Derivatives of vitamins D2 and D3 activate three MAPK pathways and upregulate pRb expression in differentiating HL60 cells

Analogs of vitamin D have been synthesized which have reduced calcemic activities yet increased anti-proliferative and differentiation-inducing properties, raising expectations that they will be useful for treatment of human neoplastic diseases. In the present study we compared the abilities of three such analogs, 24a, 24b-dihomo-1,25-dihydroxyvitamin D(3) (PRI-1890), 24-ene-1,25-dihydroxyvitamin D(2) (PRI-1906) and (24R)-1,24-dihydroxyvitamin D(3) (PRI-2191) to induce markers (CD14, CD11b and MSE) of differentiation, G(1) phase block, and associated molecular events in human promyeloblastic leukemia cells HL60. We found that the potencies of the analogs to induce differentiation paralleled their activation of Erk, JNK and p38 mitogen-activated protein kinase (MAPK) pathways, and the anti-proliferative activity closely correlated with the extent of hypophosphorylation of retinoblastoma protein (pRb). Interestingly, low concentrations of derivatives of vitamin D, which were insufficient to induce any detectable changes in the cell cycle traverse, markedly increased the levels of total pRb, which was highly phosphorylated. These results suggest that pRb may have an unsuspected role in monocytic differentiation, perhaps to increase the sensitivity of the G(1) checkpoint, by increasing the amount of substrate for cyclin-dependent kinases.     

Loss of Deoxycytidine Kinase Expression and Tetraploidization of HL60 Cells Following Long-Term Culture in 1,25-Dihydroxyvitamin D3

Development of drug resistance is a major problem in attempts to control the growth of neoplastic cell populations. The resistance can be either inherent or acquired by an exposure to a chemotherapeutic drug. The available models for study of these phenomena have not led to major improvements in the therapy for human cancers. Therefore, in order to develop a new model for such studies, we have exposed human myeloid leukemia cells HL60 to increasing concentrations of 1,25-dihydroxyvitamin D₃(1,25D₃) and characterized the emerging new phenotypes of these cells over a period of 4 years. During the stepwise development of resistance only cells which did not adhere to the flask walls were passaged. Beginning at 30 nM1,25D₃the sublines became resistant to the differentiation-inducing and growth-retarding properties of 1,25D₃even at 400 nM.Also, their growth rates in 1,25D₃-free media increased. In addition, beginning at 40 nM1,25D₃the sublines acquired resistance to 5-β- -arabinocytosine (araC) due to the lack of expression of the deoxycytidine kinase gene. The araC-resistant sublines were also near-tetraploid, as judged by their DNA content. When grown in 1,25D₃-free long-term culture the phenotype was essentially stable. The development of cross-resistance to araC during growth in the presence of an unrelated compound (i.e., 1,25D₃) shows that in some instances an apparently inherent drug resistance may in fact be due to a metabolic defect resulting from an exposure to another agent.     

核壳结构CdS-TiO_2纳米颗粒体外光催化灭活白血病HL60细胞实验研究

本文研究了核壳结构CdS-TiO_2纳米颗粒光对HL60细胞光催化灭活作用,并初步探讨了CdS量子点增强TiO_2光催化灭活效率的作用机理。实验利用超声法制备了核壳结构CdS-TiO_2纳米颗粒,使用CCK-8法检测了其对白血病HL60细胞的光催化灭活效果,并采用活性氧分析和荧光发光光谱等分析手段对CdS量子点增强TiO_2光催化灭活效率的作用机理进行了分析。细胞实验结果表明,在暗室条件下,随着CdS包裹的TiO_2外壳的增厚,细胞的暗室存活率从28.5%逐渐上升至80%;在可见光照下,细胞的存活率显著下降。CdS-TiO_2纳米颗粒对HL60细胞的光催化灭活率均超过60%,其中CdS-TiO_2样品0.6对HL60细胞的光催化灭活效率最高,达到95%。根据荧光光谱和活性氧含量分析的结果推测,这可能是由于核壳结构的CdS内核与TiO_2外壳之间产生了有效的电子转移,抑制了TiO_2的空穴电子的复合,增强了TiO_2外壳的光催化活性,最终增加了TiO_2对HL60细胞的PDT灭活效果。     

Zinc Inhibits H2O2-Induced MC3T3-E1 Cells Apoptosis via MAPK and PI3K/AKT Pathways

Zinc has been shown to increase bone mass and promote bone cell proliferation and differentiation. We, therefore, hypothesized that zinc might be cytoprotective for bone cells during oxidative stress. The cells were divided into H(2)O(2), zinc and zinc+H(2)O(2) groups. In the present study, zinc was found to inhibit H(2)O(2)-induced apoptosis in MC3T3-E1 cells, as shown by analysis of Annexin V/PI double staining. Western blot data showed that in zinc+H(2)O(2)-treated cells, zinc decreased the levels of AIF, Bax and active caspase-9 and -3, which are pro-apoptotic factors. And zinc inhibited release of cytochrome c from mitochondria to cytosol in zinc+H(2)O(2)-treated cells. Further investigation shows that protection is via activation of PI3K/Akt/mTor and MAPK /ERK pathways and inhibition of MAPK/P38 and MAPK/JNK pathways. Protecting osteoblast cells from oxidative damage presents a potential application in the treatment of osteoporosis.     

基于L-半胱氨酸修饰的Fe3O4@TiO2复合纳米颗粒体外光动力疗法灭活HL60细胞的试验研究

本文采用共沉淀法制备了L-半胱氨酸(L-Cys)修饰的Fe3O4包裹TiO2(Fe3O4@TiO2/L-Cys)复合纳米粒子。通过透射电子显微镜(TEM),X射线衍射(XRD)和傅立叶变换红外光谱仪(FTIR)对复合纳米粒子进行了表征,并讨论了复合纳米粒子对HL60细胞体外光动力疗法(PDT)灭活的影响。并对其PDT灭活机制进行了初步探索。试验表明,Fe3O4@TiO2/L-Cys复合纳米粒子分散性高,生物相容性好,对细胞的暗毒性更低,并可以有效增强靶向性,提高PDT灭活效率,在410nm波长的光激发下,光照剂量为18J/cm^2的情况下,当TiO2与Fe3O4的比例为1∶3时,整体PDT效率最高。PDT灭活效率可达69.36%。     

IGF-1 Reduces BACE-1 Expression in PC12 Cells via Activation of PI3-K/Akt and MAPK/ERK1/2 Signaling Pathways

Insulin-like growth factor 1 (IGF-1) stimulates α-secretase processing of amyloid precursor protein (APP) and decreases Aβ production. Little is known about the relationship between IGF-1 and β-site amyloid precursor protein cleaving enzyme 1 (BACE-1), the protease essential for the production of β-amyloid peptides (Aβ). Here, we investigated the effect of IGF-1 on BACE-1 in PC12 cells. Quantitative polymerase chain reaction analysis and western blot showed that treatment of cells with IGF-1 significantly decreased the levels of BACE-1 mRNA and protein. Furthermore, IGF-1 increased the phosphorylation of Akt and ERK1/2. The presence of the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 and the mitogen-activated protein kinase kinases (MEK) inhibitor PD98059 blocked the effect of IGF-1 on BACE-1. Our data indicated that IGF-1-induced reduction of BACE-1 might involve the PI3-K/Akt and MAPK/ERK1/2 signaling pathways.     

掺铁TiO2纳米颗粒与恒定磁场对HL60白血病肿瘤细胞的灭活效果

通过研究不同浓度、不同磁场作用下TiO2、掺铁TiO2纳米颗粒对HL60白血病细胞活性的影响,以及在接受光照和不接受光照条件下的细胞活性,探讨基于TiO2、掺铁TiO2纳米颗粒作为光敏剂的光动力疗法(PDT)灭活白血病肿瘤细胞的可行性.实验结果表明,纳米颗粒对细胞具有一定的抑制/毒性作用,纳米浓度越大,抑制/毒性作用越明显;磁场对细胞的毒性/抑制作用跟掺铁的浓度以及磁感应强度有关,掺铁纳米组在强磁场作用下对细胞抑制/毒性作用明显;此外,添加了纳米颗粒的PDT灭杀效率要比不添加纳米颗粒的PDT灭杀效率高.     

Protein Kinase C-δ Associates with Vimentin Intermediate Filaments in Differentiated HL60 Cells

The subcellular localization of protein kinase C (PKC)-δ was determined in HL60 cells differentiated toward monocytes/macrophages by treatment with TPA. PKC-δ was detected in the nucleus and cytoplasm of differentiated HL60 cells and, more specifically, associated with structures resembling intermediate filaments. Indirect immunostaining revealed that PKC-δ colocalized with vimentin in the cytosol and perinuclear region of these cells. Immunoprecipitation studies showed that PKC-δ was in an active (autophosphorylated) state in differentiated HL60 cells and that vimentin immunoprecipitated from these cells was also phosphorylated. Treatment of HL60 cells with the PKC-specific inhibitor chelerythrine decreased the phosphorylation of vimentin. These data suggest that vimentin is a substrate for PKC-δ and that this PKC isoenzyme may play a specific role in the regulation of shape change and cell adhesion during HL60 differentiation.     

Particulate Matter Facilitates C6 Glioma Cells Activation and the Release of Inflammatory Factors Through MAPK and JAK2/STAT3 Pathways

It has been widely accepted that astrocytes, play a role in regulating almost every physiological system. In the present study, we investigated the role of particulate matter (PM) in regulating activation of astrocytes. The glial cell strain C6 was cloned from a rat glioma which was induced by N-nitrosomethylurea. The C6 cells were plated at a density of 5 × 10⁶ cells/10 cm diameter dish and incubated with different concentrations (0, 12, 25, 50, 100, 200, and 400 μg/mL) of PM for 24 h and different time (0, 1, 3, 6, 8,12, and 24 h) with 100 μg/mL at 37 °C. The study revealed that PM stimulated the expression of inducible nitric oxide synthase (iNOS) as well as the production of IL-1β in a dose- and time-dependent manner. Furthermore, activation of JAK2/STAT3 and p38/JNK/ERK MAPKs was found in astrocytes following PM treatment. Blockage of JAK and p38/JNK/ERK MAPKs with their specific inhibitors, AG490, SB202190, SP600125 and U0126 significantly reduced PM-induced iNOS expression and IL-1β production. In addition, it was demonstrated that inhibition of p38, JNK and JAK prevented STAT3 tyrosine phosphorylation induced by PM, while blocking ERK did not. MAPKs (p38 and JNK) could regulate tyrosine STAT3 phosphorylation, which suggested that the JAK2/STAT3 pathway might be the downstream of p38/JNK MAPK pathways.     

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.     

Zinc Upregulates the Expression of Osteoprotegerin in Mouse Osteoblasts MC3T3-E1 Through PKC/MAPK Pathways

Zinc is an essential element for bone formation; however, its role in osteoblast has not been well understood. In the present study, we hypothesized that zinc could increase osteogenetic function by stimulating osteoblast proliferation and osteoprotegerin (OPG) activity. To test this hypothesis, osteoblastic MC3T3-E1 cells were cultured and treated with various concentrations of zinc (0, 10, 30, 50, 70, 110, 130, and 150 μM) for 24 and 48 h. 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide assay showed that cell proliferation was significantly stimulated with 50 μM zinc treatment. Furthermore, under the same treatment condition, OPG expression was significantly increased as evidenced by the results of RT-PCR and ELISA. However, the zinc-induced OPG expression was significantly attenuated when MC3T3-E1 cells were co-treated with either protein kinase C (PKC) inhibitor, GF109203X, or the Inhibitor of mitogen-activated extracellular signal-regulated kinase 1 (MEK1), PD98059. Moreover, OPG expression was further increased when MC3T3-E1 cells were treated with PMA (the activator of protein of kinase C) in the presence of zinc. These results suggested that zinc would increase osteogenic function by stimulating PKC and MAPK signaling pathways.     

Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways

Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited in vitro in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS.     

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.     

Propofol Treatment Inhibits Constitutive Apoptosis in Human Primary Neutrophils and Granulocyte-Differentiated Human HL60 Cells

Apoptosis regulation is essential for neutrophil homeostasis. We previously demonstrated that a process involving glycogen synthase kinase (GSK)-3β determines neutrophil apoptosis. As for this apoptotic process, an overdose of propofol (2,6-Diisopropylphenol; 25 μg/ml or 140 μM) also causes GSK-3β-mediated macrophage apoptosis; however, the early deactivation of GSK-3β with low-dose propofol has been shown. Therefore, we hypothesize that low-dose propofol may induce neutrophil survival via GSK-3β inactivation. Following in vitro culture, the therapeutic concentration of propofol (10 μg/ml or 56 μM) treatment decreased constitutive apoptosis in isolated human primary neutrophils and in granulocyte-differentiated HL60 cells after all-trans retinoic acid (1 μM) treatment. The inactivation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT and the activation of GSK-3β results in myeloid cell leukemia 1 (Mcl-1) down-regulation, the loss of the mitochondrial transmembrane potential, and caspase-3 activation in these cells, which is accompanied by apoptosis. Notably, propofol treatment attenuates these effects in a PI3-kinase-regulated manner. We found that propofol initiates PI3-kinase/AKT-mediated GSK-3β inactivation and Mcl-1 stabilization, rescuing the constitutive apoptosis in primary neutrophils and granulocyte-differentiated acute promyelocytic leukemia HL60 cells.