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1
Cruz-Cruz, R., A. Salgado, C. Sánchez-Soto, L. Vaca, and M. Hiriart. "Thapsigargin-sensitive cationic current leads to membrane depolarization, calcium entry, and insulin secretion in rat pancreatic β-cells." American Journal of Physiology-Endocrinology and Metabolism 289, no. 3 (September 2005): E439—E445. http://dx.doi.org/10.1152/ajpendo.00082.2005.
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Glucose-induced insulin secretion by pancreatic β-cells depends on membrane depolarization and [Ca2+]i increase. We correlated voltage- and current-clamp recordings, [Ca2+]i measurements, and insulin reverse hemolytic plaque assay to analyze the activity of a thapsigargin-sensitive cationic channel that can be important for membrane depolarization in single rat pancreatic β-cells. We demonstrate the presence of a thapsigargin-sensitive cationic current, which is mainly carried by Na+. Moreover, in basal glucose concentration (5.6 mM), thapsigargin depolarizes the plasma membrane, producing electrical activity and increasing [Ca2+]i. The latter is prevented by nifedipine, indicating that Ca2+ enters the cell through L-type Ca2+ channels, which are activated by membrane depolarization. Thapsigargin also increased insulin secretion by increasing the percentage of cells secreting insulin and amplifying hormone secretion by individual β-cells. Nifedipine blocked the increase completely in 5.6 mM glucose and partially in 15.6 mM glucose. We conclude that thapsigargin potentiates a cationic current that depolarizes the cell membrane. This, in turn, increases Ca2+ entry through L-type Ca2+ channels promoting insulin secretion.
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Lunde, Christopher S., Stephanie R. Hartouni, James W. Janc, Mathai Mammen, Patrick P. Humphrey, and Bret M. Benton. "Telavancin Disrupts the Functional Integrity of the Bacterial Membrane through Targeted Interaction with the Cell Wall Precursor Lipid II." Antimicrobial Agents and Chemotherapy 53, no. 8 (May 26, 2009): 3375–83. http://dx.doi.org/10.1128/aac.01710-08.
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ABSTRACT Telavancin is an investigational lipoglycopeptide antibiotic currently being developed for the treatment of serious infections caused by gram-positive bacteria. The bactericidal action of telavancin results from a mechanism that combines the inhibition of cell wall synthesis and the disruption of membrane barrier function. The purpose of the present study was to further elucidate the mechanism by which telavancin interacts with the bacterial membrane. A flow cytometry assay with the diethyloxacarbocyanine dye DiOC2(3) was used to probe the membrane potential of actively growing Staphylococcus aureus cultures. Telavancin caused pronounced membrane depolarization that was both time and concentration dependent. Membrane depolarization was demonstrated against a reference S. aureus strain as well as phenotypically diverse isolates expressing clinically important methicillin-resistant (MRSA), vancomycin-intermediate (VISA), and heterogeneous VISA (hVISA) phenotypes. The cell wall precursor lipid II was shown to play an essential role in telavancin-induced depolarization. This was demonstrated both in competition binding experiments with exogenous d-Ala-d-Ala-containing ligand and in experiments with cells expressing altered levels of lipid II. Finally, monitoring of the optical density of S. aureus cultures exposed to telavancin showed that cell lysis does not occur during the time course in which membrane depolarization and bactericidal activity are observed. Taken together, these data indicate that telavancin's membrane mechanism requires interaction with lipid II, a high-affinity target that mediates binding to the bacterial membrane. The targeted interaction with lipid II and the consequent disruption of both peptidoglycan synthesis and membrane barrier function provide a mechanistic basis for the improved antibacterial properties of telavancin relative to those of vancomycin.
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Bedut, Stephane, Christine Seminatore-Nole, Veronique Lamamy, Sarah Caignard, Jean A. Boutin, Olivier Nosjean, Jean-Philippe Stephan, and Francis Coge. "High-throughput drug profiling with voltage- and calcium-sensitive fluorescent probes in human iPSC-derived cardiomyocytes." American Journal of Physiology-Heart and Circulatory Physiology 311, no. 1 (July 1, 2016): H44—H53. http://dx.doi.org/10.1152/ajpheart.00793.2015.
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Cardiomyocytes derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) are increasingly used for in vitro assays and represent an interesting opportunity to increase the data throughput for drug development. In this work, we describe a 96-well recording of synchronous electrical activities from spontaneously beating hiPSC-derived cardiomyocyte monolayers. The signal was obtained with a fast-imaging plate reader using a submillisecond-responding membrane potential recording assay, FluoVolt, based on a newly derived voltage-sensitive fluorescent dye. In our conditions, the toxicity of the dye was moderate and compatible with episodic recordings for >3 h. We show that the waveforms recorded from a whole well or from a single cell-sized zone are equivalent and make available critical functional parameters that are usually accessible only with gold standard techniques like intracellular microelectrode recording. This approach allows accurate identification of the electrophysiological effects of reference drugs on the different phases of the cardiac action potential as follows: fast depolarization (lidocaine), early repolarization (nifedipine, Bay K8644, and veratridine), late repolarization (dofetilide), and diastolic slow depolarization (ivabradine). Furthermore, the data generated with the FluoVolt dye can be pertinently complemented with a calcium-sensitive dye for deeper characterization of the pharmacological responses. In a semiautomated plate reader, the two probes used simultaneously in 96-well plates provide an easy and powerful multiparametric assay to rapidly and precisely evaluate the cardiotropic profile of compounds for drug discovery or cardiac safety.
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Crnich, Rachael, Gregory C. Amberg, M. Dennis Leo, Albert L. Gonzales, Michael M. Tamkun, Jonathan H. Jaggar, and Scott Earley. "Vasoconstriction resulting from dynamic membrane trafficking of TRPM4 in vascular smooth muscle cells." American Journal of Physiology-Cell Physiology 299, no. 3 (September 2010): C682—C694. http://dx.doi.org/10.1152/ajpcell.00101.2010.
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The melastatin (M) transient receptor potential (TRP) channel TRPM4 mediates pressure and protein kinase C (PKC)-induced smooth muscle cell depolarization and vasoconstriction of cerebral arteries. We hypothesized that PKC causes vasoconstriction by stimulating translocation of TRPM4 to the plasma membrane. Live-cell confocal imaging and fluorescence recovery after photobleaching (FRAP) analysis was performed using a green fluorescent protein (GFP)-tagged TRPM4 (TRPM4-GFP) construct expressed in A7r5 cells. The surface channel was mobile, demonstrating a FRAP time constant of 168 ± 19 s. In addition, mobile intracellular trafficking vesicles were readily detected. Using a cell surface biotinylation assay, we showed that PKC activation with phorbol 12-myristate 13-acetate (PMA) increased (∼3-fold) cell surface levels of TRPM4-GFP protein in <10 min. Similarly, total internal reflection fluorescence microscopy demonstrated that stimulation of PKC activity increased (∼3-fold) the surface fluorescence of TRPM4-GFP in A7r5 cells and primary cerebral artery smooth muscle cells. PMA also caused an elevation of cell surface TRPM4 protein levels in intact arteries. PMA-induced translocation of TRPM4 to the plasma membrane was independent of PKCα and PKCβ activity but was inhibited by blockade of PKCδ with rottlerin. Pressure-myograph studies of intact, small interfering RNA (siRNA)-treated cerebral arteries demonstrate that PKC-induced constriction of cerebral arteries requires expression of both TRPM4 and PKCδ. In addition, pressure-induced arterial myocyte depolarization and vasoconstriction was attenuated in arteries treated with siRNA against PKCδ. We conclude that PKCδ activity causes smooth muscle depolarization and vasoconstriction by increasing the number of TRPM4 channels in the sarcolemma.
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Byrd, J. C., R. Lapalombella, A. Ramanunni, L. A. Andritsos, J. M. Flynn, P. Baum, P. Thompson, and N. Muthusamy. "Effect of CD37 small modular immuno-pharmaceutical (SMIP) on direct apoptosis in chronic lymphocytic leukemia cells via transcriptional up-regulation of the BH3 family member BIM." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): 3035. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.3035.
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3035 Background: CD37 is a tetraspan transmembrane family protein selectively expressed on normal and transformed B-cells. A novel CD37SMIP was previously demonstrated to mediate superior direct apoptosis and NK-cell mediated killing of chronic lymphocytic leukemia (CLL) and other B-cell malignancies. Methods: Given the superior in vitro apoptosis observed with CD37SMIP treatment and early clinical activity observed in highly refractory CLL patients, we hypothesized that a unique mechanism of cell killing was utilized by CD37SMIP. This was pursued in preclinical studies outlined below. Results: Unlike many other agents utilized to treat CLL, death mediated by CD37SMIP does not depend upon caspase activation. Nonetheless, CD37SMIP treatment of CLL cells promotes time-dependent induction of mitochondrial membrane depolarization, mitochondrial translocation of Bax, and up-regulation of Bim protein. CD37SMIP Bim protein induction occurred concomitantly with an increase in BIM mRNA levels. Electrophoretic mobility shift assay using oligonucleotides of the BIM promoter demonstrated increased protein binding activity in nuclear extracts derived from CD37SMIP treated cells and the physical interaction of FoxO3a transcription factor with the FoxO3a responsive element in the BIM promoter was demonstrated using a “protein pull down” assay and confirmed by chromatin immunoprecipitation assays. Furthermore, CD37SMIP treatment significantly increased BIM promoter regulated luciferase reporter expression in B-CLL cells. Consistent with a primary role of Bim up-regulation in mitochondrial membrane destabilization and apoptosis, transfection of CLL cells with BIM siRNA resulted in inhibition of CD37SMIP-induced mitochondrial membrane depolarization and apoptosis. Conclusions: These studies demonstrate CD37SMIP mediated apoptosis in CLL cells occurs via FoxO3a-dependent transcriptional up-regulation of BIM protein. This distinct mechanism of apoptosis utilized by CD37SMIP contrasts it with other agents used for CLL treatment. Additionally, it provides a mechanism for the promising clinical activity of TRU-016 (humanized CD37SMIP) observed to date in refractory CLL patients. [Table: see text]
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Wang, Lijun, Chengbiao Zhang, Xiaotong Su, and Daohong Lin. "Kcnj10 is a major type of K+ channel in mouse corneal epithelial cells and plays a role in initiating EGFR signaling." American Journal of Physiology-Cell Physiology 307, no. 8 (October 15, 2014): C710—C717. http://dx.doi.org/10.1152/ajpcell.00040.2014.
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We used primary mouse corneal epithelial cells (pMCE) to examine the role of Kcnj10 in determining membrane K+ conductance and cell membrane potential and in regulating EGF/TGFA release. Western blot, immunostaining, and RT-PCR detected the expression of Kcnj10 in mouse cornea. The single channel recording identified the 20-pS inwardly rectifying K+ channels in pMCE of WT mice, but these channels were absent in Kcnj10 −/−. Moreover, the whole cell recording demonstrates that deletion of Kcnj10 largely abolished the inward K+ currents and depolarized the cell membrane K+ reversal potential (an index of the cell membrane potential). This suggests that Kcnj10 is a main contributor to the cell K+ conductance and it is pivotal in generating membrane potential in cornea. Furthermore, to test the hypothesis that Kcnj10 expression plays a key role in the stimulation of growth factors release, we employed an immortalized human corneal epithelial cell line (HCE) transfected with siRNA-Kcnj10 or siRNA-control. Levels of TGFA and EGF secreted in the medium were measured by ELISA. Coimmunoprecipitation, biotinylation, and pull-down assay were used to examine the expression of EGFR and the GTP bound form of Rac1 (active Rac1). Downregulation of Kcnj10 activated Rac1 and enhanced EGF/TGFA release, which further contributed to the upregulation of EGFR phosphorylation and surface expression. We conclude that Kcnj10 is a main K+ channel expressed in corneal epithelial cells and the inhibition of Kcnj10 resulted in depolarization, which in turn induced an EGF-like effect.
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de Sousa Portilho, Adrhyann Jullyanne, Emerson Lucena da Silva, Emanuel Cintra Austregésilo Bezerra, Carinne Borges de Souza Moraes Rego Gomes, Vitor Ferreira, Maria Elisabete Amaral de Moraes, David Rodrigues da Rocha, Rommel Mário Rodriguez Burbano, Caroline Aquino Moreira-Nunes, and Raquel Carvalho Montenegro. "1,4-Naphthoquinone (CNN1) Induces Apoptosis through DNA Damage and Promotes Upregulation of H2AFX in Leukemia Multidrug Resistant Cell Line." International Journal of Molecular Sciences 23, no. 15 (July 23, 2022): 8105. http://dx.doi.org/10.3390/ijms23158105.
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The multidrug resistance (MDR) phenotype is one of the major obstacles in the treatment of chronic myeloid leukemia (CML) in advantage stages such as blast crisis. In this scenario, more patients develop resistance mechanisms during the course of the disease, making tyrosine kinase inhibitors (TKIs) target therapies ineffective. Therefore, the aim of the study was to examine the pharmacological role of CNN1, a para-naphthoquinone, in a leukemia multidrug resistant cell line. First, the in vitro cytotoxic activity of Imatinib Mesylate (IM) in K-562 and FEPS cell lines was evaluated. Subsequently, membrane integrity and mitochondrial membrane potential assays were performed to assess the cytotoxic effects of CNN1 in K-562 and FEPS cell lines, followed by cell cycle, alkaline comet assay and annexin V-Alexa Fluor® 488/propidium iodide assays (Annexin/PI) using flow cytometry. RT-qPCR was used to evaluate the H2AFX gene expression. The results demonstrate that CNN1 was able to induce apoptosis, cell membrane rupture and mitochondrial membrane depolarization in leukemia cell lines. In addition, CNN1 also induced genotoxic effects and caused DNA fragmentation, cell cycle arrest at the G2/M phase in leukemia cells. No genotoxicity was observed on peripheral blood mononuclear cells (PBMC). Additionally, CNN1 increased mRNA levels of H2AFX. Therefore, CNN1 presented anticancer properties against leukemia multidrug resistant cell line being a potential anticancer agent for the treatment of resistant CML.
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Baxter, Deborah F., Martin Kirk, Amy F. Garcia, Alejandra Raimondi, Mats H. Holmqvist, Kimberly K. Flint, Dejan Bojanic, Peter S. Distefano, Rory Curtis, and Yu Xie. "A Novel Membrane Potential-Sensitive Fluorescent Dye Improves Cell-Based Assays for Ion Channels." Journal of Biomolecular Screening 7, no. 1 (February 2002): 79–85. http://dx.doi.org/10.1177/108705710200700110.
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The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC4(3) has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. Here, we report the characterization of the FLIPR Membrane Potential Assay Kit (FMP) in cells expressing voltage- and ligand-gated ion channels. The steady-state and kinetics fluorescence properties of FMP were compared with those of DiBAC4(3), using both FLIPR and whole-cell patch-clamp recording. Our experiments with the voltage-gated K+ channel, hElk-1, revealed that FMP was 14-fold faster than DiBAC4(3) in response to depolarization. On addition of 60 mM KCl, the kinetics of fluorescence changes of FMP using FLIPR were identical to those observed in the electrophysiological studies using whole-cell current clamp. In addition, KCl concentration-dependent increases in FMP fluorescence correlated with the changes of membrane potential recorded in whole-cell patch clamp. In studies examining vanilloid receptor-1, a ligand-gated nonselective cation channel, FMP was superior to DiBAC4(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of KATP1 and hERG.2 Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.
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Martin, M. A., W. M. Nauseef, and R. A. Clark. "Depolarization blunts the oxidative burst of human neutrophils. Parallel effects of monoclonal antibodies, depolarizing buffers, and glycolytic inhibitors." Journal of Immunology 140, no. 11 (June 1, 1988): 3928–35. http://dx.doi.org/10.4049/jimmunol.140.11.3928.
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Abstract The anti-neutrophil mAb PMN 7C3 and IIC4 inhibited the respiratory burst of neutrophils as measured by the generation of superoxide anion or hydrogen peroxide in response to PMA, serum-treated zymosan, and FMLP. To examine the effect of these mAb on neutrophil transmembrane potential, a fluorescent probe was used in a continuous assay. Compared with control cells, antibody-treated neutrophils were partially depolarized at rest and had a blunted response when stimulated. The F(ab)2 fragment of PMN 7C3 had similar effects on both the respiratory burst and transmembrane potential, whereas the Fab fragment did not. The unrelated antineutrophil mAb 31D8 had no effect on either the respiratory burst or on transmembrane potential. Neutrophils suspended in high potassium buffers also exhibited partial depolarization of the resting cell membrane and a blunted depolarization response to stimuli and produced less superoxide anion and hydrogen peroxide in response to stimuli than did control cells in physiologic buffer. Exposure of neutrophils to 2-deoxy-D-glucose resulted in dose- and time-dependent depression of the respiratory burst. 2-Deoxy-D-glucose also caused depolarization of the resting membrane and impaired subsequent stimulus-induced depolarization. Similar effects were seen with addition of iodoacetamide or depletion of glucose. The parallel effects of anti-neutrophil mAb, depolarizing buffers, and glycolytic inhibitors on both neutrophil membrane depolarization and activation of the respiratory burst indicate a close association between these two events. The evidence suggests that the inhibitory effects of these antibodies are mediated through partial membrane depolarization which interferes with signal transduction on subsequent stimulation of the cells. The impairment in oxidative responses to phorbol esters as well as to receptor-dependent activating agents points to interruption at a distal step, e.g., subsequent to Ca2+ mobilization.
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Zhang, Yongli, Xiangsheng Wang, Wei Fang, Xiaoyan Cai, Fujiang Chu, Xiangwen Liao, and Jiazheng Lu. "Synthesis andIn VitroAntitumor Activity of Two Mixed-Ligand Oxovanadium(IV) Complexes of Schiff Base and Phenanthroline." Bioinorganic Chemistry and Applications 2013 (2013): 1–14. http://dx.doi.org/10.1155/2013/437134.
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Two oxovanadium(IV) complexes of [VO(msatsc)(phen)], (1) (msatsc = methoxylsalicylaldehyde thiosemicarbazone, phen = phenanthroline) and its novel derivative [VO (4-chlorosatsc)(phen)], (2) (4-chlorosatsc = 4-chlorosalicylaldehyde thiosemicarbazone), have been synthesized and characterized by elemental analysis, IR, ES-MS,1H NMR, and magnetic susceptibility measurements. Their antitumor effects on BEL-7402, HUH-7, and HepG2 cells were studied by MTT assay. The antitumor biological mechanism of these two complexes was studied in BEL-7402 cells by cell cycle analysis, Hoechst 33342 staining, Annexin V-FITC/PI assay, and detection of mitochondrial membrane potential (ΔΨm). The results showed that the growth of cancer cells was inhibited significantly, and complexes1and2mainly caused in BEL-7402 cells G0/G1 cell cycle arrest and induced apoptosis. Both1and2decreased significantly the ΔΨm, causing the depolarization of the mitochondrial membrane. Complex2showed greater antitumor efficiency than that of complex1.
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Hosseini Rad, A., G. Min Yi Tan, A. Poudel, and A. McLellan. "P06.02 Enhancing CAR T cell persistence and memory through modulating mitochondrial function." Journal for ImmunoTherapy of Cancer 8, Suppl 2 (October 2020): A42.1—A42. http://dx.doi.org/10.1136/jitc-2020-itoc7.81.
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BackgroundCAR T cell therapy for solid tumours has achieved limited success compared to its application to B cell malignancies. One reason for this failure is the low differentiation rate to memory subsets and low persistence of CAR T cells due to activation-induced cell death (AICD) in lymphoid tissue and the tumour microenvironment. In this study, we have expressed the MCL1 gene within CAR T cells to overcome losses by AICD in adoptively transferred T cells. The MCL1 gene expresses two isoforms; the long isoform localises to the outer membrane of mitochondria and inhibits the CD95 signalling death pathway, while the short isoform localises to the inner membrane of mitochondria to enhance mitochondrial oxidation, phosphorylation and fusion. In addition, we have also utilized a microRNA (miR) 429 to promote memory T cell formation through the suppression of genes such as T-cell-restricted intracellular antigen-1 (TIA-1), T cell activation inhibitor, mitochondrial (TCAIM) and mitochondrial fission factor (MFF).Materials and MethodsOverexpression of MCL1 was confirmed at both mRNA and protein level by real time RT-PCR (qPCR) and western blot. Similarly, overexpression of miR-429 was measured by qPCR and specific binding of miR-429 to the 3′ UTR of target genes was confirmed by luciferase reporter assay. Mitochondrial depolarization and cell viability were assessed by TMRE mitochondrial membrane potential assay (flow-cytometry) and resazurin assay. The effect of MCL1 or miR429 overexpression on HER2-CAR T cells was determined by flow cytometry. Soluble leucine-zipper CD95L (https://www.addgene.org/104349/) was expressed and purified from Expi293 cells.ResultsOverexpression of MCL1 in both Jurkat T cells and primary human T cells protected cells against mitochondria depolarization as well as the loss of cell viability in response to CD95L-triggering. Expression of miR429 downregulated TIA1, TCAIM and MFF. A HER2-CAR construct with either MCL1 or miR429 in a lentiviral system was successfully designed and transduced into primary T cells. Mitochondria in transduced T demonstrated enlarged and fusion morphology - a classic feature of memory T cells.ConclusionsOverexpressing MCL1 or miR429 significantly improves mitochondrial function in T cells. This approach will be used to increase persistence of adoptively transferred CAR T cells.Disclosure InformationA. Hosseini Rad: None. G. Min Yi Tan: None. A. Poudel: None. A. McLellan: None.
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Florian, Amy E., Christopher K. Lepensky, Ohyun Kwon, Mark K. Haynes, Larry A. Sklar, and Adam Zweifach. "Flow Cytometry Enables a High-Throughput Homogeneous Fluorescent Antibody-Binding Assay for Cytotoxic T Cell Lytic Granule Exocytosis." Journal of Biomolecular Screening 18, no. 4 (November 15, 2012): 420–29. http://dx.doi.org/10.1177/1087057112466697.
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We developed a homogeneous phenotypic fluorescence end-point assay for cytotoxic T lymphocyte lytic granule exocytosis. This flow cytometric assay measures binding of an antibody to a luminal epitope of a lysosomal membrane protein (LAMP-1) that is exposed by exocytosis to the extracellular solution. Washing to remove unbound antibody is not required. Confirming the assay’s ability to detect novel active compounds, we screened at a concentration of 50 µM a synthetic diversity library of 91 compounds in a 96-well plate format, identifying 17 compounds that blocked by 90% or more. The actions of six structurally related tetracyano-hexahydroisoindole compounds that inhibited by ~90% at a concentration of 10 µM were investigated further. Four reduced elevations in intracellular Ca2+; it is likely that depolarization of the cells’ membrane potential underlies the effect for at least two of the compounds. Another compound was found to be a potent inhibitor of the activation of the mitogen-activated protein (MAP) kinase ERK. Finally, we transferred the assay to a 384-well format and screened the Prestwick Compound Library using high-throughput flow cytometry. Our results indicate that our assay will likely be a useful means of screening libraries for novel compounds with important biological activities.
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Heid, Michelle, Salony Maniar, Simon Watkins, and Russell Salter. "NLRP3 inflammasome dependent loss of mitochondrial and cell membrane integrity in bone marrow derived macrophages (114.3)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 114.3. http://dx.doi.org/10.4049/jimmunol.188.supp.114.3.
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Abstract The NLRP3 inflammasome is a multiprotein complex that includes the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which binds with caspase-1 via CARD-CARD interactions and is involved with caspase-1 activation. Pore forming toxins, such as tetanolysin O (TLO), as well as the potassium ionophore nigericin, stimulate the NLRP3 inflammasome. We have observed that NLRP3 knockout macrophages are more resistant to the loss of membrane integrity compared to wild type macrophages exposed to nigericin or TLO, as assessed by a dye uptake assay. Furthermore, this NLRP3 dependent loss of membrane integrity corresponds to the loss of mitochondrial membrane integrity as demonstrated via live cell microscopy. The loss of mitochondrial membrane integrity, but not mitochondrial depolarization was found to be dependent on the presence of NLRP3. Furthermore, the loss of mitochondrial integrity and the loss of cell membrane integrity suggest a potential mechanistic pathway for the effects of NLRP3 inflammasome activation in BMDM. These results further our understanding of how NLRP3 functions in macrophages exposed to stressful stimuli such as bacterial toxins.
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Kantapan, Jiraporn, Siwaphon Paksee, Pornthip Chawapun, Padchanee Sangthong, and Nathupakorn Dechsupa. "Pentagalloyl Glucose- and Ethyl Gallate-Rich Extract from Maprang Seeds Induce Apoptosis in MCF-7 Breast Cancer Cells through Mitochondria-Mediated Pathway." Evidence-Based Complementary and Alternative Medicine 2020 (April 22, 2020): 1–19. http://dx.doi.org/10.1155/2020/5686029.
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Bouea macrophylla Griffith, locally known as maprang, has important economic value as a Thai fruit tree. The maprang seed extract (MPSE) has been shown to exhibit antibacterial and anticancer activities. However, the bioactive constituents in MPSE and the molecular mechanisms underlying these anticancer activities remain poorly understood. This study aims to identify the active compounds in MPSE and to investigate the mechanisms involved in MPSE-induced apoptosis in MCF-7 treated cancer cells. The cytotoxic effect was determined by MTT assay. The apoptosis induction of MPSE was evaluated in terms of ROS production, mitochondrial membrane potential depolarization, and apoptosis-related gene expression. The compounds identified by HPLC and LC/MS analysis were pentagalloyl glucose, ethyl gallate, and gallic acid. MPSE treatment decreased cell proliferation in MCF-7 cells, and MPSE was postulated to induce G2/M phase cell cycle arrest. MPSE was found to promote intracellular ROS production in MCF-7 treated cells and to also influence the depolarization of mitochondrial membrane potential. In addition, MPSE treatment can lead to increase in the Bax/Bcl-2 gene expression ratio, suggesting that MPSE-induced apoptosis is mitochondria-dependent pathway. Our results suggest that natural products obtained from maprang seeds have the potential to target the apoptosis pathway in breast cancer treatments.
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Malaisse, Willy J., Leonard C. Best, André Herchuelz, Marcia Hiriart, Hassan Jijakli, Marcel M. Kadiata, Elena Larrieta-Carasco, et al. "Insulinotropic action of β-l-glucose pentaacetate." American Journal of Physiology-Endocrinology and Metabolism 275, no. 6 (December 1, 1998): E993—E1006. http://dx.doi.org/10.1152/ajpendo.1998.275.6.e993.
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The metabolism of β-l-glucose pentaacetate and its interference with the catabolism ofl-[U-14C]glutamine, [U-14C]palmitate,d-[U-14C]glucose, andd-[5-3H]glucose were examined in rat pancreatic islets. Likewise, attention was paid to the effects of this ester on the biosynthesis of islet peptides, the release of insulin from incubated or perifused islets, the functional behavior of individual B cells examined in a reverse hemolytic plaque assay of insulin secretion, adenylate cyclase activity in a membrane-enriched islet subcellular fraction, cAMP production by intact islets, tritiated inositol phosphate production by islets preincubated with myo-[2-3H]inositol, islet cell intracellular pH, 86Rb and 45Ca efflux from prelabeled perifused islets, and electrical activity in single isolated B cells. The results of these experiments were interpreted to indicate that the insulinotropic action of β-l-glucose pentaacetate is not attributable to any nutritional value of the ester but, instead, appears to result from a direct effect of the ester itself on a yet unidentified receptor system, resulting in a decrease in K+ conductance, plasma membrane depolarization, and induction of electrical activity.
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Lockerbie, R. O., V. E. Miller, and K. H. Pfenninger. "Regulated plasmalemmal expansion in nerve growth cones." Journal of Cell Biology 112, no. 6 (March 15, 1991): 1215–27. http://dx.doi.org/10.1083/jcb.112.6.1215.
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To study the mechanisms underlying plasmalemmal expansion in the nerve growth cone, a cell-free assay was developed to quantify membrane addition, using ligand binding and sealed growth cone particles isolated by subcellular fractionation from fetal rat brain. Exposed versus total binding sites of 125I-wheat germ agglutinin were measured in the absence or presence of saponin, respectively, after incubation with various agents. Ca2(+)-ionophore A23187 in the presence of Ca2+ increases the number of binding sites (Bmax) but does not change their affinity (KD), indicating that new receptors appear on the plasma membrane. Similarly, membrane depolarization by high K+ or veratridine significantly induces, in a Ca2(+)-dependent manner, the externalization of lectin binding sites from an internal pool. Morphometric analysis of isolated growth cones indicates that A23187 and high K+ treatment cause a significant reduction in a specific cytoplasmic membrane compartment, thus confirming the lectin labeling results and identifying the plasmalemmal precursor. The isolated growth cones take up gamma-amino-butyric acid and serotonin, but show no evidence for Ca2(+)-dependent transmitter release so that transmitter exocytosis is dissociated from plasmalemmal expansion. The data demonstrate that plasmalemmal expansion in the growth cone is a regulated process and identify an internal pool of precursor membrane.
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Deb, Nilanjana, Anita Hansda, Soumyasree Dutta, Ashok Pattanaik, and Shila Elizabeth Besra. "LAWSONIA ALBA LEAVES INDUCE APOPTOSIS AND CELL CYCLE ARREST IN B16F10 MELANOMA CELLS." International Journal of Pharmacy and Pharmaceutical Sciences 10, no. 5 (May 1, 2018): 96. http://dx.doi.org/10.22159/ijpps.2018v10i5.24526.
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Objective: The present study was designed to investigate the anti-melanoma activity of the ethyl acetate fraction of Lawsonia alba lam leaves (ELA) against B16F10 cells.Methods: Cytotoxicity of ELA on B16F10 cells was determined by MTT assay and supported with the morphology of apoptotic and necrotic cells under phase-contrast microscope, fluorescence microscopy with AO/EtBr, confocal microscopy with PI, Agarose gel electrophoresis and Annexin V-FITC/PI staining, mitochondrial membrane potential and cell cycle arrest by FACS was also performed on B16F10 cells.Results: Cytotoxic effect of ELA on B16F10 melanoma cell was confirmed by MTT assay with an IC50 value of 14.10μg/ml. Morphological study showed arrays of both the nuclear changes including chromatin condensation and apoptotic body formation indicating that the treatment with ELA and 5-Fluorouracil (standard) causes apoptotic changes in the melanoma cells compared to the untreated control. Agarose gel electrophoresis study showed fragmented DNA in the form of ladder. The depolarization of mitochondrial membrane potential was confirmed. Flow cytometric analysis showed appreciable number of cells in early apoptotic stage. The cells were arrested mostly in G0/G1 phase of cell cycle.Conclusion: Ethyl acetate fraction of Lawsonia alba L. leaves possesses potent apoptotic activity against B16F10 cells.
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Alves, Celso, Joana Silva, Susete Pinteus, Eva Alonso, Rebeca Alvariño, Adriana Duarte, Diorge Marmitt, et al. "Cytotoxic Mechanism of Sphaerodactylomelol, an Uncommon Bromoditerpene Isolated from Sphaerococcus coronopifolius." Molecules 26, no. 5 (March 4, 2021): 1374. http://dx.doi.org/10.3390/molecules26051374.
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Marine natural products have exhibited uncommon chemical structures with relevant antitumor properties highlighting their potential to inspire the development of new anticancer agents. The goal of this work was to study the antitumor activities of the brominated diterpene sphaerodactylomelol, a rare example of the dactylomelane family. Cytotoxicity (10–100 µM; 24 h) was evaluated on tumor cells (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, SK-ML-28) and the effects estimated by MTT assay. Hydrogen peroxide (H2O2) levels and apoptosis biomarkers (membrane translocation of phosphatidylserine, depolarization of mitochondrial membrane potential, Caspase-9 activity, and DNA condensation and/or fragmentation) were studied in the breast adenocarcinoma cellular model (MCF-7) and its genotoxicity on mouse fibroblasts (L929). Sphaerodactylomelol displayed an IC50 range between 33.04 and 89.41 µM without selective activity for a specific tumor tissue. The cells’ viability decrease was accompanied by an increase on H2O2 production, a depolarization of mitochondrial membrane potential and an increase of Caspase-9 activity and DNA fragmentation. However, the DNA damage studies in L929 non-malignant cell line suggested that this compound is not genotoxic for normal fibroblasts. Overall, the results suggest that the cytotoxicity of sphaerodactylomelol seems to be mediated by an increase of H2O2 levels and downstream apoptosis.
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Nakao, Ryoma, Tsuyoshi Ikeda, Soichi Furukawa, and Yasushi Morinaga. "Curry Leaf Triggers Cell Death of P. gingivalis with Membrane Blebbing." Pathogens 10, no. 10 (October 6, 2021): 1286. http://dx.doi.org/10.3390/pathogens10101286.
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Periodontal disease has become a serious public health problem, as indicated by accumulating evidence that periodontal disease is not only a major cause of tooth loss but is also associated with various systemic diseases. The present study assessed the anti-bacterial activities of three herbal products (curry leaf, clove, and cinnamon) against Porphyomonas gingivalis, a keystone pathogen for periodontal diseases. The curry leaf extract (CLE) showed the strongest growth inhibitory activity among them, and the activity was maintained even after extensive heat treatment. Of note, while clove and cinnamon extracts at sub-minimum inhibitory concentrations (sub-MICs) significantly enhanced the biofilm formation of P. gingivalis, CLE at sub-MIC did not have any effect on the biofilm formation. The MIC of CLE against P. gingivalis was higher than those against a wide range of other oral bacterial species. P. gingivalis cells were completely killed within 30 min after treatment with CLE. Spatiotemporal analysis using high-speed atomic force microscopy revealed that CLE immediately triggered aberrant membrane vesicle formation on the bacterial surface. Bacterial membrane potential assay revealed that CLE induced depolarization of the bacterial membrane. Taken together, these findings suggest the mechanism behind early bactericidal activity of CLE and its therapeutic applicability in patients with periodontal diseases.
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Kwak, Ah-Won, Woo-Keun Kim, Seung-On Lee, Goo Yoon, Seung-Sik Cho, Ki-Taek Kim, Mee-Hyun Lee, et al. "Licochalcone B Induces ROS-Dependent Apoptosis in Oxaliplatin-Resistant Colorectal Cancer Cells via p38/JNK MAPK Signaling." Antioxidants 12, no. 3 (March 7, 2023): 656. http://dx.doi.org/10.3390/antiox12030656.
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Licochalcone B (LCB) exhibits anticancer activity in oral cancer, lung cancer, and hepatocellular carcinoma cells. However, little is known about its antitumor mechanisms in human oxaliplatin-sensitive and -resistant colorectal cancer (CRC) cells. The purpose of the present study was to investigate the antitumor potential of LCB against human colorectal cancer in vitro and analyze its molecular mechanism of action. The viability of CRC cell lines was evaluated using the MTT assay. Flow cytometric analyses were performed to investigate the effects of LCB on apoptosis, cell cycle distribution, reactive oxygen species (ROS), mitochondrial membrane potential (MMP) dysfunction, and multi-caspase activity in CRC cells. The results demonstrated that LCB induced a reduction in cell viability, apoptosis, G2/M cell cycle arrest, ROS generation, MMP depolarization, activation of multi-caspase, and JNK/p38 MAPK. However, p38 (SB203580) and JNK (SP600125) inhibitors prevented the LCB-induced reduction in cell viability. The ROS scavenger N-acetylcysteine (NAC) inhibited LCB-induced reduction in cell viability, apoptosis, cell cycle arrest, ROS generation, MMP depolarization, and multi-caspase and JNK/p38 MAPK activities. Taken together, LCB has a potential therapeutic effect against CRC cells through the ROS-mediated JNK/p38 MAPK signaling pathway. Therefore, we expect LCB to have promising potential as an anticancer therapeutic and prophylactic agent.
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Gerencser, Akos A. "Bioenergetic Analysis of Single Pancreatic β-Cells Indicates an Impaired Metabolic Signature in Type 2 Diabetic Subjects." Endocrinology 156, no. 10 (July 23, 2015): 3496–503. http://dx.doi.org/10.1210/en.2015-1552.
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Impaired activation of mitochondrial energy metabolism by glucose has been demonstrated in type 2 diabetic β-cells. The cause of this dysfunction is unknown. The aim of this study was to identify segments of energy metabolism with normal or with altered function in human type 2 diabetes mellitus. The mitochondrial membrane potential (ΔψM), and its response to glucose, is the main driver of mitochondrial ATP synthesis and is hence a central mediator of glucose-induced insulin secretion, but its quantitative determination in β-cells from human donors has not been attempted, due to limitations in assay technology. Here, novel fluorescence microscopic assays are exploited to quantify ΔψM and its response to glucose and other secretagogues in β-cells of dispersed pancreatic islet cells from 4 normal and 3 type 2 diabetic organ donors. Mitochondrial volume densities and the magnitude of ΔψM in low glucose were not consistently altered in diabetic β-cells. However, ΔψM was consistently less responsive to elevation of glucose concentration, whereas the decreased response was not observed with metabolizable secretagogue mixtures that feed directly into the tricarboxylic acid cycle. Single-cell analysis of the heterogeneous responses to metabolizable secretagogues indicated no dysfunction in relaying ΔψM hyperpolarization to plasma membrane potential depolarization in diabetic β-cells. ΔψM of diabetic β-cells was distinctly responsive to acute inhibition of ATP synthesis during glucose stimulation. It is concluded that the mechanistic deficit in glucose-induced insulin secretion and mitochondrial hyperpolarization of diabetic human β-cells is located upstream of the tricarboxylic acid cycle and manifests in dampening the control of ΔψM by glucose metabolism.
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Durusu, Ipek, Hazal Hepsen Husnugil, Heval Atas, Aysenur Biber, Selin Gerekci, Aliye Ezgi Gulec, and Can Ozen. "Anti-Cancer Effects of Clofazimine As a Single Agent and in Combination with Cisplatin in Multiple Myeloma." Blood 128, no. 22 (December 2, 2016): 5897. http://dx.doi.org/10.1182/blood.v128.22.5897.5897.
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Abstract Multiple myeloma (MM) is a malignant neoplasm of bone marrow plasma B cells with high morbidity. Clofazimine (CLF) is an FDA-approved leprostatic, anti-tuberculosis, and anti-inflammatory drug that was previously shown to have growth suppression effects on various cancer types such as hepatocellular, lung, cervix, esophageal, colon, and breast cancers as well as melanoma, neuroblastoma, and leukemia cells. The objective of this study was to evaluate the anticancer effect of CLF on U266 resistant MM cell line. The relative cell viability of a panel of hematological cell lines (Jurkat, U266, Namalwa, K562, HL60) treated with 10 µM CLF after 24 h of treatment significantly reduced the viability in all cell lines, with percentages ranging between 28% (U266) and 38% (Jurkat) (p<0.001). IC50 value of CLF was found as 9.8 ± 0.7 µM on the U266 cell line. Previous studies showed that this level of CLF does not inhibit growth of healthy cells, which supports safety of CLF. CLF had both dose (2, 5, 10 µM) and time (12, 24, and 48 h) dependent growth inhibitory effect. Combination chemotherapy is an approach to increase the effectiveness of chemotherapeutics as well as overcome drug resistance and suppresses side effects of drugs. Therefore, we evaluate the combination effect of CLF in U266 cells and showed that combination with cisplatin led to a synergistic interaction between two compounds in all tested dose regimes, resulting in a 2.5-7.1 fold marked increase in cell death. Importantly this synergism was observed in U266 cells, which have mutant p53 at A161T showing resistance to cytotoxic agents such as platinum analogs (cisplatin etc.). <>Depolarization of the mitochondrial membrane is one of the first events in apoptosis. JC-1 is a lipophilic and cationic dye that reversibly changes color from green to red as the mitochondrial membrane potential increases (depolarization). JC-1 assay used in both flow cytometry analyses and fluorescence microscopy images have shown that relative to the control, CLF treatment results in the depolarization of mitochondrial membrane 15, 20.5, 14.3 fold respectively at 12, 24, and 48 h in U266 cell line (Figure 1). The caspase family of cysteine proteases plays an important role in apoptosis. Caspase-3 is a major protease activated during the early stages of programmed cell death. 10 µM CLF was applied for 12, 24, and 48 h and anti-active caspase-3 PE stained U266 cells were analyzed by flow cytometry. Caspase-3 activity is enhanced 5.6, 24.5 and 13.6-fold relative to untreated controls at 12h, 24h and 48 h respectively. Phosphatidylserine (PS) translocation to the outer leaflet of the cellular membrane is one of the key steps in early stages of apoptosis. To support our previous findings on apoptotic effect of CLF, we employed Annexin-V assay. CLF treatment caused a significant increase in the percentage of early and late apoptotic cells at 12 h (2.1 and 1.8 fold respectively), 24 h (4.1 and 12.3 fold) and 48 h (10.1 and 11.5 fold). Fluorescence microscopy images also supported flow cytometry data (Figure 2). Collectively, all three apoptosis assay results show that CLF significantly induces apoptosis in U266 cells. Our study is the first to show apoptotic and growth inhibitory effects of CLF on a p53-mutant resistant MM cell line U266. Our results also proved that combined therapy employing CLF together with chemotherapeutics seems to be a possible future therapeutic approach for MM. Further in vivo and clinical studies are warranted to evaluate its therapeutic potential for resistant MM treatment. Figure 1 Effect of 10 µM CLF on mitochondrial membrane potential. Flow cytometry fluorescence intensity A) Dot plots B) Bar plots of cells stained with JC-1 (n=3). C) Fluorescence microscopy image of JC-1-stained untreated cells indicating healthy mitochondria (red), D) In CLF-treated cells, green color shows diffusion of JC-1 from damaged mitochondria. Figure 1. Effect of 10 µM CLF on mitochondrial membrane potential. Flow cytometry fluorescence intensity A) Dot plots B) Bar plots of cells stained with JC-1 (n=3). C) Fluorescence microscopy image of JC-1-stained untreated cells indicating healthy mitochondria (red), D) In CLF-treated cells, green color shows diffusion of JC-1 from damaged mitochondria. Figure 2 Flow cytometry analysis of Annexin V-PE/7-AAD stained U266 cells treated with 10 µM CLF. A) Representative dot plots of Annexin V-PE vs 7-AAD signals gated as live, early apoptotic and late apoptotic quadrants B) Cell population bar graphs of corresponding dot plot quadrants (n=3). C) Early apoptotic U266 cell (right) stained with Annexin V-PE (green) and a late apoptotic U266 cell (left) stained with both Annexin V-PE (green) and nuclear dye PI (red) D) Close-up micrograph (160X) of a late apoptotic U266 cell. Figure 2. Flow cytometry analysis of Annexin V-PE/7-AAD stained U266 cells treated with 10 µM CLF. A) Representative dot plots of Annexin V-PE vs 7-AAD signals gated as live, early apoptotic and late apoptotic quadrants B) Cell population bar graphs of corresponding dot plot quadrants (n=3). C) Early apoptotic U266 cell (right) stained with Annexin V-PE (green) and a late apoptotic U266 cell (left) stained with both Annexin V-PE (green) and nuclear dye PI (red) D) Close-up micrograph (160X) of a late apoptotic U266 cell. Disclosures No relevant conflicts of interest to declare.
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Maqoud, Fatima, Angela Curci, Rosa Scala, Alessandra Pannunzio, Federica Campanella, Mauro Coluccia, Giuseppe Passantino, Nicola Zizzo, and Domenico Tricarico. "Cell Cycle Regulation by Ca2+-Activated K+ (BK) Channels Modulators in SH-SY5Y Neuroblastoma Cells." International Journal of Molecular Sciences 19, no. 8 (August 18, 2018): 2442. http://dx.doi.org/10.3390/ijms19082442.
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The effects of Ca2+-activated K+ (BK) channel modulation by Paxilline (PAX) (10−7–10−4 M), Iberiotoxin (IbTX) (0.1–1 × 10−6 M) and Resveratrol (RESV) (1–2 × 10−4 M) on cell cycle and proliferation, AKT1pSer473 phosphorylation, cell diameter, and BK currents were investigated in SH-SY5Y cells using Operetta-high-content-Imaging-System, ELISA-assay, impedentiometric counting method and patch-clamp technique, respectively. IbTX (4 × 10−7 M), PAX (5 × 10−5 M) and RESV (10−4 M) caused a maximal decrease of the outward K+ current at +30 mV (Vm) of −38.3 ± 10%, −31.9 ± 9% and −43 ± 8%, respectively, which was not reversible following washout and cell depolarization. After 6h of incubation, the drugs concentration dependently reduced proliferation. A maximal reduction of cell proliferation, respectively of −60 ± 8% for RESV (2 × 10−4 M) (IC50 = 1.50 × 10−4 M), −65 ± 6% for IbTX (10−6 M) (IC50 = 5 × 10−7 M), −97 ± 6% for PAX (1 × 10−4 M) (IC50 = 1.06 × 10−5 M) and AKT1pser473 dephosphorylation was observed. PAX induced a G1/G2 accumulation and contraction of the S-phase, reducing the nuclear area and cell diameter. IbTX induced G1 contraction and G2 accumulation reducing diameter. RESV induced G2 accumulation and S contraction reducing diameter. These drugs share common actions leading to a block of the surface membrane BK channels with cell depolarization and calcium influx, AKT1pser473 dephosphorylation by calcium-dependent phosphatase, accumulation in the G2 phase, and a reduction of diameter and proliferation. In addition, the PAX action against nuclear membrane BK channels potentiates its antiproliferative effects with early apoptosis.
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Nascimento da Cruz, Anne Cecília, Dalci José Brondani, Temístocles I´talo de Santana, Lucas Oliveira da Silva, Elizabeth Fernanda da Oliveira Borba, Antônio Rodolfo de Faria, Julianna Ferreira Cavalcanti de Albuquerque, et al. "Biological Evaluation of Arylsemicarbazone Derivatives as Potential Anticancer Agents." Pharmaceuticals 12, no. 4 (November 17, 2019): 169. http://dx.doi.org/10.3390/ph12040169.
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Fourteen arylsemicarbazone derivatives were synthesized and evaluated in order to find agents with potential anticancer activity. Cytotoxic screening was performed against K562, HL-60, MOLT-4, HEp-2, NCI-H292, HT-29 and MCF-7 tumor cell lines. Compounds 3c and 4a were active against the tested cancer cell lines, being more cytotoxic for the HL-60 cell line with IC50 values of 13.08 μM and 11.38 μM, respectively. Regarding the protein kinase inhibition assay, 3c inhibited seven different kinases and 4a strongly inhibited the CK1δ/ε kinase. The studied kinases are involved in several cellular functions such as proliferation, migration, cell death and cell cycle progression. Additional analysis by flow cytometry revealed that 3c and 4a caused depolarization of the mitochondrial membrane, suggesting apoptosis mediated by the intrinsic pathway. Compound 3c induced arrest in G1 phase of the cell cycle on HL-60 cells, and in the annexin V assay approximately 50% of cells were in apoptosis at the highest concentration tested (26 μM). Compound 4a inhibited cell cycle by accumulation of abnormal postmitotic cells at G1 phase and induced DNA fragmentation at the highest concentration (22 μM).
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Baggaley, Erin M., Austin C. Elliott, and Jason I. E. Bruce. "Oxidant-induced inhibition of the plasma membrane Ca2+-ATPase in pancreatic acinar cells: role of the mitochondria." American Journal of Physiology-Cell Physiology 295, no. 5 (November 2008): C1247—C1260. http://dx.doi.org/10.1152/ajpcell.00083.2008.
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Impairment of the normal spatiotemporal pattern of intracellular Ca2+ ([Ca2+]i) signaling, and in particular, the transition to an irreversible “Ca2+ overload” response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca2+ overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H2O2) evokes a Ca2+ overload response and inhibition of plasma membrane Ca2+-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938–C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca2+]i clearance assay in which mitochondrial Ca2+ uptake was blocked with Ru-360, H2O2 (50 μM–1 mM) markedly inhibited the PMCA activity. This H2O2-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H2O2-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca2+ handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca2+ overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.
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Bernardes-Oliveira, Emanuelly, Dayanne Lopes Gomes, Gustavo Martelli Palomino, Kleber Juvenal Silva Farias, Wilmar Dias da Silva, Hugo Alexandre Oliveira Rocha, Ana Katherine Gonçalves, Matheus de Freitas Fernandes-Pedrosa, and Janaina Cristiana de Oliveira Crispim. "Bothrops jararacaandBothrops erythromelasSnake Venoms Promote Cell Cycle Arrest and Induce Apoptosis via the Mitochondrial Depolarization of Cervical Cancer Cells." Evidence-Based Complementary and Alternative Medicine 2016 (2016): 1–9. http://dx.doi.org/10.1155/2016/1574971.
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Bothrops jararaca(BJ) andBothrops erythromelas(BE) are viper snakes found in South-Southeast and Northeast regions of Brazil, respectively. Snake venoms are bioactive neurotoxic substances synthesized and stored by venom glands, with different physiological and pharmacological effects, recently suggesting a possible preference for targets in cancer cells; however, mechanisms of snakes have been little studied. Here, we investigated the mechanism responsible for snake crude venoms toxicity in cultured cervical cancer cells SiHa and HeLa. We show that BJ and BE snake crude venoms exert cytotoxic effects to these cells. The percentage of apoptotic cells and cell cycle analysis and cell proliferation were assessed by flow cytometry and MTT assay. Detection of mitochondrial membrane potential (Rhodamine-123), nuclei morphological change, and DNA fragmentation were examined by staining with DAPI. The results showed that both the BJ and BE venoms were capable of inhibiting tumor cell proliferation, promoting cytotoxicity and death by apoptosis of target SiHa and HeLa cells when treated with BJ and BE venoms. Furthermore, data revealed that both BJ venoms in SiHa cell promoted nuclear condensation, fragmentation, and formation of apoptotic bodies by DAPI assay, mitochondrial damage by Rhodamine-123, and cell cycle block in the G1-G0 phase. BJ and BE venoms present anticancer potential, suggesting that bothBothropsvenoms could be used as prototypes for the development of new therapies.
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Kadir, Mohamad F. A., Shatrah Othman, and Kavitha Nellore. "Dihydroorotate Dehydrogenase Inhibitors Promote Cell Cycle Arrest and Disrupt Mitochondria Bioenergetics in Ramos Cells." Current Pharmaceutical Biotechnology 21, no. 15 (December 23, 2020): 1654–65. http://dx.doi.org/10.2174/1389201021666200611113734.
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Background: The re-emerging of targeting Dihydroorotate Dehydrogenase (DHODH) in cancer treatment particularly Acute Myelogenous Leukemia (AML) has corroborated the substantial role of DHODH in cancer and received the attention of many pharmaceutical industries. Objective: The effects of Brequinar Sodium (BQR) and 4SC-101 on lymphoblastoid cell lines were investigated. Methods: DHODH expression and cell proliferation inhibition of lymphoblastoid and lymphoma cell lines were analyzed using Western blot analysis and XTT assay, respectively. JC-1 probe and ATP biochemiluminescence kit were used to evaluate the mitochondrial membrane potential and ATP generation in these cell lines. Furthermore, we explored the cell cycle progression using Muse™ Cell Cycle Kit. Results: Ramos, SUDHL-1 and RPMI-1788 cells are fast-growing cells with equal expression of DHODH enzyme and sensitivity to DHODH inhibitors that showed that the inhibition of DHODH was not cancer-specific. In ATP depletion assay, the non-cancerous RPMI-1788 cells showed only a minor ATP reduction compared to Ramos and SUDHL-1 (cancer) cells. In the mechanistic impact of DHODH inhibitors on non-cancerous vs cancerous cells, the mitochondrial membrane potential assay revealed that significant depolarization and cytochrome c release occurred with DHODH inhibitors treatment in Ramos but not in the RPMI-1788 cells, indicating a different mechanism of proliferation inhibition in normal cells. Conclusion: The findings of this study provide evidence that DHODH inhibitors perturb the proliferation of non-cancerous cells via a distinct mechanism compared to cancerous cells. These results may lead to strategies for overcoming the impact on non-cancerous cells during treatment with DHODH inhibitors, leading to a better therapeutic window in patients.
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Raafat, Dina, Kristine von Bargen, Albert Haas, and Hans-Georg Sahl. "Insights into the Mode of Action of Chitosan as an Antibacterial Compound." Applied and Environmental Microbiology 74, no. 12 (May 2, 2008): 3764–73. http://dx.doi.org/10.1128/aem.00453-08.
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ABSTRACT Chitosan is a polysaccharide biopolymer that combines a unique set of versatile physicochemical and biological characteristics which allow for a wide range of applications. Although its antimicrobial activity is well documented, its mode of action has hitherto remained only vaguely defined. In this work we investigated the antimicrobial mode of action of chitosan using a combination of approaches, including in vitro assays, killing kinetics, cellular leakage measurements, membrane potential estimations, and electron microscopy, in addition to transcriptional response analysis. Chitosan, whose antimicrobial activity was influenced by several factors, exhibited a dose-dependent growth-inhibitory effect. A simultaneous permeabilization of the cell membrane to small cellular components, coupled to a significant membrane depolarization, was detected. A concomitant interference with cell wall biosynthesis was not observed. Chitosan treatment of Staphylococcus simulans 22 cells did not give rise to cell wall lysis; the cell membrane also remained intact. Analysis of transcriptional response data revealed that chitosan treatment leads to multiple changes in the expression profiles of Staphylococcus aureus SG511 genes involved in the regulation of stress and autolysis, as well as genes associated with energy metabolism. Finally, a possible mechanism for chitosan's activity is postulated. Although we contend that there might not be a single classical target that would explain chitosan's antimicrobial action, we speculate that binding of chitosan to teichoic acids, coupled with a potential extraction of membrane lipids (predominantly lipoteichoic acid) results in a sequence of events, ultimately leading to bacterial death.
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El Gamal, Heba, Ali Hussein Eid, and Shankar Munusamy. "Renoprotective Effects of Aldose Reductase Inhibitor Epalrestat against High Glucose-Induced Cellular Injury." BioMed Research International 2017 (2017): 1–11. http://dx.doi.org/10.1155/2017/5903105.
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Diabetic nephropathy (DN) is the leading cause of end stage renal disease worldwide. Increased glucose flux into the aldose reductase (AR) pathway during diabetes was reported to exert deleterious effects on the kidney. The objective of this study was to investigate the renoprotective effects of AR inhibition in high glucose milieu in vitro. Rat renal tubular (NRK-52E) cells were exposed to high glucose (30 mM) or normal glucose (5 mM) media for 24 to 48 hours with or without the AR inhibitor epalrestat (1 μM) and assessed for changes in Akt and ERK1/2 signaling, AR expression (using western blotting), and alterations in mitochondrial membrane potential (using JC-1 staining), cell viability (using MTT assay), and cell cycle. Exposure of NRK-52E cells to high glucose media caused acute activation of Akt and ERK pathways and depolarization of mitochondrial membrane at 24 hours. Prolonged high glucose exposure (for 48 hours) induced AR expression andG1 cell cycle arrest and decreased cell viability (84% compared to control) in NRK-52E cells. Coincubation of cells with epalrestat prevented the signaling changes and renal cell injury induced by high glucose. Thus, AR inhibition represents a potential therapeutic strategy to prevent DN.
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Hunakova, Luba, Eva Horvathova, Karolina Majerova, Pavel Bobal, Jan Otevrel, and Julius Brtko. "Genotoxic Effects of Tributyltin and Triphenyltin Isothiocyanates, Cognate RXR Ligands: Comparison in Human Breast Carcinoma MCF 7 and MDA-MB-231 Cells." International Journal of Molecular Sciences 20, no. 5 (March 9, 2019): 1198. http://dx.doi.org/10.3390/ijms20051198.
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The cytotoxicity of two recently synthesized triorganotin isothiocyanate derivatives, nuclear retinoid X receptor ligands, was tested and compared in estrogen-receptor-positive MCF 7 and -negative MDA-MB-231 human breast carcinoma cell lines. A 48 h MTT assay indicated that tributyltin isothiocyanate (TBT-ITC) is more cytotoxic than triphenyltin isothiocyanate (TPT-ITC) in MCF 7 cells, and the same trend was observed in the MDA-MB-231 cell line. A comet assay revealed the presence of both crosslinks and increasing DNA damage levels after the 17 h treatment with both derivatives. Differences in cytotoxicity of TBT-ITC and TPT-ITC detected by FDA staining correspond to the MTT data, communicating more pronounced effects in MCF 7 than in the MDA-MB-231 cell line. Both derivatives were found to cause apoptosis, as shown by the mitochondrial membrane potential (MMP) depolarization and caspase-3/7 activation. The onset of caspase activation correlated with MMP dissipation and the total cytotoxicity more than with the amount of active caspases. In conclusion, our data suggest that the DNA damage induced by TBT-ITC and TPT-ITC treatment could underlie their cytotoxicity in the cell lines studied.
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Zhu, Wenhe, Huiyan Wang, Wei Zhang, Na Xu, Junjie Xu, Yan Li, Wensen Liu, and Shijie Lv. "Protective effects and plausible mechanisms of antler-velvet polypeptide against hydrogen peroxide induced injury in human umbilical vein endothelial cells." Canadian Journal of Physiology and Pharmacology 95, no. 5 (May 2017): 610–19. http://dx.doi.org/10.1139/cjpp-2016-0196.
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Antler velvet polypeptide (VAP) is a prominent bioactive component of antler velvet. Whereas uncharacterized crude extracts have typically been used in pharmacological studies, in this study, the velvet polypeptide was isolated and purified by acid water extraction, ethanol precipitation, ammonium sulfate fractionation and precipitation, and chromatography, progressively. Human umbilical vein endothelial cells (HUVECs) were induced with H2O2 followed purified polypeptide treatment. Cell viability was evaluated by MTT assay. The apoptosis of cells was detected by fluorescence microscopy and flow cytometry. A cell analyzer was used to measure the mitochondrial membrane potential. The intracellular reactive oxygen species (ROS) levels were determined by flow cytometry. Oxidative stress related biochemical parameters were detected, and the expression of apoptosis-related proteins was examined by Western blot analysis. The results indicated that a 7.0 kDa polypeptide (VAP II) was isolated from antler velvet. VAP II enhanced cell viability, decreased cell apoptosis, reversed depolarization of mitochondrial membrane potential, decreased ROS levels, inhibited oxidative stress, and regulated the downstream signaling apoptotic cascade expression caused by H2O2. The protective effects of VAP II on HUVECs suggests a potential strategy for the treatment of cardiovascular disease.
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Kauerová, Tereza, Tomáš Goněc, Josef Jampílek, Susanne Hafner, Ann-Kathrin Gaiser, Tatiana Syrovets, Radek Fedr, Karel Souček, and Peter Kollar. "Ring-Substituted 1-Hydroxynaphthalene-2-Carboxanilides Inhibit Proliferation and Trigger Mitochondria-Mediated Apoptosis." International Journal of Molecular Sciences 21, no. 10 (May 12, 2020): 3416. http://dx.doi.org/10.3390/ijms21103416.
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Ring-substituted 1-hydroxynaphthalene-2-carboxanilides were previously investigated for their antimycobacterial properties. In our study, we have shown their antiproliferative and cell death-inducing effects in cancer cell lines. Cell proliferation and viability were assessed by WST-1 assay and a dye exclusion test, respectively. Cell cycle distribution, phosphatidylserine externalization, levels of reactive oxygen or nitrogen species (RONS), mitochondrial membrane depolarization, and release of cytochrome c were estimated by flow cytometry. Levels of regulatory proteins were determined by Western blotting. Our data suggest that the ability to inhibit the proliferation of THP-1 or MCF-7 cells might be referred to meta- or para-substituted derivatives with electron-withdrawing groups -F, -Br, or -CF3 at anilide moiety. This effect was accompanied by accumulation of cells in G1 phase. Compound 10 also induced apoptosis in THP-1 cells in association with a loss of mitochondrial membrane potential and production of mitochondrial superoxide. Our study provides a new insight into the action of salicylanilide derivatives, hydroxynaphthalene carboxamides, in cancer cells. Thus, their structure merits further investigation as a model moiety of new small-molecule compounds with potential anticancer properties.
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Baker, Brett. "1289. Pravibismane is a Potent, Broad Spectrum Anti-Infective Small Molecule that Rapidly Disrupts Bacterial Bioenergetics and Halts Bacterial Growth." Open Forum Infectious Diseases 7, Supplement_1 (October 1, 2020): S659—S660. http://dx.doi.org/10.1093/ofid/ofaa439.1472.
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Abstract Background The rise in resistance to existing antimicrobials has prompted a need for the development of novel antibiotics. Microbion has identified a novel compound, pravibismane, with potent broad spectrum anti-infective and anti-biofilm activity. Methods Here we used a variety of assays, including Bacterial Cytological Profiling (BCP), to analyze pravibismane in E.coli to gain insight into its likely mechanism of action (MOA). The BCP profile of pravibismane suggested it rapidly shut down cell growth, potentially by turning off cellular gene or protein expression. This was confirmed using a plasmid based GFP induction assay in E.coli tolC that showed pravibismane strongly reduced expression of GFP. The kinetics, reversibility and MOA of pravibismane was further characterized by using time-lapse microscopy, wash out experiments and measurements of both membrane potential and relative intracellular ATP levels. Results We found that pravibismane acts rapidly (within 30 mins) to completely halt cell growth rather than causing immediate cell lysis such as that observed with non-specific cell damaging agents bleach or detergent. Inhibitor wash out experiments in which cells were exposed to pravibismane for 2 hours, washed to remove the compound, and then observed using time-lapse microscopy revealed that the effect of pravibismane is reversible and that cells recovered 8-12 hrs after removing the compound. Wash out experiments with an E.coli tolC strain carrying a plasmid with an IPTG inducible GFP demonstrated that transcription and translation ultimately resumed in most cells after washout. The bioenergetics of the membrane was measured using DiBAC 4(5), a membrane potential sensitive dye which can enter depolarized cells, which revealed that pravibismane caused depolarization of the membrane within 30 mins of exposure in a concentration dependent manner. Finally, a luciferase assay determined pravibismane reduced ATP levels (resulting in decreased luminescence) within 15 mins of exposure in a concentration dependent manner unlike antibiotic controls that had modest or no effect on luminescence. Conclusion Our results suggest that pravibismane acts rapidly to disrupt cellular bioenergetics, resulting in the immediate cessation of cell growth and protein expression. Disclosures Brett Baker, M.Sc., D.C., Microbion Corporation (Board Member, Employee)
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Delgado, Claudia, Gina Mendez-Callejas, and Crispin Celis. "Caryophyllene Oxide, the Active Compound Isolated from Leaves of Hymenaea courbaril L. (Fabaceae) with Antiproliferative and Apoptotic Effects on PC-3 Androgen-Independent Prostate Cancer Cell Line." Molecules 26, no. 20 (October 12, 2021): 6142. http://dx.doi.org/10.3390/molecules26206142.
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Cancer treatment frequently carries side effects, therefore, the search for new selective and effective molecules is indispensable. Hymenaea courbaril L. has been used in traditional medicine in South America to treat several diseases, including prostate cancer. Leaves’ extracts from different polarities were evaluated using the 3-(4,5-methyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) cell viability assay to determine the cytotoxicity in prostate p53-null cells, followed by bio-guided fractionations to obtain the most cytotoxic fraction considering the selectivity index. The most cytotoxic fraction was analyzed by GC/MS to identify the active compounds. The majority compound, caryophyllene oxide, induced early and late apoptosis, depolarized the mitochondrial membrane, leading to several morphological changes and shifts in apoptotic proteins, and caspases were evidenced. Depolarization of the mitochondrial membrane releases the pro-apoptotic protein Bax from Bcl-xL. The apoptosis process is caspase-7 activation-dependent. Caryophyllene oxide is a safe anti-proliferative agent against PC-3 cells, inducing apoptosis with low toxicity towards normal cells.
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Maciel, Leide Laura Figueiredo, Marina Barreto Silva, Rafaela Oliveira Moreira, Ana Paula Cardoso, Christiane Fernandes, Adolfo Horn, João Carlos de Aquino Almeida, and Milton Masahiko Kanashiro. "In Vitro and In Vivo Relevant Antineoplastic Activity of Platinum(II) Complexes toward Triple-Negative MDA-MB-231 Breast Cancer Cell Line." Pharmaceutics 14, no. 10 (September 22, 2022): 2013. http://dx.doi.org/10.3390/pharmaceutics14102013.
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Two platinum complexes [Pt(HL3)Cl]·H2O (3) and [Pt(HL4)Cl]·H2O (4) containing α- and β-naphthyl groups, respectively, were investigated in more detail in vitro and in vivo for antineoplastic activity. The cytotoxicity activity induced by these platinum(II) compounds against breast cancer (MDA-MB-231 and MCF-7), lung (A549), prostate (PC3), pancreas (BXPC-3), and normal peripheral blood mononuclear (PBMC) cells were evaluated by MTT assay. The cell viability MTT assay showed that complex (4) was more cytotoxic to all cancer cell lines tested and less cytotoxic against human PBMC. Therefore, complex (4) was selected to further investigate the mechanism of cytotoxic effects involved against MDA-MB-231 cell line (human triple-negative breast cancer). Sub-G1 analysis of the cell cycle showed that this complex induces cell death by apoptosis due to the cell loss of DNA content detected in flow cytometry. The cytotoxic effect induced by complex (4) was associated with the capability of the complex to induce mitochondrial membrane depolarization, as well as increase ROS levels and caspase activation, as a result of the activation of both extrinsic and intrinsic apoptosis pathways. Ultrastructural alterations were observed using scanning and transmission electron microscopy (SEM and TEM), such as membrane blebbing, filopodia reduction, empty mitochondrial matrix, and DNA fragmentation. Furthermore, complex (4) was tested in an MDA-MB-231 tumor nodule xenograft murine model and demonstrated a remarkable reduction in tumor size in BALB/c nude mice, when compared to the control animals.
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Alabsi, Aied M., Kai Li Lim, Ian C. Paterson, Rola Ali-Saeed, and Bushra A. Muharram. "Cell Cycle Arrest and Apoptosis Induction via Modulation of Mitochondrial Integrity by Bcl-2 Family Members and Caspase Dependence inDracaena cinnabari-Treated H400 Human Oral Squamous Cell Carcinoma." BioMed Research International 2016 (2016): 1–13. http://dx.doi.org/10.1155/2016/4904016.
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Dracaena cinnabariBalf.f. is a red resin endemic to Socotra Island, Yemen. Although there have been several reports on its therapeutic properties, information on its cytotoxicity and anticancer effects is very limited. This study utilized a bioassay-guided fractionation approach to determine the cytotoxic and apoptosis-inducing effects ofD. cinnabarion human oral squamous cell carcinoma (OSCC). The cytotoxic effects ofD. cinnabaricrude extract were observed in a panel of OSCC cell lines and were most pronounced in H400. Only fractions DCc and DCd were active on H400 cells; subfractions DCc15 and DCd16 exhibited the greatest cytotoxicity against H400 cells andD. cinnabariinhibited cells proliferation in a time-dependent manner. This was achieved primarily via apoptosis where externalization of phospholipid phosphatidylserine was observed using DAPI/Annexin V fluorescence double staining mechanism studied through mitochondrial membrane potential assay cytochromecenzyme-linked immunosorbent and caspases activities revealed depolarization of mitochondrial membrane potential (MMP) and significant activation of caspases 9 and 3/7, concomitant with S phase arrest. Apoptotic proteins array suggested that MMP was regulated by Bcl-2 proteins family as results demonstrated an upregulation of Bax, Bad, and Bid as well as downregulation of Bcl-2. Hence,D. cinnabarihas the potential to be developed as an anticancer agent.
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Shou, Di-Wen, and Yue-Lin Zheng. "Antitumor activity, cell cycle arrest and apoptosis induction in human colon cancer cell line by Primula macrophylla extracts." Bangladesh Journal of Pharmacology 12, no. 2 (April 4, 2017): 4. http://dx.doi.org/10.3329/bjp.v12i2.27606.
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<p class="Abstract">The primary objective of the current work was to investigate the antitumor potential of <em>Primula macrophylla</em> extracts in human colon cancer cell line (Colo-205) along with evaluating the effects on apoptosis induction, cell cycle arrest and mitochondrial membrane potential. Cell viability was assessed by tetrazolium-based MTT assay. Flow cytometry measurement was carried out to assess the effect of the extract on cell cycle phase distribution and mitochondrial transmembrane potential. Results showed that <em>P. macrophylla</em> methanol extract was effective and exhibited highest cell growth inhibition (IC<sub>50</sub> value, 26.17 μg/mL). Methanol extract significantly increased the side-scattering profile of Colo-205 cells in concentration-dependent pattern. Exposure of Colo-205 cells with different concentrations of the methanol extract (0-80 μg/mL) caused dose-dependent G0/G1 cell cycle arrest along with inducing apoptotic cascade by increasing the population of cells at G0/G1 phase. Furthermore, methanol extract treatment caused an increase in mitochondrial membrane depolarization in Colo-205 cells.</p><p class="Abstract"><strong>Video Clip of Methodlogy</strong> (3 min 17 sec): <a href="https://youtube.com/v/yy-rCjDN830">Click</a> <a href="https://youtube.com/watch?v=yy-rCjDN830">If failed</a></p>
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Chang, Jui-Chih, Chih-Feng Lien, Wen-Sen Lee, Huai-Ren Chang, Yu-Cheng Hsu, Yu-Po Luo, Jing-Ren Jeng, Jen-Che Hsieh, and Kun-Ta Yang. "Intermittent Hypoxia Prevents Myocardial Mitochondrial Ca2+ Overload and Cell Death during Ischemia/Reperfusion: The Role of Reactive Oxygen Species." Cells 8, no. 6 (June 9, 2019): 564. http://dx.doi.org/10.3390/cells8060564.
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It has been documented that reactive oxygen species (ROS) contribute to oxidative stress, leading to diseases such as ischemic heart disease. Recently, increasing evidence has indicated that short-term intermittent hypoxia (IH), similar to ischemia preconditioning, could yield cardioprotection. However, the underlying mechanism for the IH-induced cardioprotective effect remains unclear. The aim of this study was to determine whether IH exposure can enhance antioxidant capacity, which contributes to cardioprotection against oxidative stress and ischemia/reperfusion (I/R) injury in cardiomyocytes. Primary rat neonatal cardiomyocytes were cultured in IH condition with an oscillating O2 concentration between 20% and 5% every 30 min. An MTT assay was conducted to examine the cell viability. Annexin V-FITC and SYTOX green fluorescent intensity and caspase 3 activity were detected to analyze the cell death. Fluorescent images for DCFDA, Fura-2, Rhod-2, and TMRM were acquired to analyze the ROS, cytosol Ca2+, mitochondrial Ca2+, and mitochondrial membrane potential, respectively. RT-PCR, immunocytofluorescence staining, and antioxidant activity assay were conducted to detect the expression of antioxidant enzymes. Our results show that IH induced slight increases of O2−· and protected cardiomyocytes against H2O2- and I/R-induced cell death. Moreover, H2O2-induced Ca2+ imbalance and mitochondrial membrane depolarization were attenuated by IH, which also reduced the I/R-induced Ca2+ overload. Furthermore, treatment with IH increased the expression of Cu/Zn SOD and Mn SOD, the total antioxidant capacity, and the activity of catalase. Blockade of the IH-increased ROS production abolished the protective effects of IH on the Ca2+ homeostasis and antioxidant defense capacity. Taken together, our findings suggest that IH protected the cardiomyocytes against H2O2- and I/R-induced oxidative stress and cell death through maintaining Ca2+ homeostasis as well as the mitochondrial membrane potential, and upregulation of antioxidant enzymes.
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Otsubo, Yuri, Hiroki Shimada, Atsushi Yokoyama, and Akira Sugawara. "LBODP025 A Novel Drug Discovery For Primary Aldosteronism Targeting Aldosterone Synthase (cyp11b2) And Kcnj5 Mutant." Journal of the Endocrine Society 6, Supplement_1 (November 1, 2022): A234. http://dx.doi.org/10.1210/jendso/bvac150.480.
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Abstract Primary aldosteronism (PA) is a common cause of secondary hypertension. PA induces damage to cardiovascular and renal blood vessels. Therefore, PA is regarded as a problem because it increases the risk of stroke, cardiomyopathy, kidney disease, etc. Aldosterone-producing adenomas (APA) are one of the major causes of PA, and gene mutations are often identified in tumor cells. KCNJ5 mutation is well known as a gene mutation that causes the loss of ion selectivity in APA. KCNJ5 is an inwardly rectifying potassium channel. KCNJ5 induces sodium ion influx due to mutations in APA. The sodium ion influx induces depolarization, and which overexpresses CYP11B2. CYP11B2 is the rate-determining enzyme for aldosterone synthesis, overexpression of CYP11B2 induces aldosterone overproduction. Examples of therapeutic drugs for PA and APA include calcium channel blockers, aldosterone antagonist and β-blockers. However, therapeutic drugs for PA and APA targeting cell membrane depolarization have not been developed yet. We aim to find compounds that suppress CYP11B2 expression by membrane depolarization to develop novel therapeutic drugs for PA and APA. By developing a therapeutic drug that targets depolarization, we hope to establish an effective therapeutic method for APA caused by gene mutations such as KCNJ5 mutation. We performed high-throughput screening (HTS) using Tohoku University Compound Library (6080 compounds). First, we generated H295R-CYP11B2 N-Luc cells in which the luciferase gene is inserted downstream of the CYP11B2 locus. The cells H295R-CYP11B2 N-Luc were stimulated with KCl to cause depolarization, after which candidate compounds were added and HTS was performed by Luc assay. As a result, 101 compounds were selected. Furthermore, 25 of 101 compounds were selected by examining the cytotoxicity and dose response. Also, 11 of the 25 compounds suppressed CYP11B2 mRNA expression in KCl stimulated H295R cells. Secondary, we generated KCNJ5 mutant cells that expressed the KCNJ5 mutant (L168R) in a Doxycycline-inducible manner. 3 of 11 compounds that suppressed the expression of CYP11B2 mRNA were selected by examination using KCNJ5 mutant cells. After that, 2 of 3 compounds decreased the aldosterone production. These compounds have a different structure from existing therapeutic drugs such as calcium channel blockers. Since these can be novel therapeutic drugs for PA and APA caused by depolarization such as KCNJ5, we are examining the detailed mechanism of action of the compounds. Presentation: No date and time listed
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ISHII, Masaru, Satoru FUJITA, Mitsuhiko YAMADA, Yukio HOSAKA, and Yoshihisa KURACHI. "Phosphatidylinositol 3,4,5-trisphosphate and Ca2+/calmodulin competitively bind to the regulators of G-protein-signalling (RGS) domain of RGS4 and reciprocally regulate its action." Biochemical Journal 385, no. 1 (December 14, 2004): 65–73. http://dx.doi.org/10.1042/bj20040404.
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RGS (regulators of G-protein signalling) are a diverse group of proteins, which accelerate intrinsic GTP hydrolysis on heterotrimeric G-protein α subunits. They are involved in the control of a physiological behaviour known as ‘relaxation’ of G-protein-gated K+ channels in cardiac myocytes. The GTPase-accelerating activity of cardiac RGS proteins, such as RGS4, is inhibited by PtdIns(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) and this inhibition is cancelled by Ca2+/calmodulin (CaM) formed during membrane depolarization. G-protein-gated K+ channel activity decreases on depolarization owing to the facilitation of GTPase-activating protein activity by RGS proteins and vice versa on hyperpolarization. The molecular mechanism responsible for this reciprocal control of RGS action by PtdIns(3,4,5)P3 and Ca2+/CaM, however, has not been fully elucidated. Using lipid–protein co-sedimentation assay and surface plasmon resonance measurements, we show in the present study that the control of the GTPase-accelerating activity of the RGS4 protein is achieved through the competitive binding of PtdIns(3,4,5)P3 and Ca2+/CaM within its RGS domain. Competitive binding occurs exclusively within the RGS domain and involves a cluster of positively charged residues located on the surface opposite to the Gα interaction site. In the RGS proteins conserving these residues, the reciprocal regulation by PtdIns(3,4,5)P3 and Ca2+/CaM may be important for their physiological regulation of G-protein signalling.
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Haslberger, A., C. Romanin, and R. Koerber. "Membrane potential modulates release of tumor necrosis factor in lipopolysaccharide-stimulated mouse macrophages." Molecular Biology of the Cell 3, no. 4 (April 1992): 451–60. http://dx.doi.org/10.1091/mbc.3.4.451.
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Lipopolysaccharide (LPS)-mediated synthesis of macrophage gene products such as tumor necrosis factor (TNF) is controlled by different signaling pathways. We investigated intracellular free Ca2+ (Ca2+ic) and the membrane potential as early cellular responses to LPS and their role in the synthesis and release of TNF. In peritoneal macrophages and in the RAW 269 mouse macrophage cell line, LPS and its biologically active moiety lipid A stimulated TNF synthesis but exerted no significant effects on these early cellular responses using Fura-2/Indo-1 to measure Ca2+ic and bis-oxonol, as well as the patch-clamp technique to monitor membrane potential. In contrast, the platelet-activating factor transiently induced both an increase in Ca2+ic and cell membrane depolarization but no significant TNF release. Increased extracellular K+ concentrations or K(+)-channel blockers, such as quinine, tetraethylammonium, or barium chloride, inhibited the LPS-stimulated release of TNF alpha, as well as the accumulation of cell-associated TNF alpha as found by enzyme-linked immunosorbent assay analysis, but did not inhibit TNF alpha mRNA accumulation. Concentrations of quinine (greater than 125 microM) or of enhanced extracellular K+ (25-85 mM) required to inhibit TNF production both significantly depolarized macrophages. These results indicate a lack of ion transport activities as early cellular responses of macrophages to LPS but suggest an important regulatory role of the membrane potential on the posttranscriptional synthesis and release of TNF in macrophages.
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Jiang, Zhangyu, Yanqing Wang, Xiuli Xi, Weibin Cai, Changhui Liu, Ran Ye, Liu Yang, et al. "Regulation of the Keap1-Nrf2 Signaling Axis by Glycyrrhetinic Acid Promoted Oxidative Stress-Induced H9C2 Cell Apoptosis." Evidence-Based Complementary and Alternative Medicine 2022 (August 27, 2022): 1–12. http://dx.doi.org/10.1155/2022/2875558.
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Excessive reactive oxygen species (ROS) could interfere with the physiological capacities of H9C2 cells and cause cardiomyocyte apoptosis. Glycyrrhetinic acid (GA), one of the main medicinal component of Glycyrrhetinic Radix et Rhizoma, shows toxic and adverse side effects in the clinic setting. In particular, some studies have reported that GA exerts toxic effects on H9C2 cells. The purpose of this study is to assess the effect of GA-induced oxidative stress on cultured H9C2 cells and reveal the relevant signaling pathways. LDH assay was used to assess cell damage. Apoptosis was detected using Hoechst 33242 and a propidium iodide (PI) assay. An Annexin V-fluorescein isothiocyanate/PI double-staining assay was utilized to investigate GA-induced apoptosis in H9C2 cells. The expression level of specific genes/proteins was evaluated by RT-qPCR and Western blotting. Flow cytometry and DCFH-DA fluorescent testing were used to determine the ROS levels of H9C2 cells. The potential mechanism of GA-induced cardiomyocyte injury was also investigated. GA treatment increased ROS generation and mitochondrial membrane depolarization and triggered caspase-3/9 activation and apoptosis. GA treatment also caused the nuclear translocation of NF-E2-related factor 2 after its dissociation from Keap1. This change was accompanied by a dose-dependent decline in the expression of the downstream target gene heme oxygenase-1. The findings demonstrated that GA could regulate the Keap1-Nrf2 signaling axis and induce oxidative stress to promote the apoptosis of H9C2 cells.
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Krishnaveni, Marimuthu, Kathiresan Suresh, and Ramu Arunkumar. "Anti-proliferative and apoptotic effects of quinine in human Hep-2 laryngeal cancer and KB oral cancer cell." Bangladesh Journal of Pharmacology 11, no. 3 (June 8, 2016): 593. http://dx.doi.org/10.3329/bjp.v11i3.26961.
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<p>The present study evaluated the anti-proliferative and apoptotic effect of quinine on oral cancer cells Hep-2 and KB. Cell inhibition, apoptosis and anti-inflammatory effects were explored by nuclear DNA cleavage, condensation, change in membrane potential of mitochondria. Meanwhile, inflammatory and apoptosis-related mRNA and proteins expressions such as iNOS COX-2, IL-6, Bcl-2, mutant p53, Bax, caspase-3 and NF-κB were determined by RT-PCR and Western blotting assays. Results showed that, quinine treatment significantly inhibited the cell viability and colony formation, inhibited cell proliferation lead to increased generation of reactive oxygen species induction of MMP depolarization, morphological changes and DNA damage in dose- and time-dependent manner. Moreover, quinine significantly decreased the iNOS, COX-2, IL-6, Bcl-2 and mutant p53 simultaneously up-regulated Bax, caspase-3 expressions through the inhibition of NF-κB suggest that quinine may serve as a potential candidate in the prevention of cell proliferation and enhances apoptosis via inhibiting up-stream signalling.</p><p><strong>Video Clip</strong></p><p><a href="https://youtube.com/v/83HX2fFTXkg">Programmed cell death assay by dual staining:</a> 17 min 25 sec</p><p> </p>
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Martins de Lima, Thais, Maria Fernanda Cury-Boaventura, Gisele Giannocco, Maria Tereza Nunes, and Rui Curi. "Comparative toxicity of fatty acids on a macrophage cell line (J774)." Clinical Science 111, no. 5 (October 13, 2006): 307–17. http://dx.doi.org/10.1042/cs20060064.
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In the present study, the cytotoxicity of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on a macrophage cell line (J774) was investigated. The induction of toxicity was investigated by changes in cell size, granularity, membrane integrity, DNA fragmentation and phosphatidylserine externalization by using flow cytometry. Fluorescence microscopy was used to determine the type of cell death (Acridine Orange/ethidium bromide assay). The possible mechanisms involved were examined by measuring mitochondrial depolarization, lipid accumulation and PPARγ (peroxisome-proliferator-activated receptor γ) activation. The results demonstrate that fatty acids induce apoptosis and necrosis of J774 cells. At high concentrations, fatty acids cause macrophage death mainly by necrosis. The cytotoxicity of the fatty acids was not strictly related to the number of double bonds in the molecules: palmitic acid>docosahexaenoic acid>stearic acid=eicosapentaenoic acid=arachidonic acid>oleic acid>linoleic acid. The induction of cell death did not involve PPARγ activation. The mechanisms of fatty acids to induce cell death involved changes in mitochondrial transmembrane potential and intracellular neutral lipid accumulation. Fatty acids poorly incorporated into triacylglycerol had the highest toxicity.
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Kawasaki, Keisuke, Yoshiaki Suzuki, Hisao Yamamura, and Yuji Imaizumi. "Development of a Novel Cell-Based Assay System for High-Throughput Screening of Compounds Acting on Background Two-Pore Domain K+ Channels." SLAS DISCOVERY: Advancing the Science of Drug Discovery 24, no. 6 (February 25, 2019): 641–52. http://dx.doi.org/10.1177/2472555219829745.
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Two-pore domain K+ (K2P) channels are thought to be druggable targets. However, only a few agents specific for K2P channels have been identified, presumably due to the lack of an efficient screening system. To develop a new high-throughput screening (HTS) system targeting these channels, we have established a HEK293-based “test cell” expressing a mutated Na+ channel (Nav1.5) with markedly slowed inactivation, as well as a K+ channel (Kir2.1) that sets the membrane potential quite negative, close to K+ equilibrium potential. We found in this system that Kir2.1 block by 100 μM Ba2+ application consistently elicited a large depolarization like a long-lasting action potential. This maneuver resulted in cell death, presumably due to the sustained Na+ influx. When either the TWIK-related acid-sensitive K+ (TASK)-1 or TASK-3 channel was expressed in the test cells, Ba2+-induced cell death was markedly weakened. Stronger activation of TASK-1 by extracellular acidification further decreased the cell death. In contrast, the presence of K2P channel blockers enhanced cell death. IC50 values for TASK-1 and/or TASK-3 blockers acquired by measurements of relative cell viability were comparable to those obtained using patch-clamp recordings. Both blockers and openers of K2P channels can be accurately assessed with high efficiency and throughput by this novel HTS system.
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Shoeib, Amal M., Lance N. Benson, Shengyu Mu, Lee Ann MacMillan-Crow, and Paul L. Prather. "Non-Canonical Cannabinoid Receptors with Distinct Binding and Signaling Properties in Prostate and Other Cancer Cell Types Mediate Cell Death." International Journal of Molecular Sciences 23, no. 6 (March 11, 2022): 3049. http://dx.doi.org/10.3390/ijms23063049.
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Cannabinoids exert anti-cancer actions; however, the underlying cytotoxic mechanisms and the cannabinoid receptors (CBRs) involved remain unclear. In this study, CBRs were characterized in several cancer cell lines. Radioligand binding screens surprisingly revealed specific binding only for the non-selective cannabinoid [3H]WIN-55,212-2, and not [3H]CP-55,940, indicating that the expressed CBRs exhibit atypical binding properties. Furthermore, [3H]WIN-55,212-2 bound to a single site in all cancer cells with high affinity and varying densities. CBR characteristics were next compared between human prostate cancer cell lines expressing low (PC-3) and high (DU-145) CBR density. Although mRNA for canonical CBRs was detected in both cell lines, only 5 out of 15 compounds with known high affinity for canonical CBRs displaced [3H]WIN-55,212-2 binding. Functional assays further established that CBRs in prostate cancer cells exhibit distinct signaling properties relative to canonical Gi/Go-coupled CBRs. Prostate cancer cells chronically exposed to both CBR agonists and antagonists/inverse agonists produced receptor downregulation, inconsistent with actions at canonical CBRs. Treatment of DU-145 cells with CBR ligands increased LDH-release, decreased ATP-dependent cell viability, and produced mitochondrial membrane potential depolarization. In summary, several cancer cell lines express CBRs with binding and signaling profiles dissimilar to canonical CBRs. Drugs selectively targeting these atypical CBRs might exhibit improved anti-cancer properties.
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Du, Lin, April L. Risinger, Carter A. Mitchell, Jianlan You, Blake W. Stamps, Ning Pan, Jarrod B. King, et al. "Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides." Proceedings of the National Academy of Sciences 114, no. 43 (October 10, 2017): E8957—E8966. http://dx.doi.org/10.1073/pnas.1707565114.
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Mass-spectrometry-based metabolomics and molecular phylogeny data were used to identify a metabolically prolific strain ofTolypocladiumthat was obtained from a deep-water Great Lakes sediment sample. An investigation of the isolate’s secondary metabolome resulted in the purification of a 22-mer peptaibol, gichigamin A (1). This peptidic natural product exhibited an amino acid sequence including several β-alanines that occurred in a repeatingααβmotif, causing the compound to adopt a unique right-handed 311helical structure. The unusual secondary structure of 1 was confirmed by spectroscopic approaches including solution NMR, electronic circular dichroism (ECD), and single-crystal X-ray diffraction analyses. Artificial and cell-based membrane permeability assays provided evidence that the unusual combination of structural features in gichigamins conferred on them an ability to penetrate the outer membranes of mammalian cells. Compound 1 exhibited potent in vitro cytotoxicity (GI500.55 ± 0.04 µM) and in vivo antitumor effects in a MIA PaCa-2 xenograft mouse model. While the primary mechanism of cytotoxicity for 1 was consistent with ion leakage, we found that it was also able to directly depolarize mitochondria. Semisynthetic modification of 1 provided several analogs, including aC-terminus-linked coumarin derivative (22) that exhibited appreciably increased potency (GI505.4 ± 0.1 nM), but lacked ion leakage capabilities associated with a majority of naturally occurring peptaibols such as alamethicin. Compound 22 was found to enter intact cells and induced cell death in a process that was preceded by mitochondrial depolarization.
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Kumar, Ravi, Pratibha Kumari, Swapnil Pandey, Shravan Kumar Singh, and Raj Kumar. "Amelioration of Radiation-Induced Cell Death in Neuro2a Cells by Neutralizing Oxidative Stress and Reducing Mitochondrial Dysfunction Using N-Acetyl-L-Tryptophan." Oxidative Medicine and Cellular Longevity 2022 (November 26, 2022): 1–22. http://dx.doi.org/10.1155/2022/9124365.
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The deleterious effects of ionizing radiation on the central nervous system (CNS) are poorly understood. Radiation exposure during an accidental nuclear explosion, nuclear war, or radiotherapy causes severe brain damage. As a result, the current work is carried out to assess the radioprotective potential of N-acetyl-L-tryptophan (L-NAT) in neuronal cells. Radiation-induced cell death and its amelioration by L-NAT pretreatment were investigated using MTT, SRB, CFU, and comet assays. Flow cytometric and microscopic fluorescence assays were used to investigate radiation-induced oxidative stress, alteration in mitochondrial redox, Ca2+ homeostasis, depolarization of mitochondrial membrane potential, and its prevention with L-NAT pretreatment. Western blot analysis of Caspase-3, γ-H2aX, p53, ERK-1/2, and p-ERK-1/2 expression was carried out to identify the effects of L-NAT pretreatment on radiation-induced apoptosis and its regulatory proteins expression. The study demonstrated (MTT, SRB, and CFU assay) significant (~80%; p <0.001%) radioprotection in irradiated (LD50 IR dose) Neuro2a cells that were pretreated with L-NAT. In comparison to irradiated cells, L-NAT pretreatment resulted in significant (p <0.001%) DNA protection. A subsequent study revealed that L-NAT pretreatment of irradiated Neuro2a cells establishes oxidative stress by increasing antioxidant enzymes and mitochondrial redox homeostasis by inhibiting Ca2+ migration from the cytoplasm to the mitochondrial matrix and thus protects the mitochondrial membrane hyperpolarization. Caspase-3 and γ-H2aX protein expression decreased, while p-ERK1/2 and p53 expression increased in L-NAT pretreated irradiated cells compared to irradiated cells. Hence, L-NAT could be a potential radioprotective that may inhibit oxidative stress and DNA damage and maintain mitochondrial health and Ca2+ levels by activating p-ERK1/2 and p53 expression in Neuronal cells.
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Rehfeldt, Stephanie Cristine Hepp, Joana Silva, Celso Alves, Susete Pinteus, Rui Pedrosa, Stefan Laufer, and Márcia Inês Goettert. "Neuroprotective Effect of Luteolin-7-O-Glucoside against 6-OHDA-Induced Damage in Undifferentiated and RA-Differentiated SH-SY5Y Cells." International Journal of Molecular Sciences 23, no. 6 (March 8, 2022): 2914. http://dx.doi.org/10.3390/ijms23062914.
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Luteolin is one of the most common flavonoids present in edible plants and its potential benefits to the central nervous system include decrease of microglia activation, neuronal damage and high antioxidant properties. The aim of this research was to evaluate the neuroprotective, antioxidant and anti-inflammatory activities of luteolin-7-O-glucoside (Lut7). Undifferentiated and retinoic acid (RA)-differentiated SH-SY5Y cells were pretreated with Lut7 and incubated with 6-hydroxydopamine (6-OHDA). Cytotoxic and neuroprotective effects were determined by MTT assay. Antioxidant capacity was determined by DPPH, FRAP, and ORAC assays. ROS production, mitochondrial membrane potential (ΔΨm), Caspase–3 activity, acetylcholinesterase inhibition (AChEI) and nuclear damage were also determined in SH-SY5Y cells. TNF-α, IL-6 and IL-10 release were evaluated in LPS-induced RAW264.7 cells by ELISA. In undifferentiated SH-SY5Y cells, Lut7 increased cell viability after 24 h, while in RA-differentiated SH-SY5Y cells, Lut7 increased cell viability after 24 and 48 h. Lut7 showed a high antioxidant activity when compared with synthetic antioxidants. In undifferentiated cells, Lut7 prevented mitochondrial membrane depolarization induced by 6-OHDA treatment, decreased Caspase-3 and AChE activity, and inhibited nuclear condensation and fragmentation. In LPS-stimulated RAW264.7 cells, Lut7 treatment reduced TNF-α levels and increased IL-10 levels after 3 and 24 h, respectively. In summary, the results suggest that Lut7 has neuroprotective effects, thus, further studies should be considered to validate its pharmacological potential in more complex models, aiming the treatment of neurodegenerative diseases.
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Kim, Heesu, and Dong Gun Lee. "Lupeol-induced nitric oxide elicits apoptosis-like death within Escherichia coli in a DNA fragmentation-independent manner." Biochemical Journal 478, no. 4 (February 24, 2021): 855–69. http://dx.doi.org/10.1042/bcj20200925.
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Lupeol is known to be plentiful in fruits or plant barks and has an antimicrobial effect, however, its mode of action(s) has yet to be determined. To elucidate lupeol generates nitric oxide (NO), which is recognized for possessing an antimicrobial activity, intracellular NO was measured in Escherichia coli using DAF-FM. Using the properties of NO passing through plasma membrane easily, increased malondialdehyde levels have shown that lupeol causes lipid peroxidation, and the resulting membrane depolarization was confirmed by DiBAC4(3). These data indicated that lupeol-induced NO is related to the destruction of bacterial membrane. Further study was performed to examine whether NO, known as a cell proliferation inhibitor, affects bacterial cell division. As a result, DAPI staining verified that lupeol promotes cell division arrest, and followed by early apoptosis is observed in Annexin V/PI double staining. Even though these apoptotic hallmarks appeared, the endonuclease failed to perform properly with supporting data of decreased intracellular Mg2+ and Ca2+ levels without DNA fragmentation, which is confirmed using a TUNEL assay. These findings indicated that lupeol-induced NO occurs DNA fragmentation-independent bacterial apoptosis-like death (ALD). Additionally, lupeol triggers DNA filamentation and morphological changes in response to DNA repair system called SOS system. In accordance with the fact that ALD deems to SOS response, and that the RecA is considered as a caspase-like protein, increase in caspase-like protein activation occurred in E. coli wild-type, and no ΔRecA mutant. In conclusion, these results demonstrated that the antibacterial mode of action(s) of lupeol is an ALD while generating NO.