Browsing by Subject "PI3K/Akt/mTOR"

Now showing 1 - 2 of 2

Open Access
Effects of PI3K/AKT/MTOR and VEGFR pathway inhibitors on liver cancer stem cells and bioactivities of novel pyrazolic chalcone derivatives on liver cancer
(2017-12) Kahraman, Deniz Cansen
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.
Open Access
Targeting PI3K/AKT/mTOR pathway identifies differential expression and functional role of IL8 in liver cancer stem cell enrichment
(American Association for Cancer Research, 2019) Kahraman, D. C.; Kahraman, Tamer; Çetin-Atalay, R.
Activation of the PI3K/Akt/mTOR pathway is an important signaling mechanism involved in the development and the progression of liver cancer stem cell (LCSC) population during acquired Sorafenib resistance in advanced hepatocellular carcinoma (HCC). Therefore, identification of novel therapeutic targets involving this pathway and acting on LCSCs is highly essential. Here, we analyzed the bioactivities and the molecular pathways involved in the action of small-molecule PI3K/Akt/mTOR pathway inhibitors in comparison with Sorafenib, DNA intercalators, and DAPT (CSC inhibitor) on CD133/EpCAM-positive LCSCs. Sorafenib and DNA intercalators lead to the enrichment of LCSCs, whereas Rapamycin and DAPT significantly reduced CD133/EpCAM positivity. Sequential treatment with Rapamycin followed by Sorafenib decreased the ratio of LCSCs as well as their sphere formation capacity, as opposed to Sorafenib alone. Under the stress of the inhibitors, differential expression analysis of 770 cancer pathway genes using network-based systems biology approach singled out IL8 expression association with LCSCs. Furthermore, IL8 secretion and LCSC enrichment ratio was also positively correlated. Following IL8 inhibition with its receptor inhibitor Reparixin or siRNA knockdown, LCSC features of HCC cells were repressed, and sensitivity of cells to Sorafenib increased significantly. Furthermore, inflammatory cytokines (IL8, IL1β, and IL11) were also upregulated upon treatment with HCC-approved kinase inhibitors Sorafenib and Regorafenib. Hence, chemotherapeutic stress alters inflammatory cytokine gene expression in favor of hepatic CSC population survival. Autocrine IL8 signaling is identified as a critical event, and its inhibition provides a promising complimentary therapeutic approach for the prevention of LCSC population enrichment.