Pancreatic cancer (PaCa) is a fatal human cancer due to its exceptional resistance to all current anticancer therapies. The cytoprotective enzyme heme oxygenase-1 (HO-1) is significantly ...overexpressed in PaCa and seems to play an important role in cancer resistance to anticancer treatment. The inhibition of HO-1 sensitized PaCa cells to chemo- and radiotherapy in vitro. Therefore, we investigated the effects of HO-1 and its metabolites biliverdin, carbon monoxide and iron on PaCa cells. PaCa cell lines with divergent HO-1 expression patterns were used in a murine orthotopic cancer model. HO-1 expression and activity was regulated by zinc (inhibition) and cobalt (induction) protoporphyrin. Furthermore, the influence of cellular HO-1 levels and its metabolites on effects of standard chemotherapy with gemcitabine was tested in vivo and in vitro.
High HO-1 expression in PaCa cell lines was associated with increased chemoresistance in vitro. Chemoresistance to gemcitabine was increased during HO-1 induction in PaCa cells expressing low levels of HO-1. The inhibition of HO-1 activity in pancreatic tumors with high HO-1 boosted chemotherapeutic effects in vivo significantly. Furthermore, biliverdin and iron promoted PaCa resistance to chemotherapy. Consequently, specific iron chelation by desferrioxamine revealed profound anticancerous effects.
In summary, the inhibition of HO-1 and the chelation of iron in PaCa cells were associated with increased sensitivity and susceptibility of pancreatic tumors to chemotherapy in vivo. The metabolites biliverdin and iron seem to be involved in HO-1-mediated resistance to anticancer treatment. Therefore, HO-1 inhibition or direct interference with its metabolites may evolve new PaCa treatment strategies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background: It has been suggested that Crohn's disease (CD) is associated with an exaggerated T‐helper 1 cytokine response manifested by increased production of interleukin (IL)‐12. IL‐12 is a ...heterodimeric protein comprising 2 disulfide‐linked subunits designated p35 and p40. Recently, IL‐12‐related cytokines, IL‐23 and IL‐27, were described. Biologically active IL‐23 is a heterodimer whose p40 subunit is identical to IL‐12p40 whereas its p19 subunit is distantly related to IL‐12p35. IL‐27 consists of EBI3, an IL‐12p40‐related protein, and p28, a newly discovered IL‐12p35‐related polypeptide.
Aim: We sought to determine whether mucosal expression of IL‐23p19 and IL‐27p28 transcripts correlate with the inflammatory activity in inflammatory bowel disease (IBD).
Patients/Methods: Messenger RNA expression in colonic mucosa from patients with Crohn's disease (CD; n = 37) and ulcerative colitis (UC; n = 19), and in non‐IBD control subjects (specific colitis SC; n = 16) and normal, nondiseased control patients (n = 12) was measured by reverse‐transcribed real‐time polymerase chain reaction.
Results: IL‐23p19 was significantly increased in inflamed mucosa in CD (P = 0.0377) and to a lesser extent also in UC patients but not in SC patients. Elevation of IL‐23p19 transcript levels in CD correlated with the severity of endoscopic lesions. IL‐27p28 transcripts and EBI3 transcripts were significantly elevated only in active CD.
Discussion: IL‐23p19, IL‐27p28, and EBI3 transcripts are strongly up‐regulated in CD. The stimulatory effects of these cytokines on naive T cells in addition to a strongly synergistic action with IL‐12 to trigger interferon‐γ production may contribute to the perpetuation of the inflammatory process in patients with CD. Notably, increased expression of IL‐23 and IL‐27 transcripts in CD suggests a T helper 1‐dominated immunologic function in this disease.
The formation of supramolecular activation clusters within the immunological synapse, crucial for sustained signaling and T lymphocyte activation, requires costimulation-dependent reorganization of ...the actin cytoskeleton. Here we have identified the actin-remodeling protein cofilin as a key player in this process. Cell-permeable peptides that block costimulation-induced cofilin/F-actin interactions in untransformed human T lymphocytes impair receptor capping and immunological synapse formation at the interface between T cells and antigen-presenting cells. As a consequence, T cell activation, as measured by cytokine production and proliferation, is inhibited.
Cofilin, an actin‐depolymerizing protein, is essential for the functional dynamics of the actin cytoskeleton and for cell viability. In unstimulated human peripheral blood T lymphocytes cofilin is ...phosphorylated and localized in the cytoplasm. Following co‐stimulation through accessory receptors (e.g. CD2 or CD28) – however, not following TCR/CD3 stimulation alone – cofilin undergoes dephosphorylation. The subcellular localization as well as the actin‐binding activity of cofilin are regulated by the phosphorylation state of serine‐3. Thus, only the dephosphorylated form of cofilin associates with the actin cytoskeleton and possesses the capability to translocate into the nucleus. Recently, LIM‐kinase 1 was shown to inactivate cofilin through phosphorylation. Here, we have identified the functional counterparts of LIM‐kinase 1: the serine/threonine phosphatases of type 1 and type 2A not only associate with cofilin but also dephosphorylate this 19‐kDa protein and thereby mediate cofilin activation. In malignant T lymphoma cells, activation of these phosphatases occurs spontaneously, independent of external stimuli. In untransformed human peripheral blood T lymphocytes, these phosphatases function through a cyclosporin A/FK506‐resistant co‐stimulatory signaling pathway which is common for the accessory receptors CD2 and CD28. This co‐stimulatory signaling pathway is also not affected by a series of other clinically established immunosuppressive drugs (i.e. rapamycin, dexamethasone, leflunomide or mycophenolic acid).
The potential advantage of using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) methodology to detect metastasis in sentinel lymph nodes (SLNs) of breast cancer (BC) patients ...was evaluated in this prospective study. We measured the expression of relevant gene transcripts in SLNs using an innovative algorithm and compared the results of single-marker assays versus multi-marker assays with conventional histological detection methods. SLNs from women aged ≥18 years diagnosed with unilateral BC were examined by haematoxylin-eosin staining and immunohistochemistry and analysed for transcripts of several relevant genes using qRT-PCR (learning group). Four candidate panels of expressed transcript combinations with high sensitivity and specificity were selected for further investigation. The candidate panels were then validated using SLNs from a second group of BC patients (validation group). In the learning group, 74/314 SLN sections from 150 patients were positive for metastasis by histology. The transcripts analysed showed the following individual sensitivities/specificities: cytokeratin 19 (CK19) 94.6%/97.9%; mammaglobin 1 (MGB1) 82.4%/91.7%; mammaglobin 2 (MGB2) 82.4%/96.7%; carcinoembryonic antigen (CEA) 71.6%/97.5%; EPCAM (epithelial cell adhesion molecule) 91.9%/97.1%; and NY-BR-1 82.4%/93.8%. The optimal panel based on the predefined criteria comprised four markers: CK19, MGB1, EPCAM, and NY-BR-1, of which ≥2 had to be positive (95.9% sensitivity, 95.0% specificity, 85.5% positive predictive value (PPV), and 98.7% negative predictive value (NPV)). Overall concordance with histology was 95.2%. In the validation group, 84/315 SLN sections from 235 patients were histologically positive, and panel sensitivity, specificity and overall accuracy were 88.1, 95.2 and 93.3%, respectively, at the SLN section level. In conclusion, molecular staging using expression patterns of relevant transcripts in SLNs could serve as a useful complement to standard diagnostic work-up in BC patients. The proposed flexible multi-parametric approach does not improve the overall accuracy compared with the single-marker approach. However, it overcomes several limitations of the previously reported molecular assays for SLN diagnosis.
A dysregulated secretion of contra-inflammatory cytokines such as interleukin-10 (IL-10) could play a role in the pathogenesis of inflammatory bowel disease (IBD). We have investigated the expression ...of IL-10 in gut tissues from patients with Crohn's disease (CD), ulcerative colitis (UC) and controls by mRNA
in situhybridization and immunohistochemistry. Intestinal epithelial cells were found to express IL-10 mRNA and IL-10 protein in all of the tissues investigated without any major differences in the expression patterns. However, compared with noninflamed gut, significantly increased numbers of mononuclear cells (MNCs) producing IL-10 were present in inflamed gut, both in CD and UC. This cytokine was expressed most prominently by inflammatory infiltrates enriched in macrophages, although T cells seem to contribute to its production as well. Elevated IL-10 expression in IBD was mainly detected in the submucosa, whereas IL-10 production by lamina propria cells remained comparably low. In contrast, the expression of IL-1β mRNA was preferentially increased in the lamina propria. Our data argue against a general deficiency in IL-10 production in IBD. The results suggest rather that the local production of IL-10 by mucosal MNCs in IBD is insufficient to down-regulate pro-inflammatory cytokines such as IL-1β in the lamina propria compartment.
Immunoregulatory properties of cytokines may contribute to pathological immune reactions in inflammatory bowel disease. There is an urgent need for a simple and dependable means for quantitating ...inflammatory activity in mucosal biopsies and assessing relapse risk particularly in patients with active Crohn's disease (CD).
Cytokine and chemokine transcripts were quantified using real-time PCR in mucosal biopsy specimens from 70 patients with active inflammatory bowel disease (CD, n=45; ulcerative colitis n=25) and 16 patients with specific colitis (ischemic colitis, infectious colitis). Controls were 12 patients with noninflammatory conditions. CD patients with steroid-induced remission (n=20) were followed for up to 12 months.
Compared to not-inflamed mucosa the vast majority of active CD tissue samples expressed significantly elevated transcript levels of IL-1beta, IL-8, IL-23, MRP-14, MIP2alpha, and MMP-1. Moreover, increased cytokine transcript levels were detected in both active ulcerative colitis and specific colitis. Importantly, TNF-alpha, IFN-gamma, CD40L, and IL-23 transcripts increased in active CD only. Transcript levels (MRP-14, IL-8, MMP-1, MIP2alpha) were correlated with clinical disease activity (CDAI) and endoscopic scoring indices. Medical treatment induced stable remission in 14 of 20 patients which was paralleled by a reduction in increased transcript levels. All six patients without normalization of MIP2alpha, MRP-14, TNF-alpha, and IL-1beta transcripts developed an early relapse (n=5) or chronic activity (n=1) during follow-up.
Elevated proinflammatory cytokine transcripts in active CD may underlie disease reactivation and chronicity. Real-time PCR quantification is a simple and objective method for grading inflammation of intestinal mucosa and may be useful in identifying patients who would benefit from anti-inflammatory remission maintenance.
Introduction
Hyporesponsiveness of human lamina propria immune cells to microbial and nutritional antigens represents one important feature of intestinal homeostasis. It is at least partially ...mediated by low expression of the innate response receptors CD11b, CD14, CD16 as well as the cystine‐glutamate transporter xCT on these cells. Milieu‐specific mechanisms leading to the down‐regulation of these receptors on circulating monocytes, the precursor cells of resident macrophages, are mostly unknown.
Methods
Here, we addressed the question whether the short chain fatty acid n‐butyrate, a fermentation product of the mammalian gut microbiota exhibiting histone deacetylase inhibitory activity, is able to modulate expression of these receptors in human circulating monocytes.
Results
Exposure to n‐butyrate resulted in the downregulation of CD11b, CD14, as well as CD16 surface expression on circulating monocytes. XCT transcript levels in circulating monocytes were also reduced following exposure to n‐butyrate. Importantly, treatment resulted in the downregulation of protein and gene expression of the transcription factor PU.1, which was shown to be at least partially required for the expression of CD16 in circulating monocytes. PU.1 expression in resident macrophages in situ was observed to be substantially lower in healthy when compared to inflamed colonic mucosa.
Conclusions
In summary, the intestinal microbiota may support symbiosis with the human host organism by n‐butyrate mediated downregulation of protein and gene expression of innate response receptors as well as xCT on circulating monocytes following recruitment to the lamina propria. Downregulation of CD16 gene expression may at least partially be caused at the transcriptional level by the n‐butyrate mediated decrease in expression of the transcription factor PU.1 in circulating monocytes.
Hyporesponsiveness of human lamina propria immune cells to microbial and nutritional antigens represents one important feature of intestinal homeostasis. The intestinal microbiota may support symbiosis with the human host organism by n‐butyrate mediated downregulation of protein and/or gene expression of innate response receptors as well as xCT on PBMO following recruitment to the lamina propria.
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