AIM To investigate the effects of plecanatide and dolcanatide on maintenance of paracellular permeability, integrity of tight junctions and on suppression of visceral hypersensitivity. METHODS Transport of fluorescein...AIM To investigate the effects of plecanatide and dolcanatide on maintenance of paracellular permeability, integrity of tight junctions and on suppression of visceral hypersensitivity. METHODS Transport of fluorescein isothiocyanate(FITC)-dextran was measured to assess permeability across cell monolayers and rat colon tissues. Effects of plecanatide and dolcanatide on the integrity of tight junctions in Caco-2 and T84 monolayers and on the expression and localization of occludin and zonula occludens-1(ZO-1) were examined by immunofluorescence microscopy. Anti-nociceptive activity of these agonists was evaluated in trinitrobenzene sulfonic acid(TNBS)-induced inflammatory as well as in non-inflammatory partial restraint stress(PRS) rat models. Statistical significance between the treatment groups in the permeability studies were evaluated using unpaired t-tests.RESULTS Treatment of T84 and Caco-2 monolayers with lipopolysaccharide(LPS) rapidly increased permeability, which was effectively suppressed when monolayers were also treated with plecanatide or dolcanatide. Similarly, when T84 and Caco-2 monolayers were treated with LPS, cell surface localization of tight junction proteins occludin and ZO-1 was severely disrupted. When cell monolayers were treated with LPS in the presence of plecanatide or dolcanatide, occludin and ZO-1 were localized at the cell surface of adjoining cells, similar to that observed for vehicle treated cells. Treatment of cell monolayers with plecanatide or dolcanatide without LPS did not alter permeability, integrity of tight junctions and cell surface localization of either of the tight junction proteins. In rat visceral hypersensitivity models, both agonists suppressed the TNBS-induced increase in abdominal contractions in response to colorectal distension without affecting the colonic wall elasticity, and both agonists also reduced colonic hypersensitivity in the PRS model. CONCLUSION Our results suggest that activation of GC-C signaling might be involved in maintenance of barrier function, possibly through regulating normal localization of tight junction proteins. Consistent with these findings, plecanatide and dolcanatide showed potent antinociceptive activity in rat visceral hypersensitivity models. These results imply that activation of GC-C signaling may be an attractive therapeutic approach to treat functional constipation disorders and inflammatory gastrointestinal conditions.展开更多
Colorectal cancer(CRC) is a major cause of cancerrelated mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, a...Colorectal cancer(CRC) is a major cause of cancerrelated mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C(GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin(GUCA2A) and uroguanylin(GUCA2B), which bind and activate GUCY2 C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2 C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2 C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C's role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2 C ligand linaclotide(Linzess^(TM)). Here we review the known contributions of the GUCY2 C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention.展开更多
AIM To evaluate the effect of orally administered plecanatide on colorectal dysplasia in Apc^(+/Min-FCCC)mice with dextran sodium sulfate(DSS)-induced inflammation.METHODS Inflammation driven colorectal carcinogenesis...AIM To evaluate the effect of orally administered plecanatide on colorectal dysplasia in Apc^(+/Min-FCCC)mice with dextran sodium sulfate(DSS)-induced inflammation.METHODS Inflammation driven colorectal carcinogenesis was induced in Apc^(+/Min-FCCC)mice by administering DSS in their drinking water.Mice were fed a diet supplemented with plecanatide(0-20 ppm)and its effect on the multiplicity of histopathologically confirmed polypoid,flat and indeterminate dysplasia was evaluated.Plecanatide-mediated activation of guanylate cyclase-C(GC-C)signaling was assessed in colon tissues by measuring cyclic guanosine monophosphate(cG MP)by ELISA,protein kinase G-II and vasodilator stimulated phosphoprotein by immunoblotting.Ki-67,c-myc and cyclin D1 were used as markers of proliferation.Cellular levels and localization of b-catenin in colon tissues were assessed by immunoblotting and immunohistochemistry,respectively.Uroguanylin(UG)and GC-C transcript levels were measured by quantitative reverse transcription polymerase chain reaction(RT-PCR).A mouse cytokine array panel was used to detect cytokines in the supernatant of colon explant cultures.RESULTS Oral treatment of Apc^(+/Min-FCCC)mice with plecanatide produced a statistically significant reduction in the formation of inflammation-driven polypoid,flat and indeterminate dysplasias.This anti-carcinogenic activity of plecanatide was accompanied by activation of cG MP/GC-C signaling mediated inhibition of Wnt/b-catenin signaling and reduced proliferation.Plecanatide also decreased secretion of pro-inflammatory cytokines(IL-6,IL-1 TNF),chemokines(MIP-1,IP-10)and growth factors(GCSF and GMCSF)from colon explants derived from mice with acute DSS-induced inflammation.The effect of plecanatidemediated inhibition of inflammation/dysplasia on endogenous expression of UG and GC-C transcripts was measured in intestinal tissues.Although GC-C expression was not altered appreciably,a statistically significant increase in the level of UG transcripts was detected in the proximal small intestine and colon,potentially due to a reduction in intestinal inflammation and/or neoplasia.Taken together,these results suggest that reductions in endogenous UG,accompanied by dysregulation in GC-C signaling,may be an early event in inflammation-promoted colorectal neoplasia;an event that can potentially be ameliorated by prophylactic intervention with plecanatide.CONCLUSION This study provides the first evidence that orally administered plecanatide reduces the multiplicity of inflammation-driven colonic dysplasia in mice,demonstrating the utility for developing GC-C agonists as chemopreventive agents.展开更多
基金Supported by Synergy Pharmaceuticals Inc.,which provided the funding and the plecanatide,dolcanatide and placebo used for these studies
文摘AIM To investigate the effects of plecanatide and dolcanatide on maintenance of paracellular permeability, integrity of tight junctions and on suppression of visceral hypersensitivity. METHODS Transport of fluorescein isothiocyanate(FITC)-dextran was measured to assess permeability across cell monolayers and rat colon tissues. Effects of plecanatide and dolcanatide on the integrity of tight junctions in Caco-2 and T84 monolayers and on the expression and localization of occludin and zonula occludens-1(ZO-1) were examined by immunofluorescence microscopy. Anti-nociceptive activity of these agonists was evaluated in trinitrobenzene sulfonic acid(TNBS)-induced inflammatory as well as in non-inflammatory partial restraint stress(PRS) rat models. Statistical significance between the treatment groups in the permeability studies were evaluated using unpaired t-tests.RESULTS Treatment of T84 and Caco-2 monolayers with lipopolysaccharide(LPS) rapidly increased permeability, which was effectively suppressed when monolayers were also treated with plecanatide or dolcanatide. Similarly, when T84 and Caco-2 monolayers were treated with LPS, cell surface localization of tight junction proteins occludin and ZO-1 was severely disrupted. When cell monolayers were treated with LPS in the presence of plecanatide or dolcanatide, occludin and ZO-1 were localized at the cell surface of adjoining cells, similar to that observed for vehicle treated cells. Treatment of cell monolayers with plecanatide or dolcanatide without LPS did not alter permeability, integrity of tight junctions and cell surface localization of either of the tight junction proteins. In rat visceral hypersensitivity models, both agonists suppressed the TNBS-induced increase in abdominal contractions in response to colorectal distension without affecting the colonic wall elasticity, and both agonists also reduced colonic hypersensitivity in the PRS model. CONCLUSION Our results suggest that activation of GC-C signaling might be involved in maintenance of barrier function, possibly through regulating normal localization of tight junction proteins. Consistent with these findings, plecanatide and dolcanatide showed potent antinociceptive activity in rat visceral hypersensitivity models. These results imply that activation of GC-C signaling may be an attractive therapeutic approach to treat functional constipation disorders and inflammatory gastrointestinal conditions.
基金Supported by NIH,No.R01CA170533,No.R01CA206026 and No.P30CA56036Targeted Diagnostic and Therapeutics,Inc.+2 种基金a Ruth L.Kirschstein National Research Service Award for Individual Predoctoral MD/PhD Fellows from the NIH,No.CA180500(To Blomain ES)a Ruth L.Kirschstein National Research Service Award for Individual Predoctoral MD/PhD Fellows from the NIH,No.F30 DK103492(To Merlino DJ)a Predoctoral Fellowship in Pharmacology/Toxicology from the PhR MA Foundation
文摘Colorectal cancer(CRC) is a major cause of cancerrelated mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C(GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin(GUCA2A) and uroguanylin(GUCA2B), which bind and activate GUCY2 C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2 C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2 C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C's role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2 C ligand linaclotide(Linzess^(TM)). Here we review the known contributions of the GUCY2 C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention.
基金Supported by National Cancer Institute(CA133689 to Kunwar Shailubhai,CA006927 to Fox Chase Cancer Center)an appropriation from the Commonwealth of Pennsylvania(Fox Chase Cancer Center)
文摘AIM To evaluate the effect of orally administered plecanatide on colorectal dysplasia in Apc^(+/Min-FCCC)mice with dextran sodium sulfate(DSS)-induced inflammation.METHODS Inflammation driven colorectal carcinogenesis was induced in Apc^(+/Min-FCCC)mice by administering DSS in their drinking water.Mice were fed a diet supplemented with plecanatide(0-20 ppm)and its effect on the multiplicity of histopathologically confirmed polypoid,flat and indeterminate dysplasia was evaluated.Plecanatide-mediated activation of guanylate cyclase-C(GC-C)signaling was assessed in colon tissues by measuring cyclic guanosine monophosphate(cG MP)by ELISA,protein kinase G-II and vasodilator stimulated phosphoprotein by immunoblotting.Ki-67,c-myc and cyclin D1 were used as markers of proliferation.Cellular levels and localization of b-catenin in colon tissues were assessed by immunoblotting and immunohistochemistry,respectively.Uroguanylin(UG)and GC-C transcript levels were measured by quantitative reverse transcription polymerase chain reaction(RT-PCR).A mouse cytokine array panel was used to detect cytokines in the supernatant of colon explant cultures.RESULTS Oral treatment of Apc^(+/Min-FCCC)mice with plecanatide produced a statistically significant reduction in the formation of inflammation-driven polypoid,flat and indeterminate dysplasias.This anti-carcinogenic activity of plecanatide was accompanied by activation of cG MP/GC-C signaling mediated inhibition of Wnt/b-catenin signaling and reduced proliferation.Plecanatide also decreased secretion of pro-inflammatory cytokines(IL-6,IL-1 TNF),chemokines(MIP-1,IP-10)and growth factors(GCSF and GMCSF)from colon explants derived from mice with acute DSS-induced inflammation.The effect of plecanatidemediated inhibition of inflammation/dysplasia on endogenous expression of UG and GC-C transcripts was measured in intestinal tissues.Although GC-C expression was not altered appreciably,a statistically significant increase in the level of UG transcripts was detected in the proximal small intestine and colon,potentially due to a reduction in intestinal inflammation and/or neoplasia.Taken together,these results suggest that reductions in endogenous UG,accompanied by dysregulation in GC-C signaling,may be an early event in inflammation-promoted colorectal neoplasia;an event that can potentially be ameliorated by prophylactic intervention with plecanatide.CONCLUSION This study provides the first evidence that orally administered plecanatide reduces the multiplicity of inflammation-driven colonic dysplasia in mice,demonstrating the utility for developing GC-C agonists as chemopreventive agents.
