Effect of indomethacin on E-cadherin and β-catenin expression in HT-29 colon cancer cells

Division of Molecular Medicine, Ruder Bosković Institute, Bijenicka c. 54, HR-10000 Zagreb, Croatia.
Experimental and Molecular Pathology (Impact Factor: 2.71). 03/2006; 80(1):91-6. DOI: 10.1016/j.yexmp.2005.04.008
Source: PubMed


Nonsteroidal anti-inflammatory drugs (NSAIDs) lower the incidence of and mortality from colon cancer. Although there is much evidence from epidemiological and laboratory studies that NSAIDs have antitumor activity and reduce the incidence of colon cancer, the mechanism of action remains unknown. In this paper, we present the effect of indomethacin on growth inhibition, induction of apoptosis, and alterations in the expression of several genes involved in Wnt signaling in HT-29 colon cancer cells. We have shown that indomethacin reduces the proliferation rate of HT-29 colon cancer cells and induces apoptosis. Concentrations of indomethacin from 10(-4) to 10(-3) M strongly inhibited the growth of HT-29 cells. The inhibition of growth, as well as induction of apoptosis was dose and time dependent. The treatment of cells with 4 x 10(-4) M indomethacin caused strong inhibition of cell growth (about 70%), enhanced expression of APC, decreased expression of beta-catenin and induced expression of E-cadherin proteins. Expression of beta-catenin was not markedly reduced instead, beta-catenin was translocated from the nucleus and cytoplasm to the plasma membrane. These results were confirmed by real-time RT-PCR analysis on mRNA level. At a concentration of 4 x 10(-4) M indomethacin there was increased expression of APC gene (10.9-fold induction; DeltaDeltaCt = 3.43) and E-cadherin gene (3.5-fold induction; DeltaDeltaCt = 1.79). These results suggest the antiproliferative effect of indomethacin may contribute to enhanced cell adhesion through increased expression of E-cadherin and translocation of beta-catenin from the nucleus to the cell membrane.

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    • "However, the observation of similar expression changes in three unrelated coxP CRC cell lines suggests the WNT pathway may be a universal target of COX-2 inhibition by APHS. In support of our results, increasing evidence indicates a negative effect of various NSAIDs on WNT signaling both in vitro and in vivo (Brown et al 2001; Kishimoto et al 2002; Williams et al 2003; Boon et al 2004; Maier et al 2005; Roy et al 2005; Kapitanovic et al 2006; Rao et al 2006). "
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    ABSTRACT: Cyclooxygenase-2 (COX-2), the prostaglandin (PG)-synthesizing enzyme overexpressed in colorectal cancer (CRC), has pleiotropic, cancer-promoting effects. COX-2 inhibitors (CIBs) interfere with many cancer-associated processes and show promising antineoplastic activity, however, a common mechanism of CIB action has not yet been established. We therefore investigated by microarray the global response towards the CIB APHS at a dose significantly inhibiting the growth of three COX-2-positive CRC but not of two COX-2-negative cell lines. None of the genes significantly (p = 0.005) affected by APHS were common to all three cell lines and 83% of the altered pathways were cell line-specific. Quantitative polymerase chain reaction (QPCR) on selected pathways confirmed cell line-specific expression alterations induced by APHS. A low stringency data analysis approach using BRB array tools coupled with QPCR, however, identified small expression changes shared by all COX-2-positive cell lines in genes related to the WNT pathway, the key driver of colonic carcinogenesis. Our data indicates a substantial cell line-specificity of APHS-induced expression alterations in CRC cells and helps to explain the divergent effects reported for CIBs. Further, the shared inhibition of the WNT pathway by APHS suggests one potential common mechanism behind the antineoplastic effects of COX-2 inhibition.
    Targets & therapy 07/2008; 2(2):329-37. DOI:10.2147/BTT.S2663
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    ABSTRACT: Background: In the previous study it was shown that films prepared from inulin (In) in combination with Eudragit RS (ERS) and RL (ERL) were susceptible to inulinase. Purpose: The aim of this work was to assess the suitability of these combinations for colonic delivery of indomethacin. Methods: Indomethacin was loaded onto non-pareil seeds using fluidized bed apparatus to produce pellets with 20% w/w drug load. Drug loaded pellets were coated with In-ERS in the ratios of 20:80 and 30:70, or In-ERL in the ratio of 20:80 to different coating loads. The release of drug was examined in simulated gastric (for 2 hrs) and small intestine and in the presence of inulinase in simulated colonic medium (for 12 or 24 hrs). Results: The results of this study revealed that incorporation of inulin as a bacterially degradable polysaccharide into ERS or ERL could modulate drug release. Coating level up to 15% significantly affected drug release from In-ERL or In-ERS coated pellets. However further increase in coating load to 20% had no significant effect on drug release from In-ERL coated pellets (f1=9.39). Drug release from In-ERL coated pellets was faster and showed some pH dependency. Conclusions: Formulation coated with In-ERS (20:80) and coating level of 20% was considered more appropriate for colon delivery of indomethacin, as drug release was pH independent and formulation was resistant to drug release in the upper GI media for up to 7 hrs. This formulation was also susceptible to inulinase and released about 40% of indomethacin in the simulated colonic media.
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    ABSTRACT: Colorectal cancer is among the major cancers and one of the leading causes of cancer-related deaths in Western societies. Its occurrence is strongly affected by environmental factors such as diet. Thus, for preventative strategies it is vitally important to understand the mechanisms that stimulate adenoma growth and development towards accelerated malignancy or, in contrast, attenuate them to remain in quiescence for periods as long as decades. The main objective of this study was to investigate whether diet is able to modulate β-catenin signalling related to the promotion or prevention of intestinal tumourigenesis in an animal model of colon cancer, the Min/+ mouse. A series of dietary experiments with Min/+ mice were performed where fructo-oligosaccharide inulin was used for tumour promotion and four berries, bilberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), cloudberry (Rubus chamaemorus) and white currant (Ribes x pallidum), were used for tumour prevention. The adenomas (Apc-/-) and surrounding normal-appearing mucosa (Apc+/-) were investigated separately due to their mutational and functional differences. Tumour promotive and preventive diets had opposite effects on β-catenin signalling in the adenomas that was related to the different adenoma growth effects of dietary inulin and berries. The levels of nuclear β-catenin and cyclin D1 combined with size of the adenomas in the treatment groups suggests that diets induced differences in the cancerous process. Adenomas progressing to malignant carcinomas are most likely found in the sub-groups having the highest levels of β-catenin. On the other hand, adenomas staying quiescent for a long period of time are most probably found in the cloudberry or white currant diet groups. The levels of membranous E-cadherin and β-catenin increased as the adenomas in the inulin diet group grew, which could be a result of the overall increase in the protein levels of the cell. Therefore, the increasing levels of membranous β-catenin in Min/+ mice adenomas would be undesirable, due to the simultaneous increase in oncogenic nuclear β-catenin. We propose that the decreased amount of membranous β-catenin in benign adenomas of berry groups also means a decrease in the nuclear pool of β-catenin. Tumour promotion, but not the tumour prevention, influenced β-catenin signalling already in the normal appearing mucosa. Inulin-induced tumour promotion was related to β-catenin signalling in Min/+ mice, and in WT mice changes were also visible. The preventative effects of berries in the initiation phase were not mediated by β-catenin signalling. Our results suggest that, in addition to the number, size, and growth rate of adenomatous polyps, the signalling pattern of the adenomas should be considered when evaluating preventative dietary strategies. Ruoka vaikuttaa terveyteemme. Syömällämme ruoalla on suuri merkitys erilaisten elämäntapasairauksien puhkeamiseen ja jopa syövän kehitykseen. Paksusuolisyöpä on yksi yleisimmistä syövistämme aiheuttaen valitettavan paljon syöpäkuolemia. Sen synnyssä elämän aikaisilla ruokavalinnoilla tiedetään olevan suuri merkitys, niin että tavallisimmat länsimaiset paheet, kuten runsas energian saanti yhdistettynä runsaaseen kovan rasvan, punaisen lihan, sokereiden ja alkoholin käyttöön lisää paksusuolisyövän riskiä. Toisaalta taas runsas kuitupitoisten viljojen, kasvisten ja hedelmien käyttö yhdistetään pienentyneeseen syövän riskiin. Ravinnon siis tiedetään vaikuttavan paksusuolisyövän kehitykseen, mutta tarkkaa selitystä tälle ei vielä osata antaa. Syövän ehkäisemiseksi olisi tärkeä löytää ravinnon suolistossa aiheuttamia muutoksia, jotka joko altistavat tai suojaavat syövältä. Olisi myös tärkeä ymmärtää syömämme ruoan vaikutus siihen, miksi jotkut kasvaimet muuttuvat nopeasti syöväksi, kun taas toiset kasvaimet pysyvät vuosikausia hyvänlaatuisina muuttumatta syöväksi. Syövän kehitys on monimutkainen prosessi, jossa solujen normaali kehitys muuttuu mm. siksi, että solut tulkitsevat ulkopuoleltaan tulevaa informaatiota väärin omien viestiketjujensa häiriintyessä. Solujen sisäisiä signalointireittejä tiedetään olevan huomattava määrä ja yksi selkeästi paksusuolisyövän kehitykseen liitetty on β-kateniini -signalointi. Marjo Misikankaan väitöskirjatutkimuksen tarkoituksena oli selvittää kokeellisella paksusuolisyöpämallilla voidaanko ravinnolla muuttaa suoliston solujen β-kateniini -signalointia. Signalointia tutkittiin sekä suoliston kasvaimista että näitä ympäröivästä normaalista kudoksesta. Ruokavalio muutoksella kasvaimien koko muuttui merkitsevästi verrattuna kontrolliryhmään. Kasvaimet kehittyivät suuremmiksi verrattuna kontrolliin, kun ruoka sisälsi 10 % inuliinia, jota käytetään monissa elintarvikkeissa kuidun lähteenä tai rakenteen muodostajana. Toisaalta 10 % lakkaa, puolukkaa tai valkoherukkaa sisältävä ruoka pienensi sekä kasvaimien lukumäärää että kokoa verrattuna kontrolliin. Kasvainten kasvua edistävä ja ehkäisevä ruoka aiheuttivat β-kateniini -signaloinnissa vastakkaisia muutoksia eli ruokavaliolla voitiin muuttaa suoliston solujen toimintaan ja sitä kautta mahdollista syöpäriskiä. Aktiivisimman β-kateniini -signaloinnin omaavien kasvaimien joukosta olisivat luultavimmin löytyneet syöväksi kehittyvät kasvaimet. Toisaalta lakka- ja valkoherukkaryhmän kasvaimet olisivat todennäköisimmin pysyneet hyvänlaatuisina pitkiäkin aikoja. Marjo Misikankaan väitöskirjatutkimuksen mukaan ruoka aiheuttaa selkeitä muutoksia suoliston solujen toimintaan ja sitä kautta muuttaa syöpäriskiä kokeellisessa paksusuolimallissa. Esimerkiksi suomalaisten marjojen käyttö ruokavaliossa näyttäisi olevan lupaava keino tulevaisuuden syövän ehkäisyssä. Etsittäessä ravintoperäisiä keinoja syövän ehkäisyyn tulisi kasvaimien lukumäärän, koon ja kasvunopeuden lisäksi entistä enemmän kiinnittää huomiota myös soluissa tapahtuviin signalointimuutoksiin.
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