Suppression of Proliferation of Human Glioblastoma Cells by Combined Phosphodiesterase and Multidrug Resistance-Associated Protein 1 Inhibition

The paucity of currently available therapies for glioblastoma multiforme requires novel approaches to the treatment of this brain tumour. Disrupting cyclic nucleotide-signalling through phosphodiesterase (PDE) inhibition may be a promising way of suppressing glioblastoma growth. Here, we examined the effects of 28 PDE inhibitors, covering all the major PDE classes, on the proliferation of the human U87MG, A172 and T98G glioblastoma cells. The PDE10A inhibitors PF-2545920, PQ10 and papaverine, the PDE3/4 inhibitor trequinsin and the putative PDE5 inhibitor MY-5445 potently decreased glioblastoma cell proliferation. The synergistic suppression of glioblastoma cell proliferation was achieved by combining PF-2545920 and MY-5445. Furthermore, a co-incubation with drugs that block the activity of the multidrug resistance-associated protein 1 (MRP1) augmented these effects. In particular, a combination comprising the MRP1 inhibitor reversan, PF-2545920 and MY-5445, all at low micromolar concentrations, afforded nearly complete inhibition of glioblastoma cell growth. Thus, the potent suppression of glioblastoma cell viability may be achieved by combining MRP1 inhibitors with PDE inhibitors at a lower toxicity than that of the standard chemotherapeutic agents, thereby providing a new combination therapy for this challenging malignancy.     

Probing the Membrane Vibration of Single Living Cells by Using Nanopipettes

The vibration of a cell membrane plays a key role in the regulation of cell shape and the behavior of cells. However, most existing approaches for the measurement of cell vibration require either exogenous modification or sophisticated techniques, and the main challenge lies in developing methods that can monitor membrane vibration of living cells directly. Herein, a noninvasive strategy based on ultrasmall quartz nanopipettes is introduced. With a tip size of less than 100 nm, nanopipettes can be spatially controlled for precision targeting of a specific location on the membrane of single living cells. Surprisingly, by employing a constant voltage, stable cyclic oscillations are observed from the continuous current versus time traces. The time-domain current can be decomposed into two basic waves: the high-frequency one indicates the local membrane vibration driven by the electro-osmotic flow from the nanopipette, whereas the low-frequency one indicates the natural frequency of the whole cell. This provides a simple but reliable method to test local and global membrane vibration of single living cells simultaneously with little damage, which provides a tool for the quantification of drugs, disease, or mutations of the cell structure.     

应用载体介导的RNAi技术抑制HBsAg在重组CHO细胞中的表达

目的应用载体介导的RNA干扰技术,特异地干扰重组CHO细胞中乙型肝炎病毒s基因的表达。方法设计两段靶向HBV s基因的特异性siRNA,合成两对64nt寡核苷酸,插入载体H1启动子下游,构建表达干扰性发夹状RNA(Short hairpin RNA,shRNA)的载体pHs-9和pHs-170,转染重组CHO细胞(该CHO细胞整合了HBV s基因,可稳定表达s抗原),用ELISA法检测HBsAg的表达。结果pHs-9和pHs-170转染重组CHO细胞后24~120h,在细胞培养上清中检测到HBsAg表达降低,抑制率均达到70%左右。结论pHs-9和pHs-170已成功地表达了发夹状siRNA,并抑制了重组CHO细胞中HBsAg的表达。     

Comparative Analysis of Single-Molecule Dynamics of TRPV1 and TRPV4 Channels in Living Cells

TRPV1 and TRPV4, members of the transient receptor potential vanilloid family, are multimodal ion channels activated by various stimuli, including temperature and chemicals. It has been demonstrated that TRPV channels function as tetramers; however, the dynamics of the diffusion, oligomerization, and endocytosis of these channels in living cells are unclear. Here we undertook single-molecule time-lapse imaging of TRPV1 and TRPV4 in HEK 293 cells. Differences were observed between TRPV1 and TRPV4 before and after agonist stimulation. In the resting state, TRPV4 was more likely to form higher-order oligomers within immobile membrane domains than TRPV1. TRPV1 became immobile after capsaicin stimulation, followed by its gradual endocytosis. In contrast, TRPV4 was rapidly internalized upon stimulation with GSK1016790A. The selective loss of immobile higher-order oligomers from the cell surface through endocytosis increased the proportion of the fast-diffusing state for both subtypes. With the increase in the fast state, the association rate constants of TRPV1 and TRPV4 increased, regenerating the higher-order oligomers. Our results provide a possible mechanism for the different rates of endocytosis of TRPV1 and TRPV4 based on the spatial organization of the higher-order structures of the two TRPV channels.     

Regulation of the Fructose Transporter Gene Slc2a5 Expression by Glucose in Cultured Microglial Cells

Microglia play a role in the regulation of metabolism and pathogenesis of obesity. Microglial activity is altered in response to changes in diet and the body’s metabolic state. Solute carrier family 2 member 5 (Slc2a5) that encodes glucose transporter 5 (GLUT5) is a fructose transporter primarily expressed in microglia within the central nervous system. However, little is known about the nutritional regulation of Slc2a5 expression in microglia and its role in the regulation of metabolism. The present study aimed to address the hypothesis that nutrients affect microglial activity by altering the expression of glucose transporter genes. Murine microglial cell line SIM-A9 cells and primary microglia from mouse brain were exposed to different concentrations of glucose and levels of microglial activation markers and glucose transporter genes were measured. High concentration of glucose increased levels of the immediate-early gene product c-Fos, a marker of cell activation, Slc2a5 mRNA, and pro-inflammatory cytokine genes in microglial cells in a time-dependent manner, while fructose failed to cause these changes. Glucose-induced changes in pro-inflammatory gene expression were partially attenuated in SIM-A9 cells treated with the GLUT5 inhibitor. These findings suggest that an increase in local glucose availability leads to the activation of microglia by controlling their carbohydrate sensing mechanism through both GLUT5-dependent and –independent mechanisms.     

Inhibition of Macrophage Oxidative Stress Prevents the Reduction of ABCA-1 Transporter Induced by Advanced Glycated Albumin

We investigated the role of aminoguanidine and benfotiamine on the inhibition of reactive oxygen species (ROS) generation in macrophages induced by advanced glycated albumin (AGE-albumin) and its relationship with cell cholesterol homeostasis, emphasizing the expression of the ATP binding cassette transporter A-1 (ABCA-1). AGE-albumin was made by incubating fatty acid-free albumin with 10 mM glycolaldehyde. ROS production and ABCA-1 protein level were determined by flow cytometry in J774 macrophages treated along time with control (C) or AGE-albumin alone or in the presence of aminoguanidine or benfotiamine. Mitochondrial function was evaluated by oxygraphy. Compared to C-albumin, AGE-albumin increased ROS production in macrophages, which was ascribed to the activities of NADPH oxidase and of the mitochondrial system. Mitochondrial respiratory chain activity was reduced in cells incubated with AGE-albumin. ROS generation along time was associated with the reduction in macrophage ABCA-1 protein level. Aminoguanidine prevented ROS elevation and restored the ABCA-1 content in macrophages; on the other hand, benfotiamine that promoted a lesser reduction in ROS generation was not able to restore ABCA-1 levels. Inhibition of oxidative stress induced by AGE-albumin prevents disturbances in reverse cholesterol transport by curbing the reduction of ABCA-1 elicited by advanced glycation in macrophages and therefore may contribute to the prevention of atherosclerosis in diabetes mellitus.     

Pyruvate Dehydrogenase Kinase Inhibition by Dichloroacetate in Melanoma Cells Unveils Metabolic Vulnerabilities

Melanoma is characterized by high glucose uptake, partially mediated through elevated pyruvate dehydrogenase kinase (PDK), making PDK a potential treatment target in melanoma. We aimed to reduce glucose uptake in melanoma cell lines through PDK inhibitors dichloroacetate (DCA) and AZD7545 and through PDK knockdown, to inhibit cell growth and potentially unveil metabolic co-vulnerabilities resulting from PDK inhibition. MeWo cells were most sensitive to DCA, while SK-MEL-2 was the least sensitive, with IC₅₀ values ranging from 13.3 to 27.0 mM. DCA strongly reduced PDH phosphorylation and increased the oxygen consumption rate:extracellular acidification rate (OCR:ECAR) ratio up to 6-fold. Knockdown of single PDK isoforms had similar effects on PDH phosphorylation and OCR:ECAR ratio as DCA but did not influence sensitivity to DCA. Growth inhibition by DCA was synergistic with the glutaminase inhibitor CB-839 (2- to 5-fold sensitization) and with diclofenac, known to inhibit monocarboxylate transporters (MCTs) (3- to 8-fold sensitization). CB-839 did not affect the OCR:ECAR response to DCA, whereas diclofenac strongly inhibited ECAR and further increased the OCR:ECAR ratio. We conclude that in melanoma cell lines, DCA reduces proliferation through reprogramming of cellular metabolism and synergizes with other metabolically targeted drugs.     

质粒介导的NDRG2 shRNA抑制其在人肿瘤细胞HHCC中的表达

目的　构建含有针对NDRG2的shRNA的质粒 ,抑制NDRG2在HHCC细胞中的表达。方法　用PCR方法从人基因组DNA中扩增出 336bp的U6 启动子 ,与 2 9bp的NDRG2靶序列的反向重复序列和pSNAV质粒相连。连接后的pSNAVU6 质粒转染入HHCC细胞 ,检测它们对NDRG2表达的影响。结果　pSNAVU6 重组质粒能抑制NDRG2的表达。结论　针对NDRG2的短的发夹RNA能抑制NDRG2在HHCC细胞中的表达     

Subcellular Localization of Thiol-Capped CdTe Quantum Dots in Living Cells

Internalization and dynamic subcellular distribution of thiol-capped CdTe quantum dots (QDs) in living cells were studied by means of laser scanning confocal microscopy. These unfunctionalized QDs were well internalized into human hepatocellular carcinoma and rat basophilic leukemia cells in vitro. Co-localizations of QDs with lysosomes and Golgi complexes were observed, indicating that in addition to the well-known endosome-lysosome endocytosis pathway, the Golgi complex is also a main destination of the endocytosed QDs. The movement of the endocytosed QDs toward the Golgi complex in the perinuclear region of the cell was demonstrated. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of NanoBRET-Binding Assays for FKBP-Ligand Profiling in Living Cells

FK506-binding proteins (FKBPs) are promising targets for a variety of disorders and infectious diseases. High FKBP occupancy is thought to be necessary for ligands to effectively compete with the endogenous intracellular functions of FKBPs. Here, we report the development of NanoBRET assays for the most prominent cytosolic FKBPs, FKBP12, 12.6, 51 and 52. These assays allowed rapid profiling of FKBP ligands for target engagement and selectivity in living cells. These assays confirmed the selectivity of SAFit-type ligands for FKBP51 over FKBP52 but revealed a substantial offset for the intracellular activity of these ligands compared to bicyclic ligands or natural products. Our results stress the importance to control for intracellular FKBP occupancy and provide the assays to guide further FKBP ligand optimization.     

Correlation of Dual Colour Single Particle Trajectories for Improved Detection and Analysis of Interactions in Living Cells

Interactions between objects inside living cells are often investigated by looking for colocalization between fluorescence microscopy images that are recorded in separate colours corresponding to the fluorescent label of each object. The fundamental limitation of this approach in the case of dynamic objects is that coincidental colocalization cannot be distinguished from true interaction. Instead, correlation between motion trajectories obtained by dual colour single particle tracking provides a much stronger indication of interaction. However, frequently occurring phenomena in living cells, such as immobile phases or transient interactions, can limit the correlation to small parts of the trajectories. The method presented here, developed for the detection of interaction, is based on the correlation inside a window that is scanned along the trajectories, covering different subsets of the positions. This scanning window method was validated by simulations and, as an experimental proof of concept, it was applied to the investigation of the intracellular trafficking of polymeric gene complexes by endosomes in living retinal pigment epithelium cells, which is of interest to ocular gene therapy.     

Preliminary Results for the Inhibition of Pancreatic Cancer Cells by Organotin Polymers

Pancreatic cancer kills nearly all patients within 3–6 months after detection. There is no effective chemotherapy for it. We have found a number of organotin polymers that are effective at inhibiting pancreatic cancer cell lines with Chemotherapeutic Index values greater than two with some to values of 50.     

Inhibition of Citrinin-Induced Apoptotic Biochemical Signaling in Human Hepatoma G2 Cells by Resveratrol

The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. CTN causes cell injury, including apoptosis, but its precise regulatory mechanisms of action are currently unclear. Resveratrol, a member of the phytoalexin family found in grapes and other dietary plants, possesses antioxidant and anti-tumor properties. In the present study, we examined the effects of resveratrol on apoptotic biochemical events in Hep G2 cells induced by CTN. Resveratrol inhibited CTN-induced ROS generation, activation of JNK, loss of mitochondrial membrane potential (MMP), as well as activation of caspase-9, caspase-3 and PAK2. Moreover, resveratrol and the ROS scavengers, NAC and α-tocopherol, abolished CTN-stimulated intracellular oxidative stress and apoptosis. Active JNK was required for CTN-induced mitochondria-dependent apoptotic biochemical changes, including loss of MMP, and activation of caspases and PAK2. Activation of PAK2 was essential for apoptosis triggered by CTN. These results collectively demonstrate that CTN stimulates ROS generation and JNK activation for mitochondria-dependent apoptotic signaling in Hep G2 cells, and these apoptotic biochemical events are blocked by pretreatment with resveratrol, which exerts antioxidant effects.     

Multiplex Single-Cell Analysis of Cancer Cells Enables Unbiased Uncovering Subsets Associated with Cancer Relapse: Heterogeneity of Multidrug Resistance in Precursor B-ALL

Earlier detection of biomarkers responsible for cancer relapse facilitates more rational cancer treatment regimens to be designed. Herein, we develop a mass cytometry-based strategy for unbiased mining of cell subsets that potentially contribute to cancer recurrence through panoramic examination of the immunophenotypic features and multidrug resistance characteristics. The incorporation of metal tags enables multiplexed information of single cells to be interrogated based on metal fingerprint. Using acute lymphoblastic leukemia (B-ALL) as a showcase, we show overexpressed multidrug resistance biomarkers, i. e., BCRP, Bcl-2, MRP1, and P-gp in B-ALL cells compared with healthy control, and a positive correlation among different multidrug resistance biomarkers. Different cell subsets with multidrug resistance are well-defined, featured with CD34⁺CD38⁺CD10⁻ and CD34⁺CD38^+/intCD10⁺. Importantly, we uncovered that CD34 expression level is positively correlated to multidrug resistance, indicative of a higher potential of immature cells to induce B-ALL relapse. In addition, the cell subsets positively expressing CD73 and CD304 (CD34⁺CD10⁺CD304⁺; CD34⁺CD38^+/intCD10⁺CD73⁺) also overexpress multidrug resistance biomarkers, suggesting that they may serve as additional new biomarkers for B-ALL stratification and prognosis. Our data provide the first evidence that highly expressed multidrug resistance biomarkers in certain cell subpopulations with specific immunophenotypes may potentially induce B-ALL recurrence. The incorporation of multidrug resistance features with cell phenotypes using mass cytometry proposed in this study provides a general strategy for risk assessment and the prediction of recurrence of different types of cancers.     

High Spatial Resolution Imaging of Endogenous Hydrogen Peroxide in Living Cells by Solid‐State Fluorescence

Herein, we describe selective imaging of hydrogen peroxide using a precipitating dye conjugated to a boronic acid‐based immolative linker. We achieved visualization of endogenous hydrogen peroxide in phagosomes by solid‐state two‐photon fluorescence imaging in living cells with exceptionally high spatial resolution.     

Polyamide Fluorescent Probes for Visualization of Repeated DNA Sequences in Living Cells

DNA imaging in living cells usually requires transgenic approaches that modify the genome. Synthetic pyrrole‐imidazole polyamides that bind specifically to the minor groove of double‐stranded DNA (dsDNA) represent an attractive approach for in‐cell imaging that does not necessitate changes to the genome. Nine hairpin polyamides that target mouse major satellite DNA were synthesized. Their interactions with synthetic target dsDNA fragments were studied by thermal denaturation, gel‐shift electrophoresis, circular dichroism, and fluorescence spectroscopy. The polyamides had different affinities for the target DNA, and fluorescent labeling of the polyamides affected their affinity for their targets. We validated the specificity of the probes in fixed cells and provide evidence that two of the probes detect target sequences in mouse living cell lines. This study demonstrates for the first time that synthetic compounds can be used for the visualization of the nuclear substructures formed by repeated DNA sequences in living cells.     

Encapsulation of Living Cells within Giant Phospholipid Liposomes Formed by the Inverse‐Emulsion Technique

Liposomes form spontaneously by the assimilation of phospholipids, the primary component of cell membranes. Due to their unique ability to form selectively permeable bilayers in situ, they are widely used as nanocarriers for drug and small‐molecule delivery. However, there is a lack of straightforward methodologies to encapsulate living microorganisms. Here we demonstrate the successful encapsulation of whole cells in phospholipid vesicles by using the inverse‐emulsion technique of generating unilamellar vesicles. This method of liposome preparation allows for a facile encapsulation of large biomaterials that previously was not easily attainable. Using Escherichia coli as a model organism, we found that liposomes can protect the bacterium against external protease degradation and from harsh biological environments. Liposomes prepared by the inverse‐emulsion method were also capable of encapsulating yeast and were found to be naturally susceptible to hydrolysis by enzymes such as phospholipases, thus highlighting their potential role as cell delivery carriers.     

Optimization of Caged Electrophiles for Improved Monitoring of Cysteine Reactivity in Living Cells

Cysteine residues play critical roles in protein function and are susceptible to numerous post‐translational modifications (PTMs) that serve to modulate the activity and localization of diverse proteins. Many of these PTMs are highly transient and labile, thus necessitating methods to study these modifications directly within the context of living cells. We previously reported a caged electrophilic probe, CBK1, that can be activated by UV for temporally controlled covalent modification of cysteine residues in living cells. To improve upon the number of cysteine residues identified in cellular cysteine‐profiling studies, the reactivity and uncaging efficiency of a panel of caged electrophiles were explored. We identified an optimized caged electrophilic probe, CIK4, that affords significantly improved coverage of cellular cysteine residues. The broader proteome coverage afforded by CIK4 renders it a useful tool for the biological investigation of cysteine‐reactivity changes and PTMs directly within living cells and highlights design elements that are critical to optimizing photoactivatable chemical probes for cellular labeling.