Concentrations Of DAC Research Articles

The Synergistic Antitumor Effect of Decitabine and Vorinostat Combination on HepG2 Human Hepatocellular Carcinoma Cell Line via Epigenetic Modulation of Autophagy-Apoptosis Molecular Crosstalk.

Hepatocellular carcinoma (HCC) is a worldwide health issue. Epigenetic alterations play a crucial role in HCC tumorigenesis. Using epigenetic modulators for HCC treatment confers a promising therapeutic effect. The aim of this study was to explore the effect of a decitabine (DAC) and vorinostat (VOR) combination on the crosstalk between apoptosis and autophagy in the HCC HepG2 cell line at 24 h and 72 h. Median inhibitory concentrations (IC50s) of VOR and DAC were assessed in the HepG2 cell line. The activity of caspase-3 was evaluated colorimetrically, and Cyclin D1(CCND1), Bcl-2, ATG5, ATG7, and P62 levels were assessed using ELISA at different time intervals (24 h and 72 h), while LC3IIB and Beclin-1gene expression were measured by using qRT-PCR. The synergistic effect of VOR and DAC was confirmed due to the observed combination indices (CIs) and dose reduction indices (DRIs). The combined treatment with both drugs inhibited the proliferation marker (CCND1), and enhanced apoptosis compared with each drug alone at 24 h and 72 h (via active caspase-3 upregulation and Bcl-2 downregulation). Moreover, the combination induced autophagy as an early event via upregulation of Beclin-1, LC3IIB, ATG5, and ATG7 gene expression. The initial induction of autophagy started to decrease after 72 h due to Beclin-1 downregulation, and there was decreased expression of LC3IIB compared with the value at 24 h. Herein, epigenetic modulation via the VOR/DAC combination showed an antitumor effect through the coordination of an autophagy-apoptosis crosstalk and promotion of autophagy-induced apoptosis, which ultimately led to the cellular death of HCC cancer cells.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Peripheral T-cell lymphomas (PTCL) are a rare and heterogeneous group of malignancies that share a unifying feature of epigenetic dysregulation. Recent evidence suggests that PTCL, unlike other forms of cancer or even lymphoma, exhibits a marked vulnerability to hypomethylating agents (HMAs) alone and especially in combination with HDAC inhibitors. HMAs like azacitidine (AZA) and decitabine (DAC) have been shown to reverse transcriptional repression secondary to hypermethylation. Cladribine (CLAD) is FDA-approved for the treatment of hairy cell leukemia and is postulated to inhibit both DNA and histone methylation. Here, we sought to define the mechanistic differences between different DNA HMAs in order to inform a rationale on the optimal combinations that might exploit the epigenetic vulnerabilities of PTCL. Methods: Cell viability, caspase activity assay, and western blotting (WB) were performed on a panel of T-cell lymphoma cell lines to identify the effects of AZA/DAC/CLAD on cell survival, apoptosis, and DNA methyltransferase (DNMT) levels. Liquid chromatography-based mass spectrometry (LC-MS) was done to quantify 5-methyl cytosine (5-mC) levels. Results: Cell viability analysis confirmed that the dose and time-dependent sensitivity of the six PTCL cell lines differed following exposure to AZA/DAC/CLAD, where CLAD showed the lowest IC50 value (0.1–1 µM) across all cell lines, followed by AZA (IC50: 1–10 µM), and DAC had the least effect on cell viability (IC50: >100 µM, except T-ALL). A differential threshold of caspase activation was observed across the PTCL cell lines where CLAD showed the most superior caspase activity leading to the highest apoptotic potential. However, WB analysis showed that after 24 hours of treatment, DNMT1 and DNMT3A protein levels were significantly depleted at low concentrations of DAC (<0.01 µM) compared to AZA whereas CLAD did not affect the DNMT1 and DNMT3A levels. LC-MS-based quantification of 5-mC showed a similar relationship in methylation, with initial changes observed at doses >0.01 μM of DAC and >0.1 μM of AZA/CLAD. Taking the short half-life of AZA/DAC into account, cell viability assays with daily addition of AZA/DAC showed increased cell cytotoxicity for AZA/DAC treated samples, which was comparable to CLAD. Ongoing analyses of gene expression and metabolism of the universal methyl donor S-adenosylmethionine (SAM) will help to understand the mechanistic difference between these HMAs. Conclusions: These data suggest that the HMAs have a distinct mechanism of action for hypomethylation. While AZA/DAC induces hypomethylation by depletion of DNMTs, CLAD acts in a DNMT depletion-independent pathway. A mechanistic understanding of AZA/DAC/CLAD will inform how best to use them in clinic as well as assist in the strategic development of biological correlates to further support their therapeutic application. The research was funded by: Translational Orphan Blood Cancer Research Initiative Fund and RO1 # FD-R-006814-01. Keywords: Genomics, Epigenomics, and Other -Omics, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.

Mechanism of Action of Decitabine in the Treatment of Acute Myeloid Leukemia by Regulating LINC00599.

Acute myeloid leukemia (AML) is a heterogeneous malignancy with a low long-term survival rate. The aim of this study was to investigate the effects of decitabine (DAC) treatment cell proliferation and apoptosis in AML and role of the expression of LINC00599 and, consequently, miR-135a-5p. Human promyelocytic leukemia cells (HL-60) and human acute lymphatic leukemia (CCRF-CEM) cells were treated with various concentrations of DAC. Cell proliferation in each group was detected using the cell counting kit 8. For each group, apoptosis and reactive oxygen species (ROS) levels were detected using flow cytometry. Reverse transcription polymerase chain reaction (RT-PCR) was performed to examine the expression of lncRNA LINC00599. The expression of apoptosis-related proteins was analyzed using western blotting. The regulatory relationship between miR-135a-5p and LINC00599 was verified by constructing miR-135a-5p mimics, miR-135a-5p inhibit, wild type LINC00599 3'-untranslated region (UTR), and mutant LINC00599 3'-UTR. Ki-67 expression in the tumor tissues of nude mice was detected using immunofluorescent assays. Both DAC and LINC00599 Inhibit groups were able to significantly reduce the proliferation of HL60 and CCRF-CEM cells, increase apoptosis, upregulate the expression of Bad, cleaved caspase-3, and miR-135a-5p, downregulate the expression of Bcl-2, and elevate ROS levels in cells, with these effects being more pronounced after combined treatment with DAC and LINC00599 Inhibit. In comparison to mimic NC, the miR-135a-5p mimic group significantly decreased the relative fluorescence activity ratio of LINC00599 3'-UTR wild-type CCRF-CEM cells. The LINC00599 Inhibit and miR-135a-5p mimic groups exhibited substantially reduced proliferation of HL60 and CCRF-CEM cells, increased apoptosis, upregulated Bad, cleaved caspase-3, and miR-135a-5p expression, along with downregulated Bcl-2 and LINC00599 expression and increased ROS levels in cells; these effects were more pronounced after LINC00599 Inhibit was combined with miR-135a-5p mimics. In vivo experiments revealed that both DAC and LINC00599 Inhibit were able to considerably reduce the long diameter, short meridian, volume, and mass of tumors, increase miR-135a-5p expression, and decrease LINC00599 and ki-67 expression in tumor tissues of nude mice. This effect was more pronounced when the DAC and LINC00599 Inhibit were used in combination. DAC regulates the expression of miR-135a-5p by regulating the expression of LINC00599, which in turn affects cell proliferation, apoptosis, and tumor proliferation. Our findings provide a theoretical basis for improving the clinical outcome of AML.

Karakteristik Fisikokimia dan Sensoris Abon Cemani dengan Substitusi Jantung Pisang Kepok (Musa paradisiaca)

Shredded is a food known to urban and rural communities, the variation of shredded meat sale in the markets;was shredded beef, fish and chicken, but the shredded chicken only variation of poultry. The selection of cemani chicken to increase the value and benefits of cemani chicken which has been less than optimal and the male banana flower which is still lacking and becomes a mere waste. The purpose of this study is to determine the characteristics in terms of physical, chemical, and sensory and determine the quality of shredded chicken cemani with the substitution of the heart of the banana puke produced and increase its economics value. The conducted of research in the Chemix Pratama Chemical Laboratory Yogyakarta and Nutrition Laboratory, Semarang State University. The method used in the study of the complete randomized design with treatment experiment was the comparison of cemani chicken (DAC) and male banana kepok flower (JPK) and the formulation (F2) DAC 83.3%: JPK 0%, (F2) DAC 20.85%: JPK 62.5%, and (F3) DAC 41.6%: JPK 41.6%, (F4) DAC 62.6%: JPK 20.85%. The results showed that the formulation of shredded with cemani chicken meat 20,85% and male banana kepok flower 62,5% was highest nutrition content, was energy 265,27 Kcal, carbohydrates 12,60%, fat 14,66%, protein 19,66%, water content 45,57%, crude fiber content 3,77% and ash content 3,69%. Based on the physical and organoleptic values between formulas doesn’t difference, the texture parameters with means value of 4.07 (F4), the taste was 3.93 (F2), the flavor was 3.77 (F3), the color was 3.80 (F3), and the total preference was 3.80 (F2). The conclusion of the selected formula is F2 based on chemical parameters with a DAC concentration of 20.85% and JPK of 62.5%, with energy of 265.27 cal, carbohydrates of 12.60%, fat of 14.66%, protein of 19.66%, water content of 45.57%, fiber content of 3.77% and ash content of 3.69%.&#x0D; Key words: shredded, cemani chicken

Effect of Hemodialysis on Efficacy and Pharmacokinetics of Sofosbuvir Coformulated with Either Daclatasvir or Ledipasvir in Patients with End-Stage Renal Disease

Background/Aims: Direct-acting antivirals (DAAs) play a key role in the eradication of hepatitis C virus (HCV) infection. However, limited data are available on DAA for treating HCV infection in hemodialysis (HD) patients. This study was to evaluate the pharmacokinetic characteristics and effectiveness of daclatasvir/sofosbuvir (DAC/SOF) and ledipasvir/SOF (LDV/SOF) in HD patients. Methods: Seven patients were given SOF coadministered with DAC or LDV once daily for 12 weeks. The plasma concentrations of SOF007, DAC, and LDV were determined by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Results: A sustained virologic response in week 12 (SVR12) was achieved in 6 (100%) patients, except for 1 patient dying due to severe cerebral hemorrhage not related to antiviral therapy. The extraction ratio of SOF007 was 66.67%, and the estimated HD clearance of SOF007 was 5.65 L/h. Conclusion: The combination of SOF with either DAC or LDV is well tolerated and offers high SVR12 in HD patients.

Poor-Risk AML with Complex, Monosomal Karyotype: In Vivo Therapeutic Efficacy of Decitabine (DAC) in Patient-Derived Xenograft (PDX) Mouse Models

Background: DNA-hypomethylating agents (HMAs) show an encouraging but not yet well-understood activity in AML/MDS patients (pts) with adverse cytogenetics, such as -7/del(7q) embedded in a complex, monosomal karyotype (MK). We have noted that MK pts with multiple monosomies responded better to DAC treatment than pts with only one monosomal autosome (Blum, Greve and Lübbert, Curr Opin Hematol. 2017). This important characteristic of HMAs differentiates them from cytarabine (AraC, lacking DNMT-inhibitory activity) as shown in randomized studies with DAC (Wierzbowska et al., Am. J. Hematology 2018) and AZA (Döhner et al., Leukemia 2018). The mechanism of action is currently under investigation and may involve preferential gene reactivation on monosomic chromosomes by DAC (Greve et al., ASH Annual Meeting 2017, #2612). In order to address this important difference between DAC and AraC, we compared their antileukemic activity in PDX models representing cytogenetically normal (CN) AML and adverse cytogenetics AML (complex, monosomal karyotypes including del(7q) and 1 or more monosomies). Materials and Methods: PDX models were generated for 5 CN and 3 MK AML pts with a median age of 68 years (range 55-80), a median WBC of 3.0 x 103/µl (range 1.4-139.7) and a median of 42% bone marrow (BM) blasts (range 5-97). 7/8 pts were sampled at diagnosis, i.e. prior to first-line treatment (3 pts received DAC, 3 pts induction and 1 pt upfront allografting); 1/8 pt had had multiple relapses. T-cell depleted peripheral blood or bone marrow cells (3x106 cells/mouse) were injected into NSG (NOD/Shi-scid/IL-2Rγnull) mice. Leukemic cell engraftment was determined by flow cytometry (FC) in BM, peripheral blood (PB) and spleen during the course of the experiment and at the end of a study. Leukemic cells were transplanted serially at least 4-5 times to propagate and ensure full leukemic potential of the cells. Mice were treated with low-toxic concentrations of DAC (1 mg/kg/day given intraperitoneally), AraC (15 mg/kg/day given intravenously) or vehicle (PBS) for 5 consecutive days (one cycle). Drug doses were titrated to be equitoxic and as potent as possible without causing side effects. Treatment commenced upon disease onset, as evaluated by FC positivity for human CD33, increased WBC in PB and weight loss. Overall survival served as the read-out. The study was carried out in accordance with the recommendations by the Society of Laboratory Animal Science in an AAALCA accredited animal facility. The animal experiments were approved by the regional council (Regierungspräsidium Freiburg, ref. 35, permit no. G-12/86). Results: We successfully established well-characterized PDX from pts with CN (n = 5), and with MK (n = 3) with a median of 2 autosomal monosomies (range 1-6; of chromosomes 4, 6, 7 [2x], 15, 17 [2x], 18, 21). TP53 (mutational status available for 6/8 pts) was wildtype in 4 CN pts and 1 MK pt, and mutated in 1 MK pt. All models were treated with either DAC, AraC or vehicle. Across both cytogenetic cohorts, DAC treatment significantly prolonged median survival by 36 days when compared to vehicle administration (p = 0.0002, see Fig 1A). Specifically, DAC increased median survival of CN PDX by 33 days (p = 0.0104, range 61-105 days with DAC, compared to 43-65 days with vehicle, Fig 1B), and in MK PDX by 40 days (p = 0.0197; range 72-105 days with DAC, compared to 39-50 days with vehicle, Fig 1C). Notably, disease progression in two DAC-treated PDX (one CN, one MK with 6 monosomies) was delayed to 105 days (Fig 1, marked with ⋆). AraC treatment also resulted in longer median survival across both cohorts compared to vehicle, albeit not as pronounced as with DAC, by an average of 20 days (p = 0.0066). In more detail, AraC increased median survival of CN PDX by 19 days (p = 0.0025, range 53-82 days), and, though not significantly, of MK PDX by 15 days (p = 0.2048, range 46-92 days). Conclusion: In this PDX study recapitulating two different cytogenetic risk groups of AML, DAC showed encouraging activity also in models with complex, monosomal karyotype, even in the presence of 6 autosomal monosomies, and was superior to AraC in extending survival. These data lend further support to the treatment of such adverse genetics AML patients with HMAs. Disclosures No relevant conflicts of interest to declare.

Inhibitory Effect of Decitabine on Proliferation of MDS-L Cells and Its Mechanism

To investigate the inhibitory effect of decitabine (DAC) in various dosages on the proliferention of MDS-RAEB cell line MDS-L and its mechanism. LC-MS/MS method was used to test the blood DAC concentration of 2 groups of MDS patients being treated with DAC 20 and 15 mg/m2×5 d. In according to the various blood DAC concentration levels, the MDS-L cells were treated with different DAC dosages for 24, 48, 72 and 96 h, respectively. The CCK-8 method was applied to determine the cell proliferation, the flow cytometry was used to analyze the cell cycle and cell apoptosis changes, the P15INK4B DNA methylation status was measured by methylation specific PCR using EZ DNA Methylation-Gold Kit. The blood DAC concentration of MDS patients treated with DAC 20 mg/m2×5 d was 174.08±80.15(84.7-311) ng/ml, which was significantly higher than 89.87±32.94(43.2-165)ng/ml for the group treated with 15 mg/m2×5 d (P=0.014). DAC could notably inhibit the proliferation of MDS-L cells, and the effect was in dose- and- time-dependent manner(r=0.786). However, when DAC concentration was ≥0.1 µg/ml, the proliferation inhibition rates were not significantly different between various dosages. After DAC treatment, MDS-L cells in G1 phase increased notably, while cells in S phase decreased significantly. Also, the P15INK4B DNA methylation status of MDS-L cells decreased after being treated with DAC for 96 h, but the difference was not significant between various dosages. DAC can significantly suppress MDS-L cell proliferation, block MDS-L cells in G1 phase and induce the apoptosis at low concentration (0.1-0.2 µg/ml).

Induction of an Erythroid but Not Megakaryocytic Expression Signature in Myeloid Cells By in Vitro 5-Aza-2'-Deoxycytidine Treatment

Introduction: Aberrant DNA methylation is frequently found in hematologic malignancies where it is associated with altered gene expression. DNA hypomethylating agents (DNMTi), e.g. 5-aza-2'-deoxycytidine (DAC), are used for both global and gene-specific in vivo demethylation and offer a therapeutic option in myelodysplastic syndromes (MDS) and AML. DNMTi have already been utilized to upregulate suppressed fetal hemoglobin (HbF) in adult patients (pts) suffering from hemoglobinopathies. Here we systematically investigated the potential of DAC for in vitro induction of erythroid differentiation as well as HbF expression in the bipotent myeloid leukemia cell line K562 and in vivo in a clinical treatment situation in MDS pts.Methods and Results: We treated K562 cells with non-toxic concentrations of DAC (100 nM, three 24 hour pulses), hemin (50 nM) and phorbol myristate acetate (PMA, 5 nM). DAC treatment led to morphological changes indicating erythroid but not megakaryocytic differentiation. This was confirmed by benzidine staining where DAC (13% positive cells) and hemin (58%) but not PMA treated cells (0%) became positive for hemoglobin synthesis. Lack of CD41 detection by FACS analysis for DAC and hemin indicated absence of megakaryocytic differentiation. Transcriptome profiling by mRNA expression arrays (Affymetrix GeneChip® HG U133 Plus 2.0) revealed highest similarity between hemin and DAC treatment by unsupervised hierarchical clustering, followed by vehicle control and untreated cells. The transcriptome of PMA treated cells clustered most distantly to all other treatments. Both, DAC and hemin induced moderately balanced up- and downregulation of transcripts to an almost identical extent. 1414 transcripts were >2 fold upregulated and 1505 were >2 fold downregulated upon DAC treatment, whereas 1548 were up- and 2404 were downregulated in hemin treated cells, respectively. The extent of transcriptome dynamics was considerably stronger upon PMA treatment, where 4196 and 3780 transcripts were up- and downregulated, respectively. When intersecting transcriptome changes between the 3 drug treatments (Fig. 1), 368 out of 1548 (23.7%) upregulated transcripts in hemin treated cells were concordantly upregulated upon DAC treatment. The overlap of upregulated transcript was lower compared to PMA treated cells (14.9%). GO analyses of upregulated transcripts identified terms related to erythropoesis and iron metabolism among the top regulated groups of transcripts in DAC treated cells whereas terms related to megakaryocytic differentiation did not show significance. Particularly strong differences of transcripts were observed for a1-, a2-, Ag-, e- and z-globin expression upon DAC and hemin treatment, whereas b- and d-globin were expressed at low levels. These changes were not observed for PMA treated cells. Induction of a- and Ag-globin on mRNA level resulted in enrichment of a- and Ag-globin protein to 15.8% of total cellular protein amount, and consequently in HbF formation in K562 cells as assessed by reversed phase and anion exchange chromatography. HbF levels in peripheral blood were measured from 16 MDS pts, median age 74 years (range 66-78) also treated with a 3-day DAC schedule. Median HbF fraction at baseline was 0.4% (0.1-3.9%) of total hemoglobin with 6 pts (37.5%) exhibiting increased HbF levels (>1%) already before treatment. In 13/16 (81%) pts, increase of HbF with a median increment of 1.2% (range 0.3-3.7%) was observed. In 3 pts, HbF decreased over the treatment course. Median number of courses until maximum increment was 3 (2-6). HbF levels in 2 pts with AML and 1 with pancreatic cancer treated with nucleoside analogues without demethylating activity (cytosine arabinoside and gemcitabine, respectively) according to standard chemotherapy protocols served as control group and did not show comparable increments.Conclusions: We describe an erythroid differentiation program, from transcriptome level to HbF protein formation, induced by the hypomethylating agent DAC in the bipotent cell line K562. This DAC-mediated differentiation process is specific for erythropoesis but not megakaryopoesis. This is substantiated by in vivo upregulation of HbF upon DAC adminstration in MDS pts. Therefore, we propose to utilize HbF expression as potential biomarker during DAC treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Low concentrations of 5-aza-2'-deoxycytidine induce breast cancer stem cell differentiation by triggering tumor suppressor gene expression.

BackgroundBreast cancer stem cells (BCSCs) are considered the cause of tumor growth, multidrug resistance, metastasis, and recurrence. Therefore, differentiation therapy to reduce self-renewal of BCSCs is a promising approach. We have examined the effects of 5-aza-2′-deoxycytidine (DAC) on BCSC differentiation.Materials and methodsBCSCs were treated with a range of DAC concentrations from 0.625 to 100 µM. The differentiation status of DAC-treated BCSCs was graded by changes in cell proliferation, CD44+CD24− phenotype, expression of tumor suppressor genes, including BRCA1, BRCA2, p15, p16, p53, and PTEN, and antitumor drug resistance.ResultsDAC treatment caused significant BCSC differentiation. BCSCs showed a 15%–23% reduction in proliferation capacity, 3.0%–21.3% decrease in the expression of BCSC marker CD44+/CD24−, activation of p53 expression, and increased p15, p16, BRCA1, and BRCA2 expression. Concentrations of DAC ranging from 0.625 to 40 µM efficiently induce cell cycle arrest in S-phase. ABCG2, highly expressed in BCSCs, also decreased with DAC exposure. Of particular note, drug-sensitivity of BCSCs to doxorubicin, verapamil, and tamoxifen also increased 1.5-, 2.0-, and 3.7-fold, respectively, after pretreatment with DAC.ConclusionDAC reduced breast cancer cell survival and induced differentiation through reexpression of tumor suppressor genes. These results indicate the potential of DAC in targeting specific chemotherapy-resistant cells within a tumor.

Acquired resistance to decitabine and cross-resistance to gemcitabine during the long-term treatment of human HCT116 colorectal cancer cells with decitabine.

The aim of the present study was to determine the effects of long-term exposure of decitabine (DAC) to HCT116 colorectal cancer (CRC) cells on the acquisition of resistance to DAC as well as cross-resistance to anticancer drugs used for CRC or other epigenetic modifiers. In the present study, DAC-resistant HCT116 CRC cells were established through long-term treatment with increasing concentrations of DAC (10 to 540 nM); and the cross-resistance to other drugs was subsequently examined. DAC-resistant HCT116 cells were obtained following a 104-day treatment with DAC, including DAC-free intervals. The results demonstrated that the IC50 value of DAC was increased ~100-fold in DAC-resistant HCT116 cells. Messenger (m)RNA expression of secreted frizzed-related protein 1 (SFRP1), which is regulated by DNA methylation, was not detected in DAC-resistant cells; however, SFRP1 mRNA was present in HCT116 cells treated with DAC for 52 days. DNA methyltransferase 1 (DNMT1) protein levels were slightly decreased until day 81 and then returned to control levels in DAC-resistant cells. Further experiments using DAC-resistant HCT116 cells revealed that these cells exhibited cross-resistance to gemcitabine (Gem); however, cross-resistance was not observed for other DNMT inhibitors (azacitidine and zebularine), histone deacetylase inhibitors (trichostatin A, vorinostat and valproic acid) or anticancer drugs for CRC (5-fluorouracil, irinotecan and oxaliplatin). Furthermore, the protein expression levels of cytidine deaminase (CDA) were increased, while those of deoxycytidine kinase (dCK) were decreased in DAC-resistant HCT116 cells; by contrast, the mRNA expression levels for these proteins were not significantly altered. In conclusion, the results of the present study indicated that the long-term treatment of HCT116 cells with DAC led to the acquisition of resistance to both DAC and Gem. In addition, these results may be partly attributed to changes in CDA and/or dCK, which are involved in metabolic pathways common to these two drugs.

Functional study of hENT1 on SKM-1 cell resistance to decitabine

目的观察干扰1型平衡核苷转运体（human equilibrative nucleoside transporters 1，hENT1）表达后，地西他滨（DAC）对人骨髓增生异常综合征（MDS）细胞系SKM-1细胞的增殖、凋亡和去甲基化的影响。方法采用慢病毒干扰技术沉默SKM-1细胞中hENT1，采用RT-PCR方法检测靶细胞内hENT1 mRNA表达水平，采用CCK-8法检测不同浓度DAC(0.5、1、5 µmol/L)作用24、48及72 h对hENT1沉默SKM-1细胞的增殖抑制情况，应用流式细胞术及Western blot法观察DAC诱导的凋亡情况，采用甲基化特异性PCR检测p15INK4B去甲基化水平，分析hENT1沉默后DAC的去甲基化作用。结果hENT1干扰组hENT1 mRNA表达水平（0.253±0.030）相对于对照组（1.000±0.091）显著降低（P<0.01）；经0.5、1、5 µmol/L DAC作用24、48及72 h后，相对于同时期对照组，hENT1干扰组的增殖抑制率均明显下降（P<0.05），其中5 µmol/L DAC作用72 h最为明显，hENT1干扰组和对照组分别为（49.41±4.02）%和（33.03±2.47）%（P=0.007）；hENT1干扰组Caspase-3的剪切体水平及AnnexinⅤ+细胞比例减少，并且p15INK4B去甲基化水平也显著降低。结论hENT1沉默后SKM-1细胞对DAC诱导的凋亡及去甲基化作用敏感性下降。

The Effect of Decitabine Combined with Arsenic Trioxide on DAPK Gene and HL-60 Cell Proliferation and Apoptosis

Purpose: Our study was to detect the effect of Decitabine (DAC) combined with arsenic trioxide (AS2O3) on DAPK gene and HL-60 cell proliferation and apoptosis. Methods: DAC and AS2O3 monotherapy, combination treatment and DAC pretreatment were used in this study after incubating with HL-60 cell for 24 h, 48 h, 72 h. CCK8 was used to detect the cell proliferation of HL-60 cell. Flow cytometry was used to detect the cell apoptosis. Then, we used RT-PCR to obtain the gene expression level of DAPK. Results: HL-60 cells were treated with different concentrations of DAC (20 μmol/L, 40 μmol/L, 80 μmol/L), AS2O3 (1 μmol/L, 2.5 μmol/L, 5 μmol/L) monotherapy for 24 h, 48 h, 72 h; along with the extension of the drug concentration and time, proliferation inhibition rate had gradually increased. Monotherapy of DAC, AS2O3 could inhibit the proliferation and induce apoptosis of HL-60 cells, and was time- and dose-dependent. DAC (80 μmol/L) was firstly used for pretreatment, and then, different concentrations of AS2O3 (1 μmol/L, 2.5 μmol/L, 5 μmol/L) were used for 24 h, 48 h, 72 h. It was found that cell proliferation inhibition rate and apoptosis rate had increased significantly. When the two drugs were used together, the increasing proliferation inhibition rate, apoptosis rate and DAPK had become more obvious. Conclusion: DAC and AS2O3 had a synergetic effect for the HL-60 cell proliferation inhibition, apoptosis and expression of DAPK.

Effect of combined 5-aza-2'deoxycytidine and cisplatin treatment on the P15 lung adenocarcinoma cell line.

Aberrant promoter hypermethylation resulting in the epigenetic silencing of apoptosis-associated genes is a key process in the chemotherapeutic treatment of cancer. The nucleoside analog, 5-aza-2'deoxycytidine (DAC), inhibits the activity of DNA methyltransferase enzymes and is able to restore the expression levels of genes that have been silenced by aberrant DNA methylation. The aim of the present study was to investigate the effect of combined treatment with DAC and cisplatin (CDDP) on the lung adenocarcinoma cell line, P15. Growth inhibition was examined using a clone formation assay and growth inhibitory activities by cell counting during treatment with DAC alone, CDDP alone or DAC followed by CDDP. In addition, changes in the mRNA expression levels of various apoptosis-associated genes following treatment with increasing concentrations of DAC were determined using reverse transcription-polymerase chain reaction. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis was used to detect the number of apoptotic P15 tumor cells following treatment with DAC and/or CDDP. The results indicated that DAC treatment alone restored the mRNA expression levels of p73, p16INK4a , B-cell lymphoma (Bcl)-2-associated agonist of cell death and Bcl-2-associated X protein. In addition, combined therapy with DAC and CDDP was found to significantly suppress the growth of P15 tumor cells compared with DAC or CDDP treatment alone. In conclusion, DAC may enhance the chemosensitivity of the P15 cell line to treatment with CDDP.

Novel Assay for Ex Vivo Evaluation of Antiproliferative Effect of Hypomethylating Agents 5-Azacytidine and Decitabine in AML Patient Samples

Introduction: Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and aberrant DNA methylation status. One alternative treatment for AML is hypomethylating agents such as 5-Azacytidine (AZA) and Decitabine (DAC), but just around 30% of the patients obtain a clinical response. These agents are slow acting requiring 3-4 cycles to demonstrate some efficacy, for both reasons looking for biomarkers to identify sensitive patients before treatment could be very beneficial to the patients.Objective: Design an ex-vivo model using the automated flow cytometry ExviTech® platform to identify the antiproliferative effect of hypomethylating agents such as 5-Azacytidine and Decitabine in leukemic cells from AML whole bone marrow patient samples. This assay may capture changes of hypomethylating agents effect that may potentially predict ex vivo the patient clinical outcome.Methods: Bone-marrow samples from 10 patients diagnosed of AML were sent to Vivia from 24 hospitals across Spain within 24h. Whole sample were incubated for 72h in in well plates containing 8 concentrations of AZA and DAC. To induce blast proliferation, AML cells were resuspended in RPMI medium and human cytokines including GM-DSF, IL3, SCF, G-CSF, EPO and transferrin. The number of proliferative and no proliferative live leukemic cells was determined using the CFDA dye. Blast population and viability was determined labeling with monoclonal antibodies and Annexin V. Dose-response curves of AZA and DEC were measured in these 10 patient samples.Results: To corroborate the hypothesis that AZA or DAC induce cell cycle arrest and this effect is different from cytotoxicity, we evaluated the effect of DAC in an AML cell line (KG-1) that grow spontaneously with medium and serum. As shown in figure 1, a decrease in the number of cells is observed when the AML cell line (KG-1) is incubated with DAC (blue line). Interestingly, an exactly proportional increase in the Mean Fluorescent Intensity (MFI) of CFDA is observed (red line), the symmetry between these curves indicate they measure the same effect; the drug-concentration dependent decrease in proliferating cells. Green line represents the percentage of apoptosis, as a measurement of the cytotoxic activity, clearly different that the other 2 measurements and only relevant at much higher doses, indicating that DAC has an essentially antiproliferative effect in the cell line. Once tested the hypothesis, we translate the idea to fresh AML samples inducing the blast proliferation with a very complete cytokine medium. In all patients included in this study, an average population of 47.2%±16.9% proliferating cells was identified. A clear difference in the sensitivity of no proliferative vs proliferative cell subset was observed, while DAC had no activity on the no proliferating cells (flat dose response in Figure 2), it was potently (1 uM) inhibiting almost completely proliferation in the proliferating cell subset.Conclusion: Although the mechanism of action of hypomethylating agents is not still entirely clear, we hypothesize that is associated with antiproliferative effects. In this study, using our Exvitech platform we have developed an ex-vivo model to measure the antiproliferative effect of hypomethylating agents, using whole stimulated bone marrow samples from AML patients. If this new assay would correlate with patient treatment response, it could become a valuable tool to personalize hypomethylating treatments for AML patients. [Display omitted] [Display omitted] Figure 1Figure 2 DisclosuresHernandez:Vivia Biotech: Employment. Gomez:Vivia Biotech: Employment. Robles:Vivia Biotech: Employment. Gorrochategui:Vivia Biotech: Employment. Sanchez:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Employment. Martinez:Vivia Biotech: Membership on an entity’s Board of Directors or advisory committees.

Lidocaine and ropivacaine, but not bupivacaine, demethylate deoxyribonucleic acid in breast cancer cells in vitro

Lidocaine demethylates deoxyribonucleic acid (DNA) in breast cancer cells. This modification of epigenetic information may be of therapeutic relevance in the perioperative period, because a decrease in methylation can reactivate tumour suppressor genes and inhibit tumour growth. The objectives of this study were to determine the effect of two amide local anaesthetics, ropivacaine and bupivacaine, on methylation in two breast cancer cell lines and to detect whether the combination of lidocaine with the chemotherapy agent 5-aza-2'-deoxycytidine (DAC) would result in additive demethylating effects. Breast cancer cell lines BT-20 [oestrogen receptor (ER)-negative] and MCF-7 (ER-positive) were incubated with lidocaine, bupivacaine, and ropivacaine to assess demethylating properties. Then, we tested varying concentrations of lidocaine and DAC to assess whether their demethylating effects were additive. Cell numbers and global methylation status were analysed. Lidocaine decreased methylation in BT-20 and MCF-7 cells, ropivacaine decreased methylation in BT-20 cells, and bupivacaine had no demethylating effect. When combined, lidocaine and DAC had additive demethylating effects. At clinically relevant doses, lidocaine and ropivacaine exert demethylating effects on specific breast cancer cell lines, but bupivacaine does not. The demethylating effects of lidocaine and DAC are indeed additive.

Azacitidine (AZA) and Decitabine (DAC) Show Differential Effects On DNA Methyltransferase (DNMT) Levels in Patients with High Risk (HR) Myelodysplastic Syndrome (MDS)

AbstractAbstract 4906 Background:The mechanism of action (MOA) of AZA and DAC is considered to be predominantly via DNMT inhibition. However these drugs are also cytotoxic as nucleoside analogs, and AZA inhibits RNA translation and protein synthesis. Both are clinically efficacious in HR MDS but patient responses differ. We hypothesized that the relative contributions of these functions to overall MOA may be at the basis of this clinical divergence. Previous studies by others (Hollenbach et al, PLOS One 5(2) e9001) in AML cell lines demonstrated that both drugs reduced DNMT1 levels at clinically relevant concentrations and induced apoptosis. However, they differed in effects on the cell cycle: AZA was active across cell cycle phases while DAC induced a G2-M shift. Here we apply single cell network profiling (SCNP) to analyze the effect of AZA or DAC on cell cycle and DNMT levels in bone marrow mononuclear cell (BMMC) specific subsets (CD34+, nRBC, lymphocytes, myeloid cells) from primary HR MDS and healthy donors (HEA). Methods:SCNP is a multiparametric flow cytometry-based assay that simultaneously measures, at the single cell level, extracellular surface markers and changes in intracellular proteins in response to extracellular modulators (including drugs). SCNP was used to measure cell cycle (DAPI stain), apoptosis (amine aqua and c-Parp) and levels of DNMT1, DNMT3a and DNMT3b at baseline and after treatment for 24 hours with AZA (2. 5uM) and DAC (0. 625uM) in AML cell lines (U937, TF-1), primary BMMC from HEA > 60 years of age (n=15) and HR MDS (n=10, RAEB, int-2/high) patients. The Mann-Whitney U statistic was used for analysis.Results: Cell cycle and apoptosis.Treatment of U937 cells with AZA increased the subG1 cell fraction (i. e. apoptotic cells) from 0 to 12% and decreased the percentage of cells in S phase from 37% to 24%. Conversely treatment with DAC increased the fraction of cells in S phase (from 37% to 54%), consistent with DAC intercalation into DNA, with no significant effects on the subG1 fraction. In TF-1 cells, only AZA significantly induced apoptosis as measured by both amine aqua (10%) and c-Parp (5%). Similar results were observed in primary HR MDS samples: in 4 of 8 HR MDS samples AZA treatment resulted in apoptosis in CD34+ cells (median= 20. 5% induction) while DAC did not affect CD34+ cell viability. DNMTs levels.In U937 cells AZA decreased all 3 DNMTs levels (DNMT1(41%), DNMT3a(26%), DNMT3b(40%)), more effectively than DAC (4%, 4% and 2% decrease, respectively). In primary samples, nRBC subsets from HR MDS showed higher basal levels of DNMT3a and DNMT3b compared to healthy age matched BMMC (p= 0. 003 and p=0. 029 respectively). In the latter, AZA and DAC both decreased the levels of DNMT1, DNMT3a and DNMT3b in all BMMC cell subsets (lymphocytes, nRBC, myeloid and CD34+). The effects of these drugs in HR MDS contrast with healthy samples, as summarized in Table 1. Specifically: 1) both drugs increased the levels of all three DNMTs in lymphocytes, 2) DNMT1 levels were increased by both drugs in lymphocytes, myeloid and CD34+cell subsets, and 3) effects of the two drugs diverged with respect to effects on DNMT 3a and 3b in myeloid cell subsets (Table 1). Conclusions:In vitro treatment for 24 hrs with clinically relevant concentrations of AZA or DAC decreased baseline DNMTs levels in cell lines and healthy BMMC, consistent with their role as promoters of genomic hypomethylation. However, these drugs have strikingly different effects on primary HR MDS BMMC cell subsets. While MDS is not typically considered a disorder of lymphocytes, DNMTs levels increased in response to either AZA or DAC suggesting an alteration in HR MDS lymphocytes vs. HEA lymphocytes. The combined differential activities of AZA and DAC on cell cycle, intracellular DNMTs levels and apoptosis in specific cell subsets (in particular DNMT3a levels in myeloid and CD34+ HR MDS BMMC and absence of DAC-induced apoptosis) suggest the inhibition of post-transcriptional processing by AZA contributes significantly to cytotoxicity and may explain differences in clinical activities of the 2 drugs.Table 1:Summary of Effects of AZA & DAC on DNMTs levels in HR MDSCellsLymphsnRBCMyeloidCD34+DrugAZADACAZADACAZADACAZADACBMMC SourceHEAMDSHEAMDSHEAMDSHEAMDSHEAMDSHEAMDSHEAMDSHEAMDSDNMT1↓↑↓↑----↓↑↓↑↓↑↓↑DNMT3a↓↑↓↑----↓↓↓↑↓↓↓↑DNMT3b↓↑↓↑↓↑↓↑↓--↑↓--↑HEA = Healthy Donor sample; - = no change Disclosures:Cohen:Nodality, Inc.: Consultancy. Huang:Nodality, Inc.: Employment, Equity Ownership. Hawtin:Nodality, Inc.: Employment, Equity Ownership. Ware:Nodality, Inc.: Employment, Equity Ownership. Cesano:Nodality, Inc: Employment, Equity Ownership.

Synthesis of cationic polyacrylamide by aqueous two‐phase polymerization in poly(ethylene glycol) chloride solution

AbstractAqueous two‐phase copolymerization of acrylamide(AM) and acryloyloxyethyl trimethyl ammonium chloride (DAC) was performed in poly(ethylene glycol) (PEG) solution and in PEG chloride(Cl‐PEG) solution, respectively. Series of cationic polyacrylamide(CPAM) aqueous dispersion were prepared using potassium persulfate (KPS) as initiator. The effect of total amount of monomers, the dosage of initiator, the content of dispersant, the mass ratio of AM to DAC, and the temperature on the conversion, molecular weight, cationic degree, and stability of aqueous dispersion were studied in detail. It is found that the increase of initiator and reaction temperature resulted in the increase of the final conversion, whereas the increase of DAC and PEG concentration resulted in the decrease of the final conversion. The optimum reaction conditions of synthesis were as follows: (1) PEG‐H2O system: PEG 7.5 g, AM 8 g, DAC 2 g, KPS 0.05 g, H2O 100 mL, 70°C. In this process conditions, the molecular weight of CPAM was 3.21 × 106, the cationic degree of CPAM was 24.4%, the storage stability of the aqueous dispersion was over 3 months. (2) Cl‐PEG‐H2O system: Cl‐PEG 7.5 g, AM 8 g, DAC 2 g, KPS 0.05 g, H2O 100 mL, 65°C. In this process conditions, the molecular weight was 3.68 × 106, the cationic degree was 23.3%, and the storage stability of the aqueous dispersion was over 6 months. In general, the stability of CPAM aqueous dispersion in Cl‐PEG system is much better than in PEG system. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

Synergetic effects of DNA demethylation and histone deacetylase inhibition in primary rat hepatocytes

Both, DNA methylation and histone deacetylation play a crucial role in cancer development by silencing the expression of specific tumour suppressor genes. Several studies describe the use of combinations of DNA methyltransferase inhibitors (DNMT-i) and histone deacetylase inhibitors (HDAC-i) as an improved strategy to treat neoplasms. However, no information is available concerning their biological impact on healthy, non-malignant cells, including hepatocytes. Therefore, the effects of the combination of the DNMT-i decitabine (DAC) with the HDAC-i 6-[(4-pyrrolidine-1-ylbenzoyl) amino] hexanoic acid hydroxamate (AN-8) on cell proliferation and differentiation were examined in primary rat hepatocyte cultures. We found that, upon simultaneous exposure of the cells to both compounds, a synergetic anti-proliferative outcome was achieved. This inhibition of DNA synthesis was accompanied by a reduced expression of cyclin-dependent kinase 1 (cdk1), a key cell cycle marker that controls the S/G2/M transition. Compared to exposure of the cells to each agent separately, the combination of lower concentrations of both DAC and AN-8 promoted the maintenance of the differentiated phenotype of the cells as a function of culture time. The functionality of the hepatocytes was evidenced by an increased expression of the phase I biotransformation enzyme cytochrome P 450 (CYP) 1A1 and albumin secretion capacity when both agents were used in combination.

Upregulation of MAGE-A1, MAGE-A3, NY-ESO-1 and MHC Molecules on Neuroblastoma Cells Following Exposure to Decitabine and Interferon-γ

AbstractAbstract 5179MAGE-A1, MAGE-A3, and NY-ESO-1 are cancer-testis (CT) antigens expressed on a number of malignant solid tumors, including neuroblastoma (NB), but many tumor cell lines down-regulate the expression of CT antigens as well as major histocompatibility (MHC) antigens, precluding recognition by antigen specific T cells. If expression of cancer antigens as well as MHC molecules could be enhanced pharmacologically, the efficacy of CT antigen specific immunotherapy could be improved. DNA methylation is an epigenetic mechanism used by cells to decrease the expression of several genes, including tumor suppressor genes. Decitabine (5-aza-2′-deoxycytidine, DAC) is a potent inhibitor of DNA methylation, and the doses associated with the demethylating action of this drug are much lower than those required for cytotoxicit. We have demonstrated that the expression of MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells following exposure to the DAC. When these cell lines are cultured in the presence of DAC at concentrations similar to levels achieved with clinical administration, we were able to achieve expression of NY-ESO-1, MAGE-A1, or MAGE-A3 in 11/11 neuroblastoma cell lines. Concentrations of 1uM DAC were associated with strongest expression of these antigens, which was optimal after 72 hours of drug exposure. Culture of NB cell lines with IFN-γ was also associated with an increased expression of either MHC Class I or II by cytofluorometry, and Class I upregulated in 10/11 of these cell lines, although to varying degrees. MAGE-A1, MAGE-A3, and NY-ESO-1 specific CTL were cultured from volunteer donors by stimulating peripheral blood mononuclear cells with dendritic cells pulsed with overlapping peptide mixes derived from full length proteins, and these CTL lysed HLA partially matched, DAC treated neuroblastoma as well as glioblastoma cell lines. These studies confirm that DAC and IFN-γ can be used to increase the expression of CT antigens and MHC molecules on neuroblastoma cells, and pre-treatment with these agents makes tumor cell lines more susceptible to CTL mediated killing. Disclosures:No relevant conflicts of interest to declare.

Importance of dose-schedule of 5-aza-2'-deoxycytidine for epigenetic therapy of cancer

BackgroundThe inactivation of tumor suppressor genes (TSGs) by aberrant DNA methylation plays an important role in the development of malignancy. Since this epigenetic change is reversible, it is a potential target for chemotherapeutic intervention using an inhibitor of DNA methylation, such as 5-aza-2'-deoxycytidine (DAC). Although clinical studies show that DAC has activity against hematological malignancies, the optimal dose-schedule of this epigenetic agent still needs to be established.MethodsClonogenic assays were performed on leukemic and tumor cell lines to evaluate the in vitro antineoplastic activity of DAC. The reactivation of TSGs and inhibition of DNA methylation by DAC were investigated by reverse transcriptase-PCR and Line-1 assays. The in vivo antineoplastic activity of DAC administered as an i.v. infusion was evaluated in mice with murine L1210 leukemia by measurement of survival time, and in mice bearing murine EMT6 mammary tumor by excision of tumor after chemotherapy for an in vitro clonogenic assay.ResultsIncreasing the DAC concentration and duration of exposure produced a greater loss of clonogenicity for both human leukemic and tumor cell lines. The reactivation of the TSGs (p57KIP2 in HL-60 leukemic cells and p16CDKN2A in Calu-6 lung carcinoma cells) and the inhibition of global DNA methylation in HL-60 leukemic cells increased with DAC concentration. In mice with L1210 leukemia and in mice bearing EMT6 tumors, the antineoplastic action of DAC also increased with the dose. The plasma level of DAC that produced a very potent antineoplastic effect in mice with leukemia or solid tumors was > 200 ng/ml (> 1 μM).ConclusionWe have shown that intensification of the DAC dose markedly increased its antineoplastic activity in mouse models of cancer. Our data also show that there is a good correlation between the concentrations of DAC that reduce in vitro clonogenicity, reactivate TSGs and inhibit DNA methylation. These results suggest that the antineoplastic action of DAC is related to its epigenetic action. Our observations provide a strong rationale to perform clinical trials using dose intensification of DAC to maximize the chemotherapeutic potential of this epigenetic agent in patients with cancer.