DOCSLIB.ORG

Resveratrol Protects SH-SY5Y Neuroblastoma Cells from Apoptosis Induced by Dopamine

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Resveratrol Protects SH-SY5Y Neuroblastoma Cells from Apoptosis Induced by Dopamine EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 39, No. 3, 376-384, June 2007 Resveratrol protects SH-SY5Y neuroblastoma cells from apoptosis induced by dopamine 1 2 Mi Kyung Lee , Soon Ja Kang , increase in the Bcl-2 protein, and activation of cas- 3 4 Mortimer Poncz , Ki-Joon Song pase-3. These results suggest that DA may be a 4,5 and Kwang Sook Park potential oxidant of neuronal cells at biologically relevant concentrations. Resveratrol may protect 1 Department of Genetics, College of Life Science SH-SY5Y cells against this cytotoxicity, reducing 2 Department of Science Education intracellular oxidative stress through canonical College of Education signal pathways of apoptosis and may be of bio - Ewha Womans University logical importance in the prevention of a dopami- Seoul 120-750, Korea nergic neurodegenerative disorder such as Parkin - 3 Division of Hematology, Children’s Hospital of Philadelphia son disease. PA 19104, USA 4 Keywords: Department of Microbiology and antioxidant; apoptosis; dopamine; neuro- Bank for Pathogenic Viruses blastoma; neurodegenerative diseases; resveratrol Division of Brain Korea 21 Program for Biomedical Science College of Medicine, Korea University Introduction Seoul 136-705, Korea 5 Corresponding author: Tel: 82-2-920-6164; Parkinson’s disease (PD) is a common neurode- Fax: 82-2-923-3645; E-mail: [email protected] generative disease, characterized by a selective loss of dopaminergic neurons in the substantia nigra. Accepted 11 April 2007 Many factors are speculated to operate in the me- chanism of cell death of nigrostriatal dopaminergic Abbreviations: DA, dopamine; MMP, mitochondrial membrane neurons in PD, including oxidative stress and cyto- potential; PI, propidium iodide; PS, phosphatidyl serine toxicity of reactive oxygen species (ROS), distur- bances of intracellular calcium homeostasis, exoge- Abstract nous and endogenous toxins, and mitochondrial dys- function. An endogenous neurotransmitter dopamine Dopamine (DA) is an oxidant that may contribute to (DA) is thought to be a major source of oxidative stress to these neural cells (Olanow, 1993; Offen et the degeneration of dopaminergic neurons. The al present study demonstrates that DA-induced ., 1997). DA contains an unstable catechol moiety, and it can oxidize spontaneously to form ROS, free cytotoxicity in human-derived neurotypic cells, radicals, and quinones (Cohen and Heikkila, 1974; SH-SY5Y, is prevented by resveratrol, one of the Graham, 1978; Hastings, 1995). In the human subs- major antioxidative constituents found in the skin of tantia nigra, the DA oxidation products may further grapes. SH-SY5Y cells, a neuroblastoma cell line, polymerize to form another neurotoxin, neurome- µ treated with DA at 300 and 500 M for 24 h underwent lanin (Jellinger et al., 1993). These oxidation products apoptotic death as determined by characteristic can damage cellular components such as lipids, morphological features, including nuclear conden - proteins, and DNA (Halliwell, 1992). sation, and loss of mitochondrial membrane poten - One of the plausible ways to prevent the cell tial (MMP). Flow cytometric analysis using Annexin V death induced by oxidative stress is dietary or showed that DA can induce significant and severe pharmacological intake of antioxidants. Lai and Yu apoptosis. Exposure to resveratrol (5 µM) for 1 h prior (1997) showed that DA-induced apoptosis is protec- to the DA treatment attenuated DA-induced cyto - ted by some antioxidants such as glutathione and toxicity, and rescued the loss of MMP. To investigate N-acetyl-L-cysteine. One family of naturally occurring the apoptotic signaling pathways relevant to the compounds possessing free radical scavenging pro- restoration of DA-induced apoptosis by resveratrol, perties is the polyphenols. It is found in fruit, vegeta- we carried out quantitative analysis of Bcl-2, cas- bles and plant-derived beverages and may have pase-3, and cleaved poly ADP-ribose polymerase important roles as dietary components via cytopro- (PARP) by immunoblot analysis. Resveratrol pre- tective actions in many organs (Youdim and Joseph, trans- treatment led to a decrease in cleavage of PARP, an 2001). Resveratrol (3,5,4'-tihydroxy- stilbene), a Resveratrol inhibits dopamine-induced apoptosis 377 polyphenolic phytoalexin found in the skin of grapes, and incubated in the CO2 incubator for 3 h. Plates has been reported to possess a wide range of bio- were read with a microplate reader at a wavelength logical and pharmacological activities including anti- of 490 nm. Results were expressed as the per- oxidant, anti-inflammatory, antimutagenic, and anti- centage of MTS reduction, assuming that the ab- carcinogenic effects (Jang et al., 1997; Soleas et al., sorbance of control cells was 100%. For each ex- 1997; Surh, 1999). In previous studies, antioxidant periment, all treatments were performed in triplicate properties of resveratrol attenuated hippocampal cell wells. death and intracellular ROS formation (Bastianetto et al., 2000). Neuroprotective activities against exci- totoxic brain damage (Virgili and Contestabile, 2000) Nuclear staining for assessment of apoptosis and anti-apoptotic effects by caspase activation SH-SY5Y cells were stained with the chromatin- (Nicolini et al., 2001) suggest a putative neuropro- specific dye Hoechst 33324 (Molecular Probes, tective action for resveratrol. Eugene, OR). The SH-SY5Y cells on the chamber In the present study, we have examined the po- culture slide were incubated for 2 days, and then ssible protective effect of resveratrol against apop- treated with or without the resveratrol and DA at tosis induced by DA in human derived neurotypic different concentrations. SH-SY5Y cells were fixed cell, SH-SY5Y. Our results indicate that DA indeed for 10 min with 3.5% paraformaldehyde in PBS at triggers apoptosis and this deleterious effect can be room temperature. After twice rinses with PBS, the attenuated by resveratrol. cells were stained with 10 g/ml Hoechst 33324 in o PBS for 10 min at 37 C. Mounted slides were analyzed under a fluorescence microscope (Axiovert Materials and Methods 135, ZEISS, Germany) using a mercury lamp through a 360-370 nm bandpass filter. Light emitted Materials from Hoechst dye was collected through a 420 nm MEM, FBS and penicillin and streptomycin were longpass filter. obtained from Gibco BRL (Grand Island, NY). Re- sveratrol, dopamine, DMSO, Hoechst 33254, pro- Measurement of mitochondrial membrane potential pidium iodide (PI) were obtained from Sigma (St. Louis, MO). DA and resveratrol were dissolved in The level of mitochondrial membrane potential (MMP) sterilized H2O and in DMSO, respectively. Annexin V was determined by flow cytometry and the fluore- was from BD Biosciences (Palo Alto, CA). Mono- scent dye rhodamine 123. The rhodamine 123 data clonal antibodies against caspase-3, Bcl-2, cleavage were recorded using an FL-1 photomultiplier. Sample form of PARP were obtained from Cell Signaling data (10,000 cells) were used to prepare histograms Technology (Danvers, MA). Bicinchoninic acid pro- of the Cell Quest data analysis program (Becton tein assay kit and ECL chemiluminescence system Dickinson). were obtained from Pierce (Rockford, IL). Flow cytometric analysis using Annexin V and PI Cell culture SH-SY5Y cells were centrifuged to remove the me- Catecholaminergic neuroblastoma SH-SYSY cells dium, washed with PBS and stained with Annexin were grown in MEM supplemented with 10% FBS, V-FITC and PI in binding buffer (10 mM Hepes, 140 100 U/ml penicillin, and 100 g/ml streptomycin. mM NaCl, 2.5 mM CaCl2). Ten thousand events Cells were maintained in a humidified atmosphere of were collected on each sample. Stained cells were o 5% CO2, at 37 C. The cells were plated at an appro- analyzed using a FACScalibur (Becton Dickinson, priate density according to each experimental scale. Mountain View, CA) in the FL1-H and FL2-H channels. Cell viability assay Immunoblot analysis Cell viability was determined using Cell Titer 96 Aqu- Blotting was performed essentially as described eous Non-Radioactive Cell Proliferation Assay, availa- previously (Schulz et al., 1999). The cells treated ble from Promega Corp (Madison, WI). MTS (3-(4,5- with or without resveratrol and DA, were washed dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- once with ice-cold PBS and then lysed using ice- (4-sulfophenyl)-2H-tetrazo-lium inner salt) conversion cold RIPA buffer and protease inhibitor cocktail o assay was performed according to the protocol sup- (Sigma) at 4 C for 30 min. Cell lysates were cen- plied by the manufacturer. In brief, a mixture of MTS trifuged at 13,000 rpm for 25 min, and the protein and PMS (phenazine methosulfate) was added to concentrations were determined by the bicinchoninic the treated SH-SY5Y cells cultured in 96-well plates acid method (Pierce, Rockford, IL) using BSA as 378 Exp. Mol. Med. Vol. 39(3), 376-384, 2007 standard. The protein lysates were separated by resveratrol slightly but significantly increased cell 12% SDS-PAGE and transferred to nitrocellulose proliferation and DA-induced apoptotic effects were membrane (Bio-Rad, CA). Protein expression was markedly attenuated by the co-presence of 3 or 5 M detected using primary antibodies and secondary resveratrol (Figure 1C). antibodies conjugated with HRP. Resveratrol attenuated DA-induced apoptotic cell Ressults death SH-SY5Y cells treated for 24 h with DA at con- Resveratrol protected SH-SY5Y cells against centrations of 300 and 500 M exhibited morpho- DA-induced cytotoxicity logical alterations such as cell shrinkage and mem- DA concentrations from 0 to 500 M were tested brane blebbing that are normally associated with the and demonstrated that DA is capable of induce occurrence of apoptotic cell death. Compared to the metabolic interruption in SH-SY5Y cells in a dose normal SH-SY5Y cells (Figure 2A), cells treated with range of 300-500 M (Figure 1A). DA decreased cell DA round up, detached from the bottom, and ag- viability concentration over time (Figure 1B). Prior gregated as assessed by phase-contrast microscopy studies have shown that resveratrol at up to 10 M (Figure 2B and D).
Load more

Recommended publications
  • Increased Mitochondrial Uptake of Rhodamine 123 During Lymphocyte Stimulation
    Proc. Natl. Acad. Sci. USA Vol. 78, No. 4, pp. 2383-2387, April 1981 Cell Biology Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation (flow cytometry/cycling-noncycling cells/supravital fluorescent probe) ZBIGNIEW DARZYNKIEWICZ, LISA STAIANO-COICO, AND MYRON R. MELAMED Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Communicated by Lloyd J. Old, January 5, 1981 ABSTRACT The positively charged rhodamine analog rho- trifugation on Ficoll/Isopaque (Lymphoprep; Nyegaardo, Oslo, damine 123 accumulates specifically in the mitochondria of living Norway). The mononuclear cells were suspended in Eagle's cells. In the present work, the uptake of rhodamine 123 by indi- basal medium containing 15% fetal bovine serum and subcul- vidual lymphocytes undergoing blastogenic transformation in cul- tured in plastic dishes to remove most ofthe monocytes (3). The tures stimulated by phytohemagglutinin was measured by flow nonadhering cells were then suspended in Eagle's medium con- cytometry. A severalfold increase in cell ability to accumulate rho- taining penicillin, streptomycin, and 15% fetal bovine serum damine 123 was observed during lymphocyte stimulation. Maxi- and incubated at 37°C in the presence of PHA (phytohemag- mal dye uptake, seen on the third day ofcell stimulation, coincided glutinin M, GIBCO) as described (2, 3). The samples were with- in time with the peak of DNA synthesis (maximal number of cells drawn from these cultures at daily intervals. Other details ofthe in the S phase) and mitotic activity. A large intercellular variation are elsewhere 3). among stimulated lymphocytes, with some cells having fluores- cell culture system given (2, cence increased as much as 15 times in comparison with nonstim- Cell Staining.
    [Show full text]
  • Reversal of Resistance to Rhodamine 123 in Adriamycin-Resistant Friend Leukemia Cells1
    [CANCER RESEARCH 45, 2626-2631, June 1985] Reversal of Resistance to Rhodamine 123 in Adriamycin-resistant Friend Leukemia Cells1 Theodore J. Lampidis,2 Jean-Nicolas Munck, Awtar Krishan, and Haim Tapiero Department of Oncology, University of Miami, School of Medicine, Comprehensive Cancer Center for the State of Florida, Miami, Florida 33101 [T. J. L, A. K.], and Departement de Pharmacologie Cellulaire, Moléculaireet de Pharmacocinétique, ICIG (CNRS LA-149), Hôpital Paul-Brousse, 94804 Villejuif, France [J-N. M., H. T.] ABSTRACT the calcium transport inhibitor, verapamil, on the cellular accu mulation and retention of Rho-123 and ADM as it relates to Pleiotropic resistance to rhodamine 123 (Rho-123) in Adria- resistance in these cell variants. mycin (ADM)-resistant Friend leukemia cells was circumvented by cotreatment with 10 //M verapamil. Increased cytotoxicity corresponded to higher intracellular Rho-123 levels. The vera- MATERIALS AND METHODS pamil-induced increase of drug accumulation in resistant cells is Cells and Cytotoxicity Assay. FLC were derived from a clone of accounted for at least in part by the blockage or slowing of Rho- Friend virus-tranformed 745A cells. Cells were seeded and grown as 123 efflux from these cells. In contrast, accumulation and con described previously (8). The cell variant resistant to ADM (ADM-RFLC3) sequent cytotoxicity of Rho-123 in sensitive cells are not in was derived from the above clone by continuous exposure to ADM (8). creased by verapamil. Similar results were obtained when ADM Resistant cells, grown for more than 150 passages in drug-free medium, was used in this cell system.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 7476,504 B2 Turner (45) Date of Patent: Jan
    USOO7476504B2 (12) United States Patent (10) Patent No.: US 7476,504 B2 Turner (45) Date of Patent: Jan. 13, 2009 (54) USE OF REVERSIBLE EXTENSION Glatthar, Ralf, et al. 2000. A New Photocleavable Linker in Solid TERMINATOR IN NUCLECACD Phase Chemistry for Ether Cleavage. Org. Lett. 2(15): 2315-2317. SEQUENCING Goldmacher, Victor S., et al. 1992. Photoactivation of Toxin Conju gates. Bioconjugate Chem. 3(2): 104-107. Guillier, Fabrice, et al. 2000. Linkers and Cleavage Strategies in (75) Inventor: Stephen Turner, Menlo Park, CA (US) Solid-Phase Organic Synthesis and Combinatorial Chemistry. Chem. Rev. 100(6): 2091-2157. (73) Assignee: Pacific Biosciences of California, Inc., Holmes, Christopher P. 1997. Model Studies for New o-Nitrobenzyl Menlo Park, CA (US) Photolabile Linkers: Substituent Effects on the Rates of Photochemi cal Cleavage.J. Org. Chem. 62(8): 2370-2380. (*) Notice: Subject to any disclaimer, the term of this Hu, Jun, et al. 2001. Competitive Photochemical Reactivity in a patent is extended or adjusted under 35 Self-Assembled Monolayer on a Colloidal Gold Cluster. J. Am. U.S.C. 154(b) by 365 days. Chem. Soc. 123(7): 1464-1470. Kessler, Martin, et al. 2003. Sequentially Photocleavable Protecting (21) Appl. No.: 11/341,041 Groups in Solid-Phase Synthesis. Org. Lett.5(8); 1179-1181. Kim, Hanyoung, et al. 2004. Core-Shell-Type Resins for Solid-Phase (22) Filed: Jan. 27, 2006 Peptide Synthesis: Comparison with Gel-Type Resins in Solid-Phase Photolytic Cleavage Reaction. Org. Lett. 6(19): 3273-3276. (65) Prior Publication Data Maxam, Allan M., et al. 1977. A new method for sequencing DNA.
    [Show full text]
  • Mediated Multidrug Resistance (MDR) in Cancer
    A Thesis Entitled: Differential Effects of Dopamine D3 Receptor Antagonists in Modulating ABCG2 - Mediated Multidrug Resistance (MDR) in Cancer by Noor A. Hussein Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science in Pharmaceutical Sciences Degree in Pharmacology and Toxicology _________________________________________ Dr. Amit K. Tiwari, Committee Chair _________________________________________ Dr. Frank Hall , Committee Member _________________________________________ Dr. Zahoor Shah, Committee Member _________________________________________ Dr. Caren Steinmiller, Committee Member _________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo May 2017 Copyright 2017, Noor A. Hussein This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Differential Effects of Dopamine D3 Receptor Antagonists in Modulating ABCG2 - Mediated Multidrug Resistance (MDR) by Noor A. Hussein Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science in Pharmaceutical Sciences Degree in Pharmacology and Toxicology The University of Toledo May 2017 The G2 subfamily of the ATP-binding cassette transporters (ABCG2), also known as the breast cancer resistance protein (BRCP), is an efflux transporter that plays an important role in protecting the cells against endogenous and exogenous toxic substances. The ABCG2 transporters are also highly expressed in the blood brain barrier (BBB), providing protection against specific toxic compounds. Unfortunately, their overexpression in cancer cells results in the development of multi-drug resistance (MDR), and thus, chemotherapy failure. Dopamine3 receptor (D3R) antagonists were shown to have excellent anti-addiction properties in preclinical animal models but produced limited clinical success with the lead molecule.
    [Show full text]
  • Selective Toxicity of Rhodamine 123 in Carcinoma Cells in Vitro
    [CANCER RESEARCH 43, 716-720, February 1983] 0008-5472/83/0043-0000502.00 Selective Toxicity of Rhodamine 123 in Carcinoma Cells in Vitro Theodore J. Lampidis, 1 Samuel D. Bernal, 2 lan C. Summerhayes, and Lan Bo Chen a Sidney Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 ABSTRACT ing appears to be unique, since chemotherapeutic agents currently in clinical use have not been shown to be particularly The study of mitochondria in situ has recently been facilitated selective between tumorigenic and normal cells in vitro. through the use of rhodamine 123, a mitochondrial-specific fluorescent dye. It has been found to be nontoxic when applied MATERIALS AND METHODS for short periods to a variety of cell types and has thus become an invaluable tool for examining mitochondrial morphology and Cell Cultures. All cell types and cell lines were grown in Dulbecco's function in the intact living cell. In this report, however, we modified Eagle's medium supplemented with 10% calf serum (M. A. demonstrate that with continuous exposure, rhodamine 123 Bioproducts, Walkersville, Md.) at 37 ~ with 5% CO2. The following cell selectively kills carcinoma as compared to normal epithelial lines were obtained from the American Type Culture Collection: cells grown in vitro. At doses of rhodamine 123 which were CCL 105, CCL 15, CRL 1550, CRL 1420, CCL51, CCL77, CCL185, toxic to carcinoma cells, the conversion of mitochondrial-spe- CCL 34, PtK-1, CV-1, and BSC-I. The human bladder carcinoma line cific to cytoplasmic-nonspecific localization of the drug was (E J) was provided by Dr.
    [Show full text]
  • Use of the Laser Dye Rhodamine 123
    Status of Mitochondria in Living Human Fibroblasts during Growth and Senescence in Vitro : Use of the Laser Dye Rhodamine 123 SAMUEL GOLDSTEIN and LOUISE B . KORCZACK Departments of Medicine and Biochemistry, McMaster University Medical Center, Hamilton, Ontario, Canada L8N 3Z5. Dr. Goldstein's present address is : Departments of Medicine and Biochemistry, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 Downloaded from ABSTRACT Rhodamine 123, a fluorescent laser dye that is selectively taken up into mitochondria of living cells, was used to examine mitochondrial morphology in early-passage (young), late- passage (old), and progeric human fibroblasts . Mitochondria were readily visualized in all cell types during growth (mid-log) and confluent stages . In all cell strains at confluence, mitochon- jcb.rupress.org dria became shorter, more randomly aligned, and developed a higher proportion of bead-like forms . Treatment of cells for six days with Tevenel, a chloramphenicol analog that inhibits mitochondrial protein synthesis, brought about a marked depletion of mitochondria and a diffuse background fluorescence . Cyanide produced a rapid release of preloaded mitochondria) fluorescence followed by detachment and killing of cells . Colcemid caused a random coiling on January 8, 2018 and fragmentation of mitochondria particularly in the confluent stage . No gross differences were discernible in mitochondria of the three cell strains in mid-log and confluent states or after these treatments . Butanol-extractable fluorescence after loading with rhodamine 123 was lower in all cell strains in confluent compared to mid-log stages . At confluence all three cell strains had similar rhodamine contents at zero-time and after washout up to 24 h .
    [Show full text]
  • Antioxidants
    antioxidants Article Insulin–Mimetic Dihydroxanthyletin-Type Coumarins from Angelica decursiva with Protein Tyrosine Phosphatase 1B and α-Glucosidase Inhibitory Activities and Docking Studies of Their Molecular Mechanisms Md Yousof Ali 1 , Susoma Jannat 2, Hyun Ah Jung 3,* and Jae Sue Choi 4,* 1 Department of Physiology and Pharmacology, Hotchkiss Brain Institute and Alberta Children’s Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; [email protected] 2 Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB T2N 4N1, Canada; [email protected] 3 Department of Food Science and Human Nutrition, Jeonbuk National University, Jeonju 54896, Korea 4 Department of Food and Life Science, Pukyong National University, Busan 48513, Korea * Correspondence: [email protected] (H.A.J.); [email protected] (J.S.C.); Tel.: +82-63-270-4882 (H.A.J.); +82-51-629-7547 (J.S.C.) Abstract: As a traditional medicine, Angelica decursiva has been used for the treatment of many dis- eases. The goal of this study was to evaluate the potential of four natural major dihydroxanthyletin- type coumarins—(+)-trans-decursidinol, Pd-C-I, Pd-C-II, and Pd-C-III—to inhibit the enzymes, Citation: Ali, M.Y.; Jannat, S.; Jung, protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase. In the kinetic study of the PTP1B H.A.; Choi, J.S. Insulin–Mimetic enzyme’s inhibition, we found that (+)-trans-decursidinol, Pd-C-I, and Pd-C-II led to competitive Dihydroxanthyletin-Type Coumarins inhibition, while Pd-C-III displayed mixed-type inhibition. Moreover, (+)-trans-decursidinol exhib- from Angelica decursiva with Protein ited competitive-type, and Pd-C-I and Pd-C-II mixed-type, while Pd-C-III showed non-competitive Tyrosine Phosphatase 1B and type inhibition of α-glucosidase.
    [Show full text]
  • Mitochondrial Dynamics Quantitatively Revealed by STED Nanoscopy with an Enhanced Squaraine Variant Probe
    Supplementary Information Mitochondrial dynamics quantitatively revealed by STED nanoscopy with an enhanced squaraine variant probe Xusan Yang1,3, Ɨ, Zhigang Yang2, Ɨ, *, Ying He2, Chunyan Shan4, Wei Yan2, Zhaoyang Wu1, Peiyuan Chai4, Junlin Teng4, Junle Qu2, *, Peng Xi1, * 1 Department of biomedical engineering, College of engineering, Peking University, Beijing, 100871, China 2 Key laboratory of optoelectronic devices and systems of ministry of education and Guangdong province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen, 518060, China 3 School of applied and engineering physics, Cornell University, Ithaca, 14853, United States 4 School of life Sciences, Peking University, Beijing, 100871, China *Email: [email protected], [email protected], [email protected], [email protected] Ɨ These authors contributed equally to this work Supplementary Figures Supplementary Figure 1 Co-localization experiment employing MitoTracker Green (Rhodamine 123) as golden standard for mitochondrial marker. The first column, DIC image; the second column, ER- Lyso- and Mito-Tracker green labeled HeLa cells; c, g the third column, enhanced squaraine dye labeled HeLa cells; the fourth column, merged images; the fifth column, Pearson’s coefficients, excitation wavelength: Rhodamine 123, Lyso- and ER- trackers (488 nm), MitoESq-635 (633 -nm), detect range of Rhodamine 123 Lyso- and ER-trackers (500-560 nm); MitoESq-635 (640-800 nm), Scale bar, 10 μm. From Supplementary Figure 1-3, it can be demonstrated that MitoESq-635 can preferentially target for mitochondria in live cells. HeLa cells were subjected to incubating with Mito-, Lyso-, ER- trackers and MitoESq-635, respectively. From the confocal imaging results obtained by a Leica SP8 confocal Laser scanning microscope, the probe revealed the best overlapping imaging with Mitotracker (Rho123).
    [Show full text]
  • Potent Fluorescent Probes of Mitochondria in Living C. Elegans
    ydrophobic analogues of rhodamine B and rhodamine 101: potent fluorescent probes of mitochondria in living . elegans Laurie F. Mottram1, Safiyyah Forbes1, Brian D. Ackley! and Blake R. Peterson*1 Full Research Paper Open Access Address: !eilstein J. Org. Chem. 2012, 8, 156–165. 1Department of Medicinal Chemistry, The University of Kansas, doi:10.376/bjoc.8.43 Lawrence, KS 66045, United States and !Department of Molecular Biosciences, The University of Kansas, Lawrence, KS 66045, United Received: 9 September 01 States Accepted: 09 November 01 Published: 11 December 01 Email: Blake R. Peterson* - [email protected] This article is part of the Thematic Series "Synthetic probes for the study of biological function". * Corresponding author Guest Editor: J. Aube Keywords: Caenorhabditis elegans; chemical biology; fission; fluorophores; © 01 Mottram et al; licensee Beilstein-Institut. fluorescence; fusion; imaging; in vivo; microscopy; mitochondria; License and terms: see end of document. model organisms; organelle; rhodamine; spectroscopy Abstract itochondria undergo dynamic fusion and fission events that affect the structure and function of these critical energy-producing cellular organelles. Defects in these dynamic processes have been implicated in a wide range of human diseases including ischemia, neurodegeneration, metabolic disease, and cancer. To provide new tools for imaging of mitochondria in vivo, we synthesized novel hydrophobic analogues of the red fluorescent dyes rhodamine B and rhodamine 101 that replace the carboxylate with a methyl group. Compared to the parent compounds, methyl analogues termed HRB and HR101 exhibit slightly red-shifted absorbance and emission spectra (5–9 nm), modest reductions in molar extinction coefficent and quantum yield, and enhanced partitioning into octanol compared with aqueous buffer of 10-fold or more.
    [Show full text]
  • Rhodamine 123 As a Probe of Mitochondrial Membrane Potential
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1606 (2003) 137–146 www.bba-direct.com Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F0 during ATP synthesis Alessandra Baraccaa,*,1, Gianluca Sgarbib,1, Giancarlo Solainib, Giorgio Lenaza a Department of Biochemistry ‘‘G. Moruzzi’’ Alma Mater Studiorum-University of Bologna, Via Irnerio 48, I-40126 Bologna, Italy b Scuola Superiore di Studi Universitari e di Perfezionamento ‘‘S. Anna’’, P.zza Martiri della Liberta` 33, 56127 Pisa, Italy Received 9 January 2003; received in revised form 20 May 2003; accepted 25 July 2003 Abstract Rhodamine 123 (RH-123) was used to monitor the membrane potential of mitochondria isolated from rat liver. Mitochondrial energization induces quenching of RH-123 fluorescence and the rate of fluorescence decay is proportional to the mitochondrial membrane potential. Exploiting the kinetics of RH-123 fluorescence quenching in the presence of succinate and ADP, when protons are both pumped out of the matrix driven by the respiratory chain complexes and allowed to diffuse back into the matrix through ATP synthase during ATP synthesis, we could obtain an overall quenching rate proportional to the steady-state membrane potential under state 3 condition. We measured the kinetics of fluorescence quenching by adding succinate and ADP in the absence and presence of oligomycin, which abolishes the ADP-driven potential decrease due to the back-flow of protons through the ATP synthase channel, F0. As expected, the initial rate of quenching was significantly increased in the presence of oligomycin, and conversely preincubation with subsaturating concentrations of the uncoupler carbonyl cyanide p-trifluoro-metoxyphenilhydrazone (FCCP) induced a decreased rate of quenching.
    [Show full text]
  • Uptake of Fluorescent Dyes Associated with the Functional Expression of The
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 1167-1172, February 1996 Medical Sciences Uptake of fluorescent dyes associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells (gene therapy) ROBERT P. WERSTO*t, EUGENE R. ROSENTHAL*, RONALD G. CRYSTAL*t, AND KENNETH R. SPRING§ *Pulmonary Branch and §Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 Communicated by Maurice B. Burg, National Institutes of Health, Bethesda, MD, October 12, 1995 (received for review January 3, 1995) ABSTRACT Specific mutations in the cystic fibrosis ing the full length CFTR cDNA or mock virus, respectively transmembrane conductance regulator (CFTR), the most (10), were generously provided by R. Frizzell (University of common autosomal recessive fatal genetic disease of Cauca- Alabama, Birmingham). CFPAC cells were also cultured in sians, result in the loss of epithelial cell adenosine 3',5'-cyclic- IMDM. C127 cells (mouse mammary tumor cells) stably monophosphate (cAMP)-stimulated Cl- conductance. We overexpressing wild-type CFTR and a mock-transfected con- show that the influx of a fluorescent dye, dihydrorhodamine trol (11) were generously provided by S. Cheng (Genzyme) 6G (dR6G), is increased in cells expressing human CFTR after and were maintained in IMDM containing Geneticin sulfate retrovirus- and adenovirus-mediated gene transfer. dR6G (G418, GIBCO/BRL) at 200 gg/ml. influx is stimulated by cAMP and is inhibited by antagonists Recombinant, replication-deficient adenoviral vectors con- of cAMP action. Dye uptake is ATP-dependent and inhibited taining the full-length human CFTR cDNA (12) were used to by Cl- removal or the addition of 10 mM SCN-.
    [Show full text]
  • Transport of the Cationic Fluorochrome Rhodamine 123 in an Insect's Malpighian Tubule: Indications of a Reabsorptive Function of the Secondary Cell Type
    Journal of Cell Science 101, 349-361 (1992) 349 Printed in Great Britain © The Company of Biologists Limited 1992 Transport of the cationic fluorochrome rhodamine 123 in an insect's Malpighian tubule: indications of a reabsorptive function of the secondary cell type W. MEULEMANS and A. De LOOF Zoological Institute of the University of Leuven, Naamsestraat 59, B-3000 Leuven, Belgium Summary The pathway of rhodamine 123 was examined after the lumen was found using Lucifer Yellow CH, injection into Sarcophaga flies and after in vitro labeling fluorescent dextrans and fluorescent albumin. Prior of the Malpighian tubules. After in vitro labeling the incubation with the ionophores valinomycin, nigericin, primary cells only retained this potential-sensitive dye CCCP (all 1 pg/ml), dinitrophenol (1 mM) and NaN3 for a short period while all secondary cells accumulated (10~2 M) inhibited the selective accumulation of rhoda- the dye from the tubule lumen. In vivo the secondary mine 123 to a large extent while monensin (1-5 /ig/ml) cells also accumulated rhodamine 123 from the lumen, showed little inhibitory effect. Furthermore, only cat- but the primary cells in the distal parts of all four tubules ionic and no anionic or neutral dyes were accumulated retained the dye for prolonged periods. This was most by the secondary cells. In the fleshfly Calliphora and the pronounced in the distal part of the anterior Malpighian fruitfly Drosophila, the dye rhodamine 123 also selec- tubules, where rhodamine 123 was eventually precipi- tively accumulated in the secondary cells, as well in vitro tated on the luminal concretions.
    [Show full text]