• Animal Bioscience
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• 제37권6호
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• pp.1021-1030
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• 2024

Objective: R-loops are DNA:RNA triplex hybrids, and their metabolism is tightly regulated by transcriptional regulation, DNA damage response, and chromatin structure dynamics. R-loop homeostasis is dynamically regulated and closely associated with gene transcription in mouse zygotes. However, the factors responsible for regulating these dynamic changes in the R-loops of fertilized mouse eggs have not yet been investigated. This study examined the functions of candidate factors that interact with R-loops during zygotic gene activation. Methods: In this study, we used publicly available next-generation sequencing datasets, including low-input ribosome profiling analysis and polymerase II chromatin immunoprecipitation-sequencing (ChIP-seq), to identify potential regulators of R-loop dynamics in zygotes. These datasets were downloaded, reanalyzed, and compared with mass spectrometry data to identify candidate factors involved in regulating R-loop dynamics. To validate the functions of these candidate factors, we treated mouse zygotes with chemical inhibitors using in vitro fertilization. Immunofluorescence with an anti-R-loop antibody was then performed to quantify changes in R-loop metabolism. Results: We identified DEAD-box-5 (DDX5) and histone deacetylase-2 (HDAC2) as candidates that potentially regulate R-loop metabolism in oocytes, zygotes and two-cell embryos based on change of their gene translation. Our analysis revealed that the DDX5 inhibition of activity led to decreased R-loop accumulation in pronuclei, indicating its involvement in regulating R-loop dynamics. However, the inhibition of histone deacetylase-2 activity did not significantly affect R-loop levels in pronuclei. Conclusion: These findings suggest that dynamic changes in R-loops during mouse zygote development are likely regulated by RNA helicases, particularly DDX5, in conjunction with transcriptional processes. Our study provides compelling evidence for the involvement of these factors in regulating R-loop dynamics during early embryonic development.

• 약학회지
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• 제49권6호
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• pp.511-517
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• 2005

Low sensitivity to anticancer drugs such as 5-fluorouracil (5-FU) has been associated with decreased expression of genes involved in cell proliferation, apoptosis and metastasis. Recently, it has been shown that the expression levels of some of these genes are reduced by transcription inhibition due to epigenetic silencing on CpG islands. Therefore, epigenetic therapy has been proposed, where epigenetic silencing is repressed with DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors alone or in combination with other chemotherapeutic agents. The aim of our study was to evaluate the combination effect of 5-FU and its association with the status of epigenetic silencing using methylation-specific PCR of $p14^{ARF}$ when given with S-aza-2'-deoxycytidine (5-aza-dC), a DNMT inhibitor and depsipeptide, an HDAC inhibitor in DLD-1 human colorectal cancer cells. The combination of 5-aza-dC with depsipeptide showed a synergism and induced unmethylation of $p14^{ARF}$. However, triplet combination of 5-aza-dc/depsipeptide and 5-FU resulted in antagonistic effects and abrogated unmethylation of $p14^{ARF}$. These results suggest that unfavorable interaction of 5-aza-dC/depsipeptide with 5-FU in DLD-1 cells may be related with the failure in repression of epigenetic silencing, which warrants further investigation.

• Biomolecules & Therapeutics
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• 제20권3호
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• pp.313-319
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• 2012

In recent years, inhibition of HDACs has emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer, and several classes of HDAC inhibitors have been found to have potent and specific anticancer activities in preclinical studies. But their precise mechanism of action has not been elucidated. In this study, a novel synthetic inhibitor of HDAC, 3-(4-dimethylamino phenyl)-N-hydroxy-2-propenamide [IN-2001] was examined for its antitumor activity and the underlying molecular mechanisms of any such activity on human breast cancer cell lines. IN-2001 effectively inhibited cellular HDAC activity ($IC_{50}$ = 0.585 nM) inMDA-MB-231 human breast cancer cells. IN-2001 caused a significant dose-dependent inhibition of cell proliferation in estrogen receptor (ER) negative MDA-MB-231human breast cancer cells. Cell cycle analysis revealed that the growth inhibitory effects of IN-2001 might be attributed to cell cycle arrest at $G_0/G_1$ and/or $G_2$/Mphase and subsequent apoptosis in human breast cancer cells. These events are accompanied by modulating several cell cycle and apoptosis regulatory genes such as CDK inhibitors $p21^{WAF1}$ and $p27^{KIP1}$ cyclin D1, and other tumor suppressor genes such as cyclin D2. Collectively, IN-2001 inhibited cell proliferation and induced apoptosis in human breast cancer cells and these findings may provide new therapeutic approaches, combination of antiestrogen together with a HDAC inhibitor, in the hormonal therapy-resistant ER-negative breast cancers. In summary, our data suggest that this histone deacetylase inhibitor, IN-2001, is a novel promising therapeutic agent with potent antitumor effects against human breast cancers.

• Journal of Medicine and Life Science
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• 제18권3호
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• pp.41-48
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• 2021

The prevalence of attention deficit hyperactivity disorder (ADHD), a developmental neuropsychiatric disorder, is high among children and adolescents. The pathogenesis of ADHD is mediated with genetic, biological, and environmental factors. Most therapeutic drugs for ADHD have so far targeted biological causes, primarily by regulating catecholaminergic neurotransmitters. However, ADHD drugs that are clinically treated have various problems in their addictiveness and drug stability; thus, it is recommended that efficacy and safety should be secured through simultaneous prescription of multiple drugs rather than a single drug treatment. Accordingly, it is necessary to develop drugs that newly target pathogenic mechanisms of ADHD. In this study, we attempt to confirm the possibility of developing new drugs by reviewing dopamine-related developmental mechanisms of neurons and their correlation with ADHD. Histone deacetylase inhibitors (HDACi) can regulate the concentration of intracellular dopamine in neurons by expressing vesicular monoamine transporter 2 and inducing the exocytosis of neurotransmitters to the synaptic cleft, thereby promoting the development of neurons and signal transmission. This cellular modulation of HDACi is expected to treat ADHD by regulating endogenous catecholamines such as dopamine. Although studies are still in the preclinical stage, HDAC inhibitors clearly have potential as a therapeutic agent with low addictiveness and high efficacy for ADHD treatment.

• Asian-Australasian Journal of Animal Sciences
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• 제28권12호
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• pp.1703-1712
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• 2015

The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (-) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB- oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

• Asian Pacific Journal of Cancer Prevention
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• 제13권8호
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• pp.3977-3982
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• 2012

Inhibitors of histone deacetylase activity are emerging as a potentially important new class of anticancer agents. In this study, we assessed the anticancer effects of valproic acid (VPA) on ovarian cancer in vitro and in vivo. Cultured SKOV3 cells were treated by VPA with different concentrations and time, then the effects on cell growth, cell cycle, apoptosis, and related events were investigated. A human ovarian cancer model transplanted subcutaneously in nude mice was established, and the efficacy of VPA used alone and in combination with diammine dichloroplatinum (DDP) to inhibit the growth of tumors was also assessed. Proliferation of SKOV3 cells was inhibited by VPA in a dose and time dependent fashion. The cell cycle distribution changed one treatment with VPA, with decrease in the number of S-phase cells and increase in G1-phase. VPA could significantly inhibit the growth of the epithelial ovarian cancer SKOV3 cells in vivo without toxic side effects. Treatment with VPA combined with DDP demonstrated enhanced anticancer effects. The result of flow cytometry (FCM) indicated that after VPA in vitro and in vivo, the expression of E-cadherin was increased whereas vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) were decreased. This study suggests that VPA could be a novel attractive agent for treatment of ovarian cancer.

• Molecules and Cells
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• 제39권3호
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• pp.229-241
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• 2016

We have previously reported the role of miR-326-HDAC3 loop in anti-cancer drug-resistance. CAGE, a cancer/testis antigen, regulates the response to anti-cancer drug-resistance by forming a negative feedback loop with miR-200b. Studies investigating the relationship between CAGE and HDAC3 revealed that HDAC3 negatively regulated the expression of CAGE. ChIP assays demonstrated the binding of HDAC3 to the promoter sequences of CAGE. However, CAGE did not affect the expression of HDAC3. We also found that EGFR signaling regulated the expressions of HDAC3 and CAGE. Anti-cancer drug-resistant cancer cell lines show an increased expression of $pEGFR^{Y845}$. HDAC3 was found to negatively regulate the expression of $pEGFR^{Y845}$. CAGE showed an interaction and co-localization with EGFR. It was seen that miR-326, a negative regulator of HDAC3, regulated the expression of CAGE, $pEGFR^{Y845}$, and the interaction between CAGE and EGFR. miR-326 inhibitor induced the binding of HDAC3 to the promoter sequences in anti-cancer drug-resistant $Malme3M^R$ cells, decreasing the tumorigenic potential of $Malme3M^R$ cells in a manner associated with its effect on the expression of HDAC3, CAGE and $pEGFR^{Y845}$. The down-regulation of HDAC3 enhanced the tumorigenic, angiogenic and invasion potential of the anti-cancer drug-sensitive Malme3M cells in CAGE-dependent manner. Studies revealed that $PKC{\delta}$ was responsible for the increased expression of $pEGFR^{Y845}$ and CAGE in $Malme3M^R$ cells. CAGE showed an interaction with $PKC{\delta}$ in $Malme3M^R$ cells. Our results show that HDAC3-CAGE axis can be employed as a target for overcoming resistance to EGFR inhibitors.

• 생명과학회지
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• 제17권4호
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• pp.552-560
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• 2007

본 연구에서는 인체백혈병세포 U937에서 HDAC 저해제에 의한 증식억제, 세포주기 교란 및 apoptosis 유도에 미치는 Bcl-2 유전자의 영향에 관하여 조사하였다. 이를 위하여 U937/vector 및 U937/Bcl-2 세포주를 대상으로 대표적인 HDAC 저해제인 TSA 및 Na-B 처리에 의한 세포 증식 및 생존율에 미치는 영향을 조사한 결과, TSA에 의한 U937 세포의 증식억제 및 생존율의 감소는 Bcl-2의 과발현에 의하여 차단되는 효과를 보였으나, Na-B는 U937/vector 및 U937/Bcl-2세포사이에 큰 변화를 보이지는 않았다. 세포주기 교란효과에서 Na-B는 TSA에 비하여 유의적인 차이를 보이지 못하였으며, 이는 TSA에 의한 apoptosis가 U937/Bcl-2 세포에서는 억제되었으나, Na-B에 의한 apoptosis는 Bcl-2의 과발현에 의하여 차단되지 못한 것과 연관성이 있는 결과였다. 또한 TSA에 의한 apoptosis 유발의 Bcl-2에 의한 차단 효과는 TSA에 의하여 활성화된 caspase의 활성 억제, Bcl-2 발현 자체의 완화 등 apoptosis 조절 인자들의 발현 및 활성 변화에 기인 된 것임을 알 수 있었다.

• Radiation Oncology Journal
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• 제23권2호
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• pp.116-122
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• 2005

목적: 히스톤탈아세틸화효소 억제제는 그 자체의 항암효과뿐만 아니라 방사선 감작제로서의 효과가 점차분명해져가고 있다. 최근 Class I 특이적인 히스톤탈아세틸화효소 억제제의 개발로 계층 특이적인(Class specific) 연구가 가능해짐에 따라, 본 연구에서는 서로 다른 히스톤탈아세틸화효소억제제의 방사선감작효과를 비교함과 동시에 p53 발현도의 차이가 히스톤탈아세틸화효소억제제의 방사선 감수성에 미치는 영향을 알아보고자 하였다. 대상 및 방법: 이를 위해 p53 발현도가 매우 낮은 HeLa 세포에 p53의 핵 외 수송을 억제하여 세포질 내 분해를 차단하는 Leptomycin B를 처리하여 p53의 발현도를 현저하게 높인 후, Trichostatin와 SK7041의 방사선 민감도를 비교 관찰하였다. 결과: 세포생존곡선, SER 및 SF2를 비교 분석 시, p53의 발현이 높은 Leptomycin B 처리군에서 Trichostatin A가 Class I HDAC만을 억제하는 SK7041에 비해 유의하게 높은 방사선 감작효과를 나타내었다. 이는 p53이 Class I 특이적 억제제인 SK7041과 Class I과 II를 모두 억제하는 TSA의 방사선감작효과에 미치는 영향의 차이에 기전적으로 관여함을 시사한다. 결론: Leptomycln B에 의해 유도된 p53의 발현증가는 Class I과 Class I과 II를 모두 억제하는 TSA의 방사선 감작효과를 증강시킨다.