Aldo keto redüktaz inhibitörü olan kuersetin türevi CHNQ maddesinin akr1b1 susturulmuş HCT-116 kolorektal kanseri hücrelerinde etkilerinin araştırılması.

Aldo-keto reductases (AKRs) are NAD(P)H dependent oxidoreductases that are known to be involved in the biosynthesis, metabolism and detoxification of a number of substrates including glucose. These enzymes are therefore implicated in the development of diabetic complications. Additionally, this family of enzymes, particularly AKR1B1, has been shown to be involved in pathology of inflammation- associated diseases such as atherosclerosis, asthma, uveitis, sepsis, arthritis, periodontitis and cancer, including colorectal cancer (CRC). To better understand the role of AKR1B1 in CRC, we selected the cell line HCT-116 that robustly expresses AKR1B1. Next, we either pharmaceutically inhibited AKR1B1 using 3,7-dihydroxy-2-[4-(2-chloro-1,4-naphthoquinone-3-yloxy)-3-hydroxyphenyl]- 5-hydroxychromen-4-one (CHNQ), a novel quercetin derivative and AKR1B1 inhibitor, or stably silenced the gene using shRNA. Contrary to our expectations, the cellular effects of CHNQ appeared to be unrelated to its inhibitory effects on AKR1B1; rather it had a significant pro-oxidant effect both in vitro and in vivo by virtue of its 1,4 napthoquinone moiety. Confirming this, we observed an upregulation of the ERK pathway, cell cycle arrest at G2/M phase, induction of apoptosis and decreased cell migration in HCT-116 cells. These effects were similar both in cells with a stable knockdown of AKR1B1 and control cells. On the other hand, we observed that silencing of AKR1B1 itself in HCT-116 cells resulted in decreased proliferation, delayed cell cycle progression, downregulation of the NF- κB pathway as well as impaired cell motility and migration. Taken together, this study provides new data that AKR1B1 may have oncogenic properties in CRC.M.S. - Master of Scienc

FEN BİLİMLERİ ENSTİTÜSÜ/LİSANSÜSTÜ TEZ PROJESİ

ALDO KETO REDÜKTAZ İNHİBİTÖRÜ OLAN KUERSETİN VE ONUN TÜREVİ CHNQ MADDELERİNİN AKRIBI SUSTURULMUŞ HCT-116 KOLON KANSERİ HÜCRELERİNDE ETKİLERİNİN ARAŞTIRILMAS

ROLE OF AKRIBI IN THE PATHOGENESİS OF COLORECTAL CANCER AKRIBI'IN KOLOREKTAL KANSERİ PATOJENEZİNDEKİ ROLÜ

ROLE OF AKRIBI IN THE PATHOGENESİS OF COLORECTAL CANCER AKRIBI'IN KOLOREKTAL KANSERİ PATOJENEZİNDEKİ ROL

FEN BİLİMLERİ ENSTİTÜSÜ/LİSANSÜSTÜ TEZ PROJESİ

ALDO KETO REDÜKTAZ İNHİBİTÖRÜ OLAN KUERSETİN VE ONUN TÜREVİ CHNQ MADDELERİNİN AKR1B1 SUSTURULMUŞ HCT-116 KOLON KANSERİ HÜCRELERİNDE ETKİLERİNİN ARAŞTIRILMAS

Aldo Keto Redüktaz İnhibitorü Olan Kuersetin ve Onun Türevi CHNQ Maddelerinin Karakterizasyonu ve Kolon Kanser Üzerine Fonksiyonel Etkilerinin Araştırılması

TÜBİTAK SBAG Proje01.07.2016Aldo-keto redüktazlar (AKR) glikoz indirgeyici ajanlar olarak çok iyi karakterize edilmiş ve diyabet patofizyolojisinde yer alan NAD(P)H’a bağımlı oksidoredüktazlardır. Bu enzimlerin, inflamatuar tepkiyi başlatabilen lipit peroksidasyon ürünlerinin üretimine aracılık yaptığı da bilinmektedir. Kolondaki kronik inflamasyon çoğunlukla kanser gelişimi ile devam etmektedir. AKR’lar inflamasyonda yer almaktadır ve son çalışmalar bu enzimlerin kanser ilerlemesindeki önemli rolünü ortaya çıkarmıştır. Bu projede, multidisipliner bir yaklaşım ile AKR1B1 ve yakından ilişkili AKR1B10’u hedefleyen doğal bir madde olan kuersetinin, yarı sentetik türevlerinin detaylı karakterizasyonu ve bunların kolon kanseri etiyolojisindeki çeşitli inflamatuar yolaklar üzerindeki etkilerinin araştırılması amaçlanmıştır. HCT-116 kolon kanseri hücre hattında, AKR1B1 geni başarılı bir şekilde susturulmuş, AKR1B10 geni de aşırı olarak ifade edilmiştir. AKR1B1 ve AKR1B10 enzimlerini ifade eden yaban tipi HCT-116 hücreleri, AKR1B1 ifadesi susturulmuş ve AKR1B10’u aşırı ifade eden HCT-116 hücreleri hücreler, bu iki farklı AKR enzimi ifadelediği bilinen ve yaban tipi HT-29 hücreleri ile birlikte CHNQ (3,7-dihydroxy-2-[4-(2-chloro-1,4-naphthoquinone-3-yloxy)- 3-hydroxyphenyl]-5-hydroxychromen-4-one) ile muamele edilmiştir. CHNQ muamelesi, bütün hücrelerde hücre canlılığını azaltmıştır. Bu durum, apoptozda artış ve G2/M fazında tutuklanmayla takip edilmiştir. Bütün bu etkiler, parental bileşik kuersetine kıyasla çok daha güçlü bir şekilde gerçekleşmiş olup, ilginç bir şekilde, AKR1B1’in susturulmasından veya AKR1B10 ‘un aşırı ifadesinden bağımsız olarak ilerlemiştir. Dolayısıyla, çalışmada, CHNQ’nun diğer etkileri yalnızca yaban tipi hücrelerde incelenmiştir. Öncelikle, CHNQ, 1,4 naftokinon yapısına sahip olduğu için, bu bileşiğin redoks sinyal yolaklarında görev alabileceği hipotez edilmiştir. Hipotez ile tutarlı olarak, reaktif oksijen türleri (ROT) seviyelerinde güçlü bir artış ile birlikte mitojen aktive protein kinaz (MAPK) sinyal yolağında artış gözlenmiştir. Aynı zamanda, CHNQ muamelesinden dolayı görülen ölümlerin ROS kaynaklı otofajiden dolayı olduğu in vitro olarak iki farklı kolon kanseri hücre hattında ve in vivo olarak S. cerevisiae maya hücrelerinde gösterilmiştir. Bunun ardından, AKR’lerin kolon kanseri üzerindeki kendi başına etkileri incelenmiştir. HCT116 hücrelerinde AKR1B1 susturulması, ROT üretiminde azalmaya sebep olmuş ve bundan dolayı hücre çoğalmasında, hücre hareketliliği ve migrasyonunda, MAPK ve NF-κB sinyal yolaklarında azalmaya ve hücre döngüsünde gecikmeye neden olmuştur. Bütün bu bulgular, AKR1B1 in kolon kanserinde onkojenik özelliklere sahip olduğunu işaret etmektedir. Öte yandan, HCT-116 hücrelerinde AKR1B0 ‘un aşırı ifadelenmesi hücre çoğalması ve hücre xii döngüsünde bir değişime sebep olmazken, NF-κB’nin nükleer translokasyonunda, DNA bağlanmasında ve transkripsiyonel aktivitesinde önemli azalmalar ile sonuçlanmıştır. Bulgularımız, AKR enzimlerinin ve bu enzimlerin inhibitörlerinin çeşitli fonksiyonlara sahip olduklarını göstermektedir. İleriye yönelik çalışmalar, kolon kanseri hasta örneklerinde, AKR enzimlerinin ifadelenmesinin, aktivitesinin ve altında bulunan sinyal yolaklarını anlamak yolunda ve muhtemel terapötik yaklaşımlar geliştirmeyi amaçlamaktadır.Aldo-keto reductases (AKR) are NAD(P)H dependent oxidoreductases that have been best characterized as glucose reducing agents, and have been implicated in diabetic pathophysiology. These enzymes are also known to mediate the generation of lipid peroxidation metabolic products that can induce an inflammatory response. Chronic inflammation in the colon is very frequently followed by the development of carcinogenesis. Recent studies have revealed that pharmacological inhibition or genetic ablation of these enzymes could prevent colon cancer. The project encompassed a multidisciplinary approach for the detailed characterization of naturally derived quercetin semi synthetic derivative that target AKR1B1 and the closely related AKR1B10 as well as their effects on various inflammatory pathways involved in the etiology of colon cancer. We have successfully silenced AKR1B1 and overexpressed AKR1B10 in the colon cancer cell line HCT-116. These cells, wild-type HCT-116 cells and wildtype HT-29 cells were treated with CHNQ (3,7-dihydroxy-2-[4-(2-chloro-1,4-naphthoquinone3-yloxy)-3-hydroxyphenyl]-5-hydroxychromen-4-one). CHNQ treatment resulted in a significant loss of viability in all cells tested. This was accompanied by an increase in apoptosis and cell cycle arrest at G2/M. All of these effects were induced far more strongly than the parental compound quercetin and, interestingly, were independent of the overexpression or silencing of AKRs. Therefore, we have examined the effects of CHNQ alone on wild-type colon cancer cells. Since CHNQ has a 1,4 napthoquinone structure, we hypothesized that the compound could be involved in redox signaling. We observed a robust induction of reactive oxygen species (ROS) along with an increase in signaling through the mitogen activated protein kinase (MAPK) pathway. Cell death from CHNQ treatment also resulted from ROS induced autophagy both in vitro in colon cancer cell lines and in vivo in S. cerevisiae cells. We next examined the effects of AKRs per se on colon cancer cell lines. Silencing of AKR1B1 in HCT-116 cells resulted in a decrease in the production of ROS, along with a decrease in proliferation, cell motility and migration, reduced MAPK and NF-κB signaling and a delay in cell cycle progression. All of these data pointed towards AKR1B1 having oncogenic properties in colon cancer cell lines. On the contrary, overexpression of AKR1B10 in HCT-116 cells resulted in no change in proliferation or cell cycle progression, however, a robust inhibition in the nuclear translocation, DNA binding and transcriptional activity of NF-κB was seen. These data point towards a tumor suppressive function of AKR1B10 in colon cancer. xiv Our data indicate diverse functions of AKRs and their inhibitors in colon cancer. Future studies are aimed towards understanding the expression, activity and downstream signaling pathways of AKRs in colon cancer patient samples and possible therapeutic approaches

CHNQ, a novel 2-Chloro-1,4-naphthoquinone derivative of quercetin, induces oxidative stress and autophagy both in vitro and in vivo

Quercetin (Qc) shows strong antitumor effects but has limited clinical application due to poor water solubility and bioavailability. In a screening of novel semi-synthetic derivatives of Qc, 3,7-dihydroxy-2-[4-(2-chloro-1,4-naphthoquinone-3-yloxy)-3-hydroxyphenyl]-5-hydroxychromen-4-one (CHNQ) could ameliorate acetic acid induced acute colitis in vivo more efficiently than Qc. Since inflammation contributes to colorectal cancer (CRC), we have hypothesized that CHNQ may have anti-cancer effects. Using CRC cell lines HCT-116 and HT-29, we report that CHNQ was three-fold more cytotoxic than Qc along with a robust induction of apoptosis. As expected from naphthoquinones such as CHNQ, a strong induction of oxidative stress was observed. This was accompanied by reactive oxygen species (ROS) induced autophagy marked by a dramatic increase in the lipidation of LC3, decreased activation of Akt/PKB, acidic vesicle accumulation and puncta formation in HCT-116 cells treated with CHNQ. Interestingly, an incomplete autophagy was observed in HT-29 cells where CHNQ treatment led to LC3 lipidation, but not the formation of acidic vacuoles. CHNQ-induced cytotoxicity, ROS formation and autophagy were also detected in vivo in Saccharomyces cerevisiae strain RDKY3615 (WinstonS288C background). Overall, we propose that CHNQ can induce cancer cell death through the induction of oxidative stress, and may be examined further as a potential chemotherapeutic drug