OBJECTIVETo investigate the effect of all transretinoicacid(ATRA) combined with decitabine (5-Aza-2'-deoxycytidine;DAC) on DNA methylation and gene expression of p16INK4a (p16) and retinoic acid receptor β (RARβ), and to explore their combined anti neoplastic effect on U937 cells and newly diagnose delder acute myeloid leukemia(AML) patients.

METHODSThe expression levels of p16 and RARβ were determined by qRT-PCR and Western blot. Methylation-specific PCR was used to analyze their methylation status. WST-1 and flow cytometry were performed to detect growth inhibition, differentiation, apoptosis and cell cycle of U937 cells respectively.

RESULTSThe expression p16 and RARβ was down-regulated by promoter hypermethylation in newly diagnose delder AML patients and U937 cells. Combination treatment of ATRA and DAC induced DNA hypomethylation as well as gene expression of p16 and RARβ, which contributed to the growth inhibition, differentiation, apoptosis and cell cycle arrest of U937 cells. In addition for elder AML patients intolerable to standard chemotherapy, the combination regimen of ATRA and DAC showed antineoplastic activity accompamied by up-regulation of p16 and RARβ expression and decrease of bone marrow blast, moreover the parients showed good tolerence to the reginen.

CONCLUSIONThe regimen of ATRA combined with DAC as the combination therapeutic strategy for inducing differentiation and demethylation possesses the anti-AML potency, and contributes to optimizing the therapeutic strategy for elder AML patients and promoting the clinical prognosis.

Azacitidine ; analogs & derivatives ; Decitabine ; Humans ; Leukemia, Myeloid, Acute ; Tretinoin ; U937 Cells

OBJECTIVETo investigate the safety and efficacy of decitabine combined with CAG regimen in the treat-ment of newly diagnosed elderly patients with acute myeloid leukemia(AML).

METHODSFourty-nine patients with newly diagnosed acute myeloid leukemia (except M3) who were admitted to our hospital were selected. All the patients were older than 50 years old, and allogeneic hematopoietic stem cell transplantation could not be performed for various reasons. Decitabine-based chemotherapy regimens were used during induction therapy including single decitabine therapy(DAC), decitabine combined with CAG regimen(DAC-CAG) and decitabine combined with HAAG regimen(DAC-HAAG). Most of patients continued to use the original treatment after complete remission, while others were given the standard "3+7" regimen chemotherapy. A total of 2-4 courses of treatment was conducted in the majority of patients.

RESULTSAll of the 49 patients completed the induction therapy, in which 26 cases achieved complete remission(CR), 7 cases achieved partial remission(PR) and no response(NR) existed in 16 cases. The complete remission and the overall response rate(ORR) were 53% and 67% respectively. The overall response rate of DAC group, DAC-CAG group and DAC-HAAG group were 17%, 77% and 63% respectively. 14 patients were infected and 1 patients died of pulmonary infection during the induction therapy. The median number of suspended red blood cells and platelet infused were 9 units and 69 units respectively. Neutrophil recovery time was 15.1 days while the platelet recovery time was 20.1 days during the induction therapy. The mean follow-up time was 21 months. Overall survival(OS) was 75% at 6 months, 30% at 1 year, and 26% at 2 year, while disease-free survival(DFS) was 83% at 3 months, 54% at 1 year, and 47% at 2 year. The induction therapy could reach CR that was an independent prognostic factor, however, the initial white blood cell count, platelet count, age, chemotherapy regimen, prognostic stratification and whether complical by pnenmonia during chemotherapy were not independent prognostic factors.

CONCLUSIONThe induction efficacy of decitabine combined with chemotherapy is superior to that of decitabine alone. The outcome of induction chemotherapy is an independent prognostic factor, however, the high white blood cell count, poor karyotype, complications and AML with myelodysplasia-related changes do not affect long-term survival. DAC-CAG regimen is effective and have relatively few adverse reactions in AML. It is suitable for the patients who are ineligible for conventional chemotherapy.

Aged ; Antineoplastic Combined Chemotherapy Protocols ; Azacitidine ; analogs & derivatives ; Cytarabine ; Decitabine ; Humans ; Induction Chemotherapy ; Leukemia, Myeloid, Acute ; Middle Aged ; Remission Induction ; Treatment Outcome

PURPOSE: Decitabine, a DNA hypomethylating agent, was recently approved for use in Korea for older adults with acute myeloid leukemia (AML) who are not candidates for standard chemotherapy. This study aimed to evaluate the role of decitabine as a first-line treatment for older adults with AML. MATERIALS AND METHODS: Twenty-four patients with AML who received at least one course of decitabine (20 mg/m²/d intravenously for 5 days every 4 weeks) as a first-line therapy at Severance Hospital were evaluated retrospectively. RESULTS: The median age of the patients was 73.5 years. The longest follow-up duration was 502 days. A total of 113 cycles of treatment were given to 24 patients, and the median number of cycles was four (range, 1–14). Thirteen patients dropped out because of death, no or loss of response, patient refusal, or transfer to another hospital. Twenty-one (87.5%) and 12 (50%) patients completed the second and fourth cycles, respectively, and responses to treatment were evaluated in 17. A complete response (CR) or CR with incomplete blood-count recovery was achieved in six (35.3%) patients, and the estimated median overall survival was 502 days. Ten patients developed grade >2 hematologic or non-hematologic toxicities. In univariate analysis, bone marrow blasts, lactate dehydrogenase, serum ferritin level, and bone marrow iron were significantly associated with response to decitabine. CONCLUSION: Five-day decitabine treatment showed acceptable efficacy in older patients with AML who are unfit for conventional chemotherapy, with a CR rate 35.3% and about a median overall survival of 18 months.
Aged ; Antimetabolites, Antineoplastic/administration & dosage/*therapeutic use ; Azacitidine/*analogs & derivatives/therapeutic use ; DNA Methylation ; Female ; Humans ; Leukemia, Myeloid, Acute/*drug therapy/mortality ; Male ; Middle Aged ; Remission Induction ; Republic of Korea ; Retrospective Studies ; Treatment Outcome

OBJECTIVETo evaluate the clinical efficacy and safety of decitabine and cyclosporine for treatment of low-risk and intermediate-risk-1 myelodysplastic syndrome (MDS) patients.

METHODSThe clinical data of 27 cases of low risk and intermediate-risk-1 MDS during the past 3 years in Tongji hospital were analyzed retrospectively. These MDS patients were divided into 2 groups: decitabine group (11 cases) and cyclosporine group (16 cases). The MDS patients in the 2 groups were treated with ultra low dose of decitabine and cyclosporine A; the curetive efficacy and adverse reactions were evaluated.

RESULTSIn the 11 patients with low-risk and intermediate-risk-1 MDS treated with 2 courses of ultra-low-dose decitabine, 4 cases (36.4%) achieved a hematological improvement, 7 cases (63.6%) showed ineffective, including non-remission in 6 cases (54.5 %) and death in 1 patient (9.1%), total effective rate were 36.4%; 3 cases died within the first year and the overall survival (OS) rate was 72.7%. The causes of death mainly were severe myelosuppression and the associated infection and bleeding. In the 16 patients with low-risk and intermediate-risk-1 MDS treated with cyclosporine (CsA), 10 cases (62.5%) achieved a hematological improvement, 6 cases (37.5%) showed ineffective, the total efficiency of 62.5%; no patients died within 1 year, the 1-year OS was 100%. Changes in neutrophils, hemoglobin and platelet were not significantly different between the two group.

CONCLUSIONThe clinical efficacy of decitabine on low-risk and intermediate-risk-1 MDS has not confirmed to be superior to cyclosporine (P = 0.252). However, the side effects of serious infection and myelosuppression were more severe in decitabine group than that in the cyclosporine group. Moreover, the 1-year overall survival rate in decitabine group is much lower than that in the cyclosporine group (P = 0.027). In regard to the small number of cases and short follow-up time in our this study, the more patients and longer follow-up time are needed to study.

Azacitidine ; administration & dosage ; analogs & derivatives ; therapeutic use ; Cyclosporine ; administration & dosage ; therapeutic use ; Humans ; Myelodysplastic Syndromes ; drug therapy ; Pancytopenia ; Retrospective Studies ; Survival Rate ; Treatment Outcome

OBJECTIVETo study the promoter methylation status of SFRP genes and the effect of 5- aza- 2'- deoxycytidine (5- Aza- CdR)induced apoptosis via Wnt/β- catenin pathway by demethylation in Jurkat cells.

METHODSJurkat cells were treated with different concentrations of 5- Aza- CdR. The cell proliferation level of Jurkat cells was detected by MTT assay. Apoptosis was evaluated by flow cytometry. Methylation- spcific PCR (MSP) was used to determine the methylation status of SFRP genes. The expressions of SFRP genes were detected by real time fluorescence quantitative PCR. The mRNA expression levels of survivin, c- myc and cyclin- D1 were analyzed by RT- PCR. Western blot was used to detect the levels of β-catenin protein.

RESULTSCompared with control group, the different concentrations of 5-Aza-CdR could significantly inhibit the proliferation of Jurkat cells in a time-dose dependent manner (P<0.05). After being treated by 5- Aza- CdR for 48 hours, the cell early apoptosis rate in experiment group was significantly higher than that in control group (P<0.05). The promoters of SFRP1, SFRP2, SFRP4, SFRP5 genes were hypermethylation state in the control group, after being treated by 5-Aza-CdR for 72 hours, the brightness of SFRP1, SFRP2, SFRP4, SFRP5 genes' methylation strips weakened in a dose- dependent manner. SFRP mRNA expression increased (P<0.05) when 5- Aza- CdR concentration increased, and the level of β- catenin protein was dampened in a dose- dependent manner (P<0.05). As compared to the control group, the mRNA expressions of associated apoptosis genes survivin, c-myc and cyclin- D1, respectively were obviously down- regulated in a dose- dependent manner (P<0.05).

CONCLUSIONThe effect of demethylation could up- regulate SFRP genes expressions by reversing its hypermethylation and induced apoptosis by down-regulation of β-catenin and associated apoptosis genes.

Apoptosis ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Proliferation ; DNA Methylation ; Down-Regulation ; Gene Expression ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; Jurkat Cells ; Membrane Proteins ; genetics ; Promoter Regions, Genetic ; Wnt Signaling Pathway ; beta Catenin ; metabolism

OBJECTIVETo explore the effect of decitabine on Dickkopf-1 (DKK1) gene expression level and its downstream Wnt signaling pathway in acute myeloid leukemia (AML) cell line HL-60.

METHODSFlow cytometry and DNA ladder analysis were performed to detect apoptosis in HL-60 cell treated with different concentration of decitabine. Methylation-specific polymerase chain reaction (MS-PCR) was used to examine the methylation status of DKK1 gene. The expressions of mRNA and protein were determined by qRT-PCR and Western blot, respectively.

RESULTSFlow cytometric detection showed that after treating HL-60 cell line with decitabine of different concentrations for 48 h, the early apoptosis of HL-60 cells increased significantly as compared with control group (P < 0.05). DNA ladder analysis showed that the DNA ladder and demethylation of DKK1 gene appeared. RT-PCR and Western blot showed that the expressions of mRNA and protein increased. The protein expressions of β-catenin and C-MYC decreased.

CONCLUSIONThe decitabine can promote the apoptosis of HL-60 cells throngh demethylation of DDK1 gene and inhibition of Wnt signalling pathway.

Apoptosis ; Azacitidine ; analogs & derivatives ; pharmacology ; DNA Methylation ; Gene Expression Regulation, Bacterial ; Genes, myc ; HL-60 Cells ; drug effects ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Leukemia, Myeloid, Acute ; pathology ; RNA, Messenger ; Wnt Signaling Pathway ; beta Catenin ; metabolism

No abstract available.
Aged, 80 and over ; Antimetabolites, Antineoplastic/*therapeutic use ; Azacitidine/*analogs & derivatives/therapeutic use ; Biomarkers, Tumor/analysis/genetics ; Biopsy ; Bone Marrow Examination ; Cell Lineage ; Female ; Humans ; Leukemia, Biphenotypic, Acute/*drug therapy/genetics/pathology ; Phenotype ; Remission Induction ; Treatment Outcome

OBJECTIVETo investigate the reversal effects of different concentrations of DNA methylation inhibitor, 5-aza-2-deoxycytidine, on the hypermethylation of maternally expressed gene 3 (MEG3) gene promoter, and then the inhibitory effect of restoration of MEG3 expression on the proliferation of ovarian cancer cells.

METHODSHuman ovarian cancer OVCAR3 cells were treated with various concentration of 5-aza-2-deoxycytidine (0, 1, 5, 10, 20 µmol/L, respectively) for 6 days. Then the methylation status of MEG3 promoter was detected by methylation specific PCR (MSP). The alteration of MEG3 gene expression was detected by RT-PCR. Cell proliferation was determined by MTT assay and EdU incorporation assay.

RESULTSAfter treated with 5-aza-2-deoxycytidine, the methylation status of MEG3 in the 0, 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine groups were 1.00 ± 0.00, 0.79 ± 0.00, 0.67 ± 0.00, 0.65 ± 0.03 and 0.61 ± 0.01 folds, respectively (P < 0.05 for all). The relative expressions of MEG3 mRNA in the 0, 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine groups were 1.00 ± 0.00, 2.04 ± 0.16, 2.44 ± 0.17, 3.19 ± 0.34 and 5.34 ± 0.39, respectively (P < 0.05 for all). In contrast to the negative control, the inhibition rates of the OVCAR3 cell growth were increased significantly when treated with 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine in 2, 4 and 6 days. There were (40.78 ± 0.80)%, (35.65 ± 0.33)%, (31.81 ± 0.66)%, (27.33 ± 1.27)% and (17.75 ± 1.85)% of EdU-positive cells in the 0, 1, 5, 10 and 20 µmol/L 5-aza-2-deoxycytidine groups (P < 0.01 for all).

CONCLUSIONSMaternally expressed gene 3 promoter hypermethylation is reversed by 5-aza-2-deoxycytidine in ovarian cancer cells. The downregulation of MEG3 gene might be resulted from the methylation, and the re-expression of MEG3 partly contribute to the growth inhibition of epithelial ovarian cancer cells.

Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; DNA Methylation ; Female ; Humans ; Neoplasms, Glandular and Epithelial ; Ovarian Neoplasms ; Promoter Regions, Genetic ; RNA, Messenger

OBJECTIVETo investigate the inhibitory effect of classic demethylating drug 5-aza-2'-deoxycytidine (5-Aza-CdR) on the growth of human lung adenocarcinoma cells in nude mouse xenograft models, and to observe its effect on methylation status and expression of TFPI-2 gene in the nude mouse xenograft tissues.

METHODSThe nude mouse xenograft model was established by subcutaneous inoculation of human lung adenocarcinoma A549 cells. According to different doses of 5-Aza-CdR, the tumor-bearing nude mice were randomly divided into experimental groups (0.5 mg/kg group, 1 mg/kg group, 2 mg/kg group) and control group (0 mg/kg group). The tumor growth in the nude mice was observed. The methylation status and the expression of TFPI-2 gene mRNA and protein were detected by methylation specific polymerase chain reaction, real-time fluorescent quantitative polymerase chain reaction and Western blot assay.

RESULTSThe nude mice were euthanized at 28 days after intraperitoneal injection of 5-Aza-CdR. The body weight of tumor-bearing nude mice was (27.12 ± 0.38) g in the 0 mg/kg group, (26.80 ± 0.18) g in the 0.5 mg/kg group, (26.67 ± 0.28) g in the 1 mg/kg group, and (26.50 ± 0.26) g in the 2 mg/kg group, showing no significant difference among them (P > 0.05). The volume of xenograft tumors in the 0 mg/kg group was (709.22 ± 2.87)mm³, (400.67 ± 2.68)mm³ in the 0.5 mg/kg group, (285.71 ± 2.91)mm³ in the 1 mg/kg group, and (230.44 ± 3.15)mm³ in the 2 mg/kg group, showing a significant difference (P < 0.05). There were complete methylation of TFPI-2 gene in the 0 mg/kg group, incomplete methylation in the 0.5 and 1 mg/kg groups, and unmethylation in the 2 mg/kg group. The relative mRNA level in the 0, 0.5, 1, 2 mg/kg groups were 1.00 ± 0.00, 1.67 ± 0.07, 3.40 ± 0.24, and 5.55 ± 0.61, respectively (P < 0.05). The relative expression level of TFPI-2 protein in the 0, 0.5, 1, 2 mg/kg groups was 0.18 ± 0.02, 0.36 ± 0.01, 0.64 ± 0.02, and 0.81 ± 0.20, respectively (P < 0.05).

CONCLUSIONS5-Aza-CdR suppresses the tumor growth of human lung adenocarcinoma cells in nude mouse xenograft models, and induces expression of TFPI-2 gene in the xenograft tumor cells. The mechanism might be that 5-Aza-CdR induces re-expression of demethylated TFPI-2 gene by demethylation, and thus inhibits the growth and proliferation of human lung adenocarcinoma cells.

Adenocarcinoma ; drug therapy ; pathology ; Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Proliferation ; drug effects ; DNA Methylation ; Disease Models, Animal ; Gene Expression Regulation, Neoplastic ; Glycoproteins ; genetics ; Heterografts ; Humans ; Lung Neoplasms ; drug therapy ; pathology ; Mice ; Mice, Nude ; RNA, Messenger ; metabolism ; Random Allocation

OBJECTIVETo evaluate significance of ID4 gene mehtylation in demethylating myelodysplastic syndrome(MDS) cell Line MUTZ1 and 2 patients with MDS.

METHODSThe methylation-specific PCR (MS-PCR) and reverse transcription-PCR (RT-PCR) were applied to identify the methylation status and gene expression of ID4 gene in MDS cell line MUTZ1, a patient with aplastic anemia(AA) and a donor with normal bone marrow (NBM). RT-PCR was applied to detect the ID4 gene expression status in MUTZ1 cell line treated with decitabine at 3 different concentrations. Then bisulfite sequencing PCR (BSP) was applied to detect ID4 gene methylation status in 2 MDS parients treated with decitabine.

RESULTSThe MDS cell line MUTZ-1 displayed a complete methylation of ID4 gene promoter with little mRNA expression. Inversely, bone marrow of an AA patient and NBM showed complete unmethylation of this gene with intensity mRNA expression. With the increase of decitabine concentration, ID4 gene mRNA expression was more and more increased. After decitabine treatment, ID4 gene methylation-positive frequencies of both the 2 MDS patients were much more decreased than that of the first treatment. So, ID4 gene mRNA expression inhibited by promoter hypemethylation could be recovered by using demethylation medicine.

CONCLUSIONID4 as a new potential anti-oncogene suggests that its methylation may become a marker for selection and assessment of therapeutic schedules in patients with MDS.

Anemia, Aplastic ; Azacitidine ; analogs & derivatives ; Bone Marrow ; Cell Line ; DNA Methylation ; Gene Expression ; Genes, Tumor Suppressor ; Humans ; Inhibitor of Differentiation Proteins ; Myelodysplastic Syndromes ; Polymerase Chain Reaction ; Promoter Regions, Genetic