Search

Additional file 1. contains the IUPAC name, chemical structures, NMR spectrums, free energy data, Crystal binding mode and ADME-T properities of the synthesized molecules.

Additional file 1. Supporting ResultsIC50 values, RNA-seq quality, optimal PCST networks and clustering and enrichment analysis of PCST generated networks are listed and figured in this additional file.

Additional file 1. The chemical properites of 5b-5u compounds. Figure S1-S23: Chemical structure, NMR and IR spectrums of all syntheszied compounds. Table S1: IC50 values (��M) of selected compounds on immortalized normal human epithelial breast cell line, MCF12A. Figure S24. Full images of blots represented in Figure 3B and 4C. Images are obtained with Odyssey�� CLx instrument using 700 nm (red) or 800 nm (green) channels.

Hepatocellular carcinoma (HCC) is resistant to both conventional and targeted chemotherapy due to its highly heterogenous nature. Activation of RTK pathways is an important signaling mechanism involved in the development and the progression of liver cancer stem cell (LCSC) population in response to the drug resistance. Therefore, it is crucial to identify novel targets and therapeutic molecules against HCC. This study focuses on the effects of two groups of small molecule inhibitors on liver cancer stem cells. First group of molecules involve a series of novel NSAID triazolothiadiazine derivatives. Following the bioactivity screening of 32 triazolothiadiazine derivatives on 9 different HCC cell lines, the most potent compound, 7b (IC50 = 0.2–1.0 μM) was identified to cause G2/M phase cell cycle arrest and apoptosis in Huh7, HepG2, Mahlavu, FOCUS, SNU475 and SNU387 cells. This was due to the reactive oxygen species (ROS) accumulation involving JNK protein activation. The upstream ASK1 and MKK7 and downstream c-Jun proteins were also involved in the actions of 7b. Furthermore, 7b treatment reduced the tumor size and prolonged the disease-free survival of nude mice significantly. Additionally, compound 7b inhibited liver cancer stem cell enrichment and sphere formation capacity of HCC cells compared to Sorafenib. The anti-tumor effects of compound 7b approves that this small molecule can be considered as an anti-cancer agent for liver cancer therapeutics. The second group of molecules involve 10 different PI3K/Akt/mTOR pathway inhibitors which were exploited as an alternative to clinically approved drugs Sorafenib and Regorafenib. We compared the bioactivities of small molecule PI3K/Akt/mTOR pathway inhibitors with Sorafenib, DNA intercalators and DAPT (CSC inhibitor) on CD133/EpCAM positive LCSCs. LCSCs were enriched upon treatment with Sorafenib, and DNA intercalators, but not with Rapamycin and DAPT. Differential expression analysis of 770 cancer pathway genes using network-based systems biology methods allowed us to associate IL-8 with LCSC enrichment. Upon IL-8 signaling inhibition with IL-8R inhibitor Reparixin, LCSC-sphere formation capacity and stemness-related protein levels in liver cancer cells were significantly reduced. This study indicates the involvement of autocrine inflammatory cytokines in hepatic CSC population survival under chemotherapeutic stress.

With the aim of achieving new compounds possessing both anti-inflammatory and antiplatelet activities, we synthesized (E)-3-[3-(pyridin-3/4-yl)-1-(phenyl/sulfonylmethylphenyl)-1H-pyrazol-4-yl]acrylamides, and evaluated their COX-1 and COX-2 inhibitory and antiplatelet activities. Since COX-2 inhibitory and antiplatelet compounds have anticancer potential, we also screened their antiproliferative effects against three human cancer cell lines. Compounds 5n, 5p, 5s, 10d, 10g and 10i were determined as dual COX-2 inhibitor/antiplatelet compounds. Compound 10h appeared to be a compound that exhibited antiplatelet activity without inhibiting the COX enzyme. Compounds 5h, 10a and 10i were the most effective derivatives which displayed antiproliferative activity against Huh7, MCF7 and HCT116 cells. Particularly, compound 10i, as the compound exhibiting the highest cytotoxic, antiplatelet and COX-2 inhibitory activity, was remarkable.

Hepatoselüler karsinom (HSK) kansere bağlı ölüm nedenlerinde en ön sıralarda yer almaktadır. Dünyada meydana gelen kanser ölümleri arasında ikinci sırada yer almaktadır. Heterojen yapısı ve agresif davranışı sebebiyle, kemoterapötik ilaçlara ve FDA onaylı, çoğalma ve anjiyojenez yolaklarını hedef alan, multikinaz inhibitörü Sorafenib ve Regorafinibe karşı dirençlidir. Sorafenibe karşı gelişen direncin arkasında yer alan mekanizmalar, PI3K/Akt/Mtor, JAK/STAT gibi dengeleyici yolakların aktivasyonu, epitelyal mesenkimal geçiş (EMG), mikroçevre ve kanser kök hücrelerinin varlığı olarak açıklanmaktadır. Karaciğer kanser kök hücreleri (KKKH) hasara uğramış ve değişime uğramış hepatik öncü hücrelerden meydana gelmişlerdir ve bu hücreler kemoterapi direnci, tümörün nüksetmesi ve metastazı gibi olaylardan sorumludurlar. Bu sebeple, PI3K/Akt/mTOR yolağı inhibitörleri, Sorafenib ve DNA interkalatörlerinin KKKH üzerindeki etkileri incelenmiştir. Bu inhibitörler ile muamele edilmiş HSK hücre hatlarındaki CD133+/EpCAM+ popülasyonu akış sitometrisi ile analiz edilmiş, KKKH'lere karşı etkili olabilen inhibitörler Sorafenib ile kombinasyon deneylerindeki potansiyelleri belirlenmek üzere test edilmiştir. Sonuçlar göstermiştir ki, Sorafenib veya DNA interkalatörleri ile muamele edilen hücrelerde KKKH'leri zenginleşirken, Rapamisin (mTOR inhibitörü), LY294002 (PI3K inhibitörü) CD133+/EpCAM+ popülasyonu oranını düşürüp KKKH zenginleşmesine engel olmuştur. Kombinasyon çalışmaları ile hücrelerin öncelikle Rapamisin ile daha sonra ise Sorafenib ile muamele edilmeleri ile , hem KKKH oranının hem de küre oluşturma kapasitlerinin yalnızca Sorafenib ile muamele edilen hücrelere göre azaldığı gösterilmiştir. İnhibitörlerin hücrelerdeki gen ifade seviyelerinde sebep oldukları değişimleri anlamak için, Nanostring nCounter teknolojisinden faydalanarak, kanser yolaklarını regüle eden genlerin analizi yapılmıştır. Cytoscape Score Flow algoritması uygulaması ile gerçekleştirilen sistemik yolak analizi, kök hücreleşme ile ilgili genlerdeki ifade farklılıklarının belirlenmesine yardımcı olmuştur. Rapamisin veya DAPT (Notch yolağı inhibitörü) ile muamele sonrası kök hücrelerin regülasyonunda yer alan genlerin (Wnt ve Notch yolakları) ifadelerinin azaldığı gösterilmiş, fakat Sorafenib ile muamele sonucunda bu yolaklardaki genlerin ifadelerinde farklılıklar (örn. JAG1 geninde artış) gözlemlenmiştir. İlginç bir şekilde, IL-8 ifadesi Sorafenib ile muamele sonucu artış göstermiş, fakat DAPT veya Rapamisin ile muamele sonucu düşüş göstermiştir. IL-8 yolağının inhibe edilmesi, hem KKKH oranında hem de HSK hücrelerinin sfer oluşturma kapasitelerinde azalmaya sebep olmuştur. Bu da IL-8 yolağının KKKH'ların kök hücre özelliklerini koruyucu bir rölü olduğunu işaret ediyor olabilir. Bu sebeple, IL-8 sinyal yolağının bloke edilmesinin HSK terapisi için umut verici bir yaklaşım olduğu belirtilmiştir.Bu tezde ayrıca, VEGFR2 tirozin kinaz (TK) inhibitörlerinin ve quinoidlerin karaciğer kanseri hücreleri ile beraber KKKH'lerini de inhibe edebilme potansiyelleri ile ilgili çalışmalara odaklanılmıştır. Sorafenib gibi diger VEGFR TK inhibitörleri çoğu kanser çeşidinin tedavisi için yaygın olarak çalışılmaktadır. Fakat, daha önce de belirtildiği üzere, klinik çalışmalar anti-VEGF veya anti-VEGFR terapilerinin farklı kanser türlerinde hastalığı stabile edip daha sonra ilerlemesine sebep olduğu bulguları içermektedir. Son yıllarda anti-anjiyojenik ajanların tümörde hipoksi oluşturarak kanser kök hücre popülasyonunu arttırdığı gösterilmiştir. Buna karşılık olarak, quinoidlerin hipoksik ortama duyarlı ve seçici aktif bileşiklerdir. Bu sebeple, bu çalışmadaki temel amacımız, bu iki grup bileşiklerin HSK hücreleri üzerindeki biyoaktivitelerini belirlemek ve aynı zamanda KKKH'lerinin zenginleşmesi üzerindeki etkilerini analiz etmektir. Sonuçlar göstermiştir ki, VEGFR2 TK inhibitörleri quinoidlere göre çok daha düşük konsantrasyonlarda sitotoksik etkilerini göstermektedir. Buna rağmen, VEGFR2 TK inhibitörlerinin KKKH hücrelerini zenginleştirdiği, quinoidlerin bazılarının ise bu oranı azaltabildiği gösterilmiştir. Bu veriler ile, bir bileşiğin daha agresif bir kansere sebep olma potansiyelini tanımlayan ve `agresiflik faktörü` adı verilen yeni bir kavram ortaya konulmuştur. Tezin son bölümünde ise pirazolik kalkon türevlerinin HSK hücre hatları üzerindeki biyoaktiviteleri ve etki mekanizmaları araştırılmıştır. Kalkonlar ve pirazolik yapılar anti-kanser özellikleri ile bilinen bileşikleridir. Yeni sentezlenmiş 41 adet pirazolik kalkon türevleri ilk önce farklı kanser hücreleri üzerinde test edilmiş, ve aralarından IC50 değerlerine bağlı olarak seçim yapılarak, bileşiklerin etkileri HSK hücre paneli üzerinde test edilmiştir. Sonuçlar, 39, 42, 49 ve 52 bileşiklerinin 5 µM dan küçük konsantrasyonlarda anti-proliferatif etkilerini göstermesi ile en etkili türevler olduğunu göstermiştir. Hücre döngüsü ve hücre ölümü mekanizmalarının araştırılması ile 42 ve 52 bileşiklerinin G2/M fazında areste ve apoptotik hücre ölümüne sebep olduğu ortaya çıkmıştır. Buna ek olarak, bileşikler iler muamele sonucunda p21, CDK1 ve fosforile-CyclinB1 gibi hücre döngüsünde yer alan proteinlerin seviyelerinin azaldığı gösterilmiştir. Anahtar Kelimeler: Hepatoselüler karsinom, PI3K/Akt/mTOR sinyal yolağı, Sorafenib, karaciğer kanseri kök hücresi, ilaç direnci, VEGFR2 TK inhibitörleri, quinoid, pirazolik kalkon türevleri, biyoaktivite, hücre döngüsü, apoptoz Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality, such that it the second most frequent cause of cancer death worldwide. Due to its heterogeneous composition and aggressive behavior, it is resistant to conventional therapies and also Sorafenib and Regorafenib which are FDA-approved multikinase inhibitors targeting pathways involved in angiogenesis and proliferation. The mechanisms behind the acquired resistance to Sorafenib were described as activation of compensatory pathways such as PI3K/Akt/mTOR, JAK-STAT, epithelial to mesenchymal transition (EMT), microenvironment and presence of cancer stem cells. Liver cancer stem cells originate from damaged and transformed hepatic progenitor cells (HPCs) which are found responsible for chemo-resistance, tumor relapse, and metastasis. For this reason, the effects of PI3K/Akt/mTOR inhibitors, Sorafenib and DNA intercalators on the enrichment of LCSCs were investigated. CD133+/EpCAM+ population from HCC cells were analyzed by flow cytometry after treatment with inhibitors, and effective inhibitors against LCSCs were further tested for their potential combinatorial effects together with Sorafenib. It was shown that upon treatment with Sorafenib or DNA intercalators the LCSCs were enriched, whereas Rapamycin (mTOR inhibitor), LY294002 (PI3K inhibitor) were able to inhibit the enrichment of LCSCs and reduced the CD133+/EpCAM+ population ratio. Combination studies revealed that when cells are treated initially with Rapamycin and then with Sorafenib, both the LCSC ratio and the sphere formation capacity of cells were reduced compared to cells treated with Sorafenib alone. To understand the alterations in gene expression induced by the inhibitors, a large panel of genes involved in regulation of cancer pathways were analyzed using Nanostring nCounter Technology. Systematic pathway analysis using Cytoscape Score Flow algorithm application allowed us to identify differential response genes involved in stemness. It was shown that genes involved in regulation of stem cells (Wnt and Notch pathway) were downregulated upon treatment with Rapamycin and DAPT (Notch pathway inhibitor), yet Sorafenib treatment resulted in differential regulation of these pathways, where JAG1 gene was found to be up-regulated. Interestingly, IL-8 expression was upregulated dramatically upon treatment with Sorafenib, but downregulated upon DAPT or Rapamycin treatment. Inhibition of IL-8 signaling resulted in reduction in both LCSC ratio and sphere formation capacity of HCC cells, which could be indicating the role of IL-8 signaling in the conservation of stemness features of LCSCs. For this reason, blockade of IL-8 signaling was suggested to be a promising therapeutic approach for HCC.Another topic in this thesis focuses on the potential of VEGFR2 TKIs and quinoids to inhibit both liver cancer cells and liver cancer stem cells. VEGFR TKIs such as Sorafenib, are widely studied for the treatment of many cancers, yet as mentioned above, there are many clinical studies providing the evidence that anti-VEGF or anti-VEGFR therapies lead to stable disease, which is then followed by disease progression in different cancer types. In recent years it has also been shown that antiangiogenic agents are increasing cancer stem cell population via generation of tumor hypoxia. Quinoids, on the other hand, are compounds that are selectively active in hypoxic conditions. Thus, the main aim of this study was to evaluate the bioactivities of compounds from each group on liver cancer cells and also to analyze their effects on the enrichment of LCSCs. Our results have shown that VEGFR2 TKIs were cytotoxic at lower concentrations compared to quinoids. However, it was shown that VEGFR2 TKIs are more likely to enrich LCSC population whereas some of the quinoids were able to reduce this ratio. With this information, a new concept called `aggressiveness factor`, which defines the potential of a compound to cause more aggressive cancer, was introduced. In the last part of this thesis, bioactivities of pyrazolic chalcone derivatives on HCC cell lines and their mechanism of action were investigated. Chalcones and pyrazolic structures are well known for their anti-cancer activities. Newly synthesized pyrazolic chalcone derivatives were tested against different cancer cells, and selection based on the IC50 values of compounds was made to analyze their effect on a panel of HCC cells. Results have shown that, compounds 39, 42, 49 and 52 were the most effective derivatives which had anti-proliferative activities in less than 5 µM concentrations. Further investigation of cell cycle progression and cell death mechanisms have revealed that compounds 42 and 52 caused cell cycle arrest at the G2/M phase and induced apoptotic cell death. Also, levels of cell cycle proteins, p21, CDK1, and phospho-CyclinB1 were shown to decrease upon treatment with these compounds. Keywords: Hepatocellular carcinoma, PI3K/Akt/mTOR, pathway, Sorafenib, liver cancer stem cells, drug resistance, VEGFR-TKI, quinoids, pyrazolic chalcone derivatives, bioactivity, cell cycle, apoptosis 193