inflammatory cytokines (such as TNFα) in early stages of CAC, Wnt/β‐catenin signaling later appears to function as a key regulator of CAC progression. However, the exact mechanism responsible for the cross‐regulation between these 2 pathways remains unclear. Here, we found reciprocal inhibition between NF‐κB and Wnt/β‐catenin signaling in CAC samples, and the Dvl2, an adaptor protein of Wnt/β‐catenin signaling, is responsible for NF‐κB inhibition. Mechanistically, Dvl2 interacts with the C‐terminus of tumor necrosis factor receptor 1 (TNFRI) and mediates TNFRI endocytosis, leading to NF‐κB signal inhibition. In addition, increased infiltration of the pro‐inflammatory cytokine interleukin‐13 (IL‐13) is responsible for upregulating Dvl2 expression through STAT6. Targeting STAT6 effectively decreases Dvl2 levels and restrains colony formation of cancer cells. These findings demonstrate a unique role for Dvl2 in TNFRI endocytosis, which facilitates the coordination of NF‐κB and Wnt to promote CAC progression.

Decoding the Role of DVL1 in Intracranial Meningioma.

In the search for molecular candidates for targeted meningioma therapies, increasing attention has been paid to the role of signaling pathways in the development and progression of intracranial meningiomas. Although it is well known that the Wnt signaling pathway is involved in meningioma progression, the role of its central mediator, DVL1, is still unclear. In order to investigate the influence of DVL1 gene alterations on the progression of human intracranial meningioma, we focused on its central PDZ domain, which is responsible for DVL interaction with the Fzd receptor and the phosphorylation of DVL mediated through the casein kinases CK1 and CK2. A genetic analysis of genomic instability revealed the existence of microsatellite instability in 9.09% and the loss of heterozygosity in 6.06% of the samples. The sequencing of the PDZ gene region showed repetitive deletions of two bases located in intron 7 and exon 8, and a duplication in intron 8 in most samples, with different outcomes on the biological function of the DVL1 protein. Immunohistochemistry revealed that the nuclear expression of DVL1 was significantly correlated with a higher expression of active β-catenin (p = 0.029) and a higher meningioma grade (p = 0.030), which leads to the conclusion that it could be used as biomarker for meningioma progression and the activation of the Wnt signaling pathway.

Characterization of aromatase in testes and potential functional analysis with letrozole administration in male Chinese soft-shelled turtle (Pelodiscus sinensis)

Aromatase, encoded by Cyp19a1, is essential in estrogen biosynthesis and crucial in sex differentiation and ovary development. Although aromatase regulates the proliferation, development, maturation and functional maintenance of male germ cells during mammalian spermatogenesis, its role in nonmammalian vertebrates is unclear. In the current study, a polyclonal antibody was prepared and the function of aromatase in testes was determined by administering letrozole. With ELISA detection, the titer of the antibody prepared in this study was more than 1:30000. Western blot analysis showed that the antibody could bind with cyp19a1 specifically. Aromatase was detected in Leydig cells and spermatogonia by immunohistochemistry. Thirty turtles were randomly assigned into three groups and received either letrozole treatment at 0 mg/kg (in control) body weight (BW), 5 mg/kg BW, or 10 mg/kg BW each day for 8 weeks. Histological analysis demonstrated that sperm count decreased in testis after letrozole treatment, and increasing levels of apoptosis were revealed by TUNEL staining. Quantitative real time PCR showed that Esr1, Esr2, and Gper exhibited different expression patterns after letrozole treatment. Dvl2, Cdc20, Dmc1, Sycp3, and Casp3 expression was significantly upregulated with the 10 mg/kg treatment (P < 0.05). Efnb1, Spata6, and Stra8 expression was significantly downregulated with the 5 mg/kg and 10 mg/kg treatments. These findings demonstrate that aromatase might play a functional role in spermatogenesis in the Chinese soft-shelled turtle, Pelodiscus sinensis.

Synergistic effect of toosendanin and regorafenib against cell proliferation and migration by regulating WWOX signaling pathway in hepatocellular carcinoma.

Regorafenib (RGF), a second-line multi-kinase inhibitor in the treatment of HCC (hepatocellular carcinoma) after sorafenib failure, exposes to the risk of drug resistance and subsequent progression of HCC patients. Toosendanin (TSN), a triterpenoid has presented excellent inhibition on several tumors. The purpose of this study is to investigate the inhibitory effect of the combination of TSN and RGF on HCC cells. We identified that TSN and RGF combination (TRC) synergistically inhibited the proliferation and migration of MHCC-97L cells. The upregulation of WWOX (WW-domain containing oxidoreductase) played a vital role in the HCC cell growth treated with TRC. TRC suppressed the phosphorylation of Stat3 and expression of DVL2, negatively regulated the activity of β-catenin by promoting the phosphorylation of GSK3β. In addition, the intranuclear proteins, including MMP2, MMP9, and C-MYC were significantly inhibited by TRC. The in vivo xenograft models confirmed that TRC effectually prevented the tumor growth through upregulating WWOX. Therefore, the treatment of TRC may be a potential solution of RGF resistance and promising therapeutic method in malignant HCC.

Silencing of KIF3B Suppresses Breast Cancer Progression by Regulating EMT and Wnt/β-Catenin Signaling.

Breast cancer is the most common malignant tumors in women. Kinesin family member 3B (KIF3B) is a critical regulator in mitotic progression. The objective of this study was to explore the expression, regulation, and mechanism of KIF3B in 103 cases of breast cancer tissues, 35 metastatic lymph nodes and breast cancer cell lines, including MDA-MB-231, MDA-MB-453, T47D, and MCF-7. The results showed that KIF3B expression was up-regulated in breast cancer tissues and cell lines, and the expression level was correlated with tumor recurrence and lymph node metastasis, while knockdown of KIF3B suppressed cell proliferation, migration, and invasion both in vivo and in vitro. In addition, UALCAN analysis showed that KIF3B expression in breast cancer is increased, and the high expression of KIF3B in breast cancer is associated with poor prognosis. Furthermore, we found that silencing of KIF3B decreased the expression of Dvl2, phospho-GSK-3β, total and nucleus β-catenin, then subsequent down-regulation of Wnt/β-catenin signaling target genes such as CyclinD1, C-myc, MMP-2, MMP-7 and MMP-9 in breast cancer cells. In addition, KIF3B depletion inhibited epithelial mesenchymal transition (EMT) in breast cancer cells. Taken together, our results revealed that KIF3B is up-regulated in breast cancer which is potentially involved in breast cancer progression and metastasis. Silencing KIF3B might suppress the Wnt/β-catenin signaling pathway and EMT in breast cancer cells.

MiR-29c-3p reduces bone loss in rats with diabetic osteoporosis via targeted regulation of Dvl2 expression.

The aim of this study was to investigate the influence of micro ribonucleic acid (miR)-29c-3p on rats with diabetic osteoporosis (DOP) and its underlying mechanism. A total of 30 specific pathogen-free (SPF)-grade male Wistar rats aged 6-week-old were randomly selected and divided into three groups according to different intervention means, including: NC group (control rats only injected with normal saline), DOP group (rats with DOP induced by injection of streptozotocin), and ME group (DOP rats injected with miR-29c-3p agonist for 4 consecutive weeks). The changes in blood glucose and body weight were recorded in the rats of each group every week. Enzyme-linked immunosorbent assay (ELISA) was applied to detect the content of bone turnover markers (BTMs) in serum, such as alkaline phosphatase (ALP), osteocalcin (OC), and procollagen type I N-terminal propeptide (PINP). The variations in serum calcium (Ca) and phosphorus (P) levels in the abdominal aorta were determined using an atomic absorption spectrometer in the three groups. Meanwhile, bone mineral density (BMD) of femur and lumbar vertebra (L1-L4) were examined. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the changes in messenger RNA (mRNA) expressions of miR-29c-3p and Disheveled 2 (Dvl2) in the bone tissues of intervened rats. In addition, the staining and expression changes of Dvl2 protein in bone tissues were determined via immunohistochemistry. The rats in NC group had normal behavioral activities, normally increased body weight, sensitive responses, as well as normal and stable blood glucose. In DOP group, the rats manifested clinical symptoms of diabetes mellitus (DM) (i.e., polydipsia, polyphagia, polyuria, and weight loss), lackluster hairs, decreased behavioral activities, slow responses, and blood glucose at a concentration higher than 16.7 mmol/L. However, the blood glucose rose first, and then, declined and it was maintained at a level higher than normal concentration in ME group. Meanwhile, the rate of weight loss significantly decreased. The results of qRT-PCR indicated that the relative expression level of miR-29c-3p in bone tissues of DOP group was remarkably lower than that in NC group (p<0.01). However, it was significantly higher in ME group than that in DOP group (p<0.05). DOP group exhibited significantly upregulated serum BTMs (ALP, CTX-1, OC, TRACP-5b, and PIPN) when compared with NC group (p<0.05) and ME group (p<0.05). Furthermore, femoral BMD decreased in DOP group (p<0.05) while increased in ME group, showing statistically significant difference between the two groups (p<0.05). Immunohistochemistry results indicated that the bone tissues of DOP rats were deeply stained, and protein expression of Dvl2 protein was significantly higher in comparison with NC group. The bone tissues were lightly stained in ME group, and the protein expression of Dvl2 was lower than that in DOP group. Besides, qRT-PCR results demonstrated that the mRNA expression changes of Dvl2 were consistent with its protein expression trends. MiR-29c-3p reduces bone loss in rats with DOP via targeted regulation of Dvl2 expression.

Dishevelled family proteins (DVL1-3) expression in intrauterine growth restriction (IUGR) placentas.

Dishevelled family proteins (DVL1, DVL2, and DVL3) are cytoplasmic proteins that are involved in canonical and non-canonical Wnt signaling pathway during embryonic development. The role of DVL proteins in the placental tissue remains mostly unknown. In the current study, we explored the role of Dishevelled proteins in naturally invasive tissue, trophoblast. Formalin-fixed paraffin-embedded samples of 15 term placentas from physiologic term pregnancies and 15 term placentas from pregnancies complicated with intrauterine growth restrictions (IUGR) were used for the study. Expression levels of mRNA for DVL1, DVL2, and DVL3 in placentas were analyzed by quantitative real-time PCR (qRT-PCR). DVL1, DVL2, and DVL3 protein expression were semi-quantitatively analyzed using immunohistochemistry. The expression of DVL3 protein was significantly higher in trophoblasts and endothelial cells in placental villi from IUGR pregnancies compared with the control group of term placentas, while DVL2 protein expression was significantly higher in trophoblasts in placental villi from IUGR pregnancies compared with normal term placentas. The observed differences at protein levels between normal and IUGR placentas were not confirmed at the mRNA levels of DVL genes. Our data indicate the active involvement of DVL proteins in IUGR-related placentas. No significant changes were observed in DVL mRNA levels between the two groups of placentas. Further studies are required to explore the clinical relevance of these observations.

The Role of Wnt/β-Catenin Pathway Mediators in Aortic Valve Stenosis.

Aortic valve stenosis (AVS) is a prevailing and life-threatening cardiovascular disease in adults over 75 years of age. However, the molecular mechanisms governing the pathogenesis of AVS are yet to be fully unraveled. With accumulating evidence that Wnt signaling plays a key role in the development of AVS, the involvement of Wnt molecules has become an integral study target in AVS pathogenesis. Thus, we hypothesized that the Wnt/β-catenin pathway mediators, SFRP2, DVL2, GSK3β and β-catenin are dysregulated in patients with AVS. Using immunohistochemistry, Real-Time qPCR and Western blotting, we investigated the presence of SFRP2, GSK-3β, DVL2, and β-catenin in normal and stenotic human aortic valves. Markedly higher mRNA and protein expression of GSK-3β, DVL2, β-catenin and SFRP2 were found in stenotic aortic valves. This was further corroborated by observation of their abundant immunostaining, which displayed strong immunoreactivity in diseased aortic valves. Proteomic analyses of selective GSK3b inhibition in calcifying human aortic valve interstitial cells (HAVICs) revealed enrichment of proteins involved organophosphate metabolism, while reducing the activation of pathogenic biomolecular processes. Lastly, use of the potent calcification inhibitor, Fetuin A, in calcifying HAVICs significantly reduced the expression of Wnt signaling genes Wnt3a, Wnt5a, Wnt5b, and Wnt11. The current findings of altered expression of canonical Wnt signaling in AVS suggest a possible role for regulatory Wnts in AVS. Hence, future studies focused on targeting these molecules are warranted to underline their role in the pathogenesis of the disease.

The role of WNT/β-catenin signaling pathway and glutamine metabolism in the pathogenesis of CCl4-induced liver fibrosis: Repositioning of niclosamide and concerns about lithium

BackgroundLiver fibrosis is a serious health problem which may lead to advanced liver cirrhosis and hepatocellular carcinoma. ObjectiveThe present study aimed to investigate the role of Wnt/β-catenin signaling pathway and glutamine aminohydrolase enzyme (l-glutaminase) in the pathogenesis of liver fibrosis and the potential benefits of niclosamide in treating liver fibrosis. MethodsNinety male Albino rats were divided into 6 equal groups (n = 15) as follows: a normal control group (NC), CCl4-only treated group (Fib.) which received 1 mg/kg CCl4 two times weekly, niclosamide-treated group (Niclo.) which received 5 mg/kg of niclosamide one time daily, lithium chloride-treated group (LiCl) which received 100 mg/kg of LiCl one time daily, niclosamide-and-CCl4-treated group (Niclo. + Fib.) which received same doses of niclosamide and CCl4 given to other groups, and finally lithium chloride-and-CCl4-treated rat group (LiCl + Fib.) which received same doses of LiCl and CCl4 given to other groups. All treatments were administered orally for 8 weeks. Liver tissue was assessed for l-hydroxyproline, beta-catenin (β-catenin), l-glutaminase activity, as well as the gene expression of transforming growth factor beta-1 (TGF-β1) and Dishevelled-2 (Dvl2). Histopathological and immunohistochemical analyses of alpha smooth muscle actin α-SMA were performed. Serum alanine transaminase (ALT), aspartate transaminase (AST), and total bilirubin were measured. ResultsThe group of niclosamide-and-CCl4-treated rats showed a significant decrease in total bilirubin, ALT and AST, β-catenin, l-hydroxyproline, l-glutaminase activity, and gene expression of TGF-β1 and Dvl2. Moreover, the liver tissue in this group of rats showed mild α-SMA reactivity compared with the rats treated with CCl4 only (fibrosis group). On the other hand, lithium chloride-and-CCl4-treated rats showed a significant increase in liver indices, TGF-β1 expression, β-catenin, l-hydroxyproline, and l-glutaminase activity with severe α-SMA reactivity and apoptosis in the liver tissue. ConclusionsNiclosamide protected rats against liver fibrosis by inhibiting the Wnt/β-catenin pathway and glutaminolysis.

6-Gingerol delays tumorigenesis in benzo[a]pyrene and dextran sulphate sodium-induced colorectal cancer in mice

Colorectal cancer (CRC) has been linked to dietary consumption of benzo[a]pyrene (B[a]P). 6-Gingerol (6-G), a component of ginger has been reported to possess anti-inflammatory and antioxidant activities, but little is known regarding the mechanism of 6-G in CRC chemoprevention. We therefore investigated the effect of 6-G on B[a]P. and dextran sulphate sodium (DSS) induced CRC in mice. Mice in Group I and Group II received corn oil and 6-G orally at 2 ml/kg and 100 mg/kg, respectively for 126 days. Group III were administered 125 mg/kg of B[a]P for 5 days followed by 3 cycles of 4% dextran sulphate sodium (DSS). Group IV received 6-G for 7 days followed by co-administration with 125 mg/kg of B[a]P. for 5 days and 3 cycles of 4% DSS. Tumor formation was reduced and expression of Ki-67, WNT3a, DVL-2 and β-catenin following 6-G exposure. Also, 6-G increases expression of APC, P53, TUNEL positive nuclei and subsequently decreased the expression of TNF-α, IL-1β, INOS, COX-2 and cyclin D1. 6-G inhibited angiogenesis by decreasing the concentration of VEGF, Angiopoietin-1, FGF and GDF-15 in the colon of B[a]P. and DSS exposed mice. Overall, 6-G attenuated B[a]P and DSS-induced CRC in mice via anti-inflammatory, anti-proliferative and apoptotic mechanisms.