Celková úmrtnost na rakovinu v USA podle typu 200 ooo 150 000 ,_ 100 000 50 000 plíce kolon slinivka prostata prs vaječník Germany Austria Portugal Denmark Italy-France Belgium European Union The Netherlands United Kingdom Sweden Luxembourg Ireland Spain Finland Greece 0 Incidence Mortality 20 HI 40 ] W3 W3 M3 El 60 Incidence per 100,000 people 80 Figure 1 | Colorectal cancer incidence in males in the European Union. Rates of colorectal cancer by incidence, per 100,000 people, and mortality during 1996. Data were collected from Eucan — a service that provides data on the incidence and mortality of 24 key cancers in 15 member states of the European Union2. 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) Výskyt kolorektalnich nádoru (per 100 000 obyvatel; 1988-1992) Country Male Female Croatia 17.0 17.7 31.7 25.0 Denmark 31.5 32.4 Finland 15.7 18.6 GDR 20.1 24.7 Latvia 13.4 14.7 Sweden 28.6 28.2 UK, Engl.&Wales 27.9 26.9 USA 39.9 29.9 Úmrtnost na kolorektalni nádory (per 100 000 obyvatel; 1987-1988) Country Male Female Australia 26.1 14.2 CzechRepublic 29.4 16.4 Denmark 23.6 17.5 Finland 11.9 8.7 GDR 20.1 15.2 Canada 18.1 12.9 Sweden 14.7 11.2 UK, Engl.&Wales 20.2 14.2 USA 17.2 12.0 střevní epitel Sebeobnovná tkáň s unikátní topologií - dvourozměrná struktura: Proliferativní krypty a diferencované klky (villi). Jedno vršte vná bariera mezi lumen a vnitřním prostředím. TENKÉ STŘEVO - krypty - dolní část - kmenové a Panethovy buňky, proliferující „transit-amplifying" diferencující se buňky postupují k vrcholu, klky z diferencovaných buněk na vrcholu se odlupuj í cích. TLUSTÉ STŘEVO - nejsou klky, na dně krypt kmenové buňky (nejsou zde P. buňky), 2/3 krypty proliferující buňky, 1/3 diferencované b. na konci se odlupují do lumenu (apoptóza - anoikis). 2 hlavní linie buněk: Enterocyty - absorbtivní linie, nejpočetnější Sekreční linie: goblet buňky (sekretují protektivní muciny - přibývají směrem ke kolonu) Enteroendokrinní buňky (asi 1%, sekretují hormony - serotonin, sekretin) Panethovy buňky - jen v t. střevě - sekretují antimikrobiální látky - kontrola mikrob, obsahu ve střevě. Aktivní migrace buněk doprovázená diferenciací a odlupovaním do lumenu 3-5 dní ceil shedding Differentiation and migration 24-48 hr Lamina propria Differentiated cells Crypt-villus junction Goblet Entero- Absorptive cells endocrine epithelial cells cells .ví: «r- • *• Stem cell «vÜ Panem ceil Fig. 1. The anatomy of the small intestinal epithelium. The epithelium is shaped into crypts and villi (left). The lineage scheme (right) depicts the stem cell the transit-amplifying cells, and the two differentiated branches. The right branch constitutes the enterocyte Lineage; the left is the secretory lineage. Relative positions along the crypt-villus axis correspond to the schematic graph of the crypt in the center. LUMEN OF GUT epithelial cell migration Vom "birth" at the bottom of the crypt to loss at the "op of the villus (transit time is 3-5 days) epithelia cells crypt loose connective issue villus (no cell divisioi cross sečti* of villus cross section of crypt villus absorptive brush-border cells mucus-secreting goblet cells direction of movement (A) nondividing differentiated Paneth cells nondividing differentiated- cells rapidly dividing cells (cycie time - 2 hours slowly dividing ste cells (cycle time > 24 hours crypt 100 ^im Příčný řez částí stěny střeva LUMEN OF GUT Ismooth muscle epithelium —Q connective tissue circular fibers longitudinal fibers " connective tissue epithelium epithelial cell fibroblast s moot h I muscle cells riTHnH" epithelial cell |Figure 19-1. Molecular Biology of the Cell, 4th Edition. Každá tkáň je organizovaným seskupením buněk držených pohromadě buněčnými adhezemi, ECM nebo oběma. Tkáně jsou spojeny dohromady v různých kombinacích a tvoří funkční jednotky - orgány NÁDORY KOLOREKTA (CRC) Výskyt industrializované země (životní styl, výživa) ČR (třetí nejčastější příčina úmrtí na rakovinu) věková distribuce (muži nárůst případů od 60 let; ženy od 70 let) Epitel kolorekta střevní krypty (část proliferační a diferenciační) výměna epitelu (zrání buněk, odumírání apoptózou-anoikis (detachment-induced apoptosis) koncentrace růstových faktorů v kryptách (v proliferační části více buněk produkujících GF) Kolorektální karcinogeneze porušení rovnováhy mezi proliferací a diferenciací v kryptě hyperproliferativní krypta, adenom, adenokarcinom, karcinom, metastázy a I no klence r a t es of c olorecta I c ancer i i b Estimated red-m eat consumption (grammes/day) Figure 5 | Colorectal cancer incidence and red-meat consumption worldwide in men. a /Countries with a high incidence of colon cancer (cases per 100,000 people) are indicated with blue (North America, Australia); countries with moderate levels in pink or red; and countries with low incidence in green (Asia, Africa). Colon cancer incidence is correlated with red-meat intake. b | Countries that consume the most red meat, in g/day, are indicated in blue (North and South America, Australia); countries with moderate levels of consumption in pink or red; and countries with the lowest levels of red-m eat intake in green (Africa, Asia). Figure adapted with permission from Ref. 1 © (2003) I ARC Press. Fig, 5. Diagram showing a longitudinal section through a small intestinal crypt illustrating how the cell positions are numbered from the base. The diagram shows a typical distribution for the DNA synthesising (S-phase) cells in the crypt (shaded nuclei). An actual set of experimental data are illustrated (left) together with a labelling index frequency plot where labelling index as a percentage is plotted against cell position. This approach can be used to measure any parameter associated with crypt cells including the distribution of dead or dying apoptotic cells. These eel] positional distributions are commonly presented with the frequency plotted on the vertical scale and cell position plotted on the horizontal scale with the base of the crypt on the left. Crypt (a) Model (b) Growth factor profile (c) 0.6 0.4 0.2 0. Growth factor post-in itotická zóna *-j zona epiteliálně- mesenchymální interakce APOPTOZA anoikis (programovaná 1 uněčná sm terminálni diferenciace - ( APOPTOZA bunky Normální epitel a adenomy v myším tenkém a tlustém střevě vülus crypt } Surface epithelium > crypt Small intestine colon Adenoma in small intestine 73s Aberrant crypt focus in colon Fig. 2. Comparison of normaL epitheLium and adenomas in murine smaLL intestine and coLon. (A) SmaLL intestinaL crypt and villus. (B) CoLonic crypt and surface epitheLium. ProLiferative ceLLs are stained for the ceLL cycLe marker Ki67 (brown nucLei) in (A) and (B). (C) An adenoma residing inside a villus of the smaLL intestine of a min mouse. (D) A small aberrant crypt focus in the colon of a min mouse. (C) and (D) are stained for ß-catenin. Note the presence of ß-catenin (in brown) in the cell boundaries of aLL nondiseased epithelial ceLLs and the accumulation of ß-catenin throughout the ceLLs in the adenoma and aberrant crypt focus. A, B - normální epitel - proliferující buňky pozitivní pro marker cyklujících buněk Ki67(hnědá barva) C, D - adenom v tenkém střevě a fokusy aberantních krypt v kolonu min myší. Barveno na přítomnost beta-kateninu. normální buňky - beta-katenin na hranici mezi buňkami adenom a aberantní krypty - beta-katenin v celé buňce In vivo progression of colon epithelial cells GUT-LUMEN APOPTOTIC CELLS \ EPITHELIAL CELLS CRYPT BUTYRATE PRODUCTION I BY I COLON BACTERIA PROLIFERATIVE PART GUT-WALL Birth of an Epithelial Cancer EPiTHFUiuy PROLIFERATION. An initial mutation in a tingle cell [highlighted disrupts signal transduction or the cilh cyde, and the errant c«! I divides more frequently than it? neighbors The changed «It and its descendants oo-nstrrute dyspia sia. 3 precancerous growth whose cells bjí stih" differentiated and confined to the bottom cell layer Time pas-ses. A second mutation occurs in an already affected cell. ushering jn sweeping changes in gene-expression that modify metabolism, growth characteristics, and cell shape, as adhesion and anchorage slip. The efcoubty mutant ceľl and its descendants are slightly less specialized than thek neighbors. Further eSCJJpe ficin growth Constraints. cuľiipÓ Uy j third mutitlun, pertuibi The Cell £ydt irt ä different Way. .il OWN■ ... ■ i:mí: éVpartSiOrt u'' I'.i- growth. A v n i L- - ľ ■ activity Jumpi, ípeciiFi;at|ůn* cent mu ľ to rade away as :-.'.' cells fCftm Their nuclei appear enlarged and I fťiUShflpŕrt, refleCLlng the dhaůi U ith irl With still more mutations, the increasingly aggressive groivlh is now a misshapen, irstill microscopic mass. Yet jr. remains contained within the epithelium bounded by th* ba&ement membrane. The tiny tumor may remain here, as m situ carckno-ma, 1or yeajs The abnormal cells now occupy all celt levels, and aFl are de-differentiated to some degree. lNU4flON. As (he dividing cirKír wäli irtend through the bni*-men I membrane *nto ihr ^rounding Krom», maflf nyitcy beg^m- The tum-gr and ítr^ma cortimUnkale, as the cancer EgcrH|1:i^-. nvrr Mr-ni.vh I-. f.;n^rr qcH: varíte ŕibrobfíit growth fador =ind vaítvlar rnd^chc^al growih factor, whkh ?ubvert normal ang-logenesn? t* FKrinl eapilíariej,. wh-kh irtakff W\ inet argund the tumoí- Snrnp cpneer íplk ÉľSCp ilnpg (he řmnrj?|ed blodd vc^eJ-s; othert ícjueeíe Ofliwpen the lining;«Hi to entrr thr- circpfaTinn. Spread begm?. METATA5IS. Supplied wjth nutrients, oxygen, a waste removal service and -conduits, feraciou-s cancer cells exit and home to lymph nodes and more distant sites, the trajectory characteristic of tumor type: icrdney or breast to lung; proslate to bone. Here, further mutations and changes in gene expression ensue, often rendering the original tumor's offspring so different that treatments-that once worked now feik SfljBUtHtU* Ü: řWJ riiT sm-iurt« i sa Kolorektální nádory vznikají progresivní akumulací genetických a epigenetických změn vedoucích k transformaci normálního střevního epitelu do adenokarcinomu. Molekulární mechanismy kontrolující homeostázu jsou terčem změn podílejících se na vzniku nádorů. Molekulárně genetické poznatky ► Mnohastupňová progrese na molekulární i morfologické úrovni. ► Genetické (mutační aktivace onkogenů a inaktivace nádorově supresorových genů) a epigenetické změny (metylace) podporují tvorbu nádoru poskytujíce klonální růstovou výhodu změněným buňkám. ► Klíčovým molekulárním krokem je ztráta genomové stability. ► Dědičné nádorové syndromy často odpovídají formám klíčových genetických defektů u zárodečných linií, jejichž somatický výskyt nastartuje sporadické nádory kolonu. Ztráta genomové stability je klíčovým molekulárním a patogenetickým krokem vyskytujícím se na počátku nádorového procesu a vytváří permisivní prostředí pro výskyt změn onkogenů a nádorově supresorových genů. 3 hlavní formy: ►Nestabilita mikrosatelitů (MSI) ►Nestabilita chromozómů (CIN) - zisk či ztráta úseků chromozómů, aneuploidie) ► Chromozomální translokace Dědičné poruchy predisponující jedince k nádorům autozomálně dominantní typ dědičnosti ► polypózní formy (familiární adenomatózní polypóza FAP) asi 1% mutace APC (adenomatous polyposis coli) genu tisíce adenomatózních polypu ve střevě - riziko vzniku nádoru téměř 100%. APC gen ► nepolypózní formy (heredit. nepolyp. kolorektální karcinom - HNPCC), Lynch syndrom asi 15%, zvýšené riziko dalších typů nádorů, mutace genů pro MMR enzymy (mismatch DNA repair), množství mutací v repetitivních sekvencích DNA - mikrosatelitech Stádia vývoje nádoru epitelu děložního krčku (A) 50 [im NORMAL EPITHELIUM (B) (F) 50 um LOW-GRADE INTRAEPITHELIAL NEOPLASIA I 50 jim HIGH-GRADE INTRAEPITHELIAL NEOPLASIA 200 |jm INVASIVE CARCINOIVIA Figure 23-9 part 2 of 2. Molecular Biology of the Cell, 4th Edition. Familial Sporadic Modifier genGs Genes Environment Genetická podmíněnost CRC FAP, Lynch syndrom atd. Převažující dědičná složka Jiné mutace zvyšují náchylnost při působení environm. faktorů Interakce genů a e. faktorů. Další tzv modifikující geny mohou dále ovlivňovat účinky jak genů tak e. faktorů. Přesné rozlišení mezi tzv. sporadickými a familiárními a mezi genetickými a environmentálními faktory predisponuj ícími k CRC není striktní. Rgure 1 | A global view of the genetic contribution to colorectal cancer. The highly penetrant causative mutations in familial adenomatous polyposis (FAP). Lyn oh syndrome, the hamartornatous polyposis syndromes and other familial conditions underlie oases of colorectal cancer (CRC) that have a strong hereditary component, with little environmental influence. However, there are also several low-penetrance mutations that contribute to CRC susceptibility in an additive way. involving interactions between genes and with environmental factors. As well as accounting for cases of hereditary CRC, these mutations are also likely to contribute to cases of CRC that are classified as 'sporadic1. In addition, although none has been identified so far. modifier genes are also likely to influence the effects of genetic and environmental factors that contribute to CRC. Therefore, the distinction between "sporadic' and 'familial1 cases and bet/^en 'genetic' and 'environmental' predisposing factors has become blurred and might be better thought of as a continuum of risks contributing to CRC development. APC. adenomatous polyposis coli; BLM. Bloom syndrome; MMR. mismatch repair; TGFßR2, transforming growth factor-ß receptor 2 Table 1 | Heritability of selected cancers Cancer type Study 1 family risk ratios* Study 2 family risk ratios* Proportion of variance due to heritable factors* Testicular 8.57 8.50 ND Thyroid 8.48 12.42 ND Laryngeal 8.00 ND ND Multiple myeloma 4.29 5.62 ND Lung 2.55 3.16 0.26 Colorectal 2.54 4.41 0.35 Kidney 2.46 5.26 ND Prostate 2.21 9.41 0.42 Melanoma 2.10 3.41 ND Breast 1.83 2.01 0.27 The ratios shown here were in part recalculated by Risch97. Study 1 was carried out in Utah98. Ratios are based on all first-degree relatives; first-degree relatives of 35,228 probands with cancer were studied. Study 2 was carried out in Sweden". Ratios are based on siblings; data comprised from 435,000 parents with cancer who had 5,520,756 offspring, 71,424 of whom had cancer. * Based on a twin study comprising 44,788 pairs100. ND, not determined. Sporadická forma nádorů kolonu - nedědičná, postupný vývoj řadu let Na vzniku se podílí rovněž vnější faktory (dieta, životní styl) Pozitivní korelace - spotřeba tuku, červeného masa, alkohol, kouření Negativní korelace - zelenina, ovoce, vláknina, NSAIDs Rodinný výskyt „sporadického" kolorektálního karcinomu - kombinace genetických predispozic se zevními faktory Potřeba pravidelných vyšetření od určitého věku (okultní krev, Sigmoidoskopie, kolonoskopie) Normal DNA K 1 , \ x Error-free repair2 DNAAddtiď N T Surveillance for damage and/or cell cycle checkpointst* Apoptotic deaths + Error-prone or failed repair 4 Genomic instability5 I Cancer Figure 2. Diagrammatic representation of a model that could account for control of mutations contributing to colorectal oncogenesis. The three shaded boxes represent key events in the process that act to control the consequences of DIMA adduct formation. The three heavy arrows indicate the major outcomes of inherent surveillance mechanisms for controlling DIMA fidelity in response to adduct formation. Failed repair results in adduct "fixation" as a mutation that is passed on to cell progeny. Genomic instability can itself compromise all control mechanisms. The numbered superscripts represent points subject to environmental regulation by a variety of mechanisms. Epigenetic regulation can apply at all of these. Geny zahrnuté v kolorektální karcinogenezi Onkogeny (ras, c-myc, c-myb, hst-1, trk, c-raf, c-src, c- myb, Her2-neu) • Proteiny H-ras, K-ras, N-ras aktivované přes receptory spojené s G proteiny a s tyrosin kinázami - aktivace drah kináz RAF, MEF, MAPK přechod adenom - karcinom Nádorově supresorové geny p53 - mutace či delece u 70-80% nádorů, poruchy apoptózy, OHO/ crns^itmÁ n rlaľamilnrví ninmA] Irány Wnt a chromosomámí nestabilitou. Chyby spojení mikrotubulu a kinetochoru - abnormální segregace chromosomů - Polyploidie DCC - deletovaný gen u 70-80% nádorů, úloha v zástavě G2/M a apoptóze Geny reparace DNA - MMR mismatch repair (hMSH2, hMLHl) oce? S/MD* TGFßRU P5S Ě3X N ► + Caret noma MSH2 MLH1 4- OCO? r K. 22q 17q 17p 14p Sp Gq 1p Lrte lrwjaori3nd Fig. 2. The adenoma to carcinoma sequence. The development of colorectal cancer is an excellent example of the complex multistage process of turn oogenesis and most colorectal carcinomas are thought to develop from adenomas. Fearon and Vogelstein (1990) first proposed that colorectal cancer cells must acquire 4-6 genetic defects including either mutation or deregulation of proto-oncogenes (such as k-ras and c-myc), and tumour suppressor gene inactivation [such as adenomatous polyposis coli (Ape) and p53]. For example, k-ras and Ape gene mutations have been found to be involved in the early stages k DtugResia of colon carcinogenesis, while alterations of p53 and are involved in the later stages. Although this model has survived revision, it should be emphasised that the natural history of no two colorectal cancers has been found to be the same. Adapted from [19-21]. Key: DCC: Deleted in Colorectal Cancer T-PA üPA E*Caúherin AÍVÍ23 Genetické změny spojené s kolorektální karcinogenezi (chromosome Sq |_*l«or»üon_J LOF genes APC Normal epithelium 1 h perproliferative epithelium 12p GOE7 K-ras 18q LOF nec SMADs I7p LOF L> Karly ailenimia intermetliiile adenoma Late adenoma Carcinoma MMR genes: M4SH2 hMSH3 HMSU6 hMLHl hPMSl hPMS2 Poruchy „mismatch repair' Metastasis Fig. /. Genetic changes associated with colorectal tumoi igcncsis. This process is accelerated by MMR deficiency (sec text for details). Abbreviations: LOK loss of function; GOK gain of function; MMR. mismatch repair. Reproduced from Kin/Jer & Vogelstcin (2) with modifications. Genetický model kolorektální karcinogeneze Histological StSQB Normal epithelium Dysplastic crypts Tubular adenoma Dysplastic adenoma Carcinoma Metastasis Genetic regulation APC ß-catenin K-ras DCC p53 [»» p n P jo p p II Reproduced from Sharma eŕ a/., Eur. J. Cancer 2001 Funkce APC (adenomatous polyposis coli) proteinu 300kD cytoplasmatický protein kódovaný APC genem - často mutovaný v prvotních stadiích CRC (u adenomů) APC interaguje s řadou bun. proteinů a drah a přispívá tak k regulaci diferenciace, migrace, proliferace a adheze. Jeho mutace tak ovlivňuje všechny tyto procesy. ► Regulace signálu indukovaného beta-kateninem (regulace Wnt dráhy) ► Regulace buněčné adheze prostřednictvím beta-kateninu a E-kadherinu ► Regulace migrace buněk interakcemi s mikrotubuly a F-aktinem ► Blok buněčného cyklu zřejmě přímou inhibicí jeho komponent Mutace genu APC vede ke změnám cytoskeletu a deregulaci beta-kateninu. Ovlivnění migrace buněk a mitotického vřeténka -aneuploidie. Deregulace beta-kateninu -poruchy diferenciace a genové exprese - transformace. Zvýšená hladina beta-kateninu neschopnost APC vazby na mikrotubuly - deregulace migrace buněk a segregace chromozómů. Truncation of APC Accumulation Aneuploidy Transformation Cell Chromosome Deregulation migration segregation of ß-catenin Mitogenic signals Ras UNA damage Bcl-2 h NF-kB p53<«- Caspase 9 Bad Nur77. IKKot *—AKT Mdm2 Anti-apoptosis Pro-apoptosis Fig. 3. Mitogenic signals Eire transduced by Ras which inhibits the retinoblastoma (Rb) protein allowing E2F and My c to promote cell cycle progression, pi 6 is n tumour suppressor whasc mutation permits eyclin D dependent kinase (CDK) to inhibit Rb. High levels of E2F or Myc nctivntcs pi 4""^^ and thereby p53 which has multiple outputs. A series of proapoptotic bcl-2 family members are stimulated and via PTEN the antiapoptotic actions of AKT are inhibited. Note that p53 can still respond to DNA damage, and therefore chemotherapy, if the Ras/Rb pathway is disabled. Interakce buněk kolonových krypt s látkami vznikajícími v krvi nebo v lumenu ► Mutace APC (adenomatous polyposis coli gene) v kmenových buňkách jako výsledek působení látek z krve nebo zárodečné mutace, produkuje abnormality v buněčné proliferativ migraci a adhezi ► Abnormální buňky se akumulují na vrcholu krypt, tvoří se aberantní fokusy krypt (ACF), které vyčnívají do proudu stolice ► Zvyšuje se pravděpodobnost dalších mutací kontaktem proliferujících buněk s fekálními mutageny a adenomy se tvoří postupnou klonální expanzí Epigenetické změny Hypo- nebo hypermetylace promotorů Hypometylace - obecný a raný děj - odpovědná např. za overexpresi k-ras Hypermetylace - inaktivace nád. supresorových genů Deregulace růstových faktorů TGF beta - negativní růstových faktor epiteliálních buněk - zástava v Gl fázi, receptor I a II signálování přes SMAD proteiny Inaktivační mutace signální dráhy - poruchy apoptózy- progrese adenom- karcinom. Zánětlivé onemocnění střeva (IBD) Nádory často vznikají v prostředí zánětu Produkce prozánětlivých cytokinů - TNF alfa, IL-1, -6, -8, ROS, prostaglandiny - podpora, poškození DNA, angiogeneze, inhibice apoptózy a invaze. Úloha transkripčního faktoru NF kB Faktory vnějšího prostredí ► Výživa - celkový kalorický příjem a frekvence příjmu potravy - obsah a kvalita tuků v potravě (působení žlučových kyselin, obsah a kvalita nasycených a nenasycených tuků, lipidová peroxidace, zvýšená tvorba prostaglandinů) - ochranný vliv vlákniny (vazba karcinogénu, zkrácení doby tranzitu střevem, snížení pH) - vitaminy a další mikrokomponenty živin (vit. A, C a E a selen jsou antioxidanty) - konzumace alkoholu a kávy kouření (hlavně doutníky a dýmky) - potravinové mutageny (zejména heterocyklické aminy ve vařeném a pečeném mase a tucích) - konzumace masa a vajec (vyšší konzumace je riziková - vepřové, hovězí, jehněčí) ► Fyzická aktivita nedostatek je rizikovým faktorem předpoklad modifikace diety s vysokým obsahem tuků ► Profesionální faktory profese zdrojem látek zvyšujících riziko nádorů kolorekta (zejména kovoprůmysl, automobilový a dřevařský průmysl) ► Věk (zvýšený výskyt s věkem) ► Neefektivní imunitní systém Chemoprevence nesteroidní protizánětlivé léky (NSADs); antioxidanty; vápník; selen ; folát MASTNE KYSELINY S KRÁTKYM ŘETĚZCEM (SCFA) ► C2-5 organické mastné kyseliny (acetát, propionát, butyrát) ► vznikají bakteriální fermentací vlákniny a účastní se regulace funkcí a cytokinetiky v kolonu ► butyrát slouží jako zdroj energie pro normální epiteliální buňky a indukuje diferenciaci a apoptózu nádorových buněk střeva CYTOKINY Důležité endogenní faktory ovlivňující kolorektální karcinogenezi TNF-family (TNF-a, Fas ligand, TRAIL - TNF relating apoptosis inducing factor) TGF-family (TGF-ß) EGF - epidermální růstový faktor Tumour necrosis factor-alpha (TNF- a), interleukiny ► multifunkční cytokin ► jeden z hlavních mediátorů zánětu ► TNF- a je produkován makrofágy a dalšími buňkami imunitníhp systému ► koncentrace TNF- a v kolonu je zvýšena během chronického zánětu (ulcerativní kolitida nebo Crohnova choroba) ► úloha v nádorové kachexii ► existuje interakce mezi cytokiny a dietetickými faktory - mastné kyseliny a eikosanoidy Figure L Mechanism oi" Signaling by TGFß superfamily members. Binding oi TGFß superiamily ligands results in activation oi' a heteromeric receptor complex comprised oi type I and type II receptors. The activated receptor complex then phosphorylates specific R-Smads. These R-Smads associate with the common Smad. Smad4 and then translocate to the nucleus where they interact with a variety oi' DNA binding partners to regulate gene expression. Model signálů dráhy Wnt Normální stav Regulace transkripce drahou beta-kateninu. Komplex APC, axin GSK3 Fosforylace a dgradace beta-k. no Wnl signal LR P Ubiquitin-öepentlent dsgratŕon Wnl signa] Tfoflíl r u ti LR P NvcieíirHri^^ioCíir-on pora karcinogeneze Deregulace: Vazba Wnt na Frizzeled receptory Stabilizace beta-k. Akumulace v jádře Aktivace LEF1/TCF transkripčních faktoru Figure 2. A model tď Wnl signaling, Tn the absence t>f Wnl ligand (Itíft panel) APC, Axin and GSK3 ľomi n complex lhal results in JS-caienin phosphorylation and degradation. Binding ol" Writ to the Frizzled receptors (right panel) results in stabilization oi" Ji-eatenin thai then acciimmulalcs in the nucleus where it associates with LEFI/TCF transcription factors to regulate gene expression- Wnt Fig. 1, Schematic presentation of the canonical Wnt signal pathway. The left side shows the normal adult tissues where phosphorylation of ß-catenin target serine^ threonine residues are phosphorylated and degraded rapidly by ubiquitination. The right side indicates the transcriptional activation of the Wnt target genes bd unphosphorylated and therefore stabilized ß-catenin. APC, adenomatous polyposis coli. Regulace (deregulace) transkripčních faktorů u střevních buněk Wnt Signalling APi Am CK1 gsk % GfQUCÍJo"V TCF .,,, /J If Differentiation fldfäpi m ■ [i-cat c TCF r .v ^ ,v ^ Crypl proliferation SMPSignailng njinffetjiMuifrtlfiinnfin rfriii^p^n% R-SMAD:^ Co-SMAO Ä fSílC^V Mu 11 pie ectopic crypts Normal crypt formation Fig. 3. Wrrtr BMPr and Notch pathways control target gene transcription. (Left) Wnt-responsive celts carry a receptor complex consitir^g of a frizzled seven-transmembrane receptor (Fz) and Lrp5 or LrpG. In the absence of secreted Wnt factor (Left)r the destruction complex (APCr axin, and the kinases CK1 and GSK3 Ji) induces degradation of cytoplasmic Ji-catenin. Tcf completed to compressors such as groucho represses specific Wnt target genes. Receptor engagement (right) blocks the destruction complex; Ji-catenin accumulates and binds to Tcf in the nucleus to activate transcription of Wnt target genes. (Center) Type I and type II BMP receptors are not complexed in the absence of signal. Secreted BMP factors bring the two receptors together, ultimately Leading to the phosphorylation of R-SMADs, their association with co-SMADr translocation to the nucleus, and subsequent activation of BMP target genes in the nucleus. (Right) When Notch receptor meets its cell-bound Ligand (jagged or delta), sequential proteolytic steps Lead to the release of its intracellular domain (NICD)r which travels to the nucleus, where it complexes with the transcription factor CSL to activate Notch target gene transcription. Stem ceils Myofibroblasts Muscularis mucosae BMP antagonists Kosinski C. PNAS 2007 Dlfferentiative Compartment (activa BM3 signaling) Proliferative Compartment (active WNT Signaling) Stem Cell Niche (ISEMF + SMC prcvide source of BMP antagonists) BMP pathway bmp\ 2, 5,7 BMPR2. SMAD7 NOTCH pathway A I JAG1 /^ WNT pathway WNT5BrAPC, TCF4 Eph'ephrln pathway ĚFNA1. tr/VB£, EPHA.2. A5 Myc noiwn rk MAD MAX. MXI1 BMP pathway GÄEMJ, 2 CHRDL1 NOTCH pathway NOTCH t, 2, 3 RBPSUH. Tim WNT pathway FZÖi. í, 7. a TCľa. DKK3. SFRF1, 2 Ephi'ephrin pathway EPHA1, 4, T ČPHR1. 2, 3. 4. 6 Myc network MYC Fig. 5. Graphical view of human colon intestinal epithelial cell development and stem cell niche maintenance. Only genes with significant differential expression in paired ŕtest (P< 0.05) are listed. ISEMF, intestinal subepithelial myofibroblast; SMC, smooth muscle cell. Mechanismy působení vysoce nenasycených mastných kyselin (PUFAs) zahrnuté v kolorektální karcinogenezi METABOLISMUS KYSELINY ARACHIDONOVÉ (AA) COX-2 u kolorektálních karcinomů je zvýšena exprese COX-2 a množství PGE2 PGE2 stimuluje růst a inhibuje apoptózu nádorových buněk PGE2 působí prozánětlivě a reguluje funkce imunitních buněk (imunosuprese) nesteroidní antiflogistika (NSAIDs) snižují riziko kolorektálních nádorů a zánět inhibicí COX-2 LOX (5-, 12, -15) u kolorektálních karcinomů zvýšená produkce 12- a 15- HPETE Ovlivnění cytokinetiky, adhezivity a invazivity Změny genové exprese - aktivace specifických transkripčních faktorů (PPAR,NFkB,AP1) Účinky lipidové peroxidace (LP) Produkty LP mohou mít genotoxické účinky a mohou ovlivňovat buněčný cyklus. Během LP jsou produkovány reaktivní kyslíkové radikály (ROS) ROS mohou aktivovat NF-kB COX-1 COX-2 rN—^^VCOOH OH OH TXB2 Phospho lipids I CQO\J. Amchidoníc A cid 1 Dietary Polyunsaturated" Fatty rtcíds(DHrt. EPA) _ÖpX - 2+AS>K Í7^hydro*y ser ies cf ^_ ^-^1 dwasorwids (Reaolvins) 15Ä-HETÍ—► 15-epJ-IJpo^ns Arachidonyl Ethanetamide (Anandamide) CŮOH COX-Z Pe "^ ^ 5JycEfö1 «tors and ethanůJdffiidéS CGC'l-i HO OH ŮK 6-keto-P6Ft(T h-? HÚ OH peFío Dráha 5-lipoxygenázy - vznik leukotrienů 4rachidoníc Acid I CŮOH 5-LO 12-LO 15-LO ľjl- LXA„ :■.!■ i.i 4ÍMM LTB4 / '&futamyf-transpepŕtda$£ I 1^**^^ LTÍ>4 ■> CVUH COX-2 i 5-LPO stimulují buněčnou proliferativ inhibují apoptózu a indukují neoangiogenezi Cancer cell pro iteration COX-2 5-LO Apaptosis oo oo COX-2 5-LO 1 1 l>3> Neo - sngiogenssis nox - 2 5-LO I Arachidonic Acid Platelets E ndot helium GJ tract Kidney Tissue Homeostasis (-) NSAIDs Celecoxib Rofecoxib cytokines, growth factors ^tumor promoters Stromal mononuclear cells Figure I. COX isoforms include constitutive COX-1 which is involved in normal tissue homeostasis and inducible COX-2 which is upregulated at sites of inflammation and in colorectal neoplasms. NSA1D inhibit both COX isoforms, whereas COX-2 inhibitors are selective for the COX-2 enzyme. TxA2 = -thromboxane. COX-2 je nadměrně exprimována u 40-90% kolorektálních adenomů a u 90% adenokarcinomů Täblé 1. C0X2 expression in malignant úľ premalignant human tumours Prem all gnant or ma lig nant lesl on COX2 ex pressl on (%) Colorectal 80-00 Gastric 80 Oesophageal 70 H ep atocel lular (liver cirrhosis) 54 (8 1) Pancreatic 67 Head and neck 80 N on -smal l-cel 11 ung cancer 70 B reast (d uctal care Inorn a-1 n-s Itu) 40 (60) Prostatic 83-03 Bladder S6 Cervix 43 Endometrial 37 Cutaneous basal cell 25 Cutaneo us sq uamo us c el I 80 pPNET 100 G Ho b lastoma rn ultlforme 71 -74 Anaplastic astrocytoma flow grade) 44 (30) References available at http://lmage.thelanc-et.corm/extras/03oncl205webfr,pdf Některé inhibitory COX-1 a COX-2 (NSAIDs) TABLE 1. Structural class Members COX-1- nonselective COX-2- selective alkanones nabumetone anthranilic acids meclofenamic acid, mefenamic acid meclofenamate esters and amides arylpropionic acids ibuprofen, flurbiprofen, ketoprofen, naproxen, diarylheterocycles SC560 celecoxib, etoricoxib, parecoxib, rofecoxib, valdecoxib di-tcrt-butyl phenols darbufelone enolic acids piroxicam, tenoxicam, phenylbutazone meloxicam heteroaryl acetic acids diclofenac, ketorolac, tolmetin lumiracoxib indole and indene acetic acids indomethacin, sulindac etodolac, indomethacin amides (and esters) para-aminophenol derivatives acetaminophen salicylic acid derivatives aspirin, diflunisal, sulfasalazine o-(acetoxyphenyl) hept-2-ynyl sulfide (APHS) sulfanilides nimesulide, flosulide bg UCK ratio, COX-2/1) i i sVJ lumiracoicib rofecoxib etoricoxib valtecoxib do do lac me I oxi cam nimesulide celeooxib diclofenac sulindac meclofenamate tomoxiprol Piroxicam di fluni sal sodium salicylate niflomíc zomepirac fenoprofen ampyrone ibuppofeui toi metin naproxen aspirin iTidomethacirt ketoprofen suprofcn flurbiprofen ketorolac •ji h» i o « ls> « _l________l_ _l w n tí s Schematické dráhy některých funkčních efektů inhibice COX-1 a COX-2 A upper Gl tract decreased mucosal defence Z tumour increased ulcers z increased apoptosis decreased tumour growth asthmatic lung i z broncho-constriction ktdney {fluid/salt d ep feted) increased Leukotriene production i decreased GFR & Na reabsorptior I edema: increased bio od pressure blood vessels piateiets decreased PGlz production decreased TkAz produclion \ pro-thrombotic? anti-1hrorirtb0tiC TABLE 2. COX-2-selective agenta compared to traditional NSAIDs inflammation pain (arthritic, inflammatory, surgical) Alzheimer's disease Cancer Asthma gastrointestinal toxicity, minor such as dyspepsia, diarrhoea gastrointestinal toxicity, major such as perforations, obstructions and bleeds reproduction thrombosis Therapeutic indication equi-effective equi-effective Other beneficial effects NSAJD benefit shown from epidemiology but no current evidence for effectiveness of COX-2 selectives Both groups reduce development of colon cancer and possibly esophageal cancer; both groups effective in animal models of cancers in lung and pancreas Side effect no evidence for COX-2- selectives causing asthma attacks in NSAID- sensitive individuals similar effects COX-2- selectives produce less than NSAIDs both groups may delay ovulation, implantation, and preterm labor some suggestion that COX-2- selectives may increase thrombotic events at supra therapeutic doses Phospholipase A2 Phospholipids Arachidonic acid %7 Peroxidase Isomerase PGG2 PGH2 coxibs COX Prostaglandins ô ô A* Thromboxanes O o Irvaslveness EG FR modul aticn Aromatase modulation Inflammation Figure í. Tho pathways v/hfch stlmufate tumour growth through CCX2 and ihG mechanisms of action c f coxibs. Mechanismy účinků exprese COX-2 na vývoj kolo rekta I n ich nádorů: Účinky nezávislé na produkci prostaglandinů (PGE2) Aktivace karcinogénu Produkce malondialdehydu Redukce hladiny volné AA Účinky závislé na produkci PGE2: Indukce buněčné proliferace Inhibice apoptózy Indukce angiogeneze Zvýšení buněčné motility Zvýšené metastatického potenciálu Indukce lokální imunosuprese Cell-autonomous effect Cell migration | Endothelial ceil Irnmunoregulation I Aromatase modulation * FGF modulation Angiogenics Figure 3, COX2 fs overexpressed in several ceJi types, such as macrophages, synoviocytes, fibroblasts, osteoblasts, tumour endothelial cells, and "activated" endothelial celfs, and may contribute to tumour growth through several mechanisms: COX2-dependent -prostaglandins can stimulate intracellular receptors ftntracrfne mechanism) r setf-prostagiandln membrane receptors (autocrine mechanism), and prostaglandin membrane receptors of different ceflsP such as endothelial cells, with proangfogenic effects paracrine or landscaping effect), Zvýšená exprese COX-2 u řady buněčných typů - makrofágy, fibroblasty, osteoblasty, endoteliální b. - podporuje růst nádoru řadou mechanismů: a) PGs závislé na COX-2 stimulují vnitorobuněčné receptory (intrakrinní mechanismus), b) PG receptory (autokrinní mechanismus) a PG receptory jiných buněk -endoteliální b. - proangiogenní efekty (parakrinní mechanismus) Fig. 8. A proposed mec h ĺiti ism for how increased expression o fa specific hP reeeplur may facilitate colorectal lumori genesis, possibly via. a positive feedback loop involving enhanced COX-2 expression. We have previously observed lhal when low COX-2 expressing color eel a] lumourcellsfsuch as adenoma cells) are exposed lo high d-oses of PGE: their growlh is inhibiledn whilsl high COX-2 expressing carcinoma cells are growlh stimulated [152]. This could suggesl lhal a more normal colorectal epithelial cell EP receptor expression profile is biased toward growth inhibitory signals al higheiPGE: levels. However, in order lo proliferate in response lo increasing doses of PGE^, cells require a predominant growlh signal (such as EP4-medialed ERK-1/2 aclivalion). It is therefore possible lhal increased EP4 reeeplur expression-1"signalling (possibly via transcriptional upregulalion, activating mulalion or chromosomal amplification), may not only be required for PGE^-medialed growlh responses in colorectal carcinoma cells, bul may also be responsible, al least in part, for PGEt upregulalion. This may result in a positive feedhack loop, applying a selective pressure for the survival of those cells wilh the highesl hP4 receptor expression, and hence perpetually accelerating carcinoma growlh in an autonomous manner whilsl simultaneously inhibiting normal or adenoma cell growlh. Il is therefore interesting lo nole the positive correlation belween ihe stage of an intestinal tumour, ihe level of its COX-2 and mPGES expression, and ils capacity to produce PGE2- Zvýšená exprese specifických receptoru pro PG (EP4) usnadňuje kolorektální karcinogenezi - vznik pozitivní zpětné smyčky zvyšující expresi COX-2. Normální b. - nízká exprese EP, nízká exprese COX-2, - inhibice růstu při vysoké kone. PGE2 XT L A_______L 1. _____'¥___L ____________T^T^fc A_____'¥___L ____________nrw ^> _ j_______1___T^/^T^^> __9. „j_____L _j_!_____1_____ EP2 Signalling EP4 Signalling PI-3-K HpERK NucJbu* Racycted Akí Přenos signálů receptoru EP2 a EP4 Aktivace adenylát cyklázy přes G proteiny - stimulace produkce cAMP a PAK, aktivace transkr. Faktoru CREB - cAMP response-element (CRE) - genová transkripce. EP2/EP4 aktivují paralelně Tcf/Lef signální dráhu nezávisle na APC. EP4 aktivuje PI-3-K, Akt a ERK1/2 kinázy a tr. faktor EGR1 regulující expresi genů pro PGE, TNF, cyklinDl. Fig. 6. EP2 Ĺind EP4 receptor signal transduction. The EP2 and EP4 receptors activate adenylate cyclase activity through binding Os proteins. This leads lo the stimulation of c AMP production and in turn the activation of the c AM P fiependen I protein kinase, PKA. The subsequent activation of the CREB transcription factor induces c AM P-re spon se e lem en t (CR E)-dep enden t gene transcription. In parallel, the EP2 and EP4 receptors can also activate the Tcf/Lef signal ling pathway through PKA-dep enden I and -independentmechanisms, respectively [65,140]. Evidence is also presented that EP2/EP4 receptor activation can stimulate the fcf-'Lef signalling pathway independently of.APC [65]. Although it is interesting to note that EP2 receptor mediated c.AMP increases (being up to 10 limes greater than EP4-mediated c AM P increases lo the same doses of ligand [123]) impair MAP kinase activation through direct inhibition of Raf, whereas the EP4 receptor is known to have PI-3-K dependent effects on Akt andERK-1/2 activation [110]. Unlike EP4 receptor signalling, the EP2 dependent pathway is therefore also thought lo inhibit expression of the immediate early genes c-Jun and Jun B, an effect thought to be downstream of Raf inhibition, as well as EGR-1, which can regulate the expression of genes including PGES, tumour necrosis faclor-a (TNF-a) and cyclin-D1 [110]. Arrows indicate activation by phosphorylation, blocked arrows indicate inhibition of phosphorylation, and dashed arrows indicate translocation. Illustration modified from [110]. ChellS. etalBBA2006 Ceil surface F:g. 5. The EPl receptor signals via coupling Lo an as yet unohanclerised G prolein. The blocked arrow indicates the potential inhibitory effect on PGE: signalling through a variant EPl receptor such as found in the rat (see text). Molekulární mechanismy COX-2 a NSAIDs Cell membrane GMP Apaptosis phospholipids _ | Arachadonic acid rostagiandtn eceptar Prostaglandin Receptor is. A, G Protein i cAMPof Ca2* Fig, 4, Molecular median ism s for C OX -2 and NSAlDs, The right part of the model illustrates the prostaglandin synthesis pathway as well as the subsequent receptor signaling—the specific prostaglandin receptors as well as the non-canonical EGF receptor pathway. As the result of inhibiting COX enzymes, accumulation of a rach ad o nic acid would directly piomotc apoptosis and attenuation of positive feedback to proliferation and survival through receptors. The rest of the figure demonstrates several COX-2 independent mechanisms proposed for NSAJDs, Since, not all NSAJDs arc able to act through these mechanisms in every cell type, a brief tabic is attached to summarize the particular NSAJDs used in each experiment as well as the cell lines involved. Anti-Neoplastic actione of N S Al Os involving apoptosis CD95 EGF Cytokines Apoptosis Fig. 6. A siimmary of the known actions of NSAIDs relevant to prevention of colorectal cancer. Red arrows and blocks indicate actions of T^SAIDi. Mechanismy účinků některých NSAIDs No Mechanism N SAID (concentration) Cell line svslem Reference 1 Accumulation of AA causes apoplosis Sulindac (200 uM), indomťthacin (300 |iM) HT29, HEK293 [141] 2 Servť as ligands for Pl'ARy Indomťthacin (40uML Hufťnamic acid (IOOiiM}% fťnoprofťn (100 uM], ihupmfcn (100|iM) Fibroblast (C3H10T1/2) [145] 3 Inhibits PED Sulindac sulfnnť(l65uM) SW480 [140] 4 Inhibits I-ic B kinasť \] Aspirin, sulindac suliitlť, not indomťthacin HCTI6, Cos, etc. [146] 5 Blocks DNA binding of WAR 5/p Sulindac sulfide (100-250 (iM) HCT116.SW480 1136] 6 Suppresses Bd-xl Sulindac (120 pM) HCT116 [137] 7 Blocks AkL activation Celecoxib (25-50 pM) PC-3, LNCaP [1471 COX-2 v angiogenezi T Angiogenesis factor secretion t COX-2 and PG production T anti-apoptotic factors Stroma Cells -From constitutive low expression of COX-1 from all cell types -From transient high expression COX-2 from inflammatory cells and tumor cells. Cancer Cells Angiogenesis factors Prostaglandin pool 0 o Oo^°ň>8bo ° oou o °^r & go *••«•*• š?s& -r T Migration T Permeability T Neovascular formation J Macrophages I n fl a mnnatory eel I s o I Pro-inflammatory molecules Blood Vessels -positive feedbacks increase the secretion of pro-inflammatory molecules Fig, 1, COX-2 in angiogenesis. This figure models the interactive relationship among cancer cells, endothelial cells and infiltrating inflammatory cells al the site of tu mori gene s is. The prostaglandin pool is contributed to by all thiee different cell types and occasionally stromal cells. The positive feedback through prostaglandin receptors increases COX-2 expression and ensures the continued generation of prostaglandins. In the cancer cell, prostaglandin signaling also results in the production of multiple angiogenesis factors, through which they stimulate neovascular formation at the site of tumorigencsis, In inflammatory cells, prostaglandin signaling stimulates the generation of proinflammatory molecules such as IL-2, which fuither recruits additional circulating monocytes and amplifies the inflammatory response. As a response to increased levels of prostaglandins, angiogenesis factors and pro-inflammatory molecules, endothelial cells proliferate, migrate and undergo tubal formation, providing additional nutrients for oncogenesis as well as a potential route for metastasis. Figure 2, increased expression ofCOX2 in human cancers is fikely to occur ťfô several pathways: mitogen-activated protein kinases (MAPKs), protein kinase Gi(PKCt)j c-Jun N-terminal kinase {JNK)r p38r and protein kinase A (PKA)r that induce cAMP synthesis and activation ofNFxB and NF-iL6r as WQff as thQ CRE promoter site, COX2 gene transcription is induced through AFkS by an Gxtraceffufar-signai-roiat&d kfnass (ERK2)r p33f and JNKf through NF-ÍL6 via p38} and through CR E via ERK2 and JNK pathways, PKG± seems to mediate COX2 transcription through ail the three promoter sites, COX-2 is transcriptionally downnegutetied by APC and upregulated öy c-Myö, and nuclear accumulation offi-catenin, through the Wnt-sfgnaffing pathwayr in human colon and fiver carcinogenesisr whereas K-ras induces CCX2 mRNA stabilisation, DRr death receptor; F ADD, Fas-associated death domair protein. Vliv různé intenzity apoptózy na homeostázu Rychlost buněčné proliferace Intenzita (rychlost) apoptózy ^♦^♦^♦^ ♦^♦^♦^♦^♦^♦^♦^♦. akumulace buněk ^♦^♦^♦^♦wO ♦^♦^♦X+. >Xt. >X*X*X+X*^ ^♦^♦X+X^ homeostaza >X>w^ I+X+X+^ ^♦^♦^♦1 Zt^+2 úbytek buněk Vliv narušení (stimulace/inhibice) průběhu apoptozy v rámci procesu vícestupňové karcinogeneze stimulace apoptozy oprava poškozeni hepatocyt s poškozenou DNA iniciovaný hepatocyt pozmenené ložisko jaterní tkáně Růstové zvýhodnění a genetická nestabilita inhibice apoptozy Schematic representation of apoptosis, oncosis and necrosis budding APOPTOSIS-*- NECROS ONCOSIS PHAGOCYTOSIS BY MACROPHAGES OR NEARBY CELLS blebbing PHAGOCYTOSIS, INFLAMMATION Fig. 3- Schematic representation of apoptosis, oncosis, and necrosis, according to taxonomy of cell death proposed by Majno and Joris (67), The early stages of apoptosis arc characterized by a relatively intact plasma membrane and intracellular changes as described in the legend to Figure 1 and in the text During the late stage (apoptotic necrosis) the plasma membrane transport function fails resulting in cells that cannot exclude trypan blue or PI, and the remains of the apoptotic cell are engulfed by neighboring cells. During oncosis, cell mitochondria swell concomitant with a distortion of the mitochondrial structure and swelling of the whole cell. For some period of time, however, other vital cell functions are preserved albeit to different degrees. Rupture of the plasma membrane leads to a necrotic stage (oncotic necrosis) which is associated with local inflammation (modified, after Majno and Joris, ref. 67). Stage of apoptosis viewed by confocal fluorescence microscopy Viable cell Early stage of apoptosis Mid-stage of apoptosis Analysis of DNA fragmentation of apoptosis Fig. 4—Analysis of DNA fragmentation of apoptosis from three cell lines, {a) HL-60 cells, exposed to camplothecin (Ú*5jUM). Lanes; M = marker la n e containing a I kb ladder of DNA Fragments from 05 lo 12'Okb; l=controk ume 0; 2= + camptothecin, time 6 h; 3~+camptothecin, lime 12 h; 4=+campiolhecin4 time 24 h; 5=+campiothecin. lime 30 h; 6= + camplothecin. lime 48 h, íb) HL-60 cells exposed to EPA (ÍOO^M), Laněš: M = marker lane; 1= control, time 6 h: 2 = -EPA, iime 6 h: 3 = +EPA. time 12 b; 4- + EPA, time 24 h; 5 = + EPA, time 30 b; 6= +EPA, time 49 h: 7=control, time 49 h. (c) c-myŕ-transfected fibroblasts after serum withdrawal í lanes 1-5) and Mia-Pa-Ca-2 cells exposed to 100/; m EPA f lanes 6 and 7 J, Lanes: M = mar ker lane as above: ^fibroblasts detaching between 23 and 35 h; 2 = fihroblasis attached at 48 h; 3=ribroblasts detaching between 35 and 48 h; 4 = fibroblasts attached at 74 h: 5 = fibioblasts detaching between 48 and 74 h; É-Mia-Pa-Ca-2 cells detaching between 23 and 35 h showing a 'chromatin ladder of apoplosis; 7 = Mia-Pa-Ca-2 cells detaching between 35 and 4S h Viable and apoptotic cells on electron microscopy Death Receptors FasL TNF TRAIL DNA Damage Growth Factor Loss Upstream Caspases Flips lame: Ion channels PT Pores zVAD-fmk BCL-2 Family BCL-2 jfRCL-X. |Caspase3 Pro Caspase Apaf-1 Caspases Cyto-C Caspase9 Pro Ca++ Oxidants Free radicals ■ Superoxides j Lipid peroxidation ATP Depletion NECROSIS Pathways controlling apoptosis and necrosis. Activation of death receptors, DNA damage, growth factor loss, radio- or chemotherapy can result in acitvation of upstream caspases, activation of mitochondria, release of cytochrome c, activation of Apaf-1, subsequent activation of downstream caspases, and finally DNA fragmentation and apoptosis. The central role of anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-XL) and of inhibitors like IAP (inhibitors of apoptosis proteins) is demonstrated. Mitochondrial activation resulting in release of Ca++, generation of free radicals, lipid peroxidation and ATP-depletion leads to necrosis. ___I__ 1 8 /Ftfl.TPt>raní ÜK3 <■.-■*;! 1 JI>eK2l UK J I ^t! I------1 Mih, II niŕApj h-haj- L 1 SErL f ^>fí TSt- 't t -111*»!-"-IK I J_____; 'f M-kll {pWfrgOm.iVr] Cjgjg) rp{A=tiM^-); v j-&firTrx nm] ^_rKc-Fi.ip[^WĚi] x ,H[^"l»ľ Hl]----- M.lľ ^7TT)= __.____ Ľull: íl-SSl mí.,: "■ "^frulTni |^__F-C>{Íľ\PK | I *-2 1~!^ ■i ■ t^sKr-:i ľ ^'} k^j-otĽff^l jŕ.azil wfJW-yil T (ostľi ■"■■ 7^ V fTT^jT^TTTT^j i- 1< ■■ ^ťgisjcrMijj ť 11 ■" J F CE^Mj i;^i f i-(ppieiak^a|- i>ti p.asfl i ^ Qnvh^oJ k rb-fc-ťwl^^ľ; (|ffi.V,]-JJ' Ě pjT^rcjMiiTwn ťyWMinfi;: iĽlľl I l.4-4._ |-1tJ Ehh___I t>ťDJ [M"l riíKi|_ .u JLP It a rov rj. (>AK[ť.tt-. ĽirQ (Ctik+ |l Míli ÍKTkťPKii) ' I í V ^1IAJ>JM ^^^ ťľlffLl.uBJ J pol . * * + f- A [jaMfriri.s»l]-*--------1—I | i. 4 -(f-My» I1.S8.1 -l*j|J I »[A|IJl- l)^-------*----»(\-H^»^|'i^ll j —->|\FOPTO£TS| Molekulárni interakční mapa drah spojených s apoptózou, u nichž byly pozorovány rozdíly v genové expresi. Molekuly podporující apoptozu - červená Molekuly potlačující apoptozu - zelená Exprese mRNA se mění očekávaným směrem - žlutě, opačným směrem - modře Fas. Fas L Bax, Noxa* pig3 Mitochondria C y to. C Caspase-8 CZ) Caspase-3Ť 7 Apaf* 1 i Apoptosis <------------------------- AÍF Figure 2. ľuKsíhlť utr^etirty pnini of Lirukipopioik rügcrsJs ťrcmi NF-kES. Iiiirin-vEi, -open arrows i and i-xirinsm < filled arrows k n|x>piasLs pathways j re di'piu*rci The tíffcčlbř ťítfřpťJiíť^ sudí as t.;i*p:iM_--^ .md cLLMpjiStr-", Liat: nclivuttľd by upslrvum iniikiiur fitspasi:*, <:isp:t,se-8 and ciipase-y. Tflť Inici-LiQir ciispLJSĽS EltĽtiijftj'K'írS are LUiivaied byfitlicTli^anth binding lo the den 1.11 rťťcp[i Nr i (implex ■ >r takk bpuffiti C l^lcsfí^sid ffftfW tkinin^ťd iniitx ÍK>rHlri;i- Ao :inii-;ipopiO[k ľfTW'i ->i _\T-kH i> :ichiťVLxJ through ils up regulation c if ÍÁĚs tivM inhibits casuist's and lk"1-xf that prouvi* ŕňltochoiulrira froEii fuitlmr daniLA^in^.—*, aĽtivmitMi; -, inhibition. Phosphorylation, ubiquitination and degradation of IkB Nuclear translocation and DNA binding of NF-kB Conformational changes Modulation of nuclear import, DNA binding, protein-protein interactions with coactivators or co-repressors, effects on transactivation FKB-dependent gene expressio Regulace aktivity NF-kB závislá a nezávislá na IkB. (a) NFkB je aktivován po aktivaci IkB kinázy (IKK). Tyto kinázy fosforylují IkB, což vede k jeho degradaci a jaderné translokaci uvolněného NF-kB. (b) Zároveň samotný NF-kB je fosforylován cytosolovými nebo jadernými protein kinázami, což zvyšuje účinnost genové exprese indukované NF-kB. IkB, inhibitor NF-kB; NF-kB, jaderný faktor kB. Molekulární interakční mapa NFkB/I kB B D 3 CytosoJ p- > jr j > j r-rr Nucleus 15 > - - .- ■- ■ 12 r IkBci*^-----1 ■■-.-> Ť ■■ '- ■ ---------- 13 ■NF-kB IicBß*-e 16 4S -—'---------------------"2 -■-":■■-__- ._"_ —r~i Kli TAP**, t-FLIP AĽBH-1, Bcl-xL TRAF-1, TRAF-2 T APOPTOSIS NSAIDs kB NFkB target genes PPARö target genes Figure 2. (1) NSAlDs inhibit activity of IkB kinase ß (licKß) which inhibits NFkB signaling by blocking the degradation of IkB and thereby, preventing the translocation of NFkB to the nucleus. (2) NSA1D (sulindac) can inhibit the DNA binding activity of PPARö. (3) NSAIDs trigger both the mitochondrial and death receptor-mediated apoptotic pathways with resultant cytochrome c (cyt c) release and DR5 up-regulation, respectively. NSAIDs also inhibit the anti-apoptotic Bel- XL protein resulting in an increase in the ratio of pro-apoptotic BAX: Bcl-Xt Oxidatívni stres jako mediator apoptózy Mnoho látek, které indukují apoptózu jsou buď oxidanty nebo stimulátory buněčného oxidativního metabolismu. Naopak řada inhibitorů apoptózy má antioxidační účinky. Možné mechanismy: ► Bcl-2 protein (produkt bcl-2 onkogenu) - v mitochondriích, endopl. retikulu a jaderné membráně - regulace ROS ► Aktivace poly-ADP-riboso-transferasy a akumulace p53 - polymerizace ADP-ribosy s proteiny vyúsťuje v rychlou ztrátu zásoby NAD/NADH, kolaps zásob ATP a smrt buňky. ► Oxidace lipidů v bun. membránách - mediatory apoptózy HPETE (po působení TNF a) ► Aktivace genů odpovědných za apoptózu přes aktivaci specifických transkripčních faktorů jako je NFkB - rozporná úloha. ► AP-1, antioxidant-responsivní faktor může také přispívat k regulaci apoptózy. Fyziologicky se ROS se tvoří v: Peroxisomech - rozklad mastných kyselin (MK) - peroxid Kataláza využívá peroxid v detoxifikačních reakcích Mitochondriích - respirační cyklus a katabolismus MK. Mn superoxid dismutasa a další antioxidanta v mitochondriích udržují nízkou hladinu těchto ROS. Byla prokázána silně inverzní korelace mezi produkcí ROS mitochondriemi a délkou existence savčího druhu. Mikrosomální systém transportu elektronů (cytochrome P450) - vyžaduje elektrony z NADPH k produkci částečně redukovaných kyslíkových druhů. ROS vznikají jen za přítomnosti selektovaných xenobiotik - superoxidový radikál - konverze na reaktívnej ší hydroxylový radikál Mimobuněčné děje - oxidatívni vzplanutí aktivovaných makrofágů - NADPH- oxidáza -superoxid. Antioxidační obranný systém: ► neenzymatický: molekuly jako vit E, vit C a glutation působící přímo na ROS ► enzymatický: superoxid dismutáza (SOD), kataláza (CAT), GSH peroxidasa (GSH-Px) a GSH S transferasa (GST). Mohou buď přímo odstraňovat ROS nebo působit recyklaci neenzymatických molekul. OH* Hydroxy I řadič aí o2 Molecular oxygen o2- Su peroxide H202 Hydrogen peroxide HOCI Hypochlorous acid a1o2 Singlet oxyg en Antibody Figure 1 | Reactive oxygen species. Superoxide is generated from various sources, which include the NADPH oxidase (NOX) enzymes (such as the phagocyte NOX, Phox). Two molecules of superoxide can react to generate hydrogen peroxide (H202) in a reaction known as dismutation, which is accelerated by the enzyme superoxide dismutase (SOD). In the presence of iron, superoxide and H202 react to generate hydroxyl radicals. In addition to superoxide, H202 and hydroxyl radicals, other reactive oxygen species (ROS) occur in biological systems. In inflamed areas, these include hypochlorous acid (HOCI), formed in neutrophils from H202 and chloride by the phagocyte enzyme myeloperoxidase (MPO); singlet oxygen, which might be formed from oxygen in areas of inflammation through the action of Phox and MPO-catalyse d oxidation of halide ions64; and ozone, which can be generated from singlet oxygen by antibody molecules65-66. The last reaction is likely to be important in inflamed areas in which antibodies bound to microorganisms are exposed to ROS produced by phagocytes. The colour coding indicates the reactivity of individual molecules (green, relatively unreactive; yellow, limited reactivity; orange, moderate reactivity; red, high reactivity and non-specificity). For further details see BOXl. Hlavní komponenty antioxidační sítě v buňce 02+ H20 o,~ NADPH NADP+ AOX AOX' AOX Boonstra and Post, Gene 2004 NAD PH NADP+ Fig. 1. Schematic represenlalion of the major players of die cellular anli-oxidanl network. The superoxide anion (O*-) is di^mulaled by superoxide dismulase (SOD)n present in mitochondria arid the cvlosol. The produced P^O^, which could give rise lo ihc formation of ihc extremely noxious hydroxy Iradieal, can be neutralized by calalase (in ihe peroxysoines) and by the cytoscdic and mitochondrial glutathione peroxidase (GP*}. The latter enzyme removes H202 by oxidizing glutathione (GSH) to GSSG, which is subsequently reduced tc its original from by Glutathion Reductase (OR), at Lhe expanse of NA DPH. A second forrr. of GP\ can reduce more complex hydroperoxides, such as lipid-hydroperoxides (LÜOII). Lov^ molecular weight antioxidants or ^cavengsrs, such as tu ľ up h e rol, ascorbale and glutathione, can neutralize radicals (for instance the peToxylradicil (LOO ) and other radicals (R )) and are often subsequently regenerated by oneor more other antioxidants (AOX and GSH) Tocopherol (Toe) is an AOX that resides incellulaT membranes f green circle), whereas other AOXs, such as ascuibale and GSHn tue located in the cylosol. Foi čiu extensive ievit;w of the cellulai antioxidant uelwoik one \^ icfcucd to Halliwell and Gutteiidee, 1999. Nucleus Antioxidant enzyme (catalase, SOD) I ^ H202 02- í _ _ -FeCu NF^BÍ FÖ3/JUN f DNA damage Mutation Lipid peroxidation Antioxidant i GST-rc, GSH etc.) ***^ Protein ^ damage Protease inhibitor damage Protease Genomic instability Chemotherapy Resistance Invasion Metastasis Schematický přehled úlohy reaktivních kyslíkových radikálů v karcinogenezi. SOD, superoxide dismutase; .OH, hydroxyl radical; ADF, adult T-cell leukemia-derived factor; GTS, glutathione S-transferase; GHS, glutathione. Pravděpodobný mechanismus chemopreventivního účinku vitamínu C v karcinogenezi karcinogenní poškození potenciace systému antioxidačních enzymů (GPx, GST, QR, SOD, CAT, atd.) inaktivni produkty iniciace promoce progrese m L normální buňka iniciovaná preneoplastické neoplastické buňka buňky . buňky modifikace epigenetického působení (protizánětlivé, obnovení mezibuněčné komunikace, atd.) Oxidatívni stres Aktivace karcinogénu Trvalý oxidatívni stres Poškození DNA: změny struktury a mutace genů Inhibice mezibuněčné komunikace Abnormální genová exprese Abnormální enzymatická aktivita Rezistence k chemoterapii Buněčná proliferace Dědičné mutace Expanze klonů Metastáze a invazivita Iniciační stádium —* Stádium promoce Stadium progrese Plasma membrane Oo or Oo- NO, ROS, ONOO Nitrosation Oxidation Nitration Metal complexes Protein kinases SAPK, p38, JAK, ERK Protein phosphatases MKP-1,PTP IRAS Nucleus Transcription factors NFkB, AP-1, C/EBP, Sp-1, RXR y^\J^\Jf\Jf^ Gene transcription Hypotetické schéma ilusturující modulaci signálů oxidem dusíku (NO) vedoucí ke změně aktivity transkripčních faktorů a exprese genů. (ap-i activator protein 1, erk extracellular signal-regulated kinases, JAK Janus protein kinases, MKP-1 mitogen-activated protein kinase phosphatase-1, NFkB nuclear factor kB, NO nitric oxide, 02- superoxide, ONOO- peroxy nitrite, p38 p38 mitogen-activated protein kinases, PTP protein tyrosine phosphatase, Ras small GTP-binding protein, ROS reactive oxygen species, RXR retionid X receptor, SAPK stress-activated protein kinases) Figuře 2 | Transmembrane topology and domain structure of NOX and DUOX enzymes. NADPH oxidase 1 (N0X1)r NOX3 and NOX4 are similar in size and domain structure to the well-studied gp91 phoxr also known as NOX2. They contain an amino-terminal hydrophobic domain that is predicted to form six transmembrane a-helices. This region contains five conserved histidine residuesr four of which provide binding sites for two haems. Haem is an iron-containing prosthetic group found in enzymes, electron transfer proteins and oxygen-binding pigments such as haemoglobin. The iron in haems is capable of undergoing reduction and ne-oxidationr thereby functioning as an electron carrier The two haems are located approximately within the two leaflets of the membrane bilayer, and together provide a channel for electrons to pass across the membrane. The carboxy-terminal portion of the molecule folds into an independent cytoplasmic domain that contains binding sites forthe co-enzym e s flavin adenine dinucleotide (FAD) and NADPH. The NOX enzymes catalyse the NADPH-dependent reduction of oxygen to form su peroxid e r which can react with itself to form hydrogen peroxide (H£02). For gp91 phox, the H202 serves as a substrate for myeloperoxidase (MPO)r but it is not known whether other NOX enzymes provide H202 for separate peroxidase enzymes. NOX5 contains the same gp91 phox-like catalytic corer plus an amino-terminal calcium-binding domain. The dual oxidase (DUOX) enzymes build on the N0X5 structure by adding at the amino terminus an extra transmembrane ot-helix followed by a domain that is homologous to peroxidases such as MPO. This peroxidase-homology domain is predicted to be localized on the outside of the membrane, where it can use ROS generated by the catalytic core to generate more powerful oxidant species that then oxidize extracellular substrates (R). OXIDATÍVNI STRES A REDOXNI NEROVNOVÁHA VE STREVE Lipid peroxide Subtoxic dose Oxidative stress Cytotoxic dose Thiol redox imbalance Mild Substantial A proliferative genes c-myc, cyclins, cdk retinoblastoma Modulates NF-kB activity A apoptotic genes p53, p21, bax, be1-2 Proliferation Apoptosis Hypotéza buněčné proliferace a apoptozy indukované lipidovou peroxidací. NF-kB, jaderný transkripční faktor kB. Ovlivnění přenosu signálů a účinky ROS na buněčný cyklus Boonstra and Post, Gene 2004 necrosis Signal transduction ^ DNA, protein and lipid damage f* p53 / p21 * G1^5/G2/M arrest apoptüüis Signal transduction ^ DNA damaged p53 -> p21 * Signal transduction ^ -^AP-1/Spl ■» p21 ^ -[transient]-------[ permanent i S/G2/M arrest G1 arrest permanent senescence" Signal transduction ^ Prolonged signal tranducMon ^ G1/S/G2 arrest transient Enhanced proliferation G1 arrest permanent ("differentiation"! Fig. 3. Scheme, representing ihe mullilude of effects lhal KOS can have on signal transduction and cell cycle progression. Fot a given cell and ROS lype ihe efFecls depend on ihe amounl of ROS and ihe duration of exposure of ihe cells lo ROS. A shorl exposure lo relatively low doses resulls in an aclivalion or enhancement of signal transduction pathways leading lo (enhanced) cell pro l i feral ion. Prolonged exposure lo Ihese ROS concentrations will resull in prolonged activation of these signal transduction pathways, comparable lo the effects of differentiation factors, which will result in a Gl arrest. Al higher cone en tral ion s and possibly depending on the cellular localization of the ROS, damage lo DNA might occur, resulting in an induction of p53 activity and consequently in expression of p21. During the suhsequenl cell cycle arrest DNA repair will otcur after which cell proliferation will resume. Alternatively p21 may become expressed due to the AP-1 orSpl sites, which are redox sensitive, resulting in a transient or permanent Gl arrest. If ihe amounts of ROS are again higher, eilher due lo increase concentrations or prolonged exposure, all changes descrihed above will lake place, logelher with structural damage lo proleins and lipids. Under these conditions, cells will arrest in all phases of the cell cycle, especially in the Gl and G2 phases and the cells will undergo apoplosis. Upon sever damage the cells may directly undergo necrosis. OXIDATÍVNI STRES A REDOXNI NEROVNOVÁHA VE STREVE Quiescence Proliferation Apoptosis Necrosis Transformed i^— N u A K A S j / / 5 o Mitotic competent /^Stimulus ■■ ^ _______ ^^^^^^^^^^^^^^^^* Differentiated Redox status Reductants Oxidants Buněčná odpověď na oxidatívni stres a oxidačně-redukční (redox) stav. Křivky představují terminálne diferencované, mitoticky kompetentní a transformované buněčné typy. Srovnání obnovy buněk ve střevě, kůži a jaterních proliferonech INTESTINE LIVER ACINUS C 1 YEAR} n CRYPT BASAL LAYER PORTAL ZONE O UJ cc UJ Ll_ O O I— o LU CC Comparison of celt renewal in intestine, skin, and liver proliferons. Normal cell turnover in the gastrointestinal tract, skin, and liver appears to proceed similarly, but at greatly different rates. The small arrows and asterisks denote the location of proliferating stem cells. Proliferating cells may hr> dpmnnsrrat^l in crypts of the gastrointestinal tract up to the opening of the crypt, limited to the basal layer of the skin, and rarely in the liver. Toxic or destructive events may increase the proliferation rate in these organs so that proliferating cells may be seen in higher layers in the skin and in the hepatic cords. Infiuction of proliferation of hepatic stem cells requires either massive loss of hepatocytes or inhibition of hepatocyte proliferation by a necrotic dose of genotoxic carcinogen. o v KARCINOGENEZE KUZE Epidermis je vysoce účinné signální rozhraní mezi vnějším prostředím a tělem. Tzv. hyperplastická transformace zahrnuje obranné reakce jako je zánět a protektivní a reparační procesy jako je vývoj hyperplasie a hojení ran. Keratinocyty epidermis jsou nejvíce exponované a mají hlavní kontrolní funkci. Po podráždění a poškození velmi rychle reagují (aktivují se) a uvolňují řadu signálních molekul, jako jsou cytokiny, růstové faktory a prozánětlivé mediatory. Význam metabolismu AA - eikosanoidy působí jako tkáňové mediatory a účastní se kontroly proliferace a diferenciace, apoptózy, zánětu, invaze leukocytu apod. Bylo prokázáno, že tvorba eikosanoidů je důležitým dějem při rozvoji nádorů. Interakce s cytokiny In vivo - vícestupňový model a Synergismus genotox. a negenotox. faktorů. Iniciace je navozena genotox. látkou a k promoci dochází opakovanou indukcí regenerativní hyperproliferační odpovědi po působení buď nádorovými promotory jako je TPA nebo po mechanickém poranění. Proces začíná reverzibilní hyperplasií kůže, následuje objevení se klonálních preneoplastických poškození (revers, nebo irevers. papilomy) a končí vznikem invazívních a metastázujících karcinomů kůže. Důležitou roli zde hrají ROS, které vznikají z velké části oxidativním metabolismem lipidů. Antioxidanta a vychytávače radikálů mohou karcinogenezi kůže inhibovat, tj. působí chemopre ventivně. HAIR FOLLICULE BULGE CELLS f~\ BASAL CELLS LOWER TRANSIT-AMPLIFYING KJ TRICHOEPITHELIOMA ^O CELLS UPPER TRANSIT-AMPLIFYING CELLS BASAL CELL CARCINOMA Q SQUAMOUS CELL CARCINOMA Ö PAPILLOMAS TERMINALLY DIFFERENTIATED KERATINIZED CELL Fig. 6. Skin cell lineage and cancer type. The phenotype of epidermal carcinomas is related to the stage of differentiation of the cell types in the skin where the malignant phenotype is expressed. TLe most primitive cell is in the bulb of the hair follicle^ and the most differentiated cell is the temimally differentiated keiatimzed cell. Epidermální karcinomy jsou často obklopeny oblastí morfologicky pozměněných buněk, často s mutacemi (p53) vedoucími k abnormální proliferaci. Další mutace (např- c-myc pak vedou k maligní transformaci. NORMAL SKIN FIRST MUTATION (PREMALIGNANT} e.g. p53 mutation SECOND MUTATION CANCER e.g. c-myc Fig. 7. Field Cancerization. Epidermal carcinomas are frequently found to be siirrounded by a "field" of morphologically altered cells. These c ell 5 are believed be changed by mutation or loss of a geiie such as p 53, which produces abnormalities in proliferation. It is postulated that a second mutation,. stich as in c-tnyc^ then leads to malignant transformation. Úloha metabolismu AA ► enzymy se indukují a AA a eikosanoidy se uvolňují po působení nádorových promotorů ► zatímco indukce je přechodná v normální tkáni, v neoplastických místech je konstitutivní ► díky mutaci v ras onkogenu a dalším genetickým defektům působí na neoplastické buňky i některé autokrinně stimulované faktory jako např. TGFa, který dále indukuje uvolňování AA z fosfolipidů a de novo syntézu kritických enzymů metabolismu AA ► nádorech kůže je zvýšené množství prostaglandinů a 8- a 12- HETE ► dochází k aberantní expresi enzymů jako je PGH syntáza 2 (COX 2) a 8- a 12- lipoxygenáza. ► chemoprevence - využití inhibitorů eikosanoidů HEPATOKARCINOGENEZE Játra - klidový orgán s velmi nízkou bazálni hladinou replikace DNA. V odpověď na specifické stimuli reagují rychlou proliferací zprostředkovanou pravděpodobně novou expresí genů. Dediferenciace, změny v regulaci bun. cyklu, působení růst. faktorů HGF(hepatocyte growth f.) a EGF(epidermal growth f.) K regenerativní hyperplasii dochází při reparaci poškození jater v důsledku chirurgické resekce, částečné hepatektomie nebo po působení toxických látek. Negenotoxické karcinogény např. peroxisom. proliferátory (PP) nebo phenobarbital -přímý mitotický stimul in vivo stimulující asi 30% buněk během 48h. Ke stimulaci dochází i u hepatocytů kultivovaných in vitro. Molekulární mechanismy nejsou zcela objasněny. Silná korelace mezi indukcí s. DNA a následnou hepatokarcinogenitou. Tento proces však dále závisí na ploiditě. U jaterních buněk endoreplikace -polyploidizace. Narušení regulace bun. cyklu - buňky citlivější k působení chem. látek. Negenotoxické karcinogény působí u hepatocytů též supresi apoptózy. Poškozené buňky pak persistují v populaci a po dalším mitogenním působení negenotox. karcinogénu z nich mohou vznikat nádory. Při působení např. PP hrají důležitou úlohu receptory PPARa, jejichž kvantitativní exprese je pravděpodobně odpovědná za rozdíly v citlivosti mezi hlodavci a jinými živ. druhy i člověkem. Proliferace hepatocytů může být zprostředkována cytokiny TNFoc a IL-6 - přechod G0/G1 Hepatocyte growth factor (HGF), epidermal growth f. (EGF) a TGFa - přechod mezi střední a pozdní Gl fází Signály mezi různými typy buněk_-Kupfferovy buňky (jaterní makrofágy) po stimulaci PP uvolňují TNFoc a IL-6 —> aktivace specifických transkripčních faktorů jako je NFkB nebo STAŤ proteinů (přenos signálů a aktivace transkripce) v hepatocytech. U lab. zvířat sledovány počty a velikost morfologicky a enzymaticky změněných fokusů (v nich s. DNA a apoptóza). Další stupeň hepatocelulární adenomy a karcinomy. HEMATOPOIETIC STEM CELL O CD-ETHIONINE/AAF PERIDUCTULAR STEM CELL SOLT-FARBER "BIPOLAR" D U CTU L AR PROGENITOR CELL "O HEPATOCELLULAR CARCINOMA DIETHYLNITROSAMINE •^nHEPATOCYTE Fig. 8. Postulated stages of the liepatocytic Lineage that may respond to liver inj my or carcinogenic protocols. Following various models of liver injury or chemical liepatocarcinogenesis different cell types in the liepatocytic lineage may respond: 1 Undifferentiated periductiilar oval cells, which may arise from circulating bone marrow precursor cells. 2. Periductiilar cells intrinsic to the liver, 3. Bipolar ductal progenitor cells, or 4. Mature liepatocytes, which retain the potential to divide. Periductiilar cells respond to periportal injury induced by allyl alcohol or to choline denciency-ethionine carcinogenesis. Bipolar ductal progenitor cells respond to injury and to carcinogenic regimens, such as the Solt-Farber model, when proliferation of liepatocytes is inhibited. Hepatocytes respond to partial liepatectomy and to carcinogensis by diethyliiitrosamine (DEN) (from [132,315]). ATYPICAL HYPERPLASIA CARCINOGENIC EVENT HEPATOCELLULAR CARCINOMA STEM CELL TRANSmON DUCT CELL HEPATOBLASTOMA COMBINED HEPATO-CHOLANGIOCARC1NOMA CHOtANGMDFlBROSIS CHOLANGiOCARCINOMA Postulated levels of expression of carcinogenic events during hepatocarcinogenesis. The stem cell model of hepatocarcinogenesis postulates that carcinogenic events occur in proliferating cells at some stage during differentiation, resulting in expression of the malignant phenotype (blocked ontogeny). Because carcinogenesis most likely results from the accumulation of more than one mutation, it is likely that the first mutation (initiation) takes place at the level of the stem cell and that later mutations occurring at the level of the transition duct cells or in aberrantly differentiating cells (atypical hyperplasia or cholangiofibrosis) direct the level of expression of malignancy. Hepatoblastoma may represent tumors that arise because of multiple muUtions at the stem cell level Tumors with combined features of hepatocytes and bile ducts (hepatocholangiocarcinomas) may arise from multiple mutations at a later stage of differentiation. Hepatocellular carcinomas arise from a still later stage of differentiation. Peroxisome proliferators (fibrates, phtalates, etc.). 9-c/s-RA Nutrition PPAR Fatty acids (PGJ2, LTB4) RXR Target genes Transcription Proliferation CELL SPECIFIC RESPONSES I Differentiation and maturation Apoptosis * Clonal expansion of I preadipocytes promoting adipogenesis (participation on PPARy.) * Hypothetical risk in man of cell growth stimulation by activation of PPARs. MEDICAL RELEVANCE * Monocyte / macrophage differentiation (implication of PPAR7) leading to accelerated atherosclerosis. * Protective effects of PPARa. * Adipocyte differentiation responsible of obesity and other related disorders (implication of PPARa.) - Enhanced PPARg expression could lead to tumoral cell apoptosis and represents a therapeutical approach in malignant disease. Importance of PPARs in cell proliferation, differentiation and apoptosis. After activation, PPAR and RXR form heterodimers which bind to DNA regulatory sequences of target genes through interaction with PPRE. The control by PPARs of the transcriptional activity af target genes gives rise to biological effects which may have consequences for human health. LTB4, leukotriene B4; PGJ2, prostagladin J2; PP, peroxisome proliferator; PPAR, peroxisome prolifera-tor-activated receptor; PPRE, peroxisome proliferator responsive element; 9-cis-RA, 9-cis-retinoic acid; RXR, 9-cis-retinoic acid receptor. Schéma signálních drah PPAR CoRep? PPARl RXR CoAct CoRep CoAct PPARiiiiMll£ PPAR I RXŔ PPRE wwmiwi PPRE PPARs fungují jako heterodimery s jejich obvyklým partnerem - retinoidním receptorem (RXR). Kupffer CGlls RodGnt llver d Mechanism IncreasGd DNA napllcatlon IncrGased prolifGration DGcrGasGd apoptoaia Reactive oxygen species (DNA damage: proliferation) b Short-term response Transcriptional activation of genes that are Involved In Tatty-acid metabolism. In the cell cycle and In degradation or endogenous and exogenous compounds (cytochrome p450 family) Peroxisome proliferation Cell proliferation Uver hypertrophy c Long-term response Hepatocellular carcinoma Figure 2 | Consequences of Ppara activation by PP in the liver and proposed underlying mechanisms. Long-term chronic activation of p e nox iso me- prol iterator-activate d neceptor-ö (Ppara) in the hepatocytes by its ligands (initial event; a) induces a short-term pleiotrope response (b) followed by hepatocellular carcinomas in both rats and mice (c). The short-term response includes transcriptional activation of enzymes that are involved in fatty-acid metabolism (fatty-acid ß-oxidation, fatty-acid transporters and cytoplasmic liverfatty-acid-bincling protein (L-FABP)), of genes that are involved in cell-cycle control and of genes coding for enzymes of the cytochrome p450 family (second-line events)14: peroxisome and cell proliferation (third-line events); and liver hypertrophy and hyperplasia (fourth-line events). The long-term consequence of these events is the development of hepatocellular carcinomas in rodents. d | Several underlying mechanisms are being debated10,16.Peroxisome proliferates (PPs) induce DNA replication and proliferation of hepatocytes in a P para-dependent manner1922. Furthermore, PPs repress spontaneous and induced hepatocyte apoptosisr in vitro and in vivo. As well as controlling of the cell cycler the production of reactive oxygen species in response to Ppara agonists might damage DNA and promote hepatocyte proliferation., but the implication of Ppara in this effect remains to be proven. Additionally non-he pa tocyte cells, such as Kupffer cells, might participate in the short-term cascade of events by promoting hepatocyte proliferation31. Lipoxygenase Ärachidonic ac Cyclooxygenase 12-,15-Lipoxygenase g^ PPRE Figure 1 | Schematic representation of the PPAR signalling pathways, a | Endogenous agonists of peroxisome-proliferator-activated receptors (PPARs). PPARs are I igand-inducible receptors, which can be activated by fatty acids — such as arachidonic or linoleic acids — and their derivatives. The fatty-acid metabolites that activate PPARs are mainly derived from arachidonic or linoleic acids through the cyclooxygenase or the lipoxygenase pathways. The best characterized at the moment are leukothene B4 (LTB4) and ßS-HETE (hydroxyeicosatetraenoic acid), which preferentially activate PPARö; 15-deoxy-prostaglandin J2 (15-dPGJ2) and 15-HETE, which are PPARy-selective ligands; and the prostaglandin I2 (PGI2r also called prostacyclin), which is probably a PPARß/ö natural ligand. PPARyis also activated by 9-HODE (hydroxyoctadecadienoic acid) and 13-HODEH either derived from linoleic acid or as components of oxidized low-density lipoprotein (oxLDL). b | PPARs function as heterodimers with their obligate partner retinoid receptor (RXR). The dimer probably interacts with co-regulatorsr either co-activators (CoAct) or co-repressors (CoRep). In the unligandedform, PPARß/6-RXR heterodimer in contrast to PPARa-RXR and PPARy-RXR heterodimers, recrufcs co-repressers and represses the activity of P PA Ret and PPARy target genes by binding to the peroxisome proliferator response element (PPRE) that is present in their promoters67. In their liga n ded form, the PRA R-RXR heterodimers interact with co-activatorsr bind to the PPRE that is present in the promoters of their target genes and activate t heir transcription. T^J PPRE \^W% Proliferation in keratinocytes Resistance to apoptosis Migration Differentiation in inflammatory conditions Differe?itiation Cell-cycle withdrawal Preadipocytes v__ * Myeloid J ce" Liposarcoma Wound bed Adipocyte precursor Resting macrophage Adipocyte Lipid-loaded macrophage Figure 3 | PPARß/Ö and PPARy f unctions that relate to their carcinogenic properties. a | As demonstrated in a mouse-skin wound-healing model, Pparß/6 inhibits keratinocyte proliferation and participates in inflammation-induced keratinocyte differentiation, which are anti-carcinogenic actions. However, it also increases both migration and keratinocyte resistance to Tnf-a-induced apoptosis. b | PPARy is implicated in the differentiation of pre-adipocytesto adipocytes and of monocytes to macrophages. In the presence of PPARyand retinoid receptor (RXR) ligands, myeloid-cell precursors become resting macrophages, which can be turned to lipid-loaded macrophages, when PPARyand RXR ligands are maintained. PPARy can also withdraw liposarcoma-derived cells from cell division to trigger their differentiation to adipocytes. The Chromosomal Instability Pathway Other genetic errors, Bub1, hCDC4 Chromosomal instability Aneuploidy, LOH Adenoma/carcinoma transitions APC ___* TTCF/LEF1 -^ TSurvivin . mutation gene T c-Myc transcription Ť Myelin D1 ^ p53|LOH Ras mutations Inflammation ^"-^-.__ Progressive loss of apoptosis Fig. 2. The chromosomal instability pathway to colorectal cancer. MMPs C el I-surface j-L. proteins p"l Fas ligand Fas (CD95) E-cadhenn Tumor growth factor-a IGFBP-3 Resistance to chemotherapy Induces apoptosis resistance Enhances adhesion & metastasis Activates EGFR Promotes survival Figure 1. Cleavage of cell-surface proteins other than the ECM components is crucial for MMP-mediated CRC tumorigenesis. Normal flora Wild typ«___________ Tissue macrophages Not accumulated yet Not activated yet Helicobacter Wild t l-L COX2/mPGES 1 PGE2 TNF-a Macrophage accumulation * 4 Macrophage activation Proinflammatory cytokines andi GFs Normal flora T^K19C2mE_ Í 00000000 C0X-2/mPGES-1 PGE2 I TNF-a Macrophage accumulation Macrophage activation Proinflammatory cytokines and iGF's :J' Mitotic catastrophe Figure 21 Model for survivin-med i ate d protection against mitotic catastrophe. Cellular stresses induce both DNA damage and defects in the mitotic machinery (mitotic damage). DNA damage can lead to cell-cycle arrest, apoptosis or aberrant mřtosis. Mitotic damage can lead to eel I-cycle arrest, apoptosis or mitotic catastrophe through aberrant mitosis. In situations in which p53 activity has induced apoptosis, BCL2 expression can rescue cells from death. p53 — and Its target genes that encode WAF1 and 14-3-3o" — also help to prevent aberrant mitosis and mitotic catastrophe. The normal function of survivin i3to maintain the integrity of the mitotic spindle and promote mitotic progression. Loss of survivin induces cell-cycle arrest and cell death by mitotic catastrophe in a manner that is independent of both p53 and BCL2. However, loss of survivin also induces p53 and WAF1 expression, perhaps indirectly triggering p53-dependenrt apoptosis (dashed line).