维甲酸和维生素C对HL-60细胞诱导分化的研究

[目的]探讨全反式维甲酸(ATRT)和维生素C(V鄄C)对人早幼粒白血病细胞(HL鄄60)的分化作用。[方法]用1μmol/LATRT和0.5mmol/LV鄄C单独和联合处理HL鄄60细胞后,观察细胞形态改变,测定细胞增殖速度、硝基四氮唑蓝(NBT)还原反应、过氧化酶染色反应。[结果]两者都能使HL鄄60细胞形态向成熟粒细胞转变,对细胞增殖有明显的抑制作用,前者强后者弱;维甲酸使细胞的NBT反应显著增强(P<0.01),过氧化酶染色为弱阳性或阴性;V鄄C对HL鄄60细胞也有NBT反应和染色反应,但改变不明显(P<0.05),两者联合作用,分化作用增强。[结论]维甲酸有很强的诱导HL鄄60细胞分化的作用,V鄄C的分化作用不明显,但有增强维甲酸分化的作用。     

Cell Cycle-Independent Down-Regulation of BCL-2 Protein Expression in Differentiating HL-60 Cells

To understand the relationship between bcl-2 protein expression and the cell cycle during the processes of differentiation, we examined bcl-2 protein levels expressed during cell cycle phases in differentiating HL-60 human leukemia cells by using a two-color flow cytometric method. In exponentially proliferating HL-60 cells bcl-2 protein was constitutively expressed throughout the cell cycle phases, but a small population of G0/G1 cells expressed decreased levels of protein as compared with other cell cycle phases. HL-60 cells can be induced to differentiate to granulocytic pathway by retinoic acid or dimethylsulfoxide, and to monocytic/macrophagic pathway by 1, 25 dihydroxyvitamin D3 or 12-O-tetradecanoylphorbol-13-acetate. During treatment with any of these inducing agents, bcl-2 protein expression was time-dependently down-regulated after 24h. A two-color flow cytometric analysis revealed that this down-regulation occurred throughout cell cycle phases, indicating that bcl-2 protein expression was down-regulated in cell cycle-independent manner during induction of differentiation in HL-60 cells. To our knowledge, this is the first demonstration suggesting that the regulation of bcl-2 protein expression is not related to the cell cycle during induction of differentiation in HL-60 cells.     

鲎血细胞多肽诱导HL-60细胞凋亡

目的：研究鲎血细胞多肽（下称鲎血多肽）诱导HL－60细胞凋亡的作用。方法：MTT法测定鲎血多肽对HL－60细胞的毒性和HL－60细胞的相对存活率,荧光显微镜观察HL－60细胞形态的变化,流式细胞仪鉴定细胞凋亡和分析细胞周期,扫描电镜观察细胞膜的改变。结果：鲎鱼多肽对HL－60细胞有明显的细胞毒性,IC50值为24μg/ml;荧光显微镜观察到凋亡细胞主要表现为细胞体积缩小,核染色质固缩,荧光染色增强。50－100μg/ml的鲎血多肽处理6h,HL－60细胞主要表现为细胞凋亡;而鲎血多肽浓度为200μg/ml时的主要为细胞坏死。50μg/ml鲎血多肽处理HL－60细胞0－12h,流式细胞仪检测到凋亡细胞亚二倍体峰出现,细胞周期分析G1期细胞减少,G2期细胞增加。鲎血多肽浓度为25－100μg/ml时,细胞凋亡率随浓度增加而增加,鲎血多肽为200μg/ml时,则主要为细胞坏死,凋亡细胞减少。扫描电镜观察发现,HL－60细胞用鲎血多肽处理后,细胞膜损伤,出现空洞。结论：鲎血细胞多肽能诱导HL－60细胞凋亡;凋亡的发生较早,与细胞膜受损有一定的关系;G1期细胞对鲎血多肽更敏感。     

全反式维甲酸诱导前列腺癌DU-145细胞凋亡的研究

目的 :应用全反式维甲酸作用于前列腺癌细胞系DU 145细胞 ,观察维甲酸对前列腺癌细胞生长的影响。方法 :应用全反式维甲酸 (10 -5mol L)分别作用于前列腺癌DU 145细胞 36h及 72h ,分别应用光学显微镜、电子显微镜观察细胞形态及超微结构的改变 ,并用流式细胞仪检测细胞周期的改变。结果 :全反式维甲酸作用前列腺癌DU 145细胞 36h后 ,细胞生长开始减缓 ,肿瘤细胞超微结构出现细胞核固缩、染色质凝集于核膜周边等细胞凋亡早期改变。作用 72h后 ,流式细胞仪检测肿瘤细胞出现典型的亚二倍体“凋亡小峰” ,部分肿瘤细胞出现凋亡。结论 :全反式维甲酸可以诱导前列腺癌细胞产生凋亡 ,凋亡的产生与药物的作用时间密切相关     

Retinoic acid promotes phorbol ester-initiated macrophage differentiation in HL-60 leukemia cells without disappearance of protein kinase C

The ability of retinoic acid (RA) to promote 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-initiated macrophage differentiation was examined in human promyelocytic leukemia cell line HL-60. One-hour exposure to 10 nM TPA and subsequent exposure for 48 h to 1 μM RA following removal of TPA rapidly induced the macrophage phenotype in 65% of the cells. This effect was comparable to continuous exposure for 48 h to TPA alone, but contrasted with the absence of macrophage-like cells after RA treatment alone or the induction of 10% of the cell population to a macrophage phenotype after 1-h exposure to TPA. The effect of TPA + RA was accompanied by increased cell adherence and increased nonspecific esterase activity but not by a change in the reduction of nitroblue tetrazolium. Protein kinase C (PK-C) activity was increased 35–40% in cells treated for 1 h with TPA alone or after subsequent exposure to RA. Cells treated for 48 h with RA exhibited a 2-fold increase in PK-C activity while cells exposed to TPA for 48 h lost all PK-C activity. The changes in PK-C activity in TPA + RA-treated cells were accompanied by Ca²⁺/phospholipid(PL)-dependent phosphorylation in vitro of pp38 which is characteristic of treatment with RA alone, as well as the Ca ²⁺/PL-independent phosphorylation in vitro of pp82 and pp130 (vinculin) which is prevalent in cells treated continuously with TPA alone and is absent in RA-treated cells. These results indicate that the macrophage phenotype induced by TPA + RA is similar to that produced by continuous exposure to TPA alone with respect to their in vitro phosphoprotein patterns, cytochemical markers, cell adherence and morphology, but that the disappearance of PK-C is not an obligatory characteristic of these cells.     

细胞因子联合人参皂甙及三氧化二砷诱导HL-60细胞向树突状细胞分化的实验研究

目的研究HL-60细胞在体外经细胞因子联合人参皂甙及三氧化二砷(AS2O3)诱导向树突状细胞(DC)分化的情况。方法选用HL-60细胞与重组人粒单核细胞集落刺激因子(rh-GM-CSF)、白细胞介素4(IL-4)、γ干扰素(INF-γ)、TSPG及AS2O3共培养。根据细胞因子、TSPG及AS2O3不同组合分为12组。在培养后7天及14天以光学和电子显微镜观察细胞的形态学特征，用HLA-DR、CD1a、CD86等五种单克隆抗体标记流式细胞术检测细胞表型，用ELISA法测定HL-60-DC培养上清液中的IL-12及INF-γ的量，培养14天后的细胞再继续以1：10的比例与HL-60细胞共培养24h，观察HL-60-DC对白血病细胞的杀伤情况，用流式细胞仪检测凋亡率。结果HL-60细胞在组合细胞因子并分别加入TSPG及AS2O3后，均可诱生出不同比例的DC。光镜及电镜下均有典型的树突状细胞的形态学特征，细胞表达DC的表面分化抗原，诱生的DC培养上清中可测出不同量的IL-12及INF-γ。细胞凋亡率均明显高于对照组，联合中药各组的凋亡率均高于GM-CSF与IL-4组。结论①GM—CSF、IL-4与HL-60细胞共培养可诱导出HL-60-DC，在此基础上加入适当浓度的TSPG、AS2O3可使树突状细胞的特异性抗原表达加强。②在DC诱生过程中，GM—CSF联合IL-4组DC特异性抗原表达率高于IL-4联合INF组，证明GM—CSF在DC诱生过程中起重要作用。③单独应用TSPG，也可出现部分DC特异性抗原表达，但表达率明显低于联合用药组。④诱生后的HL-60-DC具有自分泌IL—12及INF-γ的功能。⑤诱生后的DC具有抗白血病细胞作用，与HL-60细胞共培养后可使白血病细胞凋亡明显增强。     

全反式维甲酸联合顺铂在诱导人头颈癌 TCA8113细胞凋亡中的作用

目的：研究全反式维甲酸（ATRA）与顺铂对人头颈癌 TCA8113细胞凋亡的影响。方法 ATRA 和顺铂单独及联合作用于 TCA8113细胞,采用 MTT 法检测细胞生长,并观察细胞形态变化;使用流式细胞仪检测细胞周期及凋亡;通过 western blot 实验检测 caspase-3表达情况。结果 ATRA 作用于 TCA8113细胞后,细胞生长明显受到抑制,细胞形态发生改变,细胞凋亡比例明显增加,caspase-3蛋白表达下调,ATRA 与顺铂联合作用于肿瘤细胞后,其抑癌作用加强。结论 ATRA 联合顺铂对人头颈癌 TCA8113细胞具有协同抑癌作用。     

Antiproliferative effect of a vitamin D3 analog, EB1089, on HL-60 cells by the induction of TGF-beta receptor

EB1089 is a novel 1,25(OH)₂D₃ analog that has more potent antitumor properties with reduced hypercalcemic effects than 1,25(OH)₂D₃. We investigated the role of transforming growth factor-β1 (TGF-β1) in the growth inhibition of human acute myeloid leukemia cell line, HL-60, by EB1089. Clonal growth of HL-60 cells was inhibited in a dose-dependent manner by EB1089. Although TGF-β1 alone slightly inhibited proliferation of HL-60 cells, the addition of TGF-β1 into culture treated with 10⁻⁸ M of EB1089 showed a significant synergistic antiproliferative effect in a dose-dependent manner. EB1089 up-regulated the expression of TGF-β receptor type I (TGF-β RI), type II (TGF-β RII) and TGF-β1. Antiproliferative effect of EB1089 was partially reversed by TGF-β1 neutralizing antibody (anti-TGF-β1). Vitamin D receptor (VDR) expression was increased by TGF-β1, suggesting synergistic action of TGF-β1 and EB1089. Combined treatment of EB1089 and TGF-β1 resulted in an increased expression of cyclin-dependent kinase inhibitor (CDKI), p27 protein, compared to either ligand alone. Up-regulation of p27 protein expression by either TGF-β1 or EB1089 was reduced by anti-TGF-β1. These findings suggest that TGF-β1 is involved in the antiproliferative effect of EB1089 on HL-60 cells.     

Antiproliferative effect of a vitamin D3 analog, EB1089, on HL-60 cells by the induction of TGF-β receptor

EB1089 is a novel 1,25(OH)₂D₃ analog that has more potent antitumor properties with reduced hypercalcemic effects than 1,25(OH)₂D₃. We investigated the role of transforming growth factor-β1 (TGF-β1) in the growth inhibition of human acute myeloid leukemia cell line, HL-60, by EB1089. Clonal growth of HL-60 cells was inhibited in a dose-dependent manner by EB1089. Although TGF-β1 alone slightly inhibited proliferation of HL-60 cells, the addition of TGF-β1 into culture treated with 10⁻⁸ M of EB1089 showed a significant synergistic antiproliferative effect in a dose-dependent manner. EB1089 up-regulated the expression of TGF-β receptor type I (TGF-β RI), type II (TGF-β RII) and TGF-β1. Antiproliferative effect of EB1089 was partially reversed by TGF-β1 neutralizing antibody (anti-TGF-β1). Vitamin D receptor (VDR) expression was increased by TGF-β1, suggesting synergistic action of TGF-β1 and EB1089. Combined treatment of EB1089 and TGF-β1 resulted in an increased expression of cyclin-dependent kinase inhibitor (CDKI), p27 protein, compared to either ligand alone. Up-regulation of p27 protein expression by either TGF-β1 or EB1089 was reduced by anti-TGF-β1. These findings suggest that TGF-β1 is involved in the antiproliferative effect of EB1089 on HL-60 cells.     

Synergistic Effects of Valproic Acid and Mitomycin C in Adenocarcinoma Cell Lines and Fresh Tumor Cells of Patients with Colon Cancer

Abstract

Valproic acid has been demonstrated to mediate cytotoxic effects against tumor cells by acting as a histone-deacetylase inhibitor. However, to date, there are only limited data on the effects of valproic acid in colon cancer. Moreover, information regarding combinations of the drug with chemotherapeutic agents is very limited. The latter is of interest as there is increasing evidence for synergism between socalled “molecular targeting drugs” and chemotherapy.

We first demonstrated that valproic acid dose-dependently reduced the viability of adenocarcimona cell lines. After co-incubation with a variety of chemotherapeutic agents, only valproic acid in combination with mitomycin C consistently induced synergistic growth inhibition in all cell lines. To confirm these results in an ex vivo situation, five samples of fresh colon cancer cells were studied. Again, the effect of valproic acid on the viability of the fresh tumor cells was dose dependent. In four of five samples of freshly isolated colon cancer cells, the synergistic effect of valproic acid and mitomycin C on the inhibition of cell growth was confirmed by calculation of the combination index by multiple drug effect analysis.

In conclusion, this is the first demonstration that valproic acid as a model substance for histone-deacetylase inhibitors is effective in tumor cells freshly isolated from patients with colon cancer and that the combination of mitomycin C and valproic acid synergistically decreases viability of colon cancer cells.