Molecular pharmacology

Molecular pharmacology. energy. Normal cells produce ATP in the mitochondria through oxidative phosphorylation (OXPHOS), whereas under hypoxia, LDE225 Diphosphate glucose is converted to lactate LDE225 Diphosphate through glycolysis to produce ATP (Cairns et al., 2011; Kroemer and Pouyssegur, 2008). Glucose oxidation starts from your irreversible decarboxylation of glycolytic intermediate pyruvate to acetyl-CoA in mitochondria by pyruvate dehydrogenase complex (PDC), a large complex of three functional enzymes: E1, E2 and E3. PDC is organized around a 60-meric dodecahedral core created by dihydrolipoyl transacetylase (E2) and E3-binding protein (E3BP) (Hiromasa et al., 2004), which binds pyruvate dehydrogenase (PDH; E1), dihydrolipoamide dehydrogenase (E3) as well as pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP) (Read, 2001). PDH is the first and most important enzyme component of PDC that converts pyruvate to acetyl-CoA, which, along with the acetyl-CoA from your fatty acid -oxidation, enters the Krebs cycle to produce ATP and electron donors including NADH. Thus, PDC links glycolysis to the Krebs cycle and thus plays a central role in glucose homeostasis in mammals (Harris et al., 2002). Since PDH catalyzes the rate-limiting step during the pyruvate Bmp3 decarboxylation, activity of PDH determines the LDE225 Diphosphate rate of PDC flux. The current understanding of PDC regulation involves the cyclic phosphorylation/dephosphorylation of PDH catalyzed by specific PDKs and PDPs, respectively (Holness and Sugden, 2003). PDK1 is a Ser/Thr kinase that inactivates PDC by phosphorylating at least one of three specific serine residues (Sites 1, 2 and 3 are S293, S300, and S232, respectively) of PDHA1 while dephosphorylation of PDHA1 by PDP1 restores PDHA1 and subsequently PDC activity (Roche et al., 2001). The Warburg effect describes the observation that cancer cells take up more glucose than normal tissue and favor aerobic glycolysis more than mitochondrial oxidation of pyruvate (Kroemer and Pouyssegur, 2008; Vander Heiden et al., 2009; Warburg, 1956). An emerging concept suggests that the metabolic change in cancer cells to reply more on glycolysis may be due in part to attenuated mitochondrial function through inhibition of PDC. In consonance with this concept, gene expression of PDK1, in addition to diverse glycolytic enzymes, is upregulated by Myc and HIF-1 LDE225 Diphosphate in cancer cells (Kim et al., 2007; Kim et al., 2006a; Papandreou et al., 2006). Moreover, we recently also reported that diverse oncogenic tyrosine kinases (TKs), including FGFR1, are localized to different mitochondrial compartments in cancer cells, where they phosphorylate and activate PDK1 to inhibit PDH and consequently PDC, providing a metabolic advantage to tumor growth (Hitosugi et al., 2011). Here we report a mechanism where lysine acetylation of PDHA1 and PDP1 contributes to inhibitory regulation of PDC, providing complementary insight into the current understanding of PDHA1 regulation through the phosphorylation/dephosphorylation cycle. RESULTS K321 and K202 acetylation inhibits PDHA1 and PDP1, respectively Our recent finding that tyrosine phosphorylation activates PDK1 (Hitosugi et al., 2011) suggests an important role for post-translational modifications in PDC regulation. To examine the potential effect of lysine acetylation on PDC activity, we treated lung cancer H1299 cells that overexpress FGFR1 (Marek et al., 2009) with deacetylase inhibitors nicotinamide (NAM) and Trichostatin A (TSA) for 16 hours, which led to increased global lysine acetylation in cells without affecting cell viability (Figure S1A). NAM+TSA treatment resulted in decreased PDC flux rate in isolated mitochondria from H1299 cells (Figure 1A), suggesting alteration of global lysine acetylation levels leads to PDC inhibition in human cancer cells. Interestingly, multiple proteomics-based studies performed by our collaborators at Cell Signaling Technology (CST) identified key components of PDC including PDHA1 ( and PDP1 (, but not PDK1 (, as acetylated at a group of lysine residues in human cancer cells. To test the hypothesis that lysine acetylation might directly affect PDHA1 and PDP1 activity, we incubated recombinant FLAG-tagged PDHA1 and PDP1 with cell lysates from NAM+TSA treated H1299 cells. Such treatment results in increased lysine acetylation of PDHA1 (Figure 1B; test. The error bars represent mean.


EJ, CP, JM, CX and AT contributed to the design and completed experiments

EJ, CP, JM, CX and AT contributed to the design and completed experiments. DMSO controls are shown, with SEM represented by error bars and individual experimental repeats plotted. A representative western image of this result is also shown (assessments were performed, and values for the SR10067 differences between cell lines are shown. (Scale bars?=?250?m (inset image scale bars?=?50?m) Using multicellular spheroids of the MCF7 and MCF7-HER2 cell lines, we were able to compare the expression of hypoxia response proteins across a 3D cellular structure through immunohistochemistry. Once again, whilst the SR10067 expression of HIF-1 was comparable between cell lines, HIF-2 protein levels were significantly higher in the context of HER2 overexpression (values and Pearsons correlation to HER2 expression in the cell lines data set are shown HER2-overexpressing SR10067 breast cancer cell lines display increased sensitivity to HIF-2 inhibition Having established a role for HER2 overexpression in driving an exacerbated hypoxic response and the increased expression of HIF-2, we investigated whether HER2-positive cell lines were more sensitive to specific inhibition of HIF-2. The growth of MCF7 and MCF-HER2 cell lines was compared in response to HIF-2-specific knock-down by siRNA. Western blotting was used to confirm the HIF-2-specific effect of two siRNA treatments; a single siRNA targeting HIF-2 (siRNA #4) and a pool of four individual HIF-2 targeting siRNAs (SMARTpool siRNA). Both treatments reduced HIF-2 to less than 10% of the level seen in untreated cells, mock transfected cells or cells treated with non-targeting siRNA; no discernible effect on HIF-1 was seen (Fig.?7a). In addition, these siRNAs were also able to reduce the levels of HIF-2 induced by hypoxia to levels below the detectable limit in MCF7-HER2 cells (Fig.?7b). Transfection of MCF7 and MCF7-HER2 cell lines with these siRNAs in sulforhodamine B (SRB) growth assays performed in normoxia or hypoxia over 5?days demonstrated an increased sensitivity in the HER2-overexpressing cell line to HIF-2 knock-down (Fig.?7c). MCF7-HER2 cells showed reduced cell density after treatment with either HIF-2-specific siRNA in normoxia or hypoxia, whilst MCF7 cells were generally unaffected showing reduced cell density with just one of the NMYC siRNAs only in normoxia. MCF7-HER2 were significantly more sensitive to siRNA treatment than MCF7 cells in all treatment categories, indicating an increased dependence on HIF-2 in HER2-overexpressing cells in normoxia and hypoxia. Open in a separate window Fig. 7 HER2-overexpressing cell lines are more sensitive to HIF-2 inhibition. a Western blot showing siRNAs knock-down of HIF-2 in MCF7-HER2 in normoxia. SiRNa knock-down was performed with 25?M of four different siRNAs as well as 5C100?M of SMARTpool, combined siRNAs. Protein level was reduced to


J Virol

J Virol. unique heterodimeric receptor complex consisting of IFN- receptor 1 and the IL-10 receptor subunit 2 [9]. However, unlike the receptors for type I IFNs, which are broadly expressed on virtually all cell types, IFN-III receptors exhibit a more restricted tissue distribution [6, 10]. Because of the use of distinct receptors, types I and III IFNs likely do not signal identical biological outcomes in anti-viral and anti-cancer activities [6]. The activity of IFN- is highly prominent in barrier epithelia compared with other cell types [11]. In addition IFN- has lower toxicity than IFN- [12]. Interestingly IFN- has been recently shown to exert antitumor effects in both murine and human models. This has been shown to occur through direct effects on target tumor cells as well as through indirect-immune-mediated responses [13, 14]. Recently human intestinal enteroids (HIEs) that exhibit a similar cellular composition to Defactinib hydrochloride the intestinal epithelium have been established, and used to study viral epithelial interactions [15]. Using this model system Saxena have shown that rotavirus infection of human intestinal epithelial cells induces type III IFN as the dominant transcriptional response over type 1 IFN [16]. Such a conclusion was also reached by Pervolaraki [17] who state that type III IFN is the frontline of antiviral response in the human gut. Interestingly viroplasm-free dsRNA is present in the cytoplasm of rotavirus-infected cells and is a key intermediate in the replication cycle of many viruses, including other major human enteric viral pathogens [16, 18]. In this context, it is worth noting that the type III IFN response to rotavirus was also obtained using the dsRNA analog poly-IC [16]. Finally, it can be speculated that Defactinib hydrochloride human intestinal epithelial cells are programmed to respond to viral dsRNA with type III IFN [16]. Other experiments have shown that the dsRNA analog poly-IC induces crypt cell death in murine enteroids [19]. In the same way poly-IC Defactinib hydrochloride administered to mice induced intestinal epithelial cell death HSP70-1 within a few hours (3 to 6 h) [20]. Apoptotic deletion of infected epithelial cells translates into pathological cell shedding [21]. Taken together, these findings Defactinib hydrochloride strongly suggest that the dsRNA analog poly-IC is able to trigger a dual effect in normal intestinal cells, i.e. an immunoadjuvant effect represented by IFN- production and epithelial cell shedding. In this context, we hypothesized that human gastrointestinal carcinoma cells could maintain these dual functions upon intracellular treatment by the dsRNA analog poly-IC. Our aim was twofold: i) determine concomitantly both IFN- secretion and cell proliferation/shedding upon poly-IC treatment in several human gastrointestinal carcinoma cell lines; and ii) evaluate whether these two parameters are connected via a common pathway using NFB signaling as a probe. RESULTS Intracellular poly-IC induces IFN- production in human gastrointestinal cancer cell lines As shown in Figure ?Figure1A,1A, T84 cancer cells exposed intracellularly to Poly-IC produced huge amounts of IFN- in a time-dependent manner. The kinetics of IFN- production shows two phases: a steep rise in IFN- accumulation in the medium, significant at time point 6 h, peaking at 72 h and followed by a plateau up to 96 h. In addition, IFN- production was almost undetectable when T84 cells were treated with extracellular poly-IC for 72 h (Figure ?(Figure1B1B). Open in a separate window Figure 1 Intracellular Poly-IC elicits IFN- production in gastrointestinal cancer cell lines as measured by ELISA in culture supernatants(A) Time-dependent effect of intracellular Poly-IC on T84 cells. Proliferating T84 cells, maintained in 6-well plates, were treated for the indicated time points with 0.64 g/ml poly-IC in presence of Dharmafect (intracellular Poly-IC). Each symbol represents the mean sem of 3 experiments performed in triplicate. (B) T84 cells were incubated with extracellular (extra) or intracellular (intra) poly-IC (0.64 g/ml) for 72 h, or with medium (control) or vehicle alone (Dharm). Mean sem of 3 experiments performed in triplicate. (poly-IC intra vs Dharm: < 0.0001; poly-IC extra vs control: NS). (C) Gastrointestinal cell lines or Jurkat cells were treated with intracellular Poly-IC for 72 h. Mean sem of 3 experiments performed in triplicate. We then determined the kinetics of committment to IFN- production. To this end, a variable exposure time to poly-IC (3 h, 6 h, 9 h) was followed by replacement of the poly-IC-containing medium by fresh medium. The read-out of results was the determination of IFN- concentration at time point 72 h. These experiments showed that an exposure time of 3 h to.