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AIM: To explore the improvement effect and mechanism of crocin on cognitive impairmrnt of Alzheimer's disease (AD) rats. METHODS: The hippocampus of SD rats were injected with Aβ 25-35 to establish AD model, then rats were randomly divided into AD group, AD + low, medium, high dose of crocin groups (10, 20, 40 mg/kg) and AD + donepezil group (1 mg/kg), intraperitoneal injection treatment for 4 weeks, set sham group. Dark avoidance test and water maze test were used to evaluate the learning and memory abilities of rats, ELISA was used to detect serum Aβ content, HE staining and Tunel staining were used to determine pathological changes and neuronal apoptosis of hippocampus of rats, immunohistochemistry was used to detect the expression of Brdu, Dcx and NeuN in hippocampus of rats, and Western blot was used to detect the protein expression of Aβ, DKK3, β-catenin, p-GSK-3β/GSK-3β, Caspase-3, Bax, Bcl-2 in hippocampus of rats. RESULTS: Compared to sham group, the learning and memory abilities of AD group rats were decreased, serum Aβ content increased, the pathological change in hippocampus was serious, neuronal apoptosis was increased, the expression of Brdu, Dcx, NeuN were decreased, the protein expression of Aβ, DKK3, p-GSK-3β/GSK-3β, Caspase-3, Bax were increased, protein expression of β-catenin, Bcl-2 were decreased (P<0.01). Compared to AD group, after the treatment of doses of crocin and donepezil, the learning and memory abilities of AD rats were improved, serum Aβ content were increased, and the pathological change in hippocampus were alleviated, neuronal apoptosis were reduced, the expression of Brdu, Dcx, NeuN were decreased, the protein expression of Aβ, DKK3, p-GSK-3β/ GSK-3β, Caspase-3, Bax were decreased, the protein expression of β-catenin, Bcl-2 were increased, notely, dose-dependent effect of crocin was significant. CONCLUSION: Crocin reduced neuronal apoptosis and mediated DKK3 to regulate GSK-3β/ β-catenin pathway to improve the cognitive impairment of AD rats.
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Objective:To study the clinical value of induced sputum DKK3 gene methylation in the evaluation of disease condition and prognosis for non-small cell lung cancer (NSCLC).Methods:Eighty NSCLC patients (observation group) and 50 benign lung disease patients (control group) who were treated in Linshu County People′s Hospital of Shandong Province from January 2015 to December 2017 were enrolled. Methylation specific polymerase chain reaction (PCR) was used to detect the induced sputum DKK3 gene methylation. The DKK3 gene methylation rates in different clinicopathological factors were compared.Results:The DKK3 gene methylation rate in observation group was significantly higher than that in control group: 81.3%(65/80) vs. 2.0%(1/50), the difference was statistically significant ( P<0.05). The DKK3 gene was methylated in lung cancer cells, and was unmethylated in normal lung epithelial cells BEAS-2B. The DKK3 gene methylation rates had correlation with pleural effusion, degree of differentiation, tumor diameter, lymph node metastasis, distant metastasis and TNM staging ( P<0.05). The R0 radicalresection, 3-year survival rate and total survival time in patients with DKK3 gene methylated were significantly lower than those in patient with DKK3 gene unmethylated: 53.8%(35/65) vs. 15/15, 28.1% vs. 37.9%, (1.8 ± 0.3) years vs. (2.1 ± 0.6) years, the differences were statistically significant ( P<0.05). Conclusions:The induced sputum DKK3 gene methylation rate in NSCLC patients is significantly higher and is related with prognosis. The induced sputum DKK3 gene methylation may provide basis for evaluating of disease condition and prognosis for NSCLC patients.
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To explore the effect and mechanisms of demethylation drug zebularine on esophageal cancer cells apoptosis, ECA109 cells and KYSE170 cells were treated with zebularine at different concentrations (25, 50, 100, 200, and 400 μmol·L-1). The cell viability was measured by CCK-8. Flow cytometry was used to detect the cell apoptosis rate, Western blot was performed to determine the expression of apoptosis protein (Bcl-2, Bax, cleaved-caspase-3, and cleaved-PARP) and Wnt signal pathway molecules (β-catenin, cyclin D1, and c-Myc), real-time quantitative PCR was used to detect the expression level of negative regulatory genes of Wnt signaling pathway, methylation specific PCR (MSP) was used to detect the methylation status of secreted frizzled related protein 2 (SFRP2) and dickkopf 3 (Dkk3) genes. After knockdown of SFRP2 and Dkk3, the effect of zebularine on apoptosis was detected. The studies showed that zebularine could inhibit the activity of ECA109 and KYSE170 cells in a dose-dependent and time-dependent manner; zebularine could induce cell apoptosis, down-regulate the expression of Bcl-2 protein, up-regulate the expression of Bax, cleaved-caspase-3, and cleaved-PARP protein, and inhibit the expression of β-catenin, cyclin D1, and c-Myc protein (P < 0.05); the mRNA expression levels of Dkk3 and SFRP2 were significantly up-regulated by zebularine, while the methylation levels of SFRP2 and Dkk3 promoters were decreased; knockdown of SFRP2 and Dkk3 could reduce the apoptosis induced by zebularine. In summary, zebularine could reduce the methylation level of SFRP2 and Dkk3 gene promoter, promote the expression of SFRP2 and Dkk3 gene, and then induce the apoptosis of esophageal cancer cells by inhibiting Wnt/β-catenin signaling pathway.
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Dickkopf-3 (DKK3) , as a critical inhibitor of the Wnt/p-catenin signaling pathway, may he involved in melanogenesis.In the current study, we investigated the effects of DKK3 on melanogenesis in melanocytes of alpaca.Overexpression of DKK3 in alpaca melanocytes, the expression of Wntl, Lefl , Myc and the major target genes termed microphthalmia-associated transcription factor (M1TF) and its downstream genes, including tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1) and tyrosinase- related protein 2 (TYRP2) were significantly decreased at both mRNA and protein levels (P<0.05); total alkali melanin, pheomelanin and eumelanin were decreased by 80.30%, 72.17% and 64.60% (P <0.05), respectively.In contrast, in the melanocytes transfected with siRNA-DKK3 (a small interference RNA targeting DKK3) , the expression of Wntl, Lefl, Myc, MITF, TYR, TYRPl and TYRP2 were significantly increased at both mRNA and protein levels (P<0.05) ; total alkali melanin, pheomelanin and eumelanin were significantly increased by 1.65 folds, 1.25 folds and 1.21 folds (P< 0.05) , respectively.These results indicate that DKK3 regulates melanogenesis in alpaca melanocytes via the Wnt/p-catenin signaling pathway and down-regulates MITF.
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Objective To study the correlation between status of methylation of DKK3 gene promoter and expression of its mRNA in gastric cancer.Methods Methylation of DKK3 gene promotor and its mRNA expression in 76 gastric cancer specimens and paired adjacent non-cancerous tissues were detected by methylation-specific PCR(MSP) and RT-PCR respectively.Results The expression of DKK3 mRNA in gastric cancer tissues was lower than that of adjacent non-cancerous tissues (43％ vs.60％,x2 =5.663,P =0.017).29 cases had methylation of DKK3 gene promoter for adjacent non-cancerous tissues.In gastric cancer tissues,however,44 cases had methylation of DKK3 gene promoter (38％ vs.58％,x2 =19.405,P =0.000).There were significant difference in depth of invasion (P =0.014),TNM stage (P =0.032),pathological grading (P =0.016) and lymphatic metastasis (P =0.007).There was distinct negative correlation between methylation of DKK3 gene promoter and expression of its mRNA (x2 =64.286,P =0.000).Conclusions Abnormal methylation of DKK3 gene promoter is an important mechanism of its inactivation,and might play an important role in carcinogenesis and progression of gastric cancer.
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Objective To study the expression of DKK3 protein and mRNA levels in gastric cancer and its correlation with the clinical prognosis of gastric cancer.Methods The expression of DKK3 protein in 158 gastric cancer specimens and paired adjacent non-cancerous tissues were detected by immunohistochemistry.The relationship between the protein expression and clinicopathological features was analyzed.Immunoblotting and real time quantitative PCR were used to examine the expression of DKK3 protein and mRNA levels in 76 paired fresh gastric cancer and adjacent non-cancerous tissues.Results The expression of DKK3 in gastric cancer tissues was lower than that in adjacent non-cancerous tissues (40.5％ vs.55.1％,x2 =41.442,P =0.000).DKK3 protein and mRNA levels in 76 cases were lower than that in adjacent non-cancerous tissues (1.096 ± 1.110 vs.3.476 ± 1.119,t =3.332,P =0.017;1.136 ± 1.013 vs.3.314 ± 1.105,t =2.673,P =0.022).There were close correlations between DKK3 expression and depth of tumor invasion (P =0.006),TNM stage (P =0.005) and lymphatic metastasis (P =0.009).The expression of DKK3 in gastric cancer was positively related with overall survival rate (47.4％ vs.28.0％,P =0.011),as an independent prognosis predictor (P =0.016).Conclusions DKK3 is an independent prognostic factor of gastric cancer,its decreased expression is significantly related with shortened survival time of gastric cancer patients.
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BACKGROUND: miR-214 was demonstrated to be upregulated in models of renal disease and promoted fibrosis in renal injury independent of TGF-ß signaling in vivo. However, the detailed role of miR-214 in acute kidney injury (AKI) and its underlying mechanism are still largely unknown. METHODS: In this study, an I/R-induced rat AKI model and a hypoxia-induced NRK-52E cell model were used to study AKI. The concentrations of kidney injury markers serum creatinine, blood urea nitrogen, and kidney injury molecule-1 were measured. The expressions of miR-214, tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, were detected by RT-qPCR. The protein levels of Bcl-2, Bax, Dickkopf-related protein 3, ß-catenin, c-myc, and cyclinD1 were determined by western blot. Cell apoptosis and caspase 3 activity were evaluated by flow cytometry analysis and caspase 3 activity assay, respectively. Luciferase reporter assay was used to confirm the interaction between miR-214 and Dkk3. RESULTS: miR-214 expression was induced in ischemia-reperfusion (I/R)-induced AKI rat and hypoxic incubation of NRK-52E cells. Overexpression of miR-214 alleviated hypoxia-induced NRK-52E cell apoptosis while inhibition of miR-214 expression exerted the opposite effect. Dkk3 was identified as a target of miR-214. Anti-miR-214 abolished the inhibitory effects of DKK3 knockdown on hypoxia-induced NRK-52E cell apoptosis by inactivation of Wnt/ß-catenin signaling. Moreover, miR-214 ameliorated AKI in vivo by inhibiting apoptosis and fibrosis through targeting Dkk3 and activating Wnt/ß -catenin pathway. CONCLUSION: miR-214 ameliorates AKI by inhibiting apoptosis through targeting Dkk3 and activating Wnt/ß -catenin signaling pathway, offering the possibility of miR-214 in the therapy of ischemic AKI.
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Animals , Male , Rats , Intercellular Signaling Peptides and Proteins/metabolism , MicroRNAs/metabolism , Catenins/metabolism , Acute Kidney Injury/metabolism , Wnt Signaling Pathway/genetics , Rats, Sprague-Dawley , Chemokines , Intercellular Signaling Peptides and Proteins/genetics , MicroRNAs/genetics , Adaptor Proteins, Signal Transducing , Cell Proliferation , Disease Models, Animal , Catenins/genetics , Acute Kidney Injury/chemically inducedABSTRACT
Purpose To investigate the expression of Dkk3 and Cyclin D1 protein in human hepatocellular carcinoma (HCC) and clinical significance.Methods Immunohistochemistry was used to detect Dkk-3 and Cyclin D1 protein expression level in 80 cases of hepatocellular carcinoma and corresponding para-cancer tissue.Results The expression of Dkk-3 in hepatocellular carcinoma was significantly lower than those in corresponding para-cancer tissue (P ＜ 0.05) and the expression of Cyclin D1 in hepatocellular carcinoma was significantly higher than those in CoTesponding para-cancer tissue (P ＜ 0.05).The up-regulation of Cyclin D1 and the down-regulation of Dkk-3 proteins were correlated with pathologic differentiation degree (P ＜0.05).There was a significant inverse correlation between Dkk3 and Cyclin D1 expression (P =0.044,rs =-0.226).Conclusion The abnormal expression of Dkk-3 and Cyclin D1 gene in human hepatocellular carcinoma suggest that Dkk-3 and Cyclin D1 gene may play an important role in the development and progression of the cancer.The combination deteetion of the two biomarkers may provide valuable data for diagnosis and prognosis estimation of HCC.
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Objective To investigate the expression of Dickkopf-3 (DKK3) in human malignant melanoma cell lines and tissues,and to evaluate effects of DKK3 on the proliferation and apoptosis of malignant melanoma cell line A375.Methods Reverse transcription PCR (RT-PCR) and real-time fluorescence-based quantitative PCR (qRT-PCR) were performed to measure the mRNA expression of DKK3 in human malignant melanoma cell lines HM,A375,WM451,WM35,SK-MEL-1,Hs-695T and MDA-MB-435s,as well as in 38 primary melanoma tissues,4 metastatic melanoma tissues and 20 pigmented nevus tissues.Cultured malignant melanoma A375 cells were divided into 2 groups to be transfected with pcDNA3.1 (+)-Flag-DKK3 (experiment group) and pcDNA3.1 (+)-Flag-Vector (control group) respectively.The overexpression of DKK3 was verified by RT-PCR.Cell counting kit-8 (CCK8) assay and plate colony formation assay were performed to evaluate the proliferative activity of A375 cells,flow cytometry was conducted to detect apoptosis of A375 cells,and Western blot analysis was performed to determine the expression of cell cycle-and cell apoptosis-related proteins.Results The mRNA expression of DKK3 was downregulated in WM35 cells,absent in HM cells,A375 cells,WM451 cells,SK-MEL-1 cells and Hs-695T cells,but upregulated in MDA-MB-435s cells.Compared with pigmented nevus tissues,the mRNA expression of DKK3 was significantly decreased in malignant melanoma tissues (P ＜ 0.001).Compared with the control group (100％),cell colony formation was markedly suppressed in the experiment group (23.22％ ± 3.55％),and the proliferative activity of A375 cells was also significantly inhibited in the experiment group 24,48,72 hours after the transfection (all P ＜ 0.05).Flow cytometry showed that compared with the control group,A375 cells were significantly arrested in G1 phase (48.68％ ± 3.92％ vs.25.38％ + 2.92％,P ＜ 0.001),and the apoptosis rate of A375 cells was significantly increased in the experiment group (P ＜ 0.001).Compared with the control group,the experiment group showed significantly higher expression of p21,Bax,cleaved-parp and cleaved-casp3,but significantly lower expression of cyclin D1 and Bcl2 (all P ＜ 0.001).Conclusion DKK3 expression is downregulated in human malignant melanoma tissues,so it may serve as a potential tumor suppressor gene involved in the development of cutaneous malignant melanoma.
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Objective: To investigate the expressions of serum DKK (Dickkopf)-1 and DKK-3 of Dickkopf family in patients with PC (prostate cancer) and the patients with BPH (benign prostatic hyperplasia). To explore the correlation of their expressions with the Gleason score and bone metastasis. Methods: Serum DKK-1 and DKK-3 levels in 40 patients with prostate cancer and 20 patients with BPH were detected by ELISA (enzyme-linked immunosorbent assay) and compared by using Spearman's correlation analysis to evaluate the intercorrelation of DKK-1 and DKK-3 expressions and their relationship with clinicopathological characteristics. The diagnostic performance of PSA (prostate specific antigen), DKK-1 and DKK-3 were determined by binary logistic regression method to draw the ROC (receiver operating characteristic) curves. Results: Serum DKK-1 and DKK-3 levels in patients with PC were significantly higher than those in patients with BPH. The serum DKK-1 level in PC with bone metastasis was higher than that in non-bone metastatic prostate cancer (P = 0.043). The predictive value of DKK-1 in diagnosing bone metastasis was slightly higher than PSA (P = 0.045). Serum DKK-3 level in high-risk PC with Gleason scores 8-9 was higher than that in moderate-risk PC with Gleason scores 6-7 (P = 0.036). The predictive value of DKK-3 in distinguishing the grade of cancer was higher than PSA (P = 0.039). There were no ralativeships between DKK-1 and bone metastasis, DKK-3 and Gleason grade, and DKK-1 and DKK-3. Conclusion: Serum DKK-1 and DKK-3 expressions are closely related to the occurrence of PC. DKK-1 may contribute to diagnose PC with bone metastasis. DKK-3 may support to distinguish the grade of PC. Copyright © 2013 by TUMOR.
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OBJECTIVES: The inhibitory effect of Dickkopf (Dkk)-1 on osteoblastic differentiation through blocking Wnt signaling has been well studied. However, the role of other members of the subfamily of Dkks remains unclear. We have examined the role of different Dkks on osteoblastic differentiation of mesenchymal progenitor cells and apoptosis of osteoblasts. METHODS: Osteoblastic differentiation was induced by treatment of Wnt-3a with Dkks or vehicle in C3H10T1/2 cells and alkaline phosphatase (ALP) activity was measured. Serum deprivation induced apoptosis was performed with pre-treatment of Dkks or vehicle in MC3T3-E1 cells and methyl thiazolyl tetrazolium (MTT) assay was done. RESULTS: Dkk-2 at low concentrations (5 and 20 nM) and Dkk-3, -4 at any concentrations (5 to 100 nM) significantly increased Wnt-3a-induced ALP activity, whereas Dkk-2 at high concentration (100 nM) significantly reduced. Treatment of Dkk-2, -3 and -4 at high concentration (100 nM) showed significant decreases of Wnt/beta-catenin transcriptional activity, whereas no effects were seen at low concentration (20 nM). In parallel experiments, treatment of Dkk-1 showed robust dose dependent inhibition not only in ALP activity but also in Wnt/beta-catenin transcriptional activity. Dkk-2, -3 and -4 increased serum deprivation-induced apoptosis in MC3T3-E1 mouse osteoblasts, while Dkk-1 had no effect. CONCLUSIONS: We found that unlike Dkk-1, Dkk-3 and -4 stimulated early osteoblastic differentiation at various concentrations regardless of their inhibitory effects on Wnt/beta-catenin transcriptional activity at high concentration. Dkk-2 had a biphasic effect where the lower doses significantly increased ALP activity while the high dose was inhibitory. Dkk-2, -3 and -4 stimulated osteoblast apoptosis whereas Dkk-1 had no effect.
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Animals , Mice , Alkaline Phosphatase , Apoptosis , Mesenchymal Stem Cells , OsteoblastsABSTRACT
Background and purpose:The expression of dickkopf homolog 3 gene(DKK3)is always reduced or absent in tumors,instead part of the tumor vascular endothelial expressed DKK3.vWF is a macromolecular glycoprote synthesized and released by vascular endothelial cells and megakaryocytes.However,vWF was also expressed by tumor.The relationship between these 2 factors and the occurrence of cancer is still unclear.The purpose of this study was to observe the expression of DKK3 and vWF proteins in colorectal carcinoma and determine their clinical significance through finding their association with MVD and correlation with each other.Methods:Immunohistochemistry staining was used to detect the expression of DKK3,vWF proteins and MVD in the colorectal carcinoma tissue microarrays that contained 94 colorectal carcinoma specimens.Results:The expression of DKK3 in colorectal carcinoma was lower than or nonexistent compared to that in normal tissues(P＜0.05).The expression of vWF in colorectal carcinoma was higher than that in normal tissues(P＜0.05).Expression of DKK3 and vWF in colorectal carcinoma were not correlated to the age or gender of the patients,invasive depth,or tumor locus of the colorectal carcinoma (P＞0.05).Correlations with the expression of DKK3 and vWF in colorectal carcinoma were only found with differentiation and iynphnode metastasis (P＜0.05).However,the expression of DKK3 and vWF in colorectal carcinoma was not correlated to MVD(P＞0.05).The expression of DKK3 was not correlated to the expression of vWF in coiorectal carcinoma(r=0.1310,P=0.2090).Conclusion:A lowered expression of DKK3 and higher expression of vWF may be associated with the carcinogenesis,various biological behaviors and metastasis of colorecml carcinoma.These 2 factors can be used as important biological markers for colorectal cancer.