Mechanisms Of Synergistic Antileukemiamic Interactions Between Cytarabine Combined With Valproic Acid And Vorinostat

In contrast to the tremendous success in treating acute lymphoblastic leukemiaover the last3decades（>80%cure rate）, improvements in AML therapy have beenlimited. Patients with acute myeloid leukemia (AML) remain inadequate with anoverall survival of40–45%reported in younger adults,resistance to cytarabine,. is amajor cause of treatment failure.How to improve the remission rate, to avoidrecurrence, strive for long-term survival is difficult in the treatment of AML. Researchon variety of drug applied alone or in combination, in order to improve the therapeuticresponse rate and long-term survival, but compared with the standard solution, has notyet found effective drug can improve therapeutic effect..Therefore,new therapies forchildren with AML are urgently needed.Acute megakaryocytic leukemia (AMKL; M7) is a biologically heterogeneousform of AML.This subtype of leukemia chemotherapy effect is poor, considered as avery high-risk subgroup with event-free survival (EFS) rates of <35%. Remarkably,Down syndrome (DS) children with AMKL, having extremely high EFS rates ofapproximately80%,increased overall survival relative to non-DS pediatricpatients.The reason is somatic mutation in exon2of the transcription factor GATA-1gene.It has been previously shown that acute myeloid leukemia (AML) patients withhigher levels of GATA1expression have poorer outcomes. Further, earlier studiesdemonstrated a worse prognosis for AML patients whose blast cells expressedhigher levels of GATA1than patients whose blasts expressed lower levels of GATA1.Collectively, these studies suggest that GATA1may contribute to chemotherapyresistance.Multicenter studies prove the existence of AML genetic and epigeneticabnormalities, providing theoretical basis for new treatment use.At present, severalHDACIs are currently being tested in clinical trials, and encouraging results have been reported for their use in treating both hematologic malignancies and solidtumors.Studies have confirmed that treatment of the Meg-01cells with the histonedeacetylase inhibitor valproic acid resulted in down-regulation of GATA1andsignificantly enhanced cytarabine sensitivity.In conclusion, based on the detection of transcription factor GATA-1expressionand cytarabine metabolic enzyme changes in the level of gene expression, discussesthe mechanism of histone acetylation enzyme inhibitors improve chemotherapysensitivity.Objective:To determine the possibility of synergistic antileukemic activity and theunderlying molecular mechanisms associated with cytarabine combined with valproicacid and SAHA in pediatric acute myeloid leukemia (AML),provideing theoreticaland experimental basis for the treatment of acute megakaryocyte leukemia celllines.Further investigate whether valproic acid and SAHA have relevation with theexpression of GATA-1level and the mechanism of improving leukemia cell’sresponse to cytarabine.Method:1. Dami cells were seeded in96-well plates and were allocated to control group,VPA group, SAHA group and Ara-c group. The growth state of cells was observedunder microscopy and the inhibition rates of each group were measured with MTTassay.2. MTT method is used to measured the inhibition rates of Dami cell, calculatedjoint index CI value, with different concentrations of VPA combined with Ara-c,SAHA combined with Ara-c.3. MTT method is used to measured the inhibition rates of Dami cell, with VPAand SAHA combined with Ara-c.4. Detecting cell cycle distribution of Dami by Propidium iodide (piodize, PI)single staining,after exposed to VPA and SAHA group alonely and combinedly24hand48h.5. With real-time quantitative RT-PCR method,the mRNA level changes oftranscription factors GATA-1, cytidine deaminase （CDA） and deoxidizationcytidine kinase（DCK）, after exposed to VPA and SAHA group respectively andjointly. Results：1. VPA、SAHA and Ara-c alone inhibited leukemia Dami cell proliferation,promoted apoptosis, inhibition with prolonged and/or dose increases with theincrease.2. VPA combined with Ara-c, SAHA combined with Ara-c respectively inhibitedleukemia Dami cell proliferation, inhibition of proliferation effect is significantlyhigher than the drug used alone (P <0.01), CI value were less than1, a collaborative.3. VPA and SAHA combined with Ara-c application respectively inhibitedleukemia Dami cell proliferation, inhibition of proliferation effect is significantlyhigher than VPA and Ara-c joint application, SAHA and Ara-c joint application（P＜0.01）.4. VPA and SAHA can block cell cycle phenomenon, mainly blocked in G1phase.Compared with control group, the proportion of each dose group of cells in G1phase increased significantly, the proportion of S phase decreased significantly (P <0.01), the proportion of G2has no obvious difference (P>0.05).24h cell cycleresults suggest: the combination group G1phase cell proportion and each drug usedalonely have no significant difference (P>0.05).48h cell cycle results suggest: thecombination group G1phase cells is higher than the proportion of SAHA group (P <0.05), but no significant difference with VPA group(P>0.05). With the extension oftime, the G1phase cell percentage is on the rise.5. The CDA、GATA-1mRNA expression level of VPA group, SAHA group andcombination group is lower than the control group (P <0.05); The VPA group DCKmRNA expression level is higher than normal group (P <0.05). The SAHA group hasno significant difference with the normal group(P>0.05). The Combination groupDCK mRNA expression level is higher than the VPA group and SAHA group (P <0.05), but CDA、GATA-1mRNA expression level has no significant difference (P>0.05).Conclusion1. VPA and SAHA combined with Ara-c joint application respectively inhibitingleukemia Dami cell proliferation, inhibition of proliferation effect is significantlyhigher than VPA combined Ara-c and SAHA combined Ara-c.2. VPA, SAHA appears the phenomenon of cell cycle arrest mainly in G1phase. With the extension of time, the G1phase cell percentage is on the rise.48h, thecombination group G1phase cells is higher than the proportion of SAHA group, butno significant difference with VPA group.3. VPA and SAHA alone and combine can reduce the mRNA expression levelof GATA-1and CDA. The VPA group DCK mRNA expression level increased, butno obvious difference was found between SAHA group and normal group. VPAcombining SAHA can significantly increase DCK mRNA expression level, but theGATA-1, CDA mRNA expression level with each drug alone no significantdifference.