| ObjectiveIn recent years, the study found that tumors occur not only with genetics and epigenetic closely, and epigenetic had became a hot issue for the current study. Epigenetics refered to that under the situation of the genomic DNA sequence does not change, the function of genes occur genetic changes, and the phenotypic change finally. Including DNA methylation, histone modifications, and RNA interference. Histone acetylation levels had a close relation with the occurrence of tumor. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) were the key enzymes of regulating histone acetylation state. The balance of acetylation and deacetylation played a key role in gene transcription and different cell protein function. At present, the study confirmed that histones in tumor cells mostly showed low acetylation state, the imbalance of histone acetylation state was closely linked with tumorigenesis. HDAC1,which is the first HDAC of mammals,was found in1996by the Taunton. The study showed that HDAC1had the important function of inhibiting of gene transcription, regulating of cell cycle etc. HDAC1mRNA and protein in the tissue of breast cancer, gastric cancer, colon cancer, liver cancer and pancreatic carcinoma had high expression [1,2,4,5]. The study based on lymphoma found that the expression of HDAC1protein may be an important factor of HDAC inhibitors (HDACi) sensitivity. HDACi can inhibit HDAC activity in cells, so that increase histone acetylation levels in cells, increase the expression of certain tumor suppressor genes, thereby inhibiting tumor cell proliferation and inducing tumor cell differentiation and apoptosis. Sodium valproate (valproic acid, VPA) is clinically used antiepileptic drugs, in recent years, studies confirmed that sodium valproate induced cell differentiation to promote apoptosis on a variety of solid tumors and malignant hematopathy. In this study, mainly by cellular morphology, WST-1colorimetry and flow cytometry of sodium valproate on the inhibition of K562cells, by the application of RT-PCR method of sodium valproate on K562cells HDAC1mRNA expression and immunohistocemistry was used to detect the impact of sodium valproate on K562cells HDAC1protein expression. Thus we can observe the induction of apoptosis and inhibition of proliferation of K562cells, and the impact of HDAC1gene and protein about sodium valproate, investigate the mechanism of action of sodium valproate treatment of leukemia.Method1. Leukemic cell line K562is provided by Stem Cell Center of Zhengzhou University.The cells were routinely cultured in10%fetal bovine serum (FBS),100U/ml streptomycin and100U/ml penicillin RPMI1640medium at a37℃,5%CO2.2. WST-1detect cells proliferation:K562cell was cultured and treated with200μg/ml,400μg/ml,800μg/ml of VPA for12h,24h,36h,48h and60h., The proliferation of cells were examined by WST-1assay. Then the growth inhibitory rates were counted and the optimal concentration and time was found.3. Observe cell morphology:K562cells was treated with400μg/ml of VPA for48h, morphological changes and growth state of cell were observed under the microscope.4. Flow cytometry analyze cells apoptosis rate:K562cells was treated with400μg/ml of VPA for48h, applicate of flow cytometry Annexin V FITC/PI double-labeled to analyze cell apoptosis rate. 5. RT-PCR analyze the expression of the HDAC1mRNA:The expression of the HDAC1gene was analyzed by RT-PCR in K562cells, which treated with200μg/ml,400μg/ml,800μg/ml of VPA for48h.6. Immunohistochemistry analyze the expression of HDAC1protein:The expression of the HDAC1protein was assayed by immuno-histochemistry in K562cells treated with400μg/ml of VPA for48h.7. Data analyzed:They were using software SPSS17.0. The quantative data were presented as mean±standard difference. The counting data were analyzed with the X2test.Taking a=0.05as the significant standard of test.Results1. WST-1results showed that:K562cell was cultured and treated with200μg/ml,400μg/ml,800μg/ml of VPA for48h,the inhibition rates of cell proliferation respectively are (31.7±0.46)%,(55.1±0.82)%,(60.4±1.64)%, respectively compared with the control group there were significant differences (P<0.05); between any two of the VPA group there existed significant differences (P<0.05);the IC50value of48h is423μg/ml. Then K562cell was cultured and treated with400μg/ml of VPA for12h,24h,36h,48h and60h, the inhibition rates of cell proliferation respectively were (14.8±0.21)%,(26.9±1.27)%,(34.6±0.30)%,(55.1±0.82)%,(57.6±0.79)%, respectively compared with the control group there were significant differences (P<0.05);between any two group, compared to thel2h,24h,36h of VPA,the K562cell suppression rate of VPA48h had significant differences (P <0.05), but the inhibition rate between48h and60h had no significant difference (P>0.05).2. Cell morphology:K562cells was treated with a final concentration of400μg/ml of VPA for48h, observed under the microscope:cell morphological changes,showing reduced colony,incomplete membrane,irregular cell shape,the increased cell debris,chromatin enrichment,nuclear condensation and nuclear fragmentation;the cell morphology was normal in control group, cells growed like pellet, good transmittance. 3. flow cytometry results showed that:K562cells was treated with400μg/ml of VPA for48h, the rate of cell apoptosis by Annexin V FITC/PI double-labeled was (12.27±1.6)%,higher than the control group (0.29±0.7)%,P<0.05.4. RT-PCR results showed that:The expression of the HDAC1gene mRNA was assessed by RT-PCR amplification in K562cells treated with200μg/ml,400μg/ml,800μg/ml of VPA for48h,respectively,the gray scale ratio were0.72±0.023,0.52±0.041,0.32±0.035, Compared to the control group0.81±0.027, there existed significant differences, p<0.05; the gray scale ratio of HDAC1mRNA between different concentration VPA had a significant difference (P<0.05).5. Immunohistochemical results showed that:The expression rate of the HDAC1protein was (8.73±5.68)%which assayed by immunohistochemistry in K562cells treated with a final concentration400μg/ml of VPA for48h,compared to the control group (46.72±5.26)%, there was a significant reduction (P<0.05).The VPA group HDAC1protein positive average score was25.60±4.31, compared with the control group of167.33±10.64, there was a significant difference (P<0.05).Conclusion1. Valproic acid can inhibit the proliferation of K562cells, in time-and dose-dependent manners.2. Valproic acid induces apoptosis of K562cells.3. Valproic acid can down-regulate expression of HDAC1mRNA and protein in K562cells, which may be the mechanism of its inhibition of cell proliferation and cell apoptosis increasing. |
PDF Full Text Request