RESUMEN
Phosphodiesterase-5 inhibitors (PDE5i) are under investigation for repurposing for colon cancer prevention. A drawback to conventional PDE5i are their side-effects and drug-drug interactions. We designed an analog of the prototypical PDE5i sildenafil by replacing the methyl group on the piperazine ring with malonic acid to reduce lipophilicity, and measured its entry into the circulation and effects on colon epithelium. This modification did not affect pharmacology as malonyl-sildenafil had a similar IC50 to sildenafil but exhibited an almost 20-fold reduced EC50 for increasing cellular cGMP. Using an LC-MS/MS approach, malonyl-sildenafil was negligible in mouse plasma after oral administration but was detected at high levels in the feces. No bioactive metabolites of malonyl-sildenafil were detected in the circulation by measuring interactions with isosorbide mononitrate. The treatment of mice with malonyl-sildenafil in the drinking water resulted in a suppression of proliferation in the colon epithelium that is consistent with results previously published for mice treated with PDE5i. A carboxylic-acid-containing analog of sildenafil prohibits the systemic delivery of the compound but maintains sufficient penetration into the colon epithelium to suppress proliferation. This highlights a novel approach to generating a first-in-class drug for colon cancer chemoprevention.
Asunto(s)
Neoplasias del Colon , Inhibidores de Fosfodiesterasa 5 , Ratones , Animales , Inhibidores de Fosfodiesterasa 5/farmacología , Citrato de Sildenafil/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Cromatografía Liquida , Espectrometría de Masas en Tándem , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/prevención & control , Proliferación Celular , GMP Cíclico/metabolismoRESUMEN
The epidermal growth factor receptor (EGFR) is commonly upregulated in multiple cancer types, including breast cancer. In the present study, evidence is provided in support of the premise that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitates the escape of breast cancer cells from BimELdependent apoptosis, conferring resistance to therapy. This conclusion is based on the findings that ectopic BimEL cDNA overexpression and confocal imaging studies confirm the proapoptotic role of BimEL in ERα expressing breast cancer cells and that upregulated EGFR/MEK1/MAPK1/2 signaling blocks BimEL proapoptotic action in an antiestrogenresistant breast cancer cell model. In addition, the present study identified a prosurvival role for autophagy in antiestrogen resistance while EGFR inhibitor studies demonstrated that a significant percentage of antiestrogenresistant breast cancer cells survive EGFR targeting by prosurvival autophagy. These preclinical studies establish the possibility that targeting both the MEK1/MAPK1/2 signaling axis and prosurvival autophagy may be required to eradicate breast cancer cell survival and prevent the development of antiestrogen resistance following hormone treatments. The present study uniquely identified EGFR upregulation as one of the mechanisms breast cancer cells utilize to evade the cytotoxic effects of antiestrogens mediated through BimELdependent apoptosis.
Asunto(s)
Apoptosis , Neoplasias de la Mama , Resistencia a Antineoplásicos , Moduladores de los Receptores de Estrógeno , Femenino , Humanos , Apoptosis/efectos de los fármacos , Proteína 11 Similar a Bcl2/efectos de los fármacos , Proteína 11 Similar a Bcl2/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos/fisiología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Moduladores de los Receptores de Estrógeno/farmacología , Moduladores de los Receptores de Estrógeno/uso terapéutico , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Regulación hacia Arriba , Transducción de SeñalRESUMEN
A large body of evidence has demonstrated that cyclic-guanosine monophosphate (cGMP), signaling has anti-tumor effects that might be used for colon cancer prevention. The tumor-suppressive mechanism and the signaling components downstream of cGMP remain largely unknown. The present study has characterized the expression of cGMP-dependent protein kinases (PKG1, PKG2) in normal and cancerous tissue from human colon. PKG1 was detected in both normal and tumor tissue, where it localized exclusively to the lamina propria and stroma (respectively). In contrast, PKG2 localized specifically to the epithelium where its expression decreased markedly in tumors compared to matched normal tissue. Neither PKG isoform was detected at the RNA or protein level in established colon cancer cell lines. To test for a potential tumor-suppressor role of PKG2 in the colon epithelium, Prkg2 knockout (KO) mice were subjected to azoxymethane/dextran sulfate-sodium (AOM/DSS) treatment. PKG2 deficiency was associated with crypt hyperplasia (Ki67) and almost twice the number of polyps per mouse as wild-type (WT) siblings. In vitro culture of mouse colon epithelium as organoids confirmed that PKG2 was the only isoform expressed, and it was detected in both proliferating and differentiating epithelial compartments. Colon organoids derived from Prkg2 KO mice proliferated more rapidly and exhibited a reduced ability to differentiate compared to WT controls. Taken together our results highlight PKG2 as the central target of cGMP in the colon, where it suppresses carcinogenesis by controlling proliferation in an epithelial-cell intrinsic manner.
Asunto(s)
Colon , Neoplasias del Colon , Proteína Quinasa Dependiente de GMP Cíclico Tipo II , Animales , Azoximetano , Carcinogénesis/patología , Proliferación Celular , Colon/patología , Neoplasias del Colon/patología , GMP Cíclico/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/genética , Sulfato de Dextran , Epitelio/patología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
There is growing interest in the potential use of phosphodiesterase (PDE) inhibitors for colorectal cancer (CRC) prevention and treatment. The present study has tested the idea that PDE inhibitors inhibit growth and viability of CRC cell lines by increasing cyclic guanosine monophosphate (cGMP) and activating cGMP-dependent protein kinase (PKG). Colon cancer cell lines and those with ectopic PKG2 expression were treated with membrane-permeable 8Br-cGMP or inhibitors of PDE5, PDE9, and PDE10a. Levels of cGMP capable of activating PKG were measured by immunoblotting for phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The effects of treatment on CRC cell proliferation and death were measured using hemocytometry with trypan blue. Treatment with 8Br-cGMP had no effect on CRC cell proliferation or death. Endogenous PKG activity was undetectable in any of the CRC cells, but expression of ectopic PKG2 conferred modest inhibition of proliferation but did not affect cell death. Extremely high concentrations of all the PDE inhibitors reduced proliferation in CRC cell lines, but none of them increased cGMP levels, and the effect was independent of PKG expression. The inability of the PDE inhibitors to increase cGMP was due to the lack of endogenous cGMP generating machinery. In conclusion, PDE inhibitors that target cGMP only reduce CRC growth at clinically unachievable concentrations, and do so independent of cGMP signaling through PKG. SIGNIFICANCE STATEMENT: A large number of in vitro studies have reported that PDE inhibitors block growth of colon cancer cells by activating cGMP signaling, and that these drugs might be useful for cancer treatment. Our results show that these drugs do not activate cGMP signaling in colon cancer cells due to a lack of endogenous guanylyl cyclase activity, and that growth inhibition is due to toxic effects of clinically unobtainable drug concentrations.
Asunto(s)
Neoplasias del Colon , Inhibidores de Fosfodiesterasa , Transformación Celular Neoplásica , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Humanos , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Transducción de SeñalRESUMEN
A leading cause of bacterial gastroenteritis, Campylobacter jejuni is also associated with broad sequelae, including extragastrointestinal conditions such as reactive arthritis and Guillain-Barré Syndrome (GBS). CbrR is a C. jejuni response regulator that is annotated as a diguanylate cyclase (DGC), an enzyme that catalyzes the synthesis of c-di-GMP, a universal bacterial second messenger, from GTP. In C. jejuni DRH212, we constructed an unmarked deletion mutant, cbrR-, and complemented mutant, cbrR+. Motility assays indicated a hyper-motile phenotype associated with cbrR-, whereas motility was deficient in cbrR+. The overexpression of CbrR in cbrR+ was accompanied by a reduction in expression of FlaA, the major flagellin. Biofilm assays and scanning electron microscopy demonstrated similarities between DRH212 and cbrR-; however, cbrR+ was unable to form significant biofilms. Transmission electron microscopy showed similar cell morphology between the three strains; however, cbrR+ cells lacked flagella. Differential radial capillary action of ligand assays (DRaCALA) showed that CbrR binds GTP and c-di-GMP. Liquid chromatography tandem mass spectrometry detected low levels of c-di-GMP in C. jejuni and in E. coli expressing CbrR. CbrR is therefore a negative regulator of FlaA expression and motility, a critical virulence factor in C. jejuni pathogenesis.
RESUMEN
BACKGROUND: NF-κB is a key link between inflammation and cancer. Previous studies of NF-κB have largely focused on tumor cells, and the intrinsic function of NF-κB in T cells in tumor development and response to immunotherapy is largely unknown. We aimed at testing the hypothesis that NF-κB1 (p50) activation in T cells underlies human colon cancer immune escape and human cancer non-response to anti-PD-1 immunotherapy. METHODS: We screened NF-κB activation in human colon carcinoma and used mouse models to determine p50 function in tumor cells and immune cells. RNA-Seq was used to identify p50 target genes. p50 binding to target gene promoters were determined by electrophoresis mobility shift assay and chromatin immunoprecipitation. A p50 activation score was generated from gene expression profiling and used to link p50 activation to T-cell activation and function pre-nivolumab and post-nivolumab immunotherapy in human patients with cancer. RESULTS: p50 is the dominant form of NF-κB that is highly activated in immune cells in the human colorectal carcinoma microenvironment and neighboring non-neoplastic colon epithelial cells. Tumor cell intrinsic p50 signaling and T-cell intrinsic p50 signaling exert opposing functions in tumor growth control in vivo. Deleting Nfkb1 in tumor cells increased whereas in T cells decreased tumor growth in preclinical mouse models. Gene expression profiling identified Gzmb as a p50 target in T cells. p50 binds directly to a previously uncharacterized κB sequence at the Gzmb promoter in T cells, resulting in repression of Gzmb expression in tumor-infiltrating cytotoxic T lymphocytes (CTLs) to induce a dysfunctional CTL phenotype to promote tumor immune escape. p50 activation is inversely correlated with both GZMB expression and T-cell tumor infiltration in human colorectal carcinoma. Furthermore, nivolumab immunotherapy decreased p50 activation and increased GZMB expression in human patients with melanoma. CONCLUSIONS: Inflammation activates p50 that binds to the Gzmb promoter to repress granzyme B expression in T cells, resulting in CTL dysfunction to confer tumor immune escape and decreased response to anti-PD-1 immunotherapy.
Asunto(s)
Inmunoterapia/métodos , Linfocitos T Citotóxicos/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Escape del TumorRESUMEN
Intestinal exocrine secretory cells, including Paneth and goblet cells, have a pivotal role in intestinal barrier function and mucosal immunity. Dysfunction of these cells may lead to the pathogenesis of human diseases such as inflammatory bowel disease (IBD). Therefore, identification and elucidation of key molecular mechanisms that regulate the development and function of these exocrine cells would be crucial for understanding of disease pathogenesis and discovery of new therapeutic targets. The Ufm1 conjugation system is a novel ubiquitin-like modification system that consists of Ufm1 (Ubiquitin modifier 1), Uba5 (Ufm1-activating enzyme, E1), Ufc1 (Ufm1-conjugating enzyme, E2) and poorly characterized Ufm1 E3 ligase(s). Recent mouse genetic studies have demonstrated its indispensable role in embryonic development and hematopoiesis. Yet its role in other tissues and organs remains poorly defined. In this study, we found that both Ufl1 and Ufbp1, two key components of the Ufm1 E3 ligase, were highly expressed in the intestinal exocrine cells. Ablation of either Ufl1 and Ufbp1 led to significant loss of both Paneth and goblet cells, which in turn resulted in dysbiotic microbiota and increased susceptibility to experimentally induced colitis. At the cellular and molecular levels, Ufbp1 deficiency caused elevation of endoplasmic reticulum stress and activation of the Unfolded Protein Response (UPR) and cell death program. Administration of small molecular chaperone partially prevented loss of Paneth cells caused by acute Ufbp1 deletion. Taken together, our results have provided unambiguous evidence for the crucial role of the Ufm1 E3 ligase in maintenance of intestinal homeostasis and protection from inflammatory diseases.
RESUMEN
IL-10 functions as a suppressor of colitis and colitis-associated colon cancer, but it is also a risk locus associated with ulcerative colitis. The mechanism underlying the contrasting roles of IL-10 in inflammation and colon cancer is unknown. We report here that inflammation induces the accumulation of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) that express high levels of IL-10 in colon tissue. IL-10 induces the activation of STAT3 that directly binds to the Dnmt1 and Dnmt3b promoters to activate their expression, resulting in DNA hypermethylation at the Irf8 promoter to silence IRF8 expression in colon epithelial cells. Mice with Irf8 deleted in colonic epithelial cells exhibit significantly higher inflammation-induced tumor incidence. Human colorectal carcinomas have significantly higher DNMT1 and DNMT3b and lower IRF8 expression, and they exhibit significantly higher IRF8 promoter DNA methylation than normal colon. Our data identify the MDSC-IL-10-STAT3-DNMT3b-IRF8 pathway as a link between chronic inflammation and colon cancer initiation.
Asunto(s)
Carcinogénesis/metabolismo , Colitis/complicaciones , Neoplasias del Colon/etiología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Silenciador del Gen , Factores Reguladores del Interferón/genética , Interleucina-10/biosíntesis , Células Supresoras de Origen Mieloide/metabolismo , Animales , Neoplasias del Colon/genética , Neoplasias del Colon/patología , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Inflamación/patología , Factores Reguladores del Interferón/metabolismo , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Factor de Transcripción STAT3/metabolismo , Regulación hacia Arriba , ADN Metiltransferasa 3BRESUMEN
Constipation is an important health burden that reduces the quality of life for countless millions of people. Symptom-centric therapeutics are often used to treat constipation due to unknown etiology, but in many cases, these drugs are either inadequate or have significant side effects. More recently, synthetic peptide agonists for epithelial guanylyl cyclase C (GC-C) have been developed which are effective at treating constipation in a sub-population of adult constipation patients. The first to market was linaclotide that is structurally related to the diarrheagenic enterotoxin, but this was followed by plecanatide, which more closely resembles endogenous uroguanylin. Both the drugs exhibit almost identical clinical efficacy in about 20% of patients, with diarrhea being a common side effect. Despite the potential for reduced side effects with plecanatide, detailed analysis suggests that clinically, they are very similar. Ongoing clinical and preclinical studies with these drugs suggest that treating constipation might be the tip of the iceberg in terms of clinical utility. The expression of cGMP signaling components could be diagnostic for functional bowel disorders, and increasing cGMP using GC-C agonists or phosphodiesterase inhibitors has huge potential for treating enteric pain, ulcerative colitis, and for the chemoprevention of colorectal cancer.
RESUMEN
The cGMP signaling axis has been implicated in the suppression of intestinal cancers, but the inhibitory mechanism and the extent to which this pathway can be targeted remains poorly understood. This study has tested the effect of cGMP-elevating agents on tumorigenesis in the ApcMin/+ mouse model of intestinal cancer. Treatment of ApcMin/+ mice with the receptor guanylyl-cyclase C (GCC) agonist linaclotide, or the phosphodiesterase-5 (PDE5) inhibitor sildenafil, significantly reduced the number of polyps per mouse (67% and 50%, respectively). Neither of the drugs affected mean polyp size, or the rates of apoptosis and proliferation. This was possibly due to increased PDE10 expression, as endogenous GCC ligands were not deficient in established polyps. These results indicated that the ability of these drugs to reduce polyp multiplicity was primarily due to an effect on nonneoplastic tissues. In support of this idea, ApcMin/+ mice exhibited reduced levels of endogenous GCC agonists in the nonneoplastic intestinal mucosa compared with wild-type animals, and this was associated with crypt hyperplasia and a loss of goblet cells. Administration of either sildenafil or linaclotide suppressed proliferation, and increased both goblet cell numbers and luminal apoptosis in the intestinal mucosa. Taken together, the results demonstrate that targeting cGMP with either PDE5 inhibitors or GCC agonists alters epithelial homeostasis in a manner that reduces neoplasia, and suggests that this could be a viable chemoprevention strategy for patients at high risk of developing colorectal cancer. Cancer Prev Res; 11(2); 81-92. ©2018 AACR.
Asunto(s)
Poliposis Adenomatosa del Colon/prevención & control , Transformación Celular Neoplásica/efectos de los fármacos , GMP Cíclico/metabolismo , Agonistas de la Guanilato Ciclasa C/farmacología , Neoplasias Intestinales/prevención & control , Lesiones Precancerosas/prevención & control , Poliposis Adenomatosa del Colon/metabolismo , Poliposis Adenomatosa del Colon/patología , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Femenino , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Péptidos/farmacología , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Citrato de Sildenafil/farmacologíaRESUMEN
Carbidopa is a drug that blocks conversion of levodopa to dopamine outside of central nervous system (CNS) and thus inhibits unwanted side effects of levodopa on organs located outside of CNS during management of Parkinson's Disease (PD). PD is associated with increased expression of inflammatory genes in peripheral and central nervous system (CNS), infiltration of immune cells into brain, and increased numbers of activated/memory T cells. Animal models of PD have shown a critical role of T cells in inducing pathology in CNS. However, the effect of carbidopa on T cell responses in vivo is unknown. In this report, we show that carbidopa strongly inhibited T cell activation in vitro and in vivo. Accordingly, carbidopa mitigated myelin oligodendrocyte glycoprotein peptide fragment 35-55 (MOG-35-55) induced experimental autoimmune encephalitis (EAE) and collagen induced arthritis in animal models. The data presented here suggest that in addition to blocking peripheral conversion of levodopa, carbidopa may inhibit T cell responses in PD individuals and implicate a potential therapeutic use of carbidopa in suppression of T cell mediated pathologies.
Asunto(s)
Antiparkinsonianos/farmacología , Autoinmunidad/efectos de los fármacos , Carbidopa/farmacología , Dopaminérgicos/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Experimental/patología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Mediadores de Inflamación/metabolismo , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/metabolismoRESUMEN
Intestinal cyclic guanosine monophosphate (cGMP) signaling regulates epithelial homeostasis and has been implicated in the suppression of colitis and colon cancer. In this study, we investigated the cGMP-elevating ability of the phosphodiesterase-5 (PDE5) inhibitor sildenafil to prevent disease in the azoxymethane/dextran sulfate sodium (AOM/DSS) inflammation-driven colorectal cancer model. Treatment of mice with sildenafil activated cGMP signaling in the colon mucosa and protected against dextran-sulfate sodium (DSS)-induced barrier dysfunction. In mice treated with AOM/DSS, oral administration of sildenafil throughout the disease course reduced polyp multiplicity by 50% compared with untreated controls. Polyps that did form in sildenafil treated mice were less proliferative and more differentiated compared with polyps from untreated mice, but apoptosis was unaffected. Polyps in sildenafil treated mice were also less inflamed; they exhibited reduced myeloid-cell infiltration and reduced expression of iNOS, IFNγ, and IL6 compared with untreated controls. Most of the protection conferred by sildenafil was during the initiation stage of carcinogenesis (38% reduction in multiplicity). Administration of sildenafil during the later promotion stages did not affect multiplicity but had a similar effect on the polyp phenotype, including increased mucus production, and reduced proliferation and inflammation. In summary, the results demonstrate that oral administration of sildenafil suppresses polyp formation and inflammation in mice treated with AOM/DSS. This validation of PDE5 as a target highlights the potential therapeutic value of PDE5 inhibitors for the prevention of colitis-driven colon cancer in humans. Cancer Prev Res; 10(7); 377-88. ©2017 AACRSee related editorial by Piazza, p. 373.
Asunto(s)
Carcinogénesis/efectos de los fármacos , Colitis/tratamiento farmacológico , Colon/efectos de los fármacos , Neoplasias Colorrectales/prevención & control , Mucosa Intestinal/efectos de los fármacos , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Administración Oral , Animales , Apoptosis/efectos de los fármacos , Azoximetano/toxicidad , Colitis/inducido químicamente , Colitis/complicaciones , Colon/patología , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/patología , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Sulfato de Dextran/toxicidad , Humanos , Inmunohistoquímica , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/etiología , Neoplasias Experimentales/patología , Neoplasias Experimentales/prevención & control , Pólipos/etiología , Pólipos/patología , Pólipos/prevención & control , Transducción de Señal/efectos de los fármacos , Citrato de Sildenafil/uso terapéuticoRESUMEN
Guanylyl cyclase-C (GC-C) agonists increase cGMP levels in the intestinal epithelium to promote secretion. This process underlies the utility of exogenous GC-C agonists such as linaclotide for the treatment of chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C). Because GC-C agonists have limited use in pediatric patients, there is a need for alternative cGMP-elevating agents that are effective in the intestine. The present study aimed to determine whether the PDE-5 inhibitor sildenafil has similar effects as linaclotide on preclinical models of constipation. Oral administration of sildenafil caused increased cGMP levels in mouse intestinal epithelium demonstrating that blocking cGMP-breakdown is an alternative approach to increase cGMP in the gut. Both linaclotide and sildenafil reduced proliferation and increased differentiation in colon mucosa, indicating common target pathways. The homeostatic effects of cGMP required gut turnover since maximal effects were observed after 3 days of treatment. Neither linaclotide nor sildenafil treatment affected intestinal transit or water content of fecal pellets in healthy mice. To test the effectiveness of cGMP elevation in a functional motility disorder model, mice were treated with dextran sulfate sodium (DSS) to induce colitis and were allowed to recover for several weeks. The recovered animals exhibited slower transit, but increased fecal water content. An acute dose of sildenafil was able to normalize transit and fecal water content in the DSS-recovery animal model, and also in loperamide-induced constipation. The higher fecal water content in the recovered animals was due to a compromised epithelial barrier, which was normalized by sildenafil treatment. Taken together our results show that sildenafil can have similar effects as linaclotide on the intestine, and may have therapeutic benefit to patients with CIC, IBS-C, and post-infectious IBS.
Asunto(s)
Estreñimiento/tratamiento farmacológico , Tránsito Gastrointestinal/efectos de los fármacos , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Citrato de Sildenafil/uso terapéutico , Administración Oral , Animales , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , GMP Cíclico/metabolismo , Sulfato de Dextran , Evaluación Preclínica de Medicamentos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Péptidos/administración & dosificación , Péptidos/uso terapéutico , Inhibidores de Fosfodiesterasa 5/administración & dosificación , Citrato de Sildenafil/administración & dosificaciónRESUMEN
Signaling through cGMP has therapeutic potential in the colon, where it has been implicated in the suppression of colitis and colon cancer. In this study, we tested the ability of cGMP and type 2 cGMP-dependent protein kinase (PKG2) to activate forkhead box O (FoxO) in colon cancer cells and in the colon epithelium of mice. We show that activation of PKG2 in colon cancer cells inhibited cell proliferation, inhibited AKT, and activated FoxO. Treatment of colon explants with 8Br-cGMP also activated FoxO target gene expression at both RNA and protein levels, and reduced epithelial reduction-oxidation (redox) stress. FoxO3a was the most prominent isoform in the distal colon epithelium, with prominent luminal staining. FoxO3a levels were reduced in Prkg2-/- animals, and FoxO target genes were unaffected by 8Br-cGMP challenge in vitro. Treatment of mice with the phosphodiesterase-5 inhibitor vardenafil (Levitra) mobilized FoxO3a to the nucleus of luminal epithelial cells, which corresponded to increased FoxO target gene expression, reduced redox stress, and increased epithelial barrier integrity. Treatment of human colonic biopsy specimens with 8Br-cGMP also activated catalase and manganese superoxide dismutase expression, indicating that this pathway is conserved in humans. Taken together, these results identify a novel signaling pathway in the colon epithelium, where FoxO tumor suppressors could provide protection from redox stress. Moreover, this pathway is regulated by endogenous cGMP/PKG2 signaling, and can be targeted using phosphodiesterase-5 inhibitors.
Asunto(s)
Antioxidantes/metabolismo , Neoplasias del Colon/metabolismo , Proteína Forkhead Box O3/metabolismo , Mucosa Intestinal/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Línea Celular Tumoral , GMP Cíclico/metabolismo , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TranscriptomaRESUMEN
Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), found in cooked meat, is a known food carcinogen that causes several types of cancer, including breast cancer, as PhIP metabolites produce DNA adduct and DNA strand breaks. Curcumin, obtained from the rhizome of Curcuma longa, has potent anticancer activity. To date, no study has examined the interaction of PhIP with curcumin in breast epithelial cells. The present study demonstrates the mechanisms by which curcumin inhibits PhIP-induced cytotoxicity in normal breast epithelial cells (MCF-10A). Curcumin significantly inhibited PhIP-induced DNA adduct formation and DNA double stand breaks with a concomitant decrease in reactive oxygen species (ROS) production. The expression of Nrf2, FOXO targets; DNA repair genes BRCA-1, H2AFX and PARP-1; and tumor suppressor P16 was studied to evaluate the influence on these core signaling pathways. PhIP induced the expression of various antioxidant and DNA repair genes. However, co-treatment with curcumin inhibited this expression. PhIP suppressed the expression of the tumor suppressor P16 gene, whereas curcumin co-treatment increased its expression. Caspase-3 and -9 were slightly suppressed by curcumin with a consequent inhibition of cell death. These results suggest that curcumin appears to be an effective anti-PhIP food additive likely acting through multiple molecular targets.
Asunto(s)
Antioxidantes/farmacología , Curcumina/farmacología , Células Epiteliales/efectos de los fármacos , Imidazoles/toxicidad , Glándulas Mamarias Humanas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citoprotección , Aductos de ADN/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A have been the subject of extensive studies; however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wild-type mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoter to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoter to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer, and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development.
Asunto(s)
Carcinoma/genética , Neoplasias del Colon/genética , Metilación de ADN/genética , Proteína p300 Asociada a E1A/genética , Interferón gamma/inmunología , Receptores Acoplados a Proteínas G/genética , Receptores Nicotínicos/genética , Acetilación , Animales , Apoptosis , Línea Celular Tumoral , Modelos Animales de Enfermedad , Genes Supresores de Tumor , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones NoqueadosRESUMEN
Signaling through cGMP has emerged as an important regulator of tissue homeostasis in the gastrointestinal tract, but the mechanism is not known. Type 2 cGMP-dependent protein kinase (PKG2) is a major cGMP effector in the gut epithelium, and the present studies have tested its importance in the regulation of proliferation and differentiation in the mouse colon and in colon cancer cell lines. Tissue homeostasis was examined in the proximal colon of Prkg2(-/-) mice using histological markers of proliferation and differentiation. The effect of ectopic PKG2 on proliferation and differentiation was tested in vitro using inducible colon cancer cell lines. PCR and luciferase reporter assays were used to determine the importance of Sox9 downstream of PKG2. The colons of Prkg2(-/-) mice exhibited crypt hyperplasia, increased epithelial apoptosis, and reduced numbers of differentiated goblet and enteroendocrine cells. Ectopic PKG2 was able to inhibit proliferation and induce Muc2 and CDX2 expression in colon cancer cells, but did not significantly affect cell death. PKG2 reduced Sox9 levels and signaling, suggesting possible involvement of this pathway downstream of cGMP in the colon. The work presented here demonstrates a novel antiproliferative and prodifferentiation role for PKG2 in the colon. These homeostatic functions of PKG2 were reproducible in colon cancer cells lines where downregulation of Sox9 is a possible mechanism. The similarities in phenotype between PKG2 and GCC knockout mice positions PKG2 as a likely mediator of the homeostatic effects of cGMP signaling in the colon.
Asunto(s)
Diferenciación Celular/fisiología , Proliferación Celular , Colon/enzimología , Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Mucosa Intestinal/enzimología , Animales , Apoptosis/genética , Factor de Transcripción CDX2 , Diferenciación Celular/genética , Línea Celular Tumoral , Colon/citología , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Proteína Quinasa Dependiente de GMP Cíclico Tipo II , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Proteínas de Homeodominio/análisis , Humanos , Mucosa Intestinal/citología , Ratones , Ratones Noqueados , Mucina 2/análisis , Factor de Transcripción SOX9/análisis , Factores de Transcripción/análisisRESUMEN
The NAD-dependent histone deacetylase silent information regulator 1 (SIRT1) is overexpressed and catalytically activated in a number of human cancers, but recent studies have actually suggested that it may function as a tumor suppressor and metastasis inhibitor in vivo. In breast cancer, SIRT1 stabilization has been suggested to contribute to the oncogenic potential of the estrogen receptor α (ERα), but SIRT1 activity has also been associated with ERα deacetylation and inactivation. In this study, we show that SIRT1 is critical for estrogen to promote breast cancer. ERα physically interacted and functionally cooperated with SIRT1 in breast cancer cells. ERα also bound to the promoter for SIRT1 and increased its transcription. SIRT1 expression induced by ERα was sufficient to activate antioxidant and prosurvival genes in breast cancer cells, such as catalase and glutathione peroxidase, and to inactivate tumor suppressor genes such as cyclin G2 (CCNG2) and p53. Moreover, SIRT1 inactivation eliminated estrogen/ERα-induced cell growth and tumor development, triggering apoptosis. Taken together, these results indicated that SIRT1 is required for estrogen-induced breast cancer growth. Our findings imply that the combination of SIRT1 inhibitors and antiestrogen compounds may offer more effective treatment strategies for breast cancer.