• Asian Pacific Journal of Cancer Prevention
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• 제16권4호
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• pp.1599-1603
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• 2015

Background: Epigenetic silencing of tumor suppressor genes due to promoter hypermethylation is one of the frequent mechanisms observed in cancers. Hypermethylation of several tumor suppressor genes involved in cell cycle regulation has been reported in many types of tumors including oral squamous cell carcinomas. LATS1 (Large Tumor Suppressor, isoform 1) is a novel tumor suppressor gene that regulates cell cycle progression by forming complexes with the cyclin dependent kinase, CDK1. Promoter hypermethylation of the LATS1 gene has been observed in several carcinomas and also has been linked with prognosis. However, the methylation status of LATS1 in oral squamous cell carcinomas is not known. As oral cancer is one of the most prevalent forms of cancer in India, the present study was designed to investigate the methylation status of LATS1 promoter and associate it with histopathological findings in order to determine any associations of the genetic status with stage of differentiation. Materials and Methods: Tumor chromosomal DNA isolated from biopsy tissues of thirteen oral squamous cell carcinoma biopsy tissues were subjected to digestion with methylation sensitive HpaII enzyme followed by amplification with primers flanking CCGG motifs in promoter region of LATS1 gene. The PCR amplicons were subsequently subjected to agarose gel electrophoresis along with undigested amplification control. Results: HpaII enzyme based methylation sensitive PCR identified LATS1 promoter hypermethylation in seven out of thirteen oral squamous cell carcinoma samples. Conclusions: The identification of LATS1 promoter hypermethylation in seven oral squamous cell carcinoma samples (54%), which included one sample with epithelial dysplasia, two early invasive and one moderately differentiated lesions indicates that the hypermethylation of this gene may be one of the early event during carcinogenesis. To the best of our knowledge, this is the first study to have explored and identified positive association between LATS1 promoter hypermethylation with histopathological features in oral squamous cell carcinomas.

• 한국발생생물학회지:발생과생식
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• 제17권1호
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• pp.9-16
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• 2013

Coactivator-associated arginine methyltransferase 1 (CARM1) is included in the protein arginine methyltransferase (PRMT) family, which methylates histone arginine residues through posttranslational modification. It has been proposed that CARM1 may up-regulate the expression of pluripotency-related genes through the alteration of the chromatin structure. Mouse embryonic stem cells (mESCs) are pluripotent and have the ability to self-renew. The cells are mainly used to study the genetic function of novel genes, because the cells facilitate the transmission of the manipulated genes into target mice. Since the up-regulated methylation levels of histone arginine residue lead to the maintenance of pluripotency in embryos and stem cells, it may be suggested that CARM1 overexpressing mESCs elevate the expression of pluripotency-related genes in reconstituted embryos for transgenic mice and may resist the differentiation into trophectoderm (TE). We constructed a fusion protein by connecting CARM1 and 7X-arginine (R7). As a cell-penetrating peptide (CPP), can translocate CARM1 protein into mESCs. CPP-CARM1 protein was detected in the nuclei of the mESCs after a treatment of 24 hours. Accordingly, the expression of pluripotency-related genes was up-regulated in CPP-CARM1-treated mESCs. In addition, CPP-CARM1-treated mESC-derived embryoid bodies (EBs) showed an elevated expression of pluripotency-related genes and delayed spontaneous differentiation. This result suggests that the treatment of recombinant CPP-CARM1 protein elevates the expression of pluripotency-related genes of mESCs by epigenetic modification, and this protein-delivery system could be used to modify embryonic fate in reconstituted embryos with mESCs.

• Biomolecules & Therapeutics
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• 제20권1호
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• pp.81-88
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• 2012

Histone deacetylases (HDACs) are enzymes involved in the remodelling of chromatin, and have a key role in the epigenetic regulation of gene expression. Histone deacetylase (HDAC) inhibitors are emerging as an exciting new class of potential anti-cancer agents. In recent years, a number of structurally diverse HDAC inhibitors have been identifi ed and these HDAC inhibitors induce growth arrest, differentiation and/or apoptosis of cancer cells in vitro and in vivo. However, the underlying molecular mechanisms remain unclear. This study aimed at investigating the anti-tumor activity of various HDAC inhibitors, IN-2001, using T47D human breast cancer cells. Moreover, the possible mechanism by which HDAC inhibitors exhibit anti-tumor activity was also explored. In estrogen receptor positive T47D cells, IN-2001, HDAC inhibitor showed anti-proliferative effects in dose-and time-dependent manner. In T47D human breast cancer cells showed anti-tumor activity of IN-2001 and the growth inhibitory effects of IN-2001 were related to the cell cycle arrest and induction of apoptosis. Flow cytometry studies revealed that IN-2001 showed accumulation of cells at $G_2$/M phase. At the same time, IN-2001 treatment time-dependently increased sub-$G_1$ population, representing apoptotic cells. IN-2001-mediated cell cycle arrest was associated with induction of cdk inhibitor expression. In T47D cells, IN-2001 as well as other HDAC inhibitors treatment significantly increased $p21^{WAF1}$ and $p27^{KIP1}$ expression. In addition, thymidylate synthase, an essential enzyme for DNA replication and repair, was down-regulated by IN-2001 and other HDAC inhibitors in the T47D human breast cancer cells. In summary, IN-2001 with a higher potency than other HDAC inhibitors induced growth inhibition, cell cycle arrest, and eventual apoptosis in human breast cancer possibly through modulation of cell cycle and apoptosis regulatory proteins, such as cdk inhibitors, cyclins, and thymidylate synthase.

• Biomolecules & Therapeutics
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• 제28권5호
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• pp.389-396
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• 2020

Valproic acid is a clinically used mood stabilizer and antiepileptic drug. Valproic acid has been suggested as a teratogen associated with the manifestation of neurodevelopmental disorders, such as fetal valproate syndrome and autism spectrum disorders, when taken during specific time window of pregnancy. Previous studies proposed that prenatal exposure to valproic acid induces abnormal proliferation and differentiation of neural progenitor cells, presumably by inhibiting histone deacetylase and releasing the condensed chromatin structure. Here, we found valproic acid up-regulates the transcription of T-type calcium channels by inhibiting histone deacetylase in neural progenitor cells. The pharmacological blockade of T-type calcium channels prevented the increased proliferation of neural progenitor cells induced by valproic acid. Differentiated neural cells from neural progenitor cells treated with valproic acid displayed increased levels of calcium influx in response to potassium chloride-induced depolarization. These results suggest that prenatal exposure to valproic acid up-regulates T-type calcium channels, which may contribute to increased proliferation of neural progenitor cells by inducing an abnormal calcium response and underlie the pathogenesis of neurodevelopmental disorders.

• Journal of Applied Biological Chemistry
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• 제62권2호
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• pp.157-165
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• 2019

Di- and tri-methylation of lysine 4 on histone H3 (H3K4me2 and H3K4me3, respectively) are epigenetic markers of active genes. Complex associated with Set1 (COMPASS) mediates these H3K4 methylations. The involvement of COMPASS activity in secondary metabolite (SM) biosynthesis was first demonstrated with an Aspergillus nidulans cclA knockout mutant. The cclA knockout induced the transcription of two cryptic SM biosynthetic gene clusters, leading to the production of the cognate SM. Monascus spp. are filamentous fungi that have been used for food fermentation in eastern Asia, and the pigment Monascus azaphione (MAz) is their main SM. Monascus highly produces MAz, implying that the cognate biosynthetic genes are highly active in transcription. In the present study, we examined how COMPASS activity modulates MAz biosynthesis by inactivating Monascus purpureus cclA (Mp-cclA) and swd1 (Mp-swd1). For both ${\Delta}Mp-cclA$ and ${\Delta}Mp-swd1$, a reduction in MAz production, accompanied by an abated cell growth, was observed. Suppression of MAz production was more effective in an agar culture than in the submerged liquid culture. The fidelity of the ${\Delta}Mp-swd1$ phenotypes was verified by restoring the WT-like phenotypes in a reversion recombinant mutant, namely, trpCp: Mp-swd1, that was generated from the ${\Delta}Mp-swd1$ mutant. Real-time quantitative Polymerase chain reaction analysis indicated that the transcription of MAz biosynthetic genes was repressed in the ${\Delta}Mp-swd1$ mutant. This study demonstrated that MAz biosynthesis is under the control of COMPASS activity and that the extent of this regulation is dependent on growth conditions.

• Molecules and Cells
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• 제44권8호
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• pp.602-612
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• 2021

DNA methylation is an important epigenetic mechanism affecting genome structure, gene regulation, and the silencing of transposable elements. Cell- and tissue-specific methylation patterns are critical for differentiation and development in eukaryotes. Dynamic spatiotemporal methylation data in these cells or tissues is, therefore, of great interest. However, the construction of bisulfite sequencing libraries can be challenging if the starting material is limited or the genome size is small, such as in Arabidopsis. Here, we describe detailed methods for the purification of Arabidopsis embryos at all stages, and the construction of comprehensive bisulfite libraries from small quantities of input. We constructed bisulfite libraries by releasing embryos from intact seeds, using a different approach for each developmental stage, and manually picking single-embryo with microcapillaries. From these libraries, reliable Arabidopsis methylome data were collected allowing, on average, 11-fold coverage of the genome using as few as five globular, heart, and torpedo embryos as raw input material without the need for DNA purification step. On the other hand, purified DNA from as few as eight bending torpedo embryos or a single mature embryo is sufficient for library construction when RNase A is treated before DNA extraction. This method can be broadly applied to cells from different tissues or cells from other model organisms. Methylome construction can be achieved using a minimal amount of input material using our method; thereby, it has the potential to increase our understanding of dynamic spatiotemporal methylation patterns in model organisms.

• Biomolecules & Therapeutics
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• 제31권3호
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• pp.253-263
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• 2023

The biogenesis and biological roles of extracellular vesicles (EVs) in the progression of liver diseases have attracted considerable attention in recent years. EVs are membrane-bound nanosized vesicles found in different types of body fluids and contain various bioactive materials, including proteins, lipids, nucleic acids, and mitochondrial DNA. Based on their origin and biogenesis, EVs can be classified as apoptotic bodies, microvesicles, and exosomes. Among these, exosomes are the smallest EVs (30-150 nm in diameter), which play a significant role in cell-to-cell communication and epigenetic regulation. Moreover, exosomal content analysis can reveal the functional state of the parental cell. Therefore, exosomes can be applied to various purposes, including disease diagnosis and treatment, drug delivery, cell-free vaccines, and regenerative medicine. However, exosome-related research faces two major limitations: isolation of exosomes with high yield and purity and distinction of exosomes from other EVs (especially microvesicles). No standardized exosome isolation method has been established to date; however, various exosome isolation strategies have been proposed to investigate their biological roles. Exosome-mediated intercellular communications are known to be involved in alcoholic liver disease and nonalcoholic fatty liver disease development. Damaged hepatocytes or nonparenchymal cells release large numbers of exosomes that promote the progression of inflammation and fibrogenesis through interactions with neighboring cells. Exosomes are expected to provide insight on the progression of liver disease. Here, we review the biogenesis of exosomes, exosome isolation techniques, and biological roles of exosomes in alcoholic liver disease and nonalcoholic fatty liver disease.

• BMB Reports
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• 제56권6호
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• pp.347-352
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• 2023

The protein family of poly (ADP-ribose) polymerases (PARPs) is comprised of multifunctional nuclear enzymes. Several PARP inhibitors have been developed as new anticancer drugs to combat resistance to chemotherapy. Herein, we characterized PARP4 mRNA expression profiles in cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines. PARP4 mRNA expression was significantly upregulated in cisplatin-resistant ovarian cancer cell lines, and this upregulation was associated with the hypomethylation of specific cytosine-phosphate-guanine (CpG) sites (cg18582260 and cg17117459) on its promoter. Reduced PARP4 expression was restored by treating cisplatin-sensitive cell lines with a demethylation agent, implicating the epigenetic regulation of PARP4 expression by promoter methylation. Depletion of PARP4 expression in cisplatin-resistant cell lines reduced cisplatin chemoresistance and promoted cisplatin-induced DNA fragmentation. The differential mRNA expression and DNA methylation status at specific PARP4 promoter CpG sites (cg18582260 and cg17117459) according to cisplatin responses, was further validated in primary ovarian tumor tissues. The results showed significantly increased PARP4 mRNA expressions and decreased DNA methylation levels at specific PARP4 promoter CpG sites (cg18582260 and cg17117459) in cisplatin-resistant patients. Additionally, the DNA methylation status at cg18582260 CpG sites in ovarian tumor tissues showed fairly clear discrimination between cisplatin-resistant patients and cisplatin-sensitive patients, with high accuracy (area under the curve = 0.86, P = 0.003845). Our findings suggest that the DNA methylation status of PARP4 at the specific promoter site (cg18582260) may be a useful diagnostic biomarker for predicting the response to cisplatin in ovarian cancer patients.

• Journal of Plant Biotechnology
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• 제50권
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• pp.190-199
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• 2023

Date palm (Phoenix dactylifera L.: Arecaceae) is a dioecious species where only female trees bear fruits. In their natural state, date palms produce dates once a year. However, in Thailand, some trees were observed to produce dates during the off-season, despite no variations in morphology. The availability of such off-season fruits can significantly increase their market value. Interestingly, most female off-season date palms investigated in this study were obtained through micropropagation. Hence, there is an urgent need for genetic markers to distinguish female offseason flowering plantlets within tissue culture systems. In this study, we aimed to develop random amplification of polymorphic DNA-sequence characterized amplified region (RAPD-SCAR) markers for the identification of female off-season flowering date palms cultivated in Thailand. A total of 160 random decamer primers were employed to screen for specific RAPD markers in off-season flowering male and female populations. Out of these, only one primer, OPN-02, generated distinct genomic DNA patterns in female off-season flowering (FOFdp) individuals compared to female seasonal flowering genotypes. Based on the RAPD-specific sequence, specific SCAR primers denoted as FOFdpF and FOFdpR were developed. These SCAR primers amplified a single 517-bp DNA fragment, predominantly found in off-season flowering populations, with an accuracy rate of 60%. These findings underscore the potential of SCAR marker technology for tracking offseason flowering in date palms. Notably, a BLAST analysis revealed a substantial similarity between the SCAR marker sequence and the transcript variant mRNA from Phoenix dactylifera encoding the SET DOMAIN GROUP 40 protein. In Arabidopsis, this protein is involved in the epigenetic regulation of flowering time. The genetic potential of the off-season flowering traits warrants further elucidation.

• International Journal of Oncology
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• 제57권4호
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• pp.1027-1038
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• 2020

Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies and is a leading cause of cancer-related mortality worldwide. Histone deacetylases (HDACs) are a class of enzymes responsible for the epigenetic regulation of gene expression. Some HDAC inhibitors have been shown to be efficient agents for cancer treatment. The aim of the present study was to discover a novel, potent HDAC inhibitor and demonstrate its anticancer effect and molecular mechanisms in CRC cells. A novel fluorinated aminophenyl-benzamide-based compound, CBUD-1001, was designed to specifically target HDAC1, and it was then synthesized and evaluated. CBUD-1001 exerted a potent inhibitory effect on HDAC enzyme activity and exhibited anticancer potency against CRC cell lines. Molecular docking analysis rationalized the high potency of CBUD-1001 by validating its conformation in the HDAC active site. Further investigation using CRC cells demonstrated that CBUD-1001 inhibited HDAC activity by hyper-acetylating histones H3 and H4, and it exerted an apoptotic effect by activating a mitochondrial-dependent pathway. Of note, it was found that CBUD-1001 attenuates the cell motility of CRC cells by downregulating the EMT signaling pathway. Thus, CBUD-1001 may prove to be a promising novel drug candidate for CRC therapy.