Buněčná a molekulární fyziologie lipidů Doc. Jiřina Hofmanová Změny cytokinetiky epiteliálních buněk kolonu indukované nutričními faktory in vitro Interakce endogenních regulátorů a lipidových složek výživy, molekulární mechanizmy Vlokmelihy Kslvakňl« ÚEtciw fiv£nr BRNO Nerovnováha mezi příjmem a výdejem energie ve vztahu k obezitě a chronickým onemocněním Gene-Nutrient Imbalance Energy expenditure Energy intake Physical activity Wholegrains Fruits Vegetables Soy protein High fat/calories High n-6fats Growth factors Genetic Susceptibility ; Polymorphisms) Genetické polymorfismy posouvají rovnováhu mezi příjmem a výdejem energie. Vyšší příjem energie, nízký poměr nenasycené/nasycené tuky, inzulínová resistence, a sedavý životní styl jsou rizikovými faktory vedoucími k obezitě a chronickým onemocněním (srdeční choroby, diabetes a nádory). To je vyvažováno fyz. aktivitou a dietou bohatou na celá zrna, ovoce, zeleninu, soju atd., která snižuje riziko těchto chorob. (Trujillo E., J Amer Diet Assoc, 106, 2006) príčiny vzniku kardiovaskulárních a nádorových onemocnení genetické +faktory životního stylu TABAK a VYZIVA (podílejí se až na 2/3 úmrtí a jsou nejvíc ovlivnitelné) Tučná jídla (zvýšené riziko), ovoce a zelenina (prevence) i Strategie minimalizace rizika vzniku onemocnění FYZICKÁ AKTIVITA, RELAXACE, „MÍR NA DUŠI", OMEZENÍ STRESU, ŽÁDNÉ KOUŘENÍ, SPRÁVNÁ VÝŽIVA ostatní 24,74% ledviny a moč. ústrojí 6,54 % moc. v I * v mecnyr 5,87% žaludek 5,38% prostata 11,14% melanom 2,47 % tlusté střevo 8,36 % konečník 8,05 % pankreas 3,45 % lorynx 2,22 % plíce 21,79% B ostatní 33,41 % vaječník 5,56 % děloha 6.85 % žaludek 4,16% hrdlo děložní 5,02 % tlusté střevo 8,44 % prs 19,74% konečník 5,48 % žluč ní k 3,32% plíce 5,37 % melanom 2,65 % Struktura hlášených onemocnění novotvary bez dg. C44. A - muži; B - ženy (podle ÚZIS) INICIACE MUTAGENY RADIACE VÍRUSY.... GEN0T0XICITA PROMOCE NEGENOTOXICKE KARCINOGÉNY GENOTOXICKE +NEGENOTOXICKÉ FAKTORY NORMÁLNI BUŇKA INICIOVANÁ. BUŇKA PRENEOPLASTICKE ZMĚNY MALIGNÍ NÁDOR METASTÁZY AKTIVACE PROTO-ONKOGENU INAKTIVACE NÁDOROVĚ SUPRESOR. GENŮ INAKTIVACE ANTIMETASTAT. GENŮ Mnohostupnový proces karcinogeneze Carcinogen metabolism Inflammatory respo Hormone regulation Figure 3. Bioactive food components can influence genetic and epigenetic events associated with a host of disease processes. Trujillo E., J Amer Diet Assoc, 106, 2006 OMICS Ve VZtahll k Výživě (farmakogenomika, nutrigenomika) Faktory výživy přispívají různými způsoby k výslednému fenotypu Nulrigenetic* Nutritional Epigenetics Nutritional Transcriptomics Proteomics Meta bol o m i es Using the "omics" of nutrition to identify how dietary factors contribute to establishing a phenotype. Trujillo E., J Amer Diet Assoc, 106, 2006 VÝŽIVA hraje roli v mnoha typech onemocnění včetně nádorových a to řadou různých mechanizmů. Je prokázáno, že vysoký příjem kalorií a tvorba tukových zásob je rizikovým faktorem. Příjem, absorpce a metabolismus velkého množství potravy vyžaduje oxidatívni metabolismus a produkuje více reaktivních kyslíkových radikálů, které poškozují DNA a mají další negativní dopady na metabolismus. Ukázalo se, že příjem tuků, zejména živočišných zvyšuje riziko kardiovaskulárních a nádorových onemocnění. Epidemiol, studie předpokládají pozitivní korelaci mezi příjmem tuků a nádory prsu, kolonu a prostaty. Navzdory dlouhé historie studií tuků a nádorů, zůstává řada protikladů. Ukazuje se, že nejen kvantita, ale i kvalita hraje důležitou roli a že se zde uplatňují i tuky rostlinné a rybí olej, zejména vysoce nenasycené mastné kyseliny (PUFAs) tříd n-3, n-6, olivový olej atd. LIPIDOVE SLOŽKY VYZIVY VICE, NEZLÍ JEN ZDROJ ENERGIE!!!! ♦ strukturální a regulační úloha ♦ dopad na fyziologické funkce organizmu ► účinky na imunitní systém ► regulace proliferace, diferenciace a apoptozy ► úloha v karcinogenezi (etiologie nádorů tlustého střeva, prostaty, prsu) aboralery yfekinelics Změny membránových fosfolipidů přímo ovlivňují syntézu lipidových mediátorů typu eikosanoidů, PAF a sekundárních přenašečů diacylglycerolu a ceramidu. Lipidové mediatory ovlivňují produkci a funkci cytokinů. To má důležitý dopad na řadu imunitních a buněčných funkcí včetně proliferace, diferenciace a apoptózy Imbalance v lipidovém metabolismu hraje roli u mnoha závažných onemocnění ► Vysoká hladina cholesterolu je spojena s kardiovaskulárními chorobami, které jsou nejčastější příčinou úmrtí v populaci. ► Lipidy produkované buňkami imunitního systému jsou zahrnuty v zánětlivých onemocněních jako je revmatoidní artritida, sepse, astma: zánětlivé onemocnění střeva. ► Lipidy hrají úlohu také v psychických a neurodegenerativních onemocněních (deprese, schizofrenie, Alzheimerova choroba) ► Lipidy ovlivňují počátek a rozvoj nádorových onemocnění Relativní procento různých mastných kyselin v potravě a změny způsobené průmyslovým zpracováním potravin 40 30 a S 20 o u 10 0 Hunter Gatherer I Agricultural | Industrial Vitamin C -4 x 10* 10,000 li8oo Years 1,900 2,000 Fig. I. Hypothetical scheme of fat, fatty acid (ü>6, ü>3, rraus and total) intake (as percentage of calories from fat) and intake of vitamins E and C (mg/d). Dc\[c\ were extrapolated from cross-sectional analyses of contemporary hunter-gatherer populations and from longitudinal observations and their putative changes during the preceding 100 years [75]. VYSOCE NENASYCENÉ MASTNÉ KYSELINY (VNMK) (Polyunsaturated fatty acids - PUFAs) - mastné kyseliny s 2 i více dvojnými vazbami. Esenciální prekurzorové kyseliny řady n-6 a n-3 CH, rostl.oleje 18:2n-ó COOH Linoleic acid —4— l:3n-3 ^AAAVW CHj COOH a-linolenic acid desaturase" m f------ cVvwwwwvCOOH 18:3n-6 l Eíongase 20:3n-6 Dihomtvy-linolenic acid (DGLA) A5 desaturase 18:4n-3 i 20:4n-3 CH, Vyw^^/wxA COOH \ 20:4n-6 A=/WWWWV\ COOJI CH3 Arachidonic acid (AAY^ QEicosapentaenoic acid (EPA) I "^ Eíongase 22:4n-6 \ \ :ooh Eíongase, A6 desaturase and peroxisomal ß-oxidation 22:6n-3 /W\AAAA3/V :WWWWWW\f 22:5n-6 Docosapentaenoic acid (DPA) ^^Docosahexaenoic acid (DHA) ,COOH . C.H., Původ n-3 and n-6 nenasycených mastných kyselin, biosynteza eikosanoidů z kys. arachidonove a eikosapentaenové PUKNT METABOLISM Acetyl-CoA t Plastids Oleic acid i Endoplasmic reticulum ,COOH Llnoleic acid ((o -6) v Chloroplast H3 C vc^ä^vavavav/ COOH a-Linolenic acid (co-3) w Marine algae ! Plankton I Fish H3C>^v^^^vvCOOH Eicosapentaenoic acid 1 Marine algae T Plankton I Fish vegetable foods \ \ marine foods COOH Docosahexaenoic acid MAMMALIAN METABOLISM COOH Arachidonic acid 02—4 Leukotrienes 4 Leukotrienes 5 02 A H3CWVWW Eicosapentaenoic acid COOH. Prostaglandin G 2 o2 O \A^wv CH3 ° J ^OH V PG I 2 TXA 2 PG I 3 (TXA3) °2 o\ / V fc|/>r~/VN COOH *" | V^V*3^ CH3 O \)H Prostaglandin G 3 Důležitý je poměr n-3: n-6 VNMKÜ! WänSKSĽBiiGM kyselina arachidonová (AA, 20:4), rostlinné oleje zdroj eikosanoidú (prostaglandiny, leukotrieny) význam u různých nádorů. V experimentálních systémech často podpůrný účinek pro vznik a rozvoj nádorů kys. eikosapentaenova (20:5) a dokosahexaenova (22:6) z rybích a některých rostl, olejů (pupalka, len, rakytník) V experimentálních systémech často inhibiční účinek pro vznik a rozvoi nádorů stné kyseliny s krátkým řetězcem- BUTYRAT ► produkován anaerobní mikrobiální fermentací vlákniny ve střevě ► zdroj energie pro normální kolonocyty ► významný pro udržení homeostázy ve střevní tkáni regulací exprese genů spojených s regulací proliferace, diferenciace a apoptózy (microarray analýza - změny exprese 19 400 genů), exportní protein MCT1 ► butyrát sodný (NaBt) snižuje proliferaci a indukuje diferenciaci a apoptozu neoplastických kolonocytů in vitro a in vivo Prevence NÁDORŮ TLUSTÉHO STŘEVA regulátory růstu, diferenciace a apoptózy (cytokiny, růstové faktory, induktory apoptózy - zejména vliv na cytokinetiku, odhalování mechanizmů) - terapeutické plikace, lipidové výživy ► Výzkum interakce VNMK s environmentálními polutanty (rozpustnost v tucích, aktivace metabolismu lipidů a jejich úloha v působení polutantů - cytokinetika, transdukce signálů, mezibuněčná komunikace) ► Výzkum interakce VNMK s vybranými farmaky (mechanismy působení NSAID-nesteroidních antiflogistik, mechanismy a modulace účinků cytostatik - terapeutické aplikace) SIGNAL (např. cytokiny) Složky lipidového metabolismu v buněčných signalizacích Mediatory a modulátory Biofyzikálni vlastnosti membrán Lipidový metabolismus Aktivace fosfolipáz Uvolňování a metabolizaceAA eikosanoidy Oxidatívni metabolismus Transdukce signálů (kinázy, fosfatázy) Aktivace membrán, i vnitrobun. receptoru - tr. faktorů Exprese proteinů Exprese genů - mRNA membránová fluidita interakce ligand-receptor inhibice (NSAID) n-6 PUFA LA,AA Korelace s cytokinetikou Posloupnost dějů IT Phospholipids lacking omega-3 fatty acids t Phospholipids containing omega-3 fatty acids Dietary omega-3 fatty acids incorporate into cell membranes Modulation of bioactive lipid mediators Signalling molecules (cytokines, etc*) Omega-3 fatty acid-derived signalling molecules ALtered membrane protein activity Signalling molecules Modified protein expression Figure i: Cell membrane showing omega-3 fatty acids incorporated into the phospholipid bilayer. Omega-3 f"at*y ^cids can modify gene and p rot e i n ex p re ss ionř m od n I ate m e m b r a n e protein activity and act as a reservoir for bioactive molecules. 69 BUNEČNÝCH MEMBRAN ► provázejí procesy diferenciace a apoptózy savčích buněk ► pozorovány rozdíly u • nádorových a normálních buněk • nádorových buněk senzitivních a rezistentních k cytostatika m ► souvisejí do značné míry s modulacemi ve složení, struktuře, symetrii a metabolismu buněčných lipidů. Detekce těchto změn a jejich korelace s dalšími parametry odrážejícími diferenciaci a apoptózu přispívá k objasnění • posloupnosti a regulace jednotlivých kroků těchto dějů • rozdílů mezi normálními a nádorovými buňkami • příčin rezistence nádorových buněk k terapii EPITEL TLUSTÉHO STŘEVA (kolonu) DIFFERENTIATION ♦ kontinuálně se obnovující buněčné populace ♦ řada zásadních fyziologických funkcí ♦ dynamická rovnováha mezi přírůstkem buněk na bázi krypty (proliferace) a úbytkem (apoptóza-anoikis) na povrchu ♦ regulace endogenními faktory (hormones and cytokines), ale rovněž složkami diety přítomnými v lumen střeva ■ 44 IÍH Huropean Journal of Cancer TNF-st modulates the differentiation induced by butyrate in the HT-29 human colon adenocarcinoma cell line M. Kovaříková^, J. Richcrnik'1, J. Hofmanová^, Z.Zadákb?A.ltoziibik»> 'iafiumaiarj.-^T-tľj.-ijAiŕacric«, .Auxin» üf £kifrky*ics, .t-mA-tuy tif Scic/aacs ü/lbe riůiJb fít/mhůt, CZ-/ÍJ2 ,U lir/K-, ťľudb A/whStc bŮÉ/wjHJ«fj ů/MtiahůJk Cart aNdCtfúHiúiůgy.Chafkji Ľuiitrsliy, TtaJiiag fbsplial, HraJtt K/AJúvi, Cnrfi fttpvbJk ftacn-red J Úddfav 1444; racaned m revised íonn 3 Moidh 200Ö; acceded 3 Afril 2000 .Um Inci I"Ji■_■ aim íxľ this SLildj1 UftiS 14 dŕlemtinií WheLhKí and hůW iLLrtůiLr neLxůSiS larlůi a]pha fTNÍF-ů:) mííd ufalus bu ly na li? L-JtLirls. A ÍIlt Uil- in-ihmiU ol liuin.ui o.-k-n .idukv.in.iik-in.1 LIT-25) luDě WiLh aůdiuin bULJ-iale (Kb&LL, TNF*« ůí WiLh Lheii LůrtbinaliůnS We dbfLtfCLtfd ltíJJ ĽJŕfc (AůW L"y LúUeĽry), ľxiII prob kraben (aniidobjaĽ-k and M'I'I'jwjjv. Ihr auoUnl ofdrad |Aualin£> and apúpLoliť CifJlS (AůW L"ylůfůfĽry and niLuiLfuLTincv imL-rusĽ^y). and Ili-_ Jl-vl-J úľ -h J ti? run Lin Li on by aJkaJnu? pho^ihati ä? (ALI1? Jŕliu\\ (ipňť LropíhůloirfciLjy), iŕdaiívii F-aŕlih eomiínl (óůnfůa] ba?r SLannnuj míľKXíoopy anaJysKj and fi-ĽadliLTnL LMprvsaon fWsSLern Hůl analysis), toLh TM'-i and NaBl dm.-rraät?d ul-JJ grow Ni m j tlocwi-d^iKkiil ma m u-r. Aľ'n.T «HnhnuxJ lira I men L ôJ I li>_ Ľrils WiLh böLh agmiLS iLSťd, either none or addilittr efltľĽ-LS Were otaerwd as Ľsxrnpa rud wilJi NaBl lira I men I aknie. The leVel ofdrad and -i p.-pi ol if ĽeUs WaS důůř^liípiíndmiJjy : hlt Lias ed after [Jna «snibnurd Irralmenl. [n LionLlraal. ITS rr'-a^upprLiuLxJ ALL1 aclivily and r>-airlm .ilvuiiiiiLiIioii HiduLxd by N.ilil. 'I'liL n.-.nil-, -.iiľ^ľl-.! Mul IM-idom íiol ihIIuľiua: iinniiiiĽaniJy iJieanl^ioJiferaliveeJťecLH ůY h-.iLil bul. '.hJiLlJLiiv I« ila jäMl-ilIss I»*u -.-I jiAřpIíMia. iL rairkt-dJj irdilĽrá NaĎĽindilĽcd dihtncai.ua Lŕuai. 1e-lik^e, dillcr-o m Li led phenol ype in response to various inducers of 04J4.S044.i00jS • n kati nnrifer .fr 3000 Sfcrá Scioioe Lt3. Al ň$is reserved. PII: S04J4.a044í00W0l7S.7 DiIľc\mnLia.Lion í2004) 72:23-3 L Inlcrnalbnal SoticLy of DifTcrcnliiLion 2MJ4 ORIGINAL ARTICLE Martina Kovaříková - Jiřina Hofmanová K arci Souček ■ Alois Ko/uhík The effects of TNF-a and inhibitors of arachidonic acid metabolism on human colon HT-29 cells depend on differentiation status RcĽekcd July 2. 2 I i s h c--:1 Ľdlinc: LuManh 2\Xrt JlHUr. 1. ] [oJmanDi4.PbD llHj ■ A.VkuLotí ■ A. KdíuLhV l.abDcalDcj tiľ Cylnkinrlks InjliLiLffDl Hitipbpiii:*, AmduEnrnr fcienns aí Lhc üueb Republic IrflmrapnlMkilH í-li-r- Hrcici.CIzcĽh JlcpubliĽ ll-Mkil: híilniunĽTUíí'ibpjci A. Qijefc l.abnralnrj ol J-ľec Aad icaJ ľnLhophfniĽ'hnf lnrf.iL.iLrĽ'1 HicipbfiiĽ^AcadEíiif o\ SciffnĽes of Lhc Cinh Republic H rnti, CiEĽb Republic treatment and prevention strategics for cancer. Arm or the study The effects of two types of polyunsaturated fatty acids (FUFAs) -arachidonic r'AA, 20:4, n-6] or do-cosahciacnoic (DHA, 22:6hn-J) -on the response of human colon adenocarcinoma HT-2y cells to sodium butyrate (NaBt) were Investigated. Methodi The parameters rcHectlrift c ell pro! I feral ion and cell death vře re studied together with oxidative response* ml-tocho n d ria I m embrane potcn tial fMMF) and L-hajigc-5 of selected Le^uJatory molecules associated with cell cycle (p27Kt" and p2ict.iflmj=ij aní3 ap0ptosisicas_ pase-*, caspasc-y, poly (ADF-ri-Lose) polymerase - FARPh Bcl-2h B ax, Bak.Mcl-U.iíeAJdrís Vife demonstrated that pre-t reatmen t with citherAAorDHA attenuated cell cycle axres t caused by NaBt which Is associated with modulation of p27KiP'h but not p21^iPl™l|:| protein expression. On the other hand, FUEte sensitised HT-29 cells to NaBt-Lnduced apoptosis. An increased amount of floating cells and ceils in the subCu/C| population was associated with Increased reactive oxygen species produ c-tionjipid peroxidations decrease of MMF, activation of caspasc-^ and -9, FARP cleavageh and decrease in the ciprcssion of anti-apoptotic Mcl-1 protein. The observed effects were modulated by the addition of a protein synthesis inhibiton. cyclohcxlmide, a n d partially reversed by the antioxidant Trolox. Go?Tchfjio?T$ FUFAs- itijy tiave benchcia.1 effects in the colon enhancing apoptosis induced by NaBt. Alte rat ion of cell membrane řid co mposit ion and potent iation oiidalivc processes accompanied by changes in mitochondria followed by stimulation of apop-totic cascade components play a role in these L-l'lV.>_lř-. Key word 5 colon ^ ncer - diet -polyunsatu ratet! ba tty acids-butyrate -apoptosis Abbreviations PCM i'.:s MMF AA aiachidonic acid NaBt CHX cydoheilmide HARP CL eardiohpin DAFI 4.6-diamidino-2- ph en vi-indole PL FUFAs DHA doccsahciacnoic acid KOS DHE-12* dinvdnorhodaminc TBARs flow cytometry foetal calf serum mitochondrial membrane potential sodium butyrate polyi ADP-ribosc) polymerase phospholipids polyunsaturated fatty acids reactive orygen species thiobarbituric acid reactive substances ľ.E.Sŕ.Ylľ:R Ava ut=i bie on 11 rw aft www. s c\ e n ce d irect .í. am * n i ■ N C m. /£p öiflicT & NCER »»»jlhŕVlSjľiJB-itjCiIil'i-.l.ld-.i Polyunsaturated fatty adds sensitize human colon adenocarcinoma HT-29 cells to death receptor-med Í at cd apoptosis Jif?Uia KofmüLinvü1*, Alena Vaculová, Aloi* KozubíTt CZ-ÓUÓSButa, Cptk Jhpubll* Almlrai-I Th; pniV Nixtiv: ajid ipirplcitic k.íjkiihľ' 1íi TNT ac j.ikJ ojiti Fľí ajriitidy (Cľll ] ]J in hunům onJnn od.iiociudiKiinj. I IT 29 cull-; -WX-; i^iJiiLkJ by pn;tii;j.1iTu;iTl -uŕfli ajachidanic (AA, 20:^, u -áj cit daoíKahhLiiíiiaic (ľJIIA, 22:ň, u —3) Tally; aciK ■wliic}i j.knĽ iikíT'j-LvJ'J IĽaJCti■^\.■■ oiygLTi íípL^i-tí prcicnjclian qjiľJ Ľpid p:n.i\jdilk.iii. jjiJ d^í.-ii;j.*;d 1h; .S-plu.-;^ íiľlh; cuTJ cycí. Th: hjqJr-i-Lniínml íiľ II■:nim q cllJJ^ -.ídbť*|tf j i p.ipíĽlKiTi and apopViťc ccrĽa -±1 ■:■:%:d in piu tiu-alĽd o:TJ.-; wi; p.iLnbJ.k:'J by cyctihŕjinudĽ-. TJiľ- ľ-ITľcIh ciľ Cl I II -wljl- asíodatŕd -uŕOi aclňation aľ caspaíĽ S, í), ajid .^, dĽaiagĽ ciľ pah-í ADP ribciÄ.L^paIymi.'jaííLL PAR P. ajid dĽciĽaííĽd nuVicliciiidiiiJ m^mbujii: polĽJľtiaJ (VIWP>, hu1 IIľíľ- paranujti-; ivltľ i*i1 K^niJicajnh,' chjj^^d aľti PLJTA piuliuatnKnl. C 20aa D.*;ví;t IiuIjjiJ LtJ. AJ] r^hl.-; n;.-;^n>;d. L LÉLť4HduťLkm fi-upplernemtíiiq>nq>ľLeII l-uItuícs iA^nrtííff IlvJíiiu anima b w hh m 3 m m f> p>l>^n.-4ai^Jiaied ľaii> 3l- ids IPUFAi^ Jed U) an iíuaease oľinese L*L'FAi m -.-ell .■'iW'.r/i.'.-.-.'.'.-.'.ŤJ.- AA. XJĽhXicSĽ jľmÍ. C'JIJť. Ľ>LÍ.tĽ.íĽiuLL. DAťl. 4ju-diaintíM>-Í-ptaH>J.ÄfcJc; DJ IA >ix»- J-k ucwil jľíJ. K.'JvL ilůft cyĽcnccf>:; FCfi. íaal calí ^Biľn; JAJAť. jiiiL'L±j-.'Ji±-jjJ uuzntťjac pousoaJ, MjH-l «.-Jnuň bu^TJCc; ťAKR půN^AIJť-rtiĎJi | pohzHíEim; VĽhA*. puNT^JEĽTJml íain* j:í1t. Jt(ŕi. iľjlDhij ůxygjca hpcĽĽ,; ľKAiU. [bouhirticĽnc júd nojen sľ luJtiuBcei; TMJiJv u[ranu[ta(\-Jrb>icnBC iibftJ üjľľ pcEbJurae. »CxiBfEH^ aul«. TeL -1431 Hllrtlgt; íoľ lite ÜIJIIJ4J. f-mal J aiJ/iit- LJnfcj^uŕifLiľi (IL Hľ*b*j^tí>. memrwane phs*J>holipidi |l^] and ma> inňuenee membrane jwwporiiei \3). Misrcincr, ŕĽFAíandíheií meiarÄslhei, eie^Manísidi, are úMiiidered as impitfiam mediana and m^xl-u laH](W 5>ľ íhe imracel lular neivrtsít (>ľsignals |H, ine> enan^e eidame meiarBSliim \51 andma> have a diíeei eílfeei (Milene ejífMesikui wnen aei ivaí infi íhe íísoeilie nuelcaí íccopuMS and íianseíijí-íiqui faeiqwi |f>,71. Cena in ༝Ai íeipLViall> ^i 1 ívfíei) vrtiie íeptfied ío inijMove imm^jní> k>^ i^d I íCijSMiie |ftl, jwevem fírt>l i ľeíai i^>n and iniiiaie apq>p-toaJs |*H, kill niJTurf ^elli in viiňs \\tti and inliibii íum^ď j^ywih in exjXíimenia I animals 111 j. The imejaeiiqMi q>f L^t'Ai ľňim dieiary fai wiih naiurallj (jecuríin^ endonerunL faenstt le^^biin^ íhe ejnitineiĽí i* *uparted panÍL-ulaíl> in íhe :uhsi wheie ephhelial eel U aíe in diicvi oq>ntd^i wiih QMM-MJMi- «e im rau « ÜDi EbwlidudUL Ali^d ismi düi: ICLlOIUjiiMfcLMtMJTTJlM Available online at www,scienoedirecr,com SCIENCE {ffV D I Al CT* ,HCI@[ CER ELSEVIER Cancer Lcttcra 2W (ZHCfi) 4.V4ÍÍ ww w. cJ sew er. c ran/] cc ate /can! c t TRAIL and docosahexaenoic acid cooperate to induce HT-29 colon cancer cell death Alena Vaculová0, Jiřina Hofmanová13, Ladislav Aiidčrab, Alois Kozubík** ALaboratoř? of C\tokineíicsr Institute of Biophysics, Academy ofSciences of the Czech Republic Královopolská 135, 612 63 Brno, Czech Republic bLaboratary of Cell Signalling and Apaptasisr institute of Molecular Genetics. Vídenská JŕJ&Jr 142 2Ü Praha 4r Czech Republic Received 6 November HX)4: received m icvised form 10 December2(304: accepted 13 December 2004 Ab*Irüf[ The resifitance of some cancer cells to TRAIL-induced apoptofiifi is a major obstacle in successful clinical application of this cytokine. Combination treatment with agents capable of sensitising the cells to TRAIL effects is beneficial for new cancer treatment strategies. Docosaheaaenoic acid iDITAi is ander intense investigation for its ability to affect cancer cell growth and apoptosis. We demonstrated a modulation of TRA IL-induced apoptosis of HT-29 human colon cancer cells by DllA on the molecular (pro-caspase-3. -H. Bid. PARP cleavage) and cellular (cell viability and adhesion) level. To conclude, TRAIL and DllA were shown to cooperate in the induction of colon cancer cell apoptosis. © "2005 Elsevier Ireland Ltd. All rights reserved. Keywords: TRAIL; DHA; Cell death; Colon; Cancer 1. IiiLroriiu'lioii The tumour neemsviü factor (TNF)-iclatfid apopto üiü inducing ligand (TRA FL J, a member of TNF family, is particularly interesting for its unique properties to induce cancer cell death while sparing most normal cells. This implies its use as a promising Abbreviations: DHA, docosahcsacnoic acid: Ft.[P. R.tCF inhibitory1 protein; MMP, mitochondrial membrane potential: PARP, polv(ADPjribofcc polymerase: ROS, je active ostyjjen ipecici; TNF, tumour necrosis factor; TRAtL, TNF-related apoptosis-inducinjr, lij;and. ♦ Conespondini; author. Tel.: +420 .W15171K2; fcx: +420 5412112*0. E-mail address: kozubikiribp.cz (A. Kozubík). anti-cancer agent [J ,21. Cross linking of the TRAIL death receptors DR4 (TRAIL RJ, APO 2) and DR5 (TRAIL R2, TRICK, Killer) results in activation of caspasc 8 at the level of the death inducing signaling complex (DISC; [3,41. The apical caspasc processing is followed by activation of cxecutioncry caspascs, e.g. caspasc 3, cleavage of death substrates Like poly (A DP riboscj polymerase (PARP J, and apoptotic cell death [51. In colon cancer cells, the TRAIL induced signal transduction pathway remains poorly defined. There is increasing evidence that a number of cancers includ ing some colon cancer cell s ate resistant to the effects of TRAIL [oJl. Sensitivity toward TRAIL induced apoptosis can be modulated at different levels 03O4-ÍS354 - sec front matter SS 2005 Elsevier Ireland Ltd. All rij^its reserved, doi: 10.1 Olo/j .canlct.2004.12 JO! o KĽBS 28* 15 FEBS Letters 577 (2004) ÍW-31Í Ethanol acts as a potent agent sensitizing colon cancer cells to the TRAIL-inchiced apoptosis Alctia Vaculová , Jiřina Hoimanová11, Karel Souček* Ladislav Andčrac, Alois Kozubík*" *Laboratory of Cytokineticrr Institute of Biophysics, Academy of Sciences ůf the Czech Republic. Královopolská i í J. ói2 65 Brno, Czech Republic Masaryk University in Brna, Faculty of Science. Department of Comparative Animal Physiology and General 'Zoology. Kotlářská 2. 611 1? Smo. Czech Republic c Laboratory of Leukocyte Antigens, institute of Mok-cuiar Cienetics, lloenská itíX3r i'42 20 Praha 4r Czech Republic Received 30 July 2004; accepted 5 October 2ft)4 Available online 1-li tícLober 2(H)4 Edited by Vladimir Skulachev Abstract I dti i [ it ic J E n 111 4i f ui4.-4.-|iJiiisins m m mJu Litimi m [Ik-' I N I'-r-L-L J[-l-ľ1 JfHif>[^taiis-uidu4.-inii linJiid iTKAlI.I-Lriduťťd Jp-np- [JISLS LS LLCLp-l IT [ J11 [ ľ-UlT ĹES fj-l IĽ -L" 11E Í LI I LIS4.' ill LI 11 [ Í-ĽLII l-Ľ-L" T t I L4ľr J f>» . 1! C11 li i n 11 -ľ li 11 induiľi:-Ľ4.-LL duJEk in litru ,hmI in i inj I ľ J Ltt-L-r-L-i i [ -i ili 11JILL i LLJ pJtlniJ^s. I ts i-tt'-tf [ in i-Jsl-KL -9L -J JťtiiätLorLs j p-[--"jp-" p-[-"[L-Ľ ISid p-r-i' [-ĺ-Li i ckayagc, adťťrtjjstJif LtLi^'tlťmiJriJl mirmbfänw.- fHittiitijL J íľJi LCLp-k-E-L.- p-nL> l ADPiritnivL- fmli tiLtrast 4: L t J i J Ü-L- ĺ. Jud disjoin.-JL-J114.-4.-JifJiitijLHiu-tiitic Mul-1 uniti-in iutí- d4.-LIHMIStrJt4.-d. UtlUlUll J4."[S JS J fHI[4ľll[ JLÍ4:IL[ S4.-JWÍlLíLH£ ťOkhJl caiLťťL 4.L-IU [4i TKAIl.-mdtiť«J apo|i[jj^K. ®2ir\}4 [■'■L'dťFjckiinjľriumiKiJii ISin4.l14.111i4.-jl *^in.i-L-ri<—. ľuUi^K-d by l!lstÚ4.T B.\. All riulits r4.-s4.T 14.d. Keywirrdx: TN h"-re Lated apoptosLs-inducing ligand; tttianol; Apoptosis; Colon; Cancer I. liitrmJuctiiilL Lthanol (CjH^OH, ethvl alcohol} lias bee n frequently used and generally aeeepted as a solmhiltzitig agent for a nutmher of different reagents used in laboratory experiments with cell cuLturei. However, previous reports nia\e demonstrated a roLe of ethanol in the apoptotic death of many celL types [1^4]. Apoptoíií has also been shown to occur in both experimental and clinical alcoholic liver disease, but the signalling pathways remain not fully understood [i| in additifin to the effects of ethanol itself, it Ls also necessary to consider its interference with other factors, such as endogenous apoptotic regulators. TNF-related apoptosLs-inducing ligand {TKAlL)ř a member of the tumour necrosis factor (TNF) family, Ls particularly interesting for its unique ability to induce cancer ceil death whiLe sparing most normal cells, which implies its use as a potent anti-cancer agent [bj. Cross-Link ing of TU Al L death reeeptots Ľ*K4 (TKALL-ťU, AKK2) and DK5 {TLtALL-RZ, TKLCiCT *Correspondinjä aiittu>r. ťax: +420-5-4L2L l±9l F-ffbiil address: kf>7u.bi.k^ibp-.cz4A. kťiziibík). Abbreviations: DK, death rcĽcptor; KLI F, KL iCt in hi bi tory protein; MMF, TnitiKihöndTial membrane pntentiaJ; FAKF. po-ly(AĽ) FJriboac polymerase; ROŕi, Teacíive oxygen species; INK, tumour necrosis factor; I'KAIL, "I'NK-related apoptosis-i u d uciu]; li^aud killer) results in activation of caspase-S at the level of the death-inducing .signal Ling complex (DISC). Activated cispase-řl then initiates the apoptosis executing caspase cascade [7,8]. However, in man v cancer cell types, resistance to T HAIL was developed. We investigated! the modulation of HT-19 human colon adenocarcinoma cell sensitivity to TllAlL by ethanol. Our results demonstrated a strong potentiation of THAlL-induced apoptosis in tne presence of ethanol and suggested some of the passible mechanisms involved in the: interference of these two agents. 2. MjE4.-riJls and itk-rltuds 2.1. Culture conditions Human colon ad e n oca nd noma HI-25 cells (A'l'tC, K43ckvillc, Ml J. USA) were cd t lined in McCoy's 5 A medium (Sigma, Germany) with ijentamycim (50 rni^/L; Siiona) and lOrVi foetal calf serum (PCS; PAN Systems, Germany) at Í? *C in 5% COi and $$>/» humidity. Ihe attached cells {24 h after seedin];) wctc treated with I'KAIL (humam killer TRAIL, 10Ů nij/mlj and eLnanol (4*'&) alone or in combination! for 4 or 24 b in the medium inn t h 5% of KCŕi. In ihec-Jiric rimcnLs us inj; etbaonl |1). 1-6%) alone, Lhe cells were treated for4Ŕb. 2.2. Stahle transfketions SS-KV.neo plasmids (L'I'R promoter, Neo resistance, L:>.ŮD (D IMi Federation of biiropein BiaĽ-hemiLul Societies. Published by tLseviei U.V. All rights tescrwd. doi: 10.10167j .fcbslet.20 04.10 JÜ15 Physiol. Res. 54: 409-418, 2005 The Effects of Parenteral Lipid Emulsions on Cancer and Normal Human Colon Epithelial Cells in vitro J. HOFMANOVÁ1, Z. ZADÄK\ R. HYŠPLERI J. MIKESKA3, P. ŽĎÄXSKÝ2, A. VACULOVÁ1. J. NETÍKOVA1. A. KOZUBÍK1 1Laboratoiy ofCytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Bino, * Department of Metabolic Care and Gerontology, Charles University, Teaching Hospital, Hradec Kiálové and Infusia Hořátev, a. s., Czech Republic Received June 14, 2004 Accepted October 19, 2004 On-line avail?/::- U- Z>ea-< n ber 9, 2004 Summary Difference5 in lipid metabolism of rumor and normal tissues suggest a distinct response to available lipid compounds. In this study, the m vitro effects of five types of commercial parenteral lipid emulsions were investigated on human cell lines derived from normal fetal colon (FHC) or colon adenocarcinoma (HT-29). Changes of the cellular lipid fatty acid content, cell oxidative response, and the cell growth and death rates were evaluated after 48 h. No effects of any type of emulsions were detected on cell proliferation and viability. Compared to the controls, supplementation with lipid emulsions resulted in a multiple increase of linoleic and linolenic acids in total cell lipids, but the content of arachidonic, eicosapentaenoic, and docosahexaenoic acids decreased particularly in HT-29 cells. The concentration of emulsions which did not affected HT-29 cells increased the percentage of floating and subG^Gi FHC cells probably due to their higher reactive oxygen species production and lipid peroxidation. C o-treatment of cells with antioxidant Trolox reduced the observed effects. Onr results imply that lipid emulsions can differently affect the response of colon cells of distinct origin. Key words Fat emulsion • Reactive oxygen • Lipid peroxidation • Apoptosis • Tumor cells Introduction Lipids in artificial nutrition play an important role in metabolic reactions and immunity of critically ill patients (Adolph 1999). Recently, it has become clear that besides direct nutritional effects, lipids play numerous structural and regulatory roles which may have an important impact on physiological functions in the organism. Different types of fatty acids may influence cell membrane fluidity, receptor mobility' and functions, signal transduction as well as eicosanoid synthesis and cytokine production and functions (Grammatikos et ol 1994a: Calder 2001, Hong et al 2002). Lipid mediators have a potent impact on a wide variety of cellular responses including cell growth, differentiation and apoptosis (Maziere et al. 1999, Rudolph et o I 2001). Certain polyunsaturated fatty acids (PLTAs) were found to inhibit tumor growth and cancer cachexia (Tisdale and Dhesi 1990. Petrik et ol 2000). Recruitment of specific hpids by rumor cells made them more sensitive to PHYSIOLOGICAL RESEARCH © 2005 Institute of Physiology, Academy of Sciences, of the Czech Republic, Prague: Czech Republic E-mail: phyaei/Ä-bioiued, caa.cz ISSN 0862-8408 Fax+420241062 164 httpi/Ávww.biomed.cas.caphyuolrei Praktické aspekty ■ Zánětlivá a nádorová onemocnění střeva ■ Farmakonutrice, „Disease specific nutrition" ► Podpůrná a kombinovaná terapie ■ Antiproliferační, diferenciační a proapoptotické účinky ■ Protizánětlivé účinky ■ Posílení imunitního systému ■ Posílení účinků chemoterapeutik a snížení vedlejších účinků ► Optimalizace lipidových výživ ■ Vývoj nových typů lipidových emulzí pro perorální použití pro prevenci a terapii vytypovaných onemocnění ONCOLOGY REPORTS 19: 5Í7-573, 20ÜS Response of normal and colon cancer epithelial cells to TNF-family apoptotic inducers JIŘINA HOťMANOVÁ, ALĽNA VACULOVÁ*. MARTINA HYZD'ALOVÁ" and ALOIS KOZUB ÍK Laboratory of Cytoldnetici, Institute of ŕJiophysics, Academy o T Science y o T the Czech Republic, v v.i., Královopolská 135, CZ-o 12 ť5 Ö mo, Čiech Kepubi ic Received September 17. 2007; AcceptedOcto^t 29, 2007 Abstract. We compared the response of normal (FHC) and canceT (HI-29} human color epithel iul tells to the important ape p tot it inducers TNJ-^ri. anti-Ris antibody andTMh-relited apoptosi^ inducing lisjand (TRAIL). The two cell lines did not respond 1o TNF-a (15 ng/ml)ľ expressed a limited sensit i vily to anti-Fas antibody (200 ng/rr.l) and a different response to TRAIL (100 ng/ml). We studied apoptosis with Tegard to the changes at the receptor level (OR, I3cR and FL:P)and at the level of mitochondria (Bid pTotein cleavage, Apo2,7 protein expression und CAspase-9 activation). Two different approaches were used to sensitize the cells to I RAIL-induced apopiosis: inhibition of protein synthesis (cyclo-hesimide, CHX) und inhibition of the pro-survival MĽK7ĽRK pathway fU012fi}, While the two cell lines were markedly sensitized to all three IN ľ family rr.e rubers by CHX, a different degree of response (especially for TRAIL) was obtained when inhibition of the MĽK/ĽKK pathway was achieved, TKAJL-induced apoptosis was significantly enhanced by UO126co-treatment in the HI-29cells,but not in (he FHC cells. The most significant differences between the HT-29 and FHC cells co-tTeaied wiih TRAIL and JO I 26 were demonstrated with regard fa the involvement of the mitochondrial apoptotic pathway, suggesting its importance in the regulation of cell sensitivity to the I RAlL-induceJ apcptosis. I ill n id ľk I u in The suppression of apoptosis of colon epithelial eel Is may cause cellular transformation and favour progression at every stage of the adenoma-carcinoma >equence (1). Cytokines of Correspondence to: Dr Alois Knzubik. Institute of Biophysics. Academy of Sciences of the Ctech Republic, CZ-612 61 Brno, Královopolská 135, Czech Republic E-mail: kuzubik@itrp.cz " Caa tri huLcd cq ua I ly Key wordw apopuiyi^TKF-ftiLtix: apoptoiiis inducing Mgaud. TN f'-at a nti -lj'aüt colon the tumour necrosis factor (TNF) family have been identified as important inducers of apoptosis* but their role in regulating epithelial cell turnover is not fully understood (2). Their effecl on colon cancer cells and the associated molecular and cellular mechanisms have yet to be elucidated (3). TNbw*. Las ligand and TNF-relited ipoptosis inducing ligand (TRAIL) induce apoptosis by binding to their respective death receptors (DRs) possessing intracellular death domains which recruit certain adaptor molecules t c form the death-inducing signalling complex ^L>!SC) activating the apoptotic caspase cascade (4). Through caspase-8 activation, subsequent downstream signals are started, either through the direct activation of effector cavpa^es (type I celU, extrinsic pathway} or by transferring a signal fa mitochondria (type II cells, intrinsi: pathway) mediated by cleavage of the Bid protein (5) Changes in mitochondria are associated with the activity of pro- and anti-apoptotic proteins of the Bcl-2 protein family and start events leading to the acüvation of caspase-9, effectoT caspases, death-subs trate cleavage and finally cell death (6). J n spite of the fact that TNF-ci. anti-bas and I RAIL can generate po Len c ant it u mo ui activity in vivo and in virro (7,8). many canceT cells are resistant to this tvpe of DR-mediuted killing. Moreover,the therapeutic: use of the I MF-n/INFR ot FasVFasL system in canceT treatment has been hampered by severe side effects. In contrast to "J'NF-ft and FasL, IRAIL induces apoptosis in a wide variety of transformed cell lines, but seems to have little or no cytotoxic effect en most normal cells in virro and in vivo. The induction of apoptO&Ü is mediated by the interaction of TKAIL with the two death receptors DR4 and DR5, and the mechanism seems to be rather different from that of TMr-tt or FasL (9). Due to the selective effects of TRAIL on cancer cells and its ability to Induce apoptosis irrespective of p53 status, it may be a safer therapeutic alternative to the other twe cytokines (10). In spite of an increase in sensitivity to TRAIL-induced apoptosis during adenoma to colon carcinoma transition being detected (I 1), many types of cancer cells become resistant to 'I RAIL (12). Some cells seem to be protected from 'IRAIL-induced apoptosis by their expression of decoy ncceptors (DcRI and DcR2). which do not transduce apoptotic signals, is well as at the level of certain molecules involved in intracell uIut signalling pa'hwavs (13;. In the present study, two different approaches were used in order to sensitize the colon cell s to the apoptotic effects of TNbM*. Fas antibody (anti-bas) and TRAIL. The cell response FEBE Letten SBO (2ÍWQ C'CS CS10 Different modulation of TRAIL-induced apoptosis by inhibition of pro-survival pathways in TRAIL-scnsítívc and TRAIL-rcsistant colon cancer cells Alena Vaculová, Jiřina Hofmanová, Karel ŕiouček, Alois Kozubik* Labůfalútyůf CylůHneftes, Institute of Biophysik Aiwdémy ofliťtetuet! &S 'A* Czech Rep-jbiic, JfráíwpůiskA I35r 612 65 Brno, Czech Republic Received 27 Octů ber 2006; accepted í November 2006 Avaüabk. ůJilijit 1 ] November 2ÍKK Edited by Lukas Huber \bstrael FvpitbeliaL cdls can be manipulated to undergo apop-Limís dependim: on tbc balince between pro-surmal ani apoptotic KÍ!;n!iK. Wc showed that TR AI L-induced apoptosis may be differential 11 regulated by inhibitors of MĽK L R K (Lfllio) or PííkľALr (I V3*UlYl3) pirhw»>- in TBAU -hmivUÍit (111-24) and TR AI ľ- resist ant (SW^iA) human epiLhelial co Lin cancer ceLLs. liJl2í> or LA'2$Í1M2 s ijín itic an L L_v enhanced TRAlL-in- duťud ^pc'pLC"-:iv Ln III —Jľ*J-ľ-ľIU. but rani in íŕWtlfl v HI -. Ä't m-port a different regulation u f the level o f in au ti-a po ptotic Mcl-I protein under Ml .K/l .KK or J'J.íK.1' \k I pathway inhibition and ■■ii!:!:Ľ--l 11 iľ iiiľľIiuiiÍ-'H]-' in-. ■ ■ I ■■ ľ 11. A -| *ľľ i u. I nil u nln-n wiis; puid to the ru le of the L R K Ml. Ak L and ^Ly ci^en sy nth ase kin ase .í ß. & 20ÜU Iľ králi i in n| Ľirope^n Biochemical Soc iet.es. ľuh-IÍ-;IjuíI b_y l!l-a;^ii;r líA . All ri^hL-s rĽitr^td. Keyword*: TRAIL; Mcl-I; GSK: ER K ApupLittis; Ci>Uín 1. Iiiiriiducliini The LumouT necrtKĽi Cacliir cůfien synthase Idna«; MMP, mi tec hond hal Hiembrane potential; PÍ IK, pnosphandjLnosJto] 3-kinase; PKC, protein kinase C; PARP. poly(ADP)ribůse půl/nierase; TRAU. TNF-related ipoptoiii-inducjng ligand; TNF, tumour necmsis factor 0O14-JT?]^]2.OO ■£ 2006 Federation of European Eioclkmical Societies, do i: 10.10 lfi/j febslet 2006.11 .»4 lines. ínLeres.mĽly, during utliPtecLal catmm^enesis, a marked increase in stnsúivúy Lín Lbs apíppLíHic efecLs ítf TRAF L, assíp-ciaLed wiL h príN^tessiím Lnim bemun Lín mahĽtianL Lumííur L^-pe bas been rep-.rUed fí]. Fíh L bis sLudy, we have used Lbe human eitlem ůdeníxatcinímiů cell line IIT-29, which we previously cbaracLeriflít as Lype H celk [7], and _Wb"20 cells, wliich are tesislanL to TRA IL-induced apiiplítsis, as mípdels fíic cítkm Failure Lín underuíN apíNpLíNsis in tespímse Lín TRAÍL LteaL-menL may tesuU in Lumour resisLanct SensiLiviLy líNwards TRAF L-meda Led apíNpLíNsis can be míNtlalaLed a L difíetenL levels in Lbe TRAF L sújnallmE pa Lb way. Pr-.NLem kinase- media Led sii^nelI I in ľ haií been described as an eiTetLive wav i>f lü recLin^ DR si ľ rials (K|. Fn ccmL nis L Lo rejrula Lion by inhibi Lířry prířLeins lít TRA FL [eceptíNTs, phíisphíhryla tiť>n-'^ased sÍĽnalli\Ľ may iPLA^LLL wLlIlíPUL L CI|LiLlCJ LUČILI ipf LLCwlj ÄJ LLIÍlCÄ LiEll pLiPLcL.li. TUĹi cíNuld be especially effecLiŕe under cíntuIílúntls when DR responses need Lín be rapidly míNtl ulaLed. Besúles iLspíNLemial Lín stlecLively kill UmiNur cells, the. phys-ÚNlíN^ical TíNleíNf TRAF L se*ms Lín bemíNte ccNmpleji. Theabilúy A if TR A f T N t prj KTTU ti ň *; I ITvi Vül ÜTlil pTJ fell \ttM lAVU Aif TU Kr-Ľ-JTlĽlKr cells bas been tepíNUed [9J. F urLbetmíNTe, in cerLain LumíNur cells, simul LaneíNus ínt cím«cuLÍve TRAľL-induced aaivaLÚm ínC apíNpLíNLic and ptíN-sutvival paLhways has been ilemíNn-sLra Led [LOJ. The balance beLween Lbese pa Lhways is LketefíNre very impíNTLanL in deLermiuin^ Lbe cell La Le. TbetefíNte, f urLber sLudúis are necessary Lín examine Lbe inLeraaúm beLween TR AF L-induced apíNpLíNLic paLhways and pri n- s ut vi val paLhways in íNider Lín predicL Lbe eflecLivenesi íNf TRAF L ir. cancer Lherapy. The miLíNuen-acLivaLed pcíNLein kinases {MAFFÍs) areserine- LUl^LPLLLLLC klLLtľNCN LIlíIL d LC d^LLVd Led Ijv pUllCt pUiPL jldL LULL LLL Lft- spímse Lín a varúäy íNf ea Lracell ular s Limuli f L L]. MAPKk play a cenLral tínIc in Lbe LransducLUNn ín f si^na's fíNT ľtínwLh and dif-ferenLiaLÚm and alsíN acL as impíNTLanL míNtlula Línts íNf variíNus ľ ľ i -i-ii: I ■:. i i r =_" signals. The eíLLracellular siunal-reLulaLed VinArfw (FEICiy?), Al1Íví*Im1 by MFKľlP, pbjn;pbjvryU\f. ahíI míNdulaLe Lbt funcLÚm íNf many regulaLíiry príNLeins. The ptíN-LecLive effecL íNf FRFÍL/2 im DR-inducíd apíNpLíNsis bas also been repoTLed [S\. PE3K/AkL is a maú>r k i jmu Min ľ. paLhway which mediales prn>-liferative siunak in Lbe in.esLiual epúhelial cd k in vitra and fn uiuo [E2). En additUm, LhíK pathway is an impíNrLariL rtĽulaLíTT of cell differ ci Lia L ú m and apíNpLíNsis [L3J. PtíNmíNLÚm íNf lkII sut-vlvjI by Lbe- jcLivjLuitl ihf Lbu; paLbway iľuitk by mbibiLum 4>f pTíN-apíNpLíNLĹ; ínt Lbe indjcLÚm of survival súmals [14,15). FF3FÍ-media Led acLivaLÚm of Ak L also resuUs in inhibi LÚm í>f k Published by Eswier B .V. All rights reserved. U buněk ovlivněných AA nebo DHA je po působení NaBt zvýšeno % plovoucích buněk a subGO/Gl populace. Spolu s dalšími detekovanými parametry ♦ snížení mit. potenciálu (FCM) ♦ vyšší produkce ROS (FCM) ♦ aktivace kaspázy 3 (Western blot) ♦ štěpení PARP (Western blot) v r v r MřA IH11111 ^TA^JCIH IH W, I SUJ UUYMl proces anoikis - apoptóza indukovaná uvolněním buněk Tento efekt je blokován inhibicí syntézy proteinů cykloheximidem (CHX) 0 AA(uJVI) NaBt(mM) JCHX^g/ml) ■ subG0/G1 D floating cells NaBt(mM) CHX^g/ml) ■ subG0/G1 D floating cells 20 - 20 - 20 5 5 - - 5 5 5 5 5 5 loborcitoř Yfcokinetiky nioFv»hölni ÜBfenv flVCIlr BnNO pro- 1 caspase-3 | PARP Bax Bak Mcl-1 control NaBt AA50 AA 50-NaBt DHA 20 DHA20-NaBt kDa m — ~ —••• M---- 32 «V •• <----- 113 <----- 89 #—— <----- 21 •ä —•— m <------ 24 - — — 1 <------ 42 A. Vaculová et xL í tEBS Letters 580 (2006) 6565-6569 Fig. 3. PARP, pro-caspase-8 and cFLIl>£ cleavage. Vlci-1 protein level, phosphorylated and total ERK1/2 levels, and phosphorylated GSK3ß in HT-29 cells prc-trcatcd with U0126 (lOjiM, 45 min) ar.d then treated with TRAIL (lOOr.g/ml, 4 h), detected by Western blotting. Results are representative of four independent experiments. An equal loading was verified using ß-actin antibody. kDa ^Qmm^* m^^ * «- 113 PAR P — _ *- 89 Caspase-6 WE SB M " *- 55,r57 gs ~ «- 41,r43 Mcl-1 *« «* «-» n «-40 PERK , _ ^ ä «-42/44 ERK S m v» mm «-42/44 pAkt *• — «-60 Akt ^ 1» mm mm «-60 pGSK ** mm mm *- 46 ß-actin w i^ i^w ^^ «- 40 / Fig. 4. PARP and pro-caspase-8 cleavage, Mcl-1 protein level. phosphorylated and total ERK1/2, Akt. and GSK3p leve:s in HT-29 ceils pre-treated with LY294002(50 ^VL 60 min) and then treated with TRAIL (100ng/mh 4n). detected by Western blotting. Results are representative of four independent experiments An equal loading was verified using ß-actin antibody. HOFMANOVÁ et all DR-MEDIATED APO PTOSIS IN CANCER AND NORMAL COLON CELLS kDa 113 89 40 X u + O 1 HT-29 -fc-4- d x < x FHC B |rňige of PARP ;md B-aclín quantification in HT-29 and FHC cells treated wílh TRAIL (100 ng/rnl), CHX (5 //g/ml), U01 26 (10 ^M) or Iheii 24 h, delecled by Weslem blolling. The results am representative of three independent experiments. (B) Expression of mil ex: h od n ill AP02.7 lili ve cells) after 24h treatment of HT-29 and FHC cells with TRAIL (100 ng/nr), CHX (5 //g/ml). LÍ0126 (10//M) or their combination; }!). versus untreated control; §. P<0.t>5 (Jt P<0.01) versus TRAIL alone; #, bel ween HT-29 and FHC cells. s»; m ■ í. vr ■ .r---'-* T- ■ . 3r?T, í ^-:v r ■'vi^'J >Cr"í' ■ í. ŕ ■ : : : ■ ;-■ ;:ff. '■':. ^ i 1 -■ JV *ŕ i «* x Mí taofaafe äHNfóg: £ w T **^- . ■ :* ■ A* T _j Tt.;^5*^ i "V = *. ■ V .- ■ *^--y & *im ■■ I j ■ ' #■* : ■ fce i ■t f íO ftiol *"b^j b - ■L i ÍTÉÉ «te-«: J? _^^^Kl n i* 1LLX*] 33 »kin eli ľ-ŕ1—WT^Sť -J W m :tóži II ■ . *-':- uk/HS-** . -:^ ■" JE FACSCali rúto ková flow) cytometrie: edna z hlavních >oužívaných netodologií laboratory tokinetics Institute of Biophysics, ßrno Academy of Sciences Czech Republic