Effects of a moderate-intensity static magnetic field and adriamycin on K562 cells

The aim of this study was to investigate whether a moderate‐intensity static magnetic field (SMF) can enhance the killing effect of adriamycin (ADM) on K562 cells, and to explore the effects of SMF combined with ADM on K562 cells. We analyzed the metabolic activity of cells, cell cycle distribution, DNA damage, change in cell ultrastructure, and P‐glycoprotein (P‐gp) expression after K562 cells were exposed continuously to a uniform 8.8 mT SMF for 12 h, with or without ADM. Our results showed that the SMF combined with ADM (25 ng/ml) significantly inhibited the metabolic activity of K562 cells (P < 0.05), while neither ADM nor the SMF alone affected the metabolic activity of these cells. Cell ultrastructure was altered in the SMF + ADM group. For example, cell membrane was depressed, some protuberances were observable, and vacuoles in the cytoplasm became larger. Cells were arrested at the G2/M phase and DNA damage increased after cells were treated with the SMF plus ADM. ADM also induced the P‐gp expression. In contrast, in the SMF group and SMF + ADM group, the P‐gp expression was decreased compared with the ADM group. Taken together, our results showed that the 8.8 mT SMF enhanced the cytotoxity potency of ADM on K562 cells, and the decrease in P‐gp expression may be one reason underlying this effect. Bioelectromagnetics 32:191–199, 2011. © 2010 Wiley‐Liss, Inc.     

Apoptosis induced by 3,7-dinitrodibenzobromonium salts in K562 cells

We evaluated the inhibitory effect of 3,7-dini-trodibenzobromonium salts (cBr) on the proliferation of human chronic myelogenous leukemia K562 cell by trypan blue exclusion test and MTT colorimetric assay.The degree of DNA damage in K562 cells treated with cBr,was detected by isotopic tracer method (3H-TdR).The morphological changes of these K562 cells were examined by fluorescence and electron microscopy.Biochemical characteristics of K562 cells were detected by flow cytome-try and 3H-thymidine incorporation assay.Findings indi-cated that cBr could significantly inhibit cell proliferation and result in DNA damage of K562 cells,cBr is a new type of immunostimulant and can induce cell apoptosis.     

Effects of a 300 mT static magnetic field on human umbilical vein endothelial cells

This study describes the effects of a static magnetic field (SMF) on cell growth and DNA integrity of human umbilical vein endothelial cells (HUVECs). Fast halo assay was used to investigate nuclear damage; quantitative polymerase chain reaction (QPCR), standard PCR, and real‐time PCR were used to evaluate mitochondrial DNA integrity, content, and gene expression. HUVECs were continually exposed to a 300 mT SMF for 4, 24, 48, and 72 h. Compared to control samples (unexposed cultures) the SMF‐exposed cells did not show a statistically significant change in their viability. Conversely, the static field was shown to be significant after 4 h of exposure, inducing damage on both the nuclear and mitochondrial levels, reducing mitochondrial content and increasing reactive oxygen species. Twenty‐four hours of exposure increased mitochondrial DNA content as well as expression of one of the main genes related to mitochondrial biogenesis. No significant differences between exposed and sham cultures were found after 48 and 72 h of exposure. The results suggest that a 300 mT SMF does not cause permanent DNA damage in HUVECs and stimulates a transient mitochondrial biogenesis. Bioelectromagnetics 31:630–639, 2010. © 2010 Wiley‐Liss, Inc.     

Simultaneous application of cisplatin and static magnetic field enhances oxidative stress in HeLa cell line

In this study, we reported the effects of simultaneous application of static magnetic field (SMF) and cisplatin as an anticancer drug on the oxidative stress in human cervical cancer (HeLa) cell line and normal skin fibroblast cells (Hu02). The cells were exposed to different SMF intensities (7, 10, and 15 mT) for 24 and 48 h. IC₅₀ concentrations of cisplatin were obtained by MTT assay. The cytotoxic effects of combined treatment were studied by measuring the intracellular reactive oxygen species content using flow cytometric method and estimation of membrane lipid peroxidation by spectrophotometry. Statistical analysis was assessed using one-way repeated measures analysis of variance (ANOVA) followed by Tukey’s test. Based on the obtained results, the highest and lowest death rate, respectively, in HeLa and Hu02 cell lines was observed at the intensity of 10 mT. Also, we found that membrane lipid peroxidation in cancer cells is higher than that of normal counterparts. SMF potently sensitized human cervical cancer cells to cisplatin through reactive oxygen species (ROS) accumulation while it had small effects on normal cells. The combination of both treatments for 48 h led to a marked decrease in the viability percentage of HeLa cells by about 89% compared to untreated cells. This study suggests that conjugation of both physical and chemical treatments could increase the oxidative stress in HeLa cell line and among three optional intensities of SMF, the intensity of 10 mT led to the higher damage to cancer cells in lower doses of drug.     

Static magnetic fields affect cell size, shape, orientation, and membrane surface of human glioblastoma cells, as demonstrated by electron, optic, and atomic force microscopy.

BACKGROUND: It is common knowledge that static magnetic fields (SMF) do not interact with living cells; thus, fewer studies of SMF compared with variable magnetic fields are carried out. However, evidence demonstrated that SMF affect cellular structures. To investigate the effect of exposure to increasing doses of SMF on cell morphology, human glioblastoma cells were exposed to SMF ranging between 80 and 3,000 G (8 and 300 mT). METHODS: Cell morphology of human glioblastoma cells, derived from a primary culture, was studied by electron and optic microscopy. FITC-phalloidin staining of actin filaments was also investigated. Finally, cell surface structure changes were detected by atomic force microscopy. RESULTS: Scanning electron microscopy demonstrated a dose-dependent cell shape modification, progressive cell detachment, loss of the long villi, and appearance of membrane roughness and blebs. FITC-phalloidin staining confirmed the villi retention and cell dimension decrease. At 3,000 G, the appearance of apoptotic morphology was also observed by transmission electron microscopy. Cell exposed to SMF showed different orientation and alignment when compared with nonexposed cells. The atomic force microscopy of the exposed cells' membrane surfaces demonstrated the disappearance of the ordered surface ripples and furrows typical of the unexposed cells, and the occurrence of surface membrane corrugation at increasing dose exposure CONCLUSIONS: Our experimental procedures demonstrated that exposure to SMF affects not only cell size, shape, and orientation but also human glioblastoma cells' membrane surfaces.     

稳恒磁场联合抗肿瘤药物协同杀伤肝癌细胞Hepa1-6

化学疗法为肿瘤临床治疗的常规方法,存在毒副作用大、抗药性强等缺陷。为了提高药物的利用效率,减少药物引起的毒副作用,将8.8 m T稳恒磁场分别与顺铂、阿霉素联用,经MTT检测发现磁场与药物联用可对肝癌细胞Hepa1-6生长具有协同抑制的效应,经HE染色发现联合处理组细胞发生明显的形态学改变。流式细胞仪检测显示磁场能增加顺铂对G2/M期细胞的滞留,而磁场与阿霉素共同作用可将细胞阻止于G1期和G2/M期。经彗星电泳检测表明磁场能够增强药物对DNA的损伤,且原子力显微镜观察发现联合处理组细胞膜表面出现较大且较深的孔洞,表面结构破坏严重。实验结果表明,抗肿瘤药物与磁场联用技术可有效抑制肿瘤细胞的生长,减少药物的使用浓度,为将抗肿瘤药物与磁场应用于临床治疗恶性肿瘤提供了一个全新的思路与策略。     

A Novobiocin Derivative,XN4, Inhibits the Proliferation of Chronic Myeloid Leukemia Cells by Inducing Oxidative DNA Damage

XN4 might induce DNA damage and apoptotic cell death through reactive oxygen species (ROS). The inhibition of proliferation of K562 and K562/G01 cells was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide). The mRNA levels of NADPH oxidase 1-5 (Nox1-5) genes were evaluated by qRT-PCR. The levels of extracellular reactive oxygen species (ROS), DNA damage, apoptosis, and cell cycle progression were examined by flow cytometry (FCM). Protein levels were analyzed by immunoblotting. XN4 significantly inhibited the proliferation of K562 and K562/G01 cells, with IC₅₀ values of 3.75±0.07 µM and 2.63±0.43 µM, respectively. XN4 significantly increased the levels of Nox4 and Nox5 mRNA, stimulating the generation of intracellular ROS, inducing DNA damage and activating ATM-γ-H2AX signaling, which increased the number of cells in the S and G2/M phase of the cell cycle. Subsequently, XN4 induced apoptotic cell death by activating caspase-3 and PARP. Moreover, the above effects were all reversed by the ROS scavenger N-acetylcysteine (NAC). Additionally, XN4 can induce apoptosis in progenitor/stem cells isolated from CML patients’ bone marrow. In conclusion, XN4-induced DNA damage and cell apoptosis in CML cells is mediated by the generation of ROS.     

Protein tyrosine kinase inhibitors modulate radiosensitivity and radiation-induced apoptosis in K562 cells.

We studied the modulating effect of protein tyrosine kinase inhibitors on the response of cells of the human chronic myelogenous leukemia cell line K562 to radiation. The radiosensitivity of the cells was increased by treatment with herbimycin A and decreased by treatment with genistein. This modulating effect of protein tyrosine kinase inhibitors on radiation sensitivity was associated with the alteration of the mode of radiation-induced cell death. After X irradiation, the cells arrested in the G(2) phase of the cell cycle, but these TP53(-/-) cells were unable to sustain cell cycle arrest. This G(2)-phase checkpoint deficit caused cell death. The morphological pattern of cell death was characterized by swelling of the cytoplasmic compartments, cytosolic vacuolation, disruption of the plasma membrane, less evident nuclear condensation, and faint DNA fragmentation, all of which were consistent with oncosis or cytoplasmic apoptosis. The nonreceptor protein tyrosine kinase inhibitor herbimycin A accelerated the induction of typical apoptosis by X irradiation, which was demonstrated by morphological assessments using nuclear staining and electron microscopy as well as oligonucleosomal fragmentation and caspase 3 activity. Herbimycin A is known to be a selective antagonist of the BCR/ABL kinase of Philadelphia chromosome-positive K562 cells; this kinase blocks the induction of apoptosis after X irradiation. Our results showed that the inhibition of protein tyrosine kinase by herbimycin A enhanced radiation-induced apoptosis in K562 cells. This effect was associated with the activation of caspase 3 and rapid abrogation of the G(2)-phase checkpoint with progression out of G(2) into G(1) phase. In contrast, the receptor-type protein tyrosine kinase inhibitor genistein protected K562 cells from all types of radiation-induced cell death through the inhibition of caspase 3 activity and prolonged maintenance of G(2)-phase arrest. Further investigations using this model may give valuable information about the mechanisms of radiation-induced apoptosis and about the radiosensitivity and radioresistance of chronic myelogenous leukemia cells having the Philadelphia chromosome.     

Enhancement of natural killer cell cytotoxicity by using static magnetic field to increase their viability

Natural killer (NK) cells are innately immune to the body’s immune system and can actively recognize and kill cancer cells. This study explores the potential for enhancing the killing ability of NK cells by co-culturing the NK cells with the target cells under a static magnetic field (SMF). In this study, NK92-MI cell lines were cultured in the presence of a 0.4-T SMF. The effect of the SMF on NK cell viability was evaluated by means of an MTT assay. Culturing tests were performed with inhibitors of the DAG/IP3, STAT3, ERK, JNK and p38 pathways in order to examine the possible signaling cascade responsible for the SMF effect on the NK92-MI cell viability. Finally, the effect of the SMF on the cytotoxicity of the NK92-MI cells was evaluated by co-culturing the NK cells with K562 leukemia cell lines. The results showed that the application of a 0.4-T SMF significantly increased (p < 0.05) the viability of the NK92-MI cells. Furthermore, the inhibitor tests indicated that the SMF affected cell viability by activating multiple MAPK signaling pathways (ERKs, JNKs, and p38-MAPK). Finally, SMF pre-exposure for 48 hr significantly improved the killing activity of the NK92-MI cells (p < 0.05). That is, pre-exposure to SMF increased the viability of the NK92-MI cells and improved their killing ability against K562 tumor cells. In general, the present results suggest that NK cells pre-exposed to 0.4-T SMF show potential as a tool for immune-therapy treatment of cancer.     

敲除LSD1基因对人慢性髓系白血病K562细胞周期的影响

为了探讨敲除LSD1基因后抑制人慢性髓系白血病 K562细胞增殖的原因,使用前期CRISPR/Cas9技术构建的人慢性髓系白血病 K562 LSD1基因敲除株,通过细胞凋亡Annexin V/PI（碘化丙啶）双染色、细胞PI染色以及流式细胞术技术,探究敲除LSD1基因后,K562细胞的凋亡水平是否改变,细胞周期是否受到影响。结果表明敲除LSD1基因后K562细胞被阻滞在G0/G1期,进入DNA复制期的细胞变少,因此导致细胞增殖速度减慢;通过细胞凋亡Annexin V/PI双染色并分析早期以及晚期凋亡细胞总比例,显示敲除LSD1基因后,不影响K562细胞的凋亡。研究结果表明,敲除LSD1基因后人慢性髓系白血病 K562细胞的增殖受到抑制,这是由于K562细胞增殖周期发生了改变,进入DNA复制期和分裂期的细胞减少;而与细胞凋亡水平的变化无关。     

Induction of apoptosis in K562 cells by jolkinolide B

Jolkinolide B, a bioactive diterpene isolated from the roots of Euphorbia fischeriana Steud, has various biological and pharmacological properties. In this study, the cytotoxicity of highly purified jolkinolide B was tested in human chronic myeloid leukemia (K562) and 2 other cell lines (human esophageal carcinoma Eca-109 and human hepatoma HepG2). The results indicate a significant decrease in the proliferation of all the 3 cell lines when treated with jolkinolide B for 24 h; the IC50 value of cytotoxicity was 12.1 microg/mL (for K562 cells), >50.0 microg/mL (for HepG2 cells), and 23.7 microg/mL (for Eca-109 cells). Further study of K562 cells involving fluorescence and transmission electron microscopy revealed characteristic apoptotic features, such as cell shrinkage, membrane blebbing, loss of microvilli, and nuclear condensation. Agarose electrophoresis of genomic DNA showed a typical fragmentation pattern for apoptotic cells. A kinetic cell-cycle analysis demonstrated that the cell cycle was arrested in the G1 phase. All these results suggest that the anti-proliferation effect of jolkinolide B on K562 cells is achieved by arresting the cell cycle in the G1 phase and subsequently inducing apoptosis.     

氯化高铁血红素诱导K562细胞红系分化对其细胞周期的影响

细胞周期的测量是细胞增殖动力学的研究基础。通过添加30μmol·L-1氯化高铁血红素(Hemin)诱导人慢性髓系白血病K562细胞红系分化,利用5-溴脱氧尿嘧啶核苷(BrdU)与7-AAD双染的方法检测Hemin诱导的K562红系分化细胞对细胞周期各期比例的影响,未诱导的K562细胞周期各期比例作为对照,检测发现Hemin诱导的K562红系分化细胞对其细胞周期相对值无明显影响。应用BrdU间隔染色结合流式细胞术的方法,通过分析BrdU间隔染色后BrdU阳性细胞群的动态变化规律,从而推算出K562红系分化细胞的倍增时间及细胞周期各期时长。根据测量结果发现,未诱导的K562细胞总倍增时间约为20 h,与通过生长曲线公式法计算倍增时间的结果相符,Hemin诱导的K562细胞的细胞周期倍增时长约为23 h。Hemin诱导的K562红系分化细胞较未诱导的K562细胞倍增时间与各期时长无明显差异。因此,Hemin诱导K562细胞红系分化对其细胞周期绝对值及相对值均无明显影响。     

Comparative study of DNA damage, cell cycle and apoptosis in human K562 and CCRF-CEM leukemia cells: role of BCR/ABL in therapeutic resistance

The Philadelphia translocation t(9;22) resulting in the bcr/abl fusion gene is the pathogenic principle of almost 95% of human chronic myelogenous leukemia (CML). Imatinib mesylate (STI571) is a specific inhibitor of the BCR/ABL fusion tyrosine kinase that exhibits potent antileukemic effects in CML. BCR/ABL-positive K562 and -negative CCRF-CEM human leukemia cells were investigated. MTT survival assay and clonogenic test of the cell proliferation ability were used to estimate resistance against idarubicin. DNA damage after cell treatment with the drug at the concentrations from 0.001 to 3 microM with or without STI571 pre-treatment were examined by the alkaline comet assay. We found that the level of DNA damages was lower in K562 cells after STI571 pre-treatment. It is suggested that BCR/ABL activity may promote genomic instability, moreover K562 cells were found to be resistant to the drug treatment. Further, we provided evidence of apoptosis inhibition in BCR/ABL-positive cells using caspase-3 activity colorimetric assay and DAPI nuclear staining for chromatin condensation. We suggest that these processes associated with cell cycle arrest in G2/M checkpoint detected in K562 BCR/ABL-positive compared to CCRF-CEM cells without BCR/ABL expression might promote clone selection resistance to drug treatment.     

A 50-Hz magnetic field induces structural and biophysical changes in membranes

The effects of a 50-Hz extremely low frequency magnetic field on cultured K562 cells growing in suspension were studied by means of scanning electron microscopy and electron paramagnetic resonance spectroscopy. Exposure of K562 cells at 2.5 mT for periods to 96 hours induced significant changes in cell-surface structure and physiology without modification of proliferative capability as indicated by quantitative analysis. Thus extremely low frequency fields seem able to induce injurious, sublethal cell alterations, and the plasma membrane seems to play an important role in this effect. © 1993 Wiley-Liss, Inc.     

低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制研究

目的初步探讨低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制。方法甲基纤维素集落形成实验检测低浓度丰加霉素对人白血病K562细胞集落形成能力的影响;CCK-8法检测低浓度丰加霉素对K562细胞的生长抑制率;AnnexinV／PI双染流式细胞仪检测低浓度丰加霉素作用下的K562细胞凋亡率;PI单染流式细胞仪检测药物作用后细胞的周期分布改变;Western免疫印迹和实时定量PCR检测周期相关分子表达水平变化。结果低浓度丰加霉素对人白血病K562细胞具有较强的集落形成抑制作用;可明显抑制K562细胞的生长,呈时间一剂量依赖性;尽管短时间（48h）的药物处理仅出现轻度的细胞凋亡和周期阻滞,但10nmol/L和30nmol/L的丰加霉素长时间（7d）作用后,K562细胞G0／G1期比例分别是（62．3±1．7）％和（76．9±0．7）％,与对照组（38．9±1．1）％相比差异具有高度统计学意义（P〈0．01）;低浓度丰加霉素长时间作用后诱导K562细胞周期相关分子P16蛋白水平和转录水平的高表达。结论丰加霉素在低浓度,长时间作用于人白血病K562细胞后,具有较强的集落形成抑制和生长抑制作用,此作用可能与诱导细胞周期相关分子p16高表达,导致细胞G0／G1期阻滞有关。     

The effect of 100 mT SMF on activation of the hsp70 promoter in a heat shock/luciferase reporter system

Human exposure to magnetic fields, increased through use of new technologies like magnetic resonance imaging (MRI), has prompted investigations into possible effects of static magnetic fields (SMFs) on cellular processes. However, controversy still remains between many studies, which likely results from a lack of uniformity across experimental parameters, including the length of magnetic field exposure, the strength of the magnetic field, and the cell type or organism under investigation. The purpose of this research was to monitor effects of SMF exposure using real‐time luminescence photometry. The study investigated the potential interaction of a 100 mT SMF on a heat shock protein (hsp70)/luciferase reporter construct in stably transfected NIH3T3 cells. Changes in heat shock promoter activation following 100 mT SMF exposure were analyzed and detected as bioluminescence in real‐time. Two heat parameters were considered in combination with sham‐ and 100 mT‐exposed experiments: no heat or 1,800 s heat. As expected, there was a significant increase in bioluminescence in response to 1,800 s of heat alone. However, no significant difference in average hsp70 promoter activation between sham and 100 mT experiments was observed for no heat or 1,800 s heat experiments. Therefore, a 100 mT SMF was shown to have no effect on the activation of the heat shock protein promoter during SMF exposure or when SMF exposure was combined with a heat insult. J. Cell. Biochem. 108: 956–962, 2009. © 2009 Wiley‐Liss, Inc.     

The short-time treatment with curcumin sufficiently decreases cell viability, induces apoptosis and copper enhances these effects in multidrug-resistant K562/A02 cells

The anti-cancer activities of curcumin (CUR), a polyphenol derived from the plant Curcuma longa, has been extensively studied. In the present study, we found that CUR displayed anti-multidrug-resistant (MDR) activity in K562/A02 cells. A short-time treatment with CUR sufficiently and equally induced DNA damage, decreased cell viability, and triggered apoptosis in parent K562 and MDR K562/A02 cells. The short-time treatment with CUR also caused decrease of pro-caspase 3 in both cell lines and decrease of pro-caspase 9, increase of PARP cleavage and the ratio of Bax/Bcl-xL in MDR K562/A02 cells. Further experiment revealed that CUR was capable of down-regulating P-glycoprotein in MDR K562/A02 cells. Moreover, we observed that Cu(2+) enhanced CUR-mediated apoptosis which was blocked by antioxidants N-acetyl-cysteine and catalase. In summary, the short-time treatment with CUR sufficiently induced DNA damage, decreased cell viability and triggered apoptosis in MDR K562/A02 cells and Cu(2+) enhanced CUR-mediated apoptosis which due to reactive oxygen species generation.