Reactive oxygen species are involved in lysophosphatidic acid-induced apoptosis in rat cerebellar granule cells

Lysophosphatidic acid (LPA) induced apoptosis in primary rat cerebellar granule cells, which was characterized morphologically by chromatin condensation and the formation of apoptotic bodies. With redox-sensitive fluorescence probes DCFH-DA and DHR123, the formation of endogenous reactive oxygen species (ROS) inside cells during the apoptosis process was monitored by laser confocal scanning microscopy (LCSM). Pretreatment with the antioxidant tetramethylpyrazine (TMP) could effectively inhibit the formation of endogenous ROS and protect neurons from apoptosis. The results suggest that ROS might be involved in LPA-induced apoptosis in neurons.     

Reactive oxygen species are involved in nitric oxide-induced apoptosis in rat cortical neurons

Nitric oxide donor SNAP induced apoptosis in primary rat cerebral cortical neurons, which was characterized morphologically by chromatin condensation and the formation of apoptotic bodies. With redox-sensitive fluorescence probes DCFH-DA and DHR123, the formation of endogenous reactive oxygen species (ROS) inside cells during the apoptosis process was monitored by laser confocal scanning microscopy (LCSM). SNAP treatment also caused the accumulation of extracellular hydrogen peroxide. Pretreatment with the nitric oxide scavenger hemoglobin could effectively inhibit the formation of endogenous ROS and protect neurons from apoptosis. The results suggested that ROS might be involved in NO-induced apoptosis in neuronal cells.     

Laminarin Induces Apoptosis of Human Colon Cancer LOVO Cells through a Mitochondrial Pathway

Many scientific studies have shown that laminarin has anti-tumor effects, but the anti-tumor mechanism was unclear. The purpose of this study was to investigate the effect of laminarin on the induction of apoptosis in human colon cancer LOVO cells and the molecular mechanism involved. LOVO cells were treated with different concentrations of laminarin at different times. Morphology observations were performed to determine the effects of laminarin on apoptosis of LOVO cells. Flow cytometry (FCM) was used to detect the level of intracellular reactive oxygen species (ROS) and pH. Laser scanning confocal microscope (LSCM) was used to analyze intracellular calcium ion concentration, mitochondrion permeability transition pore (MPTP) and mitochondrial membrane potential (MMP). Western blotd were performed to analyze the expressions of Cyt-C, Caspase-9 and -3. The results showed the apoptosis morphology, which showed cell protuberance, concentrated cytoplasm and apoptotic bodies, was obvious after 72 h treatment. Laminarin treatment for 24 h increased the intracellular level of ROS and Ca²⁺; decreased pH value; activated intracellular MPTP and decreased MMP in dose-dependent manners. It also induced the release of Cyt-C and the activation of Caspase-9 and -3. In conclusion, laminarin induces LOVO cell apoptosis through a mitochondrial pathway, suggesting that it could be a potent agent for cancer prevention and treatment.     

FITC标记重组灵芝免疫调节蛋白(rLz-8)在NB4细胞中的动态定位

实验发现, 重组灵芝免疫调节蛋白(rLz-8)可直接杀伤人急性早幼粒细胞白血病细胞株NB4. 利用异硫氰酸荧光素(FITC)标记rLz-8, 其相关的活性实验结果和晶体结构分析都表明, FITC没有影响rLz-8已知生物学功能. 通过激光共聚焦显微镜观察FITC-rLz-8在NB4细胞内的动态过程发现, FITC-rLz-8可识别细胞膜上的受体, 并可进入细胞质, 并最终富集在细胞核区域内. Annexin V-FITC双染检测结果显示, rLz-8对NB4细胞杀伤作用的可能机制是对NB4细胞凋亡的诱导作用, 在一定浓度范围内, 剂量与凋亡诱导率成正相关. 因此rLz-8能够诱导肿瘤细胞NB4发生凋亡的亚细胞学机制可定位在细胞核上.     

Role of oxidant scavengers in the prevention of Ca²+ homeostasis disorders

A number of disorders, such as Alzheimer disease and diabetes mellitus, have in common the alteration of the redox balance, resulting in an increase in reactive oxygen species (ROS) generation that might lead to the development of apoptosis and cell death. It has long been known that ROS can significantly alter Ca2+ mobilization, an intracellular signal that is involved in the regulation of a wide variety of cellular functions. Cells have a limited capability to counteract the effects of oxidative stress, but evidence has been provided supporting the beneficial effects of exogenous ROS scavengers. Here, we review the effects of oxidative stress on intracellular Ca2+ homeostasis and the role of antioxidants in the prevention and treatment of disorders associated to abnormal Ca2+ mobilization induced by ROS.     

Measurement of intracellular Ca2+ changes using novel caged cyclic nucleotides and confocal laser scanning microscopy.

The intention of this study is to explore the applicability of confocal microscopy in conjunction with the use of caged cyclic nucleotide derivatives. The methodological potential of UV laser confocal microscopy has been assessed. It is shown that illumination of a single cell or a small area of a single cell is possible, whereby the intracelluar Ca2+ signal is measured at illuminated and non-illuminated cells. Such measurements do not have a high time resolution because of the specific system parameters. However, with an N2 pulse laser (not part of the standard microscope set-up), Ca2+ signals with a time resolution of around 100 ms have been measured. This facilitates investigation of the kinetics of Ca2+ influx. Intracellular Ca2+ measurements at HEK293 and sperm cells have been made here. For sperm cells the advantages of confocal microscopy are best evidenced in conjunction with the use of caged cyclic nucleotides; a cyclic nucleotide-gated Ca2+ influx at the tail of these cells has thereby been demonstrated for the first time.     

ATP released from beta-amyloid-stimulated microglia induces reactive oxygen species production in an autocrine fashion

Present study demonstrated that fibrillar beta-amyloid peptide (fAbeta1-42) induced ATP release, which in turn activated NADPH oxidase via the P2X7 receptor (P2X7R). Reactive oxygen species (ROS) production in fAbeta1-42- treated microglia appeared to require Ca2+ influx from extracellular sources, because ROS generation was abolished to control levels in the absence of extracellular Ca2+. Considering previous observation of superoxide generation by Ca2+ influx through P2X7R in microglia, we hypothesized that ROS production in fAbeta-stimulated microglia might be mediated by ATP released from the microglia. We therefore examined whether fAbeta1-42-induced Ca2+ influx was mediated through P2X7R activation. In serial experiments, we found that microglial pretreatment with the P2X7R antagonists Pyridoxal-phosphate-6-azophenyl-2',4'- disulfonate (100 microM) or oxidized ATP (100 microM) inhibited fAbeta-induced Ca2+ influx and reduced ROS generation to basal levels. Furthermore, ATP efflux from fAbeta1-42- stimulated microglia was observed, and apyrase treatment decreased the generation of ROS. These findings provide conclusive evidence that fAbeta-stimulated ROS generation in microglial cells is regulated by ATP released from the microglia in an autocrine manner.     

荧光/MRI双模态靶向成像诊疗试剂的制备

在聚乙二醇二胺(NH_2-PEG-NH_2)修饰的石墨烯量子点(GODs)表面以酰胺键偶联二乙基三胺五乙酸(DTPA)分子,之后将Gd~(3+)离子与其进行配合,得到了GODs-Gd(DTPA)复合纳米粒子,然后再通过酰胺键在GODs-Gd(DTPA)的表面修饰叶酸(FA)靶分子,最后进一步将阿霉素(DOX)通过π-π堆垛吸附在造影剂的表面,制备了FA/GODs-Gd(DTPA)/DOX荧光/MRI双模态靶向肺癌细胞成像诊疗试剂,通过透射电子显微镜、紫外可见吸收光谱、荧光光谱和激光共聚焦扫描显微镜等手段表征了其形貌、发光性能和靶向成像性能。MRI、激光共聚焦扫描显微镜和MTT等结果表明,相对于正常的HLF细胞,所制备的FA/GODs-Gd(DTPA)/DOX纳米粒子能够靶向检测FA受体高表达的肺癌H460细胞,并具有明显的抗肿瘤活性。     

Lipoic Acid-Modified Oligoethyleneimine-Mediated miR-34a Delivery to Achieve the Anti-Tumor Efficacy

MiR-34a, an important tumor suppressor, has been demonstrated to possess great potential in tumor gene therapy. To achieve the upregulation of miR-34a expression level, an oligoethyleneimine (OEI) derivative was constructed and employed as the carrier through the modification with lipoic acid (LA), namely LA-OEI. In contrast to OEI, the derivative LA-OEI exhibited superior transfection efficiency measured by confocal laser scanning microscopy and flow cytometry, owing to rapid cargo release in the disulfide bond-based reduction sensitive pattern. The anti-proliferation and anti-migration effects were tested after the miR-34a transfection to evaluate the anti-tumor response, using human cervical carcinoma cell line HeLa as a model. The delivery of LA-OEI/miR-34a nanoparticles could achieve obvious anti-proliferative effect caused by the induction of cell apoptosis and cell cycle arrest at G1 phase. In addition, it could inhibit the migration of tumor cells via the downregulation of MMP-9 and Notch-1 level. Overall, the LA-OEI-mediated miR-34a delivery was potential to be used as an effective way in the tumor gene therapy.     

Interference study on the free calcium in the human peripheral lymphocyte by acrylamide

The role of acrylamide on the human peripheral lymphocytes was studied by laser scanning confocal microscopy technique and fluo-3. The calibration value of the apparent dissociation constant （Kd） of the fluo-3-Ca^2＋ complex was obtained as 4.83 × 10^-7 moi/L. Acrylamide （〈54 μg/mL） evoked a rise in free intracellular calcium concentration [Ca^2＋]i, in a dosedependent manner. Acrylamide induced the increase of [Ca^2＋]i was discussed in detail.     

Nonlinear dynamics of intracellular methylene blue during light activation of cell cultures

Methylene blue (MB+) is a well-known dye in medicine and has been discussed as an easily applicable drug for topical treatment in photodynamic therapy (PDT). Methylene blue can potentially be used as a redox indicator to detect the important redox reactions that are induced during PDT. The kinetics of this process was analyzed on a subcellular level with confocal laser scanning microscopy. BKEz-7 endothelial cells were incubated 4 h with 1 microM MB+. The fluorescence dynamics of MB+ during irradiation with 633 nm light was observed with subcellular resolution. Images were acquired at 0.5 s intervals (frame rate 1 image/0.5 s). Fluorescence was observed in the red channel of the laser scanning microscope. Synchronously, the phase-contrast image was visualized with the green channel. Morphological changes could therefore be correlated with the dynamics of MB+. In addition, the light-dose-dependent phototoxicity at 633 nm irradiation was determined by viable cell counting. After an induction period (phase I), fast fluorescent spikes could be observed in the whole cytoplasm, which decayed with a time constant of about 20 s (phase II), followed by a period of nearly constant fluorescence intensity (phase III) and exponential photobleaching (phase IV). Phase II exhibits highly nonlinear kinetics, which is hypothesized to correlate probably with a nonlinear quantal production of reactive oxygen species (ROS). Morphological cell changes were not observed during phase II. During phase III, a pycnotic cell nucleus developed. From the determination of viable cells we can conclude that a light dose applied within phase II was only sublethal in correlation with morphological observations. Overproduction of ROS leading finally to cell killing during phases III and IV is discussed.     

FSiNPs mediated improved double immunofluorescence staining for gastric cancer cells imaging

A fluorescent silica nanoparticles (FSiNPs) mediated double immunofluorescence staining technique has been proposed for MGC-803 gastric cancer cells imaging by confocal laser scanning microscopy. Anti-CEA antibody and anti-CK19 antibody which can be both bonded to MGC-803 gastric cancer cells were first conjugated to fluorescein isothiocyanate (FITC) doped fluorescent silica nanoparticles (FFSiNPs) and RuBPY doped fluorescent silica nanoparticles (RFSiNPs), respectively. The MGC-803 gastric cancer cells were incubated with the mixture of anti-CEA antibody-conjugated FFSiNPs and anti-CK19 antibody-conjugated RFSiNPs, and subsequently imaged using confocal laser scanning microscopy. With this method, the in vitro cultured MGC-803 gastric cancer cells lines were successfully doubled labeled and distinguished through antigen-antibody recognition, together with the green and red signal of FFSiNPs and RFSiNPs simultaneously obtained without crossreactivity by confocal laser scanning microscopy imaging. By comparison with the conventional double immunofluorescence staining using green-emitting and red-emitting dyes, the photostability of this proposed method for confocal laser scanning microscopy imaging has been greatly improved. Furthermore, the ex vivo imaging of primary MGC-803 gastric cancer cells samples came from the tumor tissues of mice bearing the MGC gastric cancer tumor xenografts by this method have also been explored. The results demonstrate that the method offers potential advantage of photostability for the confocal laser scanning microscopy imaging of MGC-803 gastric cancer cells, and is applicable to the imaging of primary MGC-803 gastric cancer cells from the tumor tissues.     

Gentle immobilization of nonionic polymersomes on solid substrates

Vesicles from Pluronic L121 (PEO5-PPO68-PEO5) triblock copolymers were first stabilized by a permanent interpenetrating polymer network and then gently immobilized onto a glass or mica surface. Fluorescence-labeled micrometer-sized vesicles were visualized with confocal laser scanning microscopy, and smaller sized capsules, around 100 nm, were probed by liquid atomic force microscopy. The immobilized vesicles were weakly attached to a negatively charged surface via negatively charged polyelectrolytes in combination with Mg2+ ions and can be reversibly detached from the surface by slightly elevated temperatures. To illustrate that the immobilized vesicles remain responsive to external stimuli, we show that it is possible to transform their shape from spherical to cylindrical by introducing a second Pluronic, namely, P123 (PEO20-PPO70-PEO20). The detailed transition process has been recorded in real time by confocal laser scanning microscopy. Electron microscopy studies confirmed that a similar morphology change also occurs in the bulk.     

Synthesis of a new ratiometric emission Ca2+ indicator for in vivo bioimaging

A novel fluorescent calcium indicator with a 490/582 nm ratiometric emission has been designed and synthesized.The indicator exhibits a highly selective ratiometric emission response to Ca²⁺ over other metal cations and a large Stokes shift of 202 nm.Moreover,its practical cell imaging capability for intracellular Ca²⁺ in the resting- and dynamic-state has been demonstrated in human umbilical vein endothelial cells using a confocal laser scanning microscope.     

Effects of low power laser irradiation on intracellular calcium and histamine release in RBL-2H3 mast cells

Although laser irradiation has been reported to promote skin wound healing, the mechanism is still unclear. As mast cells are found to accumulate at the site of skin wounds we hypothesized that mast cells might be involved in the biological effects of laser irradiation. In this work the mast cells, RBL-2H3, were used in vitro to investigate the effects of laser irradiation on cellular responses. After laser irradiation, the amount of intracellular calcium ([Ca2+]i) was increased, followed by histamine release, as measured by confocal fluorescence microscopy with Fluo-3/AM staining and a fluorescence spectrometer with o-phthalaldehyde staining, respectively. The histamine release was mediated by the increment of [Ca2+]i from the influx of the extracellular buffer solution through the cation channel protein, transient receptor potential vanilloid 4 (TRPV4). The TRPV4 inhibitor, Ruthenium Red (RR) can effectively block such histamine release, indicating that TRPV4 was the key factor responding to laser irradiation. These induced responses of mast cells may provide an explanation for the biological effects of laser irradiation on promoting wound healing, as histamine is known to have multi-functions on accelerating wound healing.     

Antitumor Effect of Sinoporphyrin Sodium‐Mediated Photodynamic Therapy on Human Esophageal Cancer Eca‐109 Cells

The aim of this study was to evaluate the photodynamic effect of Sinoporphyrin sodium (DVDMS). In this study, Eca‐109 cells were treated with DVDMS (5 μg mL^?1) and subjected to photodynamic therapy (PDT). The uptake and subcellular localization of DVDMS were monitored by flow cytometry and confocal microscopy. The phototoxicity of DVDMS was studied by MTT assay. The morphological changes were observed by scanning electron microscopy (SEM). DNA damage, reactive oxygen species (ROS) generation and mitochondria membrane potential (MMP) changes were analyzed by flow cytometry. Studies demonstrated maximal uptake of DVDMS occurred within 3 h, with a mitochondrial subcellular localization. MTT assays displayed that DVDMS could be effectively activated by light and the phototoxicity was much higher than photofrin under the same conditions. In addition, SEM observation indicated that cells were seriously damaged after PDT treatment. Furthermore, activation of DVDMS resulted in significant increases in ROS production. The generated ROS played an important role in the phototoxicity of DVDMS. DVDMS‐mediated PDT (DVDMS‐PDT) also induced DNA damage and MMP loss. It is demonstrated that DVDMS‐mediated PDT is an effective approach on cell proliferation inhibition of Eca‐109 cells.     

Long-term exposure to CdTe quantum dots causes functional impairments in live cells

Several studies suggested that the cytotoxic effects of quantum dots (QDs) may be mediated by cadmium ions (Cd2+) released from the QDs cores. The objective of this work was to assess the intracellular Cd2+ concentration in human breast cancer MCF-7 cells treated with cadmium telluride (CdTe) and core/shell cadmium selenide/zinc sulfide (CdSe/ZnS) nanoparticles capped with mercaptopropionic acid (MPA), cysteamine (Cys), or N-acetylcysteine (NAC) conjugated to cysteamine. The Cd2+ concentration determined by a Cd2+-specific cellular assay was below the assay detection limit (<5 nM) in cells treated with CdSe/ZnS QDs, while in cells incubated with CdTe QDs, it ranged from approximately 30 to 150 nM, depending on the capping molecule. A cell viability assay revealed that CdSe/ZnS QDs were nontoxic, whereas the CdTe QDs were cytotoxic. However, for the various CdTe QD samples, there was no dose-dependent correlation between cell viability and intracellular [Cd2+], implying that their cytotoxicity cannot be attributed solely to the toxic effect of free Cd2+. Confocal laser scanning microscopy of CdTe QDs-treated cells imaged with organelle-specific dyes revealed significant lysosomal damage attributable to the presence of Cd2+ and of reactive oxygen species (ROS), which can be formed via Cd2+-specific cellular pathways and/or via CdTe-triggered photoxidative processes involving singlet oxygen or electron transfer from excited QDs to oxygen. In summary, CdTe QDs induce cell death via mechanisms involving both Cd2+ and ROS accompanied by lysosomal enlargement and intracellular redistribution.     

Photodynamic effects of mTHPC on human colon adenocarcinoma cells: photocytotoxicity,subcellular localization and apoptosis

The photodynamic properties of meta-tetra(hydroxyphenyl)chlorin (mTHPC), a promising second-generation photosensitizer, were investigated using a human colon adenocarcinoma cell line (Colo 201 cells). The study on photocytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay showed that mTHPC was an effective photosensitizer on Colo 201 cells. The photocytotoxicity of mTHPC showed both drug and light dose-dependent characteristics. To reach LD50, namely, the dose at which 50% of the cells were killed, only 0.45+/-0.15 microg/mL of mTHPC and 3 J/cm2 of light dose were required. The presence of 10% fetal calf serum in culture medium significantly decreased the incorporation of mTHPC into cells and resulted in the reduction of photodynamic efficacy. Using confocal laser scanning microscopy, mTHPC was first shown to localize in lysosomes rather than in mitochondria. Furthermore, nuclear stainings demonstrated that photodynamic therapy with mTHPC induced apoptosis in Colo 201 cells.     

Effect of BCL-2 on Harringtonine-induced apoptosis and intracellular Ca2+ mobilization in human leukemia HL-60 cells

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb Cephalotaxus hainanensis Li, has been used in the clinical treatment of human glanulocytic leukemia and chromic myelocytic leukemia. In this study, we investigated the effect of Bcl-2 on HT-induced apoptosis and Ca²⁺ mobilization in human leukemia HL-60 cells. 1 g/ml HT induced the apoptosis of HL-60/Neo cells in a time-dependent manner; while 1 g/ml HT failed to induce the apoptosis of HL-60/ Bcl-2 cells. HT-, A23187- (a Ca²⁺ ionophore), Carbonyl cyanide m-chlorophenylhydrazone (CCCP,a specific releaser of Ca²⁺ from mitochondria) and thapsigargin- (an inhibitor of endoplasmic reticulum Ca^2+> -ATPase) induced changes in [Ca²⁺]_i were monitored by using Fluo 3-AM with confocal laser scanning microscopy. The results demonstrated that HL-60 cells with enforced expression of Bcl-2 (HL-60/Bcl-2 cells) had increased Ca²⁺ permeability and increased intracellular Ca²⁺ store in comparison with HL-60 cells with negative control vectors (HL-60/Neo cells), suggesting that Bcl-2 might prevent HT-induced apoptosis by increased Ca²⁺ permeability and increased intracellular Ca²⁺ buffering capacity.     

Chamaejasmine induces apoptosis in human lung adenocarcinoma A549 cells through a Ros-mediated mitochondrial pathway

In the present study, the anticancer activity of chamaejasmine towards A549 human lung adenocarcinoma cells was investigated. In order to explore the underlying mechanism of cell growth inhibition of chamaejasmine, cell cycle distribution, ROS generation, mitochondrial membrane potential (Δψ(m)) disruption, and expression of cytochrome c, Bax, Bcl-2, caspase-3, caspase-9 and PARP were measured in A549 cells. Chamaejasmine inhibited the growth of A549 cells in a time and dose-dependent manner. The IC?? value was 7.72 μM after 72 h treatment. Chamaejasmine arrested the cell cycle in the G2/M phase and induced apoptosis via a ROS-mediated mitochondria-dependent pathway. Western blot analysis showed that chamaejasmine inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade, and active-caspase-3 was involved in PARP cleavage. All of these signal transduction pathways are involved in initiating apoptosis. To the best of our knowledge, this is the first report demonstrating the cytotoxic activity of chamaejasmine towards A549 in vitro.