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18 H.J. Jang et al. / Neuroscience Letters 443 (2008) 17–22gestation as described previously [14]. Briefly, dissociated corticalcells were plated onto a previously established astroglial cell monolayerat 3 hemispheres per 24-well plate (Primaria, Falcon, USA)in plating medium (Modified Eagle medium supplemented with21 mM glucose, 26.5 mM sodium bicarbonate, 2 mM glutamine, 5%fetal bovine serum, and 5% horse serum). Cytosine arabinoside(10 M) was added 5–6 days after plating to halt the growth ofnon-neuronal cells.Treatments with taxol and other drugs were performed at DIV12–14 in serum-free MEM (minimal essential medium with Earle’ssalts). The stock solutions of drugs were made at 100× concentrationof working solution in appropriate solvent and diluted in MEMimmediately before experiments. In addition to morphologicalassessment under a phase-contrast microscopy, overall neuronalcell injury in mixed cortical cultures was quantitatively assessedby measuring lactate dehydrogenase (LDH) activity released fromdamaged cells into the bathing medium [11]. Each LDH value, aftersubtracting the mean background value in sham-washed controls(=0), was scaled to the mean value in positive control culturestreated with 500 M NMDA for 24 h (=100), which induced nearcomplete neuronal death in the absence of glial damage.SYTOX Green (Invitrogen, Carlsbad, CA, USA) staining was usedfor morphological evaluation of nuclei. After fixing the cells with4% paraformaldehyde for 50–60 min at room temperature, the cellswere permeabilized with 0.5% Triton X-100 for 10 min and treatedwith 1 M SYTOX Green stock for 15 min.Changes of microtubule network after exposure to taxol werevisualized by fluorescence immunocytochemistry for tubulin. Afterfixation with 4% paraformaldehyde for 30 min and blocking, cellswere labelled with rabbit anti-tubulin antibody (Sigma) at 1:80dilution at 4 ◦ C overnight. After washes, signals were visualizedby Cy3-conjugated affinity purified anti-rabbit IgG (1:200,Jackson ImmunoResearch, West Grove, PA) using epifluorescencemicroscopy.Intracellular free radical generation was measured using 2,7-dichlorofluorescin diacetate (DCF) (Invitrogen, Carlsbad, CA, USA)[15]. Briefly, cells were loaded with 10 M DCF for 30 min and thentreated with taxol alone or in combination with apocynin or trolox.After treatments, ROS generation was monitored at 0.5, 1, 2, 4, 6,and 8 h at 37 ◦ C in a fluorescent multiwell plate reader (FluoroskanAscent FL, Labsystems) with excitation at 485 nm and emission at530 nm.RNA was prepared with TRIZOL and reverse-transcribed intocDNA with random primer and M-MLV reverse transcriptase(Invitrogen). PCR was performed under the following conditions:denaturing for 1 min at 94 ◦ C; annealing for 1 min atFig. 1. Taxol-induced concentration-dependent neuronal apoptosis and effect of antioxidants on neuronal cell death induced taxol or vinblastine in mixed cortical cultures.(A) Concentration-dependent neuronal death in mixed cortical cultures. Neuronal death was assessed by LDH release. Each point and bar represents mean ± S.E.M. from 4to 12 wells. (B) Photomicrographs of Sytox green nuclear staining showing chromatin condensation and nuclear fragmentation (arrows) induced by 8-h exposure to 300 nMtaxol (Tax) and 100 nM vinblastine (Vin) in mouse cortical cultures. Sham-wash control (Sham). Calibration bar: 50 m. (C) Inhibitory effect of 1 g/mL cycloheximide (+CHX),100 M z-VAD-fmk (+ZVAD), 100 M trolox, 100 M ascorbic acid, and 5 mM tempol on taxol-induced neuronal death. Each column and bar is the mean ± S.E.M. from 12to 16 wells. *P < 0.05, compared with taxol-treated control group. (D) Effect of 100 M z-VAD-fmk (+ZVAD), 100 M trolox, 100 M ascorbic acid, 500 M apocynin (+Apo)and 100 nM DPI on the 100 nM vinblastine (Vin)-induced neuronal death in mixed cortical cultures. Each column and bar is the mean ± S.E.M. from 8 to 12 wells. *P < 0.05,compared with vinblastine-treated group.
H.J. Jang et al. / Neuroscience Letters 443 (2008) 17–22 1958 ◦ C for p47 phox , 57 ◦ C for gp91 phox and HPRT; extensionfor 2min at 72 ◦ C (35 cycles for p47 phox and gp91 phox ; 30cycles for HPRT). PCR products were run on 1.5% agarosegels, visualized with ethidium bromide and quantitativelymeasured with an image analyzer. Primer sequences wereas follows: 5 ′ -CAGCCAGCACTATGTGTACA-3 ′ and 5 ′ -GAACTCGTA-GATCTCGGTGAA-3 ′ for p47 phox (91 bp); 5 ′ -GGTTTATGATGAT-GGGCCTAA-3 ′ and 5 ′ -GCACTGGAACCC CTGAGAAA-3 ′ , for gp91 phox(158 bp); 5 ′ -GTAATGATCAGTCAACGGGGGAC-3 ′ and 5 ′ -CCAGCAA-GCTTGCAACCTTAACCA-3 ′ for HPRT (200 bp).For knockdown of gp91 phox expression, Stealth TM siRNA ofgp91 phox (Invitrogen) was used. The sense and antisense sequencesof gp91 phox were CCAUCCACACAAUUGCA CAUCUCUU and AAGA-GAUGUGCAAUUGUGUGGAUGG, respectively. At DIV 6, the corticalcultures were transfected with 60 pmol siRNA of gp91 phox or negativecontrol siRNA using the GeneSilencer siRNA transfectionreagent (Gene Therapy Systems, San Diego, CA) as described previously[1]. In brief, siRNA and GeneSilencer were each diluted to25 L with media and then mixed together for 10 min. Cells werefed 500 L of fresh culture medium and overlaid with the transfectionmix. After 24 h, the cells were fed with 500 L of culturemedium. Transfected cells were used for experiments at DIV 13–14.Cortical cells were lysed in lysis buffer and centrifuged at13,000 rpm for 15 min. The pellet was discarded, and the supernatantwas used for protein quantification. Fractionation of cytosoland membrane was performed as described previously [14]. Equalamounts of protein from total cell lysates or membrane or cytosolicfraction were electrophoresed on 12% SDS–PAGE and transferredto nitrocellulose membranes using Towbin buffer. The membraneswere then incubated with anti-p47 phox or anti-gp91 phox antibodies(Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnightat 4 ◦ C. Immunoreactive proteins were detected using enhancedchemiluminescence protocol (Amersham Life Science).Results are expressed as mean ± S.E.M. Significant differencesbetween means were determined by analysis of variance withTukey multiple comparisons post hoc analysis using Instat software(Graphpad Software Inc., CA, USA). P < 0.05 was consideredstatistically significant.Mixed cortical cultures were exposed to selected concentrationsof taxol for 24 h. The neurotoxicity of taxol began to appear at 30 nMwith about 10% neuronal death, and neuronal death increased to33%, 46% and 72% with increasing concentrations of 100, 300 and1000 nM, respectively (Fig. 1A). In our culture systems, taxol barelyinjured astrocytes in the 30–1000 nM concentration range (datanot shown). Taxol induced chromatin condensation and nuclearfragmentation in cortical cultures (Fig. 1B). In addition to the morphologicalapoptotic features, treatment with anti-apoptotic drugs,such as cycloheximide (1 g/mL, a protein synthesis inhibitor) andz-VAd-fmk (100 M, a pan-caspase inhibitor), also significantlyattenuated the taxol-induced neuronal death (TIND) (Fig. 1C).These data demonstrate that taxol induces neuronal apoptosis ina concentration-dependent manner in mixed cortical cultures, andthis result is in agreement with the report by Figueroa-Masot etal. [7]. To examine the involvement of oxidative stress in TIND, weinvestigated the effect of some antioxidants, such as trolox, ascorbicacid and tempol, on TIND. All three antioxidants significantlyinhibited TIND (Fig. 1C). These results indicate that oxidative stressis involved in the process of TIND. Vinblastine is a microtubuledisruptinganticancer drug like taxol, but it is different from taxolin that it causes depolymerization of microtubule [2]. Vinblastine(100 nM) induced about 50% neuronal cell death in corticalcultures. Vinblastine also induced chromatin condensation andFig. 2. Fluorescence imaging showing taxol-induced alteration of microtubule network and effects of trolox and apocynin on the changes of microbule network in corticalneurons. (A) Control cortical neurons. (B) At 80 min in presence of 300 nM taxol. Note taxol-induced neurite beadings (arrows). (C) Co-treated with 100 M trolox and taxolfor 80 min. (D) Co-treated with 500 M apocynin and taxol for 80 min. Calibration bar: 50 m.
Page 2 and 3: 56MethodsCell culture, reagents and
Page 4 and 5: 1314DiscussionThe prognosis of brea
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Page 8 and 9: ARTICLE IN PRESSBiochemical and Bio
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Page 14 and 15: 2present on the cell surface:LPA 1
Page 16 and 17: 4Table 1Taxol(nM)Effect of LPA on T
Page 18 and 19: 6Lysophosphatidate inhibits Taxol-i
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