5-aza Group Research Articles

Inflamma-miRs Profile in Myelodysplastic Syndrome Patients.

Etiological factors involved in myelodysplastic syndrome (MDS) include immunologic, oxidative stress and inflammatory factors, among others, and these are targets for microRNAs (miRNs). Here, we evaluated whether some miRNs may affect tumor development comparing untreated and 5-azacitidine (5-AZA) MDS-treated patients. Peripheral blood samples were collected from 20 controls and 24 MDS patients, and selected miRNs related to redox balance and inflammation (inflamma-miRs), including miR-18a, miR-21, miR-34a and miR-146a, were isolated and measured by quantitative real-time polymerase chain reaction (qRTPCR). A differential expression profile of miRNs was detected in untreated MDS patients and the 5-AZA group. Inflammation increases miRNs and, specifically, miR-18a, miR-21 and miR-34a were significantly overexpressed in untreated MDS, compared to controls. However, we did not observe any miRN profile alteration during the progression of the disease. On the other hand, 5-AZA treatment tends to restore miRN expression levels. Relating to prognostic risk factors, high-risk MDS groups (high Revised International Prognostic Scoring System (IPSS-R), high cytogenetic risk, high molecular risk (HMR) mutations) tended to be related with higher expression levels of miR-18a and miR-34a. Higher miRN expression is correlated with lower glutathione peroxidase activity, while they are related with a higher profile of pro-inflammatory cytokines (IL-2, IL-6, IL-8, TNF-α). Although our study was limited by the low number of MDS patients included, we identified miRN deregulation involved in MDS development that could regulate redox sensors and inflammatory responses. Finally, 5-AZA treatment is related with lower miRN expression levels in MDS patients.

MicroRNA-22-3p Regulates the Expression of Kruppel-like Factor 6 to Affect the Cardiomyocyte-like Differentiation of Bone Marrow Mesenchymal Stem Cell

Objective To explore the effect of microRNA-22-3p (miR-22-3p) regulating the expression of Kruppel-like factor 6 (KLF6) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cell (BMSC). Methods Rat BMSC was isolated and cultured,and the third-generation BMSC was divided into a control group,a 5-azacytidine(5-AZA)group,a mimics-NC group,a miR-22-3p mimics group,a miR-22-3p mimics+pcDNA group,and a miR-22-3p mimics+pcDNA-KLF6 group.Real-time fluorescent quantitative PCR (qRT-PCR) was carried out to determine the expression of miR-22-3p and KLF6 in cells.Immunofluorescence staining was employed to detect the expression of Desmin,cardiac troponin T (cTnT),and connexin 43 (Cx43).Western blotting was employed to determine the protein levels of cTnT,Cx43,Desmin,and KLF6,and flow cytometry to detect the apoptosis of BMSC.The targeting relationship between miR-22-3p and KLF6 was analyzed by dual luciferase reporter gene assay. Results Compared with the control group,5-AZA up-regulated the expression of miR-22-3p (q=7.971,P<0.001),Desmin (q=7.876,P<0.001),cTnT (q=10.272,P<0.001),and Cx43 (q=6.256,P<0.001),increased the apoptosis rate of BMSC (q=12.708,P<0.001),and down-regulated the mRNA (q=20.850,P<0.001) and protein (q=11.080,P<0.001) levels of KLF6.Compared with the 5-AZA group and the mimics-NC group,miR-22-3p mimics up-regulated the expression of miR-22-3p (q=3.591,P<0.001;q=11.650,P<0.001),Desmin (q=5.975,P<0.001;q=13.579,P<0.001),cTnT (q=7.133,P<0.001;q=17.548,P<0.001),and Cx43 (q=4.571,P=0.037;q=11.068,P<0.001),and down-regulated the mRNA (q=7.384,P<0.001;q=28.234,P<0.001) and protein (q=4.594,P=0.036;q=15.945,P<0.001) levels of KLF6.The apoptosis rate of miR-22-3p mimics group was lower than that of 5-AZA group (q=8.216,P<0.001).Compared with the miR-22-3p mimics+pcDNA group,miR-22-3p mimics+pcDNA-KLF6 up-regulated the mRNA(q=23.891,P<0.001) and protein(q=13.378,P<0.001)levels of KLF6,down-regulated the expression of Desmin (q=9.505,P<0.001),cTnT (q=10.985,P<0.001),and Cx43 (q=8.301,P<0.001),and increased the apoptosis rate (q=4.713,P=0.029).The dual luciferase reporter gene experiment demonstrated that KLF6 was a potential target gene of miR-22-3p. Conclusion MiR-22-3p promotes cardiomyocyte-like differentiation of BMSC by inhibiting the expression of KLF6.

Standard Risk-Assessment of AML Doesn't Predict the Outcome in Older AML Patients Undergoing Non-Intensive Chemotherapy

Background: Acute myeloid leukemia (AML) still has a poor prognosis for elderly patients, both because of specific characteristics of the disease in this population, and especially because of their frailty that limits the use of intensive treatments (IT). Low dose Aracytine (LDAC) and 5-Azacitidine (5-AZA) are the 2 options currently recommended for these patients, but real-life data are lacking about response rates, survival, and superiority of one treatment over the other. Methods: In our multi-center study based on the French Hauts-de-France AML observatory, we enrolled all patients with newly diagnosed AML treated in first line with LDAC and 5-AZA. The primary endpoint was the overall survival (OS). Our secondary objectives were: evaluating the response rates, identifying predicting factors of early mortality and the profile of long-term survivors (i.e alive 6 months after the diagnosis). Results: Between January 2009 and September 2019, 279 patients who received LDAC (n=87) or 5-AZA (n=192) were included. Median age was 76 years (IQR 70 - 81) (LDAC 79 years (IQR 75 - 83) and 5-AZA 74 years (IQR 68 - 79), p<0.001)) and the Charlson comorbidity index was significantly higher in the LDAC arm: 7 (IQR 6 - 8) versus 6 (IQR 5 - 7) in 5-AZA, p=0.007). Leukocytosis was significantly lower in the 5-AZA group: 3.2G/L (IQR 1.8- 7.7) versus 8.5G/L in LDAC (IQR 2.3-35), p<0.001. Patients treated with 5-AZA predominantly had AMLs associated with myelodysplasia-related changes and adverse cytogenetics according to ELN 2017, whereas those treated with LDAC predominantly had AML NOS with intermediate karyotype. After a median follow-up of 6.9 months (IQR 1.9-11.6), median OS (Figure 1A) was 7.8 months (IQR 2.5 - 13.6): 4.8 months (IQR 2.13 - 14.41) for the LDAC group versus 8.9 months (IQR 3.2 - 13.5) for the 5-AZA group (p=0.046), but there was no significant difference in OS at the end of the study (Figure1B). Complete remission rate was 27.3% without any difference between the 2 groups. At day 30, on the basis of Kaplan-Meier estimates, 25.3% of patients in the LDAC group had died compared to 9.4% in the 5-AZA group (OR 0.28; 95% CI (0.09 - 0.84), p<0.023). After six months of treatment, there was no difference in OS between LDAC and 5-AZA groups (HR 1.37; 95% CI (0.92-2.04), p= 0.116). The predicting factors of mortality at day 30, at 6 months and after 6 months are summarized in the Table 1 (multivariate analysis, odds-ratio). Interestingly, the predictive factors of early mortality were albuminemia less than 30g/L (adjusted OR 6.25; 95% CI (2.08 - 20.00); p<0.001) and treatment with LDAC (adjusted OR 3.57; 95%CI (1.19 - 11.11); p=0.023). At 6 months, ECOG 2-3, high bone marrow blast count at diagnosis and albuminemia less than 30g/L significantly impaired the OS. After 6 months, the only predictive factor of survival was albuminemia greater than 30g/L (adjusted HR 0.65, 95% CI (0.44- 0.96); p=0.030). Usual molecular markers and prognosis classifications (ELN and Lindsley) were not helpful to predict mortality (both early and late) of this specific population. Discussion: Standard prognostic classifications of AML, such as the ELN 2017 and Lindsley classifications, which have been evaluated in patients eligible for IT, do not appear to be accurate in predicting outcome in older unfit patients treated with LDAC or 5-AZA. Albumin, a simple biomarker, was the strongest predictor of early mortality and prolonged survival beyond 6 months, highlighting that nutritional status and global fitness are the key determinants of survival in older patients non eligible for IT. Overall, the low survival emphasizes the poor prognosis of AML in these patients, and the urgent need for innovative therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Azacitidine Maintenance Following Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia: A Systematic Review

Background and Objective: Disease relapse remains the primary cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (Allo-HSCT) in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Oral azacitidine (5-aza) was associated with clinical benefit as maintenance therapy in transplant ineligible patients. However, contradicting data have been reported regarding the role of 5-aza as a maintenance therapy to reduce relapse rate -post-allo-HSCT. We conducted this systematic review to describe the efficacy and safety of 5-aza in this context. Materials and Methods: We systematically searched multiple databases, including PubMed, Embase, Cochrane, and Clinicaltrials.gov. We also searched major conferences for oral or poster presentations. We used MeSH terms and keywords for MDS, AML, Allo-HSCT, and 5-aza. We included all retrospective and prospective studies of 5-aza (all formulations) published until March 2020. The primary database search yielded 1209 articles. We excluded irrelevant, duplicate, and review articles. The final search revealed 20 articles that we explored in detail for various efficacy and safety outcomes. Results: A total of 1211 patients were enrolled in 14 prospective and 6 retrospective studies. Of those, 1169 patients were evaluable. Prospective studies CALGB 100801 trial by Vij et al. (2015) reported overall survival (OS) of 45.7% and progression-free survival (PFS) of 41.2% at 24 months following reduced intensity conditioning (RIC) in a phase 2 trial of 41 patients. In a randomized control trial, Oran et al. (2018) evaluated relapse-free survival (RFS) as the primary outcome after 12 cycles of 5-aza at 32 mg/m2/day (n=187). Only 30% of the patients in the azacitidine arm completed the targeted number of cycles. The study showed a median RFS of 24.8 months in the treatment arm vs 15.3 months in the control arm (p=0.43). In a phase 2 trial conducted by Guillaume, T et al. (2019) the cumulative incidence of relapse was 27.6% in patients who received 5-aza, compared with 41.9% in 58 matched patients control group (p=0.21). Platzbecker et al. (2018-2019) studied a higher dose of azacitidine (75mg/m2/day) in a phase 2 trial as a pre-emptive strategy for patients who develop minimal residual disease (MRD) within 24 months. The 12-month OS and PFS were 94% and 44%, respectively. De Lima et al. (2018) studied the role of maintenance Oral 5-aza in Phase I/II trial (n=30). A dose-escalation design was used with a dosage ranging from 150 mg to 400 mg, received in a 7- or 14-day cycles. The 12-month PFS was 54% and 72% and estimated survival was 86% and 81% among 7-days and 14-days cycles, respectively. (Table 1) Retrospective studies: Mishra et al. (2017) reported a better OS in 14 patients who received 5-aza maintenance compared with a control arm (p-value 0.026). Cheikh et al. reported a 12-month OS of 70%. Ali, N et al. (2020) (n=107) compared 5-aza group vs. control group retrospectively. EFS was 53.1% vs. 49.5% (p=0.02) while OS was 56.8 and 53.4 months (p=0.01) in the treatment arm vs the control arm respectively. Safety: The most common grade 3 or 4 hematological adverse effect was neutropenia, while some patients also experienced grade 3 or 4 anemia, thrombocytopenia, or lymphopenia. The main non-hematological adverse effects were infections, fatigue, and gastrointestinal distress. The incidence of acute graft versus host disease varied from 13% to 50%. The most common reason for treatment discontinuation was disease relapse. A minority of patients discontinued treatment due to side effects. Conclusion: To our knowledge, this is the first comprehensive systematic review for the role of 5-aza maintenance -post-Allo-HSCT in patients with MDS or AML. The heterogeneity of the studies, in terms of dosing regimens, variable duration of treatment and patient selection, precludes definitive conclusions. Despite that, 5-aza seems to improve relapse rates and OS at least numerically but also significantly in some studies, as illustrated in this review. Low number of patients involved in most of these studies contributed to non-significant p-values. Azacitidine remains a valid and safe option, especially in patients with high risk of relapse. Further studies aiming at those high risk patients, such as AML with myelodysplasia related changes (AML-MRC) and high-risk MDS following RIC, are of utmost need. Disclosures Anwer: Incyte, Seattle Genetics, Acetylon Pharmaceuticals, AbbVie Pharma, Astellas Pharma, Celegene, Millennium Pharmaceuticals.: Honoraria, Research Funding, Speakers Bureau. Fazal:Takeda: Consultancy, Speakers Bureau; GlaxoSmithKline: Consultancy, Speakers Bureau; Agios: Consultancy, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Speakers Bureau; Janssen: Speakers Bureau; Gilead/Kite: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Stemline: Consultancy, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Karyopharm: Speakers Bureau; Jazz: Consultancy, Speakers Bureau.

Differentiation of mesenchymal stem cells into cardiomyocytes is regulated by miRNA-1-2 via WNT signaling pathway

BackgroundBone marrow derived stem cells (BMSCs) have the potential to differentiate into cardiomyocytes, but the rate of differentiation is low and the mechanism of differentiation is unclear completely. Here, we aimed to investigate the role of miR1-2 in differentiation of mouse BMSCs into cardiomyocyte-like cells and reveal the involved signaling pathways in the procedure.MethodsMouse BMSCs were treated with miR1-2 and 5-azacytine (5-aza). The expression of cardiac cell markers: NKx2.5, cTnI and GATA4 in BMSCs were examined by qPCR. The apoptosis rate was detected by flow cytometry and the activity of the Wnt/β-catenin signaling pathway was evaluated by measuring the upstream protein of this signaling pathway.ResultsAfter over-expression of miR1-2 in mouse BMSCs, the apoptosis rate was significantly lower than the 5-aza group, while the expressions of cardiac-specific genes: such as Nkx2.5, cTnI and GATA4 were significantly increased compared to the control group and the 5-aza group. Meanwhile, over-expression of miR1-2 in mouse BMSCs enhanced the expression of wnt11, JNK, β-catenin and TCF in the Wnt/β-catenin signaling pathway. Use of LGK-974, an inhibitor of Wnt/β-catenin signaling pathway, significantly reduced the expression of cardiac-specific genes and partially blocked the role of the miR1-2.ConclusionOver-expression of miR1-2 in mouse BMSCs can induce them toward promoted cardiomyocyte differentiation via the activation of the Wnt/β-catenin signaling pathway. Compared to 5-aza, miR1-2 can induce differentiation of BMSCs into cardiomyocytes more effectively with a less cytotoxicity.

Acute Myeloblastic Leukemia and Myelodysplastic Syndrome: Azacitidine for Prophylactic and Preventive Purposes after Allogeneic Hematopoietic Stem Cell Transplantation

Aim. To evaluate the effectiveness of preventive and prophylactic post-transplantation therapy using azacitidine (5-AZA) in patients at high risk of post-transplantation relapse. Methods. 136 patients were included in the study performed by the pairwise comparison: 68 of them received 5-AZA after allo-HSCT and 68 patients were included in the historical control group. 5-AZA was prescribed for prophylactic or preventive purposes. The results were assessed according to the OS, RR, EFS, DUM, and relapse-free and GVHR-free survival. Results. 1-year OS was 76 % in the 5-AZA group (95% CI 60-84 %) and 44 % in the reference group (95% CI 33-55 %) (р = 0.001); 2-year OS was 63 % (95% CI 39-67 %) and 37 % (95% CI 26-48 %) (р = 0.007), respectively. The relapse rate (RR) in the 5-AZA group was 34 % (95% CI 22-46 %) during 1 year and 51 % (95% CI 38-64 %) in the reference group (р = 0.02). 1- and 2-year disease unrelated mortality (DUM) was similar: 5 % in the 5-AZA group (95% CI 0.1-14.0 %) and 25 % (95% CI 13-37 %) in the reference group (р = 0.005). 1-year EFS was 76 % in the 5-AZA group (95% CI 61-85 %) and 44 % in the reference group (95% CI 33-55 %) (р = 0.001); 2-year EFS was 63 % (95% CI 39-67 %) and 37 % (95% CI 2648 %) (р = 0.01), respectively. 1-year relapse-free and GVHR-free survival was 55 % in the 5-AZA group (95% CI 41-69 %) and 28 % in the reference group (95% CI 17-39 %) (р = 0.001); 2-year relapse-free and GVHR-free survival was 47 % (95% CI 32-62 %) and 27 % (95% CI 17-37 %) (р = 0.002), respectively. Conclusion. The use of 5-AZA for prophylactic and preventive purposes after allo-HSCT does not increase the risk of GVHR and DUM, does not suppress the GVL effect and can be used in combination with the donor lymphocyte infusion (DLI). The therapy with 5-AZA is safe during the early period after allo-HSCT. The drug does not suppress the GVL effect and can be used in high risk patients to prevent early post-transplantation relapse. The use of 5-AZA in combination with DLI does not increase the incidence of severe GVHR.

Role of DNA methyltransferases in the pathogenesis of neuropathic pain in rats

To explore the role of DNA methyltransferases (DNMTs) in the pathogenesis of neuropathic pain (NPP) in rats following sciatic nerve chronic constriction injury (CCI). A total of 27 adult male Sprague-Dawley rats with successful implantation of lumbar intrathecal catheter were randomly divided into 3 groups: a sham + normal saline group (sham+NS group), a CCI+NS group, and a CCI+5-azacytidine group (CCI+5-AZA group) (n=9 in each group). The rats in the Sham+NS group and the CCI+NS group received NS, while the rats in the CCI+5-AZA group received 10 μmol/L of 5-AZA (a DNMTs inhibition) once a day through spinal injection from the 3th day to 14th day after CCI surgery. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of ipsilateral hinds in the 3 groups were measured before or at the 3th, 5th, 7th, 10th or 14th day after CCI surgery. At the end of experiments, all rats were killed under deep anesthesia and their lumbar spinal cords were dissected to examine the DNMT1, DNMT3a and DNMT3b expression by RT-PCR, Western blot and immunohistochemistry, respectively. Compared with the sham+NS group, the MWT and TWL in the CCI+NS group were obviously reduced from the 3th day to the 14th day after surgery (both P<0.05). Compared with the CCI+NS group, the MWT and TWL in the CCI+5-AZA group were obviously increased from the 5th day to the 14th day after surgery (both P<0.05), but they were still reduced compared with the sham+NS group (both P<0.05). The DNMT1, DNMT3a and DNMT3b were highly expressed in the lumbar spinal dorsal horn in all rats, and the positive signals were mainly located in the nucleus. The DNMT1, DNMT3a and DNMT3b levels in the CCI+NS group were increased significantly compared with that in the sham+NS group on the 14th day after surgery (all P<0.05). The DNMT1, DNMT3a and DNMT3b expressions in the CCI+ 5-AZA group were decreased significantly compared with that in the CCI+NS group (all P<0.05), but they still increased compared with that in the sham+NS group (all P<0.05). Up-regulation of DNMTs in the lumbar spinal may play an important role in the pathogenesis of NPP in CCI rats. DNMTs inhibitors (5-AZA) could reduce expression of DNMTs and attenuate CCI-induced NPP, which might be a potential therapeutic drug for NPP.

Rehmannia glutinosa oligosaccharide induces differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells

The aim of this study was to observe the effect of Rehmannia glutinosa oligosaccharide (RGO) on differentiation of bone marrow mesenchymal stem cells (MSCs) into cardiomyocyte-like cells . Rat MSCs were isolated, treated, and grouped as follows: RGO treatment group, 5-azacytidine (5-aza) treatment group, RGO + 5-aza treatment group, and control group. Following a four-week induction period, cardiac troponin I (cTnI) levels in MSCs were quantified by chemiluminescence, and the levels of myocardial enzymes creatine kinase (CK) and creatine kinase isoenzyme-MB (CK-MB) were measured using a dry chemistry analyzer. The cTnI- and connexin 43 (Cx43)-positive MSC population was identified by immunofluorescence, and expression levels of cTnI and Cx43 were analyzed by western blots. Following induction, cTnI, CK, and CK-MB levels were significantly higher in the RGO + 5-aza group as compared with the RGO and 5-aza groups (P < 0.05). In addition, fluorescence intensity of cTnI and Cx43 was higher in the RGO + 5-aza group as compared with the RGO and 5-aza groups. No cTnI- or Cx43-positive cells were detected in the control group. Western blot analysis further confirmed that cTnI and Cx43 were not expressed in the control group, while cTnI and Cx43 was higher in the RGO + 5-aza group than in the RGO and 5-aza groups. These results suggest that MSCs can be induced by RGO to differentiate into cardiomyocyte-like cells in vitro, and that RGO in combination with 5-aza enhance differentiation of MSCs.

Differentiation of Bone Marrow Mesenchymal Stem Cells to Cardiomyocyte-Like Cells Is Regulated by the Combined Low Dose Treatment of Transforming Growth Factor-β1 and 5-Azacytidine.

Bone marrow mesenchymal stem cells (BMMSCs) are used in cardiac tissue engineering for the regeneration of diseased hearts. We examined the differentiation of rat BMMSCs into cardiomyocyte-like cells when induced with a combined low dose treatment of transforming growth factor-β1 (TGF-β1) and 5-azacytidine (5-AZA). Results showed that cell proliferation in the combined low dose treatment group of TGF-β1 and 5-AZA was increased compared with the TGF-β1 group or the 5-AZA group. The cell apoptosis was relieved by combined TGF-β1 and 5-AZA treatment compared to 5-AZA treatment alone. The number of cells positive for myosin heavy chain, connexin-43, α-actin, and troponin I in the combined treatment group was higher than those observed in the TGF-β1 group or the 5-AZA group. Moreover, the combined low dose treatment group of TGF-β1 and 5-AZA reveals the strongest expression of troponin I, α-actin, and phosphorylated extracellular signal-regulated protein kinases 1 and 2 (p-ErK1/2) among the treatment groups. These results suggest that the combined low dose treatment of TGF-β1 and 5-AZA can improve the differentiation potential of rat BMMSCs into cardiomyocyte-like cells and alleviate cell damage effects in vitro. The mechanism that is involved in influencing differentiation may be associated with p-ErK1/2.

Azacitidine Improves Survival in Myelodysplastic Syndromes; Single Institute Retrospective Study

ObjectivesProspective clinical trials show that 5-Azicitidine (5-Aza) is superior to conventional care regimens in prolonging the survival of patients with high-risk myelodysplastic syndromes (MDS) (pts). To confirm the survival benefit of 5-Aza in daily clinical practice, we compared the survival of high-risk MDS patients who received parental treatment before 5-Aza approval to those who could be initially treated with 5-Aza. MethodsIn Japan, 5-Aza was approved in January 2011. We collected data of consecutive adult patients (pts) with high-risk MDS, namely refractory anemia with excess blasts-1 (RAEB-1), RAEB-2, and acute myeloid leukemia with MDS related features (AML/MRF), who received the first parental chemotherapy between January 2000 and April 2013. Survival was not calculated from the day on which the first chemotherapy was delivered, but from the day on which the diagnosis of high-risk MDS was established. Survival analysis was done by the Kaplan-Meier method. The Cox regression model was used for the analyses. A total of 107 pts with high-risk MDS were identified. They were divided into two groups (grps) on the basis of whether or not they could be initially treated with 5-Aza. In brief, pts who received the first parental treatment before January 2011 were categorized into the conventional care (CC) grp (N=75), and pts for whom treatment began after January 2011 were categorized in the 5-Aza grp (N=32). The following variables were also considered to affect survival in multivariate analysis: sex, age (>=65 years (yrs) old or < 65 yrs old), WHO classification, and IPSS at diagnosis of high-risk MDS. ResultsMedian follow-up times of CC and 5-Aza grp were 43 and 11 months, respectively. As shown in Table 1, the distributions of sex, age, WHO classification and IPSS were nearly the same in both grps. As for initial treatment, pts in the CC group mainly received low-dose cytarabine (ld-AraC), whereas most pts in the 5-Aza grp were treated with 5-Aza. Unadjusted survival curves of the two grps are shown in Figure 1. The unadjusted 2-yr survival rate was 33% in the CC grp and 52% in 5-Aza era grp. Median survival times were 11 and 24 months in CC and 5-Aza grps, respectively. Uni-variate analysis of overall survival revealed a trend for improved survival in younger pts, but this was contradicted by the results of multi-variate analysis. In uni- and multi-variate analyses, the 5-Aza group showed an overall, and statistically significant higher survival benefit (Table 2).Removing the 26 patients allografted after parental treatment (10 in 5-Aza grp and 16 in CC grp) at the time of alloSCT from the analyses did not modify the survival advantage of the 5-Aza grp. ConclusionThis retrospective analysis confirms that the introduction of 5-Aza has certainly improved the outcome of high-risk MDS in daily clinical practice.Table 1Patients characteristics at diagnosis of high-risk MDSPatients characteristics<=2010 (N=75) (N/%)>=2011 (N=32)Total (N=107)Sex(male/female)57(76)/18(24)28(88)/4(12)85(79)/22(21)Age (median)<65 >=656829(39)46(61)68.514(44)18(56)6843(40)64(60)Initial treatmentlow-dose AraC5-Azaintensive chemotherapy63(84)0(0)12(16)3(9)26(81)3(9)66(62)26(24)15 (14)WHO ClassificationRAEB1, RAEB2, AML/MRFMissing/Unevaluable19(25)31(42)15(20)10(13)7(22)13(41)8(25)4(13)26(24)44(41)23(21)14(13)IPSS lowInt-1Int-2high Missing/Unevaluable1(1)15(20)21(28)24(32)14(19)0(0)7(22)8(25)12(38)5(16)1(1)22(21)29(27)36(34)19(18) [Display omitted] Table 2Uni- and multi-variate analysis of overall survivalVariablesUnivariateMultivariateRR (95%CI)PRR (95%CI)PAge (vs.<65y)>=65y1.5(0.98-2.41)0.0611.5(0.94-2.5)0.84Sexmale0.92(0.53-1.60)0.760.73(0.39-1.4)0.33Disease status (vs.RAEB1)RAEB2AML/MRFMissing/Unevaluable0.73(0.42-1.3)0.78(0.41-1.5)0.69(0.34-1.4)0.270.450.320.72(0.37-1.4)1.3(0.51-3.4)0.76(0.25-2.3)0.330.580.62Treatment Era (vs. Conventional Care Era)5-Aza0.54(0.32-1.0)0.0270.39(0.18-0.83)0.043IPSS (vs. Low)Intermediate-1Intermediate-2HighMissing/Unevaluable2.6(0.35-20)2.3(0.31-17)2.0(0.26-15)1.9(0.24-14.)0.350.420.510.555.0(0.53-48)2.5(0.22-28) 1.3(0.099-17)3.0(0.21-44)0.160.470.840.42 Disclosures:No relevant conflicts of interest to declare.

GW24-e1012 The experimental study of Bone Mesenchymal stem cells transdifferentiation to cardiac cells by knockdown β-catenin in vitro

ObjectivesWe blocked the canonical wnt signalling pathway through knocking down β-catenin in Mesenchymal stem cells (MSCs), and investigated the role of 5-Azacytidine, salvianolic acid B and cardiomyocytes lysis in inducing...

GW24-e3756 Bone marrow mesenchymal stem cells differentiate into myocardial cells in different inducing conditions In vitro.

Objectivesthis study is to explore the effect of differentiation of BMSCs into myocardial cells in different inducing conditions In vitro.MethodsBMSCs were isolated with the density gradient centrifugation combined with adherent...

Myocardial structural protein expression in umbilical cord blood mesenchymal stem cells after myogenic induction

To assess the effects of three methods of inducing myogenic cells differentiation, umbilical blood mesenchymal stem cells (UCMSCs) from nearly full-term pregnancy mongrel dogs were purified and cultured. Fourth-passage UCMSCs were used to detect surface antigens, including CD11a, CD11b, CD29, CD34 and CD71. The cells were induced by 5-azacytidine (5-aza), myocardial lysates and myocardial induced fluid. Positive expression of Nkx2.5, α-actin, desmin, β-MHC and troponin-I (TN I) were detected after 3 weeks. The immunohistochemical results were CD11a (-), CD11b (-), CD34 (-), CD29 (+) and CD71 (+). Nkx2.5 was detected in 5-aza group, myocardial lysates group and myocardial induced fluid group. Semi-quantitative analysis showed Nkx2.5 expression significantly higher in myocardial lysates group than in the 5-aza group or myocardial-induced fluid group (P < 0.05), but there was no significant difference between the 5-aza and myocardial-induced fluid groups for Nkx2.5 expression (P > 0.05). MSCs did not express myocardial structural proteins before differentiation, but α-actin, desmin, β-MHC and troponin-I were present after differentiation. The positive expression of four proteins differed with the differentiation conditions. The UCMSCs can be differentiated into myogenic cells by three methods, but the degrees of differentiation are inconsistent. Our results show that the effects of 5-aza and myocardial lysates are better than that of myocardial induced fluid.

The combination of angiotensin II and 5-azacytidine promotes cardiomyocyte differentiation of rat bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (BMMSCs) are ideal seed cells for tissue engineering and regenerative medicine. Many studies have shown that 5-azacytidine (5-aza) can induce BMMSCs to differentiate into cardiomyogenic cells, but some issues still remain to be resolved. In this study, we investigated the effects of angiotensin II (Ang II) on the proliferation and differentiation of BMMSCs induced by 5-aza in vitro. BMMSCs were isolated from the bone marrow of Sprague-Dawley rats by density gradient centrifugation. The third-passage cells were divided into four groups: the Ang II group (0. 1 μmol/l) (group A), the 5-aza group (10 μmol/l) (group B), the Ang II combined with 5-aza group (0.1 and 10 μmol/l) (group C), and the untreated group as control. After 24 h of induction, the medium was changed to the complete culture medium without any inductor, and the cells were cultured for 3 weeks. Morphological changes were observed under a phase contrast microscope. The effect of Ang II and 5-aza on BMMSC proliferation was evaluated by the methyl thiazolyl tetrazolium (MTT) assay. Cardiomyogenic cells were identified through immunofluorescence staining, and the induction ratio was examined by flow cytometry. The level of cardiac troponin I (cTnI) was examined by western blotting, and the ultrastructures of the induced cells were viewed with a transmission electron microscope. The MTT assay showed that the cell proliferation in group C outweighed that in either group A or group B, but no significant difference existed between group A and group B. The expression of specific proteins, namely, cTnI and sarcomeric α-actin in induced BMMSCs was verified as positive. Flow cytometry showed that the induction ratio in group C was higher than that in group A or group B. The protein levels of cTnI in groups A, B, and C were significantly higher than those in the control group. Transmission electron microscopy showed that the induced cells had myofilaments, z line-like substances, desmosomes, and gap junctions. Angiotensin II and 5-azacytidine can promote the proliferation and differentiation of BMMSCs into cardiomyocyte-like cells.

Inhibition of p53-p21 pathway promotes the differentiation of rat bone marrow mesenchymal stem cells into cardiomyocytes

P53 is shown recently to play an important role in the proliferation and differentiation of bone marrow mesenchymal stem cells (BMMSCs). In this study, by inhibiting p53-p21 pathway with p53 inhibitor (p-fifty three inhibitor-alpha, PFT-α), we investigated the resulting effects on the differentiation of rat BMMSCs into cardiomyocyte-like cells. BMMSCs were isolated from bone marrow of SD rats by density gradient centrifugation. The fourth passage cells were divided into four groups: control group, PFT-α group, 5-AZA group, and PFT-α + 5-AZA group. The purified BMMSCs were identified by surface antigens and the proliferation and apoptosis of BMMSCs were examined by MTT and flow cytometry analysis. The expression of cTnI and CX-43 in BMMSCs after induction was detected by immunofluorescence and that of cTnI, p53, and p21 was detected by western blot. Our results demonstrated that PFT-α at 20 μmol/l significantly reduced the apoptosis and promoted the proliferation of BMMSCs, and induced BMMSCs to differentiate into cardiomyocyte-like cells. In conclusion, these data open up new possibility of modulating p53-p21 pathway for directed differentiation of BMMSCs into cardiomyocytes, which will be valuable for cardiovascular regenerative medicine.

E0035 The study of derivation from bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro via cardiotrophin-1

ObjectiveTo investigate the effects of CT-1 on differentiation of induced swine BMSCs in vitro independently or with 5-aza.MethodsBMSCs were divided into four groups : bland contro1; induced with CT-1; induced...

The mechanism underlying the differentiation of human umbilical cord-derived mesenchymal stem cells into myocardial cells induced by 5-azacytidine

To investigate the molecular mechanism underlying the differentiation of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) into myocardial cells induced by 5-azacytidine (5-aza), and to explore the expression and significance of DLL4-Notch signaling in this process. hUCMSCs were isolated and purified from the umbilical cords of normal or cesarean term deliveries under sterile conditions. After treatment with 5-aza for 24 h, hUCMSCs was continued to culture, the expression of GATA4 and NKx2.5 at 4 weeks after induction, DLL4 and Notch1 mRNA at 1d, 3d, 5d, 7d after induction were detected. The expression of cardiac troponin I (cTnI) after 4 weeks was determined by immunocytochemistry. hUCMSCs treated with 5-aza were stained positively for cTnI 4 weeks after induction. The expression of Notch1 and DLL4 mRNA in the 5-aza-induced group was stable and significantly higher than that in the control group (mean Ct value for the Notch1 gene: 0.51 ± 0.21 in the 5-aza-induced group vs. 7.85 ± 0.35 in the control group; mean Ct value for the DLL4 gene: 1.60 ± 0.49 in the 5-aza-induced group vs. 12.42 ± 0.73 in the control group). Similar results were observed for Nkx2.5 and GATA4 genes. The expressions of Nkx2.5 and GATA4 mRNA in the 5-aza group were 4.72 ± 0.58 and 3.76 ± 0.06 times higher than that in the control group, respectively, with statistical significance. hUCMSCs can be differentiated into myocardial cells by 5-aza induction in vitro. 5-Aza may affect this process by regulating the expression of GATA4 and Nkx2.5 genes. The DLL4-Notch signal pathway may be involved in this process.

Orotidylate decarboxylase: insights into the catalytic mechanism from substrate specificity studies.

Pyrimidine nucleotides were tested as substrates for pure yeast orotidylate decarboxylase in an attempt to gain insight into the nature of the catalytic mechanism of the enzyme. Substitutions of the 5-position in the pyrimidine ring of the orotidylate substrate resulted in compounds that are either excellent inhibitors or substrates of the enzyme. The 5-bromo- and 5-chloroorotidylates are potent inhibitors while the 5-fluoro derivative is a good substrate with a turnover number 30 times that observed with orotidylate. When carbon 5 of the pyrimidine ring is replaced by nitrogen in 5-azaorotidylate, the resulting compound is unstable in solution with a half-life of 25 min at pH 6. However, studies with freshly generated 5-azaorotidylate show that an enzyme-dependent reaction occurs, presumably decarboxylation. This enzyme reaction follows simple Michaelis-Menten kinetics. Because the 5-aza group is not electrophilic, an enzyme mechanism utilizing a nucleophilic addition of the enzyme at the 5-position is ruled out. We also present studies that are not compatible with a mechanism requiring the formation of a Schiff's base prior to decarboxylation. The enzyme is tolerant of modest substitution at the 4-position, for the 4-keto group can be replaced with a thioketone. However, no catalysis is observed when the same substitution is made at the 2-position. Similarities in the substrate specificity of orotate phosphoribosyltransferase and orotidylate decarboxylase led us to compare the amino acid sequences of the two enzymes; significant (20%) sequence homology was observed.(ABSTRACT TRUNCATED AT 250 WORDS)