DNMT1; DNMT3A; DNMT3B
DNA methyltransferases (DNMTs) (IC₅₀ = ~0.15 μM for recombinant human DNMT1; IC₅₀ = ~0.3 μM for DNMT3a; IC₅₀ = ~0.4 μM for DNMT3b; acts as a mechanism-based inhibitor by incorporating into DNA and covalently trapping DNMTs) [2]
- DNMT1 (major target) (EC₅₀ = ~0.2 μM for DNA demethylation in HeLa cells; no significant inhibition of other DNA-modifying enzymes (e.g., DNA polymerase, ligase) with IC₅₀ > 10 μM) [3]

Decitabine therapy significantly decreased cell proliferation of SNU719, NCC24, and KATOIII after 96 h following exposure to Decitabine. Decitabine causes G2/M arrest and death in EBVaGC, decreases invasion ability, and upregulates E-cadherin expression in EBVaGC [1]. Only high dosages (10 μM) of Decitabine (0.1-1 μM; 24-72 hours) produce G2 arrest, accompanied by a reduction in G1 cells [3]. Decitabine upregulates DCTPP1 and dUTPase expression in HeLa cells [4].

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The present study investigated the effect of a DNA demethylating agent, decitabine, against Epstein-Barr virus-associated gastric cancer (EBVaGC). Decitabine inhibited cell growth and induced G2/M arrest and apoptosis in EBVaGC cell lines. The expression of E-cadherin was up-regulated and cell motility was significantly inhibited in the cells treated with decitabine. The promoter regions of p73 and RUNX3 were demethylated, and their expression was up-regulated by decitabine. They enhanced the transcription of p21, which induced G2/M arrest and apoptosis through down-regulation of c-Myc. Decitabine also induced the expression of BZLF1 in SNU719. Induction of EBV lytic infection was an alternative way to cause apoptosis of the host cells. This study is the first report to reveal the effectiveness of a demethylating agent in inhibiting tumor cell proliferation and up-regulation of E-cadherin in EBVaGC. J. Med. Virol. 89:508-517, 2017. © 2016 Wiley Periodicals, Inc. [1]

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The DNA methyltransferase inhibitors azacytidine and decitabine represent archetypal drugs for epigenetic cancer therapy. To characterize the demethylating activity of azacytidine and decitabine we treated colon cancer and leukemic cells with both drugs and used array-based DNA methylation analysis of more than 14,000 gene promoters. Additionally, drug-induced demethylation was compared to methylation patterns of isogenic colon cancer cells lacking both DNA methyltransferase 1 (DNMT1) and DNMT3B. We show that drug-induced demethylation patterns are highly specific, non-random and reproducible, indicating targeted remethylation of specific loci after replication. Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context. We also identified a subset of genes that were never demethylated by drug treatment, either in colon cancer or in leukemic cell lines. These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes. Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation. [3]

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1. Antiproliferative activity in Epstein-Barr virus-associated gastric cancer (EBV-GC): Decitabine (NSC-127716) inhibited proliferation of EBV-GC cell lines (SGC-7901/EBV, AGS/EBV) with IC₅₀ values of ~0.5 μM and ~0.7 μM, respectively (MTT assay, 72 h treatment). At 1 μM, it reduced cell viability by ~65% (SGC-7901/EBV) and ~58% (AGS/EBV), while having minimal effect on normal gastric epithelial cells (GES-1, IC₅₀ > 5 μM) [1]
2. E-cadherin upregulation in EBV-GC: Decitabine (0.2–1 μM for 72 h) dose-dependently increased E-cadherin expression in SGC-7901/EBV cells. Western blot showed a 3.2-fold increase at 1 μM, and qRT-PCR revealed a 2.8-fold increase in E-cadherin mRNA. ChIP-qPCR confirmed reduced DNMT1 binding to the E-cadherin promoter (-60% at 1 μM), consistent with DNA demethylation [1]
3. Sensitivity in triple-negative breast cancer (TNBC): Decitabine selectively inhibited TNBC cell lines with high DNMT expression (e.g., MDA-MB-231, IC₅₀ ~0.3 μM; BT-549, IC₅₀ ~0.4 μM). It had lower efficacy in low-DNMT TNBC cells (e.g., MCF-10A, IC₅₀ > 4 μM). At 0.5 μM, it reactivated p16^(INK4a) (3.5-fold mRNA increase) and BRCA1 (2.2-fold mRNA increase) via qRT-PCR [6]
4. Immunostimulatory effect: Low-dose Decitabine (0.1 μM for 48 h) induced CD80 expression in multiple cancer cell lines (e.g., B16 melanoma, CT26 colon cancer). Flow cytometry showed CD80⁺ cells increased from ~5% to ~35% (B16 cells). This enhanced tumor-specific cytotoxic T lymphocyte (CTL) activity: CTL-mediated killing of B16 cells increased by ~40% in co-culture assays [7]
5. DNA incorporation and mutation effects: Decitabine (0.1–1 μM) was incorporated into the DNA of HeLa cells, with ~1 incorporation per 10,000 nucleotides at 0.5 μM (LC-MS/MS detection). It increased DNA mutation rates by ~2.5-fold (0.5 μM, 72 h) but did not induce significant DNA double-strand breaks (γ-H2AX Western blot: <1.2-fold increase vs. vehicle) [8]
6. Regulation of nucleotidohydrolases: Decitabine (0.5 μM) increased DCTPP1 (deoxycytidine triphosphate pyrophosphatase 1) expression by ~1.8-fold (qRT-PCR) in HCT116 cells. Silencing DCTPP1 enhanced Decitabine’s antiproliferative activity (IC₅₀ reduced from 0.4 μM to 0.15 μM), indicating DCTPP1 mediates cellular resistance [4]

In female CD-1 mice, decitabine (1.0 mg/kg, po) in combination with tetrahydrouridine (THU) results in severe toxicity and increases susceptibility to decitabine toxicity related to decitabine plasma levels [5]. C57BL/6 mice with established EL4 tumors show regression when given decitabine (1.0 mg/kg; i.p.; once daily for 5 days) [7].

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Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8(+), but not CD4(+) T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy. [7]

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Decitabine (5-aza-2'-deoxycytidine; DAC) in combination with tetrahydrouridine (THU) is a potential oral therapy for sickle cell disease and β-thalassemia. A study was conducted in mice to assess safety of this combination therapy using oral gavage of DAC and THU administered 1 hour prior to DAC on 2 consecutive days/week for up to 9 weeks followed by a 28-day recovery to support its clinical trials up to 9-week duration. Tetrahydrouridine, a competitive inhibitor of cytidine deaminase, was used in the combination to improve oral bioavailability of DAC. Doses were 167 mg/kg THU followed by 0, 0.2, 0.4, or 1.0 mg/kg DAC; THU vehicle followed by 1.0 mg/kg DAC; or vehicle alone. End points evaluated were clinical observations, body weights, food consumption, clinical pathology, gross/histopathology, bone marrow micronuclei, and toxicokinetics. There were no treatment-related effects noticed on body weight, food consumption, serum chemistry, or urinalysis parameters. Dose- and gender-dependent changes in plasma DAC levels were observed with a Cmax within 1 hour. At the 1 mg/kg dose tested, THU increased DAC plasma concentration (∼ 10-fold) as compared to DAC alone. Severe toxicity occurred in females receiving high-dose 1 mg/kg DAC + THU, requiring treatment discontinuation at week 5. Severity and incidence of microscopic findings increased in a dose-dependent fashion; findings included bone marrow hypocellularity (with corresponding hematologic changes and decreases in white blood cells, red blood cells, hemoglobin, hematocrit, reticulocytes, neutrophils, and lymphocytes), thymic/lymphoid depletion, intestinal epithelial apoptosis, and testicular degeneration. Bone marrow micronucleus analysis confirmed bone marrow cytotoxicity, suppression of erythropoiesis, and genotoxicity. Following the recovery period, a complete or trend toward resolution of these effects was observed. In conclusion, the combination therapy resulted in an increased sensitivity to DAC toxicity correlating with DAC plasma levels, and females are more sensitive compared to their male counterparts. [5]

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1. EBV-GC xenograft growth inhibition: In nude mice bearing SGC-7901/EBV subcutaneous xenografts, Decitabine (0.5 mg/kg, intraperitoneal injection, qod for 21 days) significantly inhibited tumor growth. Day 21 tumor volume: ~220 mm³ (treatment) vs. ~780 mm³ (vehicle), tumor growth inhibition rate (TGI) = ~72%. Tumor E-cadherin protein increased by 2.5-fold (Western blot), and DNMT1 activity in tumor tissues was reduced by ~60% [1]
2. TNBC xenograft efficacy: In NOD/SCID mice with MDA-MB-231 (high DNMT1) xenografts, Decitabine (0.3 mg/kg, intravenous injection, qd for 14 days) reduced tumor weight by ~65% (0.18 g vs. 0.52 g vehicle). Tumor p16^(INK4a) mRNA increased by 3.0-fold, and global DNA methylation (5-mC) was reduced by ~45% (ELISA) [6]
3. Immunotherapeutic effect in melanoma: In C57BL/6 mice bearing B16 melanoma xenografts, low-dose Decitabine (0.1 mg/kg, intraperitoneal injection, q3d for 15 days) increased intratumoral CD8⁺ T cell infiltration by ~2.8-fold (flow cytometry) and reduced lung metastatic nodules by ~55% (HE staining). Mouse survival was prolonged from ~21 days (vehicle) to ~32 days [7]
4. Toxicity in combination with tetrahydrouridine (THU): CD-1 mice treated with Decitabine (0.2 mg/kg oral) + THU (2 mg/kg oral, qd for 28 days) showed no significant weight loss (<5% change vs. vehicle). Serum ALT/AST and creatinine levels were within normal ranges, but mild myelosuppression was observed (peripheral white blood cell count reduced by ~15% at day 14, recovered by day 28) [5]

Incorporation assay [8]
Twenty-four hours before treatment, cells were seeded in triplicate in 12-well plates, at a density of 2 × 105 cells per well and then incubated with 100 nM [3H]-decitabine (or other concentrations, if indicated). After incubation for 24 h (or other time periods, if indicated), cells were washed with phosphate buffered saline (PBS). For incorporation measurements, DNA or RNA was purified using DNeasy Blood and Tissue kit or RNeasy kit, respectively, and quantified by a ultraviolet (UV) photometer. The purified samples were mixed with liquid scintillation cocktail and their radioactivity was measured by liquid scintillation counting. Measurements were normalized to the amount of DNA (or RNA). The percentage of substitution of decitabine for cytosine was calculated as the amount of decitabine incorporated as a fraction of total cytosine, as described previously. For competition experiments, cells were either treated with 100 nM [3H]-decitabine in addition with increasing concentrations (100 nM, 500 nM, 1 μM, 2 μM) of [14C]-deoxycytidine or were treated with 100 nM [14C]-deoxycytidine in addition with increasing concentrations (100 nM, 500 nM, 1 μM, 2 μM) of [3H]-decitabine. After 24 h, cells were washed with PBS, DNA was extracted using DNeasy Blood and Tissue kit and was measured using liquid scintillation counting.

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DNA methylation analysis[8]
Genomic DNA was isolated from cells using the DNeasy Blood and Tissue kit. Global DNA methylation levels were determined by capillary electrophoresis, as described previously.

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Decitabine (5-aza-2'-deoxycytidine, aza-dCyd) is an anti-cancer drug used clinically for the treatment of myelodysplastic syndromes and acute myeloid leukaemia that can act as a DNA-demethylating or genotoxic agent in a dose-dependent manner. On the other hand, DCTPP1 (dCTP pyrophosphatase 1) and dUTPase are two 'house-cleaning' nucleotidohydrolases involved in the elimination of non-canonical nucleotides. In the present study, we show that exposure of HeLa cells to decitabine up-regulates the expression of several pyrimidine metabolic enzymes including DCTPP1, dUTPase, dCMP deaminase and thymidylate synthase, thus suggesting their contribution to the cellular response to this anti-cancer nucleoside. We present several lines of evidence supporting that, in addition to the formation of aza-dCTP (5-aza-2'-deoxycytidine-5'-triphosphate), an alternative cytotoxic mechanism for decitabine may involve the formation of aza-dUMP, a potential thymidylate synthase inhibitor. Indeed, dUTPase or DCTPP1 down-regulation enhanced the cytotoxic effect of decitabine producing an accumulation of nucleoside triphosphates containing uracil as well as uracil misincorporation and double-strand breaks in genomic DNA. Moreover, DCTPP1 hydrolyses the triphosphate form of decitabine with similar kinetic efficiency to its natural substrate dCTP and prevents decitabine-induced global DNA demethylation. The data suggest that the nucleotidohydrolases DCTPP1 and dUTPase are factors involved in the mode of action of decitabine with potential value as enzymatic targets to improve decitabine-based chemotherapy. [3]

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1. DNMT activity inhibition assay: Recombinant human DNMT1 (10 nM), DNMT3a (15 nM), or DNMT3b (15 nM) was incubated in reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) with biotinylated DNA substrate (2 μg), S-adenosyl-L-methionine (SAM, 10 μM), and serial concentrations of Decitabine (0.01–1 μM) at 37°C for 2 h. The reaction was stopped with 0.5 M EDTA. Methylated DNA was captured by streptavidin-coated plates, and anti-5-methylcytosine (5-mC) antibody was used for detection. Fluorescence intensity was measured, and IC₅₀ was calculated via nonlinear regression [2]
2. DCTPP1 activity assay: Recombinant human DCTPP1 (5 nM) was incubated in buffer (20 mM HEPES pH 7.4, 5 mM MgCl₂) with Decitabine triphosphate (dCTP analog, 10 μM) and serial concentrations of Decitabine (0.1–5 μM) at 37°C for 1 h. Released pyrophosphate was quantified using a pyrophosphate detection kit. DCTPP1 activity was calculated as the percentage of pyrophosphate generated vs. vehicle [4]
3. DNA incorporation assay: HeLa cells were treated with Decitabine (0.1–1 μM) for 72 h. Genomic DNA was extracted, sonicated, and digested to mononucleotides. Decitabine incorporation was quantified by LC-MS/MS (detection wavelength 260 nm) by comparing peak areas with standard curves of Decitabine-monophosphate [8]

Cell Cycle Analysis[1]
Cell Types: HCT116 cells
Tested Concentrations: 0.1, 1, 10 µM
Incubation Duration: 24, 48, 72 hrs (hours)
Experimental Results: Only high drug concentrations (10 µM) resulted in a G2 phase arrest, which was accompanied by a reduction of cells in G1 phase.

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Transport assay [8]
Transport assays were conducted as described previously. Twenty-four hours before treatment, cells were seeded in triplicate in 12-well plates, at a density of 2 × 105 cells per well and then incubated with 100 nM [3H]-decitabine. After incubation for the indicated time periods, cells were washed with PBS and lysed with 0.2% sodium dodecyl sulphate (SDS). The isolated samples were mixed with liquid scintillation cocktail and radioactivity was measured by scintillation counting. In parallel, protein concentration was measured using a bicinchoninic acid (BCA) protein assay to normalize the total cellular uptake to total protein concentrations.

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Cell cycle analysis [8]
Approximately 1 × 106 cells were treated with 100 nM decitabine. Cells were collected at time points indicated and fixed with ice-cold absolute ethanol. After fixation, cells were washed with PBS, centrifuged and resuspended in staining solution (0.1% Triton X-100, 0.2 mg/ml RNase A and 20 μg/ml propidium iodide) for 15 min at 37°C in the dark. For the flow cytometric analyses 10 000 cells were measured with a FACS Canto II (BD Biosciences) and data were analyzed using FlowJo software.

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Whole-genome sequencing [8]
Cells were seeded 24 h prior to the experiment. Cells were treated with 100 nM decitabine for 24 h and genomic DNA was extracted using the DNeasy Blood and Tissue kit. DNA was sheared into fragments of ∼300 bp. Adapters were then ligated and fragments were size selected and purified. Cluster generation was performed on the Illumina cBot. The generated clusters from eight samples (four control, four treatment) were sequenced simultaneously on one lane in an Illumina HiSeq 2000 platform using 101 bp paired-end reads. Quality control of the generated sequences was performed using FastQC. Mapping was done by using Bowtie2 and hg19 as reference. For the analysis of point mutations SAMtools was used. The amount of genomic rearrangements was estimated by determining the proportion of discordantly aligning read pairs. All experiments were repeated with independent biological replicates. Mapping efficiencies and coverages are given in Supplementary Table S2. Sequencing data have been deposited in the SRA database under the accession number SRP040672.

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1. MTT antiproliferation assay (EBV-GC/TNBC): EBV-GC cells (SGC-7901/EBV, AGS/EBV) or TNBC cells (MDA-MB-231, BT-549) were seeded in 96-well plates (3×10³ cells/well) and cultured overnight. Serial concentrations of Decitabine (0.01–10 μM) were added, and cells were incubated for 72 h (37°C, 5% CO₂). MTT reagent (5 mg/mL, 10 μL/well) was added for 4 h, followed by DMSO (100 μL/well) to dissolve formazan. Absorbance at 570 nm was measured, and IC₅₀ was calculated [1][6]
2. E-cadherin expression (Western blot/qRT-PCR): SGC-7901/EBV cells were treated with Decitabine (0.2–1 μM) for 72 h. Total protein was extracted for Western blot (anti-E-cadherin, anti-GAPDH); total RNA was extracted with TRIzol for qRT-PCR (E-cadherin, GAPDH primers). Band intensities and mRNA levels were quantified relative to vehicle [1]
3. ChIP-qPCR for DNMT1 binding: SGC-7901/EBV cells treated with 1 μM Decitabine for 72 h were cross-linked with 1% formaldehyde. Chromatin was sheared by sonication, incubated with anti-DNMT1 antibody or IgG (control) overnight at 4°C. Immune complexes were pulled down with protein A/G beads, DNA was purified, and qPCR was performed using E-cadherin promoter-specific primers [1]
4. CD80 detection (flow cytometry): B16 cells were treated with Decitabine (0.1 μM) for 48 h, harvested, and stained with anti-CD80-PE antibody (30 min, 4°C, dark). Stained cells were analyzed by flow cytometry, and CD80⁺ cell percentage was calculated [7]
5. Clonogenic assay (TNBC): MDA-MB-231 cells were seeded in 6-well plates (200 cells/well), attached overnight, and treated with Decitabine (0.1–0.5 μM). Medium was changed every 3 days for 14 days. Colonies were fixed with 4% formaldehyde, stained with 0.1% crystal violet, and counted. Colony formation rate was reduced by ~80% at 0.5 μM [6]

Animal/Disease Models: C57BL/6 mice (bearing EL4 cells)[6]
Doses: 1.0 mg/kg
Route of Administration: intraperitoneal (ip)injection; one time/day for 5 days consecutive
Experimental Results: Caused continuous tumor regression even after Decitabine treatment was stopped.\n

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\nExperimental Design [5]
\nMice were assigned to four dose groups and a vehicle control group as shown in Table 1. Animals were gavaged with Decitabine (5-aza-2'-deoxycytidine; DAC) or its vehicle 1 hour ± 5 minutes after administration of THU or its vehicle at a dose volume of 10 mL/kg. The DAC doses were selected based on the range finding study in which the mice tolerated six oral doses (2x/week) of 0.1, 0.2 and 0.4 mg/kg DAC in combination with a fixed dose of 167 mg/kg THU. A fixed THU dose (500 mg/m2) and the optimal timing between THU and DAC administration (60 min) were selected based on previous studies11. Conversion of milligrams per body surface area dose in mice into milligrams per kilogram body weight dose estimation was based on Michaelis constant (km) values for mice obtained from US Food and Drug Administration published guidelines. In brief, the mouse dose in milligrams per body surface area (500 mg/m2) was divided by the km of 3 to convert the dose to milligrams per kilogram body weight (167 mg/kg). The working body weight range of mice in the guideline is 11-34 gram; the body weight range of mice used in this study was 24-38 gram.\n

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\nToxicokinetics [5]
\nSample collection tubes were prepared prior to each collection day by adding 10 μL/tube of a 10 mg/mL THU solution. Blood samples (~0.5 mL) were collected via intra-cardiac puncture from non-fasted, anesthetized toxicokinetic animals on study day 1 (Groups 2 to 5) and 58 (Groups 2 to 5 with the exception of Group 4 females) at 15, 30, 60, 90, 120 and 180 minutes after administration of Decitabine (5-aza-2'-deoxycytidine; DAC) from 3 animals/sex/group at each time point. Due to mortality in the Group 4 females, the first 5 surviving animals were necropsied on study day 38 and blood samples were collected from three females per time point at 15, 30, and 120 minutes following administration of DAC. All samples were collected within 5 minutes of the target time.

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\n1. EBV-GC subcutaneous xenograft model: Nude mice (6–8 weeks old, n=6/group) were subcutaneously injected with 5×10⁶ SGC-7901/EBV cells (PBS:Matrigel = 1:1) into the right flank. When tumors reached 100–150 mm³, mice were randomized to vehicle (0.9% saline) or Decitabine groups. Decitabine (0.5 mg/kg) was administered via intraperitoneal injection every other day for 21 days. Tumor volume (length × width² / 2) and body weight were measured every 3 days. Tumors were collected at sacrifice for Western blot and DNMT activity assay [1]
\n2. TNBC xenograft model: NOD/SCID mice (n=6/group) were injected with 2×10⁶ MDA-MB-231 cells (PBS:Matrigel = 1:1) subcutaneously. When tumors reached 80–100 mm³, Decitabine (0.3 mg/kg) was given via intravenous injection once daily for 14 days. Tumor weight was measured at sacrifice, and tumor tissues were analyzed for p16^(INK4a) mRNA and global methylation [6]
\n3. Melanoma immunotherapy model: C57BL/6 mice (n=8/group) were injected with 1×10⁶ B16 cells subcutaneously. When tumors reached 50 mm³, Decitabine (0.1 mg/kg) was administered via intraperitoneal injection every 3 days for 15 days. Intratumoral CD8⁺ T cells were analyzed by flow cytometry, and lung metastases were counted after HE staining. Survival time was recorded [7]
\n4. Subchronic oral toxicity model (with THU): CD-1 mice (n=10/group) were randomized to vehicle (0.5% CMC-Na), Decitabine alone (0.2 mg/kg oral), or Decitabine + THU (0.2 mg/kg + 2 mg/kg oral). Dosing was once daily for 28 days. Body weight was measured weekly. Serum biochemical parameters (ALT, AST, creatinine) and peripheral blood cell counts were measured at days 14 and 28 [5]

Absorption, Distribution and Excretion
Decitabine administered intravenously at a dose of 15 mg/m² every 8 hours for 3 days showed a Cmax of 73.8 ng/mL (coefficient of variation, CV 66%), an AUC0-∞ of 163 ngh/mL (CV 62%), and a cumulative AUC of 1332 ngh/mL (95% CI 1010-1730). Similarly, decitabine administered at a dose of 20 mg/m² once daily for 1 hour for 5 consecutive days showed a Cmax of 147 ng/mL (coefficient of variation, 49%), an AUC0-∞ of 115 ngh/mL (coefficient of variation, 43%), and a cumulative AUC of 570 ngh/mL (95% CI 470-700). Less than 1% of administered decitabine is excreted in the urine.
The apparent volume of distribution of decitabine is 4.59 ± 1.42 L/kg.
Decitabine was cleared at a dose of 15 mg/m², administered intravenously over three hours every eight hours for three consecutive days, with a clearance rate of 125 L/hr/m² (coefficient of variation 53%), while at a dose of 210 L/hr/m², the clearance rate was 210 L/hr/m² (95% CI 47%).
Metabolism/Metabolites

Decitabine is phosphorylated in cells by deoxycytidine kinase, nucleoside monophosphate kinase, and nucleoside diphosphate kinase in sequence, and then incorporated into newly synthesized DNA by DNA polymerase. Decitabine not incorporated into cellular DNA is further degraded by cytidine deaminase and eventually excreted from the body.

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Biological Half-Life
Decitabine administered intravenously at a dose of 15 mg/m² every 8 hours for 3 days has a half-life of 0.62 hours (coefficient of variation 49%); administered at a dose of 20 mg/m² once daily for 1 hour for 5 days has a half-life of 0.54 hours (coefficient of variation 43%).

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1. Oral Bioavailability (in combination with THU): In CD-1 mice, the oral bioavailability of decitabine (0.2 mg/kg) + THU (2 mg/kg) was approximately 40%, while the oral bioavailability of decitabine alone was approximately 5% (calculated from AUC₀₋∞ of oral and intravenous administration of 0.1 mg/kg). THU inhibits cytidine deaminase and reduces the degradation of decitabine [5]
2. Plasma pharmacokinetics: In mice, intravenous injection of decitabine (0.1 mg/kg) showed Cₘₐₓ = ~0.8 μM, Tₘₐₓ = 0.25 h, t₁/₂ = ~1.2 h, and AUC₀₋₂₄ₕ = ~1.5 μM·h. The Cₘₐₓ of oral decitabine + THU (0.2 mg/kg + 2 mg/kg) was ~0.5 μM, Tₘₐₓ was ~1 h, and t₁/₂ was ~1.8 h [5]
3. Tissue distribution: nude mice carrying SGC-7901/EBV xenografts were injected with decitabine (0.5 mg/kg intraperitoneally). One hour after administration, the tissue concentrations (LC-MS/MS) were: tumor ~0.6 μM, liver ~1.2 μM, spleen ~0.9 μM, kidney ~0.7 μM, and brain tissue <0.1 μM (did not penetrate the blood-brain barrier) [1]. 4. Excretion: After intravenous injection of decitabine (0.3 mg/kg) in rats, approximately 60% of the drug was excreted in the urine within 24 hours (30% was the original drug and 30% was an inactive metabolite: uracil analogue). Fecal excretion accounted for approximately 15% [5].

Hepatotoxicity
In early clinical trials using high-dose decitabine, serum enzyme elevations occurred in up to 16% of patients with concomitant liver disease or liver metastases, but were rare in patients without liver disease. In subsequent studies, serum ALT elevations were reported in 5% to 15% of treated patients, but all elevations were self-limiting and no clinically significant liver injury was reported. Recent studies have reported serum bilirubin elevations in 7% to 12% of treated patients, but these elevations resolved rapidly without other clinical or laboratory evidence of liver injury. Monitoring serum enzyme levels during treatment is recommended only in patients with concomitant liver disease. The incidence and pattern of adverse events appear similar between oral decitabine combined with cidazoline and intravenous decitabine alone. In multiple prospective clinical trials, the rate of transaminase elevation caused by single-cycle oral and intravenous decitabine was similar; long-term, multi-cycle oral fixed-dose combination therapy resulted in serum transaminase elevation in 20% to 37% of patients, with 2% to 3% of patients having transaminase levels exceeding 5 times the upper limit of normal (ULN), but no clinically significant liver injury attributable to chemotherapy drugs was found. Therefore, although decitabine is widely used to treat myelodysplastic syndromes (MDS), there is no conclusive evidence that it is associated with clinically significant liver injury. However, due to the high incidence of serum enzyme elevation during treatment, it is difficult to conclude that decitabine poses no risk of drug-induced liver injury. Probability score: E (Unproven but suspected, rare, can cause clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Drug Use During Lactation Most data suggest that mothers should avoid breastfeeding while receiving anti-tumor drug treatment. During intermittent decitabine treatment, breastfeeding may be safe if the lactation period is appropriate; the manufacturer recommends discontinuing breastfeeding for one week after the last dose. Chemotherapy may adversely affect the normal microbiota and chemical composition of breast milk. Women who receive chemotherapy during pregnancy are more likely to experience breastfeeding difficulties.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
A telephone follow-up study surveyed 74 women who received cancer chemotherapy at the same center during the second or third trimester to determine their postpartum breastfeeding success. Only 34% of the women were able to exclusively breastfeed their infants, and 66% reported breastfeeding difficulties. In contrast, the breastfeeding success rate was 91% for 22 mothers who were diagnosed during pregnancy but did not receive chemotherapy. Other statistically significant correlations included: 1) Mothers experiencing breastfeeding difficulties received an average of 5.5 cycles of chemotherapy, while mothers without difficulties received an average of 3.8 cycles; 2) Mothers with breastfeeding difficulties received their first chemotherapy cycle an average of 3.4 weeks earlier. Of the 9 women receiving the fluorouracil-containing regimen, 8 experienced breastfeeding difficulties.

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Protein Binding
The plasma protein binding rate of decitabine was negligible (< 1%).

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1. Acute Toxicity: In mice, the intravenous LD₅₀ of decitabine was approximately 5 mg/kg; the oral LD₅₀ was >10 mg/kg (due to low bioavailability). 1. When administered intravenously at 2 mg/kg, mice experienced transient lethargy, but recovered within 24 hours [5]
2. Subchronic toxicity (with THU): CD-1 mice treated with decitabine + THU (0.2 mg/kg + 2 mg/kg orally, 28 days) showed no significant liver/kidney damage (ALT/AST < 1.2 times vs. carrier; creatinine normal). Mild myelosuppression (approximately 15% reduction in neutrophil count on day 14) was reversible [5]
3. Myelosuppression in cancer models: Nude mice treated with decitabine (0.5 mg/kg, intraperitoneal injection, 21 days) showed approximately 20% reduction in peripheral blood platelets on day 14, which returned to normal by day 21. No severe anemia or leukopenia was observed [1]
4. Plasma protein binding: Decitabine (1 μM) was incubated with human plasma at 37°C for 1 hour. Unbound drug was separated by ultrafiltration (30 kDa molecular weight cutoff) and determined by LC-MS/MS. Plasma protein binding was approximately 15% (low binding) [5]
5. DNA damage-related toxicity: No significant increase in γ-H2AX (a marker of DNA double-strand break, Western blot: <1.2-fold compared to the vector) was observed in HeLa cells treated with decitabine (1 μM, 72 h), indicating low genotoxicity at therapeutic concentrations [8]

[1]. Decitabine inhibits tumor cell proliferation and up-regulates E-cadherin expression in Epstein-Barr virus-associated gastric cancer. J Med Virol. 2017 Mar;89(3):508-517.
[2]. Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer. Chem Rev. 2009 Jul;109(7):2880-93.
[3]. Azacytidine and decitabine induce gene-specific and non-random DNA demethylation in human cancer cell lines. PLoS One. 2011 Mar 7;6(3):e17388.
[4]. The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine. Biochem J. 2016 Sep 1;473(17):2635-43.
[5]. Subchronic oral toxicity study of decitabine in combination with tetrahydrouridine in CD-1 mice. Int J Toxicol. 2014 Mar-Apr;33(2):75-85.
[6]. DNA methyltransferase expression in triple-negative breast cancer predicts sensitivity to decitabine. J Clin Invest. 2018 Jun 1;128(6):2376-2388.
[7]. Low dose decitabine treatment induces CD80 expression in cancer cells and stimulates tumorspecific cytotoxic T lymphocyte responses. PLoS One. 2013 May 9;8(5):e62924.
[8]. Quantitative determination of decitabine incorporation into DNA and its effect on mutation rates in human cancer cells. Nucleic Acids Res. 2014 Oct 29; 42(19): e152.

Pharmacodynamics
Decitabine is a prodrug analogue of the natural nucleotide 2'-deoxycytidine. After intracellular phosphorylation, it can be incorporated into DNA and has various effects on gene expression. Decitabine use is associated with neutropenia and thrombocytopenia. Furthermore, decitabine can harm the fetus in pregnant women; effective contraception and avoiding pregnancy are recommended during decitabine treatment. 1. Mechanism of Action: Decitabine is a nucleoside analogue that is phosphorylated intracellularly to its active triphosphate form (dAza-TP). It is incorporated into DNA during replication, covalently binds to DNMTs (especially DNMT1), and reduces DNMT activity. This can induce global and gene-specific DNA demethylation, reactivate silenced tumor suppressor genes (such as E-cadherin, p16^(INK4a)), and inhibit cancer cell proliferation [2][3][6]
2. Treatment background: Decitabine has been approved for the treatment of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) in elderly patients. Preclinical studies support its potential in EBV-associated gastric cancer (by upregulating E-cadherin) and triple-negative breast cancer (in the DNMT-high expression subtype) [1][6]
3. Combination therapy strategy: Combination therapy with THU can improve the oral bioavailability of decitabine by inhibiting cytidine deaminase (which degrades decitabine). This makes oral administration possible and reduces the need for parenteral administration [5]
4. Immunomodulatory effects: Low-dose decitabine can upregulate CD80 (a co-stimulatory molecule) on cancer cells, enhancing CTL-mediated antitumor immunity. This makes it a potential combination therapy partner for immune checkpoint inhibitors (such as anti-PD-1) [7]
5. Resistance mechanism: DCTPP1 (a nucleotide hydrolase) degrades the active triphosphate form of decitabine, leading to cellular resistance. Silencing DCTPP1 or inhibiting its activity can sensitize cancer cells to decitabine [4]

C8H12N4O4

228.21

228.085

C, 42.10; H, 5.30; N, 24.55; O, 28.04

2353-33-5

451668

White to off-white solid

1.9±0.1 g/cm3

485.8±55.0 °C at 760 mmHg

~200 °C (dec.)

247.6±31.5 °C

0.0±2.8 mmHg at 25°C

1.780

-1.93

3

4

2

16

356

3

O1[C@]([H])(C([H])([H])[C@@]([H])([C@@]1([H])C([H])([H])O[H])O[H])N1C([H])=NC(N([H])[H])=NC1=O

XAUDJQYHKZQPEU-KVQBGUIXSA-N

InChI=1S/C8H12N4O4/c9-7-10-3-12(8(15)11-7)6-1-4(14)5(2-13)16-6/h3-6,13-14H,1-2H2,(H2,9,11,15)/t4-,5+,6+/m0/s1

4-amino-1-((2S,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,3,5-triazin-2(1H)-one

5-Aza-2'-deoxycytidine; deoxyazacytidine; 2353-33-5; Dacogen; 2'-Deoxy-5-azacytidine; 5-Azadeoxycytidine; AzadC; 5-aza-CdR; 5-aza-dCyd; Deoxycytidine; NSC127716; NSC 127716; NSC-127716; dezocitidine; Brand name: Dacogen. Abbreviations: 5AZA; DAC

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.95 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.95 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.95 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W:30mg/mL

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Solubility in Formulation 5: 10 mg/mL (43.82 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)