组蛋白去乙酰化酶抑制剂丙戊酸对多发性骨髓瘤细胞株KM3细胞增殖的影响
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摘要
背景:多发性骨髓瘤(MM)是一种浆细胞恶性克隆性疾病,骨髓瘤细胞分泌的血管内皮生长因子(VEGF)以自分泌的方式与MM细胞表面VEGFR-1/Flt-1结合,通过细胞外调节蛋白激酶(MEK-ERK)和蛋白激酶C(PKC)途经分别促进MM细胞增生和迁徙是MM发病机制之一。丙戊酸(VPA)是一种治疗癫痫的传统药物,近年研究发现其具有组蛋白去乙酰化酶(HDACs)抑制剂的作用,可抑制多种肿瘤细胞增殖。
目的:研究VPA对多发性骨髓瘤细胞株KM3细胞增殖的影响,观察KM3细胞中乙酰化的组蛋白H4(Ac-histone H4)和VEGF受体(VEGFR)表达的变化,探讨其抗多发性骨髓瘤细胞的分子生物学机制。
方法:采用四氮唑蓝(MTT)比色法检测KM3细胞增殖;Annexin V和PI染色后应用流式细胞仪检测细胞凋亡率;RT-PCR检测KM3细胞中VEGFR mRNA的表达;采用免疫细胞化学法观察KM3细胞VEGFR蛋白、Ac-histone H4蛋白的表达。
结果:VPA可明显抑制KM3细胞增殖,且具有时间剂量依赖性(P＜0.05);不同浓度VPA处理KM3细胞48h后,可明显诱导细胞凋亡,呈剂量依赖性(P＜0.05),不同浓度的VPA(0.5、1、2、4mmol/L)诱导KM3细胞的凋亡率分别为(11.77±4.64)%、(22.13±1.20)%、(23.95±2.57)%和(42.72±4.61)%,呈剂量依赖性(P＜0.05);RT-PCR结果显示,KM3细胞仅表达VEGFR-1/fit-1,且VPA能在mRNA水平抑制VEGFR-1的表达;免疫细胞化学结果显示,4mmol/L的VPA作用KM3细胞48h后,与未处理组KM3细胞相比较,细胞中Ac-histone H4吸光度值明显增加,同时VEGFR-1的吸光度明显降低(P＜0.05)。
结论:VPA通过上调Ac-histone H4水平、下调KM3细胞表面VEGFR-1的表达,从而阻断骨髓微环境中VEGF的自分泌促增殖作用发挥对KM3细胞的增殖抑制作用。VPA作为一种新型的组蛋白去乙酰化酶抑制剂(HDAIs)对多发性骨髓瘤细胞株KM3细胞具有抗肿瘤作用。
Background:Despite of the recent advances in the treatment of multiple myeloma(MM),it is still an incurable disease in the majority of patients.In the bone marrow microenvironment of MM,VEGF is essential for tumor growth and survival, and myeloma cells are known to produce VEGF and express VEGF receptors. Binding of VEGF_(165)to MM cells triggers VEGFR-1 tyrosine phosphorylation. Subsequently the downstream signaling pathways are activated,as follows:(1) PI3-kinase/protein kinase Cα(PKCα)-dependent cascade mediating MM cell migration on fibronectin,(2)MEK-extracellular signal-regulated protein kinase(ERK) pathway mediating MM cell proliferation.Now histone deacetylase inhibitors(HDAIs) have been regarded as a new kind of anticancer drug and several of them are currently being investigated in clinical trials.Valproic acid(VPA)is a shortchain fatty acid with a long history of clinical use as an anticonvulsant.However,VPA also inhibits histone deacetylases(HDACs)and induces apoptosis in selected solid tumors as well as in hematologic neoplasms.
Objective:To define the effects of VPA on multiple myeloma cell line KM3 cells in vitro and the underlying mechanism of VPA in the treatment of MM were investigated.
Methods:KM3 cells were cultured in RPMI1640 medium and treated with VPA in different concentration.The cell proliferation was measured by 3-[4, 5-dimehyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay;the AnnexinⅤand PI staining was used to detect the apoptosis rates;the mRNA level of VEGFR was determined by RT-PCR;and immunocytochemistry was used to detect the protein level of acetylated-histone H4(Ac-histone H4)and VEGFR.
Results:The proliferation rate of KM3 cells was decreased in VPA- treated group in a time-dose dependent manner(P＜0.05).And the AnnexinⅤand PI staining showed VPA can induce apoptosis of KM3 cells,treatment with VPA(0.5、1、2 and 4mmol/L) for 48h,the apoptosis rates of KM3 cells were(11.77±4.64)%、(22.13±1.20)%、(23.95±.2.57)%and(42.72±4.61)%respectively.The RT-PCR results demonstrated the KM3 cells only express VEGFR-1 and it was decreased in VPA(0.5、1、2 and 4 mmol/L for 48h)treated groups compared with control(P＜0.05).Results of immunocytochemistry show the protein of Ac-histone H4 was increased significantly in VPA(4 mmol/L for 48h)treated group whereas the VEGFR-1/Flt-1 was decreased (P＜0.05).
Conclusion:VPA,as an histone deacetylase inhibitor,can increase the expression of Ac-histone H4 and inhibit the expression of VEGFR-1 in KM3 cells and it plays an important role in regulating proliferation and apoptosis of multiple myeloma cell line KM3 cells in vitro.
目的:研究VPA对多发性骨髓瘤细胞株KM3细胞增殖的影响,观察KM3细胞中乙酰化的组蛋白H4(Ac-histone H4)和VEGF受体(VEGFR)表达的变化,探讨其抗多发性骨髓瘤细胞的分子生物学机制。
方法:采用四氮唑蓝(MTT)比色法检测KM3细胞增殖;Annexin V和PI染色后应用流式细胞仪检测细胞凋亡率;RT-PCR检测KM3细胞中VEGFR mRNA的表达;采用免疫细胞化学法观察KM3细胞VEGFR蛋白、Ac-histone H4蛋白的表达。
结果:VPA可明显抑制KM3细胞增殖,且具有时间剂量依赖性(P＜0.05);不同浓度VPA处理KM3细胞48h后,可明显诱导细胞凋亡,呈剂量依赖性(P＜0.05),不同浓度的VPA(0.5、1、2、4mmol/L)诱导KM3细胞的凋亡率分别为(11.77±4.64)%、(22.13±1.20)%、(23.95±2.57)%和(42.72±4.61)%,呈剂量依赖性(P＜0.05);RT-PCR结果显示,KM3细胞仅表达VEGFR-1/fit-1,且VPA能在mRNA水平抑制VEGFR-1的表达;免疫细胞化学结果显示,4mmol/L的VPA作用KM3细胞48h后,与未处理组KM3细胞相比较,细胞中Ac-histone H4吸光度值明显增加,同时VEGFR-1的吸光度明显降低(P＜0.05)。
结论:VPA通过上调Ac-histone H4水平、下调KM3细胞表面VEGFR-1的表达,从而阻断骨髓微环境中VEGF的自分泌促增殖作用发挥对KM3细胞的增殖抑制作用。VPA作为一种新型的组蛋白去乙酰化酶抑制剂(HDAIs)对多发性骨髓瘤细胞株KM3细胞具有抗肿瘤作用。
Background:Despite of the recent advances in the treatment of multiple myeloma(MM),it is still an incurable disease in the majority of patients.In the bone marrow microenvironment of MM,VEGF is essential for tumor growth and survival, and myeloma cells are known to produce VEGF and express VEGF receptors. Binding of VEGF_(165)to MM cells triggers VEGFR-1 tyrosine phosphorylation. Subsequently the downstream signaling pathways are activated,as follows:(1) PI3-kinase/protein kinase Cα(PKCα)-dependent cascade mediating MM cell migration on fibronectin,(2)MEK-extracellular signal-regulated protein kinase(ERK) pathway mediating MM cell proliferation.Now histone deacetylase inhibitors(HDAIs) have been regarded as a new kind of anticancer drug and several of them are currently being investigated in clinical trials.Valproic acid(VPA)is a shortchain fatty acid with a long history of clinical use as an anticonvulsant.However,VPA also inhibits histone deacetylases(HDACs)and induces apoptosis in selected solid tumors as well as in hematologic neoplasms.
Objective:To define the effects of VPA on multiple myeloma cell line KM3 cells in vitro and the underlying mechanism of VPA in the treatment of MM were investigated.
Methods:KM3 cells were cultured in RPMI1640 medium and treated with VPA in different concentration.The cell proliferation was measured by 3-[4, 5-dimehyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay;the AnnexinⅤand PI staining was used to detect the apoptosis rates;the mRNA level of VEGFR was determined by RT-PCR;and immunocytochemistry was used to detect the protein level of acetylated-histone H4(Ac-histone H4)and VEGFR.
Results:The proliferation rate of KM3 cells was decreased in VPA- treated group in a time-dose dependent manner(P＜0.05).And the AnnexinⅤand PI staining showed VPA can induce apoptosis of KM3 cells,treatment with VPA(0.5、1、2 and 4mmol/L) for 48h,the apoptosis rates of KM3 cells were(11.77±4.64)%、(22.13±1.20)%、(23.95±.2.57)%and(42.72±4.61)%respectively.The RT-PCR results demonstrated the KM3 cells only express VEGFR-1 and it was decreased in VPA(0.5、1、2 and 4 mmol/L for 48h)treated groups compared with control(P＜0.05).Results of immunocytochemistry show the protein of Ac-histone H4 was increased significantly in VPA(4 mmol/L for 48h)treated group whereas the VEGFR-1/Flt-1 was decreased (P＜0.05).
Conclusion:VPA,as an histone deacetylase inhibitor,can increase the expression of Ac-histone H4 and inhibit the expression of VEGFR-1 in KM3 cells and it plays an important role in regulating proliferation and apoptosis of multiple myeloma cell line KM3 cells in vitro.
引文
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[2]Glozak MA, Sengupta N, Zhang XH, et al.Acetylation and deacetylation of non-histone proteins.gene[J] .2005 ;363:15-23.
[3]Shukla PK, Tikoo KB. Histone Deacetylase' Inhibitor Inhibitors as therapeutic Target. Curr Res Info Pharmaceu Sci [J]. 2004; 5(1):9-18.
[4]Rosato RR, Maggio SC, Almenara JA, et al.The Histone Deacetylase Inhibitor LAQ824 Induces Human Leukemia Cell Death through a Process Involving XIAP Down-Regulation, Oxidative Injury, and the Acid Sphingomyelinase-Dependent Generation of Ceramide.Mol Pharmacol[J].2006;69(1): 216-225.
[5]Duan H, Heckman CA, Boxer LM. Histone Deacetylase Inhibitors Down-Regulate bcl-2 Expression and Induce Apoptosis in t(14;18) Lymphomas.Molecular and Cellular Biology[J].2005;25 (5) .1608-1619.
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