AIM: To study the effect of 5-Aza-2’-deoxycytidine (5-Aza-CdR) on heat shock protein 70 (HSP70), human leucocyte antigen-Ⅰ (HLA-Ⅰ) and NY-ESO-1 proteins in exosomes produced by hepatoma cells, HepG2 and Hep3B. METH...AIM: To study the effect of 5-Aza-2’-deoxycytidine (5-Aza-CdR) on heat shock protein 70 (HSP70), human leucocyte antigen-Ⅰ (HLA-Ⅰ) and NY-ESO-1 proteins in exosomes produced by hepatoma cells, HepG2 and Hep3B. METHODS: Exosomes derived from HepG2 and Hep3B cells treated with or without 5-aza-CdR were isolated and purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The number of exosomes was counted under electron microscope. Concentration of proteins in exosomes was measured by bicinchoninic acid protein assay. Expression of HSP70, HLA-Ⅰ and NY-ESO-1 proteins in exosomes was detected by Western blotting and immunoelectron microscopy. mRNA expression of p53 gene was detected by reverse transcription polymerase chain reaction.RESULTS: The mRNA expression of p53 gene was increased in both hepatoma cell lines after treatment with 5-Aza-CdR. The number of exosomes and the concentration of total proteins in exosomes were increased signifi cantly after treatment with 5-aza-CdR (P < 0.05). After treatment with 5-Aza-CdR, immunoelectron microscopy and Western blotting showed that the HSP70, HLA-Ⅰ and NY-ESO-1 proteins were increased in exosomes produced by both hepatoma cell lines. CONCLUSION: 5-aza-CdR, an inhibitor of DNA methyltransferase, can increase exosomes produced by hepatoma cells and immune-associated protein component of exosomes, which may be mediated by p53 gene upregulation and 5-Aza-CdR demethylation.展开更多
AIM: To investigate the effect of 5-Aza-2’-deoxycytidine(5-Aza-CdR),a DNA methyltransferase(DNMT) inhibitor,on the growth and survival of the Chinese retinoblastoma(RB) cell line HXO-RB44. ·METHODS: The DNA meth...AIM: To investigate the effect of 5-Aza-2’-deoxycytidine(5-Aza-CdR),a DNA methyltransferase(DNMT) inhibitor,on the growth and survival of the Chinese retinoblastoma(RB) cell line HXO-RB44. ·METHODS: The DNA methylation status of the Ras association domain family(RASSF1A) promoter in the presence of 5-Aza-CdR at different concentrations was analyzed by methylation-specific polymerase chain reaction(MSP). RASSF1A mRNA and protein levels were measured by semiquantitative RT-PCR and immunohistochemistry staining,respectively,when cells were treated with 5.0μmol/L of 5-Aza-CdR. The effect of 5.0μmol/L 5-Aza-CdR on the proliferation and viability of HXO-RB44 cells was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and flow cytometry. ·RESULTS: 5-Aza-CdR efficiently induced cell cycle arrest at G0 /G1 and apoptotic death in HXO-RB44 cells. MSP analysis showed that unmethylated RASSF1A DNA increased and methylated RASSF1A decreased in a dose-dependent manner in a range of 0.5-5.0μmol/L 5-Aza-CdR. Accordingly,RASSF1A expression was reactivated at both mRNA and protein levels. Incubation time of 5-Aza-CdR treatment also functioned as a factor for the demethylation status of RASSF1A promoter DNA,with a plateau on day four. 5-Aza-CdR at 5.0μmol/L completely demethylated the RASSF1A promoter in HXORB44 cells on day four,and as a result,RASSF1A expression increased significantly from day 4 to day 7.·CONCLUSION: 5-Aza-CdR inhibits the growth of the HXO-RB44 RB cell line and induces apoptosis by demethylating the RASSF1A gene.展开更多
The non-classical HLA class I antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells (DC). As a result, HLA-G expression in malignant cells may provide them wit...The non-classical HLA class I antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells (DC). As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2’-deoXycytidine (5-AC) and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5’ regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1 A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components (APM) as well as β2 microglobulin (β2-m) expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1 A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1 A cells.展开更多
Objective： To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-Cdr） and trichostatin A （TSA） combined with p53-expressing adenovirus （Ad-p53） on Hep-2 cell line in vivo and in vitro, in order to explor...Objective： To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-Cdr） and trichostatin A （TSA） combined with p53-expressing adenovirus （Ad-p53） on Hep-2 cell line in vivo and in vitro, in order to explore its possibility in biological treatment of laryngocarcinoma. Methods： Effects of 5-Aza-Cdr and TSA in combination with Ad-p53 on Hep-2 cell line in vivo were determined by Cell Counting Kit-8 （CCK-8） assay. The effect of drug combination was calculated by Jin＇s formula. Effects on the cell line in vitro were investigated by establishing the nude mice model. Results： 5-Aza-Cdr and TSA showed inhibitory effects on the proliferation of Hep-2 cells in dose- and time-dependent manner. Ad-p53 can inhibit the growth of Hep-2 cells in vivo and in vitro. However, the combination of epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 was less effective than individual use of Ad-p53. 5-Aza-Cdr and Ad-p53 inhibited the growth of transplanted tumors and reduced the volume of tumors, and the tumor volume of Ad-p53 group was significantly smaller than that of the control group （P0.05）. Conclusion： Both epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 can suppress cell proliferation on Hep-2 in vivo and in vitro and there may be some antagonistic mechanism between Ad-p53 and epigenetic reagents （5-Aza-Cdr/ TSA）.展开更多
Objective： hMLH1 protein serves to detect the DNA damage caused by cisplatin （DDP） and destroys the cell. The absence of hMLH1 expression has been correlated with acquired resistance of ovarian cancer cells to plat...Objective： hMLH1 protein serves to detect the DNA damage caused by cisplatin （DDP） and destroys the cell. The absence of hMLH1 expression has been correlated with acquired resistance of ovarian cancer cells to platinum. The aim of this study was to determine the possible role of DNA methylation and histone H3 lysine 9 （H3-K9） acetylation on the loss of hMLH1 expression, and to evaluate the reversal effects of 5-Aza-2＇-deoxycytidine （5-Aza-dC） and Trichostatin A （TSA） on DDP-resistance in ovarian cancer cell lines. Methods： Two human ovarian cancer cell lines, COC1 and its DDP-resistant subline, COCI/DDP were cultured. The two cancer cells were treated with 5-Aza-dC or TSA. Using COC1 cells as a control, we used methylation-specific PCR （MSP） to analyze DNA methylation at hMLHI gene promoter, hMLH1 mRNA and protein expressions were analyzed by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blot. Chromatin immunoprecipitation assay （CHIP） was used to test the levels of histone H3-K9 acetylation at hMLH1 gene promoter. Results： In COC1 cells, there was no DNA methylation at hMLH1 gene promoter, while there were hMLH1 mRNA and protein expression. In COC1/DDP cells, there was DNA hypermethylation at hMLH1 gene promoter, while there was no hMLH1 mRNA or protein expression. The treatment with 5-Aza-dC resulted in DNA demethylation at the promoter region, as well as restoration of hMLH1 expression in COC1/DDP cells. The treatment with TSA had no effects on DNA demethylation or restoration of hMLH1 expression in COC1/DDP cells. Conclusion： Hypermethylation of DNA at the promoter is related to the silencing of hMLH1 in COC1/DDP ovarian cancer cells. DNA methylation at hMLH1 promoter could play a significant role in determining the sensitivity of ovarian cancer to DDP. The drug resistance mediated by methylation of hMLH1 could be overcome by 5-Aza-dC.展开更多
Objective: To study the effect of 5-Aza-2’ -deoxycytidine (5-Aza-cdR) on tumour suppressor gene p16 expres- sion in hepatocellular carcinoma cells. Method: Expression of pl6 mRNA and protein in hepatocellular carcino...Objective: To study the effect of 5-Aza-2’ -deoxycytidine (5-Aza-cdR) on tumour suppressor gene p16 expres- sion in hepatocellular carcinoma cells. Method: Expression of pl6 mRNA and protein in hepatocellular carcinoma cell lines SMMC-7721 and HePG2 before and after treatment with 5-Aza-cdR were analyzed via reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistrty Results: The expression levels of p16 mRNA and protein were increased dramatically after treatment with 5-Aza-cdR. Conclusion: Our data show that, 5-Aza-2’ -deoxycytidine can increase the expression of pl6 gene both at transcription and translation. The findings suggested that 5-Aza-cdR may reactivate the pl6 gene by demethylation.展开更多
The pyrimidine analog, 5-aza-2’-deoxycytidine (5-aza-dC) is a DNA methyltransferase inhibitor that triggers DNA demethylation leading to the reactivation of epigenetically silenced tumor suppressor genes. To understa...The pyrimidine analog, 5-aza-2’-deoxycytidine (5-aza-dC) is a DNA methyltransferase inhibitor that triggers DNA demethylation leading to the reactivation of epigenetically silenced tumor suppressor genes. To understand the shift in gene expression which mediates the beneficial 5-aza-dC effects in leukemia, we have treated human myeloid derived leukemic cells with 5-aza-dC. Target genes were identified first in MV4-11 cells using a genome-wide gene expression profiling assay to detect differences in treated and untreated cells. From this analysis six genes were identified (HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2) as being significantly different expressed after treatment. To validate microarray data, we performed quantitative PCR on these genes from multiple leukemic cells. The results suggest that these genes are epigenetically regulated indicating that dysregulation of HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2 expression may play an important role in establishing the malignant phenotype in AML.展开更多
基金Supported by Capital Medical Development Scientif ic Research Fund, No. 2005-3086
文摘AIM: To study the effect of 5-Aza-2’-deoxycytidine (5-Aza-CdR) on heat shock protein 70 (HSP70), human leucocyte antigen-Ⅰ (HLA-Ⅰ) and NY-ESO-1 proteins in exosomes produced by hepatoma cells, HepG2 and Hep3B. METHODS: Exosomes derived from HepG2 and Hep3B cells treated with or without 5-aza-CdR were isolated and purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The number of exosomes was counted under electron microscope. Concentration of proteins in exosomes was measured by bicinchoninic acid protein assay. Expression of HSP70, HLA-Ⅰ and NY-ESO-1 proteins in exosomes was detected by Western blotting and immunoelectron microscopy. mRNA expression of p53 gene was detected by reverse transcription polymerase chain reaction.RESULTS: The mRNA expression of p53 gene was increased in both hepatoma cell lines after treatment with 5-Aza-CdR. The number of exosomes and the concentration of total proteins in exosomes were increased signifi cantly after treatment with 5-aza-CdR (P < 0.05). After treatment with 5-Aza-CdR, immunoelectron microscopy and Western blotting showed that the HSP70, HLA-Ⅰ and NY-ESO-1 proteins were increased in exosomes produced by both hepatoma cell lines. CONCLUSION: 5-aza-CdR, an inhibitor of DNA methyltransferase, can increase exosomes produced by hepatoma cells and immune-associated protein component of exosomes, which may be mediated by p53 gene upregulation and 5-Aza-CdR demethylation.
基金Supported by the National Natural Science Foundation of China(No.3087282No.81072221)
文摘AIM: To investigate the effect of 5-Aza-2’-deoxycytidine(5-Aza-CdR),a DNA methyltransferase(DNMT) inhibitor,on the growth and survival of the Chinese retinoblastoma(RB) cell line HXO-RB44. ·METHODS: The DNA methylation status of the Ras association domain family(RASSF1A) promoter in the presence of 5-Aza-CdR at different concentrations was analyzed by methylation-specific polymerase chain reaction(MSP). RASSF1A mRNA and protein levels were measured by semiquantitative RT-PCR and immunohistochemistry staining,respectively,when cells were treated with 5.0μmol/L of 5-Aza-CdR. The effect of 5.0μmol/L 5-Aza-CdR on the proliferation and viability of HXO-RB44 cells was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and flow cytometry. ·RESULTS: 5-Aza-CdR efficiently induced cell cycle arrest at G0 /G1 and apoptotic death in HXO-RB44 cells. MSP analysis showed that unmethylated RASSF1A DNA increased and methylated RASSF1A decreased in a dose-dependent manner in a range of 0.5-5.0μmol/L 5-Aza-CdR. Accordingly,RASSF1A expression was reactivated at both mRNA and protein levels. Incubation time of 5-Aza-CdR treatment also functioned as a factor for the demethylation status of RASSF1A promoter DNA,with a plateau on day four. 5-Aza-CdR at 5.0μmol/L completely demethylated the RASSF1A promoter in HXORB44 cells on day four,and as a result,RASSF1A expression increased significantly from day 4 to day 7.·CONCLUSION: 5-Aza-CdR inhibits the growth of the HXO-RB44 RB cell line and induces apoptosis by demethylating the RASSF1A gene.
文摘The non-classical HLA class I antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells (DC). As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2’-deoXycytidine (5-AC) and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5’ regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1 A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components (APM) as well as β2 microglobulin (β2-m) expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1 A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1 A cells.
基金supported by the National Natural Science Foundation of China (No.30772407)
文摘Objective： To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-Cdr） and trichostatin A （TSA） combined with p53-expressing adenovirus （Ad-p53） on Hep-2 cell line in vivo and in vitro, in order to explore its possibility in biological treatment of laryngocarcinoma. Methods： Effects of 5-Aza-Cdr and TSA in combination with Ad-p53 on Hep-2 cell line in vivo were determined by Cell Counting Kit-8 （CCK-8） assay. The effect of drug combination was calculated by Jin＇s formula. Effects on the cell line in vitro were investigated by establishing the nude mice model. Results： 5-Aza-Cdr and TSA showed inhibitory effects on the proliferation of Hep-2 cells in dose- and time-dependent manner. Ad-p53 can inhibit the growth of Hep-2 cells in vivo and in vitro. However, the combination of epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 was less effective than individual use of Ad-p53. 5-Aza-Cdr and Ad-p53 inhibited the growth of transplanted tumors and reduced the volume of tumors, and the tumor volume of Ad-p53 group was significantly smaller than that of the control group （P0.05）. Conclusion： Both epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 can suppress cell proliferation on Hep-2 in vivo and in vitro and there may be some antagonistic mechanism between Ad-p53 and epigenetic reagents （5-Aza-Cdr/ TSA）.
文摘Objective： hMLH1 protein serves to detect the DNA damage caused by cisplatin （DDP） and destroys the cell. The absence of hMLH1 expression has been correlated with acquired resistance of ovarian cancer cells to platinum. The aim of this study was to determine the possible role of DNA methylation and histone H3 lysine 9 （H3-K9） acetylation on the loss of hMLH1 expression, and to evaluate the reversal effects of 5-Aza-2＇-deoxycytidine （5-Aza-dC） and Trichostatin A （TSA） on DDP-resistance in ovarian cancer cell lines. Methods： Two human ovarian cancer cell lines, COC1 and its DDP-resistant subline, COCI/DDP were cultured. The two cancer cells were treated with 5-Aza-dC or TSA. Using COC1 cells as a control, we used methylation-specific PCR （MSP） to analyze DNA methylation at hMLHI gene promoter, hMLH1 mRNA and protein expressions were analyzed by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blot. Chromatin immunoprecipitation assay （CHIP） was used to test the levels of histone H3-K9 acetylation at hMLH1 gene promoter. Results： In COC1 cells, there was no DNA methylation at hMLH1 gene promoter, while there were hMLH1 mRNA and protein expression. In COC1/DDP cells, there was DNA hypermethylation at hMLH1 gene promoter, while there was no hMLH1 mRNA or protein expression. The treatment with 5-Aza-dC resulted in DNA demethylation at the promoter region, as well as restoration of hMLH1 expression in COC1/DDP cells. The treatment with TSA had no effects on DNA demethylation or restoration of hMLH1 expression in COC1/DDP cells. Conclusion： Hypermethylation of DNA at the promoter is related to the silencing of hMLH1 in COC1/DDP ovarian cancer cells. DNA methylation at hMLH1 promoter could play a significant role in determining the sensitivity of ovarian cancer to DDP. The drug resistance mediated by methylation of hMLH1 could be overcome by 5-Aza-dC.
文摘Objective: To study the effect of 5-Aza-2’ -deoxycytidine (5-Aza-cdR) on tumour suppressor gene p16 expres- sion in hepatocellular carcinoma cells. Method: Expression of pl6 mRNA and protein in hepatocellular carcinoma cell lines SMMC-7721 and HePG2 before and after treatment with 5-Aza-cdR were analyzed via reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistrty Results: The expression levels of p16 mRNA and protein were increased dramatically after treatment with 5-Aza-cdR. Conclusion: Our data show that, 5-Aza-2’ -deoxycytidine can increase the expression of pl6 gene both at transcription and translation. The findings suggested that 5-Aza-cdR may reactivate the pl6 gene by demethylation.
文摘The pyrimidine analog, 5-aza-2’-deoxycytidine (5-aza-dC) is a DNA methyltransferase inhibitor that triggers DNA demethylation leading to the reactivation of epigenetically silenced tumor suppressor genes. To understand the shift in gene expression which mediates the beneficial 5-aza-dC effects in leukemia, we have treated human myeloid derived leukemic cells with 5-aza-dC. Target genes were identified first in MV4-11 cells using a genome-wide gene expression profiling assay to detect differences in treated and untreated cells. From this analysis six genes were identified (HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2) as being significantly different expressed after treatment. To validate microarray data, we performed quantitative PCR on these genes from multiple leukemic cells. The results suggest that these genes are epigenetically regulated indicating that dysregulation of HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2 expression may play an important role in establishing the malignant phenotype in AML.
