인체대장암 세포에서 후성적 유전자 불활성화 저해제와 5-Fluorouracil의 병용효과분석

김미영(Mi Young Kim),손정규(Jung Kyu Son),이숙경(Suk Kyung Lee),구효정(Hyo Jeong Kuh) 大韓藥學會 2005 약학회지 Vol.49 No.6

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 p14ARF when given with 5-aza-2'-deoxycytidine (5-aza-dC), a DNMT inhibitor and depsipeptide, and HDAC inhibitor in DLD-1 human colorectal cancer cells. The combination of 5-aza-dC with depsipeptide showed a synergism and induced unmethylation of p14ARF. However, triplet combination of 5-aza-dC/epsipeptide and 5-FU resulted in antagonistic effects and abrogated unmethylation of p14ARF. 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.

TAp73 and ΔNp73 Have Opposing Roles in 5-aza-2'-Deoxycytidine-Induced Apoptosis in Breast Cancer Cells

Lai, Jing,Yang, Fang,Zhang, Wenwen,Wang, Yanru,Xu, Jing,Song, Wei,Huang, Guichun,Gu, Jun,Guan, Xiaoxiang Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.8

The p73 gene contains an extrinsic P1 promoter and an intrinsic P2 promoter, controlling the transcription of the pro-apoptotic TAp73 isoform and the anti-apoptotic ${\Delta}Np73$ isoform, respectively. The DNA methylation status of both promoters act equally in the epigenetic transcriptional regulation of their relevant isoforms. The aim of this study was to analyze the different effects of these p73 isoforms in 5-aza-2'-deoxycytidine (5-aza-dC)-induced apoptosis in breast cancer cells. We investigated the effects of the DNA demethylation agent, 5-aza-dC, on the T-47D breast cancer cell line, and evaluated the methylation status of the p73 promoters and expression of TAp73 and ${\Delta}Np73$. Furthermore, we assessed the expression of p53 and p73 isoforms in 5-aza-dC-treated T-47D cells and p53 knockout cells. 5-aza-dC induced significant anti-tumor effects in T-47D cells, including inhibition of cell viability, G1 phase arrest and apoptosis. This was associated with p73 promoter demethylation and a concomitant increase in TAp73 mRNA and protein expression. In contrast, the methylation status of promoter P2 was not associated with ${\Delta}Np73$ mRNA or protein levels. Furthermore, demethylation of P2 failed to inhibit the expression of ${\Delta}Np73$ with 5-aza-dC in the p53 knockdown cell model. Our study suggests that demethylation of the P1 and P2 promoters has opposite effects on the expression of p73 isoforms, namely up-regulation of TAp73 and down-regulation of ${\Delta}Np73$. We also demonstrate that p53 likely contributes to 5-aza-dC-induced ${\Delta}Np73$ transcriptional inactivation in breast cancer cells.

TAp73 and ΔNp73 Have Opposing Roles in 5-aza-2'-Deoxycytidine-Induced Apoptosis in Breast Cancer Cells

Jing Lai,Fang Yang,Wenwen Zhang,Yanru Wang,Jing Xu,Wei Song,Guichun Huang,Jun Gu,Xiaoxiang Guan 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.8

The p73 gene contains an extrinsic P1 promoter and an intrinsic P2 promoter, controlling the transcription of the pro-apoptotic TAp73 isoform and the anti-apoptotic ΔΝp73 isoform, respectively. The DNA methylation status of both promoters act equally in the epigenetic transcriptional regulation of their relevant isoforms. The aim of this study was to analyze the different effects of these p73 isoforms in 5-aza-2'-deoxycytidine (5-aza-dC)-induced apoptosis in breast cancer cells. We investigated the effects of the DNA demethylation agent, 5-aza-dC, on the T-47D breast cancer cell line, and evaluated the methylation status of the p73 promoters and expression of TAp73 and ΔNp73. Furthermore, we assessed the expression of p53 and p73 isoforms in 5-aza-dC-treated T-47D cells and p53 knockout cells. 5-aza-dC induced significant anti-tumor effects in T-47D cells, including inhibition of cell viability, G1 phase arrest and apoptosis. This was associated with p73 promoter demethylation and a concomitant increase in TAp73 mRNA and protein expression. In contrast, the methylation status of promoter P2 was not associated with ΔNp73 mRNA or protein levels. Furthermore, demethylation of P2 failed to inhibit the expression of ΔNp73 with 5-aza-dC in the p53 knockdown cell model. Our study suggests that demethylation of the P1 and P2 promoters has opposite effects on the expression of p73 isoforms, namely up-regulation of TAp73 and down-regulation of ΔΝp73. We also demonstrate that p53 likely contributes to 5-aza-dC-induced ΔNp73 transcriptional inactivation in breast cancer cells.

5-Aza-2'-deoxycytidine Induces Hepatoma Cell Apoptosis via Enhancing Methionine Adenosyltransferase 1A Expression and Inducing S-Adenosylmethionine Production

Liu, Wei-Jun,Ren, Jian-Guo,Li, Ting,Yu, Guo-Zheng,Zhang, Jin,Li, Chang-Sheng,Liu, Zhi-Su,Liu, Quan-Yan Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.11

In hepatocellular cancer (HCC), lack of response to chemotherapy and radiation treatment can be caused by a loss of epigenetic modifications of cancer cells. Methionine adenosyltransferase 1A is inactivated in HCC and may be stimulated by an epigenetic change involving promoter hypermethylation. Therefore, drugs releasing epigenetic repression have been proposed to reverse this process. We studied the effect of the demethylating reagent 5-aza-2'-deoxycitidine (5-Aza-CdR) on MAT1A gene expression, DNA methylation and S-adenosylmethionine (SAMe) production in the HCC cell line Huh7. We found that MAT1A mRNA and protein expression were activated in Huh7 cells with the treatment of 5-Aza-CdR; the status of promoter hypermethylation was reversed. At the same time, MAT2A mRNA and protein expression was significantly reduced in Huh7 cells treated with 5-Aza-CdR, while SAMe production was significantly induced. However, 5-Aza-CdR showed no effects on MAT2A methylation. Furthermore, 5-Aza-CdR inhibited the growth of Huh7 cells and induced apoptosis and through down-regulation of Bcl-2, up-regulation of Bax and caspase-3. Our observations suggest that 5-Aza-CdR exerts its anti-tumor effects in Huh7 cells through an epigenetic change involving increased expression of the methionine adenosyltransferase 1A gene and induction of S-adenosylmethionine production.

5-aza-2’-deoxycytidine처치에 의해 유도되는 B16F10 세포주의 유전자 발현

손진식,송달원,김태종,채한수,박종욱 대한이비인후과학회 2002 대한이비인후과학회지 두경부외과학 Vol.45 No.8

Background and Objectives:by a tumor, and many types of vaccines such as gene modified vaccines have been developed to increase imunogenicity of vaccine. We studied to determine whether or not 5-aza-2-deoxycytidine (ADC) can increase the immunogenicity of B16F10 melanoma cell. Materials and Method:B16F10 cell was treated with ADC for the induction of DNA demethylation. An ADC treated B16F10 melanoma cell was analyzed first using the reverse transcriptase-polymerase chain reaction (RT-PCR) (MAGE-2, MAGE-5) and immunity-enhancing cytokines (GM-CSF, IL-12), and then by flow cytometry to evaluate the expression of MHC and B7 that are responsible for antigen expres-sion and T cell activation on B16F10 cell surface. In order to evaluate vaccination effect of ADC-treated B16F10 vaccine, each mouse group were injected with PBS, ADC, B16F10 vaccine or B16F10-ADC vaccine and they were also challenged with live B16F10 cell 7 days after vaccination. On the 20th day after live B16F10 cell challenge, the tumor mass size and the Results:ADC treatment for B16F10 melanoma cell increased expresion of MHC and B7. ADC treatment also increased gene expresion of MAGE-2, MAGE-5, GM-CSF and IL-12. The growth of tumor mass was decreased and the mouse survival period was elongated in B16F10-ADC vaccine imunized group. Conclusion:ADC treatment may increase imunogenicity of B16F10 cell, and B16F10-ADC vaccine imunization can induce anti-cancer imunity in vivo. (Korean J Otolaryngol 2002;45:784-90)

마우스 유방암 모델에서 5-Aza-2'-deoxycytidine의 암줄기세포 유지 억제 효과

노경진(Kyoung-Jin Nho),양인숙(In-Sook Yang),김란주(Ran-Ju Kim),김수림(Soo-Rim Kim),박정란(Jeong-Ran Park),정지윤(Ji-Youn Jung),조성대(Sung-Dae Cho),남정석(Jeong-Seok Nam) 한국생명과학회 2009 생명과학회지 Vol.19 No.8

비정상적 DNA메칠화는 암 발생에 있어 중요한 역할을 한다. 최근 연구에 의하면 암줄기세포 유지에 있어 DNA과메칠화가 연관되어 있다고 보고하고 있다. 따라서 본 연구는 4T1 유방암 실험모델에서 demethylating agent인 AZA 처리에 따른 후성유전적 변화가 암줄기세포의 유지 및 증식에 있어 어떠한 영향을 미치는지 조사 하였다. 4T1 세포에서 AZA 처리에 따른 tumorsphere 형성이 감소 하는 것을 in vitro 실험을 통해 관찰 하였고, in vivo 실험에서는 줄기세포 조절 유전자들(Oct-4, Nanog. Sox2)의 발현이 감소 되는 것을 확인 하였다. 본 연구 결과로 볼 때 4T1 유방암 실험모델에서 AZA에 의한 후성유전적 변화는 줄기세포 조절 유전자(SRG)들의 발현을 조절하면서 암줄기세포 특성을 변화시켜 암줄기세포의 증식 및 유지를 억제 할 것으로 사료된다. 향후 이러한 DNA 메칠화억제를 항암치료에 응용하면, 암줄기세포를 파괴함으로써 암의 재발 및 악성화를 효과적으로 제어 할 수 있을 것으로 사료된다. Aberrant DNA methylation plays an important role in the development of cancer. It has been reported recently that DNA hypermethylation is involved in the maintenance of cancer stem cells. The present study was designed to test the hypothesis that the demethylating agent, 5-aza-2‘-deoxycytidine (AZA), can inhibit the potential for maintenance of cancer stem cells. To validate this hypothesis, we used 4T1 syngeneic mouse models of breast cancer. The AZA pre-treated 4T1 cells showed a dramatic inhibition of tumorsphere formation, compared to their counterparts in vitro. In addition, the AZA treatment significantly suppressed the expression of stem regulator genes, such as oct-4, nanog and sox2, compared to counterparts in vivo. Therefore, selective inhibition of DNA methylation may be useful for stem-specific cancer therapy.

5-Aza-2’-deoxycytidine Induces Cell Cycle Arrest and the Cytoskeletal Reorganization by Recovery of Chimaerin2 in 267B1/K-ras Human Prostate Cancer Cells

Ji Young Lim,Sook Jung Jeong,김보연,김군도 대한암예방학회 2010 Journal of cancer prevention Vol.15 No.2

5Aza2'deoxycytidine,a demethylating agent, induced recovery of the expression of tumor suppressor genes. Chimaerin2 among these genes down-regulated the expression of Rac, WAVE2, Arp2/3, and Villin1in Racdependent cytoskeletal pathway and led to the changes of cell morphology, actin dynamics and cell migration in 267B1/Kras. 5-Aza-2'-deoxycytidine, also, caused cell cycle arrest at G1 to S phase by the inhibition of the expression of Cyclin-dependent kinases (CDKs), and Cdc25A. The results represent that 5-Aza-2'-deoxycytidine causes cell cycle arrest and regulates Rac-dependent actin polymerization on 267B1/K-ras human prostate cancer cells. (Cancer Prev Res 15, 118-126, 2010)

Differential Promoter Methylation and Histone Modification Contribute to the Brain Specific Expression of the Mouse Mbu-1 Gene

김병탁,김선정,Seongeun Kang 한국분자세포생물학회 2012 Molecules and cells Vol.34 No.5

Mbu-1 (Csrnp-3) is a mouse gene that was identified in our previous study as showing highly restricted expres-sion to the central nervous system. In this study, to eluci-date the regulatory mechanism for tissue specificity of the gene, epigenetic approaches that identify the profiles of CpG methylation, as well as histone modifications at the promoter region were conducted. Methylation-specific PCR revealed that the CpG sites in brain tissues from embryo to adult stages showed virtually no methylation (0.052- 0.67%). Lung (9.0%) and pancreas (3.0%) also showed lower levels. Other tissues such as liver, kidney, and heart showed much higher methylation levels ranging from approximately 39-93%. Treatment of 5-aza-2-deoxycytidine (5-Aza-dC) significantly decreased promoter methylation, reactivating Mbu-1 expression in NG108-15 and Neuro-2a neuronal cells. Chromatin immunoprecipitation assay revealed that 5-Aza-dC decreased levels of acetylated H3K9 and methylated H3K4, and increased methylated H3K9. This result indicates that CpG methylation converses with histone modifications in an opposing sense of regulating Mbu-1 expression.

Methylation of the Mouse DIx5 and Osx Gene Promoters Regulates Cell Type-specific Gene Expression

Dong Sun Kim,Ji Yun Lee,Yu Mi Lee,Mi Jin Kim,Je Yong Choi,박의균,Shin Yoon Kim,Sam Poong Lee,Jae Sup Yang 한국분자세포생물학회 2006 Molecules and cells Vol.22 No.2

Expression Profiling after Induction of Demethyla-tion in MCF-7 Breast Cancer Cells Identifies Involvement of TNF-alpha Mediated Cancer Pathways

김주희,김선정,Seongeun Kang,김태우,Lihong Yin,Ran Liu 한국분자세포생물학회 2012 Molecules and cells Vol.33 No.2

Epigenetic methylation change is a major process that occurs during cancer development. Even though many tumor-related genes have been identified based on their relationship between methylation and expression, few studies have been conducted to investigate the relevant biological pathways involved in these changes. To identify essential pathways likely to be affected by methylation in breast cancer, we examined a pool of genes in which expression was upregulated after induction of demethy-lation by 5-Aza-2'-deoxycytidine (Aza) in the MCF-7 breast cancer cell line. Genome-wide demethylation was confir-med by monitoring the demethylation of a previously known gene, SULT1A1. Overall, 210 and 213 genes were found to be upregulated and downregulated (fold change > 2), respectively, in common in cells treated with 5 and 10 M of Aza. Network analysis of these 423 genes with altered expression patterns identified the involvement of a cancer related network of genes that were heavily regulated by TNF-alpha in breast tumorigenesis. Our results suggest that epigenetic dysregulation of cellular processes relevant to TNF-alpha-dependent apoptosis may be intimately involved in tumorigenesis in MCF-7 cells.