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AIM:To investigate the effect of Twist transcription factor on angiogenesis of nasopharyngeal cancer cells by regulating ERK signaling pathway in vitro.METHODS:Twist siRNA was transfected into the CNE2 cells with Lipofectamine 2000, and the CNE2 cell-conditioned medium (CM) was prepared, which was then used to treat human umbilical vein endothelial cells (HUVECs).The CCK-8 assay, Boyden chamber and capillary tube formation assay were used to evaluate the angiogenic activity of HUVECs.The mRNA expression of Twist in nasopharyngeal cancer cells was detected by real-time PCR.The protein levels of Twist, ERK, p-ERK, p38, p-p38, JNK, p-JNK and VEGF were detected by Western blot.The ERK, p38 and JNK signaling pathway inhibitors were employed to treat HUVECs, and their effect on the capillary tube formation of HUVECs was analyzed.RESULTS:The expression of Twist at mRNA and protein levels in the CNE2 cells was significantly inhibited after transfection with Twist siRNA (P<0.01).The abilities of proliferation, invasion and capillary tube formation of HUVECs were markedly suppressed after knockdown of Twist expression (P<0.01).The protein level of p-ERK was significantly elevated in the CNE2-siTwist cells, while VEGF expression was decreased.Knockdown of Twist did no change the levels of p-p38 and p-JNK.The ERK signaling pathway inhibitor partly abrogated the anti-angiogenic effect of Twist knockdown.CONCLUSION:Knockdown of Twist inhibits the angiogenesis of nasopharyngeal cancer cells in vitro partly through activation of ERK signaling pathway.
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Objective@#To investigate the significance of Twist2 in glioma and whether it is involved in the malignant transformation of glioma by epithelial-mesenchymal transition (EMT).@*Methods@#Using immunohistochemical method detected the expression level of Twist2 in 60 cases of gliomas (including WHO grades Ⅱ, Ⅲ and Ⅳ, each for 20 cases) and 20 cases of non-tumor brain tissues. Real-time fluorescence quantitative PCR and Western blot were used to detect the expression level of Twist2 mRNA and protein in 61 cases of fresh glioma tissue (WHO grade Ⅱ 16 cases, Ⅲ 21 cases, Ⅳ 24 cases) and 12 cases of adjacent tissues, and the expression levels of E-cadherin, N-cadherin and vimentin were also investigated in fresh glioma tissue.@*Results@#Immunohistochemistry results showed that the percentages of Twist2 expression in glioma was 90%(54/60) compared with 30%(6/20) in non-tumor brain tissues(P<0.01). The percentages of Twist2 expression were 75% (15/20), 95% (19/20), and 100% (20/20) in the WHO gradesⅡ, Ⅲ and Ⅳ gliomas, respectively. WHO grades Ⅳ and Ⅲ were significantly higher than that of WHO grade Ⅱ (P<0.01). There was no significant difference between WHO grade Ⅳand WHO Ⅲ glioma (P>0.05). Real-time fluorescence quantitative PCR and Western blot showed that the expression level of Twist 2 in gliomas was significantly higher than that in para-cancerous tissues (P<0.01), and those in WHO grades Ⅳ and Ⅲ gliomas were significantly higher than that in WHO grade Ⅱ glioma (P<0.01). There was no significant difference between WHO grade Ⅳand grade Ⅲ glioma (P>0.05). Detection of key protein expression in EMT by Western blot displayed that the expression of E-cadherin was negatively associated with Twist2 in glioma (r=-0.972, P<0.01). The expression of N-cadherin and vimentin was positively associated with Twist2 in glioma(r=0.971, P<0.01; r=0.968, P<0.01).@*Conclusions@#The expression of Twist2 in human glioma is positively correlated with the malignant grade of glioma, which may be involved in the malignant progression of glioma by EMT.
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BACKGROUND: Epithelial-mesenchymal transition (EMT) is associated with tumor hypoxia. EMT is regulated, in part, by the action of TWIST, which inhibits of E-cadherin expression and may interfere with the p53 tumor-suppressor pathway. METHODS: We examined the expression of TWIST, E-cadherin, hypoxia-inducible factor 1alpha (HIF1alpha), and p53 by immunohistochemistry in 123 cases of ovarian epithelial cancers (OEC) to evaluate the role of TWIST in OEC. We assessed the association between protein expression and clinicopathologic parameters. RESULTS: The expression of TWIST, E-cadherin, HIF1alpha, and p53 proteins was found in 28.5%, 51.2%, 35.0%, and 29.3% of cases, respectively. TWIST expression was associated with higher histologic grade and unfavorable survival. TWIST expression was correlated with HIF1alpha expression and reduced E-cadherin expression. The altered HIF1alpha/TWIST/E-cadherin pathway was associated with lower overall survival (OS), while the co-expression of TWIST and p53 was correlated with lower progression-free survival. In the multivariate analyses, TWIST expression was an independent prognostic factor for OS. CONCLUSIONS: Our data imply that TWIST expression could be a useful predictor of unfavorable prognosis for OEC. TWIST may affect the p53 tumor-suppressor pathway. Moreover, hypoxia-mediated EMT, which involves the HIF1alpha/TWIST/E-cadherin pathway may play an important role in the progression of OEC.
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Hypoxia , Cadherins , Disease-Free Survival , Epithelial-Mesenchymal Transition , Immunohistochemistry , Multivariate Analysis , Prognosis , Tumor Suppressor Protein p53 , Twist-Related Protein 1ABSTRACT
Objective To investigate the relationship of Twist induced epithelial mesenchymal transitions (EMT) during pancreatic carcinoma progression.Methods The expressions of Twist,N-cadherin and Vimentin proteins were tested by immunohistochemistry in 42 cases of pancreatic carcinomas and 26 cases of adjacent paracancerous tissues.Results The positive rates of Twist,N-cadherin and Vimentin in 42 cases of pancreatic carcinomas were 76.2％,59.5％ and 57.1％,respectively.The positive rate of Twist,N-cadherin and Vimentin were all significant between pancreatic carcinomas and paired tumor-adjacent paracancerous tissues (all P ＜0.001).The differences of the expression of Twist,N-cadherin and Vimentin in pancreatic carcinoma of tumor differentiation,TNM stage,neural invasion and lymph node metastasis were significant (all P ＜ 0.05).Significantly positive correlation was found between the expression of Twist and the others (N-cadherin and Vimentin) by using spearman correlation analysis (r =0.506,P =0.001 ; r =0.417,P =0.006).Significantly positive correlation was found between the expression of N-cadherin and Vimentin by using spearman correlation analysis(r =0.462,P =0.002).Conclusions Twist signaling pathway activation might mediate pancreatic epithelial cells to EMT and then promote pancreatic carcinoma invasion and metastasis.
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Objective To investigate the relationship between the expressions of Twist and clinical pathological parameters in gastric carcinoma,to explore its significance in judging the prognosis of patients,and to analyze the expression of Twist proteins in the epithelial-mesenchymal transition (EMT) phenomenon.Methods Immunohistochemistry was used to detect the expressions of Twist and EMT relative proteins E-cad,N-cad in 120 cases of gastric carcinoma specimens and corresponding adjacent normal tissue.Analysis The relationship between Twist expression and clinical pathological parameters,and the Twist expression and E-cad,N-cad were analyzed.Results In gastric carcinoma,Twist,E-cad,N-cad positive rates were 59.1％(71/120),36.6 ％ (44/120) and 47.5 ％ (57/120),and in adjacent tissues,the positive rate were 17.5 ％ (21/120),93.3 ％ (112/120) and 11.6 ％ (14/120).There were significant differences in each index between tumor tissues and adjacent tissues (P ＜ 0.05).Patients with Twist positive expression showed higher incidence rates of lymph node metastasis (P ＜ 0.05) and distant metastasis (P ＜ 0.05) than those with Twist negative expression.The expression of Twist was significantly correlated in gastric carcinoma accompanied by low expression of E-cad and abnormal high N-cad expression (P ＜ 0.05).Conclusions Compared with the adjacent tissue,the expression of Twist,E-cad and N-cad have significant differences,and the expression of Twist was closely related to the biological characteristics in gastric carcinoma.The expression of Twist in gastric cancer have negative correlation with the EMT relative factor E-cad,while positive correlation with the N-cad.
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Epithelial-mesenchymal transition (EMT) is a major mechanism in tumor metastasis.As the key regulatory factor,Twist gene plays an important role in EMT,and it can be induced by radiotherapy,chemotherapy and a variety of cytokines.Researches show that tumor cells can get stem cell-like properties via EMT,which can lead to chemo-radiotherapy resistance,tumor angiogenesis and distant metastasis.EMT has a great influence on the prognosis of tumor,and it is expected to become an important target for tumor treatment.
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Objective To investigate whether transforming growth factor-β1 (TGF-β1) could induce the expression of twist protein in the human kidney tubular epithelial cells (HKCs).Methods Human proximal HKCs were cultured in vitro and divided into three groups as follows:control group,TGF-β1group (10 ng/ml),and TGF-β1 + wortmannin group (10 ng/ml,10nmol/l,respectively).After cultured for 48 hours,cell immunofluorescene was used to observe the expression of E-cadherin and alpha-smooth muscle actin (α-SMA).Meanwhile,Western blotting was used to detect the protein expression of E-cadherin,α-SMA,and twist.Results Compared to control group,the protein expressions of E-cadherin were significantly decreased in T group (3.54 ± 0.17 vs 16.06 ± 0.50,P =0.001),whereas the protein expressions of α-SMA and twist were significantly increased in T group (α-SMA:14.78 ± 0.48 vs 3.75 ± 0.50,P=0.001 ;twist:14.24 ±0.14 vs 3.06 ±0.15,P =0.001).Compared to T group,a significant increase of the E-cadherin protein expression was detected in TGF-β1 + wortmannin group (15.88 ± 0.36,3.46 ±0.19,P =0.001),whereas,the protein expressions of α-SMA and twist were significantly decreased in TGF-β1 + wortmannin group (α-SMA:3.50 ±0.39 vs 15.0 ±0.24,P =0.001 ;twist:3.09 ±0.1 vs 14.04± 0.16,P =0.001).Cell imunofluorescence showed that the expressions of E-cadherin and α-SMA were consistent with the results of Western blotting.Conclusions TGF-β1 can induce the expression of twist protein in HKCs through Akt pathways.
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ObjectiveTo investigate the expression of Twist and N-cadherin in pancreatic cancer,and to study its relationship with clinicopathological parameters and patients' prognosis.Methods The expression of the Twist and N-cadherin in 62 tissue samples from patients with pancreatic ductal adencocarcinoma and 10 normal pancreatic tissue samples was determined by using immunohistochemistry MaxVision two step method,and the relationship with clinicopathological parameters and patients' prognosis was analyzed.ResultsThe positive expression rate of Twist in pancreatic cancer was higher than that of normal pancreatic tissues (96.8％ vs.30％,P ＜0.01 ),and the positive expression rate of N-cadherin was higher than that of normal pancreatic tissues (75.8％ vs.0,P ＜0.01),but there was no correlation between them (r =0.100,P =0.441 ).The expressions of Twist and N-cadherin was significantly correlated with TNM stage,lymph node metastasis,infiltration of portal vein or nerves and tumor location ( P ＜0.05 ),but not with age,gender and degree of differentiation ( P ＞ 0.05 ).The post-operative survival of patients decreased with the increasing Twist expression,but the survival was not associated with the expression of N-cadherin.TNM stage,the expression of Twist was independent predictive factors of prognosis for pancreatic cancer patients.ConclusionsTwist and N-cadherin are highly expressed in pancreatic cancer,and the expression was associated malignant behavior of pancreatic cancer.The abnormal expression of Twist may be a potential marker for prognosis evaluation of pancreatic cancer patients.
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Objective To detect the gene expression difference between gastric cancer tissue and lymph node metastasis,screen different express genes,and study its mechanism of metastasis and the relationship with biological behavior.Methods Using U133plus 2.0 gene chip technology,we detected the gene expression difference between gastric cancer tissue and epithelial cells of lymph node metastasis in five patients,and screened out the differentially expressed gene Twist-l.In vitro,the cell proliferation and apoptosis level were measured by using gene over-expression and gene knockout.Metastasis ability of carcinoma cells was detected by cell scratch assay.The expression changes of epithelial-mesenchymal transition (EMT)-associated protein (E-cadherin,Vimentin) in Twist-1 overexpression and gene knockout cells were determined by Western blot.Finally,we detected the expression of Twist-1 in gastric cancer tissue,and its correlation with TNM stage was analyzed.Results The expression of Twist-1 was higher in epithelial cells than in gastric cancer tissue (12.12±3.21 vs.2.07±0.71,P＜0.01).There was no correlation of the expression of Twist-1 with cell proliferation(absorbance of cell proliferation:negtive control 0.84±0.16,null vector control 0.74 ±0.06 and Twist-1 expression cell 0.71 ± 0.07) and apoptosis [cell apoptosis rate:negtive control (2.05±0.08)％,null vector control (4.31±0.07)％ and Twist-1 expression (3.95±0.09)％],but cell migration ability enhanced.In Twist-1 over-expression group,the level of E-cadherin was decreased,while vimentin increased.Conclusions Twist-1 gene changes might be correlated with the metastasis of gastric cancer by the way of EMT.
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ObjectiveTo explore the expression rule and clinic significance of Twist and E-cadherin and Vimentin in cervical squamous epithelial cancerization.MethodsChronic cervicitis were used as control group,the expression of Twist (using hybridization in situ),E-cadherin and Vimentin( SABC immunohistochemical stain) in tissues of cervical intraepithelial neoplasia (CIN) and cervical squamous carcinoma were detected,and the correlations were analyzed.ResultsThe positive rate of Twist in group of chronic cervicitis,CIN Ⅰ,CINⅡ,CIN Ⅲ and cervical squamous carcinoma was 10.0％,28.1％,29.6％,42.9％ and 64.3％,respectively.The expression rate of Twist in cervical squamous carcinoma group was higher than other groups ( P ＜ 0.05 ).The positive rates of E-cadherin and Vimentin were 80.0％,68.8％,55.6％,51.4％,39.3％ and 0,6.3％,7.4％,31.4％,32.1％,respectively.The difference of E-cadherin and Vimentin expression between the cervical squamous carcinoma ( or CIN Ⅲ group) and the chronic cervicitis group was obvious ( P ＜0.05,respectively).The expression of Twist was negatively correlated with E-cadherin( r =-0.37,P ＜0.01 ),and it was positively correlated with Vimentin in all cases of CIN and cervical squamous carcinoma( r =0.23,P ＜0.05).ConclusionsThere is a close relationship between abnormal expression of Twist and cervical squamous epithelial cancerization.Twist may participate in the genesis of cervical squamous carcinoma through mediating the expression of E-cadherin and Vimentin in cervix tissue.
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ObjectiveTo investigate the expression of Twist protein in adenocarcinoma of prostate and clinicopathological features and explore its roles in the carcinogenesis and progression of adenocarcinoma of prostate and its relationship with prognosis.MethodsThe expression of Twist was detected in 70 cases with adenocarcinoma of prostate and 30 cases with side cancer tissue by immunohistochemistry (EnlivionTM).ResultsThe expression of Twist protein in adenocarcinoma of prostate was higher than side cancer tissue.The expression of twist was related to Gleason grade,clinical stage and bony metastasis (P＜0.05),but it was not related to the age (P＞0.05).The survival rate of Twist protein positive patients was significant lower than negative patients(x2=9.52,P＜0.01).ConclusionsTwist might be an important biological marker for malignant transformation and invasion and metastasis adenocarcinoma of prostate,and the up-regulation of the expression of Twist reflects more invasion and poor prognosis.
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ObjectiveTo observe the expression of Twist protein, vascular endothelial growth factor (VEGF) mRNA and protein in laryngeal squamous cell carcinoma(LSCC) and their relation to clinical pathological characteristics, and aslo debate their effect and clinical application in carcinogenesis, developing of LSCC. MethodMolecular hybridization in situ was carried to detect VEGF mRNA, and immunohistochemistry S-P method was carried to detect the expression of Twist protein and VEGF protein in 80 cases of LSCC, 30 cases of paraneoplastic tissue. ResultsThe expression of Twist protein, VEGF mRNA and protein was related to TNM stage, T stage, thyroid cartilage involvement, lymph node metastasis and distance metastasis (P ＜ 0.05 or ＜ 0.01 ). The expression of Twist protein and VEGF protein was related to differentiated degree (P ＜ 0.01 or ＜ 0.05), however, the expression of VEGF mRNA was not related to differentiated degree (P ＞ 0.05 ). The expression of Twist protein was positively related to the expression of VEGF protein in LSCC (r =0.334, P ＜ 0.01 ) ; and the expression of VEGF mRNA was positive correlation to the expression of VEGF protein (r =0.484,P ＜ 0.01 ). ConclusionsOverexpression of Twist protein and VEGF mRNA and protein might play a joint action in the carcinogenesis, development of LSCC. Combined detection of Twist protein and VEGF mRNA and protein has vital significance in predicting the invasion and metastasis of LSCC, which might be an index to judge the biological behavior of LSCC.
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Objective To explore the expression and clinical significance of Twist. E-cadherin and N-cadherin in gallbladder carcinoma. Methods From 2000 to 2008, in Zhejiang Provincial People's Hospital, the expression of Twist, E-cadherin and N-cadherin protein were detected in 79 surgically removed gallbladder carcinoma tissue specimens in paraffin blocks and 20 normal gallbladder tissue specimens by tissue microarray technique and immunohistochemistry. Results Compared with normal gallbladder tissues, the expression of Twist and N-cadherin was up-regulated in gallbladder carcinoma tissues and the positive percentage was 68.3％ and 49.4％ respectively, while which both were 1/20 in normal gallbladder tissues. The percentage of high E-cadherin expression in gallbladder carcinoma tissues was only 27.8 ％, which was significantly lower than that in normal gallbladder tissues (20/20;X2 ＝29.31, P＜0. 05). The expression of Twist was correlated with T classification, lymph node metastasis, distant organ metastasis, hepatoduodenal ligament invasion, lymphatic invasion and UICC stage of gallbladder carcinoma (P＜0.05). The expression of E-cadherin was correlated with T classification, distant organ metastasis, hepatoduodenal ligament invasion, differentiation degree and UICC stage of gallbladder carcinoma (P＜0.05). The expression of N-cadherin was only correlated with lymphatic invasion of gallbladder carcinoma (P ＜0.05 ). There was significant negative correlation between Twist and E-cadherin expression (P＜0. 01). All the 79 gallbladder carcinoma patients were followed up after the surgery, the mean follow-up time was 30.6±14.3months. The 3-year survival rates of patients with low or high Twist expressions were 66％ and 7％ respectively,there was significant difference between the two groups(P＜0. 01). The 3-year survival rates of patients with low or high E-cadherin expressions were 25 ％ and 86 ％ respectively, the difference of two groups was significant(P＜0.01). The 3-year survival rates of patients with low or high Ncadherin expressions were 39％ and 41％ respectively, there was no significant difference between the two groups(P＞0.05). The multivariate analysis indicated that the Twist expression was one of independent prognostic factors of gallbladder carcinoma. Conclusion The abnormal expression of Twist and E-cadherin was correlated with the development and progression of gallbladder carcinoma,and Twist expression was one of the independent prognostic factors of gallbladder carcinoma.
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BACKGROUND: Epithelial-mesenchymal transition (EMT) is critical for morphogenesis during embryonic development and is also implicated in the conversion of early-stage tumors into invasive malignancies. Recently, Twist has been identified to play an important role in EMTmediated metastatic progression of several types of human cancer. The present study examined the expression of Twist and evaluated its clinicopathologic significance in urothelial carcinoma of upper urinary tract. METHODS: Immunohistochemical staining for Twist expression was performed on 70 upper urinary tract urothelial carcinomas (UUT-UCs) using tissue microarray. RESULTS: Immunohistochemical staining for Twist was positive in 31/70 cases (44.3%) of UUT-UCs. Twist expression was associated with high-grade and advanced-stage (ISUP grade, p<0.01; stage, p=0.045). The patients with Twist positive-tumors revealed lower disease free survival rate than those with Twist negative-tumors (p<0.01). The overall survival for patients with Twist positive-tumors was slightly worse than the patients with Twist negative- tumors, but the difference was not statistically significant (p=0.12). CONCLUSION: Our results suggest that Twist is a novel marker for advanced UUT-UC.