Targeting cyclin B1 inhibits proliferation and sensitizes breast ...
Targeting cyclin B1 inhibits proliferation and sensitizes breast ...
Targeting cyclin B1 inhibits proliferation and sensitizes breast ...
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56MethodsCell culture, reagents <strong>and</strong> cell synchronizationCervical cancer cell line HeLa <strong>and</strong> <strong>breast</strong> cancer cell lines MCF-7, BT-474, SK-BR-3 <strong>and</strong>MDA-MB-231 were obtained from DSMZ (Braunschweig). Fetal calf serum (FCS) waspurchased from PAA laboratories (Cölbe). Opti-MEM I, oligofectamine, glutamine,penicillin, streptomycin <strong>and</strong> trypsin were obtained from Invitrogen (Karlsruhe). Taxol wasfrom Mayne Pharma (Haar). Cells were synchronized to G1/S boundary by a doublethymidineblock. Briefly, cells were treated with 2 mM thymidine (Sigma-Aldrich,of protein expression were carried out at 24 h, 48 h, 72 h <strong>and</strong> 96 h <strong>and</strong> <strong>proliferation</strong> assayswere performed at 24 h, 48 h <strong>and</strong> 72 h after siRNA transfection in MCF-7 cells.For chemotherapeutic treatment, MCF-7 cells were at first transfected with siRNA <strong>and</strong> 4 hlater followed by treatment of taxol (3 ng/ml). For irradiation, 6 h post transfection cells wereexposed to a single dose of 8 Gy at room temperature by a linear accelerator (SL 75/5, Elekta,Crawley, UK) with 6 MEV photons/100 cm focus-surface distance <strong>and</strong> a dose rate of 4.0Gy/min. 48 h after transfection of siRNAs cells were harvested for <strong>proliferation</strong> assay, cellcycle analysis <strong>and</strong> apoptosis evaluation.Taufkirchen) for 16 h, released into fresh medium for 8 h <strong>and</strong> subjected again to thymidine forfurther 16 h. To obtain prometaphase arrest, after initial thymidine incubation <strong>and</strong> 8hreleasecells were exposed to 50 ng/ml nocodazole (Sigma-Aldrich) for 14 h.Transfection of siRNA <strong>and</strong> the combined treatment with drugs or irradiationFour siRNAs targeting <strong>cyclin</strong> <strong>B1</strong> (NCBI accession number of <strong>cyclin</strong> <strong>B1</strong>: NM 031966) weresynthesized by Dharmacon Research, Inc. (Lafayette), referred to as siRNA1-4. siRNA1against <strong>cyclin</strong> <strong>B1</strong> corresponds to positions 340-360 of the <strong>cyclin</strong> <strong>B1</strong> open reading frame,siRNA2 to positions 476-496, siRNA3 to positions 776-796 <strong>and</strong> siRNA4 to positions 1302-1322. Control siRNA targeting green fluorescent protein (siGFP) was also purchased fromDharmacon. All siRNAs were 21 nucleotides in length <strong>and</strong> contained symmetric 3’ overhangsof two deoxythymidines.Cells were transfected with siRNA using transfection reagent oligofectamine, according to themanufacturer’s instructions (Invitrogen). In brief, one day prior to transfection, cells wereseeded without antibiotics to a density of 50-60%. In all experiments cells were transfectedwith siRNA1-4 or siGFP at a concentration of 10 nM. Cells were harvested 48 h after siRNAtreatmentfor cell cycle evaluation, Western blot analysis <strong>and</strong> kinase assay. The time kineticsWestern blot analysis <strong>and</strong> kinase assay in vitroCell lysis was performed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40,0.5% Na-desoxycholate, 0.1% SDS, 1 mM Na3VO4, 1 mM phenylmethylsulphonyl-fluoride(PMSF), 1 mM Dithiothreitol (DTT), 1 mM NaF, <strong>and</strong> protease inhibitor cocktail Complete(Roche, Mannheim)). Total protein was separated by using 12% sodium dodecyl sulfatepolyacrylamidegel electrophoresis (SDS-PAGE) <strong>and</strong> then transferred to Immobilon-Pmembranes (Millipore, Bedford, MA). Membranes were exposed to corresponding antibodiesfor 1 h in PBS containing 5% slim milk, washed with phosphate-buffered saline (PBS)containing 0.2 % Tween-20, incubated subsequently with secondary antibodies for 1 h.Finally, the protein b<strong>and</strong>s were visualized with the enhanced chemiluminescence reagent(ECL, Pierce, Rockford). Mouse monoclonal antibodies against <strong>cyclin</strong> <strong>B1</strong> (1:5,000), Cdk1(1:2,000), anti-mouse secondary antibodies (1:4,000) <strong>and</strong> anti-rabbit secondary antibodies(1:4,000) were purchased from Santa Cruz (Heidelberg). Rabbit polyclonal antibodies againstPARP (poly(ADP-ribose) polymerase, 1: 1000) were from Cell Signaling Technology(Beverly). Mouse monoclonal antibodies against β-actin (1:200,000) were obtained fromSigma-Aldrich. Western blots were quantified by applying a Kodak gel documentation system7(model 1D 3.5) <strong>and</strong> st<strong>and</strong>ardized with loading control. For kinase assays in vitro, antibodiesagainst <strong>cyclin</strong> <strong>B1</strong> (Santa Cruz, Heidelberg) were used for immunoprecipitation from 600 μgof cellular extracts. 0.5 μg histone H1 (Calbiochem, Darmstadt) served as substrate for eachreaction. Kinase assays were performed as previously described [29].8(Zeiss, Oberkochen). The colony number in control sample was referred as 100 % byquantification.As to experiments in vivo, HeLa cells were treated with siRNA3 or siGFP <strong>and</strong> harvested after48 h. HeLa cells (1×10 6 ) were resuspended in 300 μl of 0.9% NaCl <strong>and</strong> subcutaneouslyinjected into both flanks of nude mice. Each group contained 4 mice. Three weeks afterCell <strong>proliferation</strong>, cell cycle analysis <strong>and</strong> apoptosis assayCell viability was assessed by trypan blue staining. The <strong>proliferation</strong> rate of cells wasdetermined at indicated time points by counting cell numbers with a hemacytometer. Allexperiments were performed in triplicate. For cell cycle analysis, cells were harvested,washed with PBS <strong>and</strong> fixed in 70% chilled ethanol at 4°C for 30 min, then treated with 1mg/ml of RNase A (Sigma-Aldrich) <strong>and</strong> stained with 100 μg/ml of propidium iodide for 30min . The DNA content of 10,000 cells was determined with a fluorescent-activated cell sorterFACScan (Becton Dickinson Biosciences, Heidelberg). The data were analysed with cellcycle analysis software ModFit LT 2.0 (Verity Software House, Topsham, ME). Most of theexperiments were performed in triplicate. Indirect immunofluorescence staining forinoculation the tumor sizes were measured every 3-4 days using callipers <strong>and</strong> the tumorvolumes were calculated according to a st<strong>and</strong>ard formula: π/6 × length × width 2 . The tumorvolumes within the group were represented by the mean value. All mice were properly treatedin accordance with the guidelines of the local animal committee.Statistic analysisFor assays in vitro, Student’s t-tests were used to evaluate the significance of differencebetween control cells <strong>and</strong> siRNAs-treated cells. Differences were considered as statisticallysignificant when p< 0.05. With xenograft mouse model, the significant difference between thesiGFP-treated group <strong>and</strong> siRNA3-treated group was analyzed by Mann-Whitney U test.subcellular <strong>cyclin</strong> <strong>B1</strong> localization <strong>and</strong> DNA were carried out as previously described [29].Apoptosis was assessed using Vybrant TM apoptosis assay kit according to the manufacturer’sinstructions (Molecular Probes, Leiden).Colony formation assay <strong>and</strong> in vivo experimentsMCF-7 cells were treated with siRNA1-3 or siGFP for 48 h <strong>and</strong> harvested for colonyformation assays. Briefly, cells were seeded in 24 well-plates at a density of 200 cells/wellinto culture medium containing 0.3% agar (Roth, Karlsruhe) overlaying 0.5% agar. Cells werecultured at 37°C with 5% CO2, <strong>and</strong> colonies were counted 4 weeks later using a microscope
9ResultsCancer cell lines lend themselves as useful models to further our underst<strong>and</strong>ing ofgynecological cancers such as <strong>breast</strong> <strong>and</strong> cervical cancer. To study the function of <strong>cyclin</strong> <strong>B1</strong>in <strong>breast</strong> cancer cells we selected cancer cell lines MCF-7, BT-474, SK-BR-3 <strong>and</strong> MDA-MB-231, as they represent the best characterized cell lines for <strong>breast</strong> cancer research. While MCF-7 <strong>and</strong> BT-474 express both estrogen receptor (ER) <strong>and</strong> progesterone receptor (PgR), SK-BR-3<strong>and</strong> MDA-MB-231 cells lack those receptors [30]. Unlike SK-BR-3 <strong>and</strong> BT-474, which areHer-2/neu-positive, MCF-7 cells only exhibit a basal level of Her-2/neu [30]. In addition,MCF-7 cells express high amounts of markers typical of the luminal epithelia phenotype of<strong>breast</strong> cells, whereas BT-474 <strong>and</strong> SK-BR-3 cells exhibit a weakly luminal epithelia-likephenotype. Distinct from the two phenotypes above, MDA-MB-231 represents a highlyinvasive “mesenchymal-like“ <strong>breast</strong> cancer cell line by expressing a high level of vimentin.In addition, the cervical cancer cell line HeLa was selected, because it represents the mostextensively studied cancer cell line thus far.Specific downregulation of <strong>cyclin</strong> <strong>B1</strong> with siRNAWe were at first interested whether <strong>breast</strong> cancer cell lines are similarly sensitive to specificdownregulation of <strong>cyclin</strong> <strong>B1</strong> by siRNA. As shown in Fig. 1A, while the protein level of<strong>cyclin</strong> <strong>B1</strong> in MDA-MB-231 was almost undetectable after treatment with siRNA1 or siRNA3,33% <strong>and</strong> 16% of <strong>cyclin</strong> <strong>B1</strong> were still detectable in MCF-7 cells after treatment with siRNA1<strong>and</strong> siRNA3, respectively, relative to the protein level of <strong>cyclin</strong> <strong>B1</strong> in control cells. In contrastto <strong>cyclin</strong> <strong>B1</strong>, β-actin was not affected. The protein level of Cdk1, the catalytic partner of<strong>cyclin</strong> <strong>B1</strong>, hardly changed, indicating <strong>cyclin</strong> <strong>B1</strong> knockdown by siRNAs was specific.Treatment of BT-474 <strong>and</strong> SK-BR-3 cells with siRNA1 <strong>and</strong> siRNA3 also reduced <strong>cyclin</strong> <strong>B1</strong>levels, albeit to a lower extent as compared to MDA-MB-231 <strong>and</strong> MCF-7 cells.10staining with monoclonal specific antibodies against <strong>cyclin</strong> <strong>B1</strong> (data not shown). Furthermore,in consistence with downregulation of <strong>cyclin</strong> <strong>B1</strong> protein, the kinase activity of Cdk1/<strong>cyclin</strong><strong>B1</strong> was decreased to 28% in cellular extracts from siRNA1-treated BT-474 cells as comparedto siGFP-treated cells (Fig. 1B). Similar results were also obtained in MCF-7 cells aftersiRNA administration (data not shown).Proliferation is inhibited in <strong>breast</strong> cancer cells with reduced <strong>cyclin</strong> <strong>B1</strong>As Cdk1/<strong>cyclin</strong> <strong>B1</strong> is essential for the initiation of mitosis <strong>and</strong> required for cell division wesubsequently studied the impact of <strong>cyclin</strong> <strong>B1</strong> downregulation on cell <strong>proliferation</strong> rate.Expectedly, a reduced <strong>proliferation</strong> was observed in all four <strong>breast</strong> cancer cell lines after 48 htreatment with siRNAs as compared to control cells treated with siGFP (Fig. 2). In particular,MCF-7 cells exhibited a strong inhibition of <strong>proliferation</strong>, followed by MDA-MB-231, BT-474 <strong>and</strong> SK-BR-3 cells after 48 h siRNA1-3 treatment against <strong>cyclin</strong> <strong>B1</strong> (Fig. 2A-D, upperpanels). Analyses of cell cycle distribution displayed an accumulation of cells in G2/M phase,suggestive of a G2/M arrest, after treatment with siRNA1-3 targeting <strong>cyclin</strong> <strong>B1</strong> (Fig. 2A-D,lower panels).Time kinetics of siRNA treatment in MCF-7 cellsIn order to study the effect of cycin <strong>B1</strong> knockdown on <strong>proliferation</strong>, we subjected MCF-7cells to more detailed time dependent analysis. MCF-7 cells were treated with siRNA1 orsiRNA3 <strong>and</strong> harvested for Western blot analysis <strong>and</strong> <strong>proliferation</strong> assay at the indicated timepoints. The reduction of <strong>cyclin</strong> <strong>B1</strong> protein levels were evident 24 h after siRNA1/3 treatmentas compared to control cells (Fig. 3A). This effect increased dramatically with time <strong>and</strong> after96 h <strong>cyclin</strong> <strong>B1</strong> protein became undetectable. In line with the reduction of <strong>cyclin</strong> <strong>B1</strong>, theDownregulation of <strong>cyclin</strong> <strong>B1</strong> was also corroborated by indirect immunofluorenscence<strong>proliferation</strong> rate of MCF-7 cells was clearly inhibited at 24 h with siRNA3 treatment <strong>and</strong> theinhibition became more striking with longer exposure to siRNA1 (Fig. 3B).11investigated the combination of knockdown of <strong>cyclin</strong> <strong>B1</strong> with irradiation. Irradiationenhanced the G2/M population <strong>and</strong> induced more apoptosis in <strong>cyclin</strong> <strong>B1</strong> reduced MCF-712cells, compared to control cells (data not shown).Suppression of <strong>cyclin</strong> <strong>B1</strong> renders cells more susceptible to taxolTaxol (Paclitaxel ® ), a taxane frequently used in multidrug regiments for the therapy of severalImpaired colony-forming ability <strong>and</strong> inhibited tumor growthsolid tumors, binds to the β-subunit of tubulin,thereby impairing the dynamics ofAnchorage independent cell growth is one of the hallmarks of malignant tumor cells. Givenmicrotubules by promoting their polymerization, leading to mitotic arrest <strong>and</strong> apoptosis [31].In order to explore <strong>cyclin</strong> <strong>B1</strong> knockdown as a possible combination with taxol, we transfectedMCF-7 cells with siRNA3 <strong>and</strong> followed by further incubation with taxol. Cells wereharvested 48 h posttransfection. As depicted in Fig. 4A, <strong>cyclin</strong> <strong>B1</strong> protein level was stronglyreduced after siRNA3 treatment. Cyclin <strong>B1</strong> level was also decreased after treatment withtaxol at a low dosage of 3 ng/ml, possibly due to the induction of apoptosis, <strong>and</strong> almostdisappeared when cells were pre-treated with siRNA3 (Fig. 4A, upper panel). Indeed, PARP(poly(ADP-ribose) polymerase) was cleaved in cells treated with taxol <strong>and</strong> more stronglycleaved when siRNA3 was used together with taxol (Fig. 4A, middle panel), indicating thatthe combined treatment triggers more robust apoptotic response. In addition, <strong>proliferation</strong> wasinhibited to a higher extent after the combined treatment (Fig. 4B). The results were furthercorrelated with the cell cycle analysis: a G2/M population was more prominent in MCF-7cells after the combined treatment, compared to cells exposed to siRNA3 alone (Fig. 4C). Thedata indicate that the combined therapy activates more strongly caspase-3 independentapoptotic pathways in MCF-7 cells as the caspase-3 gene is deleted in MCF-7 cells [32].the notion of <strong>cyclin</strong> <strong>B1</strong> deregulation being involved in neoplastic transformation <strong>and</strong>associated with malignancy grade of tumors, we wondered whether knockdown of <strong>cyclin</strong> <strong>B1</strong>protein might translate into reduced colony-forming ability in MCF-7 cells. As shown in Fig.5A, the colony numbers of MCF-7 cells treated with siRNA1-3 were strongly reduced, inparticular, in MCF-7 cells treated with siRNA3, compared to controls.Cervical carcinoma HeLa cells express a high level of <strong>cyclin</strong> <strong>B1</strong> (data not shown). In cellculture, only 25-30% HeLa cells were left after 48 h treatment with siRNA3 targeting <strong>cyclin</strong><strong>B1</strong> (data not shown). To further address whether <strong>cyclin</strong> <strong>B1</strong> is required for aggressive growthof tumors in vivo, a xenograft experiment with HeLa cells was performed in nude mice. Micewere inoculated with HeLa cells treated 48 h with siRNA3 targeting <strong>cyclin</strong> <strong>B1</strong> or siGFP. Asshown in Fig. 5B, tumor growth of HeLa cells treated with siRNA3 prior to inoculation waseffectually retarded, in comparison with the growth of control HeLa tumors. The data suggestthat <strong>cyclin</strong> <strong>B1</strong> is indeed required in vivo for promoting <strong>proliferation</strong> of tumor cells <strong>and</strong> thereduction of <strong>cyclin</strong> <strong>B1</strong> slows down the tumor growth.Similar results were also obtained in MDA-MB-231 cells: while siRNA3 or taxol alonereduced <strong>proliferation</strong> by 33% <strong>and</strong> 31%, respectively, relative to siGFP treatment, thecombination of siRNA3 with taxol resulted in a 65% reduction of <strong>proliferation</strong> (data notshown). Thus, the combined action of <strong>cyclin</strong> <strong>B1</strong> knockdown together with taxol enhancesantiproliferative <strong>and</strong>proapoptotic responses in <strong>breast</strong> cancer cells. Furthermore, we
1314DiscussionThe prognosis of <strong>breast</strong> <strong>and</strong> cervical cancer patients has been improved during recent years,related partly to sophisticated surgery, radiotherapy <strong>and</strong> adjuvant systemic therapy. Despitethese advances, these cancers remain major clinical problems by causing considerablemorbidity <strong>and</strong> mortality in women worldwide. Apart from the st<strong>and</strong>ard approaches, novelpotent molecular agents for anticancer therapy are in great dem<strong>and</strong>.In this communication we show that the knockdown of <strong>cyclin</strong> <strong>B1</strong>, the regulatory subunit ofCdk1, inhibited cell <strong>proliferation</strong> <strong>and</strong> induced apoptosis in various <strong>breast</strong> <strong>and</strong> cervical cancercell lines. Importantly, siRNA mediated <strong>cyclin</strong> <strong>B1</strong> knockdown in combination withchemotherapeutical agent taxol, enhanced the antiproliferative effect on <strong>breast</strong> cancer cells.Interestingly, the reduction of <strong>cyclin</strong> <strong>B1</strong> in MCF-7 cells impaired colony-forming ability, ahallmark of malignancy in tumor cells. Moreover, while control HeLa cells wereprogressively growing, the tumor growth of HeLa cells treated with siRNA targeting <strong>cyclin</strong><strong>B1</strong> prior to inoculation was strongly inhibited in nude mice, indicating <strong>cyclin</strong> <strong>B1</strong> isindispensable for tumor growth in vivo. Taken together, the data strengthen the notion of<strong>cyclin</strong> <strong>B1</strong> being required for the survival <strong>and</strong> <strong>proliferation</strong> of <strong>breast</strong> <strong>and</strong> cervical cancer cells<strong>and</strong> depletion/downregulation of <strong>cyclin</strong> <strong>B1</strong> <strong>inhibits</strong> <strong>proliferation</strong> of cancer cells in vitro aswell as in vivo.Recent genetic evidence demonstrates that Cdk1 is the only Cdk sufficient to drive themammalian cell cycle because embryos from Cdk1 - / - mice fail to develop to the morula <strong>and</strong>blastocyst stages, whereas mouse embryos lacking all interphase Cdks (Cdk2, Cdk3, Cdk4<strong>and</strong> Cdk6) undergo organogenesis <strong>and</strong> develop to midgestation [33]. These data underscorethat Cdk1 is essential for cell cycle regulation <strong>and</strong> a major force driving cell <strong>proliferation</strong>.Cyclin <strong>B1</strong>, the regulatory subunit of Cdk1, controls the activity of Cdk1 as it associates with<strong>and</strong> thereby activates Cdk1, regulates its nuclear translocation <strong>and</strong> passively mediates itsinactivation when <strong>cyclin</strong> <strong>B1</strong> is degraded at anaphase transition. Cyclin <strong>B1</strong> is fundamental forcell <strong>proliferation</strong>. Uncontrolled expression of <strong>cyclin</strong> <strong>B1</strong> is associated with neoplastictransformation <strong>and</strong> gynecological cancer development [5,9,11,34,35]. Overexpression of<strong>cyclin</strong> <strong>B1</strong> is believed to confer therapy resistance [8,11]. Thus, targeting <strong>cyclin</strong> <strong>B1</strong>, leadingconsequently to the inactivation of Cdk1, could be a promising specific strategy for cell cycleintervention against <strong>breast</strong> <strong>and</strong> cervical cancer.In this work, as a proof-of-concept, the RNA interference was used to downregulate/deplete<strong>cyclin</strong> <strong>B1</strong> <strong>and</strong> a clear antiproliferative effect was observed in all cancer cell lines studied.Among the <strong>breast</strong> cancer cell lines investigated, MCF-7 cells exhibited the strongestinhibitory effect on cell <strong>proliferation</strong> after <strong>cyclin</strong> <strong>B1</strong> siRNA treatment, followed by MDA-MB-231, SK-BR-3 <strong>and</strong> BT-474 cells (Fig. 2), which possibly correlates with the <strong>cyclin</strong> <strong>B1</strong>level in exponential growing status of each cell line (data not shown). Although the proteinlevel of <strong>cyclin</strong> <strong>B1</strong> in MDA-MB-231 cells was nearly undetectable after siRNA1 or siRNA3transfection, the inhibitory impact was moderate (Fig. 2D), suggesting the <strong>proliferation</strong> ofMDA-MB-231 cells is not necessarily dependent on the normal level of <strong>cyclin</strong> <strong>B1</strong> <strong>and</strong> thelittle amount of remaining <strong>cyclin</strong> <strong>B1</strong> might be sufficient for the survival of MDA-MB-231cells. Finally, SK-BR-3 <strong>and</strong> BT-474 cells were also not as sensitive to siRNA treatment (Fig.2B <strong>and</strong> 2C) as HeLa or MCF-7 cells. This could be due to the cellular context of SK-BR-3<strong>and</strong> BT-474 cells, e.g. Her-2/neu+, which very often leads to a hormone-independent<strong>proliferation</strong> of cells. Thus, unlike in MCF-7 cells, targeting Her-2/neu or other factorspromoting G1/S transition could be more effective for inhibiting cell cycle progression in SK-BR-3 <strong>and</strong> BT-474 cells, which has been shown by our previous study [36]. On that account,specific targeting of oncogene(s) in individual cancer cell lines, like Her-2/neu in SK-BR-3<strong>and</strong> BT-474 cells, or <strong>cyclin</strong> <strong>B1</strong> in MCF-7, could improve <strong>breast</strong> cancer therapy. Collectively,downregulation/depletion of <strong>cyclin</strong> <strong>B1</strong> worked effectively in all gynecological cancer celllines tested. However, only in some cell lines, such as MCF-7 <strong>and</strong> HeLa, <strong>cyclin</strong> <strong>B1</strong>15knockdown resulted in a strong proliferative inhibition, most likely because <strong>proliferation</strong> inthose cell lines is more dependent on high <strong>cyclin</strong> <strong>B1</strong> levels as compared to other cell lines.Taxane drugs represent the most important class of anticancer agents <strong>and</strong> are integrated in16as well as in vivo <strong>and</strong> <strong>sensitizes</strong> <strong>breast</strong> cancer cells to taxol. The data indicate that thecombination of reducing <strong>cyclin</strong> <strong>B1</strong> with chemotherapeutic drugs could be a new strategy formolecular intervention in a subset of <strong>breast</strong> cancers.multidrug-regiments for the therapy of several solid tumors including gynecological cancers.Despite their relevant contribution in ameliorating the quality of life <strong>and</strong> overall survival ofcancer patients, drug resistance <strong>and</strong> site-effects hamper its wide usage. Therefore, it isdesirable to find new ways of lowering drug dosage without losing effectiveness to limit sideeffects<strong>and</strong> possibly also to slow down drug resistance. In this work, <strong>cyclin</strong> <strong>B1</strong> siRNA incombination with taxol, blocking entry into mitosis <strong>and</strong> targeting the transition of metaphaseto anaphase, respectively, demonstrated a high efficacy in inhibiting <strong>proliferation</strong> of MCF-7cells. The data suggest that specific targeting of <strong>cyclin</strong> <strong>B1</strong> could sensitize some gynecologicalcancer cells, like MCF-7 <strong>and</strong> MDA-MB-231 cells, to conventional chemotherapeutic agentslike taxol, thereby reducing their side-effects by lowering their dosage.Taken together, the data from this work further strengthen the notion that <strong>cyclin</strong> <strong>B1</strong> could bean attractive target for potential anticancer therapy. Inhibiting <strong>cyclin</strong> <strong>B1</strong> function incombination with chemotherapeutic drugs could reinforce the antiproliferative effect in asubset of cancers. As RNA interference still faces the major challenge of systematic delivery[37], an alternative strategy could be small molecule inhibitors targeting <strong>cyclin</strong> B, as itscrystal structure is recently published [38]. In parallel to Cdk inhibitors, which have beenextensively under clinical investigations, small molecule inhibitors against <strong>cyclin</strong> <strong>B1</strong> couldopen up a new door for specific molecular cancer therapy by interfering with its proteinstability, binding capacity to Cdk1 or its subcellular localization.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsIA <strong>and</strong> AK conducted cell cycle analyses, <strong>proliferation</strong> assays, Western blot analyses,apoptosis assays <strong>and</strong> mouse xenograft experiments in vivo. RY performed the kinetics ofMCF-7 cells <strong>and</strong> soft-agar assays. FR is involved in the combination therapy <strong>and</strong> assays. MK<strong>and</strong> RG coordinated this project. KS co-supervised this study <strong>and</strong> supported the manuscriptwriting. JY designed <strong>and</strong> supervised this study, <strong>and</strong> drafted the manuscript. All the authorsread <strong>and</strong> approved the final manuscript.AcknowledgementsThis work was supported by the Deutsche Krebshilfe (#107594). We gratefully acknowledgethe help of Dr. F. Eckerdt (Howard Hughes Medical Institute <strong>and</strong> Department ofPharmacology, University of Colorado, School of Medicine), who improved <strong>and</strong> modified themanuscript. We thank also Dr. S. Kappel for critical reading the manuscript. We are gratefulto Ms H. Beschmann <strong>and</strong> Ms S. Diehl, Department of Dermatology, University of Frankfurt,for supporting apoptotic analysis.ConclusionsThis work demonstrates that <strong>cyclin</strong> <strong>B1</strong> is required for survival <strong>and</strong> <strong>proliferation</strong> of <strong>breast</strong> <strong>and</strong>cervical cancer cells. Downregulation of <strong>cyclin</strong> <strong>B1</strong> <strong>inhibits</strong> <strong>proliferation</strong> of tumor cells in vitro
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Faltus T, Yuan J, Zimmer B, Kramer A, Loibl S, Kaufmann M, Strebhardt K:Silencing of the HER2/neu gene by siRNA <strong>inhibits</strong> <strong>proliferation</strong> <strong>and</strong> inducesapoptosis in HER2/neu-overexpressing <strong>breast</strong> cancer cells. Neoplasia 2004, 6:786-795.37. Bumcrot D, Manoharan M, Koteliansky V, Sah DW: RNAi therapeutics: a potentialnew class of pharmaceutical drugs. Nat Chem Biol 2006, 2:711-719.38. Petri ET, Errico A, Escobedo L, Hunt T, Basavappa R: The crystal structure ofhuman <strong>cyclin</strong> B. Cell Cycle 2007, 6:1342-1349.1920Figure legendsFigure 1 - Reduced <strong>cyclin</strong> <strong>B1</strong> protein <strong>and</strong> kinase activity of Cdk1/<strong>cyclin</strong> <strong>B1</strong>A. Upper panel: Breast cancer cells MCF-7, BT-474, SK-BR-3 <strong>and</strong> MDA-MB-231 cells weretreated with <strong>cyclin</strong> <strong>B1</strong> siRNA1, siRNA3, GFP siRNA (GFP) or without treatment (C) for 48h. Cells were harvested <strong>and</strong> cellular lysates were prepared for Western blot analyses withantibodies targeting cylin <strong>B1</strong>, Cdk1 <strong>and</strong> β-actin. The later served as loading control. Lowerpanel: Quantification of <strong>cyclin</strong> <strong>B1</strong> levels from Western blots (upper panel), normalized to β-actin. B. Kinase assay of Cdk1/<strong>cyclin</strong> <strong>B1</strong> in vitro. BT-474 cells were treated with <strong>cyclin</strong> <strong>B1</strong>siRNA1 or siGFP or without treatment (C). 24 h later cells were lysed <strong>and</strong> Cdk1/<strong>cyclin</strong> <strong>B1</strong>complex was immunoprecipitated by using antibodies against <strong>cyclin</strong> <strong>B1</strong> from cellularextracts. Normal IgG served as negative control. Kinase assays were carried out with theprecipitates in the presence of histone H1 as substrate.Figure 2 – Inhibited <strong>proliferation</strong> after downregulation of <strong>cyclin</strong> <strong>B1</strong>MCF-7 (A), BT-474 (B), SK-BR-3 (C) <strong>and</strong> MDA-MB-231 cells (D) were treated withsiRNA1-3, or siGFP or oligofectamine alone (OF). After 48 h cells were harvested for cellnumber counting (upper panels in Fig. 3 A-D) <strong>and</strong> for cell cycle analysis (lower panels in Fig.3 A-D). Cells without any treatment (C) served as control. The results of cell numbers areexpressed as mean ± SD (n = 3) <strong>and</strong> statistically analysed. *P < 0.05, **P < 0.01.Figure 3 - Time kinetics in MCF-7 cellsMCF-7 cells were treated with <strong>cyclin</strong> <strong>B1</strong> siRNA1, siRNA3, siGFP or without treatment (C).Treated cells were harvested at different time points as indicated for Western blot analyses
21with antibodies against <strong>cyclin</strong> <strong>B1</strong>, Cdk1 <strong>and</strong> β-actin (A) <strong>and</strong> for cell number determinations(B). The results of cell numbers are expressed as mean ± SD (n =3).Figure 4 – More sensitive to taxol in MCF-7 cells with reduced <strong>cyclin</strong> <strong>B1</strong>A. MCF-7cellsweretransfectedwithsiRNA3orsiGFP<strong>and</strong>4hlaterfollowedwithtreatmentAMCF-7BT-474SK-BR-3MDA-MB-231<strong>cyclin</strong> <strong>B1</strong>of taxol (C+, siGFP+ <strong>and</strong> siRNA3+) or without taxol (C, siGFP <strong>and</strong> siRNA3). 48 h aftertransfection cells were harvested for Western blot analyses with antibodies as indicated. β-actin served as loading control. PARP: poly (ADP-ribose) polymerase. B. Cells were treatedas in A <strong>and</strong> cell numbers were counted. The results are expressed as mean ± SD (n =3)<strong>and</strong>statistically analysed. *P < 0.05. C. MCF-7 cells were treated as in A <strong>and</strong> cell cycle wasanalyzed (upper panel) <strong>and</strong> distribution of cell cycle population was quantified (lower panel).Figure 5 - Impaired colony-forming ability <strong>and</strong> reduced tumor growthA. Colony formation assay. MCF-7 cells were treated with siRNA1-3 or siGFP. 48 h aftertransfection cells were transferred to soft-agar plates for further incubation. 4 weeks later thecolony numbers were scored with a microscope. The colony number of control cells wasregarded as 100% for calculation. The data were expressed as mean ± SD (n =3)<strong>and</strong>analysed by Student’s t-test. *P < 0.05, **P < 0.01. B. Xenograft experiment in nude mice.1×10 6 of HeLa cells treated with siRNA3 or control siGFP were subcutaneously injected intoeach flank of nude mice. Each group contained 4 mice. Tumor sizes were measured every 2-3days <strong>and</strong> tumor volumes were calculated. The data were statistically analysed by Mann-<strong>cyclin</strong> <strong>B1</strong> relativeprotein level<strong>B1</strong>,61,20,80,40Figure 1MCF-7CsiGFPsiRNA1siRNA3CsiGFPsiRNA1siRNA3relative kinase activity(%)16012080400CsiGFPsiRNA1siRNA3CCBT-474CsiGFPsiRNA1siRNA3siGFPCsiGFPsiRNA1siRNA3SK-BR-3siGFPsiRNA1siRNA3siRNA1CBT-474MDA-MB-231IgGsiGFPsiRNA1siRNA3CsiGFPsiRNA1siRNA3β-actinCdk1Whitney U test. *P < 0.05, **P < 0.01.A10 4 cells/well%201510506040200MCF-7∗ ∗∗C OF siGFP siRNA1 siRNA2 siRNA3MCF-7C OF siGFP siRNA1 siRNA2 siRNA3BG0/G1SG2/M10 4 cells/well%302520151080604020050BT-474∗C OF siGFP siRNA1 siRNA2 siRNA3BT-474C OF siGFP siRNA1 siRNA2 siRNA3A<strong>B1</strong>0.0CsiGFPCsiRNA1siRNA3siGFP24 h 48 h72 h 96 hCsiRNA1siRNA3siGFPCsiRNA1siRNA3siGFPsiRNA1siRNA3<strong>cyclin</strong> <strong>B1</strong>β-actinCdk1C10 4 cells/well161284SK-BR-3∗D10 4 cells/well302010∗MDA-MB-231∗10 4 cells/well7.55.02.524 h 48 h 72 h060%40C OF siGFP siRNA1 siRNA2 siRNA3SK-BR-3G0/G1SG2/M%06040C OF siGFP siRNA1 siRNA2 siRNA3MDA-MB-2310Figure 3CsiGFPsiRNA1siRNA3CsiGFPsiRNA1siRNA3CsiGFPsiRNA1siRNA320200Figure 2C OF siGFP siRNA1 siRNA2 siRNA30C OF siGFP siRNA1 siRNA2 siRNA3
ACsiGFPsiRNA3C+siGFP+siRNA3+<strong>cyclin</strong> <strong>B1</strong>PARPcleaved PARPA30MCF-7BC10 4 cells/well403020100CsiGFPβ-actin- - - + + + taxol (3 ng/ml)MCF-7siRNA3 C+ siGFP+ siRNA3+∗Btumor volume (x10 3 mm)colony number201004003002001000∗ ∗ ∗∗C OF siGFP siRNA1 siRNA2 siRNA3siGFPsiRNA3∗∗ P
ARTICLE IN PRESSBiochemical <strong>and</strong> Biophysical Research Communications xxx (2009) xxx–xxxContents lists available at ScienceDirectBiochemical <strong>and</strong> Biophysical Research Communicationsjournal homepage: www.elsevier.com/locate/ybbrcNon-anti-mitotic concentrations of taxol reduce <strong>breast</strong> cancer cell invasivenessTruong-An Tran a,b,1 , Ludovic Gillet a,b,1 , Sébastien Roger a,b , Pierre Besson a,b ,Edward White c , Jean-Yves Le Guennec a,b, *a Inserm, U921, 37000 Tours, Franceb Université François-Rabelais, 37000 Tours, Francec Institute of Membrane <strong>and</strong> Systems Biology, University of Leeds, LS2 9JT LEEDS, UKarticleinfoabstractArticle history:Received 9 December 2008Available online xxxxKeywords:TaxolTubulinCancer invasivenessNa V 1.5MetastasisMDA-MB-231MDA-MB-468Taxol is widely used in <strong>breast</strong> cancer chemotherapy. Its effects are primarily attributed to its anti-mitoticactivity. Microtubule perturbators also exert antimetastatic activities which cannot be explained solelyby the inhibition of <strong>proliferation</strong>. Voltage-dependent sodium channels (Na V ) are abnormally expressedin the highly metastatic <strong>breast</strong> cancer cell line MDA-MB-231 <strong>and</strong> not in MDA-MB-468 cell line. InhibitingNa V activity with tetrodotoxin is responsible for an approximately 0.4-fold reduction of MDA-MB-231 cellinvasiveness. In this study, we focused on the effect of a single, 2-h application of 10 nM taxol on the twocell lines MDA-MB-231 <strong>and</strong> MDA-MB-468. At this concentration, taxol had no effect on <strong>proliferation</strong> after7 days <strong>and</strong> on migration in any cell line. However it led to a 40% reduction of transwell invasion of MDA-MB-231 cells. There was no additive effect when taxol <strong>and</strong> tetrodotoxin were simultaneously applied.Na V activity, as assessed by patch-clamp, indicates that it was changed by taxol pre-treatment. We concludethat taxol can exert anti-tumoral activities, in cells expressing Na V , at low doses that have no effecton cell <strong>proliferation</strong>. This effect might be due to a modulation of signalling pathways involving sodiumchannels.Ó 2008 Elsevier Inc. All rights reserved.Taxol <strong>and</strong> its derivatives are widely used in the treatment of<strong>breast</strong> cancers [1]. The mechanism for their beneficial effects isthought to be the stabilization of microtubules during mitosiscausing a blockade of cell division [2]. This blockade can also involveparticular potassium channels, EAGs, known to be regulatedby the cytoskeleton <strong>and</strong> involved in the control of cell <strong>proliferation</strong>[3]. However, the effect of taxol on cell invasiveness, an importantparameter linked to the capacity of cells to produce metastases,has not been evaluated in <strong>breast</strong> cancer cells.Voltage-dependent sodium channels are known to be involvedin <strong>breast</strong> <strong>and</strong> lung cancer cell invasiveness [4,5]. These proteinshave been found in biopsies from metastatic tissues <strong>and</strong> are thusthought to be involved in the development of secondary tumours[6]. In <strong>breast</strong> cancer, the isoform expressed is Na V 1.5 [4,7]. Thischannel is expressed in the <strong>breast</strong> cancer cell line MDA-MB-231<strong>and</strong> not in MDA-MB-468 [4]. It promotes cell invasiveness <strong>and</strong> islikely to be controlled by intracellular regulators such as proteinkinases <strong>and</strong>/or cytoskeletal elements [4,8]. Depending on the cell* Corresponding author. Address: Inserm U921, N2C, Université François-Rabelais,Faculté de Médecine, 10 Bd Tonnellé, 37032 Tours, France. Fax: +33 2 47 36 6226.E-mail address: Jean-Yves.LeGuennec@Univ-Tours.Fr (J.-Y Le Guennec) .1 These authors contributed equally to this work.type in which it is expressed, Na V 1.5 is, or is not, sensitive to microtubuleperturbators [9].In the present study, we investigated whether stabilizing microtubulesby taxol regulates the invasiveness of MDA-MB-231 <strong>and</strong>MDA-MB-468 cells. Cell invasiveness was assessed by measuringtranswell migration on Matrigel Ò -coated inserts. The effect of taxolon Na V activity was evaluated in patch-clamp. The proportions offree vs polymerized tubulin were assessed by Western blot. Wefound that pre-treating the cells with a low concentration of taxol(10 nM) slightly increased microtubule polymerization. The effectsof TTX <strong>and</strong> taxol on cell invasivity were not additive. We concludethat taxol is responsible for the reduced invasion of MDA-MB-231cells through a mechanism interfering with Na V 1.5 signalling atdoses which do not interfere with cell mitosis.Materials <strong>and</strong> methodsCell culture. The human <strong>breast</strong> cancer cells MDA-MB-231 <strong>and</strong>MDA-MB-468 were obtained from the ATCC (Manassas, Virginia,USA) <strong>and</strong> cultured in Dulbecco’s modified Eagle’s medium (DMEM,Lonza, France) supplemented with 5% FCS at 37 °C in a humidified5% CO 2 incubator. For all experiments, cells were pre-treated for2 h with concentrations of taxol ranging from 10 nM (low concentration)to 10 lM (high concentration). Taxol was then removedprior to performing the different assays (<strong>proliferation</strong>, migration0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.bbrc.2008.12.073Please cite this article in press as: T. -A. Tran et al., Non-anti-mitotic concentrations of taxol reduce <strong>breast</strong> cancer cell invasiveness, Biochem.Biophys. Res. Commun. (2009), doi:10.1016/j.bbrc.2008.12.073
ARTICLE IN PRESS2 T.-A. Tran et al. / Biochemical <strong>and</strong> Biophysical Research Communications xxx (2009) xxx–xxx<strong>and</strong> invasion). Drugs <strong>and</strong> chemicals were purchased from Sigma–Aldrich (France) <strong>and</strong> Latoxan (France).Cell survival <strong>and</strong> <strong>proliferation</strong>. Cells were seeded at 4 10 4 cellsper well in 12 wells of a 24-well plate for a given condition (includinguntreated controls) on two separate experiments <strong>and</strong> weregrown for a total of 5 days. The culture medium was changed everyother day. Growth <strong>and</strong> viability of cells were measured as a wholeby the tetrazolium salt assay [10] as previously described [4]. Cell<strong>proliferation</strong> was expressed as formazan 570 nm absorbance <strong>and</strong>expressed as a ratio of treated to control values.Migration <strong>and</strong> in vitro invasion assay. To evaluate migration <strong>and</strong>invasion, we performed transwell migration assays using inserts aspreviously described [4]. In these assays, we evaluated the capacityof cells to move through holes smaller than the cell diameter undera chemotactic gradient. To distinguish between migration <strong>and</strong>invasion, inserts were either devoid of, or coated with Matrigel Ò .Therefore, in invasion assays, cells had to digest Matrigel Ò beforemigrating through the pores. In our experiments, we evaluated24 h migration <strong>and</strong> invasion. Cells were pre-treated for 2 h withthe different concentrations of taxol, washed <strong>and</strong> then seeded intothe inserts <strong>and</strong> were not in contact with taxol for the duration ofthe test. Migration was analyzed in 24-well plates with 8 lm poresize polyethylene terephtalate membrane cell culture inserts (Becton–Dickinson,France). The upper compartment was seeded with4 10 4 cells. The lower compartment was filled with culture mediumsupplemented with 10% FCS (fetal calf serum) as a chemoattractant.After 24 h at 37 °C, remaining cells were removed fromthe upper side of the membrane <strong>and</strong> cells that had migrated <strong>and</strong>were attached to the lower side were stained with haematoxylin<strong>and</strong> counted on the whole insert, using a light microscope at200 magnification [4].In vitro invasion was assessed using the same inserts <strong>and</strong> thesame protocol as above but with the membrane covered with afilm of Matrigel Ò (Becton–Dickinson, France), mimicking the extracellularmatrix. Migration <strong>and</strong> invasion assays were performed intriplicate in eight separate experiments. For comparison betweenconditions, results obtained for migration <strong>and</strong> invasion were normalizedto the control condition.Polymerization of microtubules. The evaluation of the polymerizationof microtubules was performed by Western blot analysis[11]. Specific primary monoclonal antibodies were directed towarda-tubulin (Sigma–Aldrich, France). MDA-MB-231 cells, in theirsubconfluent state, were collected by trypsinization. After centrifugation(1000g for 5 min), the culture medium was replaced by amicrotubule stabilizing buffer [11,12]. After further centrifugations(two at 30,000g for 1 h 30 min), the free <strong>and</strong> polymerized tubulinfractions were collected in the supernatants [11,12]. The total proteinconcentrations of cell lysates were determined using thebicinchoninic acid method (Bio-Rad, USA) (triplicate experiments).Protein samples (20 lg per lane) were diluted in reducing (DTT)<strong>and</strong> denaturing (SDS) sample buffer, boiled for 3 min, separatedby SDS–PAGE on 10% gels [13], then transferred onto a PVDF membrane(Millipore, USA). After saturation for 2 h in 5% non-fat milkTBS solution containing 0.5% Tween 20 <strong>and</strong> 5% non-fat milk(TBST5), the membrane was incubated overnight at 4 °C with theprimary antibody (1/1000) in a 2% non-fat milk TBST5 solution.The membrane was further incubated for 1 h at room temperaturewith a goat anti-mouse (1/1000 in TBST5 solution) horseradishperoxidase-conjugated secondary antibody (Sigma–Aldrich,France). Finally, immunoblots were revealed with the ECL immunodetectionsystem (Amersham Biosciences, UK). Pre-stainedmolecular masses were from Bio-Rad. After washing to removeECL, the membrane was incubated (2 h at room temperature) withb-actin specific monoclonal antibodies (Santa Cruz, USA) to correctfor protein quantity in each well. Immunoblots were revealed withthe ECL immunodetection system a second time.Electrophysiology. Patch pipettes were pulled from borosilicateglass to a resistance of 4–6 MX. Currents were recorded, in rupturedwhole-cell configuration, under voltage-clamp mode at room temperatureusing an Axopatch 200 B patch-clamp amplifier (AxonInstrument, USA). Analogical signals were filtered at 5 kHz, <strong>and</strong> sampledat 10 kHz using a 1322A Digidata converter. Cell capacitance<strong>and</strong> series resistances were electronically compensated by about60%. The P/2 sub-pulse correction of cell leakage <strong>and</strong> capacitancewas used to study Na + current (I Na ). Sodium currents were recordedby depolarizing the cells from a holding potential of 100 mV to amaximal test pulse of 5 mV for 30 ms every 500 ms. The protocolAMDA-MB-231Relative <strong>proliferation</strong> (%)BMDA-MB-231Relative migration/invasionCRelative invasion10080604020****** **0 ****** ** ********** **0 1 2 3 4 5 6 71.00.80.60.40.20.01.00.80.60.40.20.0ControlControlControlTaxol 10 nMTaxol 100 nMTaxol 1 µMTaxol 10 µMDays after treatment**Taxol10 nM∗∗**MDA-MB-231**Taxol100 nM10 nM taxol**MigrationInvasion**Taxol1 µMMDA-MB-468Fig. 1. Effect of taxol on <strong>breast</strong> cancer cell <strong>proliferation</strong>, migration <strong>and</strong> invasivity.Taxol, at the concentrations of 10 nM, 100 nM, 1 lM <strong>and</strong> 10 lM, was applied for 2 hthen washed off. Effect of taxol, on; (A) cell <strong>proliferation</strong>, mean of threeexperiments. (B) Cell migration (line filled bars) <strong>and</strong> invasion (hatched bars) meanof three <strong>and</strong> four experiments in triplicate, respectively. (C) Effect of 10 nM taxol onMDA-MB-231 <strong>and</strong> MDA-MB-468 cell invasiveness. Each bar represents the mean ofeight experiments performed in triplicate. ** p < 0.05 vs control (one-way ANOVA).Please cite this article in press as: T. -A. Tran et al., Non-anti-mitotic concentrations of taxol reduce <strong>breast</strong> cancer cell invasiveness, Biochem.Biophys. Res. Commun. (2009), doi:10.1016/j.bbrc.2008.12.073
ARTICLE IN PRESST.-A. Tran et al. / Biochemical <strong>and</strong> Biophysical Research Communications xxx (2009\) xxx–xxx 3used to build sodium current–voltage (I Na –V) relationships was asfollows: from a holding potential of 100 mV, the membrane wasstepped to potentials from 90 to +60 mV, with 5-mV increments,for 50 ms at a frequency of 2 Hz. Availability–voltage relationshipswere obtained by applying 50 ms prepulses using the I Na –V curveprocedure followed by a depolarizing pulse to 5 mV for 50 ms. Inthis case, currents were normalized to the amplitude of the test currentwithout a prepulse (i.e., from 100 mV). Conductance throughNa + channels (g Na ) was calculated as already described [4]. The conductance–voltage<strong>and</strong> availability–voltage relationships were fittedwith a Boltzman equation to get the EC 50 <strong>and</strong> slope of the relationship.Current amplitudes were normalized to cell capacitance <strong>and</strong>expressed as current density (pA/pF).Solutions. The physiological saline solution (PSS) had the followingcomposition (in mM): NaCl 140, KCl 4, MgCl 2 1, CaCl 2 2, D-glucose11.1 <strong>and</strong> Hepes 10, adjusted to pH 7.4 with 1 M NaOH. Theintrapipette solution had the following composition (in mM):zK-glutamate, 125, KCl 20, CaCl 2 0.37, MgCl 2 1, Mg–ATP 1, EGTA1, Hepes 10, adjusted to pH 7.2.Statistics. Data are described as means ± st<strong>and</strong>ard error of themean (n = number of cells). Kruskal–Wallis one-way ANOVA onranks followed by a Student–Newmann–Keuls test were used tocompare cell <strong>proliferation</strong>, migration <strong>and</strong> invasion in control conditionsor in the presence of different taxol concentrations. Twoways repeated measures ANOVA on ranks followed by a Student–Newmann–Keulstest was used to compare availability <strong>and</strong>conductance relationship. p < 0.05 was considered as statisticallysignificant.ResultsWe evaluated MDA-MB-231 cell viability over 7 days in controlconditions or after 2 h pre-treatment with taxol (10 nM, 100 nM,1 lM <strong>and</strong> 10 lM). As shown in Fig. 1A, taxol reduced relative cellA1.0MDA-MB-231Relative invasion0.80.60.40.2******0.0ControlTTXTaxolTTX + TaxolB4036200 pA150 pAI NaCurrent (pA/pF)32282420161285 ms5 ms4ControlTaxolCActivationControl1.0 *Taxol***0.8*0.6*0.4*0.2*DAvailability1.00.80.60.40.2ControlTaxol0.0-80 -60 -40 -20 0 20Voltage (mV)0.0-120 -100 -80 -60 -40 -20 0Voltage (mV)Fig. 2. Involvement of Na V activity in taxol-sensitive cell invasiveness. (A) Effect of 10 nM taxol <strong>and</strong> 30 lM TTX on MDA-MB-231 cell invasiveness. At this concentration TTX<strong>inhibits</strong> Na V 1.5 [4]. Inhibitors were applied separately or in combination as indicated on the graph. Each bar represents the mean of eight experiments performed in triplicate.** p < 0.05 vs control. (B) The amplitude of I Na was measured as the difference between the peak <strong>and</strong> the end-pulse current when the cell was depolarized to 5 mV for 50 msfrom a holding potential of 100 mV. The individual values obtained from control (left, n = 12 cells) <strong>and</strong> taxol (right, n = 12 cells) experiments are given <strong>and</strong> the bars indicatethe median. Representative currents in both conditions are given at the top of the panel. (C) Taxol induced a significant leftward shift of the activation properties of Na V 1.5. Incontrol conditions (filled squares), V 1/2 = 19.8 ± 0.1 mV (n = 12 cells) <strong>and</strong> slope = 6.1 ± 0.1. After the treatment with taxol (empty squares), V 1/2 = 29.3 ± 0.1 mV <strong>and</strong> slope5.9 ± 0.1 (n = 12 cells). (D) Taxol had no effect on the availability of the Na V 1.5. In control conditions (filled squares), V 1/2 = 62.3 ± 0.2 mV (n = 12 cells) <strong>and</strong> width = 9.2 ± 0.2.After the treatment with taxol (empty squares), V 1/2 = 64.7 ± 0.2 mV <strong>and</strong> width = 9.2 ± 0.2 (n = 12 cells). * p < 0.05.Please cite this article in press as: T. -A. Tran et al., Non-anti-mitotic concentrations of taxol reduce <strong>breast</strong> cancer cell invasiveness, Biochem.Biophys. Res. Commun. (2009), doi:10.1016/j.bbrc.2008.12.073
ARTICLE IN PRESS4 T.-A. Tran et al. / Biochemical <strong>and</strong> Biophysical Research Communications xxx (2009) xxx–xxxnumber for concentrations above 10 nM. A 2-h pre-treatment with10 nM taxol had no effect on cell <strong>proliferation</strong>.On this basis, we evaluated the effects of 10 nM taxol on the cancercell properties involved in the development of metastases. Thetwo parameters evaluated were cell migration <strong>and</strong> invasion.Fig. 1B shows that migration was not affected by 10 nM taxol whileit was significantly reduced by higher concentrations (22.6% ± 0.5inhibition at 100 nM <strong>and</strong> 30.1% ± 1.9 at 1 lM). For invasion, 10 nMtaxol was sufficient to reduce cell invasion by about 40%. Higher concentrationsof taxol had no greater effect on cell invasion.Since the Matrigel Ò invasion properties of MDA-MB-468 cellsare similar to those of MDA-MB-231 cells [4], we evaluated the effectof 10 nM taxol on the two cell lines invasivity. As seen onFig. 1C, 10 nM taxol (which had no effect on MDA-MB-468 cell <strong>proliferation</strong><strong>and</strong> migration, data not shown) reduced MDA-MB-231cells invasiveness while it did no affect MDA-MB-468 cellsinvasiveness.Invasiveness is under the control of Na V activity in MDA-MB-231 but not MDA-MB-468 cells [4]. Inhibition of Na V 1.5 by tetrodotoxinis known to reduce cancer cell invasiveness without affectingcell migration [4]. To evaluate if the effect of taxol involves Na V 1.5,we measured the effects of 30 lM TTX <strong>and</strong> 10 nM taxol, alone <strong>and</strong>in combination. Fig. 2A shows that 30 lM TTX, known to block Na Vactivity [4] reduced cell invasiveness by about 40% as did 10 nMtaxol. Adding 30 lM TTX after the pre-treatment with taxol hadadditional effect upon the reduction of invasiveness. This suggeststhat taxol might act through a regulation of Na V 1.5 signalling.Patch-clamp experiments were then performed to evaluate theeffects of 2 h of pre-treatment with 10 nM taxol on I Na . We havepreviously shown that maximum I Na current was obtained whencells were depolarized from 100 to 5mV[4]. We thus comparedthe amplitudes of the current elicited by a similar voltage-clampprotocol in control conditions with those of cells pre-treated withtaxol. Fig. 2B shows that the distribution of the maximal currentamplitude was not affected by taxol. Since we did not observe areduction of the current, we checked other functional parametersof Na V 1.5, namely activation <strong>and</strong> availability. A significant leftwardshift of the activation was observed with taxol (Fig. 2C) while it hadno effect on channel availability (Fig. 2D). This result indicates thatpolymerization of microtubules regulates the channel gating.To evaluate the polymerization of microtubules, we performedWestern blot analysis using antibodies directed against a-tubulinin conditions allowing the separation of free <strong>and</strong> polymerizedforms of this microtubule subunit (Fig. 3A). Fig. 3B summarisesthe results obtained in three separate experiments. While about40% of a-tubulin subunits were in a polymerized form in the controlconditions, this proportion increased in a dose-dependentmanner when cells were pre-treated with 100 nM <strong>and</strong> 1 lM taxol,indicating that a unique short application of a low dose of taxol issufficient to give a measurable effect on microtubule polymerization.At 10 nM, there was a tendency for an increased tubulin polymerizationwhich was not statistically significant.DiscussionTaxol <strong>and</strong> its derivatives, such as docetaxel, are used in <strong>breast</strong>cancer chemotherapy [1]. The classically accepted mechanism ofaction is the stabilization of the mitotic spindle, leading to an inhibitionof cell <strong>proliferation</strong>. In this study, we showed that a unique,short, 2 h long, application of low doses of taxol, ineffective uponcell <strong>proliferation</strong>, had significant anti-invasive effects on the cancercell line MDA-MB-231. This cell line expresses Na V 1.5 in contrastto another invasive cell line, MDA-MB-468 [4]. In patients, it hasbeen reported that the presence of mRNA coding for Na V 1.5 arepositively related to lymph node metastasis [14]. Thus, the effectsof low concentrations of taxol on cell invasiveness are likely to prevent/reducethe formation of metastases. This finding probably accountsfor most of the effects of taxol since it is known that areduced invasive capacity of cancer cells is associated with a reducedtumour growth in situ. We propose that taxol may reduceinvasion by interfering with Na V 1.5 signalling pathways. Indeed,we found that the reduction of invasiveness induced by taxolwas not additive with the effect of TTX. The reduction of cell migrationobserved for concentrations of taxol above 10 nM are in thesame range as the reduction of cell <strong>proliferation</strong>. The toxicologicaleffects of taxol, more than anti-migrative effects, can explain thisobservation.It has been found that gating properties of the endogenous cardiacchannels Na V 1.5 were affected by taxol [9]. This finding is inline with our results. The lack of further I Na reduction after taxoltreatment suggests that the involvement of Na V activity in thecells’ invasive properties is rather complex <strong>and</strong> does not involvesolely a direct ionic or electrical effect, taxol might affect proteinsother than Na V 1.5 <strong>and</strong> which are involved in the signalling pathwayregulated by Na V 1.5 activity. For example, it is known thatperturbation of microtubules by colchicine can modulate the activityof sodium channels in rat cardiac cells through perturbation ofGTP homeostasis [15]. It is thus possible that this kind of effect occurs<strong>and</strong> modulates Na V 1.5 activity, or other participants in theNa V 1.5 signalling pathway, to produce a reduced invasiveness.In conclusion, taxol might exert some anti-cancer propertiesthrough mechanisms other than its well-known anti-mitotic activity.A reduction of cell invasiveness can be observed in cell expressingNa V at concentrations of taxol (10 nM) having no effect on cellmitosis. At this concentration, no significant global tubulin polymerizationwas found. However, a localised, sub-membrane increasein polymerization may occur <strong>and</strong> sensitivity of membraneproteins such like Na V 1.5 to local perturbations of the cytoskeletoncould explain the observed effects upon cell invasiveness. It seemsthat this effect involves the Na V 1.5 signalling pathways but theprecise mechanisms remain to be determined. This will be doneA Free PolymerizedBkDa755037Tubulin relative proportion1.00.90.80.70.60.50.40.30.20.10.0ControlControlTaxol 1000 nMTaxol 100 nMTaxol 10 nMFree-tubulin**Polymerized-tubulinControlTaxol 1000 nMTaxol 100 nMTaxol 10nM**10 100 1000Taxol (nM)α-Tubulinβ-ActinFig. 3. Effect of treating cancer cells for 2 h with taxol on the relative concentrationof free <strong>and</strong> polymerized a-tubulin. (A) Example of gels obtained for free <strong>and</strong>polymerized a-tubulin. (B) Relative proportion of normalized free <strong>and</strong> polymerizeda-tubulin in control conditions <strong>and</strong> after pre-treatment with 10, 100 <strong>and</strong> 1000 nMtaxol, Tubulin levels were normalized to b-actin. Mean results obtained from threedifferent experiments. ** p < 0.05 vs control.Please cite this article in press as: T. -A. Tran et al., Non-anti-mitotic concentrations of taxol reduce <strong>breast</strong> cancer cell invasiveness, Biochem.Biophys. Res. Commun. (2009), doi:10.1016/j.bbrc.2008.12.073
ARTICLE IN PRESST.-A. Tran et al. / Biochemical <strong>and</strong> Biophysical Research Communications xxx (2009\) xxx–xxx 5only when we will have a better underst<strong>and</strong>ing of the molecularlink between Na V <strong>and</strong> invasiveness.AcknowledgmentsWe thank the Université François-Rabelais de Tours for fundingthe visit of EW to Inserm U921. This work was financed in part by agrant from the Ligue Contre le Cancer de la Région Centre.References[1] G. Dang, C. Hudis, Adjuvant taxanes in the treatment of <strong>breast</strong> cancer: nolonger at the tip of the iceberg, Clin. Breast Cancer 7 (2006) 51–58.[2] S. Horwitz, Taxol (Paclitaxel): mechanisms of action, Ann. Oncol. 5 (1994)S3–S6.[3] J. Camacho, A. Sanchez, W. Stühmer, L. Pardo, Cytoskeletal interactionsdetermine the electrophysiological properties of human EAG potassiumchannels, Pflügers Arch. 441 (2000) 167–174.[4] S. Roger, P. Besson, J.-Y. Le Guennec, Involvement of a novel fast inward sodiumcurrent in the invasion capacity of a <strong>breast</strong> cancer cell line, Biochim. Biophys.Acta 1616 (2003) 107–111.[5] S. Roger, J. Rollin, A. Barascu, P. Besson, P.-I. Raynal, S. Iochmann, M. Lei, P.Bougnoux, Y. Gruel, J.-Y. Le Guennec, Voltage-gated sodium channelspotentiate the invasive capacities of human non-small-cell lung cancer celllines, Int. J. Biochem. Cell Biol. 39 (2007) 774–786.[6] S. Roger, M. Potier, C. V<strong>and</strong>ier, P. Besson, J.-Y. Le Guennec, Voltage-gatedsodium channels: new targets for epithelial cancer therapy?, Curr Pharm. Des.12 (2006) 3681–3695.[7] S. Judé, S. Roger, E. Martel, P. Besson, S. Richard, P. Bougnoux, J.-Y. Le Guennec,Dietary long-chain omega-3 fatty acids of marine origin: a comparison of theirprotective effects on coronary heart disease <strong>and</strong> <strong>breast</strong> cancers, Prog. Biophys.Mol. Biol. 90 (2006) 299–325.[8] S. Roger, P. Besson, J.-Y. Le Guennec, Influence of the whole cell patch-clampconfiguration on electrophysiological properties of the voltage-dependentsodium current expressed in MDA-MB-231 <strong>breast</strong> cancer cells, Eur. Biophys. J.33 (2004) 274–279.[9] V. Maltsev, A. Undrovinas, Cytoskeleton modulates coupling betweenavailability <strong>and</strong> activation of cardiac sodium channel, Am. J. Physiol. 273(1997) H1832–H1840.[10] T. Mosmann, Rapid colorimetric assay for cellular growth <strong>and</strong> survival:application to <strong>proliferation</strong> <strong>and</strong> cytotoxicity assays, J. Immunol. Methods 65(1983) 55–63.[11] H. Tsutsui, K. Ishihara, G. Cooper 4th, Cytoskeletal role in the contractiledysfunction of hypertrophied myocardium, Science 260 (1993) 682–687.[12] M. Takahashi, H. Shiraishi, Y. Ishibashi, K. Blade, P. McDermott, D. Menick, D.Kuppuswamy, G. Cooper IV, Phenotypic consequences of b 1 -tubulin expression<strong>and</strong> MAP 4 decoration of microtubules in adult cardiocytes, Am. J. Physiol.Heart Circ. Physiol. 285 (2003) H2072–H2083.[13] U.K. Laemmli, Cleavage of structural proteins during the assembly of the headof bacteriophage T4, Nature 227 (1970) 680–685.[14] S. Fraser, J. Diss, A. Chioni, M. Mycielska, H. Pan, R. Yamaci, F. Pani, Z. Siwy, M.Krasowska, Z. Grzywna, W. Brackenbury, D. Theodorou, M. Koyuturk, H. Kaya,E. Battaloglu, M. De Bella, M. Slade, R. Tolhurst, C. Palmieri, J. Jiang, D.Latchman, R. Coombes, M. Djamgoz, Voltage-gated sodium channel expression<strong>and</strong> potentiation of human <strong>breast</strong> cancer metastasis, Clin. Cancer Res. 11(2005) 5381–5389.[15] D. Motlagh, K. Alden, B. Russell, J. Garcia, Sodium current modulation by atubulin/GTP coupled process in rat neonatal cardiac myocytes, J. Physiol. 540.1(2002) 93–103.Please cite this article in press as: T. -A. Tran et al., Non-anti-mitotic concentrations of taxol reduce <strong>breast</strong> cancer cell invasiveness, Biochem.Biophys. Res. Commun. (2009), doi:10.1016/j.bbrc.2008.12.073
Oncogene (2008), 1–12& 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00www.nature.com/oncORIGINAL ARTICLEAutotaxin protects MCF-7 <strong>breast</strong> cancer <strong>and</strong> MDA-MB-435 melanomacells against Taxol-induced apoptosisN Samadi 1 , C Gaetano 2 , IS Goping 2 <strong>and</strong> DN Brindley 21Department of Laboratory Medicine <strong>and</strong> Pathology, University of Alberta, Edmonton, Alberta, Canada <strong>and</strong> 2 Departmentof Biochemistry (Signal Transduction Research Group), University of Alberta, Edmonton, Alberta, CanadaAutotaxin (ATX) promotes cancer cell survival, growth,migration, invasion <strong>and</strong> metastasis. ATX converts extracellularlysophosphatidylcholine (LPC) into lysophosphatidate(LPA). As these lipids have been reported toaffect cell signaling through their own G-protein-coupledreceptors, ATX could modify the balance of this signaling.Also, ATX affects cell adhesion independently of its catalyticactivity. We investigated the interactions of ATX,LPC <strong>and</strong> LPA on the apoptotic effects of Taxol, which iscommonly used in <strong>breast</strong> cancer treatment. LPC had nosignificant effect on Taxol-induced apoptosis in MCF-7<strong>breast</strong> cancer cells, which do not secrete significant ATX.Addition of incubation medium from MDA-MB-435melanoma cells, which secrete ATX, or recombinat ATXenabled LPC to inhibit Taxol-induced apoptosis ofMCF-7 cells. Inhibiting ATX activity blocked this protectionagainst apoptosis. We conclude that LPC has nosignificant effect in protecting MCF-7 cells against Taxoltreatment unless it is converted to LPA by ATX. LPAstrongly antagonized Taxol-induced apoptosis throughstimulating phosphatidylinositol 3-kinase <strong>and</strong> inhibitingceramide formation. LPA also partially reversed theTaxol-induced arrest in the G2/M phase of the cell cycle.Our results support the hypothesis that therapeuticinhibition of ATX activity could improve the efficacy ofTaxol as a chemotherapeutic agent for cancer treatment.Oncogene advance online publication, 15 December 2008;doi:10.1038/onc.2008.442Keywords: ceramides; chemotherapy; chemoresistance;lysophosphatidate; lysophosphatidylcholine; phosphatidylinositol3-kinaseIntroductionCorrespondence:Professor DN Brindley, Department of Biochemistry(Signal Transduction Research Group), University of Alberta,Edmonton, Alberta, Canada T6G 2S2.E-mail:david.brindley@ualberta.caReceived 24 July 2008; revised 8 October 2008; accepted 1 November2008Breast cancer is the most common malignancy amongwomen in North America <strong>and</strong> approximately one-thirdof these women develop metastases <strong>and</strong> die (Jemal et al.,2006). Dysregulation of normal mechanisms of apoptosisplay an important role in the pathogenesis <strong>and</strong>progression of <strong>breast</strong> cancer. Importantly, the efficacy ofchemotherapy can be compromised by the survivalsignals that tumor cells receive (Krajewski et al., 1999).There is a strong association of autotaxin (ATX)expression with <strong>breast</strong> cancer cell survival, growth,migration, invasion <strong>and</strong> metastasis (Nam et al., 2000,2001; Umezu-Goto et al., 2002; Yang et al., 2002; Hamaet al., 2004). ATX was originally isolated from humanmelanoma A2058 cells (Stracke et al., 1992) <strong>and</strong> itgenerates lysophosphatidate (LPA) from circulatinglysophosphatidylcholine (LPC). Although the involvementof LPA <strong>and</strong> ATX in the invasiveness of <strong>breast</strong>cancer has been studied (Yang et al., 2002), relativelylittle is known about how ATX might confer chemoresistance.First, the substrate of ATX, LPC, hasbeen postulated to be an extracellular signaling lipidby acting on G2A <strong>and</strong> GPR4 (Kabarowski et al., 2001;Zhu et al., 2001; Rikitake et al., 2002; Lin <strong>and</strong> Ye, 2003;Radu et al., 2004; Kim et al., 2005). Unsaturated LPC issecreted by the liver (Brindley, 1993) <strong>and</strong> saturated LPCis produced by circulating lecithin:cholesterol acyltransferasein high-density lipoproteins (Aoki et al.,2002). LPC is present in blood at up to 200 mM(Moolenaar et al., 2004). ATX could, therefore, regulatecell activation through changing signaling by LPCversus LPA. Secondly, ATX decreases the adhesion ofoligodendrocytes to the extracelllular matrix through anon-catalytic mechanism involving its C-terminus, <strong>and</strong>this facilitates morphological remodeling (Dennis et al.,2005). This suggests that ATX is a matrix-cellularprotein that signals through integrin-dependent focaladhesion assembly <strong>and</strong> consequently cell interactionswith the extracellular matrix (Fox et al., 2004). This <strong>and</strong>other non-catalytic effects of ATX could contribute toits association with the aggressiveness of cancer cells.Autotaxin provides a major route for generatingextracellular LPA, which is present at up to 20 mM inblood <strong>and</strong> extracellular fluid (Moolenaar et al., 2004;Yue et al., 2004). LPA is produced by activated plateletsto facilitate wound healing <strong>and</strong> is secreted by cancer cells(Fang et al., 2000; Radeff-Huang et al., 2004). ExtracellularLPA has been implicated in the etiology ofhuman cancer, as it stimulates cell growth, <strong>proliferation</strong>,differentiation, motility <strong>and</strong> survival (Mills <strong>and</strong> Moolenaar,2003; Brindley, 2004). Diverse actions of LPA aremediated by at least six G-protein coupled receptors
2present on the cell surface:LPA 1 /EDG2, LPA 2 /EDG4,LPA 3 /EDG7, LPA 4 /GPR23/p2y9, LPA 5 /GRP92 <strong>and</strong>LPA 6 /p2y5 (Lee et al., 2006; Pasternack et al., 2008).The expression of these receptors is cell specific,enabling different cells to respond in a unique mannerthrough signaling pathways that are activated by G i ,G s ,G q <strong>and</strong> G 12/13 . These signaling cascades includephosphatidylinositol 3-kinase (PI3K) <strong>and</strong> Akt, theextracellular signal-regulated kinase (ERK) pathway,<strong>and</strong> the small G-protein, RhoA, that mediates cellsurvival (Ye et al., 2002; Tigyi <strong>and</strong> Parrill, 2003; Radeff-Huang et al., 2004). Activation of LPA receptors alsodecreases the abundance of the p53 tumor suppressor,thereby promoting cell survival <strong>and</strong> cell cycle progression(Murph et al., 2007).Lysophosphatidate levels are high in ascites fluid <strong>and</strong>plasma of patients with ovarian tumors (Xu et al., 1998;Fang et al., 2002; Mills <strong>and</strong> Moolenaar, 2003). LPApromotes ovarian tumor development through increased<strong>cyclin</strong> D expression (Chappell et al., 2001). LPA alsoincreases vascular endothelial growth factor production,which stimulates angiogenesis (So et al., 2005). In a coloncancer cell line, LPA increases the synthesis of macrophagemigration inhibitory factor, which promotestumor growth (Sun et al., 2003). However, the effect ofLPA on cell survival <strong>and</strong> apoptosis varies among celltypes. LPA mediates survival of ovarian cancer cells,macrophages, fibroblasts <strong>and</strong> neonatal cardiac myocytes,while promoting apoptosis in hippocampal neurons <strong>and</strong>PC12 cells (Fang et al., 2002; Ye et al., 2002).Taxol is a microtubule-stabilizing agent that interfereswith spindle microtubule dynamics causing cell cyclearrest <strong>and</strong> apoptosis through interaction with b-tubulin(Bergstralh <strong>and</strong> Ting, 2006). This causes lateral polymerization<strong>and</strong> microtubule stability (Snyder et al.,2001). Taxol is widely used for treating metastatic <strong>and</strong>early-stage <strong>breast</strong> cancer, with benefits in overall <strong>and</strong>disease-free survival. However, resistance to Taxol iscommon <strong>and</strong> there is a need to identify patients who willrespond to treatment (McGrogan et al., 2008).In this study, we established that ATX protects MCF-7 <strong>breast</strong> cancer cells <strong>and</strong> MDA-MB-435 melanoma cellsagainst Taxol-induced apoptosis through its productionof LPA from the LPC that bathes most cells. LPA isstrongly antagonistic to Taxol-induced apoptosis,whereas LPC has no significant effect. From our results,we predict that identifying patients who express highATX activity <strong>and</strong> then inhibiting this activity couldprovide an important adjuvant for improving theefficacy of Taxol in cancer treatment.ResultsLysophosphatidate <strong>inhibits</strong> Taxol-induced apoptosisN Samadi et alExpression of ATX in MCF-7 <strong>and</strong> MDA-MB-435 cancercellsATX expression in the MCF-7 <strong>and</strong> MDA-MB-435 cellsused in our experiments was compared. Real-timereverse transcriptase–PCR (RT–PCR) analysis showedthat MDA-MB-435 cells expressed relatively high levelsof ATX mRNA compared with MCF-7 cellsRelative fluorescence105Relative ATX mRNA6420MCF-7mRNAMDA-MB-435MCF-7MediumMDA-MB-435Western blotkDa MDA-MB-435 MCF-725015010075ATX activity00 25 50 75 100 125Time (minutes)(Figure 1a). MDA-MB-435 cells secreted ATX proteininto the serum-free medium, whereas ATX was notdetectable in the equivalent medium concentrated fromMCF-7 cells (Figure 1b). ATX activity secreted byMDA-MB-435 cells was about 24 times higher(Po0.001) than that from MCF-7 cells (Figure 1c).Therefore, MCF-7 cells provide a system for studyingapoptosis in the absence of ATX <strong>and</strong> for examining theeffects of introducing ATX.LPA protects MCF-7 cells from Taxol-induced apoptosisWe next established conditions for studying Taxolinducedapoptosis <strong>and</strong> how LPC <strong>and</strong> LPA modify this†rATXFigure 1 Differential expression of autotoxin (ATX) in MDA-MB-435 <strong>and</strong> MCF-7 cells. In (a), mRNA for ATX was determinedby real-time RT-PCR <strong>and</strong> expressed relative to that of glyceraldehyde3-phosphate dehydrogenase (GAPDH). (b) A westernblot obtained by loading 40 mg of total protein from threeindependent samples of concentrated serum-free medium obtainedfrom either MDA-MB-435 or MCF-7 cells is shown. A st<strong>and</strong>ard ofrecombinant rATX is shown on the right. In (c), 20 concentratedserum-free medium from MCF-7 <strong>and</strong> MDA MB-435 cells wasassayed for ATX activity using the FS-3 compound. Fluorescencewas measured at excitation <strong>and</strong> emission wavelengths of 485 <strong>and</strong>538 nm, respectively. The results are shown as the average ofrelative fluorescence activity±s.e.m. from three independentexperiments. w Po0.001.Oncogene
Lysophosphatidate <strong>inhibits</strong> Taxol-induced apoptosisN Samadi et al3Figure 2 Protective effect of lysophosphatidate (LPA) on Taxolinducedapoptosis in MCF-7 cells. (a) The percentage of apoptoticnuclei from MCF-7 cells that were incubated with 0–200 nM Taxolfor 24 h (&) or48h(m) is shown. (b) The percentage of viable cellsrecovered from the wells expressed relative to untreated cells isshown. (c <strong>and</strong> d) The percentage of apoptotic nuclei <strong>and</strong> viablecells, respectively, that were recovered after adding differentconcentration of LPA with 50 nM Taxol <strong>and</strong> incubating for 24 hincubation is shown. Results are expressed as means±s.e.m. fromthree independent experiments.process. Apoptosis was detected both by morphologicalexamination using 4,6-diamidino-2-phenylindole (DAPI)staining <strong>and</strong> by measuring mitochondrial tetramethlyrhodamineethyl ester (TMRE) uptake. We used mediumsupplemented with 10% charcoal-treated fetal bovineserum (FBS) to remove lipid mediators includingLPC <strong>and</strong> LPA. This treatment also removed ATX, aswe could not detect significant ATX activity in mediacontaining 10% charcoal-treated FBS (results notshown).Treating MCF-7 cells with 50 nM Taxol produced anoptimum increase in the percentage of recovered apoptoticnuclei from 0.8% to about 12% after 24 h <strong>and</strong> 26%after 48 h (Figure 2a), <strong>and</strong> a similar effect was observedwith MDA-MB-435 cells (results not shown). Thisprovides part of the picture, as treatment with 50 nMTaxol for 24 <strong>and</strong> 48 h decreased the number of cellsattached to the dishes by about 60 <strong>and</strong> 70%, respectively(Figure 2b). We could not recover most of these cellsfrom the medium presumably because of completefragmentation. These results are compatible with thoseof Sunders et al. (1997), who concluded that Taxolinduces both apoptotic <strong>and</strong> nonapoptotic death inMCF-7 cells.The next experiments determined whether LPA canprotect against apoptosis induced by 50 nM Taxol over24 h. LPA (0.5 mM) alone had no significant effect on thepercentage of apoptotic nuclei, but it produced aFigure 3 Effect of lysophosphatidate (LPA) on Taxol-inducedchange in mitochondrial potential. (a) The percentage of tetramethlyrhodamineethyl ester (TMRE)-negative MCF-7 cells thatwere obtained after incubated with 0–50 nM Taxol for 24 h (&) or48 h (m) is shown. (b) The percentage of TMRE-negative cellsobtained after adding different concentrations of Taxol (0–50 nM)with 0–20 mM LPA is shown. The results are shown as means±s.e.m. of three experiments.maximum decrease in the percentage of apoptotic cellsremaining on the plates in the presence of Taxol. Lesseffect was observed at 5 <strong>and</strong> 10 mM LPA (Figure 2c).However, 0.5–10 mM LPA increased the recovery ofviable cells from 49±4.3 to 68±6.1% (Figure 2d).The second method used flow cytometry to detectapoptotic cells by TMRE-negative staining. Optimumapoptosis in the recovered cells was detected at 20 nMTaxol <strong>and</strong> this increased from 24 to 48 h (Figure 3a).We chose to use 48 h incubations, which increasedthe population of apoptotic cells to about 55%. LPA(5–20 mM) had a significant protective effect againstdifferent concentrations of Taxol. Maximum protectionwas obtained with 5 mM LPA against 20 nM Taxol inwhich the percentage of apoptotic cells decreased(Po0.05) from 54±2 to33±3% (Figure 3b).Isobologram analysis was used to evaluate theinteractions between Taxol <strong>and</strong> LPA at the IC 50(inhibitory concentration 50%). All LPA concentrationsused produced an antagonistic effect on Taxol-inducedapoptosis with combination index values of >1(Table 1). This antagonism was particularly apparentat 10 <strong>and</strong> 20 mM LPA (Po0.01). For example, 50 nMTaxol was required when 20 mM LPA was present toproduce equivalent apoptosis to that obtained withOncogene
4Table 1Taxol(nM)Effect of LPA on Taxol-induced change in mitochondrialpotentialCombinationdoseLPA(mM)FractionalapoptosisLysophosphatidate <strong>inhibits</strong> Taxol-induced apoptosisN Samadi et alTaxol dose(nM)(alone)Dose–responseindexCombinationindex5 1 0.31 4.6 0.9 22.210 5 0.32 5.0 0.5 23120 10 0.38 8.0 0.4 163050 20 0.36 7.2 0.2 1490The dose–response interactions between different concentrations ofTaxol <strong>and</strong> LPA in Figure 3b were assessed by isobologram analysis.7.2 nM Taxol in the absence of LPA. Thus, 20 mM LPAproduced a 6.9-fold decrease at IC 50 in Taxol sensitivity.ATX protects MCF-7 <strong>and</strong> MDA-MB-435 cells againstTaxol-induced apoptosis by producing LPAWe next studied whether ATX protects MCF-7 cellsagainst Taxol-induced apoptosis by changing the balanceof cell activation by LPC versus LPA. MCF-7 cells wereincubated with 10% charcoal-treated FBS <strong>and</strong> concentratedmedium obtained from 10 6 MDA-MB-435 cells, orMCF-7 cells in the presence or absence of LPC or LPA.Concentrated medium from MDA-MB-435 cells showedan average of 32 times more ATX activity compared withequivalent medium from MCF-7 cells.Concentrated medium from MDA-MB-435 cellssupplemented with 10 mM LPC decreased percentageapoptosis measured by DAPI staining from 7.4±1.3% to4.4±0.6% (88% of the effect of 0.5 mM LPA) (Figure 4a).The percentage of viable cells recovered also increased(49±3% to 77±7%; Po0.05) (Figure 4b). Concentratedmedium from MDA-MB-435 cells appeared todecrease apoptosis (6.1±0.8%) in the absence of addedLPC or LPA, but this was not statistically significant.LPC in the absence of conditioned medium had noprotective effect (Figure 4a).We also used an ATX inhibitor, VPC8a202 (Cui et al.,2007), to determine whether the effect of concentratedmedium depended upon ATX activity. VPC8a202 (1 mM)decreased ATX activity by 87% as expected (results notshown) <strong>and</strong> it abolished the protective effect of LPC plusconcentrated media from MDA-MB-435 cells (Figure 4a).There was no increase in the percentage of viable cellsafter applying the ATX inhibitor alone (Figure 4b).The TMRE assay also showed a significant decreasein apoptosis (Po0.01) in cells treated with concentratedmedium from MDA-MB-435 cells when 200 mM LPCwas added (Figure 4c). Concentrated medium alone orLPC alone had no significant protective effect(Figure 4c). VPC8a202 (1 mM) abolished the protectiveeffect of concentrated media with LPC. As a control, theeffect of VPC8a202 was shown to be specific, as it didnot alter the protective effect of LPA on Taxol-inducedapoptosis (Figures 4b <strong>and</strong> c).To confirm that the effects of concentrated mediumcells depended on ATX activity, we used recombinantATX, which protected against Taxol-induced apoptosis<strong>and</strong> increased the number of viable cells recoveredwhen LPC was present (Figures 4d <strong>and</strong> e). BlockingATX activity with 1 mM of the inhibitors VPC8a202, orS32826 (Ferry et al., 2008), or heating at 70 1C abolishedthe protective effect of recombinant ATX. Furthermore,concentrated medium from MCF-7 cells that containedno significant ATX activity failed to provide anysignificant protection with LPC against Taxol-inducedapoptosis (Figures 5a–c).We also showed that the endogenous ATX producedby MDA-MB-435 cells protects these cells againstTaxol-induced apoptosis when LPC is present, as thiseffect is blocked by the ATX inhibitors, VPC8a202 orS32826 (Figure 6). The inhibitors had no effect of theprotection afforded by LPA.LPA protects against Taxol-induced apoptosis byincreasing PI3K/Akt <strong>and</strong> decreasing ceramide productionWe examined the roles of the PI3K-Akt <strong>and</strong> MAPK/ERK kinase-ERK pathways on apoptosis by usingLY294002 <strong>and</strong> PD98059, respectively. LY294002(50 mM) almost completely abrogated the effects ofLPA in protecting against apoptosis (Figures 7a<strong>and</strong> b). PD98059 (40 mM) showed no significant effecton the LPA protection against Taxol-induced apoptosis,although it blocked the activation of p42/44 mitogenactivatedprotein kinase (MAPK) (Figures 8a–c). Also,phospho-42/44p MAPK <strong>and</strong> total 42/44p MAPKshowed a significant increase over 12 h in response toLPA. There was no significant effect of Taxol alone onthe expression of phospho-42/44p MAPK <strong>and</strong> total42/44p MAPK.Lysophosphatidate (5 mM) increased (Po0.05) theexpression of both phospho-Akt <strong>and</strong> total Akt by abouttwofold over 12 h (Figure 8a) <strong>and</strong> the ratio of phospho-Akt/totalAkt (Figure 8d). Taxol (30 nM) alonehad no significant effect. When Taxol was added withLPA (5 mM), there was still a 50% increase in phospho-Akt <strong>and</strong> total Akt compared with nontreated cells(Figure 8a) <strong>and</strong> an increase in the phospho-Akt/totalAkt ratio (Figure 8d). Applying 50 mM LY294002abolished the effect of LPA on the elevation of activated<strong>and</strong> total Akt.To determine the protective mechanism of LPA againstTaxol-induced apoptosis, we measured ceramide mass.There was a 2.9-fold increase in ceramide accumulationin Taxol-treated cells (Po0.05) after 24 h of incubation.LPA (0.5 mM) decreased (Po0.05) the Taxol-inducedincrease in ceramides to 1.9-fold. LY294002 (50 mM)blocked the effect of LPA in decreasing ceramideaccumulation by about 80% (Figure 9).Interactions of LPA versus Taxol on the cell cycleTaxol-induced apoptosis is accompanied by increasedaccumulation of cells in the G2/M phase of the cell cycle(McGrogan et al., 2008). Under our conditions, thisrepresented an increase from about 16 to 43% (Po0.05)(Figures 10a <strong>and</strong> c), which is compatible with previousstudies (Spankuch et al., 2007; Demidenko et al., 2008).Oncogene
Lysophosphatidate <strong>inhibits</strong> Taxol-induced apoptosisN Samadi et al5Figure 4 Autotaxin protects against Taxol-induced apoptosis in MCF-7 cells. Combinations of concentrated medium from MDA-MB-435 cells (medium/435) or recombinant autotoxin (ATX) (50 ng/ml), with lysophosphatidate (0.5 mM for 4,6-diamidino-2-phenylindole (DAPI) staining <strong>and</strong> 5 mM for tetramethlyrhodamine ethyl ester (TMRE) assay), lysophosphatidylcholine (200 mM) <strong>and</strong>the ATX inhibitors, VPC8a202 (1 mM) <strong>and</strong> S32826 (1 mM), were added with 50 nM (a, b, d, e)or30nM Taxol (c). (a, b) The percentage offragmented nuclei <strong>and</strong> the relative number of viable cells on the dishes as determined by DAPI staining after a 24 h incubation isshown. (c) The percentage of TMRE-negative cells after a 48 h incubation is shown. (d, e) The percentage of fragmented nuclei <strong>and</strong> therelative number of viable cells on the dishes as determined by DAPI staining after a 24 h incubation with 50 ng/ml recombinant ATX(rATX), rATX heated for 10 min at 70 1C to inhibit ATX activity >96% <strong>and</strong> the ATX inhibitors, VPC8a202 (1 mM) <strong>and</strong> S32826(1 mM), is shown. The results are shown as means±s.e.m. of three experiments. *Po0.05.Adding 5 mM LPA decreased the cells in G2/M to 28%<strong>and</strong> this represented reversal of the Taxol-induced effectof about 44% (Po0.05) (Figures 10c <strong>and</strong> d). AddingLY294002 blocked most of the LPA effect in reducing(Po0.05) the cells trapped in G2/M in the presence ofTaxol (Figures 10d <strong>and</strong> h). As controls, we showed that50 nM LY294002 or 5 mM LPA alone or in combinationhad no significant effect on the cells in G2/M phase(Figures 10b, e <strong>and</strong> f). These results demonstrated thedominant role of the PI3K pathway in LPA protectionagainst Taxol-induced apoptosis.DiscussionEffective chemotherapy depends on successful inductionof apoptosis <strong>and</strong> defects in apoptotic signaling are amajor cause of drug resistance (Melet et al., 2008).Chemotherapeutic resistance results in poor responses<strong>and</strong> reduced overall survival in patients with metastatic<strong>breast</strong> cancer (McGrogan et al., 2008).We used doses of Taxol, LPA <strong>and</strong> LPC up to 50 nM,20 mM <strong>and</strong> 200 mM, respectively, with MCF-7 cells. TheseTaxol concentrations are used for the treatment of<strong>breast</strong> cancer patients (Marchetti et al., 2002), <strong>and</strong> theLPA <strong>and</strong> LPC concentrations are compatible with thosein the circulation <strong>and</strong> tumor microenvironment (Moolenaaret al., 2004; Yue et al., 2004). We showed thatTaxol-induced killing is strongly antagonized by LPAon the basis of measurement of nuclear fragmentation,loss of mitochondrial potential <strong>and</strong> the recovery ofviable cells. LPA (0.5 mM) partially blocked the Taxolinducedincrease in the percentage of apoptotic nuclei,but we needed 5 mM LPA to antagonize the effect ofOncogene
6Lysophosphatidate <strong>inhibits</strong> Taxol-induced apoptosisN Samadi et alFigure 6 Autotaxin protects against Taxol-induced apoptosis inMDA-MB-435 cells. Lysophosphatidate (0.5 mM) or lysophosphatidylcholine(200 mM) <strong>and</strong> autotaxin inhibitors, VPC8a202 (1 mM)<strong>and</strong> S32826 (1 mM), were added with 50 nM Taxol. (a, b) Thepercentage of fragmented nuclei <strong>and</strong> the relative number of viablecells on the dishes as determined by 4,6-diamidino-2-phenylindolestaining after a 24 h incubation, respectively, is shown. Results areshown as means±s.e.m. of three experiments. *Po0.05.Figure 5 The effect of concentrated medium from MCF-7 cells onTaxol-induced apoptosis. Combinations of concentrated mediumfrom MCF-7 cells (medium/MCF-7), together with lysophosphatidate(LPA) (0.5 mM), or lysophosphatidylcholine (200 mM) wereadded with 50 nM Taxol (a, b) or 30nM Taxol (c). (a, b) Thepercentage of apoptotic cells <strong>and</strong> the relative number of viable cellson the dishes as determined by 4,6-diamidino-2-phenylindolestaining after a 24 h incubation is shown. (c) The percentageof tetramethlyrhodamine ethyl ester-negative cells after a 48 hincubation where 5 mM LPA was used. The results are shown asmeans±s.e.m. of three experiments. *Po0.05.Taxol in increasing the proportion TMRE-negativecells. The discrepancies suggest that cells that wereprotected at low LPA concentrations, where no differencesin TMRE staining were observed, probably diedby a mitochondria-independent <strong>and</strong> nonapoptotic(necrotic) mechanism. However, another major factoris that the DAPI <strong>and</strong> TMRE measurements determineonly what happened to cells remaining on the dishes.We, therefore, do not have information concerningthe mechanisms of cell death of more than 50% of cellsthat were not recovered. Consequently, it is significantthat 0.5–10 mM LPA increased the number of cells thatwere recovered after Taxol treatment <strong>and</strong> that were thenreflected in the percentage of apoptotic cells recordedin the DAPI <strong>and</strong> TMRE staining assays.Accumulating evidence shows that LPA promotessurvival of ovarian cancer cells (Frankel <strong>and</strong> Mills,1996; Goetzl et al., 1999a), renal proximal tubular cells(Levine et al., 1997), lymphoblastoma T cells (Goetzlet al., 1999b), macrophages (Koh et al., 1998), neonatalSchwann cells (Weiner <strong>and</strong> Chun, 1999), fibroblast(Fang et al., 2000), mesangial cells (Inoue et al., 2001)<strong>and</strong> colon cancer cells (Rusovici et al., 2007) bypreventing apoptosis. Different cell types expressdifferent LPA receptors, which have specific roles, <strong>and</strong>hence the effects of LPA signaling vary from cell to cell.In contrast to the survival role of LPA 1 in Schwann cells(Weiner <strong>and</strong> Chun, 1999; Contos et al., 2000), overexpressionof LPA 1 in ovarian cancer cells inducedapoptosis <strong>and</strong> anoikis (Furui et al., 1999), whereas LPA 2appeared to mediate LPA-stimulated survival of ovariancancer cells (Goetzl et al., 1999a). In T lymphoblasts,both LPA 1 <strong>and</strong> LPA 2 promoted LPA-induced survival(Goetzl et al., 1999a). LPA also prevents apoptosis incolon cancer cells through LPA 2 receptor (Rusoviciet al., 2007).LPA 2 is the predominant LPA receptor in MCF-7cells (Chen et al., 2007). This is compatible with ourfinding that 1 mM VPC51299 (an LPA 1/3 receptorantagonist) failed to block the LPA protection againstOncogene
Figure 7 Phosphatidylinositol 3-kinase, but not extracellularsignal-regulated kinase activation, is required for lysophosphatidate(LPA)-mediated protection against Taxol-induced apoptosis.(a) The effects of 50 mM LY294002 <strong>and</strong> 40 mM PD9805 on thepercentage of apoptotic nuclei in MCF-7 cells treated for 24 h inthe presence or absence of 50 nM Taxol <strong>and</strong> 0.5 mM LPA are shown.(b) The effects of 50 mM LY294002 <strong>and</strong> 40 mM PD9805 onpercentage of tetramethlyrhodamine ethyl ester-negative cellsafter treatment in the presence or absence of 30 nM of Taxol with5 mM LPA are shown. Results are shown as means±s.e.m. of threeexperiments. *Po0.05.Taxol-induced apoptosis in MCF-7 cells (results notshown). Unfortunately, no effective specific agonist forLPA 2 receptors has been developed.Activation of PI3K-Akt protects primary lymphocyticleukemia cells, ovarian cancer cells <strong>and</strong> intestinalepithelial cells from apoptosis, whereas G i -mediatedactivation of ERK is necessary for the survival offibroblasts (Fang et al., 2000; Gauthier et al., 2001;Baudhuin et al., 2002; Dufour et al., 2004; Harnoiset al., 2004; Hu et al., 2005). In this study, inhibition ofPI3K blocked the protective effect of LPA on Taxolinducedapoptosis, whereas blocking ERK activationwith PD98059 was without any effect.Lysophosphatidylcholine is present in plasma <strong>and</strong>serum at >100 mM in a predominantly albumin-boundform (Croset et al., 2000) <strong>and</strong> it is also detected in mediafrom tumor cells (Umezu-Goto et al., 2002). However,in our work, LPC alone did not antagonize Taxolinducedapoptosis unless concentrated medium fromMDA-MB-435 cells or recombinant ATX was added.This protective effect was blocked by VPC8a202 <strong>and</strong>S32826, two ATX inhibitors (Cui et al., 2007; Ferryet al., 2008), or by heat inactivation of ATX activity.The MDA-MB-435 medium, compared with thatproduced by MCF-7 cells, contained abundant ATX.Our work establishes that LPC is inactive in protectingagainst Taxol-induced apoptosis unless it is converted toLPA by ATX. We also showed in our unpublished workLysophosphatidate <strong>inhibits</strong> Taxol-induced apoptosisN Samadi et althat LPC only stimulates the migration of MDA-MB-231 <strong>breast</strong> cancer cells in the presence of active ATX.These conclusions are compatible with the view that theputative LPC receptors, G2A <strong>and</strong> GPR4, are protonsensingreceptors whose activity is negatively regulatedby LPC (Tomura et al., 2005). Many reported biologicaleffects of LPC are probably mediated through ATX <strong>and</strong>LPA production.In vivo, ATX might also hydrolyze sphingosylphosphorylcholineto sphingosine 1-phosphate (Clair et al.,2003). LPA <strong>and</strong> sphingosine 1-phosphate show overlappingbiological activities on cell survival by actingon distinct receptor families. However, the physiologicalsignificance of ATX in producing sphingosine1-phosphate is not established.Binding of Taxol to microtubules aberrantly engagesthe mitotic checkpoint, effecting sustained mitotic arrest(McGrogan et al., 2008). Ceramide levels regulatesustained mitotic arrest, apoptosis <strong>and</strong> mitotic slippage(Asakuma, 2003), <strong>and</strong> elevated ceramide concentrationsare a known component of Taxol-mediated cell death(Charles et al., 2001; Sietsma et al., 2002; Swanton et al.,2007). Sustained ceramide elevation in the endoplasmicreticulum coordinately activates the stress responses <strong>and</strong>inactivates anti-apoptotic Akt, thus leading to apoptosis(Swanton et al., 2007). We showed that LPA attenuatedthe Taxol-induced increase in ceramides <strong>and</strong> subsequentapoptosis through PI3K/Akt activation.In conclusion, our work demonstrates that theabundant extracellular lipid, LPC, protects MCF-7<strong>breast</strong> cancer cells <strong>and</strong> MDA-MD-435 melanoma cellsagainst Taxol-induced apoptosis, but only after itsconversion to LPA by ATX. LPA itself showed strongantagonism against the apoptotic effects of Taxol. Thiswork identifies that inhibition of ATX activity couldimprove the treatment of cancers by Taxol <strong>and</strong> possiblyother chemotherapeutic agents in patients who expresshigh ATX activity. It is, therefore, hoped that biologicallystable ATX inhibitors will be developed to test thishypothesis in vivo.Materials <strong>and</strong> methodsMaterialsRPMI1640 medium, penicillin <strong>and</strong> streptomycin were obtainedfrom GIBCO (Carlsbad, CA, USA). Trypsin-ethylenediaminetetraaceticacid, TMRE, goat anti-mouse IgG, ATX primers<strong>and</strong> superscript II reverse transcriptase were obtained fromInvitrogen (Carlsbad, CA, USA). DAG kinase, PD98059 <strong>and</strong>dithiothreitol were obtained from EMD Chemicals Inc.(Gibbstown, NJ, USA). Fatty acid-free bovine serum albumin,paclitaxel (Taxol), activated charcoal (Norit), Hoechst33258,ATP, diethylenetriaminepentaacetic acid (DETAPEC), pertussistoxin, oleoyl-L-a-lysophosphatidic acid, sodium salt (LPA),oleoyl-L-a-LPC, ceramide <strong>and</strong> monoclonal anti-GAPDH werepurchased from Sigma-Aldrich (Oakville, ON, Canada).VPC51299 <strong>and</strong> VPC8a202 were provided by Drs KR Lynch<strong>and</strong> TL Macdonald (University of Virginia, Charlottesville,VA, USA). LY294002 was obtained from Biomol Int.(Plymouth Meeting, PA, USA). S32826 was provided byDr JA Boutin (Institut de Recherches Servier). [ 32 P]LPA was7Oncogene
8Lysophosphatidate <strong>inhibits</strong> Taxol-induced apoptosisN Samadi et alPhosphoAktTotal AktP-44p MAPKP-42p MAPKTotal 44p MAPKTotal 42p MAPKGAPDHP-p42 MAPK/Totalp42 MAPK0.100.050.00***Figure 9 The role of Taxol <strong>and</strong> lysophosphatidate (LPA) incontrolling ceramide concentrations. Ceramide mass was determinedin MCF-7 cells after treating with 50 nM Taxol in thepresence or absence of 0.5 mM of LPA or 50 mM LY294002 for 24 h.The mass of ceramide (pmol) was normalized to the concentrationof phospholipid phosphate (nM) in each individual lipid extract.The results are expressed as means±s.e.m. of eight individualdishes for each treatment. *Po0.05.P-p44 MAPK/Totalp44 MAPKP-Akt/Total Akt0.60.40.20.00.40.30.20.1*** * *0.0Taxol - + - + - + - + - + - +- - + + - - + + - - + +LPALY294002PD98059- - - - - - - - + + + +- - - - + + + + - - - -Figure 8 The effects of lysophosphatidate (LPA) on phosphatidylinositol3-kinase (PI3K)/AKT <strong>and</strong> mitogen-activated proteinkinase (MAPK) pathways. MCF-7 cells were incubated for 12 hwith Taxol (30 nM) <strong>and</strong> LPA (5 mM) in the presence of 50 mMLY294002 (PI3K inhibitor) <strong>and</strong> 40 mM PD98059 (MAPK/extracellularsignal-regulated kinase kinase inhibitor). Results in (a)show the western blot analyses for phospho- <strong>and</strong> total Akt,phospho- <strong>and</strong> total ERK (p42 <strong>and</strong> p44 MAPK) <strong>and</strong> glyceraldehydephosphate dehydrogenase (GAPDH), which was used as a loadingcontrol. The results are representative of three independentexperiments <strong>and</strong> they were quantified by densitometric analysisusing a Licor Imaging System. (b) The densitometric ratio ofphospho-p42MAPK/total p42MAPK is shown; (c) phosphop44MAPK/totalp44MAPK ratio is shown <strong>and</strong> in (d), phospho-Akt/total Akt is shown. The results are shown as means±s.e.m. ofthree independent experiments. *Indicates the difference (Po0.05)from the nontreated cells.synthesized as before (Jasinska et al., 1999). Rabbit polyclonalantibody against ATX <strong>and</strong> recombinant ATX were gifts fromDr T Clair, through the Intramural Research Program ofNIH, National Cancer Institute, Center for Cancer Research**(Bethesda, MD, USA). Goat anti-rabbit IgG was obtainedfrom Rockl<strong>and</strong> (Gilbertsville, PA, USA). Polyclonal anti-Akt,phospho-Akt (S473) <strong>and</strong> monoclonal phospho-p44/42 MAPkinase were obtained from Cell Signaling Technology (Danvers,MA, USA). Polyclonal anti-MAP kinase (sc-93) wasfrom Santa Cruz Biotechnology (Santa Cruz, CA, USA). FS-3compound for the ATX assay was from Echelon Company(San Jose, CA, USA). FBS was from Medicorp Inc. (Montre´ al,PQ, Canada). RNAqueous kit, DNA-free kit <strong>and</strong> Syber Greenbuffer mix were obtained from Ambion (Austin, TX, USA).Cell cultureThe MCF-7 <strong>and</strong> MDA-MB-435 cells were obtained from theAmerican Type Culture Collection. Cells were maintainedin RPMI1640 medium with 10% FBS <strong>and</strong> 1% penicillin/streptomycin in a humidified atmosphere with 5% CO 2at 37 1C.Quantification of apoptotic nuclei using DAPI stainMCF-7 cells (2 10 4 cells/well) were grown in 96-well plates.Cells (about 70% confluent) were washed with phosphatebufferedsaline <strong>and</strong> treated in serum-free media for 24 h. Cellswere then fixed in 4% paraformaldehyde for 20 min <strong>and</strong>permeabilized in 0.1% Triton X-100 for 15 min. Fixed cellswere stained with 1 mg/ml Hoechst33258 for 15 min afterevaporation of the fixing solution. Nuclear morphology of thecells was observed by fluorescence microscopy. Triplicatesamples were prepared for each treatment <strong>and</strong> at least 300 cellswere counted in r<strong>and</strong>om fields for each sample <strong>and</strong> apoptoticnuclei were identified (Sakashita et al., 2007).Measurement of LPA breakdownSix-well plates were seeded with 2 10 5 cells <strong>and</strong> these weregrown to 70% confluence. The half-life of LPA wasdetermined after adding 2 ml of medium containing 32 P-labeledLPA (100,000 d.p.m.) with 1 or 20 mM nonradioactive LPA <strong>and</strong>measuring 32 P i - <strong>and</strong> 32 P-labeled LPA during 24 h (Pilquil et al.,2006). The half-life of LPA was about 10 h <strong>and</strong> this wasindependent of the LPA concentration (results not shown).Mitochondrial membrane potential (TMRE) assayBreakdown of the mitochondrial membrane potential (DCm)was determined using TMRE staining. MCF-7 cells (2 10 5 )Oncogene
Lysophosphatidate <strong>inhibits</strong> Taxol-induced apoptosisN Samadi et alCounts1801501209060300M1Non-treated cells73%11%CountsM216% 40M113%M300 200 400 600 800 10000 200 400 600 800 1000FL2-AFL2-A20016012080M2LPA (5 µM)M375%12%Sub G1/G1SG2/M9Taxol (30 nM)Taxol (30 nM) + LPA (5 µM)Counts1801501209060300M1M2M352%5%43%0 200 400 600 800 1000FL2-ACounts1200M1M2M360%12%28%0 200 400 600 800 1000FL2-ALY-294002 (50 µM)LY-294002 (50 µM) + LPA (5 µM)15012069%15012072%Counts906030M1M2M311%23%Counts906030M1M28%20%00 200 400 600 800 1000FL2-A0M30 200 400 600 800 1000FL2-ATaxol (30 nM) + LY-294002 (50 µM)Taxol (30 nM)+ LY-294002 (50 µM)+ LPA (5 µM)Counts1501209060300M1M2M350%9%41%0 200 400 600 800 1000FL2-ACounts1501209060300M1M2M353%8%39%0 200 400 600 800 1000FL2-AFigure 10 Effects of lysophosphatidate (LPA) <strong>and</strong> Taxol on cell cycle progression of MCF-7 <strong>breast</strong> cancer cells. MCF-7 cells wereincubated for 48 h with 30 nM Taxol <strong>and</strong> 5 mM LPA. Then the cells were collected <strong>and</strong> stained with propidium iodide (PI) beforeanalysis by fluorescence-activated cell sorting. DNA histograms were measured using CellQuest software <strong>and</strong> the percentage of G0/G1,S <strong>and</strong> G2/M cells were calculated. Results are representative of two independent experiments. The action of LPA in partially reversingthe Taxol-induced G2/M arrest was also confirmed in a single experiment with a 24 h incubation.were grown to confluence in six-well plates over 48 h inRPMI1640 medium with 10% FBS. The medium was changedto RPMI1640 medium with 10% of charcoal-treated FBS.This removed >95% of the LPA as assessed after adding 32 P-labeled LPA to the FBS (results not shown). Cells wereincubated for an additional 48 h with LPA-depleted FBS withTaxol, or LPA as indicated. LPA concentrations were maintainedby adding half of the original concentration of LPA every10 h on the basis of the calculated half-life of LPA. Cells werethen tripsinized, collected in 96 V-bottomed plate <strong>and</strong> exposed toTMRE (500 nM) for30minat371C. Excess dye was removedwith phosphate-buffered saline containing 0.1% fatty acid-freeBSA <strong>and</strong> cell-associated fluorescence was measured with aCalibur Flow Cytometer (Becton Dickinson, San Jose´ , CA,USA) using Cell Quest software (Ramjaun et al., 2007).ATX expressionmRNA expression Total RNA was extracted using theRNAqueous kit, according the manufacture’s instruction.DNA-free kit was also applied to remove contaminatingDNA from RNA preparation. Total RNA was treated withOncogene
10Lysophosphatidate <strong>inhibits</strong> Taxol-induced apoptosisN Samadi et alsuperscript II reverse transcriptase. Real-time RT-PCR wasperformed with 25 ml of master mix containing 2 SyberGreen buffer mix <strong>and</strong> forward <strong>and</strong> reverse primers (Invitrogen).The internal control was the constitutively expressedhousekeeping human glyceraldehyde 3-phosphate dehydrogenase(GAPDH). Primers for human ATX were as follows:sense, 5 0 -ACAACGAGGAGAGCTGCAAT-3 0 ; antisense,5 0 -AGAAGTCCAGGCTGGTGAGA-3; <strong>and</strong> for humanGAPDH were as follows:sense, 5 0 -ACAGTCAGCCGCATCTTCTT-3 0 ; antisense, 5 0 -GACAAGCTTCCCGTTCTCAG-3 0 .Samples were assayed in triplicate on the 7500 Real Time PCRSystem (Applied Biosystems).Western blot analysis Serum-free medium from MD-MB-435cells was concentrated to 200 ml per 10 6 cells by centrifugationusing a Centricon Millipore’s Ultracel YM cellulose membrane(Millipore Corporation, Bedford, TX, USA) <strong>and</strong> analysedusing 10% SDS–polyacrylamide gel electrophoresis. Nitrocellulosemembranes were treated with Odyssey blocking bufferfor 24 h <strong>and</strong> incubated with 1:5000 dilution of ATX antibodyfor 60 min followed by four washes with phosphate-bufferedsaline þ 0.1% Tween-20. Blots were incubated in 1:10 000dilution of goat anti-rabbit IgG for 45 min. After washing fourtimes, ATX was visualized at 800 nm with the OdysseyInfrared Scanner.ATX activity assay ATX activity was assayed in 96-wellplates using 1 mM FS-3 as a substrate. Fluorescence was read at15 min intervals by a Synergy2 system (BioTek, Winooski, VT,USA) with excitation <strong>and</strong> emission wavelengths of 485 <strong>and</strong>538 nm, respectively.Ceramide mass assayCells (1 10 6 ) were plated in 10 cm culture dishes <strong>and</strong> grown toconfluence in RPMI1640 medium with 10% FBS. After 48 h,the medium was changed to RPMI1640 medium with 10% ofcharcoal-treated FBS, <strong>and</strong> cells were incubated for 48 h withthe indicated concentrations of Taxol, LPA <strong>and</strong> LY294002.After washing with phosphate-buffered saline, cell lysateswere collected in methanol <strong>and</strong> the lipids were extracted(Martin et al., 1997). Ceramide concentrations were measuredby conversion to ceramide 1-phosphate, using the diacylglycerolkinase <strong>and</strong> [g- 32 P]ATP <strong>and</strong> by comparison to st<strong>and</strong>ardsof ceramide. Results were normalized to the phospholipidconcentration of the extract (Martin et al., 1997).Statistical analysis <strong>and</strong> determination of the interaction of Taxolwith LPA on apoptosisThe interaction was evaluated by the isobologram technique(CalcuSyn software, Biosoft, Cambridge, UK). The experimentaliso-effect points were the concentrations (relative toIC 50 concentrations) of the two agents that when combinedkill 50% of the cells. A quantitative index of these interactionswas provided by the isobologram equation combinationindex ¼ (a/A) þ (b/B), where A <strong>and</strong> B represent the Taxol<strong>and</strong> LPA concentrations that alone produce a fixed levelof inhibition (IC 50 ), a <strong>and</strong> b represent the concentrationsrequired for the same effect when they were combined. CIvalues of o1 indicate synergy; 1 represents addition <strong>and</strong> 41indicate antagonism.Statistical analysis was performed by analysis of variancewith a Kruskal–Wallis post hoc test.AbbreviationsATX, autotaxin; DAPI, 4,6-diamidino-2-phenylindole; FBS,fetal bovine serum; LPA, lysophosphatidate; LPC, lysophosphatidylcholine;MAPK, mitogen-activated protein kinase;PI3K, phosphatidylinositol 3-kinase; TMRE, tetramethlyrhodamineethyl ester.AcknowledgementsWe thank Mr M Czernick <strong>and</strong> Mr J Dewald for excellenttechnical support in the fluorescence-activated cell sortinganalysis <strong>and</strong> preparing labeled LPA, respectively. We thankDr M Sawyer for help with the isobologram analysis,Dr E Posse de Chaves for advice on DAPI staining <strong>and</strong>Drs F Bamforth <strong>and</strong> GS Cembrowski for their support. Wethank Dr T Clair for providing recombinant ATX <strong>and</strong> theATX antibody. 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Neuroscience Letters 443 (2008) 17–22Contents lists available at ScienceDirectNeuroscience Lettersjournal homepage: www.elsevier.com/locate/neuletTaxol induces oxidative neuronal cell death by enhancing the activity ofNADPH oxidase in mouse cortical culturesHong Jeon Jang a,b , Shinae Hwang b , Kyu Yong Cho a , Do Kyung Kim c , Kee-Oh Chay d , Jong-Keun Kim b,∗a Department of Neurosurgery, Kwangju Christian Hospital, Gwangju 503-715, Republic of Koreab Department of Pharmacology, Chonnam National University Medical School, 5 Hak-Dong, Gwangju 501-746, Republic of Koreac Department of Oral Physiology <strong>and</strong> The second stage of BK21, Chosun University College of Dentistry, Gwangju 501-759, Republic of Koread Department of Biochemistry, Chonnam National University Medical School, Gwangju 501-746, Republic of KoreaarticleinfoabstractArticle history:Received 23 May 2008Received in revised form 16 July 2008Accepted 18 July 2008Keywords:TaxolOxidative stressNADPH oxidaseNeuronal deathWe examined the involvement of oxidative stress in neuronal cell death induced by taxol, a microtubulestabilizinganti-cancer drug <strong>and</strong> investigated whether NADPH oxidase plays a role in taxol-inducedneuronal cell death in mouse cortical cultures. Cell death was assessed by measuring lactate dehydrogenasein the bathing media after 24-h exposure to taxol. Taxol (30–1000 nM) induced theconcentration-dependent neuronal death with apoptotic features. The neuronal death induced by taxolwas significantly attenuated not only by anti-apoptotic drugs such as z-VAD-fmk <strong>and</strong> cycloheximide butalso by antioxidants such as trolox, ascorbic acid <strong>and</strong> tempol. Vinblastine, a microtubule-depolymerizinganti-cancer drug, also induced neuronal death. The neuronal cell death induced by vinblastine was alsoattenuated by z-VAD-fmk, but not by antioxidants <strong>and</strong> NADPH oxidase inhibitors. Exposure the corticalcultures to taxol for 80 min formed neurite beadings visualized by fluorescence immunocytochemistry fortubulin. Treatment with either trolox or apocynin, an NADPH oxidase inhibitor, did not affect formationof the neurite beadings. RT-PCR <strong>and</strong> Western blot analysis revealed that exposure to taxol increased theexpression of p47 phox <strong>and</strong> gp91 phox <strong>and</strong> induced translocation of the p47 phox to the membrane in corticalcultures. Exposure to taxol markedly increased cellular 2,7-dichlorofluorescin diacetate fluorescence, anindicator for reactive oxygen species. Apocynin <strong>and</strong> trolox markedly inhibited the taxol-induced increaseof the fluorescence. Moreover, treatment with NADPH oxidase inhibitors or suppression of gp91 phox bysiRNA significantly attenuated the taxol-induced neuronal death. These results indicate that taxol inducesoxidative neuronal apoptosis by enhancing the activity of NADPH oxidase.© 2008 Elsevier Irel<strong>and</strong> Ltd. All rights reserved.Taxol has been widely used as an anti-cancer drug for ovarian,<strong>breast</strong>, lung <strong>and</strong> prostate cancer [4]. It <strong>inhibits</strong> the normal functionof microtubules, which causes the failure of mitosis <strong>and</strong> the death ofproliferating cells, <strong>and</strong> also affects other cellular functions such asintracellular signaling, organelle transport <strong>and</strong> cellular locomotion[8].Taxol induces apoptosis in cortical neurons by a mechanism distinctfrom that in non-neuronal cells. In contrast to non-neuronalcells, expression of wild type Bcl-2 in cortical neurons protectsagainst taxol-induced apoptosis [7]. Moreover, taxol induces activationof N-terminal c-Jun protein kinase (JNK) <strong>and</strong> phosphorylationof Bcl-2 in cancer cells [4], whereas it induces neither JNK activationnor phosphorylation of Bcl-2 in cortical neurons [7].NADPH oxidase is a superoxide-producing enzyme that consistsof membrane (gp91 PHOX <strong>and</strong> p22 PHOX ) <strong>and</strong> cytosolic (p47 PHOX ,∗ Corresponding author. Fax: +82 62 232 6974.E-mail address: ckkim@jnu.ac.kr (J.-K. Kim).p67 PHOX , <strong>and</strong> p40 PHOX ) components [3,5]. Although NADPH oxidasewas originally characterized as part of the host defense machineryin phagocytes, all of the subunits of NADPH oxidase are alsoexpressed in cortical neurons [10,15] <strong>and</strong> are involved in ROSinducedoxidative cell death in some neurodegenerative diseases[16,18].In the present study, we examined the effects of some antioxidantson neuronal cell death induced by taxol <strong>and</strong> investigatedwhether NADPH oxidase plays a role in taxol-induced neuronaldeath.4-(2-Aminoethyl)benzenesulfonylfluoride (AEBSF), apocynin,tempol <strong>and</strong> N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone(z-VAD-fmk) were obtained from Calbiochem Corporation(San Diego, CA, USA). Fetal bovine serum <strong>and</strong> horse serum werefrom Hyclone (Logan, UT, USA). Media for cell culture were purchasedfrom Gibco BRL (Rockville, MD, USA) <strong>and</strong> other reagentswere from Sigma (St Louis, MO, USA).Mixed cortical cell cultures, containing both neurons <strong>and</strong>astroglia, were prepared from fetal ICR mice at 14–15 days of0304-3940/$ – see front matter © 2008 Elsevier Irel<strong>and</strong> Ltd. All rights reserved.doi:10.1016/j.neulet.2008.07.049
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, <strong>and</strong> 5% horse serum). Cytosine arabinoside(10 M) was added 5–6 days after plating to halt the growth ofnon-neuronal cells.Treatments with taxol <strong>and</strong> 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 <strong>and</strong> 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 <strong>and</strong> 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 <strong>and</strong> 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 <strong>and</strong> thentreated with taxol alone or in combination with apocynin or trolox.After treatments, ROS generation was monitored at 0.5, 1, 2, 4, 6,<strong>and</strong> 8 h at 37 ◦ C in a fluorescent multiwell plate reader (FluoroskanAscent FL, Labsystems) with excitation at 485 nm <strong>and</strong> emission at530 nm.RNA was prepared with TRIZOL <strong>and</strong> reverse-transcribed intocDNA with r<strong>and</strong>om primer <strong>and</strong> 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 <strong>and</strong> 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 <strong>and</strong> bar represents mean ± S.E.M. from 4to 12 wells. (B) Photomicrographs of Sytox green nuclear staining showing chromatin condensation <strong>and</strong> nuclear fragmentation (arrows) induced by 8-h exposure to 300 nMtaxol (Tax) <strong>and</strong> 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, <strong>and</strong> 5 mM tempol on taxol-induced neuronal death. Each column <strong>and</strong> 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)<strong>and</strong> 100 nM DPI on the 100 nM vinblastine (Vin)-induced neuronal death in mixed cortical cultures. Each column <strong>and</strong> 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 <strong>and</strong> HPRT; extensionfor 2min at 72 ◦ C (35 cycles for p47 phox <strong>and</strong> gp91 phox ; 30cycles for HPRT). PCR products were run on 1.5% agarosegels, visualized with ethidium bromide <strong>and</strong> quantitativelymeasured with an image analyzer. Primer sequences wereas follows: 5 ′ -CAGCCAGCACTATGTGTACA-3 ′ <strong>and</strong> 5 ′ -GAACTCGTA-GATCTCGGTGAA-3 ′ for p47 phox (91 bp); 5 ′ -GGTTTATGATGAT-GGGCCTAA-3 ′ <strong>and</strong> 5 ′ -GCACTGGAACCC CTGAGAAA-3 ′ , for gp91 phox(158 bp); 5 ′ -GTAATGATCAGTCAACGGGGGAC-3 ′ <strong>and</strong> 5 ′ -CCAGCAA-GCTTGCAACCTTAACCA-3 ′ for HPRT (200 bp).For knockdown of gp91 phox expression, Stealth TM siRNA ofgp91 phox (Invitrogen) was used. The sense <strong>and</strong> antisense sequencesof gp91 phox were CCAUCCACACAAUUGCA CAUCUCUU <strong>and</strong> 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 <strong>and</strong> GeneSilencer were each diluted to25 L with media <strong>and</strong> then mixed together for 10 min. Cells werefed 500 L of fresh culture medium <strong>and</strong> 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 <strong>and</strong> centrifuged at13,000 rpm for 15 min. The pellet was discarded, <strong>and</strong> the supernatantwas used for protein quantification. Fractionation of cytosol<strong>and</strong> membrane was performed as described previously [14]. Equalamounts of protein from total cell lysates or membrane or cytosolicfraction were electrophoresed on 12% SDS–PAGE <strong>and</strong> 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, <strong>and</strong> neuronal death increased to33%, 46% <strong>and</strong> 72% with increasing concentrations of 100, 300 <strong>and</strong>1000 nM, respectively (Fig. 1A). In our culture systems, taxol barelyinjured astrocytes in the 30–1000 nM concentration range (datanot shown). Taxol induced chromatin condensation <strong>and</strong> 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) <strong>and</strong>z-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, <strong>and</strong>this 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 <strong>and</strong> 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 <strong>and</strong>Fig. 2. Fluorescence imaging showing taxol-induced alteration of microtubule network <strong>and</strong> effects of trolox <strong>and</strong> 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 <strong>and</strong> taxolfor 80 min. (D) Co-treated with 500 M apocynin <strong>and</strong> taxol for 80 min. Calibration bar: 50 m.
20 H.J. Jang et al. / Neuroscience Letters 443 (2008) 17–22nuclear fragmentation (Fig. 1B). To examine whether oxidativestress is specific for TIND or not, we investigated the effects ofz-VAD-fmk <strong>and</strong> antioxidants on the vinblastine-induced neuronaldeaths. Treatment with z-VAD-fmk significantly attenuated thevinblastine-induced neuronal death, but antioxidants such as trolox<strong>and</strong> ascorbic acid or NADPH oxidase inhibitors such as apocynin <strong>and</strong>DPI failed to inhibit the vinblastine-induced death (Fig. 1D). Thesefindings indicate that oxidative stress is uniquely involved in TINDbut not in vinblastine-induced deaths.To delineate whether the oxidative stress is involved in upstreamor downstream of microtubule inhibition by taxol, we observedthe effects of taxol on microtubule network using fluorescenceimmunocytochemistry for tubulin. After treating neurons withtaxol for 80 min, neurite beadings were identified (Fig. 2B). So weinvestigated the effect of trolox <strong>and</strong> apocynin on the formation ofneurite beadings by taxol. Treatment with either trolox or apocynindid not affect formation of the neurite beadings (Fig. 2C <strong>and</strong>D). These results suggest that oxidative stress is downstream eventof microtubule inhibition by taxol.Based on our findings <strong>and</strong> reports that all subunits of NADPHoxidase are expressed in cultured cortical cells [10,15] <strong>and</strong> thatactivation of this enzyme is involved in ROS-induced oxidative celldeath in neurodegenerative diseases [9,18], we hypothesize thatactivation of NADPH oxidase may also be involved in TIND in mixedcortical cultures. First, we examined p47 phox <strong>and</strong> gp91 phox expressionat the mRNA level by RT-PCR analysis <strong>and</strong> at the protein levelby western blot. Whereas cortical cultures expressed low levels ofp47 phox <strong>and</strong> gp91 phox mRNA, 2-h exposure to taxol substantiallyincreased the mRNA levels for p47 phox <strong>and</strong> gp91 phox in cortical cultures(Fig. 3A). Western blot assays confirmed that taxol exposureincreased the expressions of both p47 phox <strong>and</strong> gp91 phox after 3 h(Fig. 3B) <strong>and</strong> induced translocation of the p47 phox to the membranein cortical cultures, a signature event for the activation of NADPHoxidase (Fig. 3C).Next, we examined whether the enhanced expression of NADPHoxidase contributed to ROS generation after exposure to taxol.Exposure of mixed cortical cultures to taxol for 3 h markedlyincreased cellular DCF fluorescence, an indicator for superoxide,compared with sham-washed controls. Treatment with eitherapocynin, an NADPH oxidase inhibitor [19], or trolox, a vitamin Eanalog, markedly reduced the DCF fluorescence (Fig. 4A). We monitoredthe changes in DCF fluorescence for 8 h after exposure to taxol.As shown in Fig. 4B, taxol markedly increased the fluorescence forup to 2 h <strong>and</strong> then gradually increased the fluorescence for up to6 h. Treatment with either apocynin or trolox significantly reducedthe increase of DCF fluorescence.The next question was whether ROS generated by NADPH oxidasecontributed to TIND. Treatment with known NADPH oxidaseFig. 3. Induction <strong>and</strong> activation of p47 phox <strong>and</strong> gp91 phox in mixed cortical cultures exposed to taxol. (A) RT-PCR for p47 phox <strong>and</strong> gp91 phox in sham-washed control cultures(S) <strong>and</strong> in sister cultures after 2-h exposure to 300 nM taxol (T). The panels are representative of three independent experiments. HPRT was used as a house-keeping gene.Bars denote densitometer readings of RT-PCR signals expressed as folds of respective control values (mean ± S.E.M.; n = 3). *P < 0.05, compared with respective controls. (B)Western blots for p47 phox <strong>and</strong> gp91 phox in sham-washed control cultures (S) <strong>and</strong> in sister cultures after 3-h exposure to 300 nM taxol (T). The panels are representative ofthree independent experiments. (C) Western blot for p47 phox in the cytosolic fraction <strong>and</strong> the membrane fraction of cortical cultures, 3 h after sham wash (S) <strong>and</strong> 300 nMtaxol exposure (T).
H.J. Jang et al. / Neuroscience Letters 443 (2008) 17–22 21Fig. 4. Reduction of taxol-induced ROS generation by apocynin or trolox. (A) DCF fluorescencein cortical cultures 3 h after sham wash (Sham) or 3 h exposure to 300 nMtaxol without (Tax) or with addition of 500 M apocynin (+Apo) or 100 M trolox(+Tro). (B) Relative ROS generation was analyzed by measuring DCF fluorescence incortical cultures exposed to 300 nM taxol without () or with 500 M apocynin (),100 Mtrolox(○) for the indicated times. Each point <strong>and</strong> bar is mean ± S.E.M. from6 to 12 wells. *P < 0.05, compared with taxol-treated group.inhibitors, such as apocynin, AEBSF [6] or DPI [12], significantlyreduced the neuronal cell death (Fig. 5A). Furthermore, suppressingthe expression of gp91 phox with siRNA also significantly attenuatedTIND (Fig. 5B).Taxol mainly causes peripheral neuropathy in humans <strong>and</strong> itsCNS toxicity is rare <strong>and</strong> transient [20]. In the present study, wedemonstrated that TIND is apoptotic based on morphological features,such as chromatin condensation <strong>and</strong> nuclear fragmentation,<strong>and</strong> intervention with anti-apoptotic drugs in mouse cortical cultures.Although taxol is known to induce apoptotic cell death inproliferating cells, taxol also induces apoptotic cell death in nonproliferatingcells like cortical neurons <strong>and</strong> cardiomyocytes [7,17].Interestingly, it has been suggested that taxol may be useful fortreating Alzheimer’s disease due to its stabilization of microtubules[13].The attenuation of TIND by treatment with antioxidants indicatesthat oxidative stress is involved in this process. Increasedoxidative stress is an important underlying factor for the pathogenesisof a number of neurodegenerative diseases, includingAlzheimer disease (AD), multiple sclerosis (MS) <strong>and</strong> stroke. Thepresent study has demonstrated that NADPH oxidase is expressedin mixed cortical cultures <strong>and</strong> exposure of the cortical cultures totaxol induces the expression of this enzyme. Several lines of evi-Fig. 5. Attenuation of taxol-induced neuronal death by NADPH oxidase inhibitors<strong>and</strong> knockdown of gp91 phox by siRNA. (A) Effect of 500 M apocynin, 50 M AEBSF<strong>and</strong> 100 nM DPI on the 300 nM taxol-induced neuronal death in mixed corticalcultures. Each column <strong>and</strong> bar is the mean ± S.E.M. from 8 to 12 wells. *P < 0.05,compared with taxol-treated group. (B) Effect of knockdown of gp91 phox with siRNAon the 300 nM taxol-induced neuronal death in mixed cortical cultures. Each column<strong>and</strong> bar is the mean ± S.E.M. from 8 to 12 wells. *P < 0.05, compared withtaxol-treated control group.dence indicate that NADPH oxidase participates in the process ofneuronal death in some pathologic conditions. For example, activationof NADPH oxidase was also observed in Alzheimer’s diseasebrain [16], <strong>and</strong> NADPH oxidase participates in the neuronal deathinduced by beta-amyloid peptide [9], in zinc-induced neuronaldeath [15] <strong>and</strong> in BDNF-induced neuronal death in cultured neurons[10].In the present study of mixed cortical cultures, the contributionby NADPH oxidase to TIND was directly supported by findingsthat antioxidants <strong>and</strong> inhibitors of NADPH oxidase attenuate bothROS generation <strong>and</strong> TIND. Furthermore, suppression of NADPH oxidasewith siRNA for gp91 phox also significantly reduced TIND. Thisagain supports the hypothesis that oxidative stress through activationof NADPH oxidase is a major mechanism in TIND. Consistentwith these findings, a recent study showed that taxol killed cancercells by generating ROS through enhancing the activity of NADPHoxidase [3].Vinblastine <strong>and</strong> taxol are involved in microtubule-disruptinganticancer drug, but they have opposite action on microtubule polymerization[8]. In present study, vinblastine also induced apoptotic
22 H.J. Jang et al. / Neuroscience Letters 443 (2008) 17–22neuronal cell deaths which were not attenuated by treatment withantioxidants or NADPH oxidase inhibitors. These results indicatethat oxidative stress is uniquely involved in TIND <strong>and</strong> suggest thatmechanisms of activating NADPH oxidase in TIND may be relatedto its mode of action on the microtubule polymerization.Although mechanisms underlying the activation of NADPH oxidasemust still be delineated, we report for the first time that taxolcan generate ROS through the expression <strong>and</strong> activation of NADPHoxidase <strong>and</strong> cause neuronal cell death in mixed cortical cells. ThisTIND in cortical cultures may be a useful model for neurodegenerativedisorders that are characterized by increased oxidative stresssuch as AD, MS <strong>and</strong> stroke.References[1] M. Aarts, K. Iihara, W.L. Wei, Z.G. Xiong, M. Arundine, W. Cerwinski, J.F. Mac-Donald, M. Tymianski, A key role for TRPM7 channels in anoxic neuronal death,Cell 115 (2003) 863–877.[2] J. Alex<strong>and</strong>re, Y. Hu, W. Lu, H. Pelicano, P. Huang, Novel action of paclitaxel againstcancer cells: byst<strong>and</strong>er effect mediated by reactive oxygen species, Cancer Res.67 (2007) 3512–3517.[3] B.M. Babior, NADPH oxidase: an update, Blood 93 (1999) 1464–1476.[4] M.V. Blagosklonny, T. Fojo, Molecular effects of paclitaxel: myths <strong>and</strong> reality (acritical review), Int. J. Cancer 83 (1999) 151–156.[5] F.R. DeLeo, M.T. Quinn, Assembly of the phagocyte NADPH oxidase: molecularinteraction of oxidase proteins, J. Leukoc. Biol. 60 (1996) 677–691.[6] V. Diatchuk, O. Lotan, V. Koshkin, P. Wikstroem, E. Pick, Inhibition of NADPHoxidase activation by 4-(2-aminoethyl)-benzenesulfonyl fluoride <strong>and</strong> relatedcompounds, J. Biol. Chem. 272 (1997) 13292–13301.[7] X.A. Figueroa-Masot, M. Hetman, M.J. Higgins, N. Kokot, Z. Xia, Taxol inducesapoptosis in cortical neurons by a mechanism independent of Bcl-2 phosphorylation,J. Neurosci. 21 (2001) 4657–4667.[8] S. Honore, E. Pasquier, D. Braguer, Underst<strong>and</strong>ing microtubule dynamics forimproved cancer therapy, Cell. Mol. Life Sci. 62 (2005) 3039–3056.[9] A. Jana, K. Pahan, Fibrillar amyloid-beta peptides kill human primary neuronsvia NADPH oxidase-mediated activation of neutral sphingomyelinase,Implications for Alzheimer’s disease, J. Biol. Chem. 279 (2004) 51451–51459.[10] S.H. Kim, S.J. Won, S. Sohn, H.J. Kwon, J.Y. Lee, J.H. Park, B.J. Gwag, Brain-derivedneurotrophic factor can act as a pronecrotic factor through transcriptional<strong>and</strong> translational activation of NADPH oxidase, J. Cell. Biol. 159 (2002)821–831.[11] J.Y. Koh, D.W. Choi, Quantitative determination of glutamate mediated corticalneuronal injury in cell culture by lactate dehydrogenase efflux assay, J. Neurosci.Methods 20 (1987) 83–90.[12] Y. Li, M.A. Trush, Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, alsopotently <strong>inhibits</strong> mitochondrial reactive oxygen species production, Biochem.Biophys. Res. Commun. 253 (1998) 295–299.[13] R. Nelson, Microtubule-stabilising drugs may be therapeutic in AD, Lancet Neurol.4 (2005) 83–84.[14] K.M. Noh, Y.H. Kim, J.Y. Koh, Mediation by membrane protein kinase C of zincinducedoxidative neuronal injury in mouse cortical cultures, J. Neurochem. 72(1999) 1609–1616.[15] K.M. Noh, J.Y. Koh, Induction <strong>and</strong> activation by zinc of NADPH oxidase in culturedcortical neurons <strong>and</strong> astrocytes, J. Neurosci. 20 (2000) RC111.[16] S. Shimohama, H. Tanino, N. Kawakami, N. Okamura, H. Kodama, T. Yamaguchi,T. Hayakawa, A. Nunomura, S. Chiba, G. Perry, M.A. Smith, S. Fujimoto, Activationof NADPH oxidase in Alzheimer’s disease brains, Biochem. Biophys. Res.Commun. 273 (2000) 5–9.[17] E. Skobel, H. Kammermeier, Relation between enzyme release <strong>and</strong> irreversiblecell injury of the heart under the influence of cytoskeleton modulating agents,Biochim. Biophys. Acta 1362 (1997) 128–134.[18] G.Y. Sun, L.A. Horrocks, A.A. Farooqui, The roles of NADPH oxidase <strong>and</strong> phospholipasesA2 in oxidative <strong>and</strong> inflammatory responses in neurodegenerativediseases, J. Neurochem. 103 (2007) 1–16.[19] Q. Wang, K.D. Tompkins, A. Simonyi, R.J. Korthuis, A.Y. Sun, G.Y. Sun, Apocyninprotects against global cerebral ischemia-reperfusion-induced oxidativestress <strong>and</strong> injury in the gerbil hippocampus, Brain Res. 1090 (2006)182–189.[20] C.G. Ziske, B. Schöttker, M. Gorschlüter, U. Mey, R. Kleinschmidt, U. Schlegel, T.Sauerbruch, I.G. Schmidt-Wolf, Acute transient encephalopathy after paclitaxelinfusion: report of three cases, Ann. Oncol. 13 (2002) 629–631.
BRAIN RESEARCH 1239 (2008) 216– 225available at www.sciencedirect.comwww.elsevier.com/locate/brainresResearch ReportDifferential sensitivity of human glioblastoma LN18(PTEN-positive) <strong>and</strong> A172 (PTEN-negative) cells to Taxolfor apoptosisRan Zhang a , Naren L. Banik a , Swapan K. Ray b, ⁎a Department of Neurosciences, Medical University of South Carolina, Charleston, SC 29425, USAb Department of Pathology, Microbiology <strong>and</strong> Immunology, University of South Carolina School of Medicine, 6439 Garners Ferry Road,Building 2, Room C11, Columbia, SC 29209, USAARTICLE INFOABSTRACTArticle history:Accepted 21 August 2008Available online 4 September 2008Keywords:ApoptosisCytochrome cGlioblastomaPTENTaxolVEGFGlioblastoma is the most malignant brain tumor in humans <strong>and</strong> an average survival ofglioblastoma patients hardly exceeds 12 months. Taxol is a plant-derived anti-cancer agent,which has been used in the treatments of many solid tumors. Deletion or mutation ofphosphatase <strong>and</strong> tension homolog located on chromosome ten (PTEN) occurs in more than80% of glioblastomas. We examined the sensitivity of human glioblastoma LN18 (PTENpositive)<strong>and</strong> A172 (PTEN-negative) cells to Taxol for induction of apoptosis. Wright stainingshowed morphological features of apoptosis after treatment with different doses of Taxol for24 h. Significant amount of apoptosis occurred in LN18 cells after treatment with 25 nM Taxol,while in A172 cells only after treatment with 50 nM Taxol. Western blotting with an antibodythat could specifically detect activation or phosphorylation of Akt (p-Akt) did not show any p-Akt in LN18 cells but an increase in p-Akt in A172 cells. Activation of Akt in A172 cells could bereversed by pre-treatment of the cells with the phosphatidylinositol-3-kinase (PI3K) inhibitorLY294002, indicating involvement of PI3K activity in this process. Apoptosis occurred with anincrease in Bax:Bcl-2 <strong>and</strong> mitochondrial release of cytochrome c into the cytosol leading toactivation of mitochondria-dependent caspase cascade. Taxol did not cause upregulation ofvascular endothelial growth factor (VEGF), a key mediator of angiogenesis, in LN18 cells butsubstantial upregulation of VEGF in A172 cells. After treatment with Taxol, increases in p-Akt<strong>and</strong> VEGF could maintain survival <strong>and</strong> angiogenesis, respectively, in PTEN-negativeglioblastoma. As a single chemotherapy, Taxol might be more efficacious in PTEN-positiveglioblastoma than in PTEN-negative glioblastoma. Thus, our study showed differentialsensitivity of PTEN-positive <strong>and</strong> PTEN-negative glioblastoma cells to Taxol.© 2008 Elsevier B.V. All rights reserved.1. IntroductionTaxol is one of the most powerful anti-cancer compoundsamong all chemotherapeutic drugs (Gligorov <strong>and</strong> Lotz, 2004). Itis a plant-derived anti-cancer agent that has been efficacious invarious solid tumors in preclinical studies (Rose, 1992; Geneyet al., 2005; Das et al., 2008) as well as in clinical studies(Utsunomiya et al., 2006; Veltkamp et al., 2007). Taxol has beenwidely used for induction of apoptosis in many solid tumors(Gan et al., 1998; Gag<strong>and</strong>eep et al., 1999) including glioblastoma⁎ Corresponding author. Fax: +1 803 733 3192.E-mail address: raysk8@gw.med.sc.edu (S.K. Ray).0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.brainres.2008.08.075
BRAIN RESEARCH 1239 (2008) 216– 225217(Cahan et al., 1994; Das et al., 2008). It activates an intrinsicpathway of apoptosis via mitochondrial releaseof cytochrome c into the cytosol leading to formation ofapoptosome for activation of caspases (Jordan <strong>and</strong> Wilson,2004). Taxol is not so effective for treatment of recurrentglioblastoma even though it has shown great effectiveness intreatment of high-grade glioblastoma (Glantz et al., 1999;Rosenthal et al., 2000). However, the anti-cancer efficacy ofTaxol in glioblastoma can be significantly increased incombination with other therapeutic agents (Son et al., 2006;Karmakar et al., 2008) <strong>and</strong> also by increasing its transportacross the blood-brain barrier (Fellner et al., 2002; Koziaraet al., 2004). Recently, there is an excitement about the use oftemozolomide for treatment of some glioblastomas thatcontain epigenetic silencing of the DNA repair gene O 6 -methylguanine methyltransferase (Hegi et al., 2005). Even theuse of temozolomide alone may fail (Huang et al., 2008) orshow only moderate efficacy (Yang et al., 2006) in recurrent<strong>and</strong> progressive glioblastomas. Temozolomide may alsopromote angiogenesis in glioblastomas (Fisher et al., 2007).So, research continues to advocate Taxol as an importantchoice for treatment of glioblastoma <strong>and</strong> to identify themolecular markers that can modulate (increase or decrease)Taxol efficacy in glioblastoma. Apoptosis machinery doesexist in glioblastomas (Ray et al., 2002). But recurrentglioblastomas may lack or harbor molecular markers of cellsurvival. Identification of such molecular markers thatmodulate cell survival in recurrent glioblastomas may helpdesign rational therapeutics for their treatment.Phosphatase <strong>and</strong> tensin homolog located on chromosometen (PTEN), which encodes a cytoplasmic enzyme with bothprotein <strong>and</strong> lipid phosphatase activity, is frequently mutatedor deleted at chromosome 10q23 in malignant glioblastomas(Sano et al., 1999; Ohgaki <strong>and</strong> Kleihues, 2007). Recent studiesindicate that mutation <strong>and</strong> deletion of PTEN account for morethan 80% of human glioblastomas. Glioblastoma is the mostprevalent <strong>and</strong> malignant brain tumor in humans <strong>and</strong> theaverage survival time of glioblastoma patients is less than12 months even after treatment with the currently availablebest therapeutic regimens (Jiang et al., 2007; Cloughesy et al.,2008). Angiogenesis is a crucial feature for the growth <strong>and</strong>progression of glioblastoma (Hanahan <strong>and</strong> Folkman, 1996;Kleihues et al, 1999). In comparison with other malignancieselsewhere in the body, glioblastoma is a notorious tumor dueto its high capability of angiogenesis. Vascular endothelialgrowth factor (VEGF) plays a critical role in tumor angiogenesis<strong>and</strong> the level of expression of VGEF correlates with the degreeof malignancy of glioblastomas (Fischer et al., 2005; Reardonet al., 2008). Currently, it is a popular theory that attacking thetumor vascular bed instead of tumor cells themselves is aneffective anti-tumor strategy due to low cytotoxicity <strong>and</strong> lackof drug resistance problem in this approach (Steiner et al.,2004). Transfection of PTEN to glioblastoma cells coulddecrease the secretion of VEGF (Gomez-Manzano et al., 2003).Because of the modest anti-tumor activity of Taxol in someglioblastomas, we wanted to explore any potential role for thetumor suppressor PTEN in Taxol sensitivity in glioblastomasusing human glioblastoma LN18 (PTEN-positive) <strong>and</strong> A172(PTEN-negative) cells. Our findings suggest that the dramaticdifference in Taxol sensitivity for apoptosis in these two celllines is due to their PTEN status. Presence of PTEN makesglioblastoma cells sensitive to Taxol, which also reduces theexpression of VEGF in PTEN-positive cells but not in PTENnegativecells. Notably, VEGF is an important angiogenic factor<strong>and</strong> the primary focus of anti-angiogenic interventions. Ourstudy indicates that Taxol is a promising therapeutic agent forboth induction of apoptosis <strong>and</strong> inhibition of angiogenic factorin PTEN-positive glioblastoma cells.Fig. 1 – Western blotting to assess the levels of PTENexpression <strong>and</strong> activation of Akt in LN18 <strong>and</strong> A172 cells.(A) Representative Western blots showing the levels of PTENexpression. (B) Densitometric determination of the levels ofPTEN expression. (C) Representative Western blotsshowing activation or phosphorylation of Akt (p-Akt).(D) Densitometric determination of the levels of p-Akt.Treatments (24 h): Control, 25 nM Taxol, 50 nM Taxol, <strong>and</strong>20 μM LY294002 (1 h pre-treatment)+50 nM Taxol.Significant difference from control value was indicated by*p
218 BRAIN RESEARCH 1239 (2008) 216– 2252. Results2.1. Levels of PTEN expression in human glioblastomaLN18 <strong>and</strong> A172 cell linesOverexpression of the tumor suppressor PTEN in malignantcells contributes to apoptosis <strong>and</strong> suppression of survivalsignaling. We examined the levels of PTEN expression in LN18<strong>and</strong> A172 cells after treatments with 25 nM <strong>and</strong> 50 nM Taxol(Fig. 1). The results demonstrated that treatments of LN18 cellswith 25 nM <strong>and</strong> 50 nM Taxol induced PTEN expression (Fig. 1A)significantly (p
BRAIN RESEARCH 1239 (2008) 216– 22521925 nM <strong>and</strong> 50 nM Taxol significantly (p
220 BRAIN RESEARCH 1239 (2008) 216– 225active 37 kDa caspase-9 in LN18 <strong>and</strong> A172 cells after Taxoltreatments (Fig. 5B). Further, colorimetric assay confirmedsignificant (p
BRAIN RESEARCH 1239 (2008) 216– 225221(Fig. 7B) <strong>and</strong> caspase-3-specific 120 kDa SBDP (Fig. 7C),respectively, after Taxol treatments.2.6. Changes in expression of VEGF <strong>and</strong> bFGFVEGF is a key factor for changing tumor microenvironment<strong>and</strong> tumor angiogenesis. It has been reported previously thatthe expression of VEGF in glioblastoma is regulated in a PI3Kdependentmanner (Maity et al., 2000). Because PTEN controlsPI3K activity, the level of PTEN may alter expression of VEGF<strong>and</strong> any other angiogenic factor. Expression of bFGF alsocontributes to tumor angiogenesis. We used Western blottingto examine any influence of PTEN status in changing levels ofthe potential angiogenic factors (VEGF <strong>and</strong> bFGF) in LN18 <strong>and</strong>A172 cells after Taxol treatments (Fig. 8). Western blotting (Fig.8A) showed that 50 nM Taxol caused a significant decrease inexpression of VEGF (Fig. 8B) <strong>and</strong> no change in expression ofbFGF (Fig. 8C) in PTEN-positive LN18 cells. On the other h<strong>and</strong>,Taxol treatments increased the levels of VEGF significantly(Fig. 8B) <strong>and</strong> bFGF non-significantly (Fig. 8C) in the PTENnegativeA172 cells.Fig. 8 – Western blotting to estimate the levels of VEGF <strong>and</strong>bFGF in LN18 <strong>and</strong> A172 cells. Treatments (24 h): Control,25 nM Taxol, <strong>and</strong> 50 nM Taxol. (A) Representative Westernblots to show the changes in the levels of VEGF <strong>and</strong> bFGF.Expression of β-actin was examined to ensure almostuniform protein loading in all lanes. (B) Bar diagrams to showdensitometric determination of the levels of VEGF. (C) Bardiagrams to show densitometric determination of the levelsof bFGF. Significant difference from control value wasindicated by *p
222 BRAIN RESEARCH 1239 (2008) 216– 225treatment (Fig. 5). We performed Western blotting <strong>and</strong>colorimetric assay to observe increases in active 37 kDacaspase-9 fragment <strong>and</strong> caspase-9 activity, respectively,after Taxol treatments. The low dose of Taxol was sufficientto cause the highest increases in caspase-9 activation <strong>and</strong>activity in LN18 cells while Taxol dose-dependently increasedthe caspase-9 activation <strong>and</strong> activity in A172 cells (Fig. 5). Wealso examined the increases in active 20 kDa caspase-3fragment <strong>and</strong> caspase-3 activity following Taxol treatments(Fig. 6). The low <strong>and</strong> high doses of Taxol were most efficaciousin LN18 <strong>and</strong> A172 cells, respectively, for increasing thecaspase-3 activation <strong>and</strong> activity. Similarly, the low <strong>and</strong> highdoses of Taxol were the most effective in LN18 <strong>and</strong> A172 cells,respectively, for proteolysis of 270 kDa α-spectrin to generatethe calpain-specific 145 kDa SBDP <strong>and</strong> caspase-3-specific120 kDa SBDP (Fig. 7). These results demonstrated that bothlow <strong>and</strong> high doses of Taxol in LN18 <strong>and</strong> A172 cells caused thehighest increases in proteolytic activities of calpain <strong>and</strong>caspase-3. Therefore, the presence of PTEN in LN18 cellsappeared to help increase the sensitivity to Taxol for activationof proteolytic activities for apoptosis.Because VEGF <strong>and</strong> bFGF are the critical angiogenic factors forpromoting growth in glioblastoma, we examined the changes inthe levels of VEGF <strong>and</strong> bFGF in LN18 <strong>and</strong> A172 cells after Taxoltreatments (Fig. 8). Taxol treatments significantly decreasedexpression of VEGF in LN18 cells but increased in A172 cells. Ithas previously been shown that transfection of PTEN into theglioblastoma U87MG cells using retroviral vector could suppresstumor angiogenesis due to decrease in VEGF at the mRNA levelin vitro as well as in vivo (Wen et al., 2001; Abe et al., 2003).When compared with the PTEN-positive cells, the PTENnegativecells showed high secretion of VEGF protein (Zundelet al., 2000; Edwards et al., 2008). Integrin-linked kinase (ILK) is atherapeutic target in cancer treatment. Studies demonstratedthat the PTEN-positive cells were sensitive to ILK inhibitorsthan the PTEN-negative cells <strong>and</strong> also suggested that ILKinhibitors decreased VEGF secretion due to induction of PTENexpression (Edwards et al., 2008). Our data indicated that Taxolcould decrease expression of VEGF significantly <strong>and</strong> bFGF tosome extent in PTEN-positive LN18 cells but increased theirlevels in PTEN-negative A172 cells.In conclusion, our current study demonstrated differentialanti-cancer effects of Taxol in human glioblastoma LN18(PTEN-positive) <strong>and</strong> A172 (PTEN-negative) cells. Activation ofthe cell survival PI3K/Akt signaling pathway <strong>and</strong> lastingexpression of the anti-apoptotic Bcl-2 in PTEN-negativeglioblastoma cells could confer resistance to Taxol chemotherapy.This study implies that the absence of PTEN inrecurrent glioblastomas may help avoid apoptosis <strong>and</strong> promoteangiogenesis leading to overall poor prognosis of theseglioblastomas even after treatment with a powerful chemotherapeuticdrug such as Taxol.4. Experimental procedures4.1. MaterialsHuman glioblastoma cell lines LN18 <strong>and</strong> A172 were purchasedfrom the American Type Culture Collection (ATCC, Rockville,MD, USA). We obtained polyclonal IgG antibodies against PTENfrom Cell Signaling Technologies (Danvers, MA, USA), cytochromec from BD Biosciences (San Jose, CA, USA), <strong>and</strong> Bax,Bcl-2, VEGF, <strong>and</strong> bFGF from Santa Cruz Biotechnology (SantaCruz, CA, USA). Monoclonal IgG antibody against α-spectrinwas received from Affiniti (Exeter, UK).4.2. Cell cultureHuman glioblastoma LN18 <strong>and</strong> A172 cell lines were separatelygrown in DMEM supplemented with 10% fetal bovine serum(FBS) <strong>and</strong> 1% penicillin <strong>and</strong> streptomycin. Cells were seeded in75-cm 2 flasks <strong>and</strong> incubated at 37 °C in a fully-humidifiedatmosphere with 5% CO 2 . Once the cells were 80% confluent,they were starved in DMEM with 1% FBS for 24 h <strong>and</strong>maintained in this low serum condition in the course of alltreatments. Cells were treated with 25 nM <strong>and</strong> 50 nM Taxol for24 h. After the treatments, cells were processed for assessmentsof residual cell viability, morphological <strong>and</strong> biochemicalfeatures of apoptosis, apoptosis-related proteins byWestern blotting, <strong>and</strong> caspase activities by colorimetricassays.4.3. Cell viability assayAfter the treatments of cells with Taxol, trypan blue dyeexclusion test was performed to evaluate the residual cellviability (Ray et al., 1999; 2006). Viable cells maintainedmembrane integrity <strong>and</strong> did not take up trypan blue. Cellswith compromised cell membranes took up trypan blue <strong>and</strong>therefore were counted as dead. At least 600 cells in eachtreatment were counted in four different fields <strong>and</strong> thenumber of viable cells was calculated as percentage of thetotal cell population.4.4. Wright staining for morphological featuresof apoptosisCells from all treatments were harvested <strong>and</strong> washed in PBS,pH 7.4, <strong>and</strong> sedimented onto the microscopic slides using theCentra CL2 centrifuge (IEC, Needham Heights, MA, USA) at1000 rpm for 5 min. Cells were fixed for Wright staining, as wereported previously (Das et al., 2006). Cellular morphology wasexamined under the light microscopy to examine amount ofapoptosis. Cells were considered apoptotic with the characteristicssuch as reduction in cell volume, condensation of thechromatin, <strong>and</strong>/or the presence of cell membrane blebbing.About 600 cells were counted in each treatment <strong>and</strong> thepercentage of apoptotic cells was calculated.4.5. ApopTag peroxidase assay for detection of apoptoticDNA fragmentationFor detection of DNA fragmentation as a biochemical markerof apoptosis, glioblastoma cells on the microscopic slides weresubjected to the TdT-mediated dUTP nick-end labeling(TUNEL) using the ApopTag peroxidase assay kit (Intergen,Purchase, NY, USA), as we reported (Ray et al., 2006). In TUNELstaining, we used 3,3′-diaminobenzidine (DAB) as a peroxidasesubstrate. Cells with DNA fragmentation produced a brown
BRAIN RESEARCH 1239 (2008) 216– 225223product from oxidative polymerization <strong>and</strong> cyclization of DABin the course of the ApopTag peroxidase assay. ApopTagpositivecells were brown in a pale green background <strong>and</strong> wereconsidered as apoptotic cells. Experiments were conducted intriplicate <strong>and</strong> the percentage of ApopTag-positive cells wasdetermined by counting the brown cells from r<strong>and</strong>omlyselected fields under the light microscope.4.6. Protein extraction <strong>and</strong> Western blottingAfter the treatments, cells were lysed in a buffer composed of50 mM Tris–HCl, pH 7.4, 0.1 mM phenylmethylsulfonyl fluoride(PMSF), <strong>and</strong> 5 mM EGTA for extraction of cellular proteins.Concentration of total proteins was determined colorimetricallyusing Coomassie-Plus protein assay reagent (Pierce,Rockford, IL, USA). The samples were mixed with an equalvolume of 2× loading buffer [125 mM Tris–HCl, pH 6.8, 4%sodium dodecyl sulfate (SDS), 20% glycerol, 200 mM 1,4-dithio-DL-threitol (DTT), <strong>and</strong> 0.02% bromophenol blue], boiled for5 min, <strong>and</strong> loaded (40 μg/lane) onto the 4–20% gradient gels forthe SDS-polyacrylamide gel electrophoresis (SDS-PAGE). AfterSDS-PAGE, the gels were blotted to Immunobilon-P nylonmembrane. The blots were blocked in 5% non-fat milk, 0.1%Tween, Tris–HCl, pH 7.8, for 2 h at room temperature. Then theblots were incubated with a specific primary IgG antibody for2 h at room temperature or overnight at a cold room followedby alkaline horseradish peroxidase-conjugated secondary IgGantibody for 1 h. Blots were developed using the enhancedchemiluminescence (ECL) or ECL-Plus reagents (AmershamPharmacia, Buckinghamshire, UK). The ECL autoradiogramswere scanned on a PowerLook Scanner (Umax Technologies,Fremont, CA, USA) using Photoshop software (Adobe Systems,Seattle, WA, USA) <strong>and</strong> the optical density (OD) of each b<strong>and</strong>was determined using Quantity One software (Bio-Rad,Hercules, CA, USA).4.7. Analysis of mitochondrial release of cytochrome c intothe cytosolCells from each treatment were harvested, washed once withice-cold PBS, <strong>and</strong> gently lysed for 1 min in 50 μl ice-cold lysisbuffer (250 mM sucrose, 1 mM EDTA, 0.05% digitonin, 25 mMTris–HCl, pH 6.8, 1 mM DTT, 1 μg/ml leupeptin, 1 μg/mlpepstatin, 1 μg/ml aprotinin, 1 mM benzamidine, <strong>and</strong> 0.1 mMPMSF) following the st<strong>and</strong>ard procedure (Piqué et al., 2000).Lysates were centrifuged at 12,000 ×g in cold (4 °C) for 3 minto obtain a pellet (the fraction containing mitochondria) <strong>and</strong>supernatant (the cytosolic extract without mitochondria).Pellet <strong>and</strong> supernatant were analyzed by Western blottingusing cytochrome c antibody.4.8. Colorimetric assays for measurement of caspase-9<strong>and</strong> caspase-3 activitiesMeasurements of caspase-9 <strong>and</strong> caspase-3 activities in cellswere performed with the commercially available colorimetricassay kits (Sigma Chemical, St. Louis, MO, USA). Proteolyticactivities of caspase-9 <strong>and</strong> caspase-3 were assayed based onthe release of p-nitroanilide (p-NA) from LEHD-p-NA <strong>and</strong>DEVD-p-NA, respectively. The concentration of the p-NAreleased from the substrate was calculated from the absorbanceat 405 nm.4.9. Statistical analysisResults were analyzed using StatView software (AbacusConcepts, Berkeley, CA, USA) <strong>and</strong> compared using one-wayanalysis of variance (ANOVA) with Fisher's post hoc test.Data were presented as mean±st<strong>and</strong>ard deviation (SD) ofseparate experiments (n ≥ 3). A significant difference fromcontrol value was indicated by ⁎(p
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