Cisplatin increases self-renewal ability of oral squamous cell carcinoma stem cells through Notch4 signaling pathway / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To study the effect of Notch4 signaling pathway on the self-renewal capacity of cancer stem cells (CSCs) of oral squamous cell carcinoma PJ15 and PJ41 cells and its mechanism. Methods: The expression of Notch4 gene in head and neck tumors was analyzed using TCGA database. 2 μmol/L cisplatin was used to treat oral squamous cell carcinoma PJ15 and PJ41 cells for 48 h, following with the treatment of Notch pathway inhibitor DAPT for 24 h. Flow cytometry was used to detect the ratio of CSCs, Western blotting and qPCR were used to detect the expressions of CSCs markers (Sox2, Bmi-1, Oct4, Nanog) and downstream genes in Notch4 signaling pathway (JAG1, HEY1, HEY2, HES1, HES2, DLL4, etc.). Notch4 was knocked down by siRNA technology. Notch4 was silenced with siRNA technology. Western blotting and qPCR were used to detect the effect of si-Notch4 and cisplatin on the expression level of CSCs markers. The stem cell pelletingtestwasusedtodetecttheself-renewalabilityof CSCs. Results: Notch4 gene was highly expressed in head and neck tumor tissues (P=0.046). After cisplatin treatment, compared with the control group, expressions of NICD4 and CSCs markers (Sox2, Bmi-1, Oct4, Nanog) as well as downstream genes of Notch4 signaling pathway (JAG1, HEY1, HEY2, HES1,HES2, DLL4, etc.) increased significantly (all P<0.05), and the proportion of ALDH1 positive cells increased significantly (P<0.05); with the further addition of DAPT, the expressions of the above genes decreased significantly (P<0.05 or P<0.01). After knocking down Notch4, compared with the control group, the size and number of spheres of PJ15 cells [1.33±0.47] vs [8.00±0.82], P<0.01) and PJ41 cells ([1.00±0.82] vs [7.67±1.25], P<0.01) were significantly lower than that of the control group; after the addition of 2 μmol/L DDP for 24 h, there was no significant difference in the expressions of Nanog and Bmi-1 gene between si-Notch4 group and control group. Conclusion:Activation of Notch4 signaling pathway can enhance the self-renewal ability of CSCs in oral squamous cell carcinoma PJ15 and PJ41 cells.

Notch4 affects migration and invasion of pancreatic cancer cells by regulating activated transcription factor 2 and its possible mechanism / 中国肿瘤生物治疗杂志

@#Objective: To investigate the effect of Notch4 regulatingATF2 (activating transcription factor 2) on the invasion and migration of pancreatic cancer (PC) cells and its possible mechanism. Methods:Atotal of 60 pairs of PC tissues and corresponding para-cancerous tissues that surgically resected at Taizhou University Hospital during February 2015 and July 2019 were collected for this study. The expression of Notch4 was detected by immunohistochemistry. siRNA technology was used to knock down Notch4 gene expression in PC cell lines (MiaPaCa-2 and PANC-1). Transwell assay was used to analyze the effect of Notch4 knockdown on cell invasion and migration. qPCR and Western blotting (WB) were used to detect the effects of Notch4 knockdown on mRNA and protein expressions of Notch4 and ATF2. Results: Compared with para-cancerous tissues, the expression of Notch4 in PC tissues significantly higher (P<0.01). After Notch4 siRNA transfection, the mRNA and protein expressions of Notch4 and ATF2 in MiaPaCa-2 and PANC-1 cells significantly decreased (all P<0.01). Compared with Control siRNA group, the migration and invasion ability of PC cells in Notch4 siRNA groupsignificantlyreduced(allP<0.01).Conclusion:Notch4ishighlyexpressedinPCtissues.Knockdown of Notch4 can down-regulate the expression ofATF2 at the transcriptional level, thereby inhibiting the invasion and migration ability of PC cells.

Downregulation of Notch4 - a prognostic marker in distinguishing oral verrucous carcinoma from oral squamous cell carcinoma / Regulação descendente de Notch4 - um marcador prognóstico na distinção entre carcinoma verrucoso oral e carcinoma de células escamosas oral

Abstract Introduction: Oral verrucous carcinoma is a special form of well-differentiated squamous cell carcinoma which possesses specific clinical, morphologic and cytokinetic features that differ from other types of oral cancers and hence diagnosis requires immense experience in histopathology. Hence it is certainly important to distinguish such a lesion from other oral tumors as treatment strategies vary widely between them. Objective: In search of a critical diagnostic marker in distinguishing oral verrucous carcinoma from oral squamous cell carcinoma, Notch4 receptor, one of the key regulatory molecules of the Notch signaling family has been aberrantly activated in the progression of several types of tumors. However its function in oral verrucous carcinoma remains unexplored. Thus the present study aims in determining the differential expression pattern of Notch4 in oral verrucous carcinoma and oral squamous cell carcinoma. Methods: Ten patients reported positive for oral cancer (5 patients with oral verrucous carcinoma and 5 patients with oral squamous cell carcinoma). Five normal tissue samples were also obtained and evaluated for clinicopathological parameters and immunohistochemistry, western blotting and real time polymerase chain reaction for Notch4 expression. Results: Our results reveal that the expression of Notch4 was considerably high in oral squamous cell carcinoma lesions compared to normal tissue, whereas in oral verrucous carcinoma, irrespective of the clinicopathological features, complete regulação descendente of Notch4 was observed. Conclusions: These preliminary findings strongly support the fact that Notch4 is downregulated in oral verrucous carcinoma and could be considered as a suitable prognostic marker in distinguishing oral verrucous carcinoma from oral squamous cell carcinoma. This distinguishing marker can help in improving therapeutic options in patients diagnosed with oral verrucous carcinoma.

Resumo Introdução: O carcinoma verrucoso de cavidade oral é uma forma especial de carcinoma de células escamosas bem diferenciada que tem características clínicas, morfológicas e citocinéticas específicas que diferem de outros tipos de cânceres orais. Por essa razão, o diagnóstico requer grande experiência em histopatologia. Portanto, é certamente importante distingui-lo de outros tumores orais, pois as respectivas estratégias de tratamento variam muito. Objetivo: Em busca de um marcador de diagnóstico crítico na distinção entre o carcinoma verrucoso e o carcinoma de células escamosas de cavidade oral, o receptor Notch4, uma das principais moléculas reguladoras da família de sinalizadores Notch, foi ativado de maneira anormal na progressão de vários tipos de tumores. No entanto, sua função no carcinoma verrucoso permanece inexplorada. Assim, o presente estudo tem como objetivo determinar o padrão de expressão diferencial de Notch4 no carcinoma verrucoso e de células escamosas de cavidade oral. Método: Dez pacientes tiveram resultado positivo para câncer oral (cinco pacientes com carcinoma verrucoso e cinco pacientes com carcinoma de células escamosas) e cinco amostras normais foram também obtidas. Além da avaliação dos parâmetros clínico-patológicos, foram feitos análise imuno-histoquímica, Western Blot e reação de polimerase em cadeia em tempo real para a expressão de Notch4. Resultados: Nossos resultados revelam que a expressão de Notch4 foi consideravelmente alta em carcinomas de células escamosas em comparação com os tecidos normais, enquanto que no carcinoma verrucoso, independentemente das características clínico-patológicas, observou-se regulação descendente completa de Notch4. Conclusão: Esses achados preliminares apoiam fortemente o fato de que Notch4 estava regulado para baixo no carcinoma verrucoso oral e poderia ser considerado um marcador prognóstico adequado para distinguir entre carcinoma verrucoso e carcinoma de células escamosas de cavidade oral. Esse marcador distintivo pode ajudar a melhorar as opções terapêuticas em pacientes com diagnóstico de carcinoma verrucoso oral.

Effects of Buyang Huanwu Decoction on Endothelial-mesenchymal Transformation of Lung Tissue in Idiopathic Pulmonary Fibrosis Model Rats and Mechanism Study / 中国药房

OBJECTIVE： To investigate the effects of Buyang huanwu decoction on endothelial-mesenchymal transformation （EndMT） of lung tissue in idiopathic pulmonary fibrosis （IPF） model rats， and to explore its potential mechanism. METHODS： Male SD rats were randomly divided into normal group， model gorup， dexamethasone group [0.405 mg/（kg·d）]， Buyang huanwu decoction low-dose， medium-dose and high-dose groups [6.435， 12.87， 25.74 g/（kg·d）， by raw material]， with 8 rats in each group. Except for normal group， other groups were given endotracheal injection of bleomycin to induce IPF model. On the second day after modeling， normal group and model group were given water intrgastrically [10 mL/（kg·d）]； administration groups were given relevant medicine intragastrically， once a day， for consecutive 28 days. 24 h after last medication， the expression of endothelial cell markers [platelet endothelial cell adhesion molecule 1， vascular endothelial cell cadherin] and interstitial cell markers [α-smooth muscle actin， fibroblast specific protein 1] were detected by immunohistochemistry method. The expression of Notch4 and DLL4 in lung tissue of rats were detected by Western blotting assay. RESULTS： Compared with normal group， the expression of endothelial cell markers were decreased significantly in lung tissue of model group， while the expressipon of interstitial cell markers， Notch4 and DLL4 were increased significantly （P＜0.01）. Compared with model group， the expression of endothelial cell markers in lung tissue of rats were increased significantly in administration groups， while Buyang huanwu decoction low-dose group was significantly lower than dexamethasone group； the expression of interstitial cell markers， Notch4 and DLL4 were decreased significantly， while Buyang huanwu decoction low-dose group was significantly higher than dexamethasone group （P＜0.05 or P＜0.01）. CONCLUSIONS： Buyang huanwu decoction can relieve IPF of model rats by intervening in EndMT， the mechanism of which may be associated with inhibiting DLL4/Notch4 singaling pathway.

Expression of Notch4 in renal cell carcinoma and its relationship with the microvascular density / 实用医学杂志

Objective To investigate the expression of Notch4 protein and to analyze its correlation with the clinical parameters and the microvessel dentisty in renal cell carcinoma. Methods The expression of Notch4 was examined in 60 cases of renal cell carcinoma and the para-carcinoma tissue by SP immunohistochemical stain-ing ,and CD34 detection was used for counting microvessel density. Statistical analysis was performed to reveal the correlation with clinicopathological parameters ,microvessel density and prognosis. Results The positive rate of Notch4 protein expression was 75%(45/60)in para-carcinoma tissue,and was 43.3%(26/60)in renal cell car-cinoma,with significant difference on tumor grade and Lymph node metastasis(P<0.05). The microvessel densi-ty in Notch4 positive tissues was significant lower than that in the negative samples(P<0.05). The survival time of patients with Notch4 positive expression was significantly longer than that of patients with Notch4 negative expres-sion(P<0.05). Conclusion Notch4 protein plays an important role in the development of renal cell carcinoma. Notch4 expression might both attenuate the malignant biological characteristics and suppress the angiogenesis dur-ing tumor development.

Notch4 Inhibition Suppresses Invasion and Vasculogenic Mimicry Formation of Hepatocellular Carcinoma Cells / 华中科技大学学报（医学）（英德文版）

Vasculogenic mimicry (VM) is a process by which aggressive tumor cells generate non-endothelial cell-lined channels in malignant tumors including hepatocellular carcinoma (HCC).It has provided new insights into tumor behavior and has surfaced as a potential target for drug therapy.The molecular events underlying the process of VM formation are still poorly understood.In this study,we attempted to elucidate the relationship between Notch4 and VM formation in HCC.An effective siRNA lentiviral vector targeting Notch4 was constructed and transfected into Be17402,a HCC cell line.VM networks were observed with a microscope in a 3 dimensional cell culture system.Cell migration and invasion were evaluated using wound healing and transwell assays.Matrix metalloproteinases (MMPs) activity was detected by gelatin zymography.Furthermore,the role of Notch4 inhibition in Be17402 cells in vivo was examined in subcutaneous xenograft tumor model of mice.The results showed that downregulation of Notch4 destroyed VM network formation and inhibited migration and invasion of tumor cells in vitro (P＜0.05).In vivo,tumor growth was also inhibited in subcutaneous xenograft model (P＜0.05).The potential mechanisms might be related with down-regulation of MT1-MMP,MMP-2,MMP-9 expression and inhibition of the activation of MMP2 and MMP9.These results indicated that Notch4 may play an important role in VM formation and tumor invasion in HCC.Related molecular pathways may be used as novel therapeutic targets for HCC antiangiogenesis therapy.

Investigation of the expression of Notch 4 and its relationship to invasion 、metastasis and prognostic in squamous cell carcinoma of the esophagus after esophagectomy / 中华胸心血管外科杂志

Objective To investigate the expression of Notch 4 in esophagus squamous cell carcinoma and its relationship with clinicopathological significance and prognosis.Methods A retrospective study was performed of 97 patients with midthoracic esophageal squamous carcinoma who accepted surgical treatment from 2007 to 2010.The expression of Notch 4 was examined by immunohistochemistry.The survival rate was calculated by Kaplan-meier method and the difference was determined by the log-rank analyze.The cox regression analysis was performed to determine the independent risk factors for lymph-metastastic recurrence.Results Notch 4 protein expression was detected in 42 patients of 97 with positive rate of 45.2％.The positive rate of Notch 4 in T1,T2,T3 and T4 patients was separately 66.7 ％ 、54.5 ％ and 28.6％ with significant difference(P =0.009).The positive rate of Notch 4 expression in NO and N1 patients was separately 57.1％ and 29.2％ with significant difference(P =0.005).the positive rate of Notch 4 expression in low-middle differentiation and high differentiation patients was separately 31.3 ％ and 56.3 ％ with significant difference (P =0.006).the 5-year survival rate of patients with Notch 4expression and without Notch 4 expression was separately 47.6％ and 18.2％ with significant difference(P =0.000).The Cox regression analysis showed that the negative expression of Notch 4 and the lymphometastasis were independent risk factors.Conclusion The low expression of Notch 4 protein was detected in esophagus squamous cell carcinoma and had a correlation with the differentiation,T classification and lmphometastasis.Cox regression analysis showed that the low expression of Notch 4protein is an independent risk factor and can be a biomarker of poor prognosis.

Association Study between Two Single Nucleotide Polymorphisms in the NOTCH4 Gene and Schizophrenia in Koreans / 신경정신의학

OBJECTIVES: Previous studies on NOTCH4 gene and schizophrenia have not produced consistent results, and more studies with various ethnicities and populations were warranted. This study was performed with Korean population to find the role of the NOTCH4 gene in the development of schizophrenia. METHODS: 235 schizophrenics and 236 normal controls participated in the study. Two SNPs (-1725 A/G and -25 T/C) on the NOTCH4 gene were investigated. Genotyping was done by Taqman assay, and statistical analysis was done by contingency chi-square test for the allele and genotype frequencies and PowerMarker V3.0 for the haplotype. RESULTS: The two SNPs did not deviate from Hardy-Weinberg equilibrium in neither schizophrenics or normal controls. Two groups were not different in terms of allele and genotype distribution for both SNPs. Two SNPs were found to be in linkage disequilibrium. Haplotype analysis could not find an association between schizophrenia and these two SNPs. There was no association between the age at onset and the genotypes for both SNPs. CONCLUSION: We could not find any significant association between schizophrenia and the NOTCH4 gene in this Korean population. Although there are limitations in this study, this result supports the conclusion that the NOTCH4 gene is less likely to play a major role on the development of schizophrenia in the Asian population.

Expression of Notch2,Notch4 in Hyperoxia Induced Lung Injury in Premature Rats / 实用儿科临床杂志

Objective To explore the roles of Notch2,Notch4 in hyperoxia induced lung injury in premature rats.Methods At the postnatal 1 day Sprague-Dawley premature rats were randomly assigned to about 85% hyperoxia group and air group.At the 1,7,14,21 days after exposed,8 rats of each group were used to evaluate expressions of Notch2,Notch4 in lungs by immunohistochemistry and the level of Nothch2,Notch4 mRNA by reverse transcription polymerase chain reaction(RT-PCR).Results Expressions of Notch2,Notch4 had their rules in rats′ different stages of development;85% oxygen exposed would change their tracks.Conclusion Prolonged 85% oxygen exposure result in abnormal expressions of Notch2 and Notch4 ,which is likely to lead to the pathogenesis of hyperoxic lung injure in premature rats.