The assessment of Dickkopf-1 and Dickkopf-2 protein concentration in different subtypes of non-small cell lung cancer subtypes.

Introduction: Lung cancer is one of the most prevalent cancers worldwide. Dickkopf-1 (DKK-1) and -2 (DKK-2) are important proteins for the regulated Wnt signalling pathway. Alternations in the Wnt pathway are associated with tumour progression. The aim of the study was to analyse the concentration of DKK-1 and DKK-2 in tumour and matched non-tumour (NT) samples of 65 patients with non-small cell lung cancer (NSCLC), including 3 subtypes: adenocarcinoma (AC), squamous cell carcinoma (SCC), and large cell carcinoma (LCC). Material and methods: The protein concentration was measured by enzyme-linked immunosorbent assay (ELISA) in homogenates. Results: The difference between the level of DKK-1 in tumour and NT specimens was not significant for the whole NSCLC group and SCC and LCC subtype, while in AC samples they were significantly higher (p = 0.028). The highest concentration of DKK-1 was found in the advanced NSCLC samples, with the T4 parameter as well as stage III. Significantly decreased DKK-2 concentrations were detected in all NSCLC subtypes (p < 0.05). Moreover, the DKK-2 level was higher in non-smokers than in smokers. The results indicate that concentrations of DKKs were different in relation to subtypes as well as clinical and socio-demographic parameters. The concentration of DKKs could be associated with the progression of NSCLC. Conclusions: We suggest that DKK-1 could play an oncogenic role in AC, while DKK-2 could be a tumour suppressor in all NSCLC subtypes. Dickkopf-1 and DKK-2 proteins could have differential roles in the Wnt signalling pathway, which is important in many cellular processes, such as proliferation and apoptosis.

Dickkopf-1 (DKK1) blockade mitigates osteogenesis imperfecta (OI) related bone disease.

BACKGROUND: The current treatment of osteogenesis imperfecta (OI) is imperfect. Our study thus delves into the potential of using Dickkopf-1 antisense (DKK1-AS) to treat OI. METHODS: We analysed serum DKK1 levels and their correlation with lumbar spine and hip T-scores in OI patients. Comparative analyses were conducted involving bone marrow stromal cells (BMSCs) and bone tissues from wild-type mice, untreated OI mice, and OI mice treated with DKK1-ASor DKK1-sense (DKK1-S). RESULTS: Significant inverse correlations were noted between serum DKK1 levels and lumbar spine (correlation coefficient = - 0.679, p = 0.043) as well as hip T-scores (correlation coefficient = - 0.689, p = 0.042) in OI patients. DKK1-AS improved bone mineral density (p = 0.002), trabecular bone volume/total volume fraction (p < 0.001), trabecular separation (p = 0.010), trabecular thickness (p = 0.001), trabecular number (p < 0.001), and cortical thickness (p < 0.001) in OI mice. DKK1-AS enhanced the transcription of collagen 1α1, osteocalcin, runx2, and osterix in BMSC from OI mice (all p < 0.001), resulting in a higher von Kossa-stained matrix area (p < 0.001) in ex vivo osteogenesis assays. DKK1-AS also reduced osteoclast numbers (p < 0.001), increased ß-catenin and T-cell factor 4 immunostaining reactivity (both p < 0.001), enhanced mineral apposition rate and bone formation rate per bone surface (both p < 0.001), and decreased osteoclast area (p < 0.001) in OI mice. DKK1-AS upregulated osteoprotegerin and downregulated nuclear factor-kappa B ligand transcription (both p < 0.001). Bone tissues from OI mice treated with DKK1-AS exhibited significantly higher breaking force compared to untreated OI mice (p < 0.001). CONCLUSIONS: Our study elucidates that DKK1-AS has the capability to enhance bone mechanical properties, restore the transcription of osteogenic genes, promote osteogenesis, and inhibit osteoclastogenesis in OI mice.

Bone Remodeling Markers in Children with Acute Lymphoblastic Leukemia after Intensive Chemotherapy: The Screenshot of a Biochemical Signature.

PURPOSE: to investigate the effects of intensive chemotherapy and glucocorticoid (GC) treatment on bone remodeling markers in children with acute lymphoblastic leukemia (ALL). METHODS: A cross-sectional study was carried out in 39 ALL children (aged 7.64 ± 4.47) and 49 controls (aged 8.7 ± 4.7 years). Osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), osteocalcin (OC), C-terminal telopeptide of type I collagen (CTX), bone alkaline phosphatase (bALP), tartrate-resistant acid phosphatase 5b (TRACP5b), procollagen type I N-terminal propeptide (P1NP), Dickkopf-1 (DKK-1), and sclerostin were assessed. Statistical analysis was conducted using the principal component analysis (PCA) to study patterns of associations in bone markers. RESULTS: ALL patients showed significantly higher OPG, RANKL, OC, CTX, and TRACP5b than the controls (p ≤ 0.02). Considering ALL group, we found a strong positive correlation among OC, TRACP5b, P1NP, CTX, and PTH (r = 0.43-0.69; p < 0.001); between CTX and P1NP (r = 0.5; p = 0.001); and between P1NP and TRAcP (r = 0.63; p < 0.001). The PCA revealed OC, CTX, and P1NP as the main markers explaining the variability of the ALL cohort. CONCLUSIONS: Children with ALL showed a signature of bone resorption. The assessment of bone biomarkers could help identify ALL individuals who are most at risk of developing bone damage and who need preventive interventions.

Micro Ribonucleic Acid-29a (miR-29a) Antagonist Normalizes Bone Metabolism in Osteogenesis Imperfecta (OI) Mice Model.

Osteogenesis imperfecta (OI) is not curative nowadays. This study tried to unriddle the therapeutic potential of micro ribonucleic acid-29a (miR-29a) antagonist in treating OI in a mouse animal model (B6C3Fe a/a-Col1a2oim/J). We showed that the expression levels of miR-29a were higher in bone tissues obtained from the OI mice than from wild-type mice demonstrated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and in situ hybridization assay. We established lentivirus-shuttled vector expressing miR-29a antisense oligonucleotide (miR-29a-AS) and miR-29a precursors (pre-miR-29a), showing that the inferior bony architecture in micro-computed tomography and pertinent morphometric parameters could be rescued by miR-29a-AS and deteriorated by pre-miR-29a. The decreased proliferating cell nuclear antigen (PCNA), increased Dickkopf-1 (DKK1), and decreased ß-catenin expression in OI mice could be accentuated by pre-miR-29a and normalized by miR-29a-AS. The decreased osteogenesis and increased osteoclastogenesis in OI mice could also be accentuated by pre-miR-29a and normalized by miR-29a-AS. miR-29a-AS did not seem to possess severe hepatic or renal toxicities.

Effect of Dickkopf-1 regulated Wnt pathway on transdifferentiation of hypoxia-induced lens epithelial cells / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To scrutinize the role of the Wnt/β-catenin signaling pathway in the epithelial-mesenchymal transition(EMT)of lens epithelial cells under hypoxic conditions, and to further analyze the effect of Dickkopf-1(DKK-1)expression on EMT of lens epithelial cells.METHODS: Human lens epithelial cells(HLEB3 cells)were propagated in vitro and then separated into two groups: one exposed to standard oxygen levels, added DMEM culture solution containing 10% FBS(normoxic group)and another subjected to low oxygen levels(hypoxic group). The hypoxic condition was emulated by applying a concentration of 100 μmol/L cobalt chloride(CoCl2)for 6, 12, 24, and 48h. The utilization of immunofluorescence staining enabled the detection of Wnt3a and DKK-1 expressions, along with the expression and localization of β-catenin protein in these groups. The expression of DKK-1 mRNA was discerned by quantitative real-time polymerase chain reaction(qRT-PCR).RESULTS: Immunofluorescence assays indicated an escalating trend in the Wnt3a and DKK-1 protein expression, which corresponded with the increasing duration of hypoxia. Likewise, an intensified nuclear accumulation of β-catenin protein was observed to be directly proportional to the length of hypoxia treatment. The qRT-PCR demonstrated that the difference in DKK-1 mRNA expression between the normoxic group and the group exposed to hypoxia for 6h was not statistically significant(P&#x003E;0.05), whereas the DKK-1 mRNA expression of the 12, 24, and 48h hypoxia groups were significantly increased(P&#x003C;0.001).CONCLUSION: Hypoxia can activate Wnt/β-catenin pathway in lens epithelial cells and induce the expression of DKK-1, thus regulating the Wnt/β-catenin pathway and affecting the EMT process.

Dickkopf-1 exerts protective effects by inhibiting PANoptosis and retinal neovascularization in diabetic retinopathy.

Diabetic retinopathy (DR) is a key reason for legal blindness worldwide. Currently, it is urgently necessary to determine the etiology and pathological molecular mechanism of DR to search for resultful therapies. Dickkopf-1 (DKK1) is inhibitive for canonical Wnt signaling via negative feedback, and has been reported as a biomarker for DR. However, the related mechanisms are still unclear. In this work, our data showed that DKK1 was decreased in the vitreous tissues at an early stage of diabetes triggered by streptozotocin (STZ) injection in rats. We subsequently found that DKK1 intravitreal injection significantly ameliorated the physiological function of retina in STZ-challenged rats, accompanied by improved retinal structure. Surprisingly, our results indicated that DKK1 injection remarkably suppressed PANoptosis in retinal tissues of STZ-challenged rats with DR, as proved by ameliorated pyroptosis, apoptosis and necroptosis, which were mainly through the blockage of cleaved Gasdermin-D (GSDMD), Caspase-3 and receptor-interacting protein kinase-3 (RIPK3). Additionally, Wnt signaling including the expression of Wnt, ß-catenin and LDL receptor-related protein 5/6 (LRP5/6) was also highly prohibited in retina of DKK1-injected rats with DR. Furthermore, retinal neovascularization and acellular vessel in DR rats were also considerably abolished after DKK1 injection, accompanied by reduced expression levels of retinal vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9). More in vitro experiments showed that DKK1 treatment markedly repressed the proliferative and migratory ability of endothelial cells via inhibiting angiogenesis-related molecules. Together, all our results broaden the knowledge of the correlation between DKK1 and DR, and then provide a novel therapeutic strategy for the suppression of management of DR.

Comparison of Maternal Serum Levels and Placental mRNA Levels of Dickkopf-1 in Preeclamptic and Normal Pregnant Women at Delivery.

Background Preeclampsia remains a major cause of perinatal and maternal mortality and morbidity worldwide. Wnt/ß-catenin signaling is known to be critically involved in placenta development processes. Dickkopf-1 (DKK1) is a key regulator of this transduction pathway. The aim of this study is to compare maternal serum DKK1 levels and placental mRNA levels of DKK1 and ß-catenin in preeclamptic and normal pregnant women at delivery. Methods The present study included 30 women with preeclampsia and 30 women with normal pregnancy. Maternal serum DKK1 levels were measured by ELISA. Placental mRNA levels of DKK1 and ß-catenin were detected using RT-PCR. Results Decreased maternal serum DKK1 levels were associated with worse maternal and fetal complications including HELLP syndrome, determination of one or more pathological symptom and IUGR diagnosis. No significant difference in maternal serum DKK1 levels was reported between preeclamptic women and women with normal pregnancy. Placental mRNA DKK1 levels were lower in preeclamptic women compared with normal pregnant women. Placental mRNA ß-catenin levels showed no significant difference between two groups. Conclusions Our findings reported the aberrant placental mRNA DKK1 levels in patients with preeclampsia. In addition, worse preeclampsia features were associated with decreased maternal serum DKK1 levels. Hence, aberrant Wnt/ß-catenin signaling might present a plausible mechanism in preeclampsia pathogenicity. Dysregulated expression of DKK1 at gene level in the placenta but not at protein level in the maternal serum might confirm the notion that preeclampsia is a type of placenta-derived disease.

NLRP3 inflammasome activation in alveolar epithelial cells promotes myofibroblast differentiation of lung-resident mesenchymal stem cells during pulmonary fibrogenesis.

Idiopathic pulmonary fibrosis (IPF) is a lethal and agnogenic interstitial lung disease, which has limited therapeutic options. Recently, the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome has been demonstrated as an important contributor to various fibrotic diseases following its persistent activation. However, the role of NLRP3 inflammasome in pulmonary fibrogenesis still needs to be further clarified. Here, we found that the activation of the NLRP3 inflammasome was raised in fibrotic lungs. In addition, the NLRP3 inflammasome was found to be activated in alveolar epithelial cells (AECs) in the lung tissue of both IPF patients and pulmonary fibrosis mouse models. Further research revealed that epithelial cells, following activation of the NLRP3 inflammasome, could induce the myofibroblast differentiation of lung-resident mesenchymal stem cells (LR-MSCs). In addition, inhibiting the activation of the NLRP3 inflammasome in epithelial cells promoted the expression of dickkopf-1 (DKK1), a secreted Wnt antagonist. DKK1 was capable of suppressing the profibrogenic differentiation of LR-MSCs and bleomycin-induced pulmonary fibrosis. In conclusion, this study not only provides a further in-depth understanding of the pathogenesis of pulmonary fibrosis, but also reveals a potential therapeutic strategy for disorders associated with pulmonary fibrosis.

The diagnostic value of golgi protien-73 and DICKKOPF-1 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Early detection of HCC is essential for improved prognosis and long-term survival. Golgi protein-73 (GP73) and Dickkopf-1 (DKK1), can be used as noninvasive biomarkers for early diagnosis of HCC. Aim of the work: measure and assess the clinical utility of Golgi protein-73 and Dickkopf-1 as noninvasive biomarkers for early diagnosis of HCC. This study included 60 patients: 30 patients with HCC, 30 patients with cirrhosis together with 30 healthy controls. There was a highly significant difference in GP73 and DKK1 between all the studied groups. There was a highly significant positive correlation between GP73 and DKK1 and a significant negative correlation between (GP73 and albumin) and (DKK1 and ALT) in group I. GP73, DKK-1 correlated with the number, overall size, and lymph node metastasis. GP73 and DKK1 can serve as diagnostic serologic markers for early HCC as they had high sensitivity and specificity, correlated with the aggressiveness of the tumor superior to AFP. The combined use of GP73, DKK-1, and AFP increased the sensitivity and specificity for the diagnosis of HCC than each one alone.

Circulating osteocyte-related biomarkers (vitamin D, sclerostin, dickkopf-1), hepcidin, and oxidative stress markers in early breast cancer: Their impact in disease progression and outcome.

Breast cancer (BC) is a major concern to female health worldwide. We assessed the circulating osteocyte-related biomarkers, hepcidin, and oxidative stress status among early-stage BC patients in aspects of clinical severity and impact on the outcome. The study incorporated 73 patients categorized into 57 early-stage BC and 16 benign breast diseases and 30 healthy controls. Serum 25-hydroxyvitamin D [25(OH)D], sclerostin (SOST), dickkopf-1(DKK1), and hepcidin were measured using ELISA, while, serum oxidative stress markers were assessed by spectrophotometry. Our results show that patients with BC showed significant increase in the mean levels of DKK1, SOST, hepcidin, and LPER and significant decrease in the mean levels of 25(OH)D, SOD, GPx, and Hb when compared with controls and benign breast diseases. Significantly higher DKK1, hepcidin, and SOD levels among benign breast diseases were found in comparison to control group. There were significantly lower levels of 25(OH)D, SOD, and Hb and significantly higher levels of SOST, DKK1, hepcidin, No, and LPER with advanced grade. Lower levels of 25(OH)D, SOD and higher levels of SOST, hepcidin were observed with increasing the malignant stage. Reduced levels of 25(OH)D, and SOD were significantly associated with poor prognosis and were strong predictors among BC. There were significant negative correlations between 25(OH)D with LPER, SOST, and hepicidin. We conclude that low 25(OH)D, high SOST, DKK1, and hepcidin, and dysregulated oxidative stress could be helpful in early detection and assessment of BC. 25(OH)D, and SOD were the most relevant to tumor progression and prognosis which indicate a significant role in the BC pathogenesis and could be promising targets in management. Our research paves the way to disrupt vicious circle between these biomarkers to obtain the best care of BC.

Bone, a Secondary Growth Site of Breast and Prostate Carcinomas: Role of Osteocytes.

Bone is the primarily preferred site for breast and prostate cancer to metastasize. Bone metastases are responsible for most deaths related to breast and prostate cancer. The bone's particular microenvironment makes it conducive for the growth of cancer cells. Studies on bone metastasis have focused on the interaction between cancer cells and the bone microenvironment. Osteocytes, the most common cell type of bone tissue, have received little attention in bone metastasis, although they are master signal sensors, integrators, and skeleton transducers. They play an important role in regulating bone mass by acting on both osteoblasts and osteoclasts, through the release of proteins such as sclerostin, Dickkopf-1 (DKK-1), and fibroblast growth factor 23 (FGF23). Osteocytes have been extensively re-evaluated, in light of their multiple functions: with different experimental approaches, it has been shown that, indeed, osteocytes are actively involved in the colonization of bone tissue by cancer cells. The present review focuses on recent research on the role that osteocytes play in bone metastasis of breast and prostate cancers. Moreover, the studies here summarized open up perspectives for new therapeutic approaches focused on modulating the activity of osteocytes to improve the condition of the bone metastatic patients. A better understanding of the complex interactions between cancer cells and bone-resident cells is indispensable for identifying potential therapeutic targets to stop tumor progression and prevent bone metastases.

Joint remodeling outcome of serum levels of Dickkopf-1 (DKK1), cartilage oligomeric matrix protein (COMP), and C-telopeptide of type II collagen (CTXII) in rheumatoid arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) is the most widespread chronic inflammatory rheumatic disease over the world. It is characterized by chronic proliferation of synovium, cartilage destruction, and periarticular erosion/bone loss. We investigated the serum levels of the C-telopeptide of type II collagen (CTX-II), Dickkopf-1 (DKK1), and cartilage oligomeric matrix protein (COMP) in relationship to the disease activity. MATERIAL AND METHODS: Serum COMP, CTX-II, and DKK1 levels were measured in 63 RA patients and 50 person age and gender matched as a healthy controls by ELISA test. Disease activity score (DAS) were calculated. RESULTS: The mean level of and COMP and CTX-II were significantly higher in patients with RA than in healthy controls (5.71 ±7.04 vs. 2.70 ±1.31 ng/ml, and 0.45 ±0.27 vs. 0.23 ±0.16 ng/ml, respectively; p < 0.001). Also, DKK1 serum levels were significantly higher in patients with RA than in healthy controls (6970.68 ±7566.68 vs. 3276.96 ±1306.77 pg/m; p < 0.001). There was a positive significant correlation between DKK1 and swollen joint (r = 0.42, p < 0.001). There were no significant differences in the number of patients, gender, the duration of RA disease, DAS, and RF. Sensitivity was 58.7% and specificity was 85.7% at a cut-off point (> 3.6 ng/ml) for serum COMP in RA patients, while, sensitivity was 100% and specificity was 52.4% at a cut-off point (> 0.15 ng/ml) for serum CTX-II and sensitivity was 68.3% and specificity was 95.2 % at a cut-off point (> 4876 pg/ml) for serum DKK1. CONCLUSIONS: Measurement of some serological biomarkers such as CTX-II, COMP, and DKK1 that reflect bone and cartilage destruction in RA patients could be used to indicate disease activity and early joint affection.

Analyzing the prognostic value of DKK1 expression in human cancers based on bioinformatics.

BACKGROUND: The Dickkopf1 (DKK1) gene encodes a protein that belongs to the Dickkopf family. The protein can inhibit the Wnt signaling pathway which plays a key role in the carcinogenesis and progression of various types of cancers. Based on this, we hypothesized that the differential expression of DKK1 may figure significantly in cancers by regulating Wnt signaling pathway transduction. In this study, we conducted bioinformatics analysis to evaluate the prognostic and therapeutic value of DKK1 expression level in human cancers. METHODS: The expression level was analyzed by using the Oncomine database and Gene Expression Profiling Interactive Analysis tool. The analysis of prognosis was conducted by using the UALCAN, Gene Expression Profiling Interactive Analysis (GEPIA), and DriverDBv3 databases. We also investigated using DKK1 promoter methylation to define cancer types through the UALCAN database. Meanwhile, the related functional networks of DKK1 were analyzed by using the GeneMANIA interactive tool and Cytoscape software. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis was conducted using the Metascape online website, and we used the cBioPotartal database to explored DKK1 expression, aberrant information, and the co-expression genes in the subgroups of lung cancer. Finally, we performed the overall survival (OS) meta-analysis of the DKK1 expression in lung squamous cell carcinoma (LUSC) via the Lung Cancer Explorer (LCE). RESULTS: DKK1 was differentially expressed in different types of human cancers. DKK1 was overexpressed in human cancers including head and neck squamous cell carcinoma (HNSC), LUSC, and pancreatic adenocarcinoma (PAAD). Overexpression of DKK1 indicated adverse OS in bladder urothelial carcinoma (BLCA), HNSC, and PADD, but no difference in OS was found between the LUSC and healthy groups. The high expression of DKK1 was also associated with shorter disease-free survival (DFS) in HNSC, LUSC, and PAAD. Gene regulation network analysis indicated that DKK1 was mainly involved in Wnt signaling pathways and several other signaling pathways. CONCLUSIONS: Our findings showed that DKK1 is significantly expressed in various cancers and could be a biomarker for targeted therapy and a predictor for prognosis of these specific cancers. The bioinformatics analysis revealed a significant overexpression of DKK1 in HNSC, LUSC, and PAAD, with DKK1 overexpression being associated with adverse outcome in these patients, but how DKK1 expression levels relate to hematological malignancies and prognosis is still unclear. These new insights into the function of DKK1 may provide a basis for new targeted drug therapy and an avenue for further investigation into the mechanisms underlying carcinogenesis of DKK1 in different cancer types.

DKK1 expression in head and neck squamous cell carcinoma: analysis of its clinical value and regulatory factors / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To explore the expression and regulation mechanism of Dickkopf-1 (DKK1) in head and neck squamous cell carcinoma (HNSCC) tissues. Methods: Based on the TCGA database, the relationship of DKK1 expression in HNSCC tissues and its methylation site with patients’prognosis was analyzed. GO and KEGG gene enrichment method were used to analyze the signaling pathways of DKK1 enrichment. STRING was used to analyze the interaction between DKK1 protein and other proteins. TargetScan was used to analyze the miRNAs that regulate the expression of DKK1, and the transcription factors of DKK1 were analyzed with the TRRUST website. Results: DKK1 gene was highly expressed in HNSCC tissues (P<0.01), and its expression level was significantly correlated with the HPV status, age, pathological grade, and clinical stage of patients (all P<0.05); the prognosis of HNSCC patients with high DKK1 expression was poorer than those with low DKK1 expression (P<0.01). There were 19 methylation sites in DKK1, 12 of which were significantly different between cancer tissues and normal tissues (P<0.05), and 11 sites were significantly related to the prognosis of HNSCC (P<0.05). In addition, miRNA, circRNA, lincRNA and transcription factors, etc. also participated in the regulation of DKK1. A total of 5 DKK1-related PPI networks that may involve in the occurrence, development, invasion and metastasis of HNSCC were obtained. Conclusion: DKK1 is highly expressed in HNSCC tissues and is a risk factor for poor prognosis of HNSCC patients. DKK1 plays an important role in the pathogenesis of HNSCC and is expected to become a potential target for HNSCC treatment.

Sclerostin rather than Dickkopf-1 is associated with mSASSS but not with disease activity score in patients with ankylosing spondylitis.

OBJECTIVE: To determine the serum levels of Dickkopf-1 (DKK-1) and sclerostin, as well as their correlations with the structural damage assessed by modified stoke ankylosing spondylitis spine score (mSASSS) and the disease activity evaluated by ankylosing spondylitis disease activity score (ASDAS) in patients with ankylosing spondylitis (AS). METHODS: Eighty-eight AS patients, 26 rheumatoid arthritis (RA) patients, and 26 age- and gender-matched healthy controls (HC) were collected from rheumatic clinic of the Second Affiliated Hospital of Zhejiang University, School of Medicine, between March 2015 and July 2015. Demographic data, parameters of ASDAS, and image evaluations of spine (i.e., mSASSS) were collected. The serum levels of DKK-1 and sclerostin were measured using commercially available ELISA kits. RESULTS: Both DKK-1 and sclerostin were significantly higher in the AS patients than in the controls (1855 ± 84.58 vs. 1406 ± 99.76 pg/ml and 106 ± 6.75 vs. 62.78 ± 6.39 pmol/l, respectively, P < 0.05). The correlation analysis suggested a negative correlation between serum sclerostin and mSASSS (P = 0.019, r2 = 0.062). DKK-1 had a trend of positive correlation with mSASSS, but was not statistically significant (P > 0.05). There was no association between the serum levels of DKK-1 or sclerostin and disease activity assessed by ASDAS (P > 0.05). DKK-1 and sclerostin had a negative correlation (P = 0.013, r2 = 0.07). CONCLUSION: In the present study, the expressions of serum DKK-1 and sclerostin were independent of disease activity. Sclerostin was negatively correlated with the mSASSS, which suggests that sclerostin may be a potential marker indicating the spine ossification process in AS. The specific mechanism remains to be investigated.

Effect of DKK1 on proliferation, cell cycle and apoptosis of gastric cancer AGS cells / 中国肿瘤生物治疗杂志

@# Objective： ：To study the effect of silencing DKK1 (Dickkopf1) gene on the proliferation, cell cycle and apoptosis of gastric cancer AGS cells and the action mechanism. Methods： ：The DKK1-shRNA vector was constructed and transfected into AGS cells. The stably transfected cell lines were screened. The total protein and RNAof the transfected cells were extracted and the mRNAand protein expressions of DKK1 were detected by qPCR and WB, respectively. The experiment was divided into blank control group (Control), negative control group (shNC) and DKK1 silence group (DKK1-shRNA). CCK8 assay was used to detect the proliferation ofAGS cells of each group cultured for 0, 24, 48, 72, 96, 120 and 144 h, and flow cytometry was used to analyze the cell cycle and apoptosis in each group. The relationship between DKK1 and clinicopathological features of gastric cancer was analyzed after searching HPA database. Results：The gastric cancer AGS cells with stable DKK1 gene knockdown was successfully established, and it was confirmed that the mRNA and proteinexpressions of DKK1 in DKK1-shRNA group decreased by 72% and 47%, respectively, compared to shNC group (all P<0.05). The cell proliferation curve showed that, the cell proliferation in DKKl-shRNAgroup significantly decreased after 72 hour of culture compared with that in control and shNC groups (P<0.05). The cell number of S phase decreased from 32.06% to 25.87%, while the number of G2/M phase increased from 8.49% to 21.26% compared with shNC group (all P<0.05). The number of apoptotic cells also statistically increased from 10.34% to 20.65% (all P<0.05). The data of HPAdatabase showed that DKK1 mRNAlevel in gastric cancer tissues was significantly higher than that in normal tissues, and the high expression of DKK1 mRNAwas negatively correlat ed with the survival rate of gastric cancer patients. Conclusion: : Silencing DKK1 gene can inhibit the proliferation of gastric cancer cells, arrest cells in G2/M phase and promote cell apoptosis. DKK1 plays a pro-carcinogenic effect in gastric cancer.

Dickkopf-1 (Dkk1) protein expression in breast cancer with special reference to bone metastases.

Dysregulation of the Wnt inhibitor dickkopf-1 protein (Dkk1) has been reported in a variety of cancers. In addition, it has been linked to the progression of malignant bone disease by impairing osteoblast activity. This study investigated serum- and tissue levels of Dkk1 in breast cancer patients with- or without bone metastases. Serum Dkk1 levels were measured by ELISA in 89 breast cancer patients and 86 healthy women. Tissue levels of Dkk1 and ß-catenin, a major downstream component of Wnt transduction pathway, were tested with immunohistochemical staining in 143 different tissues, including adjacent non-tumoral breast tissues, primary breast tumours, lymph nodes metastases, and bone metastases. Serum levels of Dkk1 were significantly increased in breast cancer patients without metastases compared with healthy controls and even more increased in patients with bone metastases. Tissue expression of Dkk1 was positive in 70% of tested primary breast cancer tissues and demonstrated significant correlation with histological type and PR status. Less frequent expression of Dkk1 was found in lymph nodes metastases and bone metastases compared with adjacent non-tumoral breast tissues and primary breast tumours. Tissue expression of ß-catenin was positive in the vast majority of all tested tissue types indicating activated Wnt/ß-catenin signalling. Our results suggested that Wnt/ß-catenin signalling in breast tumours and their secondary lymph nodes- and bone metastases is dysregulated and this could be related to aberrant Dkk1 expression levels. Hence, Dkk1 protein might provide insights into the continued development of novel comprehensive and therapeutic strategies for breast cancer and its bone metastases.

BPI-9016M, a c-Met inhibitor, suppresses tumor cell growth, migration and invasion of lung adenocarcinoma via miR203-DKK1.

Activation of c-Met plays a critical role in tumorigenesis, migration and invasion in lung cancer. Here, we explored the therapeutic efficacy of a novel small-molecule c-Met inhibitor (BPI-9016M) in lung adenocarcinoma and investigated the underlying molecular mechanisms. Method: BPI-9016M, a c-Met tyrosine kinase receptor inhibitor, was used to treat patient-derived xenografts (PDX) from lung adenocarcinoma in NOD/SCID mice. Immunohistochemistry and Western blot analysis were used to determine the expression of c-Met and its downstream signaling molecules. CCK8, wound healing, and trans-well assays were used to analyze cell proliferation, spreading, migration and invasion. RNA sequencing and quantitative real-time PCR (qPCR) was used to screen and validate the expression of downstream genes in lung adenocarcinoma cells treated with BPI-9016M. Luciferase reporter assay was used to detect the interaction between miRNA and the targeted gene. Results: BPI-9016M significantly suppressed growth in three out of four lung adenocarcinoma PDX models, particularly in the tumors with high expression of c-Met. In lung adenocarcinoma cell lines, BPI-9016M treatment resulted in increased miR203, which reduced migration and invasion and also repressed Dickkopf-related protein 1 (DKK1) expression. Forced overexpression of DKK1 or down-regulation of miR203 reversed the inhibitory effect of BPI-9016M on migration and invasion. C-Met was verified to positively and negatively associate with DKK1 and miR203, respectively. High expression of c-Met/DKK1 or low expression of miR203 related to poor outcome of lung adenocarcinoma patients. Furthermore, we observed significantly enhanced tumor cell growth inhibition upon combining BPI-9016M treatment with miR203 mimics or DKK1 siRNA. Conclusion: Our data indicated that BPI-9016M is an effective agent against lung adenocarcinoma, particularly in tumors with c-Met activation, and likely functions through upregulation of miR203 leading to reduced DKK1 expression.

Combined detection of serum Dickkopf-1 and its autoantibodies to diagnose esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) can be treated effectively if diagnosed at an early stage. We evaluated whether measurement of Dickkopf-1 (DKK-1) in combination of DKK-1 autoantibodies in serum may benefit early diagnosis of ESCC. Serum DKK-1 and DKK-1 autoantibodies were measured by enzyme-linked immunosorbent assay in a training cohort (185 ESCC samples vs. 97 normal controls) and validated in a validation cohort (104 ESCC samples vs. 53 normal controls). Receiver operating characteristic (ROC) was applied to calculate diagnostic accuracy. Testing of DKK-1 and DKK-1 autoantibodies together could differentiate ESCC from normal controls (area under the ROC curve [AUC] 0.769, 95% confidence interval (CI), 0.715-0.823, 50.3% sensitivity, and 90.7% specificity in the training cohort; AUC 0.752, 95% CI, 0.675-0.829, 50.0% sensitivity, and 84.9% specificity in the validation cohort). Importantly, the diagnostic performance of the combination of DKK-1 and DKK-1 autoantibodies persisted in early ESCC patients (AUC 0.780, 95% CI, 0.699-0.862, 50.0% sensitivity, and 90.7% specificity in the training cohort; AUC 0.745, 95% CI, 0.626-0.865, 53.8% sensitivity, and 84.9% specificity in the validation cohort). Furthermore, the levels of serum DKK-1 or DKK-1 autoantibody after surgical resection were lower, respectively, compared with the corresponding preoperative samples (P < 0.05). Our results suggest that measurement of DKK-1 combined with DKK-1 autoantibodies is a potentially valuable tool for the early detection of ESCC.

[Analysis of serum levels of Dickkopf-1 (DKK-1) in monoclonal gammopathy of undetermined significance and multiple myeloma]. / Analýza sérových hladin Dickkopf-1 (DKK-1) u monoklonální gamapatie nejistého významu a mnohocetného myelomu.

BACKGROUND: Several recent studies aim at the detection of biological parameters that enable more precise diagnostics and stratification of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). The objective of our study was to assess the potential contribution of serum levels of Dickkopf-1 (DKK-1) in MGUS and MM from the point of more specific differentiation of both conditions, and the relationship of DKK-1 to selected laboratory parameters, individual forms and clinical stages of both conditions. METHODS AND RESULTS: The analyzed cohort consisted of 46 individuals with MGUS and 152 patients with MM at the time of diagnosis. For the assessment of serum levels of DKK-1 we used ELISA method. We assessed also serum levels of free light chains (FLC) κ and λ using the Freelite system, and ß2-microglobulin (ß2-M) using the Immulite 1000 method. For statistical estimation we used: Pearson χ2-test, U-test according to Mann-Whitney and Kruskal-Wallis test. Our analysis revealed that there was no significant difference between the levels of DKK-1 in MGUS risk groups (0-3) and between the states with different FLC concentration including the κ/λ index of monoclonality. In MM there was a significant relationship of DKK-1 to the level of hemoglobin (p<0.008) but not to the levels of FLC, creatinine or ß2-microglobulin. Within the Durie-Salmon staging system, there were significant differences of DKK-1 between the stages I vs. III (p=0.001) and I vs. II+III (p=0.002). In the International Staging System (ISS) there were significant differences only between stages 1 vs. 2+3 (p=0.045). Although there was no overall significant difference of DKK-1 levels between MGUS and MM, there was a difference between MGUS vs stage III (p=0.001) and II+III (p=0.001) according to Durie-Salmon, and also MGUS vs. stage 2 (p=0.005) and vs. stages 2+3 (p=0,012) according to ISS. There were no significant differences in DKK-1 between MGUS and initial/asymptomatic form of MM (stage I). CONCLUSION: Although there was a significant difference of serum levels of DKK-1 between MGUS and initial/asymptomatic stage of MM when compared to advanced stage MM, and in patients with different Hb levels, we do not find the evaluation of serum levels of DKK-1 useful for routine discrimination of MGUS and MM, and for the specification of temporary stratification systems.