Stereo-Specific Modulation of the Extracellular Calcium-Sensing Receptor in Colon Cancer Cells

Pharmacological allosteric agonists (calcimimetics) of the extracellular calcium-sensing receptor (CaSR) have substantial gastro-intestinal side effects and induce the expression of inflammatory markers in colon cancer cells. Here, we compared the effects of both CaSR-specific (R enantiomers) and -unspecific (S enantiomers) enantiomers of a calcimimetic (NPS 568) and a calcilytic (allosteric CaSR antagonists; NPS 2143) to prove that these effects are indeed mediated via the CaSR, rather than via off-target effects, e.g., on β-adrenoceptors or calcium channels, of these drugs. The unspecific S enantiomer of NPS 2143 and NPS S-2143 was prepared using synthetic chemistry and characterized using crystallography. NPS S-2143 was then tested in HEK-293 cells stably transfected with the human CaSR (HEK-CaSR), where it did not inhibit CaSR-mediated intracellular Ca2+ signals, as expected. HT29 colon cancer cells transfected with the CaSR were treated with both enantiomers of NPS 568 and NPS 2143 alone or in combination, and the expression of CaSR and the pro-inflammatory cytokine interleukin 8 (IL-8) was measured by RT-qPCR and ELISA. Only the CaSR-selective enantiomers of the calcimimetic NPS 568 and NPS 2143 were able to modulate CaSR and IL-8 expression. We proved that pro-inflammatory effects in colon cancer cells are indeed mediated through CaSR activation. The non-CaSR selective enantiomer NPS S-2143 will be a valuable tool for investigations in CaSR-mediated processes

Nutritional and pharmacological targeting of the calcium-sensing receptor influences chemically induced colitis in mice

The calcium-sensing receptor (CaSR) is the main regulator of extracellular Ca2+ homeostasis. It has diverse functions in different tissues, including the intestines. Intestine-specific knockout of the CaSR renders mice more susceptible to dextran sulphate sodium (DSS)-induced colitis. To test our hypothesis that the CaSR reduces intestinal inflammation, we assessed the effects of nutritional and pharmacological agonists of the CaSR in a colitis model. We treated female Balb/C mice with dietary calcium and protein (nutritional agonists of the CaSR) or pharmacological CaSR modulators (the agonists cinacalcet and GSK3004774, and the antagonist NPS-2143; 10 mg/kg), then induced colitis with DSS. The high-protein diet had a strong pro-inflammatory effect—it shortened the colons (5.3 ± 0.1 cm vs. 6.1 ± 0.2 cm normal diet, p < 0.05), lowered mucin expression and upregulated pro-inflammatory cytokines, such as interferon-γ, (4.2-fold, p < 0.05) compared with the normal diet. Cinacalcet reduced mucin expression, which coincided with an increase in tumor necrosis factor-α (4.4-fold, p < 0.05) and IL-6 (4.9-fold, p < 0.05) in the plasma, compared with vehicle. The CaSR antagonist, NPS-2143, significantly reduced the cumulative inflammation score compared with the vehicle control (35.3 ± 19.1 vs. 21.9 ± 14.3 area under the curve, p < 0.05) and reduced infiltration of inflammatory cells. While dietary modulation of the CaSR had no beneficial effects, pharmacological inhibition of the CaSR may have the potential of a novel add-on therapy in the treatment of inflammatory bowel diseases

The calcium-sensing receptor modulates the prostaglandin E 2 pathway in intestinal inflammation

Introduction: The prostaglandin E2 (PGE2) pathway is one of the main mediators of intestinal inflammation. As activation of the calcium-sensing receptor (CaSR) induces expression of inflammatory markers in the colon, we assessed the impact of the CaSR on the PGE2 pathway regulation in colon cancer cells and the colon in vitro and in vivo. Methods and Results: We treated CaSR-transfected HT29 and Caco-2 colon cancer cell lines with different orthosteric ligands or modulators of the CaSR and measured gene expression and PGE2 levels. In CaSR-transfected HT29CaSR-GFP and Caco-2CaSR-GFP cells, the orthosteric CaSR ligand spermine and the positive allosteric CaSR modulator NPS R-568 both induced an inflammatory state as measured by IL-8 gene expression and significantly increased the expression of the PGE2 pathway key enzymes cyclooxygenase (COX)-2 and/or prostaglandin E2 synthase 1 (PGES-1). Inhibition of the CaSR with the calcilytic NPS 2143 abolished the spermine- and NPS R-568-induced pro-inflammatory response. Interestingly, we observed cell-line specific responses as e.g. PGES-1 expression was affected only in HT29CaSR-GFP but not in Caco-2CaSR-GFP cells. Other genes involved in the PGE2 pathway (COX-1, or the PGE2 receptors) were not responsive to the treatment. None of the studied genes were affected by any CaSR agonist in GFP-only transfected HT29GFP and Caco-2GFP cells, indicating that the observed gene-inducing effects of spermine and R-568 were indeed mediated by the CaSR. In vivo, we had previously determined that treatment with the clinically approved calcimimetic cinacalcet worsened symptoms in a dextran sulfate sodium (DSS)-induced colitis mouse model. In the colons of these mice, cinacalcet significantly induced gene expression of PGES-2 and the EP3 receptor, but not COX-2; while NPS 2143 increased the expression of the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Importantly, neither treatment had any effect on the colons of non-DSS treated mice. Discussion: Overall, we show that activation of the CaSR induces the PGE2 pathway, albeit with differing effects in vitro and in vivo. This may be due to the different microenvironment in vivo compared to in vitro, specifically the presence of a CaSR-responsive immune system. Since calcilytics inhibit ligand-mediated CaSR signaling, they may be considered for novel therapies against inflammatory bowel disease

Calcium-sensing receptor in colorectal inflammation and cancer : Current insights and future perspectives

The extracellular calcium-sensing receptor (CaSR) is best known for its action in the parathyroid gland and kidneys where it controls body calcium homeostasis. However, the CaSR has different roles in the gastrointestinal tract, where it is ubiquitously expressed. In the colon, the CaSR is involved in controlling multiple mechanisms, including fluid transport, inflammation, cell proliferation and differentiation. Although the expression pattern and functions of the CaSR in the colonic microenvironment are far from being completely understood, evidence has been accumulating that the CaSR might play a protective role against both colonic inflammation and colorectal cancer. For example, CaSR agonists such as dipeptides have been suggested to reduce colonic inflammation, while dietary calcium was shown to reduce the risk of colorectal cancer. CaSR expression is lost in colonic malignancies, indicating that the CaSR is a biomarker for colonic cancer progression. This dual anti-inflammatory and anti-tumourigenic role of the CaSR makes it especially interesting in colitis-associated colorectal cancer. In this review, we describe the clinical and experimental evidence for the role of the CaSR in colonic inflammation and colorectal cancer, the intracellular signalling pathways which are putatively involved in these actions, and the possibilities to exploit these actions of the CaSR for future therapies of colonic inflammation and cancer.(VLID)467278

Nutritional and Pharmacological Targeting of the Calcium-Sensing Receptor Influences Chemically Induced Colitis in Mice

The calcium-sensing receptor (CaSR) is the main regulator of extracellular Ca2+ homeostasis. It has diverse functions in different tissues, including the intestines. Intestine-specific knockout of the CaSR renders mice more susceptible to dextran sulphate sodium (DSS)-induced colitis. To test our hypothesis that the CaSR reduces intestinal inflammation, we assessed the effects of nutritional and pharmacological agonists of the CaSR in a colitis model. We treated female Balb/C mice with dietary calcium and protein (nutritional agonists of the CaSR) or pharmacological CaSR modulators (the agonists cinacalcet and GSK3004774, and the antagonist NPS-2143; 10 mg/kg), then induced colitis with DSS. The high-protein diet had a strong pro-inflammatory effect&mdash;it shortened the colons (5.3 &plusmn; 0.1 cm vs. 6.1 &plusmn; 0.2 cm normal diet, p &lt; 0.05), lowered mucin expression and upregulated pro-inflammatory cytokines, such as interferon-&gamma;, (4.2-fold, p &lt; 0.05) compared with the normal diet. Cinacalcet reduced mucin expression, which coincided with an increase in tumor necrosis factor-&alpha; (4.4-fold, p &lt; 0.05) and IL-6 (4.9-fold, p &lt; 0.05) in the plasma, compared with vehicle. The CaSR antagonist, NPS-2143, significantly reduced the cumulative inflammation score compared with the vehicle control (35.3 &plusmn; 19.1 vs. 21.9 &plusmn; 14.3 area under the curve, p &lt; 0.05) and reduced infiltration of inflammatory cells. While dietary modulation of the CaSR had no beneficial effects, pharmacological inhibition of the CaSR may have the potential of a novel add-on therapy in the treatment of inflammatory bowel diseases

“Reflecting on Style and Surtitles: The Case of Falstaff by Giuseppe Verdi”

The aim of this work is to integrate a more traditional description of what opera surtitling is – a professional practice first and foremost, relatively recently turned into the object of academic research, which should deserve further attention by scholars and professionals alike, also due to its strategic importance in terms of social inclusion and accessibility – with a reflection on style in translation: in other words, this contribution intends to verify whether, and to what extent, opera surtitles should be “condemned” to be a highly summarised and abridged form of translation, or whether, instead, they can be respectful and accurate in reproducing the stylistic features and rhetorical effects of the original libretto, in spite of their brevity and conciseness. The selection of the linguistic materials fell on Falstaff by Giuseppe Verdi, based on Arrigo Boito’s libretto because, due to the sparking creativity and huge variety of his lexical and stylistic choices, producing English surtitles from this libretto is a stimulating task which requires not only a sound professional background, but also a refined artistic sensibility. Therefore, many significant examples are available to those who want to investigate such issues as surtitling and stylistics, since these surtitles are a successful illustration of a stylistically-oriented translation

Image5_The calcium-sensing receptor modulates the prostaglandin E2 pathway in intestinal inflammation.TIF

Introduction: The prostaglandin E2 (PGE2) pathway is one of the main mediators of intestinal inflammation. As activation of the calcium-sensing receptor (CaSR) induces expression of inflammatory markers in the colon, we assessed the impact of the CaSR on the PGE2 pathway regulation in colon cancer cells and the colon in vitro and in vivo.Methods and Results: We treated CaSR-transfected HT29 and Caco-2 colon cancer cell lines with different orthosteric ligands or modulators of the CaSR and measured gene expression and PGE2 levels. In CaSR-transfected HT29CaSR-GFP and Caco-2CaSR-GFP cells, the orthosteric CaSR ligand spermine and the positive allosteric CaSR modulator NPS R-568 both induced an inflammatory state as measured by IL-8 gene expression and significantly increased the expression of the PGE2 pathway key enzymes cyclooxygenase (COX)-2 and/or prostaglandin E2 synthase 1 (PGES-1). Inhibition of the CaSR with the calcilytic NPS 2143 abolished the spermine- and NPS R-568-induced pro-inflammatory response. Interestingly, we observed cell-line specific responses as e.g. PGES-1 expression was affected only in HT29CaSR-GFP but not in Caco-2CaSR-GFP cells. Other genes involved in the PGE2 pathway (COX-1, or the PGE2 receptors) were not responsive to the treatment. None of the studied genes were affected by any CaSR agonist in GFP-only transfected HT29GFP and Caco-2GFP cells, indicating that the observed gene-inducing effects of spermine and R-568 were indeed mediated by the CaSR.In vivo, we had previously determined that treatment with the clinically approved calcimimetic cinacalcet worsened symptoms in a dextran sulfate sodium (DSS)-induced colitis mouse model. In the colons of these mice, cinacalcet significantly induced gene expression of PGES-2 and the EP3 receptor, but not COX-2; while NPS 2143 increased the expression of the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Importantly, neither treatment had any effect on the colons of non-DSS treated mice. Discussion: Overall, we show that activation of the CaSR induces the PGE2 pathway, albeit with differing effects in vitro and in vivo. This may be due to the different microenvironment in vivo compared to in vitro, specifically the presence of a CaSR-responsive immune system. Since calcilytics inhibit ligand-mediated CaSR signaling, they may be considered for novel therapies against inflammatory bowel disease.</p

Table1_The calcium-sensing receptor modulates the prostaglandin E2 pathway in intestinal inflammation.DOCX

Introduction: The prostaglandin E2 (PGE2) pathway is one of the main mediators of intestinal inflammation. As activation of the calcium-sensing receptor (CaSR) induces expression of inflammatory markers in the colon, we assessed the impact of the CaSR on the PGE2 pathway regulation in colon cancer cells and the colon in vitro and in vivo.Methods and Results: We treated CaSR-transfected HT29 and Caco-2 colon cancer cell lines with different orthosteric ligands or modulators of the CaSR and measured gene expression and PGE2 levels. In CaSR-transfected HT29CaSR-GFP and Caco-2CaSR-GFP cells, the orthosteric CaSR ligand spermine and the positive allosteric CaSR modulator NPS R-568 both induced an inflammatory state as measured by IL-8 gene expression and significantly increased the expression of the PGE2 pathway key enzymes cyclooxygenase (COX)-2 and/or prostaglandin E2 synthase 1 (PGES-1). Inhibition of the CaSR with the calcilytic NPS 2143 abolished the spermine- and NPS R-568-induced pro-inflammatory response. Interestingly, we observed cell-line specific responses as e.g. PGES-1 expression was affected only in HT29CaSR-GFP but not in Caco-2CaSR-GFP cells. Other genes involved in the PGE2 pathway (COX-1, or the PGE2 receptors) were not responsive to the treatment. None of the studied genes were affected by any CaSR agonist in GFP-only transfected HT29GFP and Caco-2GFP cells, indicating that the observed gene-inducing effects of spermine and R-568 were indeed mediated by the CaSR.In vivo, we had previously determined that treatment with the clinically approved calcimimetic cinacalcet worsened symptoms in a dextran sulfate sodium (DSS)-induced colitis mouse model. In the colons of these mice, cinacalcet significantly induced gene expression of PGES-2 and the EP3 receptor, but not COX-2; while NPS 2143 increased the expression of the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Importantly, neither treatment had any effect on the colons of non-DSS treated mice. Discussion: Overall, we show that activation of the CaSR induces the PGE2 pathway, albeit with differing effects in vitro and in vivo. This may be due to the different microenvironment in vivo compared to in vitro, specifically the presence of a CaSR-responsive immune system. Since calcilytics inhibit ligand-mediated CaSR signaling, they may be considered for novel therapies against inflammatory bowel disease.</p

Image3_The calcium-sensing receptor modulates the prostaglandin E2 pathway in intestinal inflammation.TIF

Introduction: The prostaglandin E2 (PGE2) pathway is one of the main mediators of intestinal inflammation. As activation of the calcium-sensing receptor (CaSR) induces expression of inflammatory markers in the colon, we assessed the impact of the CaSR on the PGE2 pathway regulation in colon cancer cells and the colon in vitro and in vivo.Methods and Results: We treated CaSR-transfected HT29 and Caco-2 colon cancer cell lines with different orthosteric ligands or modulators of the CaSR and measured gene expression and PGE2 levels. In CaSR-transfected HT29CaSR-GFP and Caco-2CaSR-GFP cells, the orthosteric CaSR ligand spermine and the positive allosteric CaSR modulator NPS R-568 both induced an inflammatory state as measured by IL-8 gene expression and significantly increased the expression of the PGE2 pathway key enzymes cyclooxygenase (COX)-2 and/or prostaglandin E2 synthase 1 (PGES-1). Inhibition of the CaSR with the calcilytic NPS 2143 abolished the spermine- and NPS R-568-induced pro-inflammatory response. Interestingly, we observed cell-line specific responses as e.g. PGES-1 expression was affected only in HT29CaSR-GFP but not in Caco-2CaSR-GFP cells. Other genes involved in the PGE2 pathway (COX-1, or the PGE2 receptors) were not responsive to the treatment. None of the studied genes were affected by any CaSR agonist in GFP-only transfected HT29GFP and Caco-2GFP cells, indicating that the observed gene-inducing effects of spermine and R-568 were indeed mediated by the CaSR.In vivo, we had previously determined that treatment with the clinically approved calcimimetic cinacalcet worsened symptoms in a dextran sulfate sodium (DSS)-induced colitis mouse model. In the colons of these mice, cinacalcet significantly induced gene expression of PGES-2 and the EP3 receptor, but not COX-2; while NPS 2143 increased the expression of the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Importantly, neither treatment had any effect on the colons of non-DSS treated mice. Discussion: Overall, we show that activation of the CaSR induces the PGE2 pathway, albeit with differing effects in vitro and in vivo. This may be due to the different microenvironment in vivo compared to in vitro, specifically the presence of a CaSR-responsive immune system. Since calcilytics inhibit ligand-mediated CaSR signaling, they may be considered for novel therapies against inflammatory bowel disease.</p