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• W2081005846 abstract "We have studied the potential effect against human malignant mesotheliomas(MM) of α-tocopheryl succinate (α-TOS), a redox-silent vitamin Eanalog with strong pro-apoptotic and anti-cancer activity. α-TOS atsub-apoptotic levels inhibited proliferation of MM cell lines, while beingnontoxic to nonmalignant mesothelial cells. Because MM cells are typified by ahighly metastatic phenotype, we investigated the effect of α-TOS ongenes playing a major role in MM progression. Of these, α-TOS downregulated fibroblast growth factor (FGF)-1 and, in particular, FGF-2 on thetranscriptional level in MM cells, and this was not observed in theirnonmalignant counterparts. FGF-2 short interfering RNA suppressedproliferation of MM cells. Down-regulation of FGF-2 was likely because ofinhibition of the egr-1 transcription activity that was decreased in MM cellsvia oxidative stress induced by α-TOS, as evidenced by EPR spectroscopy,whereas nonmalignant cells did not show this response. Treatment of MM cellswith egr-1 short interfering RNA suppressed proliferation, which wasoverridden by exogenously added recombinant FGF-1 and, in particular, FGF-2.An analog of coenzyme Q targeted to mitochondria and superoxide dismutaseoverrode inhibition of MM cell proliferation by α-TOS as well asα-TOS-induced inhibition of egr-1-dependent transactivation. Finally,α-TOS significantly suppressed experimental MM in immunocompromisedmice. Our data suggest that α-TOS suppresses MM cell proliferation bydisrupting the FGF-FGF receptor autocrine signaling loop by generatingoxidative stress and point to the agent as a selective drug against thus farfatal mesotheliomas. We have studied the potential effect against human malignant mesotheliomas(MM) of α-tocopheryl succinate (α-TOS), a redox-silent vitamin Eanalog with strong pro-apoptotic and anti-cancer activity. α-TOS atsub-apoptotic levels inhibited proliferation of MM cell lines, while beingnontoxic to nonmalignant mesothelial cells. Because MM cells are typified by ahighly metastatic phenotype, we investigated the effect of α-TOS ongenes playing a major role in MM progression. Of these, α-TOS downregulated fibroblast growth factor (FGF)-1 and, in particular, FGF-2 on thetranscriptional level in MM cells, and this was not observed in theirnonmalignant counterparts. FGF-2 short interfering RNA suppressedproliferation of MM cells. Down-regulation of FGF-2 was likely because ofinhibition of the egr-1 transcription activity that was decreased in MM cellsvia oxidative stress induced by α-TOS, as evidenced by EPR spectroscopy,whereas nonmalignant cells did not show this response. Treatment of MM cellswith egr-1 short interfering RNA suppressed proliferation, which wasoverridden by exogenously added recombinant FGF-1 and, in particular, FGF-2.An analog of coenzyme Q targeted to mitochondria and superoxide dismutaseoverrode inhibition of MM cell proliferation by α-TOS as well asα-TOS-induced inhibition of egr-1-dependent transactivation. Finally,α-TOS significantly suppressed experimental MM in immunocompromisedmice. Our data suggest that α-TOS suppresses MM cell proliferation bydisrupting the FGF-FGF receptor autocrine signaling loop by generatingoxidative stress and point to the agent as a selective drug against thus farfatal mesotheliomas. Malignant mesothelioma(MM) 1The abbreviations used are: MM, malignant mesothelioma; BrdUrd,5-bromo-2-deoxyuridine; DCF, dihydrodichlorofluorescein diacetate; DMPO,5,5-dimethyl-1-pyrroline N-oxide; egr-1, early growth responsefactor-1; hr, human recombinant; FGF, fibroblast growth factor; FGFR, FGFreceptor; FITC, fluorescein isothiocyanate; MAP, mitogen-activated protein;mito-Q, mitochondrially targeted coenzyme Q; PI, propidium iodide; Q-PCR,quantitative real time PCR; RNAi, RNA interference; ROS, reactive oxygenspecies; siRNA, short interfering RNA; SOD, superoxide dismutase; α-TOH,α-tocopherol; TGF-β, transforming growth factor-β;α-TOS, α-tocopheryl succinate; VEGF, vascular endothelial growthfactor; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco's modifiedEagle's medium; PBS, phosphate-buffered saline.1The abbreviations used are: MM, malignant mesothelioma; BrdUrd,5-bromo-2-deoxyuridine; DCF, dihydrodichlorofluorescein diacetate; DMPO,5,5-dimethyl-1-pyrroline N-oxide; egr-1, early growth responsefactor-1; hr, human recombinant; FGF, fibroblast growth factor; FGFR, FGFreceptor; FITC, fluorescein isothiocyanate; MAP, mitogen-activated protein;mito-Q, mitochondrially targeted coenzyme Q; PI, propidium iodide; Q-PCR,quantitative real time PCR; RNAi, RNA interference; ROS, reactive oxygenspecies; siRNA, short interfering RNA; SOD, superoxide dismutase; α-TOH,α-tocopherol; TGF-β, transforming growth factor-β;α-TOS, α-tocopheryl succinate; VEGF, vascular endothelial growthfactor; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco's modifiedEagle's medium; PBS, phosphate-buffered saline. is a highlyaggressive tumor that arises from mesothelial cells of the serosal surfaces ofbody cavities. It has a very grim prognosis, as patients frequently succumbwithin months after diagnosis. The incidence of MM is increasing, and aprogressive rise has been predicted until at least the year 2020(1Steele J.P. Semin.Oncol. 2002; 29: 36-40Crossref PubMed Scopus (39) Google Scholar,2Baas P. Curr. Opin.Oncol. 2003; 15: 127-130Crossref PubMed Scopus (24) Google Scholar). The lack of effectivetreatment for MM makes studies aimed at understanding the molecular mechanismunderlying proliferation and metastasis of MM of high relevance(3Tomek S. Emri S. Krejcy K. Manegold C. Br. J. Cancer. 2003; 88: 167-174Crossref PubMed Scopus (74) Google Scholar).Of the cytokines that are known to elicit a potent mitogenic response,vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-1and FGF-2, and the transforming growth factor-β (TGF-β) are involvedin the malignant phenotype of various cancers(4Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7176) Google Scholar, 5Cross M.J. Claesson-Welsh L. Trends Pharmacol. Sci. 2001; 22: 201-207Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar, 6Kumar-Singh S. Weyler J. Martin M.J. Vermeulen P.B. Van Marck E. J. Pathol. 1999; 189: 72-78Crossref PubMed Scopus (169) Google Scholar).These cytokines have been also suggested to control proliferation andmigration of MM cells (7Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3128) Google Scholar).Active VEGF is expressed in a variety of human malignancies including MM(8Catalano A. Romano M. Martinotti S. Procopio A. Oncogene. 2002; 21: 2896-2900Crossref PubMed Scopus (55) Google Scholar). The biological effects ofVEGF are mediated via two distinct cell surface tyrosine kinase receptors, Fltand Flk. Upon VEGF binding, the receptors dimerize, and several intracellularsignaling routes that induce proliferation are activated, including themitogen-activated protein (MAP) kinase pathway(5Cross M.J. Claesson-Welsh L. Trends Pharmacol. Sci. 2001; 22: 201-207Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar,7Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3128) Google Scholar). FGF belongs to a family of23 members, of which FGF-1 and FGF-2 have been proposed to play a role in MMcell (patho)physiology (9Strizzi L. Vianale G. Catalano A. Muraro R. Mutti L. Procopio A. Int. J. Oncol. 2001; 1: 1093-1098Google Scholar).Signaling mechanisms induced by FGFs are mediated by four high affinitytyrosine kinase receptors known as FGF receptors (FGFR), which aretransmembrane proteins that, once bound to cognate FGFs, dimerize and signalvia intracellular pathways, such as the MAP kinase route, that induce cellproliferation and migration(5Cross M.J. Claesson-Welsh L. Trends Pharmacol. Sci. 2001; 22: 201-207Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). FGF receptors (FGFR)1–3 are alternatively spliced into two specific exons denoted IIIb andIIIc. The expression of FGFR 1–3 isoforms is highly controlled andessential for initiating specific cellular responses(5Cross M.J. Claesson-Welsh L. Trends Pharmacol. Sci. 2001; 22: 201-207Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). TGF-β is a potentgrowth regulatory cytokine that exerts a diverse range of effects on manytypes of cells. TGF-β has potent mitogenic effects on several types of MMcells, which produce it at relatively high levels(6Kumar-Singh S. Weyler J. Martin M.J. Vermeulen P.B. Van Marck E. J. Pathol. 1999; 189: 72-78Crossref PubMed Scopus (169) Google Scholar,10Marzo A.L. Fitzpatrick D.R. Robinson B.W. Scott B. Cancer Res. 1997; 57: 3200-3207PubMed Google Scholar). All of these cytokinesmay control proliferation of MM cells via autocrine signaling(11Bermudez E. Everitt J. Walker C. Exp. Cell Res. 1990; 190: 91-98Crossref PubMed Scopus (52) Google Scholar,12Walker C. Everitt J. Ferriola P.C. Stewart W. Mangum J. Bermudez E. Cancer Res. 1995; 55: 530-536PubMed Google Scholar).Because these inducers and mediators are essential for the process offormation and progression of MM, understanding the regulatory mechanisms oftheir expression is important, because they control pathways that may presenta target for MM treatment (6Kumar-Singh S. Weyler J. Martin M.J. Vermeulen P.B. Van Marck E. J. Pathol. 1999; 189: 72-78Crossref PubMed Scopus (169) Google Scholar,13Ohta Y. Shridhar V. Bright R.K. Kalemkerian G.P. Du W. Carbone M. Watanabe Y. Pass H.I. Br.J. Cancer. 1999; 81: 54-61Crossref PubMed Scopus (308) Google Scholar,14Nowak A.K. Lake R.A. Kindler H.L. Robinson B.W. Semin. Oncol. 2002; 29: 82-96Crossref PubMed Scopus (121) Google Scholar). Conceivably, an idealanti-cancer agent preventing progression of the metastatic disease wouldselectively down-regulate the expression of those cytokines that positivelycontrol tumor growth, while being nontoxic toward normal cells.Recent data showed that analogs of vitamin E have potent anti-proliferativeand pro-apoptotic effects on multiple cancer cell lines and inhibit cancer inpre-clinical models (15Neuzil J. Br. J.Cancer. 2003; 89: 1822-1826Crossref PubMed Scopus (103) Google Scholar).These compounds are epitomized by α-tocopheryl succinate (α-TOS),a redox-silent compound that has been reported to suppress several types ofneoplasia(16Malafa M.P. Fokum F.D. Mowlavi A. Abusief M. King M. Surgery. 2002; 131: 85-91Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 17Weber T. Lu M. Andera L. Lahm H. Gellert N. Fariss M.W. Korinek V. Sattler W. Ucker D.S. Terman A. Schroder A. Erl W. Brunk U.T. Coffey R.J. Weber C. Neuzil J. Clin. Cancer Res. 2002; 8: 863-869PubMed Google Scholar, 18Malafa M.P. Fokum F.D. Smith L. Louis A. Ann. Surg. Oncol. 2002; 9: 1023-1032Crossref PubMed Google Scholar, 19Barnett K.T. Fokum F.D. Malafa M.P. J. Surg. Res. 2002; 106: 292-298Abstract Full Text PDF PubMed Scopus (77) Google Scholar).The vitamin E analogs inhibit proliferation of cancer cells by severalmechanisms, including inhibition of DNA synthesis, induction of apoptosis andcellular differentiation, and by affecting the protein kinase C and the MAPkinase pathways(19Barnett K.T. Fokum F.D. Malafa M.P. J. Surg. Res. 2002; 106: 292-298Abstract Full Text PDF PubMed Scopus (77) Google Scholar, 20Neuzil J. Weber T. Schroder A. Lu M. Ostermann G. Gellert N. Mayne G.C. Olejnicka B. Negre-Salvayre A. Sticha M. Coffey R.J. Weber C. FASEBJ. 2001; 15: 403-415Crossref PubMed Scopus (267) Google Scholar, 21You H. Yu W. Munoz-Medellin D. Brown P.H. Sanders B.G. Kline K. Mol. Carcinog. 2002; 33: 228-236Crossref PubMed Scopus (44) Google Scholar, 22Yu W. Liao Q.Y. Hantash F.M. Sanders B.G. Kline K. Cancer Res. 2001; 61: 6569-6576PubMed Google Scholar, 23Neuzil J. Weber T. Gellert N. Weber C. Br. J. Cancer. 2001; 84: 87-89Crossref PubMed Scopus (220) Google Scholar, 24Birringer M. EyTina J.H. Salvatore B.A. Neuzil J. Br. J. Cancer. 2003; 88: 1948-1955Crossref PubMed Scopus (126) Google Scholar).More importantly, α-TOS exerts anti-proliferative/pro-apoptotic effectsin malignant cell lines but is largely nontoxic toward normal cells andtissues (17Weber T. Lu M. Andera L. Lahm H. Gellert N. Fariss M.W. Korinek V. Sattler W. Ucker D.S. Terman A. Schroder A. Erl W. Brunk U.T. Coffey R.J. Weber C. Neuzil J. Clin. Cancer Res. 2002; 8: 863-869PubMed Google Scholar,23Neuzil J. Weber T. Gellert N. Weber C. Br. J. Cancer. 2001; 84: 87-89Crossref PubMed Scopus (220) Google Scholar,25Neuzil J. Tomasetti M. Mellick A.S. Alleva R. Salvatore B.A. Birringer M. Fariss M.W. Curr.Cancer Drug Targets. 2004; 4: 267-284Crossref PubMed Scopus (91) Google Scholar). It has also been reportedthat α-TOS can modulate cytokine gene expression in cancer cells(26Turley J.M. Funakoshi S. Ruscetti F.W. Kasper J. Murphy W.J. Longo D.L. Birchenall-Roberts M.C. Cell Growth & Differ. 1995; 6: 655-663PubMed Google Scholar,27Malafa M.P. Neitzel L.T. J.Surg. Res. 2000; 93: 163-170Abstract Full Text PDF PubMed Scopus (131) Google Scholar). For example, α-TOSdown-regulates FGFR-1 in MM cells, although the precise mechanism has not beenresolved (28Stapelberg M. Tomasetti M. Gellert N. Procopio A. Neuzil J. Biochem. Biophys. Res.Commun. 2004; 318: 636-641Crossref PubMed Scopus (23) Google Scholar).Because α-TOS is a potent inducer of apoptosis in the generallyresistant MM cells (29Tomasetti M. Rippo M.R. Alleva R. Moretti S. Andera L. Neuzil J. Procopio A. Br. J.Cancer. 2004; 90: 1644-1653Crossref PubMed Scopus (54) Google Scholar) andinhibits MM in vivo(30Tomasetti M. Gellert N. Procopio A. Neuzil J. Int. J. Cancer. 2004; 109: 641-642Crossref PubMed Scopus (51) Google Scholar), we investigated theeffects of the vitamin E analog on expression of cytokines involved in controlof cancer development and progression. We show here that α-TOS disruptsthe FGF autocrine loop through suppression of egr-1 transcriptional activityin MM cells but not in their nonmalignant counterparts, further highlightingthe potential of α-TOS as a therapeutic agent.EXPERIMENTAL PROCEDURESCell Culture—Human MM cell lines, MM-B1 (biphasic), Meso-2(sarcomatose), Ist-Mes and Ist-Mes2 (both epithelioid)(31Pass H.I. Stevens E.J. Oie H. Tsokos M.G. Abati A.D. Fetsch P.A. Mew D.J. Pogrebniak H.W. Matthews W.J. Ann. Thorac. Surg. 1995; 59: 835-844Abstract Full Text PDF PubMed Scopus (119) Google Scholar), and a nonmalignantmesothelial cell line, Met-5A (ATCC), were used in our studies. The cells werecultured in DMEM supplemented with 10% fetal calf serum (both from JRHBiosciences), 2 mm l-glutamine, 100 units/ml penicillin, and 100μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C. All assays were performed when the cells reached 60–70%confluency.Cell Proliferation Assay—Cell proliferation was determinedby assessing DNA synthesis. An end point enzyme-linked immunosorbent assay(ELISA) colorimetric kit (Roche Applied Science) was used to determine cellsin the S phase of the mitotic cycle, based on incorporation of5-bromo-2-deoxyuridine (BrdUrd) into their DNA. Briefly, MM cells were seededat 104 cells per well into a 96-well plate, treated as specified,and incubated with 10 μm BrdUrd for 2 h at 37 °C. The cellswere then fixed and denatured by a 30-min incubation with Fixdenat (RocheApplied Science), incubated for 90 min with an anti-BrdUrd antibody, washed,and incubated further with a substrate solution (tetramethylbenzidine). After30 min, 1 m H2SO4 was added to each well tostop the reaction, and the absorbance was read at 450 nm using a plate reader.In some cases, cells were assessed for the expression of aproliferation-specific gene, the proliferating cell nuclear antigen by Westernblotting, using an anti-proliferating cell nuclear antigen IgG (Santa CruzBiotechnology). Where indicated, cells were exogenously supplemented withhuman recombinant (hr) FGF-1 or hrFGF-2 (both from Sigma), at 10 ng/ml 24 hprior to treatment.Cell Cycle Assay—MM and Met-5A cells were plated at105 cells per well in 24-well plates. The cells were allowed toattach overnight and were then incubated for up to 3 days with α-TOS.The floating and attached cells were collected, washed with PBS, resuspendedin buffer containing sodium citrate (1%), Triton X-100 (0.1%), RNase A (0.05μg/ml), and propidium iodide (PI) at 5 μg/ml, and incubated in the darkfor 30 min at 4 °C. The nuclear suspension was filtered through a 60-μmmesh and analyzed by flow cytometry.Apoptosis Assessment—Apoptosis was quantified by the annexinV-fluorescein isothiocyanate (FITC) method, which detects phosphatidylserineexternalized in the early phases of apoptosis(20Neuzil J. Weber T. Schroder A. Lu M. Ostermann G. Gellert N. Mayne G.C. Olejnicka B. Negre-Salvayre A. Sticha M. Coffey R.J. Weber C. FASEBJ. 2001; 15: 403-415Crossref PubMed Scopus (267) Google Scholar). Briefly, cells wereseeded at the density of 105 cells per well in 24-well plates andtreated with α-TOS after overnight recuperation. Floating and attachedcells were collected, washed with PBS, resuspended in 0.1 ml of binding buffer(10 mm HEPES, 140 mm NaCl, 5 mmCaCl2, pH 7.4), incubated for 20 min at room temperature with 2μl of annexin V-FITC supplemented with 10 μl of PI (10 μg/ml), andanalyzed by flow cytometry (FACSCalibur; BD Biosciences) using channel 1 forannexin V-FITC binding and channel 2 for PI staining.Detection of Reactive Oxygen Species (ROS)—Cellular ROS weredetected indirectly by flow cytometry and directly by EPR spectroscopy,following treatment of cells with α-TOS as indicated in the figurelegends. In some experiments, the cells were pretreated for 1 h with 2μm mitochondrially targeted coenzyme Q (mito-Q)(32Kelso G.F. Porteous C.M. Coulter C.V. Hughes G. Porteous W.K. Ledgerwood E.C. Smith R.A. Murphy M.P. J. Biol. Chem. 2001; 276: 4588-4596Abstract Full Text Full Text PDF PubMed Scopus (860) Google Scholar) or co-incubated withsuperoxide dismutase (SOD; EC 1.15.1.1; Sigma S4636) at 750 units/ml. Forindirect evaluation, cells were treated with α-TOS and reacted withdihydrodichlorofluorescein diacetate (DCF; Molecular Probes) for 30 min, andscored by flow cytometry for cells with high fluorescence, which was evaluatedon the basis of an increase in mean fluorescence intensity. EPR spectroscopyanalysis of ROS generation was based on the use of the radical trap5,5-dimethyl-1-pyrroline N-oxide (DMPO; Sigma). In brief, cells wereplated in T25 flasks and allowed to reach 60–70% confluency (∼5× 106 cells per flask). Cells were washed, overlaid with thePSS medium (33Thomas S.R. Chen K. Keaney J.F. J. Biol. Chem. 2002; 277: 6017-6024Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar), and incubatedwith 50 μm α-TOS 5 min after addition of 10 mmDMPO. Analyses of DMPO adducts were performed with samples taken from the cellsuspension as well as the cell-conditioned medium transferred into a quartzflat cell (Wilmad). The quartz cell was then placed into the cavity of theBruker EMX bench-top spectrometer set at 293 K with the following spectrometerparameters: field sweep 10 millitesla, microwave power 20 milliwatts,microwave frequency 100 kHz, modulation amplitude, 0.1 millitesla, sweep time83.9 s. The detection limit of the stable nitroxide (TEMPO) under identicalconditions was ∼50 nm.Real Time mRNA Analysis—Relative quantification of mRNAexpression was achieved using quantitative real time-PCR (Q-PCR). Briefly,this technique is based on the detection of a fluorescent signal produced bythe incorporation of the fluorescent dye SYBR-green during PCR amplification(Prism 7700 sequence detection system; Applied Biosystems). The expression ofall genes of interest was related to that of the 18 S RNA control. Total RNAwas extracted from MM cell cultures using Trizol (Invitrogen). To minimizepotential genomic contamination, RNA samples were treated with RQ1 RNase-freeDNase (Promega) and purified using the RNeasy mini kit (Qiagen). Each assaywas performed according to the manufacturer's protocol. First strand cDNA wassynthesized using the Superscript III Reverse Transcriptase kit (Invitrogen)according to the manufacturer's protocol. PCR primers were specificallydesigned for real time PCR. Particular attention was given to maintaining aconstant 60 °C annealing temperature of primer pairs, which is common forQ-PCR. Each set of primers was also designed across intron/exon boundaries todetect genomic DNA contamination.The primers used were as follows: FGF-1, 5′-GGG CTT TTA TACGGC TCA CA-3′, and 5′-GGC CAA CAA ACC AAT TCT TC-3′;FGF-2, 5′-GAC CCT CAC ATC AAG CTA CAA CT-3′, and5′-AAA GAA ACA CTC ATC CGT AAC ACA-3′; VEGF,5′-AGGCCA GCA CAT AGG AGA GA-3′, and 5′-TTT CTT GCG CTT TCG TTTTT-3′; TGF-β,5′-GAG CCT GAG GCC GAC TAC TA-3′,and 5′-TCG GAG CTC TGA TGT GTT GA-3′.RNA Interference (RNAi)—For RNAi, cells were seeded at afinal density of 5 × 104 cells per well in 12-well plates,cultured until ∼50% confluency, and then treated with egr-1, FGF-1, orFGF-2 siRNA (all designed and synthesized by Proligo) as follows: siRNA (0.5μg/ml) was combined with 100 μl of serum-free DMEM supplemented with 20μlof Oligofectamine (Invitrogen) and left for 15 min at room temperature.The transfection mixture was added to cells, which were then left in theincubator for 24 h, after which they were overlaid with complete DMEM.24–48 h later, the cells were used in experiments. Typically,90–95% of treated cells showed significant down-regulation of thetargeted genes as estimated by flow cytometric analysis (data not shown).Nonsilencing RNA was used as a negative control and FITC-tagged nonspecificRNA as a control for transfection efficacy (both Qiagen).Analysis of FGF-1 and FGF-2 Protein—The FGF-1 and FGF-2protein levels were assessed using an ELISA kit (R & D Systems) accordingto the manufacturer's instructions. In brief, cells were seeded in 24-wellplates and allowed to reach 60–70% confluency. Following treatment, 100μl of cell-conditioned medium was transferred to the ELISA 96-well plate,mixed with 100 μl of the assay diluent, and incubated for 2 h at roomtemperature. After washing, each well was supplemented with 200 μl of FGFconjugate, which was followed by a 2-h incubation at room temperature and a30-min incubation with 200 μl of the substrate solution. Absorbance at 450nm was assessed using an ELISA plate reader. The system was calibrated usinghrFGF-1 or hrFGF-2.Immunofluorescence Analysis of Protein Expression—MM andMet-5A cells were grown on coverslips and treated with α-TOS at 10 and20 μm for 24 and 48 h. The cells were then fixed in 3.6%formalin in PBS and reacted on ice for 2 h with anti-egr-1, anti-FGF-1, oranti FGF-2 IgG (all from Santa Cruz Biotechnology) diluted 1:100 in PBScontaining 2% fetal calf serum, followed by incubation with a secondary,FITC-, or Texas Red-conjugated IgG. Background fluorescence was accounted forin samples where primary antibody was omitted. The cells were then mounted in4,6-diamidino-2-phenylindole-containing Vectashield and assessed forfluorescence, reflecting protein expression, using the Leica DMI ER2fluorescence microscope. Images were taken, and the level of staining wasestimated using the Leica image analysis/deconvolution software.Assessment of egr-1 Transcription Activity—To assess whetherα-TOS affects egr-1-dependent trans-activation, cells were transientlytransfected with a plasmid comprising the egr-1-response element in thepromoter followed by the lux gene (Stratagene) and treated asdescribed in the figure legends. Luciferase activity was assessed using theLuciferase Reporter Gene Detection Kit (Sigma) according to the manufacturer'sprotocol, and the extent of luciferase activity was related to the activity inthe untreated controls.Animal Experiments—Immunocompromised (athymic) mice wereinjected subcutaneously with Ist-Met2 (2 × 106 cells peranimal). After 15 days when tumors were established, the mice were injectedinto the peritoneum with 100 μl of 200 mm α-TOS (dilutedper animal) per animal every 3 days or with the vehicle alone. Tumor volumewas estimated with calipers, and the volume was calculated by using theequation height × length × width × 0.524 as describedpreviously (34Kogure K. Manabe S. Hama S. Tokumura A. Fukuzawa K. Cancer Lett. 2003; 192: 19-24Crossref PubMed Scopus (48) Google Scholar). The growth oftumors was expressed as an increase in their volume relative to the tumorvolume at the onset of treatment.Assessment of Intracellular Levels of α-TOS andα-TOH—To assess the levels of the two vitamin E analogs,a high pressure liquid chromatography method was applied as describedelsewhere (35Fariss M.W. Merson M.H. O'Hara T.M. Toxicol. Lett. 1989; 47: 61-75Crossref PubMed Scopus (34) Google Scholar).RESULTSα-TOS Is Selectively Toxic to MM Cells—α-TOSsuppresses several types of cancer in pre-clinical models(16Malafa M.P. Fokum F.D. Mowlavi A. Abusief M. King M. Surgery. 2002; 131: 85-91Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 17Weber T. Lu M. Andera L. Lahm H. Gellert N. Fariss M.W. Korinek V. Sattler W. Ucker D.S. Terman A. Schroder A. Erl W. Brunk U.T. Coffey R.J. Weber C. Neuzil J. Clin. Cancer Res. 2002; 8: 863-869PubMed Google Scholar, 18Malafa M.P. Fokum F.D. Smith L. Louis A. Ann. Surg. Oncol. 2002; 9: 1023-1032Crossref PubMed Google Scholar, 19Barnett K.T. Fokum F.D. Malafa M.P. J. Surg. Res. 2002; 106: 292-298Abstract Full Text PDF PubMed Scopus (77) Google Scholar,27Malafa M.P. Neitzel L.T. J.Surg. Res. 2000; 93: 163-170Abstract Full Text PDF PubMed Scopus (131) Google Scholar), although the reasons forthis effect are not completely understood. Here we studied the effect ofα-TOS on several MM cell lines of different phenotypic origin and ontheir nonmalignant counterparts. α-TOS consistently induced apoptosis inall MM cell lines in a concentration-dependent manner, and the extent ofapoptosis was comparable irrespective of the cell phenotype(Fig. 1). In all cases, dosesof >30 μm were required for apoptosis induction, and suchdoses are achievable in vivo(17Weber T. Lu M. Andera L. Lahm H. Gellert N. Fariss M.W. Korinek V. Sattler W. Ucker D.S. Terman A. Schroder A. Erl W. Brunk U.T. Coffey R.J. Weber C. Neuzil J. Clin. Cancer Res. 2002; 8: 863-869PubMed Google Scholar). In contrast, andimportant to the therapeutic potential of α-TOS, the vitamin E analogwas nontoxic to Met-5A cells (Fig.1).We next tested the effect of α-TOS on cell proliferation. The cellswere treated with the agent at 10–50 μm for up to 72 h. Asshown in Fig. 2, significantinhibition of cell proliferation was observed in MM cells treated for morethan 24 h with α-TOS at concentrations between 10 and 50μm. It is noteworthy that the vitamin E analog inhibitedproliferation at levels where it did not induce apoptosis, pointing to effectsthat are apoptosis-independent. Suppression of cellular proliferation by thevitamin E analog suggests that it may inhibit cell cycle progression(Fig. 3). Indeed, treatment ofMeso-2 MM cells with a subapoptotic concentration of α-TOS resulted inaccumulation of cells in G2 phase at the expense of cells in theS-phase and, to a lesser extent, cells in G1 phase(Fig. 3B). Cell cyclearrest was comparable in the other MM cell lines tested (data not shown). Cellcycle analysis also revealed a low number of cells accumulating insub-G0 (data not shown), supporting the idea that α-TOSexhibits anti-proliferative activity in addition to induction of apoptosis.Again, little effect was observed in Met-5A cells(Fig. 3A).Fig. 2α-TOS inhibits proliferation in MM cells but not in nonmalignantmesothelial cells. Met-5A (A), MM-B1 (B), Meso-2(C), and Ist-Mes2 cells (D) were seeded in 24-well platesand left overnight to recuperate. The cells were then at ∼50% confluencyand incubated with α-TOS at concentrations shown for 24, 48, or 72 h,and the level of proliferation was estimated by the BrdUrd assay. Cellproliferation is expressed relative to proliferation of control cells at theonset of the experiment. Data shown represent mean values ± S.D.(n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3α-TOS arrests MM cells in G1. Met-5A (A)and Meso-2 cells (B) were seeded in 24-well plates and left overnightto reach ∼50% confluency. The cells were then treated with α-TOS for24, 48, and 72 h at 10 and 20 μm, harvested, and assessed forcell cycle distribution as described under “ExperimentalProcedures.” Data shown represent mean values ± S.D. (n= 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)α-TOS Selectively Suppresses Expression of FGF-1 andFGF-2—Inhibition of cell cycle progression and proliferation byα-TOS is indicative of modulation by the agent of expression of genesthat are involved in these processes. Therefore, we treated the cells withα-TOS at sub-apoptotic levels and assessed for the expression of severalgenes that have an important role in tumor growth, namely FGF-1 (acidic FGF),FGF-2 (basic FGF), TGF-β, and VEGF. Overall, the MM and nonmalignantmesothelial cells express the individual mRNAs, with the exception ofIst-Mes2, where no VEGF mRNA was detected(Fig. 4). Although the relativelevels of mRNA for TGF-β and VEGF did not differ substantially,significant differences in the expression of FGFs were observed. FGF-2 mRNAwas expressed ∼2–3-times more than FGF-1 mRNA in all cell lines.Notably, Meso-2 cells expressed the highest levels of FGF-2 mRNA, whereasthese cells expressed less FGF-1 mRNA. This pattern was similar for other MMcell lines. The nonmalignant Met-5A cells expressed relatively low levels ofFGF-2 mRNA. When cha" @default.
• W2081005846 created "2016-06-24" @default.
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• W2081005846 date "2005-07-01" @default.
• W2081005846 modified "2023-10-17" @default.
• W2081005846 title "α-Tocopheryl Succinate Inhibits Malignant Mesothelioma byDisrupting the Fibroblast Growth Factor Autocrine Loop" @default.
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• W2081005846 doi "https://doi.org/10.1074/jbc.m414498200" @default.
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