(C) mRNA expression of HCT(pTRAFNrf2/HIF/NFkB) reporter cells grown as spheroids

(C) mRNA expression of HCT(pTRAFNrf2/HIF/NFkB) reporter cells grown as spheroids. inhibitor 1B (p27); CSC, cancer stem cell; DMOG, dimethyloxalylglycine; GSH, glutathione; HIF, hypoxia inducible factor; MUC2, mucin2; NCL, nucleolin; NFE2L2 or Nrf2, nuclear factor (erythroid-derived 2)-like 2; NF-B, nuclear Hydroflumethiazide factor kappa-light-chain-enhancer of activated B cells; PHD, prolyl hydroxylase domain proteins; pTRAF, plasmid for transcription factor reporter activation based on fluorescence; ROSI, rosiglitazone; SCM, stem cell medium; XCT, cystine-glutamate exchange transporter strong class=”kwd-title” Keywords: Redox regulation, Cancer stem cells, Spheroids, Nrf2, HIF, NF-B Graphical abstract Open in a separate window 1.?Introduction Malignant tumors consist of a heterogenic mixture of cancer cells, and only a subset of undifferentiated tumor cells have clonogenic and tumor-initiating potential [1]. These cells are commonly termed cancer stem cells (CSCs) as they share many properties with normal adult and embryonic stem cells [2]. CSCs have unlimited self-renewal capacity, can differentiate asymmetrically, and are believed to drive the heterogeneous cell populations constituting a tumor. They are either slowly proliferating or fully quiescent, and are typically resistant to chemotherapy. Factors and conditions that either control maintenance of undifferentiated clonogenic CSCs or their differentiation into more mature cancer cells are Fam162a incompletely defined, but redox modulation is likely to be important. Several observations have shown that cancer cells in general have higher endogenous levels of oxidative stress than normal healthy cells [3], [4] and thus up-regulate their expression of antioxidant enzymes in order to achieve redox homeostasis and cell survival [5]. How the redox state of CSCs compares to more differentiated cells from the same original cancer cell clone is not known. Redox signaling pathways that are activated in response to growth factor stimulation are typically coupled to synthesis of H2O2 by NADPH oxidases, but also other sources of H2O2 might play a role together with peroxynitrite and lipid hydroperoxides. Many transcription factors are redox regulated, including NF-B, HIF, Nrf2, Oct-4, -catenin, Notch, and c-Myc. All of them are known to be important mediators of development Hydroflumethiazide and cellular differentiation, but also of cancer promotion [6], [7], [8]. NF-B is involved in cellular responses to inflammation [6]. Under basal conditions, NF-B is kept inactive in the cytosol by binding to IB, the inhibitor of NF-B. Upon activation, a phosphorylation cascade results in the degradation of IB and nuclear translocation of NF-B. In relation to colorectal cancer, elevated NF-B signaling enhances Wnt activation and can support tumor growth [9], [10]. Under conditions of constitutively activated Wnt signaling, Rac1-driven H2O2 production is also required for NF-B activation and initiation of colon tumorigenesis [11]. The HIF1 transcription factor consists of two subunits, HIF1 and HIF1 [12]. During normoxia, HIF1 is hydroxylated by prolyl hydroxylase domain proteins (PHD), allowing the recognition and ubiquitination of HIF1 by the Von Hippel-Lindau protein followed by proteasomal degradation. Upon hypoxia (O2 below 3%), PHDs are inactivated by a shift from Fe3+ to Fe2+ in their active center. HIF1 becomes stabilized and translocates to the nucleus, where it together with HIF1 induces HIF target genes involved in e.g. the adaptation to hypoxia, angiogenesis, glucose transport, survival and Hydroflumethiazide invasion. HIF1 is activated in many different types of cancers, mainly caused by the hypoxic core that develops when tumors grow bigger. For colorectal cancer, it has been shown that hypoxia promotes an aggressive CSC phenotype resulting in invasion and accelerated metastatic outgrowth [13]. During cell homeostasis, Nrf2 is bound to Keap1 and constantly degraded. Upon oxidative or electrophilic stress, Keap1 is modified, whereupon Nrf2 translocates to the nucleus to activate an array of antioxidant and detoxification enzymes, including important proteins of the glutathione (GSH) and thioredoxin systems [6], [14], [15]. Thus, Nrf2 provides host defense systems that can protect from cancer initiation through more efficient elimination of harmful substances. However, Nrf2 activation in cancer cells can accelerate malignant cell growth [16] and Nrf2 is typically activated in many tumors [17], [18]. In.