丙戊酸钠对K562细胞增殖、凋亡及HDAC1表达的影响
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摘要
背景
近年来研究发现,肿瘤的发生与遗传学尤其是表观遗传学有着密切的关系,因此表观遗传学成为目前肿瘤研究的热点问题之一。表观遗传学是指基因的功能在DNA序列不发生改变的情况下发生了可遗传的变化,并且最终使表型发生变化。表观遗传学主要包括DNA的甲基化,组蛋白修饰,RNA干扰等。其中组蛋白乙酰化水平与肿瘤的发生紧密相关。组蛋白乙酰化与去乙酰化的平衡在基因转录和不同细胞蛋白功能中起到关键的作用。组蛋白去乙酰化酶(histone deacetylases, HDACs)和组蛋白乙酰化转移酶(histone acetyltransferases, HATs)是调节组蛋白乙酰化状态的关键酶。目前研究证实,在肿瘤细胞中组蛋白大多呈现去乙酰化状态,这种组蛋白乙酰化状态的失衡与肿瘤的发生有密切的联系。组蛋白去乙酰化酶-1(histone deacetylases-1, HDAC1)是1996年被Taunton等发现的第一个哺乳动物的HDAC。研究显示,HDAC1具有抑制抑癌基因转录,调节细胞周期等重要作用。在乳腺癌、胃癌、结肠癌、肝癌和胰腺癌组织中HDAC1mRNA和蛋白水平均高表达。最近基于淋巴瘤的研究发现HDAC1蛋白的表达可能是对HDAC抑制剂(HDACi)敏感的重要因素。HDACi可以抑制细胞内HDAC的活性,使细胞内组蛋白乙酰化水平增高,提高某些抑癌基因的表达,进而抑制肿瘤细胞的增殖,诱导肿瘤细胞分化、凋亡。丙戊酸钠(valproic acid, VPA)是临床上常用的抗癫痫药物,近几年研究证实VPA其在抗癫痫有效治疗浓度时表现出很强的抑制组蛋白去乙酰化酶的活性,对多种实体瘤和恶性血液病细胞具有诱导分化及促进凋亡作用。本实验应用水溶性四唑盐光吸收(WST-1)法检测丙戊酸钠对K562细胞增殖的影响；观察药物作用后细胞形态的变化；应用流式细胞学技术检测丙戊酸钠对K562细胞凋亡的影响；应用RT-PCR法检测丙戊酸钠对K562细胞HDAC1mRNA表达的影响；免疫组化法检测丙戊酸钠对K562细胞HDAC1蛋白表达的影响。观察丙戊酸钠抑制K562细胞增殖,诱导其凋亡的作用以及对HDAC1基因及蛋白表达的影响,为丙戊酸钠用于白血病的治疗提供实验依据。
方法
1.细胞培养：人白血病细胞系K562细胞株由郑州大学干细胞中心保存。将细胞从液氮罐中拿出迅速复苏后,用含有10%胎牛血清(FBS)、100U/ml链霉素和100U/ml青霉素的RPMI1640培养基,在温度为37℃、C02浓度为5%、饱和湿度培养箱中进行培养。
2.实验分组：选取对数生长期的K562细胞进行实验,实验共分为两组：对照组和实验组。对照组不加药,只加入等量生理盐水,实验组加入不同浓度的丙戊酸钠。
3.水溶性四唑盐光吸收(WST-1)法检测细胞的增殖：分别应用最终浓度为200μg/ml,400μg/ml,800μg/ml的丙戊酸钠作用于K562细胞12h、24h、36h、48h、60h后,应用WST-1法检测细胞增殖的情况,计算出细胞增殖抑制率,划出生长曲线,找出丙戊酸钠的最佳药物浓度和最适作用时间。
4.观察细胞形态：终浓度为400μg/ml的丙戊酸钠处理K562细胞48h后,电子显微镜下观察细胞生长状态及形态学变化。
5.流式细胞术检测细胞凋亡：终浓度为400μg/ml的丙戊酸钠作用于K562细胞48h后,通过流式细胞仪技术(AnnexinV FITC/PI检测法)检测细胞的凋亡情况。
6. RT-PCR法检测HDAC1mRNA的表达：终浓度为200μg/ml,400μg/ml,800μg/ml丙戊酸钠分别作用于K562细胞48h后,用RT-PCR法检测丙戊酸钠对K562细胞HDAC1mRNA表达的影响。
7.免疫组化法检测HDAC1蛋白的表达：终浓度为400μg/ml丙戊酸钠作用于K562细胞48h后,应用免疫组化法检测丙戊酸钠对K562细胞HDAC1蛋白表达的影响。
8.统计学分析：所得数据应用SPSS17.0统计学软件处理,统计学数据用均数±标准差(x±s)表示。对计量资料,同一时间点各实验组与对照组之间比较用两样本t检验分析,计数资料采用χ2检验,。以a=0.05为检验水准。
结果
1.WST-1结果显示：应用终浓度为200μg/ml,400gg/ml,800μg/ml的丙戊酸钠分别作于K562细胞48h,细胞增殖抑制率分别为(31.7±0.46)%、(55.1±0.82)%、(60.4±1.64)%,分别与对照组比较存在显著差异(P<0.05)；丙戊酸钠组间两两比较,差异具有统计学意义(P<0.05),丙戊酸钠48小时IC50为423gg/m1。400μg/ml丙戊酸钠作用于K562细胞12h、24h、36h、48h、60h,细胞增殖抑制率分别为(14.8±0.21)%、(26.9±1.27)%、(34.6±0.30)%、(55.1±0.82)%、(57.6±0.79)%,与对照组相比,差异具有统计学意义(P<0.05)；组间两两比较,丙戊酸钠48h与12h、24h、36h对K562细胞增殖抑制率有显著性差异(P<0.05),48h与60h的差异无统计学意义(P>0.05)。
2.细胞形态：用终浓度为400μg/ml的丙戊酸钠处理K562细胞48h,光学显微镜下观察示细胞发生形态学改变,表现为细胞集落减少,细胞膜不完整,细胞形状不规则,细胞碎片增多,染色质浓缩,核固缩、核碎裂；对照组细胞形态正常,细胞呈团状生长,透光度好。
3.流式细胞术结果显示：浓度为400μg/ml的丙戊酸钠作于K562细胞48h,AnnexinV FITC/PI双标结果显示凋亡率为：(12.27±1.6)%,高于对照组(0.29±0.7)%,两组比较差异具有统计学意义(P<0.05)
4. RT-PCR结果显示：浓度为200μg/ml,400μg/ml,800μg/ml的丙戊酸钠分别作于K562细胞48h后,RT-PCR扩增结果显示,HDAC1mRNA灰度比值分别为0.72±0.023,0.52±0.041,0.32±0.035与对照组0.81±0.027相比有显著差异,p<0.05；不同浓度丙戊酸钠组之间HDAC1mRNA灰度比值相比,差异具有统计学意义(p<0.05)。
5.免疫组化结果显示：终浓度为400μg/ml的丙戊酸钠处理细胞48h后,经免疫组化法染色法检测结果显示：K562细胞中HDAC1蛋白阳性表达率在丙戊酸钠组为(8.73±5.68)%,与对照组(46.72±5.26)%相比显著降低(P<0.05)。丙戊酸钠组HDAC1蛋白表达阳性平均积分为25.60±4.31,较对照组167.33±10.64有显著性差异(P<0.05)。
结论
1.丙戊酸钠对K562细胞的增殖具有抑制作用,并且其抑制作用具有浓度和时间依赖性。
2.丙戊酸钠能够诱导K562细胞凋亡。
3.丙戊酸钠能够下调K562细胞HDAC1mRNA及HDAC1蛋白的表达,这可能与其抑制细胞的增殖,诱导细胞凋亡的机制有关。
Objective
In 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.
Method
1. 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.
Results
1. 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).
Conclusion
1. 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.
近年来研究发现,肿瘤的发生与遗传学尤其是表观遗传学有着密切的关系,因此表观遗传学成为目前肿瘤研究的热点问题之一。表观遗传学是指基因的功能在DNA序列不发生改变的情况下发生了可遗传的变化,并且最终使表型发生变化。表观遗传学主要包括DNA的甲基化,组蛋白修饰,RNA干扰等。其中组蛋白乙酰化水平与肿瘤的发生紧密相关。组蛋白乙酰化与去乙酰化的平衡在基因转录和不同细胞蛋白功能中起到关键的作用。组蛋白去乙酰化酶(histone deacetylases, HDACs)和组蛋白乙酰化转移酶(histone acetyltransferases, HATs)是调节组蛋白乙酰化状态的关键酶。目前研究证实,在肿瘤细胞中组蛋白大多呈现去乙酰化状态,这种组蛋白乙酰化状态的失衡与肿瘤的发生有密切的联系。组蛋白去乙酰化酶-1(histone deacetylases-1, HDAC1)是1996年被Taunton等发现的第一个哺乳动物的HDAC。研究显示,HDAC1具有抑制抑癌基因转录,调节细胞周期等重要作用。在乳腺癌、胃癌、结肠癌、肝癌和胰腺癌组织中HDAC1mRNA和蛋白水平均高表达。最近基于淋巴瘤的研究发现HDAC1蛋白的表达可能是对HDAC抑制剂(HDACi)敏感的重要因素。HDACi可以抑制细胞内HDAC的活性,使细胞内组蛋白乙酰化水平增高,提高某些抑癌基因的表达,进而抑制肿瘤细胞的增殖,诱导肿瘤细胞分化、凋亡。丙戊酸钠(valproic acid, VPA)是临床上常用的抗癫痫药物,近几年研究证实VPA其在抗癫痫有效治疗浓度时表现出很强的抑制组蛋白去乙酰化酶的活性,对多种实体瘤和恶性血液病细胞具有诱导分化及促进凋亡作用。本实验应用水溶性四唑盐光吸收(WST-1)法检测丙戊酸钠对K562细胞增殖的影响；观察药物作用后细胞形态的变化；应用流式细胞学技术检测丙戊酸钠对K562细胞凋亡的影响；应用RT-PCR法检测丙戊酸钠对K562细胞HDAC1mRNA表达的影响；免疫组化法检测丙戊酸钠对K562细胞HDAC1蛋白表达的影响。观察丙戊酸钠抑制K562细胞增殖,诱导其凋亡的作用以及对HDAC1基因及蛋白表达的影响,为丙戊酸钠用于白血病的治疗提供实验依据。
方法
1.细胞培养：人白血病细胞系K562细胞株由郑州大学干细胞中心保存。将细胞从液氮罐中拿出迅速复苏后,用含有10%胎牛血清(FBS)、100U/ml链霉素和100U/ml青霉素的RPMI1640培养基,在温度为37℃、C02浓度为5%、饱和湿度培养箱中进行培养。
2.实验分组：选取对数生长期的K562细胞进行实验,实验共分为两组：对照组和实验组。对照组不加药,只加入等量生理盐水,实验组加入不同浓度的丙戊酸钠。
3.水溶性四唑盐光吸收(WST-1)法检测细胞的增殖：分别应用最终浓度为200μg/ml,400μg/ml,800μg/ml的丙戊酸钠作用于K562细胞12h、24h、36h、48h、60h后,应用WST-1法检测细胞增殖的情况,计算出细胞增殖抑制率,划出生长曲线,找出丙戊酸钠的最佳药物浓度和最适作用时间。
4.观察细胞形态：终浓度为400μg/ml的丙戊酸钠处理K562细胞48h后,电子显微镜下观察细胞生长状态及形态学变化。
5.流式细胞术检测细胞凋亡：终浓度为400μg/ml的丙戊酸钠作用于K562细胞48h后,通过流式细胞仪技术(AnnexinV FITC/PI检测法)检测细胞的凋亡情况。
6. RT-PCR法检测HDAC1mRNA的表达：终浓度为200μg/ml,400μg/ml,800μg/ml丙戊酸钠分别作用于K562细胞48h后,用RT-PCR法检测丙戊酸钠对K562细胞HDAC1mRNA表达的影响。
7.免疫组化法检测HDAC1蛋白的表达：终浓度为400μg/ml丙戊酸钠作用于K562细胞48h后,应用免疫组化法检测丙戊酸钠对K562细胞HDAC1蛋白表达的影响。
8.统计学分析：所得数据应用SPSS17.0统计学软件处理,统计学数据用均数±标准差(x±s)表示。对计量资料,同一时间点各实验组与对照组之间比较用两样本t检验分析,计数资料采用χ2检验,。以a=0.05为检验水准。
结果
1.WST-1结果显示：应用终浓度为200μg/ml,400gg/ml,800μg/ml的丙戊酸钠分别作于K562细胞48h,细胞增殖抑制率分别为(31.7±0.46)%、(55.1±0.82)%、(60.4±1.64)%,分别与对照组比较存在显著差异(P<0.05)；丙戊酸钠组间两两比较,差异具有统计学意义(P<0.05),丙戊酸钠48小时IC50为423gg/m1。400μg/ml丙戊酸钠作用于K562细胞12h、24h、36h、48h、60h,细胞增殖抑制率分别为(14.8±0.21)%、(26.9±1.27)%、(34.6±0.30)%、(55.1±0.82)%、(57.6±0.79)%,与对照组相比,差异具有统计学意义(P<0.05)；组间两两比较,丙戊酸钠48h与12h、24h、36h对K562细胞增殖抑制率有显著性差异(P<0.05),48h与60h的差异无统计学意义(P>0.05)。
2.细胞形态：用终浓度为400μg/ml的丙戊酸钠处理K562细胞48h,光学显微镜下观察示细胞发生形态学改变,表现为细胞集落减少,细胞膜不完整,细胞形状不规则,细胞碎片增多,染色质浓缩,核固缩、核碎裂；对照组细胞形态正常,细胞呈团状生长,透光度好。
3.流式细胞术结果显示：浓度为400μg/ml的丙戊酸钠作于K562细胞48h,AnnexinV FITC/PI双标结果显示凋亡率为：(12.27±1.6)%,高于对照组(0.29±0.7)%,两组比较差异具有统计学意义(P<0.05)
4. RT-PCR结果显示：浓度为200μg/ml,400μg/ml,800μg/ml的丙戊酸钠分别作于K562细胞48h后,RT-PCR扩增结果显示,HDAC1mRNA灰度比值分别为0.72±0.023,0.52±0.041,0.32±0.035与对照组0.81±0.027相比有显著差异,p<0.05；不同浓度丙戊酸钠组之间HDAC1mRNA灰度比值相比,差异具有统计学意义(p<0.05)。
5.免疫组化结果显示：终浓度为400μg/ml的丙戊酸钠处理细胞48h后,经免疫组化法染色法检测结果显示：K562细胞中HDAC1蛋白阳性表达率在丙戊酸钠组为(8.73±5.68)%,与对照组(46.72±5.26)%相比显著降低(P<0.05)。丙戊酸钠组HDAC1蛋白表达阳性平均积分为25.60±4.31,较对照组167.33±10.64有显著性差异(P<0.05)。
结论
1.丙戊酸钠对K562细胞的增殖具有抑制作用,并且其抑制作用具有浓度和时间依赖性。
2.丙戊酸钠能够诱导K562细胞凋亡。
3.丙戊酸钠能够下调K562细胞HDAC1mRNA及HDAC1蛋白的表达,这可能与其抑制细胞的增殖,诱导细胞凋亡的机制有关。
Objective
In 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.
Method
1. 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.
Results
1. 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).
Conclusion
1. 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.
引文
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