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 Incidence Mortality 0 ZU ZZI ZJ ZZ 20 40 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 moc. ústrojí 6,54% moc mecnyr 5,87 % prostoto 11,14% žaludek 5,38 % 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 % žlučník 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, kl LUSTE STŘEVO - nejsou klky, na dně krypt kmenové buňky (nejsou zde P. buňky), 2/3 krypty proliferující buňky. 2 hlavní linie buněk: Enterocyty - absorbtivní linie, nejpočetnější Sekreční linie: goblet buňky (sekretují protektivní muciny - přibývají směrem ke kolonu) fetují hormony - serotonin ekretují antimikrobiální lať mikrob, obsahu ve střevě. Aktivní migrace buněk doprovázená diferenciací a odlupovaním do lumenu rraa«nwii í^B Ifcjiíi Fíg. 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 secth of villus villus absorptive brush-border cells cross section of crypt direction of movement (A) nondividing differentiated Paneth cells nondividing differentiated- cells rapidly dividing cells (cycle time - 2 hours slowly dividing ste ceils {cycle time > 24 hours 100 ^im Příčný řez částí stěny střeva LUMEN OF GUT Ismooth muscle epithelium -C connective tissue r circular fibers longitudinal fibers " connective tissue epithelium '"'''^rr-fBíffls&saaES5''' epithelial cell fibroblast smoothl muscle cells 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) alizované země (životní styl, výživa) vtřetí nejčastější příčina úmrtí na rakovinu) věková distribuce (muži nárůst případů od 60 let; ženy od 70 let) 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, metastá 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 meatr 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. % positional frequency Fig. 3. 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 cell 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) 20 c E 10 CO '-5 °-2 E £8 CL g r 1 ± ■ j_ _i__■' c .2 o Q. "Ô O 0.8 0.6 0.4 0.2 0. Growth factor KU IäIsb postní itotická J zóna zona proliferace epiteliálně- mesenchymální interakce APOPTOZA anoikis (programovaná unecna srn terminálni diferenciace poškozeni buňky APOPTOZA Normální epitel a adenomy v myším tenkém a tlustém střevě villus crypt } Surface epithelium V cfypt ^■I^V ^h. ^v*-* ! ■ i '•'*!'• r * TI Nř * c * r.v / . JfrJWt & .* *i ^'i-&\\. ** V ^^Phí r .* 2Z L ^-~ i • '> . ■V *_^^fcrf fek -4V Ü ijy > ■"*5 Smalt intestine colon Adenoma in small intestine Aberrant crypt focus in colon Fig. 2. Comparison of normat epithetium 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 ji-catenin. Note the presence of ji-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) adenom v tenkém střevě a fokusy aberantních krypt v kolonu min myší. Barveno na přítomnost beta-kateninu. r 1 r 1 \/ IIISIÍ!lMil!l!lA^ a aberantní krypty - beta-katenii t2^£^2a aboratory ytokinetics In vivo progression of colon epithelial cells GUT-LUMEN APOPTOTIC CELLS X EPITHELIAL CELLS CRYPT P3UTYRATE RODUCTION BY .ON BACTERIA PROLIFERATIVE PART Birth of an Epithelial Cancer PROU DERATION An initial mutation in a single cell {highlighted^ disrupts signal transduction ar the celh cyde, and the errant ceM divides more- frequently than its neighbors The changed cell and its descendant constitute dysplasia, a precancerous- growth whose cells are stilJ differentiated and confined to lha bottom ceíl layer Time passes. A second mutation occurs in an already affected ceil, usherinj in E-weeping changes in gene expression that modify meLabolism. growíh characteristics, and cell shape, as adhesion and anchorage slip. The doubly mutant cell and its descendants are shghlry less specialized than thefcr Aeighbors. forth** escape fr*m growth ců-nstrainU, caused hy b Lhitd riVjLjLirjr-., ptTluibs ihr LeM ť^dŕ in J dilTelcrtl way, all Dwirig rapid enpanLran ůf Lht growth As rn -t-j: i activity JUtfipi, SprCLdllZSLlIúrti G*HlirlUe tu Füd.- i Vid;,' ks ne* :.í-lk form. Thriť r-.U( lei appear enlarged and mü-ihjpen, refletLIrig [h* chaůl Within With sti|] more mutations.tfre increasingly aggressive growth is now a misshapen, ir still microscopic mass. Yet it remains contained within the epithelium bounded by tire basement membrane. The tiny tumor may remain here, as an situ carcinoma, for years. The abnormal cells now occupy all cell levels, end-all are de-differentiated to some degree. 52 | SrplrrriL*i-ií2.2UŮS I In- Sei rutin Invasion. A* the divid/mg rančer wlk **t*nd through th* ba«- menl rr.r-m^Mnr :n1y ihr -surrounding ^rrjma, míhg. nqncy rjigipi- Thi tumgr and stroma commUni-catc, ü thí cjnCcr lučily «>£í gver- Meanwhile, Cancer celh iecr-ct« rVbrQb^0->( growth factor and vascular rndi-thihsr growih Factor, which subvert ngrmaE angipg.*Tnrm to fKfWl capilíňriffí, Which lnak-r; in ň nd »TPtrnd th? lumnr Some cancer cell t creep algng the aiigciatod felood Vesurfi; otheri *quařie bciwren the lining cfllfi to inter Ine circurjügn, Sprsjif bflgmv METATaSiS. Supplied WHth nutrients, oxygen, a waste removaJ service and conduits, ferocious cancer ceils exit and home to iymph nodes and mo^e distant files, the Rectory characteristic of tumor type; fcrdney or breast to lung; prostate to bone. Here, further mutations a-nd changes in gEne expression ensue, often rendering tbc original tumors orTspfing so dmerentthat treatments that once worked now *ail. SrfWMiiHU.lZ.ZWi TltrSťMlHiH |5J 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 HE oi o hi tmsBšBľ raBCTSiifomaiiugřBiiKiar i yrc ;ena •fiHsga 11 ira.fBifrr supresorových í tvorbu nádoru poskytujíce gmmnm Žml! ImBB genů) a epigenetické klonální růstovou v ► Klíčovým molekul r ch genetických defektů u zárodečných linií, jejichž somatický výskyt nastartuje sporadické ládory kolonu. Ztráta genomové stability je klíčovým molekulárním a patogenetickým kroke 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 1 Dědičné poruchy predisponující jedince k nádorům autozomálně dominantní typ dědičnosti isi l°Á :q 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 50 fim NORMAL EPITHELIUM 50 (im LOW-GRADE INTRAEPITHELIAL NEOPLASIA 50 (im HIGH-GRADE INTRAEPITHELIAL NEOPLASIA 200 |jm INVASIVE CARCINOMA Figure 23-9 part 2 of 2. Molecular Biology of the Cell, 4th Edition. Modifier genus EnvlronmQnt Rgure 1 | A global view of the genetic contribution to colorectal cancer. The highly penetrant causative mutations in familial adenomatous polyposis (FAR). Lynch syndrome, the hamartomatous polyposis syndromes and other familial conditions underlie cases 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 ORG 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 between "genetic' and 'environmentalľ predisposing factors has become blurred and might be better thought of as a continuum of risks contributing to CRC development. APG, 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 Utah9S. 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 nádoru 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 ^^^ffi omu ÍSElff LÍmi fakt otřeba pravidelných vyšetření od určitéj rev, Sigmoidoskopie, kolonoskopie) ku (okultní Normal DNA K \ , \ Error-free repair2 DNA Adduct1 N * K Surveillance for damage and/or cell cycle checkpoints^ Apoptotic death3 + Error-prone or failed repair i Genomic instability5 \ Cancer Figure 2. Diagrammatic representation of a modei 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 DNA adduct formation. The three heavy arrows indicate the major outcomes of inherent surveillance mechanisms for controlling DNA fidelity in response to adduct formation. Failed repair results in adduct 'fixation1' 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 reguíatíon 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, VPC - delece či mutace, brzký děj u adenomů 80%, spojené s deregulací signální dráhy Wnt a chromosomální nestabilitou. Chyby spojení mikrotubulů 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) r 22q 17q 17p 14p £p 6q -í p ďcnoffH — Irr/Monand Fig. 2. The adenoma Id carcinoma sequence- The development of colorectal cancer is an excellent example of the complex multistage process of tumorigenesis 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, v f k-ras and Ape gene mutations have been found to be involved in the early stages Drugftesia 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 vPA E>Catitoorin Genetické změny spojené s kolorektale karcinogenezi [chromosome j 5 q | iteration 1 LOF genes A PC UildiliiiUiLb Normal epithelium >-> llypcrprolifcralive epithelium I2p GOľ K-ras 18q LOF nec SMADs I7p LOF L> Karly ariťiitmiii Intermediate adenoma Laie adenoma Carcinoma MMR genes: hMSH2 hMSH3 HMSU6 hMLHI hPMSl hPMS2 Poruchy „mismatch repair' Metastasis Fig. I. Genetic changes associated with colorectal lumorigcncsis. This process is accelerated by MMR deficiency (sec text for details). Abbreviations: LOK loss oľ function; GOK gain of function; MMR. mismatch repair. Reproduced from Kin/Jet & Vogelslein (2) with modifications. Genetický model kolorektální karcinogeneze Histological stage Normal epithelium Dysplastic crypts Tubular adenoma Dysplastic adenoma Carcinoma Metastasis Genetic regulation APC ß-catenin K-ras DCC p53 [»» p p n jo p p n Reproduced from Sharma eř 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 Truncation of APC Mutace genu APC vede ke změnám cytoskeletu a deregulaci beta-kateninu. Ovlivnění migravce 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. V Accumulation Aneuploidy Transformation Cell Chromosome migration segregation Deregulation of ß-catenin Figure 11 Truncation mutations in APC affect cytoskeletal organization and the deregulation of ^-catenin The resulting cytoskeletal changes lead to defects in cell migration and compromised mitotic spindles. This causes the inappropriate accumulation of tissue and also leads to aneuploidy. Deregulation of /S-catenin leads to defects in differentiation and gene expression, causing transformation. Elevated levels of /Katenin in cells leads to a reduction in the ability of APC to bind microtubules and perfom its function as a cytoskeletal regulator. This could contribute indirectly to defects in cell migration and chromosome segregation. \^ Mucigenic signals Ras DNA damage Caspase 9 Bad Anti-apoptosis Pfo-apoptosis Fig. 3. Mitogenic signals are transduced by Ras which inhibits the retinoblastoma (Rb) protein allowing E2F and Myc to promote cell cycle progression, pi6 is a tumour suppressor whose mutation permits cyclin D dependent kinase (CDK) to inhibit Rb. High levels of E2F or Myc activates pi4 ^ and thereby p53 which has multiple outputs. A series of pioapoptotic 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 ► Mutac «■ 331 adenomatous polypo kme nn^ng^ ilUlgŠffl , migraci a adhezi ► Abnormální buňky se akumulují na vrcholu krypt, tv< aberantní fokusy krypt (ACF), které vyčnívají do proudu stolice Bia g^ra ni vMm iiHHwiwiiijri o iiiriRiiBiRiWiMiiinBW^iiiímare rn n »iSreimimKi) 11 ra ;uněk tvoří postupnou klonální expanzí ;wnraimrn 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 - c M&S 1 5S5U iikIiEIsíwJ ice pnjrn n no ÍRiMiBBilJirfftKroľf ľ '' ► Výživa i a frekve a kvalita nasycených a nenasycených tuků, lipidová peroxidace, zvýšená tvorba prostaglandins - ochranný vliv vlák ' / «KiEtmiia vaz ÍKmSuTSlW) J mm^^ffiH wmM Bi - vitaminy a další mikrokomponenty živin (vi antioxidantyx Bi ľaEa selen j sou miirei mac gu^TO^CT^^H tfMiiaj21Ľ!ilÍ3MU ýmky - potravinové mutageny (zejména heterocyklické aminy ve vařeném a pečeném mase a tucích) - konzumace masa a vajec (vyšší konzumace je rizikov hovězí, jehněčí^ řové, ► Fyzická aktivita nedostatek je rizikovým faktorem předpoklad modifikace diety s vysokým obsahem tuků liiiMrďMIfenniEiffni ojem látek zvyšujících riziko nádoru kolorekta (zejména íobilový a dřev° řfflfl ► Věk(zvý ► Neefekt ímunimi sys 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 . GF-ß) EGF - epidermální růstový faktor Tumour necrosis factor-alpha (TNF- a), interleukiny ► multifunkční cvtoki ► jeden z hlavních mediátoru zái ► TNF- a je produkován makrofágy a dalšími buňkami imunitníhp systému ► koncentrace TNF- a v kolonu je zvýšena během chronického zánětu (ulcerativní kohtida nebo Crohnova choroba) ► úloha v nádorové kachexii dstuje interakce mezi cytokiny a dietetickými faktory - mastné Figure L Mechanism oi" Signaling by TGFß superiamily members, Binding ol" TGFß super family 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 Wn| signal LRP (nfiiV Ubiquitin-depenctent degration Wn( signai Twív Fbc LRP Nuclear translocation induction ^ora karcinogeneze Deregulace: Vazba Wnt na Frizzeled Stabilizace bet Akumulace v jádře Aktivace LEF1/TCF tramskripčních faktorů Figuře 2, A model úľ Wnt signaling. In the absence oť Wnl ligand (lell pane]) APC- Axin and GSK3 i orní a complex that results in ß-eateniu phosphorylatiO]i and degradation. Binding oi' Wnt to tile Fri/zled receptors (right panel) results in stabilization ofß-eatenin that then aecummulates in the nucleus uIlltl1 il associaĽs wAh LIT« TCI" ImiisĽnplion J'lllľms U? regulate gene expression. ß-Cat 9 ■ ■ • ■ fc ■ ■ ■ ■ ■£%■ i ■ ■ ■ ■ J** ß-Cat ß-Cat TCF ß-Cat Fig. 1, Schematic presentation of the canonical Wnt signal pathway. The left side Wnt 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 bw unphosphorylated and therefore stabilized ß-catenin. APC, adenomatous polyposis coli. Regulace (deregulace) transkripčních faktorů u střevních buněk Writ Signalling SMP Signalling fl^at ^\ Differentiator! TCF Crypl proliferation Brnp R-SMADjJ ® CO-SMAO r- •«i p //////. L> Notch Signalling Molch ÍU1% NICO VI u 11 pie ectopic crypts Normal crypt formation Fig. 3. Wnt, BMP, and Notch pathways control target gene transcription. (Left) Wnt-responsive cetts carry a receptor complex consitirig of a frizzled seven-transmembrane receptor (Fz) and LrpS or Lrp6. In the absence of secreted Wnt factor (Left), the destruction complex (APC, axin, and the kinases CK1 and GSK3 ß) induces degradation of cytoplasmic ji-catenin. Tcf completed to corepressors such as graucho represses specific Wnt target genes. Receptor engagement (right) blocks the destruction complex; ß-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-SMAD, translocation to the nucleus, and subsequent activation of BMP target genes in the nucleus. (Right) When Notch receptor meets its cell-bound Ligand (JaSSed or detta)h sequential proteolytic steps Lead to the release of its intracellular domain (NICD), which travels to the nucleus, where it complexes with the transcription factor CSL to activate Notch target gene transcription. Mechanismy působení vysoce nenasycených mastných kyselin (PUFAs) zahrnuté v kolorektální karcinogenezi METABOLISMUS KYSELINY ARACHIDONOVE (AA) nozstv Egal COX-2 u kolorektálních karcinomů je zvýšena exprese < PGE2 stimuluje růst a inhibuje apoptózu nádorových buněL PGE2 působí prozánětlivě a reguluje funkce imunitních buněk (imunosupi nesteroidní antiflogistika (NSAIDs) snižují riziko kolorektálních nádorů a zánět ibicí r iBmEttfr« ■»VllVll« Změny genové expres >PAR, NFkB, AP mvazivi livace specifických transkripčních faktorů genotoxické účinky a mohou ovlivňovat buněčný cyklus. Během LP jsou produkovány reaktivní kyslíkové radikály (ROS) ROS mohou aktivovat NF-kB Phospholipids i Diôtúry Polyunsaturated Fatty Acids (DHA. EPA) COO\J. Amchidonic Acid COX-1 COX-2 ^—x^^v^OOH TXA; OH TXB, Ufi-hydtoxy series of docosartoids (ftesolvins) 15Ä-HETÍ-----► l5-epMipoxin5 Arachidonyl Etlronotamide (Anandamide) ^jqv.2 PS nr.d TX glycerol fistfirs ^ ~"" and ethanalúFfií dis r*sa&&- „CÚQH 9v^~"^N^oh ho 5h PGE2 kí MÚ OH P6F2n lO OH v PSI* \ COOH 6-keto-PôF L,: Dráha 5-lipoxygenázy - vznik leukotrienů jAracKtdoníc Acid l cOCf- 5-LO Oi 12-LO 15-LO LXA. ■-.I- i.i LXB, ^ &WU 5(5)-HETE ^^p^ LTC4 LTB4 / '&ÍuŤGmy}-tranšpept}dQ$€. í:- ■:- /ÍŮCM Cancer cell p roí ff erat ion COX-2 I Apoptosis oo oo COX-2 1 Neo - angiogen&sis COX - 2 5-LO 1 , 5-LO 5-LO _u o ArachidonJc Acid Platelets Endothelium Gl 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 lHig. 2 COX-2 expression in tumoral cells and superficial interstitial cells {arrows) in a well differentiated colon adenocarcinoma, im m u noh i s toe hem i s try f x 2(H) Table 1. COX2 expression in malignant or premalignant human tumours Premalignant or malignant lesion COX2 expression (%) Colorectal 30-90 Gastric SO Oesophageal 70 Hepatocellular (liver cirrhosis) 54 (81) Pancreatic 07 Head and neck 80 Non-small-cell lung cancer 70 Breast (ductal carclnoma-ln-sltu) 40 (00) Prostatic 83-93 Bladder 36 Cervix 43 Endometrial 37 Cutaneous basal cell 25 Cutaneous squamous cell 80 pPNET 100 Glioblastoma multiforme 71-74 Anaplastic astrocytoma (low grade) 44 (30) References available at http://lnnage.thelancet.conn/e)ítras/03oncl205webfr.pdf Některé inhibitory COX-1 a COX-2 (NSAIDs) TABLE 1. Structural class Members COX-1- nonselective COX-2- selective alkanones nabumetone anthľanilic acids meclofenamic acid, mefenamic acid meclofenamate esters and amides arylpropionic acids ibuprofen, flurbiprofen, ketoprofen, naproxen, diarylheterocycles SC560 celecoxib, etoricoxib, parecoxib, rofecoxib, valdecoxib di-teit-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-vnvl sulfide (APHS) sulfanilides nimesulide, flosulide log CK* ratio, COX-2/1) i * >,; sV3 <-• O lumiracoKib rofecoxib etoricoxib valdecoxib do do lac me I osi cam nimesulide celecoxib diclofenac sulindac meelofftiamate tomoxiprol Piroxicam di fluni sal sodium salicylate nifiumic zomepirac fenoprofen ampyrone ibuprofen tolmetin naproxen aspirin indomeUiacin ketoprofen suprofen flurbiprofen ketorolac 51 r, n c ls> « (g ■o i __I v, ÍL n r: C E? r: - s v- I—■- Schematické dráhy některých funkčních efektů inhibice COX-1 a COX-2 decreased mucosal defence Z tumour increased ulcers z increased apoptosis asthmatic lung i křdney {fluid/salt deputed) decreased tumour growth increased [eukfttrierifc production i decreased GFR & Na re&bsorptior z broncho-constriction I edema; increased bio od pressure blood vessels piateiets decreased PGlz production decreased TxAz produclion \ pro-thrombotic? TABLE 2. COX-2-selective agents 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 Thťmpeutic indication equi-effective equi-effective Otker 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 Peroxidase I some rase Phospholipids Arachidonic acid PGG2 PGH2 coxJbs COX Prostaglandins ♦V Angiogenesis Thromboxanes Apoptosfs invasiveness EGFR modulation Aromatase modulation Inflammation Figure 7. The pathways which stimulate tumour growth through COX2 and the mochanisms of action ofcoxlbs. Mechanismy účinků exprese COX-2 na vývoj kolorektal n ich nádorů: MkiTÍEIta \ go 00^8% Cancer Cells Angiogenesis factors j£°-£# tí Macrophages Inflammatory cells T Migration T Permeability T NeovascuIar formation Proinflammatory molecules -positive feedbacks increase the secretion of pro-inflammatory molecules Fig, I. COX-2 in angiogenesis. This figure models the interactive relationship among cancer cells, endothelial cells and infiltrating i n flam ma to iy cells at the site of m mori gene s is. The prostaglandin pool is contributed to by rill three 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 rix: cancer cell, prostaglandin signaling also results in the production of multiple angiogenesis factors, through which they stimulate neovascular formation at the site of tu mori gene sis, In inflammatory ceils, prostaglandin signaling stimulates the generation of proinflammatory molecules such as IL-2, which further 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. Growth factors Figure 2. increased expression ofCOX2 In human cancers is fikeiy to occur via several pathways: mftogen-actlvated protein kinases (MARKS), protein kinase C^RKCJ, c-Jun N-terminal kinase {JNK)t p38r and protein kinase A (RKA), that induce cAMP synthesis and activation oťNFkB and NF-IL6, as well as the CR E promoter site. CÖX2 gene transcription Is Induced through NFkB by an extraceffufar-signal-reiated kinase (ERK2)> p3&, and JNK, through NF-IL6 vfap38} and through CRE via ERK2 and JNKpathways, PKC, seems to mediate COX2 transcription through aii the three promoter sites, COX-2 is transcriptionally downregufated by ARC and upregu* fated by c-Myb, and nuclear accumulation offi-catenln: through the Wnt-slgnaffing pathway. In human colon and fiver carcinogenesis, whereas K-ras induces COX2 mRNA stabilisation. DRt death receptor; FADD, Fas-associated death domain protein. Vliv různé intenzity apoptózy na homeostázu Rychlost buněčné proliferace Intenzita (rychlost) apoptózy >X+X+X+^ ♦^♦^♦^♦^♦^♦^♦^ I+w+X+^ >X+X+X+X+X+X+X+X+^ >IOIO^ >w4w4w4w^ .♦X+X+X+X+X+ akumulace buněk .♦X+X+X+. >X+X+^ L+X+, >^+^4^ >X4w^Z homeostaza >r4T4: >x+x«£ úbytek buněk Vliv narušení (stimulace/inhibice) průběhu apoptozy v rámci procesu vícestupňové karcinogeneze stimulace apoptozy oprava poškozeni U 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 eel! 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 > (jšsji&}jijä£ň WBBJHHgjEl HHj I ľ^JJa^'- ^^B R^ \£* *^B;.:,v' V- ! ■bS äEISSSi ^^P^ ^^^5 Fig. 4—Analysis of DNA I'ragmenlalion of apoptosis from three cell lines, in) HL-60 cells, exposed to camptothecin (Oó^m). Lanes; M = marker lane containing či 1 kb ladder of DNA fragments from 0 5 to 12'Okb; l=controh time 0; 2=+ camptotherin, time 6h; 3 =+campTotherin, lime 12 h;4=+camptothecin, time 24 h; 5=+campiotheciii. time 30 h; 6= + camplothecin. lime 48 h. fb} HL-60 cells exposed to EPA (IQQjiMl Lanes: M=marker lane; 1= control, time 6 h: 2 =-EPA, lime 6h:3 = + EPA. time 12 h; 4=^ĽPA, time 24 h; 5 = + EPA, time 30 h; 6=+ EPA, time 49 h: 7 = control, time 49 h. (e) e-wv;iLKv »Jl-X"^ 'í (IBVMtriA |1I -f "I 1-----------f (pmifrgfl~ N__J rcifTľci.x nm] ____l___ Hl.ti j= 8 riíii-rť <■ jy~y t tawp ; •- -.'."."i J,"; /—* ..■íl t. [LISI - _ »j jKMitiZ ^3 rnlTni *7—J 1 ■-------------------- |bmiri4 j LfOfSttuďT i.: 22 j] |^_rl>{lľvPK | I -2 ]"ilřj Lf^tKfľ^ i^.a=ll ľ-p-*p[.IM (ľ-M»e . .■■ ]| } *''■ J M"J kí A * *m tuláci lo-pi i; -"Hl 72 ■ ,. |* pni tlť i i^Tiroasij i ■. L-i^fr^i-gD diiiTii-Vihr1' i'ypC^J" il^iÜ p -*MM.-aJ- t>*pj P0T4] napu L0.551 —>JXPOPŤOŠŤŠ Molekulárni interakční mapa drah spojených s apoptózou, u nichž byly pozorovány rozdíly v genové expresi. Molekuly podporující apoptózu - červená Molekuly suprimující apoptózu - zelená Exprese mRNA se mění očekávaným směrem - žlutě, opačným směrem - modi Fas. FasL*------ p53------* Baxt Noxa, pig3 T NF-kB-------+ Bclx, Bcl2 ------1 Mitochondria ctAPs.XIAP I— Diabo/Smac^^ c c Caspase-8 CZj Caspase-3, 7 V^ Caspase-9\—H Apaf- ] Apoptosis *------------------------ AIF *- Figurt: i. S'uKsihlť uťgtrtiTití point oť jmi ;tpop[oik signals ťrotn NF-kB IiiirÍEiMi, i open arrows) and i/xlrinsic (filled arrows* j|*>plosLs pmlixv:iy< ;irt: tkpn k-ci "l"hc ťOťťlur Caspars, sftich ;tt ť;isp:isť^ Jnd t^p:istr-", !M?c ndivutcd hy (íp.sir-uMin iniikaior t^päs&jft ^ispu^e-ŕ* ütul ťJiŕipuse-V. Thť Initiator t;i\p:jst^. EhLTiiM-lves aťe iiťiiv;jlťd by t-itluT li^ilnd> híiuling to thť cíľluIi recťpti n* foniplťJí or t yUKÍlpyílrie Ľ ivk"isťd tínali ťtiiTui^cJ mirtxľl k hu triu. Art :inii iipopiock rfícvi or XF-kI1 i* :ichkrWiJ through iK up rvguíaiion JF-kB2 <5§D IkBÔ W Cylosql -------------- Nucleus 15 Ě> n___ 12 Ik5ci«<-----h 14 póSRelA i M NrF-*B c-Rel CR^T)-^^ <1uT) 6a H J >J >fr > ľ i» .- ■ .-r-- *-r 13 ■NF-kB IicBß*^ 16 -^>* 17 """i—5—'—T Reiß m kB IAP'u, C--PIJP Al/Bf]-1T Bcl-xL TRAF-1, TRAF-2 T APOPTOSIS NSAIDs NFkB target genes PPARS target genes Figure 2. (1) NSAIDs 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-regulatiom respectively. NSAIDs also inhibit the anti-apoptotic Bel- XL protein resulting in an increase in the ratio of pro-apoptotic BAX: Bcl-XL Oxidatívni stres jako mediator apoptózy Mnoho látek, které indukují apoptózu jsou buď oxidanty nebo stimulátory buněčného oxidativního metabolismu. Naopak řada inhibitorů apoptózy má antioxidační účink; ožne mechanis ► Bcl-2 protein (produkt bcl-2 onkogenu) - v mitochondriích tikulu a jaderné membráně - regulace ROS ivace poly-ADP-riboso-transferasy a akumulace p53 -polymerizace ADP-ribosy s proteiny vyúsťuje v rychlou HBSRrBEÍ iBTRI BaSiiiB HPE i v bun. ni( L po působení TNF a) ^iJíijiRiiKHt^jŤTr^fe ATP a smrt tory apoptc uwmmmmtKMjn^ EISI 3T«»l-myaiiimKi ► Aktivace genů odpovědných za apoptózu přes aktivaci speciJ 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 ■■ resnirační cvklus a katabolismus MK. Mn superoxid dismutasa x.o ROi ^relace mezi produkcí ROS mitochondriemi KMtKlRil íoxiaanta v mi Lální ^K^ Mikrosomální systém transportu elektronu (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 radť Mimobuněčné děje - oxidatívni vzplanutí aktivovaných makrofágu - NADPH- oxidáza -superoxid. Antioxidační obranný systén ► neenzymatický: molekuly jako vit E, vit C a glutation působící přímo na ► enzymatický: superoxid dismutáza (SOD), kataláza (CAT), GSH peroxi< ÍGSH-Px) a GSH S transferasa (GS™ * M mmm mĚm ■ OH* Hydroxy! radical o2 Molecular oxygen o2- Superoxide (fe^ H202 Hydrogen peroxide HOCI Hypochlorous acid Singlet oxygen Antibody o3 Ozone 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 hatide tons64; and ozone, which can be generated from singlet oxygen by antibody molecules1-^-1-10. 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 BOX 1. Hlavní komponenty antioxidační sítě v buňce NADPH NADP AOX AOX AOX" AOX' AOX GSH GSSG Boonstra and Post, Gene 2004 NADPH NADP* Fig. I. Schematic representation of the major players of the cellular anti-oxidanl network. The superoxide anion (0*~) is dismulaled by superoxide dismutase (SOD), present in mitochondria and the cylosol. The produced H202, which could give rise to the formation of the extremely noxious hydroxylradical, can he neutralized by catalase (in the peroxysomes) and by the cytosolic and mitochondrial glutathione peroxidase (GPx). The latter enzyme removes H202 by oxidizing glutathione (GSH) to GSSG, which is subsequently reduced to its original from by Glulathion Reductase (GR), at the expense of NADPH. A second form of GPx can reduce more complex hydroperoxides, such as lipid-hydroperoxides (LOOH). Low molecular weight antioxidants or scavengers, such as tocopherol, ascoľbale and glutathione, can neutralize radicals (foT instance the peroxylradical (LOO ) and other radicals (R )) and are often subsequently regenerated by one or more other antioxidants (AOX and GSH). Tocopherol (Toe) is an AOX that resides in cellular membranes (green cirele), whereas other AOXs, such as ascorbale and GSH, are located in the cylosol. For an extensive review of the cellular antioxidant network one is referred to H alii well and Gulterid^e, 1999. Nucleus NF^BÍ FÖS/JUN f Antioxidant enzyme (catalase, SOD) I ^ H202 02- í _ . -FeCu DMA damage Mutation Lipid peroxidation Protein damage Protease inhibitor Antioxidant t damage (ADF, GST-7C, GSH etc.) | 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.) maktivni produkty iniciace promoce progrese M y normální buňka iniciovaná preneoplastické neoplastické buňka buňky . buňky modifikace epigenetického působení (protizánětlivé, obnovení mezibuněčné komunikace, atd.) Oxidatívni stres Aktivace karcinogénu Poškození DNA: změny struktury a mutace genů Inhibice mezibuněčné komunikace Trvalý oxidatívni stres Abnormální genová exprese Abnormální enzymatická aktivita Rezistence k chemoterapii Buněčná proliferace Dědičné mutace Expanze klonů Metastáze a invazivita Iniciační stádium —* Stádium promoce Stadium progrese c^ " t i ^ T T 1 NO, ROS, ONOO- 1 Plasma membrane Oo or Oo- Nitrosation Oxidation Nitration Metal complexes Protein kinases SAPK, p38, JAK, ERK Protein phosphatases MKP-1, PTP RAS Nucleus Transcription factors NFkB, AP-1, C/EBP, Sp-1, RXR yjrurunup* 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, OA/OO" peroxynitrite, 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 J, NOX3 and NOX 4 are similar in size and domain structure to the well-studied gp91 phox, 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 residues, four of which provide binding sites for two haems. Haem is an iron-containing prosthetic group found in enzymes, efectron transfer proteins and oxygen-binding pigments such as haemoglobin. The řnon in haems is capable of undergoing reduction and re-oxidation, 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 for the co-enzym e s flavin adenine dinucleotide (FAD) and NADPH. The NOX enzymes catalyse the NADPH-dependent reduction of oxygen to form superoxide, which can react with itself to form hydrogen peroxide fH2Oz). For gp91 phox, the H202 serves as a substrate for myeloperoxidase (MPG), but it is not known whether other WOX enzymes provide H202 for separate peroxidase enzymes. NOX5 contains the same gp91phox-like catalytic corer plus an amino-terminal calcium-binding domain. The dual oxidase (DUOX) enzymes build on the WGX5 structure by adding at the amino terminus a n extra transmembrane a-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 cone 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 Necrosis 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 r.w.vtui Gene 2004 ^" necrosis Signal transduction ^ /-vCELLS ( ) UPPER TRANSIT-AMPLIFYING Q BASAL CELL CARCINOMA CELLS SQUAMOUS CELL CARCINOMA 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. The most primitive cell is in the bulb of the hair follicle, and the most differentiated cell is the terminally differentiated keratinized 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 surrounded by a "field" of morphologically altered cells. These celh are believed be changed by mutation or loss of a geue such as p53, which produces abnormalities in proliferation. It is postulated that a second mutation, such as in c-myc^ then leads to malignant transformation. Úloha metabolismu AA 11) flllltHt) ill BEumwiiii echodná v normální tkáni, mmvm 212^2*3 /ch místecl kon, ► d neo. niníran] i v ras o: K Kí IM X L/uňky i některé autokrinně stimulované faktory jako např. TGFoc, 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 ~ '~~ 12- lipoxygenáza. 12 (UUXH^^^H HEPATOKARCINOGENEZE Játra - klidový orgán s velmi nízkou bazálni hladinou replikace DNA. V odpověď na specifické stimuli reagují rychlou proliferací zprostředkovanou pravděpodobně novou expresí genů. in. cyklu, působení růst. faktorů HGF(hepatocyte growth f.) a EGF(epidermal growth f.) Kre* O •/ A A AAA ^ resekce, částečné hepatektomie nebo po působení toxických h Negenotoxické karcinogény např. peroxisom. proliferátory (PP) nebo phenobarbital ■ přímý mitotický stimul in vivo stimulující asi 30% buněk během 48h. Ke stimulaci dochází i u hepatocytu 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 - 1 1 • 1 • ^T \/ r 1 1 11 1 \/1 M* v«vM o 1 r 1 1 r Á polyploid] Negenotoxické karcinogény působí u hepatocytu též supresi apoptózy. Poškozené buňky pak persistují v populaci a po dalším mitogenním působení negenotox. karcinogénu z nich mohou vznikat nádo] 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 človv Proliferace hepatocytu může být zprostředkována cytokiny TNFoc a IL-6 - přechod G0/G1 Hepatocyte growth factor (HGF), epidermal growth f. (EGF) a TGFoc - 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 Mü5a8 'vace bi iJTATj i §i\ ma rei ti; Éfnm^mwttímnmtiR zni' a apoptoza). Další stupeň hepatocelulární adenomy a karcinomy. HEMATOPOIETIC STEM CELL O CD-ETHIONINE/AAF PERIDUCTULAR STEM CELL SOLT-FARBER "BIPOLAR" DUCTU L AR PROGENITOR CELL r? DIETHYLNITROSAMINE •^XHEPATOCYTE HEPATOCELLULAR CARCINOMA Fig. 8. Postulated stages of the liepatocytic Unease that may respond to liver injury or carcinogenic protocols. Following various models of liver injury or chemical hepatocarcinogenesis different cell types in the liepatocytic lineage may respond: 1 Undifferentiated periductular oval cells, which may arise from circulating bone marrow precursor cells. 2. Periductular cells intrinsic to the liven 3. Bipolar ductal progenitor cells, or 4. Mature hepatocytes. which retain the potential to divide. Periductular cells respond to periportal injury induced by allyl alcohol or to choline deficiency-ethionine carcinogenesis. Bipolar ductal progenitor cells respond to injury and to carcinogenic regimens, such as the Solt-Farber model, when proliferation of hepatocytes is inhibited. Hepatocytes respond to partial hepatectomy and to carcinogensis by dietliylnitrosamine (DEN) (from [132,315]). CHOLANGJOF1BROSIS CHOLWGíOCARCINOMA 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 mutations 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 RXR Fatty acids (PGJ2, LTB4) iPPREk/VH Target genes Transcription Proliferation CELL SPECIFIC RESPONSES 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 PPARy) 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? ppApl Wmk :■: ■: I ■: w<\ ■: -V :iXXl CoAct CoRep PPÄRI RXR PPRE PARs fungují jako heterodimery s jejich obvyklým partnerem - retinoidním receptorem (RXR). Ppar« activation Kupflteľ cells Rodent liver d Mechanism Increased DNA replication Increased proliferation Decreased apoptosls Reactive ajcygen species (DNA damage: proliferation) b Short-term response Transcriptional activation of genes that are Involved In fatty-acid metabolism. In the cell cycle and In degradation of endogenous and exogenous compounds (cytochrome p450fiamlly) Peroxisome proliferation Cell proliferation Liver 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 receptor-« (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-binding 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 proliferators (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 Arachidonic ad