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OBJECTIVE To observe the regulation effect of chidamide on energy metabolism in HCT-8 and HT-29 cells. METHODS HCT-8 and HT-29 cells were treated with chidamide 5,10 and 20 μmol · L-1. Morphological changes of these cells were observed under an ordinary optical microscope. Cell proliferation was detected by MTT. ATP production was determined by CellTiter-Glo? assay kit. Metabolic changes were tested by glycolytic stress kit. The mRNA level of lactate dehydrogenase A (LDH-A)was analyzed by real-time quantitative PCR,whereas the protein level of LDH-A was analyzed by Western blotting. RESULTS Compared with control group,cell morphology of HCT-8 and HT-29 cells in chidamide treated group was irregular,accompanied by deformation,shrinkage and cell debris, and the inhibitory rate of proliferation increased(P<0.05). There was no significant difference in ATP total content between chidamide 5 and 10 μmol · L-1 16 h treatment groups,but in chidamide 20 μmol · L-1 treatment group it was decreased(P<0.05). Chidamide 20μmol · L-1 had no effect on oxygen consumption rate, but glycolysis ATP generation rate was reduced by 30.7% and 37.9%(P<0.05),respectively. Chidamide 20μmol · L-1 had no effect on LDH-A mRNA level,but it decreased the protein level of LDH-A(P<0.01). CONCLUSION Chidamide can abate the respiratory metabolic ability of HCT-8 and HT-29 cells. The mechanism may be related to the down-regulation of LDH-A.
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Objective To verify enzyme activity inhibition of a novel histone deacetylase inhibitor ( HDACi ) JZ005 using an HDACi chemiluminescence detection kit and a cell-based screening model .Methods The plasmid with p21 gene promoter elements and luciferase reporter gene was transfected into human embryonic kidney cells 293 , and the stable transfectants were established by G418 screening.Enzyme activity inhibition of JZ005 on histone deacetylases (HDACs) was verified by the HDACi chemiluminescence detection kit and the cell-based screening model .A well-known HDACi , tri-chostatin A ( TSA) was used as the positive control .MTT assay was used to detect the protection of rat H 9c2 myocardial cells suffering from CoCl 2-induced hypoxia and treated with different concentrations of JZ 005 .The expression of acetylated histone H3 protein of normal and CoCl 2-induced hypoxia H9c2 cells before and after JZ005 treatment was assayed by West-ern blotting while the effect of drug administration on apoptosis was detected by flow cytometry ( FCM) .Results An HDA-Ci cell-based screening system targeting the p21 gene promoter was ranging established .The JZ005, a HDACi, markedly suppressed the activity of HDACs by more than 50%with the concentration ranging from 50 to 400 μmol/L.JZ005 signifi-cantly protected H9c2 cells from hypoxia injury .Cell viability was increased by 38.33%,56.00% and 35.20% compared with control,accompanied by an enhanced acetylation level of histone H 3.JZ005(25,50 and 100 μmol/L) treatment sig-nificantly decreased the number of apoptotic cells (6.63%,10.56% and 8.89%) compared to control group (12.89%). Conclusion An HDACi cell-based screening system is successfully established .JZ005 effectively protects myocardial cells against hypoxia injury while enhancing the acetylation level of histone H 3.Our results indicate that JZ005 might be developed as a potential drug for hypoxia treatment .
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Objective To examine the anticancer effect of a novel histone deacetylase inhibitor (HDACi), JZ004, on colon cancer cells HCT-8 and HT-29, and to investigate the molecular mechanisms of proliferation inhibition and apoptosis induction of cancer cells treated by JZ 004.Methods Colon cancer cells were treated with a series of concentrations of JZ004 .MTT assay was used to detect the proliferation of cancer cells .The cell cycle distribution and apoptosis were deter-mined by flow cytometry .Rhodamine 123 and DCFH-DA were applied to detect the mitochondrial membrane potential (ΔΨm) and reactive oxygen species ( ROS) production.The protein expressions of acetyl-histone H3, p21, cyclin-dependent kinase(CDK)4, Bcl-2, Mcl-1 and Bax were assayed by Western blotting .Results JZ004 was found to inhibit proliferation and induce apoptosis of colon cancer cells in a time-and dose-dependent manner , accompanied by a dose-dependent hyperacetylation of histone H3.JZ004 induced the cancer cell arrest in G 0/G1 phase by increasing the expres-sion level of p21 while CDK4 was downregulated .JZ004 also increased cellular ROS production and reduced ΔΨm by regu-lating the expressions of Bcl-2 family proteins .Conclusion As a novel HDACi , JZ004 effectively inhibits proliferation and increases ROS production to induce apoptosis of colon cancer cells .The results indicate that JZ004 is a potential compound to be developed as an anti-colon cancer agent for clinic application .