Methylation level of ASC/TMS1 and MyD88 genes in healthy larynx and laryngeal carcinoma tissue

2021 ◽  
Author(s):  
Lana Kovac Bilic ◽  
Jelena Knezevic ◽  
Maja Sutic ◽  
Srecko Branica ◽  
Krsto Dawidowsky ◽  
...  
Keyword(s):  
Protein Expression ◽  
Laryngeal Cancer ◽  
Laryngeal Carcinoma ◽  
Methylation Level ◽  
Tumor Tissue ◽  
Prognostic Biomarker ◽  
Methylation Status ◽  
Cancer Tissue ◽  
Carcinoma Tissue ◽  
Fresh Frozen

Abstract There are no biomarkers for diagnosis, prognosis and treatment of patients with laryngeal carcinoma. Methylation changes of ASC/TMS1 and MyD88 genes, in healthy and cancer tissue, might be related with development and progression of cancer. The study explored is there a difference in gene’s methylation in healthy and tumor tissue and does it correlate with protein expression. The total of 36 patients were enrolled in the study. Methylation of bisulphite converted DNA was quantified by pyrosequencing in fresh frozen cancer and adjacent non-malignant tissues. The overall methylation of MyD88 gene is significantly higher in healthy tissue and this finding correlates with protein expression and the overall methylation of ASC/TMS1 gene is unchanged but the protein expression of ASC/TMS1 is significantly higher in cancer. The methylation status of the ASC/TMS1 and MyD88 genes are promising prognostic biomarker candidates and may lead to earlier detection of laryngeal cancer.

scholarly journals Standardization of DNA amount for bisulfite conversion for analyzing the methylation status of LINE-1 in lung cancer

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0256254
Author(s):  
Duong Anh Thuy Pham ◽  
Son Duc Le ◽  
Trang Mai Doan ◽  
Phuong Thu Luu ◽  
Uyen Quynh Nguyen ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Dna Methylation ◽  
Methylation Level ◽  
Genomic Dna ◽  
Methylation Status ◽  
Cancer Tissue ◽  
Nucleotide Position ◽  
Bisulfite Conversion ◽  
Precise Location ◽  
Significant Variance

Highly methylated Long Interspersed Nucleotide Elements 1 (LINE-1) constitute approximately 20% of the human genome, thus serving as a surrogate marker of global genomic DNA methylation. To date, there is still lacking a consensus about the precise location in LINE-1 promoter and its methylation threshold value, making challenging the use of LINE-1 methylation as a diagnostic, prognostic markers in cancer. This study reports on a technical standardization of bisulfite-based DNA methylation analysis, which ensures the complete bisulfite conversion of repeated LINE-1 sequences, thus allowing accurate LINE-1 methylation value. In addition, the study also indicated the precise location in LINE-1 promoter of which significant variance in methylation level makes LINE-1 methylation as a potential diagnostic biomarker for lung cancer. A serial concentration of 5-50-500 ng of DNA from 275 formalin-fixed paraffin-embedded lung tissues were converted by bisulfite; methylation level of two local regions (at nucleotide position 300–368 as LINE-1.1 and 368–460 as LINE-1.2) in LINE-1 promoter was measured by real time PCR. The use of 5 ng of genomic DNA but no more allowed to detect LINE-1 hypomethylation in lung cancer tissue (14.34% versus 16.69% in non-cancerous lung diseases for LINE-1.1, p < 0.0001, and 30.28% versus 32.35% for LINE-1.2, p < 0.05). Our study thus highlighted the optimal and primordial concentration less than 5 ng of genomic DNA guarantees the complete LINE-1 bisulfite conversion, and significant variance in methylation level of the LINE-1 sequence position from 300 to 368 allowed to discriminate lung cancer from non-cancer samples.

scholarly journals Brusatol Inhibits Laryngeal Cancer Cell Proliferation and Metastasis Via Abrogating JAK2/STAT3 Signaling Mediated Epithelial-Mesenchymal Transition

2021 ◽  
Author(s):  
Jiangtao Zhou ◽  
Jing Hou ◽  
Jun Wang ◽  
Jiajing Wang ◽  
Jianping Gao ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Cancer Cell ◽  
Western Blotting ◽  
Laryngeal Cancer ◽  
Laryngeal Carcinoma ◽  
Epithelial Mesenchymal Transition ◽  
Migration And Invasion ◽  
Mesenchymal Transition ◽  
Stat3 Signaling

Abstract Background: Brusatol (BR) is a principal bioactive quassinoid derived from the Chinese medicinal plant Brucea javanica, which has recently been reported to exert notable cytotoxic effects against numerous cancer cell lines. However, the role that BR played in Laryngeal cancer (LC) is seldom known by the public. In the current study, we have investigated the effects of BR on human laryngeal squamous carcinoma cell (Hep-2) and explored its underlying mechanism both in vitro and in vivo experiments. Methods: In the present research, cell proliferation, apoptosis, cycle, migration and invasion assays were used to examine the anti-tumor effect of BR on Hep-2 cells. qRT-PCR, immunohistochemistry (IHC) and Western blotting were performed to study the molecular mechanisms of the action. A subcutaneous tumor-bearing model of Balb/c mice with Hep-2 cells of laryngeal carcinoma was established to observe the inhibitory effect of BR on laryngeal cancer cells in vivo. Results: The results indicated that BR markedly inhibited the viability, migration and invasion of Hep-2 cells in a dose and time-dependent manner, with no significant toxic effect on normal cells BEAS-2B. Also, BR induced cell apoptosis and the cells were blocked in the S phase to suppress cell proliferation. Moreover, the results of IHC showed that BR induction inhibited the protein expression levels of epithelial-mesenchymal transition (EMT)-related markers. Mechanistically, western blotting results exhibited that BR could suppress the protein expression of JAK2/STAT3 and their phosphorylation levels. In vivo experiments further confirmed the anti-cancer effect of BR on laryngeal carcinoma cells in vitro, BR suppressed the growth of xenograft laryngeal tumors without apparent toxicity.Conclusions: Consequently, the present study revealed that the anti-LC effect of BR might be closely relevant to abrogation of JAK2/STAT3 signaling mediated EMT process. BR may be a promising therapeutic candidate for the treatment of laryngeal cancer.

Serum Hypoxia Inducible Factor‐2: A Candidate Prognostic Biomarker for Laryngeal Cancer

2021 ◽  
Author(s):  
Görkem Eskiizmir ◽  
Gizem Çalıbaşı Koçal ◽  
Tuğba Uysal ◽  
Hülya Ellidokuz ◽  
Yasemin Başpınar
Keyword(s):  
Laryngeal Cancer ◽  
Prognostic Biomarker ◽  
Hypoxia Inducible Factor

scholarly journals COXIBs and 2,5-dimethylcelecoxib counteract the hyperactivated Wnt/β-catenin pathway and COX-2/PGE2/EP4 signaling in glioblastoma cells

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Aleksandra Majchrzak-Celińska ◽  
Julia O. Misiorek ◽  
Nastassia Kruhlenia ◽  
Lukasz Przybyl ◽  
Robert Kleszcz ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Viability ◽  
Methylation Level ◽  
Molecular Mechanisms ◽  
Methylation Status ◽  
Receptor Expression ◽  
Cell Cycle Distribution ◽  
Ep4 Receptor ◽  
Cox 2 ◽  
The Impact

Abstract Background Glioblastoma (GBM) is the deadliest and the most common primary brain tumor in adults. The invasiveness and proliferation of GBM cells can be decreased through the inhibition of Wnt/β-catenin pathway. In this regard, celecoxib is a promising agent, but other COXIBs and 2,5-dimethylcelecoxib (2,5-DMC) await elucidation. Thus, the aim of this study was to analyze the impact of celecoxib, 2,5-DMC, etori-, rofe-, and valdecoxib on GBM cell viability and the activity of Wnt/β-catenin pathway. In addition, the combination of the compounds with temozolomide (TMZ) was also evaluated. Cell cycle distribution and apoptosis, MGMT methylation level, COX-2 and PGE2 EP4 protein levels were also determined in order to better understand the molecular mechanisms exerted by these compounds and to find out which of them can serve best in GBM therapy. Methods Celecoxib, 2,5-DMC, etori-, rofe- and valdecoxib were evaluated using three commercially available and two patient-derived GBM cell lines. Cell viability was analyzed using MTT assay, whereas alterations in MGMT methylation level were determined using MS-HRM method. The impact of COXIBs, in the presence and absence of TMZ, on Wnt pathway was measured on the basis of the expression of β-catenin target genes. Cell cycle distribution and apoptosis analysis were performed using flow cytometry. COX-2 and PGE2 EP4 receptor expression were evaluated using Western blot analysis. Results Wnt/β-catenin pathway was attenuated by COXIBs and 2,5-DMC irrespective of the COX-2 expression profile of the treated cells, their MGMT methylation status, or radio/chemoresistance. Celecoxib and 2,5-DMC were the most cytotoxic. Cell cycle distribution was altered, and apoptosis was induced after the treatment with celecoxib, 2,5-DMC, etori- and valdecoxib in T98G cell line. COXIBs and 2,5-DMC did not influence MGMT methylation status, but inhibited COX-2/PGE2/EP4 pathway. Conclusions Not only celecoxib, but also 2,5-DMC, etori-, rofe- and valdecoxib should be further investigated as potential good anti-GBM therapeutics.

Methylation status of oestrogen receptor α-A: a predictor of prognosis in leukaemias

2010 ◽  
Vol 30 (4) ◽  
pp. 217-222 ◽  
Author(s):  
Jie Yao ◽  
Xiao-Bing Zhang ◽  
Xiao-li Zhang ◽  
Wei-Ling Fu
Keyword(s):  
Protein Expression ◽  
Cell Apoptosis ◽  
Thymidine Incorporation ◽  
Symptomatic Relief ◽  
Methylation Status ◽  
Dna Demethylation ◽  
Blue Staining ◽  
Chemotherapy Treatment ◽  
Propidium Iodide Staining ◽  
Epigenetic Biomarker

Many studies have shown that epigenetic regulation of ERs (oestrogen receptors) plays a key role in the pathogenesis of leukaemia. In the present study, it was found that the methylated status of ERα-A might serve as an epigenetic biomarker of leukaemias. In this study, the protein expression and cell apoptosis, cycle, proliferation and viability with and without 5-aza-dC (5-aza-2′-deoxycytidine) were evaluated with Western blotting, 3H-TdR (3H-thymidine) incorporation, propidium iodide staining and Trypan Blue staining respectively. The protein expression of ERα was significantly enhanced in all leukaemic cell lines using treatment with the DNA demethylation reagent 5-aza-dC. However, no obvious change in the protein expression of ERβ takes place with 5-aza-dC. And with 5-aza-dC, cell apoptosis, cell cycle, cell proliferation and viability were all inhibited significantly. We also tracked 40 cases of leukaemias with ERα-A methylation (95%; 38 of 40) to observe the prognosis 1 year after chemotherapy treatment. The patients with ERα-A methylation have no obvious symptomatic relief; however, two patients without ERα-A methylation have obtained effective relief. This result suggested that ERα plays a significant role in leukaemogenesis, and the methylated status of ERα-A not only might serve as an epigenetic biomarker of leukaemias for diagnosis, but also has the potential to serve as a predictor of prognosis in leukaemias.

Quantitative estimation of BRCA1 protein expression in breast cancer tissue using the method of flow cytometry

2016 ◽  
Vol 61 ◽  
pp. S183
Author(s):  
E. Shestakova ◽  
E. Dudko ◽  
A. Grishanina ◽  
V. Kirsanov ◽  
N. Vichljantzeva ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Flow Cytometry ◽  
Protein Expression ◽  
Quantitative Estimation ◽  
Breast Cancer Tissue ◽  
Cancer Tissue ◽  
Brca1 Protein

scholarly journals Methylation status and protein expression of RASSF1A in endometriosis

2016 ◽  
Vol 11 (6) ◽  
pp. 4107-4112 ◽  
Author(s):  
YU WU ◽  
MINGDE ZHANG ◽  
XIAN ZHANG ◽  
ZHENZHOU XU ◽  
WEIGUO JIN
Keyword(s):  
Protein Expression ◽  
Methylation Status

Novel Method for Simultaneous Analysis of p53 and K-ras Mutations and p53 Protein Expression in Single Histologic Sections

2001 ◽  
Vol 125 (3) ◽  
pp. 347-352
Author(s):  
Kazuya Yamashita ◽  
Tsutomu Yoshida ◽  
Hiroshi Shinoda ◽  
Isao Okayasu
Keyword(s):  
Polymerase Chain Reaction ◽  
Protein Expression ◽  
P53 Protein ◽  
Single Strand Conformation Polymorphism ◽  
Antigen Retrieval ◽  
Cancer Tissue ◽  
Polymorphism Analysis ◽  
Chain Reaction ◽  
Novel Method ◽  
Polymerase Chain

Abstract Background and Objective.—Abnormal protein expression and gene mutation should be examined on exactly identified lesions. To perform simultaneous analyses of oncogene or tumor suppressor gene mutations and related protein expression in single histologic sections, we have developed a novel method using an antigen-retrieval solution for a polymerase chain reaction template before immunohistochemical staining. Methods.—Using 20 cases of sporadic colorectal carcinoma, several kinds of antigen-retrieval solutions were tested after heating rehydrated, 4-μm-thick, formalin-fixed, paraffin-embedded histologic sections at 96°C for 20 minutes. Polymerase chain reaction–single-strand conformation polymorphism analysis was conducted for p53 (exons 5 through 9) and K-ras (exons 1 and 2), and the histologic sections were then immunostained with monoclonal antibody against p53. Results.—DNA analysis of antigen-retrieval solutions was possible in all 20 cases and revealed completely consistent results (100%) with fresh cancer tissue and microdissected cancer tissue of paraffin-embedded histologic sections. With this method, K-ras mutations were positive in 10 of 20 cases (exon 1 in 9 cases and exon 2 in 1 case) and p53 mutations were positive in 9 of 20 cases (exon 5 in 4 cases, exon 6 in 1, exon 7 in 3, and exon 8 in 1 case), with 8 of the 9 p53 mutation cases showing diffuse p53 protein expression on immunostaining. Base alterations of all abnormal conformers were confirmed with direct sequencing. For polymerase chain reaction–single-strand conformation polymorphism analysis, sodium citrate buffer (pH 6.0) was found to be the optimal antigen-retrieval solution. Conclusions.—This newly developed method can be used for routine immunostaining and genetic analysis with single histologic sections.

scholarly journals Diagnostic impact of promoter methylation and E-cadherin gene and protein expression levels in laryngeal carcinoma

2013 ◽  
Vol 3 ◽  
pp. 263-271
Author(s):  
Katarzyna Starska ◽  
Ewa Forma ◽  
Iwona Lewy-Trenda ◽  
Paweł Papież ◽  
Jan Woś ◽  
...  
Keyword(s):  
Protein Expression ◽  
Promoter Methylation ◽  
Laryngeal Carcinoma ◽  
Expression Levels ◽  
Diagnostic Impact ◽  
Gene And Protein Expression ◽  
E Cadherin
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