Elevated Expression of Notch 2 & Notch 3 is associated with Disease Progression in Colorectal Cancer

Background: The potential involvement of Notch signaling pathway in cell fate decision, tumor heterogeneity and angiogenesis in solid tumors can be explored in colorectal cancer (CRC). This might further help to improve outcomes in CRC. Here, the promoter methylation of Notch receptor gene (Notch2 and Notch3) and their co-expression with its downstream transcription factor Hes1 has been analyzed. Methods: Seventy-two CRC patients were enrolled to study the role of Notch2, Notch3 and Hes1 genes in colorectal cancer. Promoter methylation and mRNA expression in tumor and adjoining normal tissue were assessed by Methylation Specific PCR and quantitative Real time PCR respectively. Statistical correlation was done by using SPSS. Results: We found that Notch2 and Notch3 were hypomethylated in 52/72 (72.22%) and 54/72 (75%) cases respectively. Hypomethylation of Notch 2 and Notch 3 showed significant association with advanced stage (p=0.001) and (p=0.003) and nodal metastasis (p=0.036) and (p=0.012) respectively. Both Notch 2 and Notch 3 showed increased mRNA expression in 49 (68.05%) and 51(70.84%) patients with a fold change of 3.37 and 5.43 respectively. Positive correlation between hypomethylation and expression was observed for both genes. High expression of Hes1 was found in 53(73.61%) of cases which was highly relatable with over expression of notch receptor genes. Upregulation of Notch 2, Notch 3 and Hes1 showed significant association with high grade tumors, advance stage and presence of LN metastasis, additionally Notch 3 and Hes1 showed significant association with distant metastasis. Conclusion: Hypomethylation of Notch 2 and 3 receptors is playing crucial role in regulating the expression of these genes in CRC. Overexpression of Notch 2, Notch 3 and Hes1 are associated with disease progression in CRC.

Effect of azacitidine on HOXA9 gene expression and biological behaviors of A375 melanoma cells / 中华皮肤科杂志

Objective:To investigate the effect of the methyltransferase inhibitor azacitidine (5-azaC) on the expression of homeobox A9 (HOXA9) gene in, as well as proliferation, invasion and migration of A375 cells.Methods:In vitro cultured A375 cells were treated with 5-azaC at various concentrations of 1, 5, 10 and 20 μmol/L, while routinely cultured A375 cells receiving no drug intervention served as control group. Methylation-specific PCR was performed to analyze methylation status of the HOXA9 gene promoter region after the treatment with different concentrations of 5-azaC, in order to screen the optimal concentration of 5-azaC for following experiments. Cell counting kit-8 (CCK8) assay was conducted to evaluate the proliferation of A375 cells, Transwell and wound healing assays were performed to estimate the invasion and migration of A375 cells, and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were conducted to determine the mRNA and protein expression of HOXA9 in A375 cells after 5-azaC treatment. Two-independent-sample t test was used for comparisons between two groups. Results:Methylation was observed in the HOXA9 gene promoter region in A375 cells in the control group. After 5-azaC treatment, methylated and unmethylated states coexisted in the HOXA9 gene promoter region in A375 cells, and the higher the concentration of 5-azaC, the higher the degree of demethylation of the HOXA9 gene. Therefore, 20 μmol/L 5-azaC was selected to treat A375 cells for 72 hours, which served as 5-azaC treatment group in subsequent experiments. Compared with the control group, the 5-azaC treatment group showed significantly decreased cellular proliferative ability (72.46% ± 2.19% vs. 100%, t = 28.09, P < 0.001) , significantly decreased number of invasive cells (242.70 ± 29.19 vs. 466.00 ± 22.65, t = 10.47, P < 0.001) , significantly decreased migratory ability (27.56% ± 2.74% vs. 35.69% ± 2.50%, t = 3.79, P = 0.019) , significantly increased HOXA9 mRNA expression (1.73 ± 0.28 vs. 1.01 ± 0.15, t = 3.93, P = 0.017) , and significantly increased HOXA9 protein expression (0.62 ± 0.03 vs. 0.50 ± 0.01, t = 3.82, P = 0.019) . Conclusion:5-azaC can inhibit the proliferative, invasive and migratory ability of A375 melanoma cells, and one of the possible mechanisms underlying this process may be that 5-azaC reverses the methylation in the HOXA9 gene promoter region, activates HOXA9 gene expression, and participates in the regulation of biological behaviors of melanoma cells.

Promoter-associated DNA methylation & expression profiling of genes (FLT 3, EPB41L3 & SFN) in patients with oral squamous cell carcinoma in the Khasi & Jaintia population of Meghalaya, India

Background & objectives: Oral squamous cell carcinoma is one of the most lethal forms of cancer, and its aetiology has been attributed to both genetic and epigenetic factors working in liaison to contribute to the disease. Epigenetic changes especially DNA methylation is involved in the activation or repression of gene functions. The aim of this study was to investigate the DNA methylation pattern and expression profiling of the promoter regions of FMS-related tyrosine kinase 3 (FLT3), erythrocyte membrane protein band 4.1-like 3 (EPB41L3) and stratifin (SFN) genes in oral cancer within the Khasi and Jaintia tribal population of Meghalaya in North East India. Methods: Quantitative methylation analyses of the selected genes were carried out by MassARRAY platform System, and the relative expression profiling was carried out by real-time polymerase chain reaction. Results: Quantitative methylation results indicated that the level of methylation was significantly higher (hypermethylated) for FLT3 and EPB41L3 and significantly lower (hypomethylated) for SFN in tumour tissues as compared to the adjacent paired normal tissue. Expression profiling was in concurrence with the methylation data whereby hypermethylated genes showed low mRNA level and vice versa for the hypomethylated gene. Interpretation & conclusions: The findings show that hyper- and hypomethylation of the selected genes play a potential role in oral carcinogenesis in the selected Khasi and Jaintia tribal population of Meghalaya. The methylation status of these genes has not been reported in oral cancer, so these genes may serve as promising biomarkers for oral cancer diagnosis as well as in disease monitoring.

Progress in epigenetic regulation of regulatory T cell / 白血病·淋巴瘤

Regulatory T (Treg) cells exert significant influence on the control of autoimmunity and maintenance of homeostasis,while the progressive roles of Treg cells in solid tumor and hematological malignance have also attracted much attention recently.Except for the specific expression of transcription factor forkhead box protein 3 (Foxp3),natural Treg (nTreg) cells have Treg-specific hypomethylation pattern in a limited number of key genes,which is indispensable for stable Treg cells and functional nTreg cells to define the T-cell subpopulation possessing Treg-type epigenome more accurately.The expression of Foxp3 and Treg-specific hypomethylation phenotype have close relationship with the epigenetic regulation,which involves DNA methylation,histone methylation,acetylation,microRNA,etc.This review summarizes the different mechanisms of epigenetic regulation according to the two important features of Treg cells.

Research progress of azacitidine in the treatment of elderly patients with acute myeloid leukemia / 白血病·淋巴瘤

Azacitidine belongs to a novel family of demethylation antitumor drug.Foreign clinical researches have demonstrated that it may be a low toxic and effective choice for elderly patients with acute myeloid leukemia,whose physical conditions are too poor to sustain intensive chemotherapy.This review will summarize recent reports on azacitidine in the treatment of elderly patients with acute myeloid leukemia.

5-Aza-2'-deoxycytidine acts as a modulator of chondrocyte hypertrophy and maturation in chick caudal region chondrocytes in culture / 대한해부학회지

This study was carried out to explore the effect of DNA hypomethylation on chondrocytes phenotype, in particular the effect on chondrocyte hypertrophy, maturation, and apoptosis. Chondrocytes derived from caudal region of day 17 embryonic chick sterna were pretreated with hypomethylating drug 5-aza-2'-deoxycytidine for 48 hours and then maintained in the normal culture medium for up to 14 days. Histological studies showed distinct morphological changes occurred in the pretreated cultures when compared to the control cultures. The pretreated chondrocytes after 7 days in culture became bigger in size and acquired more flattened fibroblastic phenotype as well as a loss of cartilage specific extracellular matrix. Scanning electron microscopy at day 7 showed chondrocytes to have increased in cell volume and at day 14 in culture the extracellular matrix of the pretreated cultures showed regular fibrillar structure heavily embedded with matrix vesicles, which is the characteristic feature of chondrocyte hypertrophy. Transmission electron microscopic studies indicated the terminal fate of the hypertrophic cells in culture. The pretreated chondrocytes grown for 14 days in culture showed two types of cells: dark cells which had condense chromatin in dark patches and dark cytoplasm. The other light chondrocytes appeared to be heavily loaded with endoplasmic reticulum indicative of very active protein and secretory activity; their cytoplasm had large vacuoles and disintegrating cytoplasm. The biosynthetic profile showed that the pretreated cultures were actively synthesizing and secreting type X collagen and alkaline phosphatase as a major biosynthetic product.

Methylation situation of let-7a-3 in chronic myeloid leukemia and its clinical significance / 重庆医学

Objective To investigate the methylation situation of let‐7a‐3 promoter in patients with chronic myeloid leukemia (CML) and its clinical significance .Methods The methylation level of let‐7a‐3 promoter in the bone marrow mononuclear cells of 52 CML patients and 25 controls was detected by using the real‐time quantitative methylation‐specific PCR (RQ‐PCR) .Results The non-hypomethylation of let‐7a‐3 promoter was positive in 31 cases(59 .6% ) of 52 CML patients ,while only 1 case(4% ,1/25) in the control group ,the difference between the two groups were statistically significant (P0 .05) .The non-hypomethylation level of let‐7a‐3 in chronic phase and accel‐erate phase was significantly higher than that in blastic crisis of CML .Conclusion The hypomethylation level of let‐7a‐3 promoter is decreased with disease progression .

Quantitative PCR analysis for methylation level of genome: clinical implications in cancer.

Background: Cancer cells are frequently characterized by hypomethylation of the genome including repetitive sequences. This epigenetic process is believed to be associated with several biological causes and consequences in cancer. Therefore, LINE-1 repetitive sequences demethylation in cancer should result in different clinical outcomes. Objective: Recently, we have developed an improved quantitative combined bisulfite restriction analysis PCR protocol that efficiently evaluates the methylation status of LINE-1s; the method is referred to as PCR “COBRALINE-1”. This article reviewed what have been learned by applying this technique to study methylation level of repetitive sequences from several sources of genomic DNA. Results: We have found that LINE-1 methylation patterns among normal tissues are distinct. Therefore, this epigenetic event may be continuously altered in adult tissues by the process of cellular differentiation. Moreover, we confirmed that global hypomethylation is an ongoing process that develops during tumor progression, in addition to previous evidence of genomic and LINE-1 hypomethylation occurring as an early event in carcinogenesis. COBRALINE-1 is a highly effective technique for evaluating the genome-wide level of methylation, in particular from tissue samples with minute amounts of low quality DNA. The technique has been applied to study samples from micro-dissected archived paraffin-embedded tissues and sera of several types of cancer. Conclusion: The COBRALINE-1 technique demonstrated its potential to be a tumor marker and a great tool to explore the biology of global hypomethylation.

Epigenetic Changes (Aberrant DNA Methylation) in Colorectal Neoplasia

Both genetic and epigenetic events have been implicated in the stepwise histological progression involving adenoma-carcinoma and hyperplastic polyp/serrated adenoma-carcinoma sequences in the development of colorectal cancer. Genetic changes have been observed at each step in the initiation and progression of polyps to adenocarcinomas. Epigenetic changes also occur at each step in the pathogenesis of colorectal cancers and include CpG island DNA hypermethylation in the promoter region of genes resulting in transcriptional silencing through associated changes in chromatin structure and effects on binding of transcription factors, and DNA global hypomethylation which leads to chromosomal instability. Recent studies on MLH1 and APC genes indicate that epigenetic and genetic changes cooperate to facilitate tumor initiation and progression. Since aberrant CGI DNA promoter hypermethylation can be detected not only in colorectal polyps and cancers, but also in sera and stool, hypermethylated genes may serve as molecular markers for early detection, risk assessment and diagnosis. In addition, silenced genes caused by CGI DNA promoter hypermethylation can be reactivated by demethylating agents and also by both the inhibitors of DNA methyltransferases and histone deacetylases. Therefore, these epigenetically acting drugs should be evaluated for their chemopreventive and therapeutic potential for colorectal cancers.