RESUMO
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.
Assuntos
Neoplasias do Colo , Inibidores da Fosfodiesterase 5 , Camundongos , Animais , Inibidores da Fosfodiesterase 5/farmacologia , Citrato de Sildenafila/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5 , Cromatografia Líquida , Espectrometria de Massas em Tandem , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/prevenção & controle , Proliferação de Células , GMP Cíclico/metabolismoRESUMO
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.
Assuntos
Colo , Neoplasias do Colo , Proteína Quinase Dependente de GMP Cíclico Tipo II , Animais , Azoximetano , Carcinogênese/patologia , Proliferação de Células , Colo/patologia , Neoplasias do Colo/patologia , GMP Cíclico/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo II/genética , Sulfato de Dextrana , Epitélio/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
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.
Assuntos
Neoplasias do Colo , Inibidores de Fosfodiesterase , Transformação Celular Neoplásica , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Humanos , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Antioxidantes/metabolismo , Neoplasias do Colo/metabolismo , Proteína Forkhead Box O3/metabolismo , Mucosa Intestinal/metabolismo , Transdução de Sinais/fisiologia , Animais , Western Blotting , Linhagem Celular Tumoral , GMP Cíclico/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TranscriptomaRESUMO
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.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células , Colo/enzimologia , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Mucosa Intestinal/enzimologia , Animais , Apoptose/genética , Fator de Transcrição CDX2 , Diferenciação Celular/genética , Linhagem Celular Tumoral , Colo/citologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Proteína Quinase Dependente de GMP Cíclico Tipo II , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas de Homeodomínio/análise , Humanos , Mucosa Intestinal/citologia , Camundongos , Camundongos Knockout , Mucina-2/análise , Fatores de Transcrição SOX9/análise , Fatores de Transcrição/análiseRESUMO
BACKGROUND: Type 1 cyclic 3',5'-guanosine monophosphate-dependent protein kinase (PKG) has recently been reported to inhibit tumor growth and angiogenesis. These effects suggest that PKG activation may have therapeutic value for colon cancer treatment, but the signaling downstream of this enzyme is poorly understood. The present study examined the mechanism underlying the inhibition of angiogenesis by PKG. METHODS: The effect of ectopically expressed PKG on colon cancer cell adaptation to a 1% O(2) (hypoxic) environment was examined in vitro by measuring hypoxic markers, cell death/viability, and hypoxia inducible factor (HIF) activity. RESULTS: Ectopic PKG inhibited angiogenesis in SW620 xenografts and significantly attenuated hypoxia-induced increases in vascular endothelial growth factor at both the mRNA and protein levels. PKG activation also blocked hypoxia-induced hexokinase 2 expression, which corresponded with reduced cellular adenosine triphosphate levels. Moreover, PKG expression significantly reduced cell viability and promoted necrotic cell death after 2 days in a hypoxic environment. To gain some mechanistic insight, the effect of PKG on HIF activation was determined using luciferase reporter assays. PKG activation inhibited HIF transcriptional activity in several colon cancer cell lines, including SW620, HCT116, and HT29. The mechanism by which PKG can inhibit HIF activity is not known, but it does not affect HIF-1α protein accumulation or nuclear translocation. CONCLUSIONS: These findings demonstrate for the first time that PKG can block the adaptation of colon cancer cells to hypoxia and highlights this enzyme for further evaluation as a potential target for colon cancer treatment.
Assuntos
Adaptação Fisiológica , Neoplasias do Colo/patologia , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Neovascularização Patológica/prevenção & controle , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/metabolismo , Humanos , Fator 1 Induzível por Hipóxia/fisiologia , Camundongos , NecroseRESUMO
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.
RESUMO
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.
Assuntos
Imunoterapia/métodos , Linfócitos T Citotóxicos/imunologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Evasão TumoralRESUMO
Large conductance, calcium- and voltage-activated potassium (BK(Ca)) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BK(Ca) channels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BK(Ca) channel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BK(Ca) channel alpha-subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BK(Ca) channels. Results showed that mutating S1076 altered the effect of PKC activation on BK(Ca) channels in HEK-293 cells. Specifically, the phospho-deficient mutation BK(Ca)-alpha(S1076A)/beta(1) attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BK(Ca) channels, whereas the phospho-mimetic mutation BK(Ca)-alpha(S1076E)/beta(1) increased the excitatory effect of PMA on BK(Ca) channels. In addition, the phospho-null mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BK(Ca) channels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BK(Ca) channel alpha-subunits influences BK(Ca) channel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.
Assuntos
Cálcio/metabolismo , Rim/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Mutação/genética , Proteína Quinase C-alfa/genética , Células Cultivadas , Humanos , Rim/citologia , Mutagênese Sítio-Dirigida , Fosforilação , Proteína Quinase C-alfa/metabolismoRESUMO
Patients with chronic inflammatory bowel disease have a high risk of colon cancer. The molecules that initiate and promote colon cancer and the cancer pathways altered remain undefined. Here, using in vitro models and a mouse model of colitis, we show that nitric oxide (NO) species induce retinoblastoma protein (pRb) hyperphosphorylation and inactivation, resulting in increased proliferation through the pRb-E2F1 pathway. NO-driven pRb hyperphosphorylation occurs through soluble guanylyl cyclase/guanosine 3',5'-cyclic monophosphate signaling and is dependent on the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase MEK/ERK and phosphatidylinositol 3-kinase/AKT pathways. Our results reveal a link between NO and pRb inactivation and provide insight into molecules that can be targeted in the prevention of the inflammation-to-cancer sequence.
Assuntos
Colite/metabolismo , Neoplasias do Colo/metabolismo , Óxido Nítrico/farmacologia , Proteína do Retinoblastoma/metabolismo , Animais , Colite/complicações , Colite/enzimologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/etiologia , GMP Cíclico/metabolismo , Fator de Transcrição E2F1/metabolismo , Guanilato Ciclase/metabolismo , Células HCT116 , Células HT29 , Humanos , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espermina/análogos & derivados , Espermina/farmacologiaRESUMO
Apoptotic resistance is often associated with metastatic phenotype in tumor cells and is considered a hallmark of tumor progression. In this study, IFN regulatory factor 8 (IRF8) expression was found to be inversely correlated with an apoptotic-resistant and metastatic phenotype in human colon carcinoma cell lines in vitro. This inverse correlation was further extended to spontaneously arising primary mammary carcinoma and lung metastases in a mouse tumor model in vivo. Exogenous expression of IRF8 in the metastatic tumor cell line restored, at least partially, the sensitivity of the tumor cells to Fas-mediated apoptosis, and disruption of IRF8 function conferred the poorly metastatic tumors with enhanced apoptotic resistance and metastatic capability. DNA demethylation restored IRF8 expression and sensitized the metastatic tumor cells to Fas-mediated apoptosis. Analysis of genomic DNA isolated from both primary and metastatic tumor cells with methylation-sensitive PCR revealed hypermethylation of the IRF8 promoter in metastatic tumor cells but not in primary tumor cells. Taken together, our data suggest that IRF8 is both an essential regulator in Fas-mediated apoptosis pathway and a metastasis suppressor in solid tumors and that metastatic tumor cells use DNA hypermethylation to repress IRF8 expression to evade apoptotic cell death and to acquire a metastatic phenotype.
Assuntos
Adenocarcinoma/genética , Apoptose/genética , Neoplasias do Colo/genética , Metilação de DNA , Fatores Reguladores de Interferon/genética , Neoplasias Mamárias Experimentais/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Humanos , Fatores Reguladores de Interferon/biossíntese , Fatores Reguladores de Interferon/deficiência , Interferon gama/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas , Transfecção , Receptor fas/biossíntese , Receptor fas/genéticaRESUMO
PURPOSE: One of the impediments of immunotherapy against cancer is the suppression of tumor-specific CTLs in the tumor microenvironment, partly due to the selective inhibition of the perforin pathway and the emergence of Fas-resistant tumors. Therefore, we sought to identify perforin- and Fas-independent cytotoxic pathways and explored the potential of targeting LTbetaR with tumor-specific CTLs to induce tumor rejection in vivo. EXPERIMENTAL DESIGN: Fas-resistant tumors were examined for their susceptibility to perforin-deficient (pfp) CTLs via CTL adoptive transfer in mouse models of experimental lung metastasis. The specificity of LTbetaR, a cell surface death receptor, in causing tumor rejection by CTLs was analyzed by LTbetaR-specific neutralizing monoclonal antibody in vitro. The specificity and efficacy of LTbetaR in the suppression of established tumors was further investigated by silencing LTbetaR in tumor cells in vivo. RESULTS: pfp CTLs exhibited significant cytotoxicity against Fas-resistant tumors in vivo. The perforin- and Fas-independent cytotoxicity was directly mediated, at least in part, by the adoptively transferred CTLs. It was observed that LTbetaR was expressed on the tumor cell surface, and LTalpha, LTbeta, and LIGHT, all of which are ligands for LTbetaR, were either constitutively expressed or activated in the tumor-specific CTLs and primary CD8(+) T cells. Blocking LTbetaR with LTbetaR-specific neutralizing monoclonal antibody decreased CTL cytotoxicity in vitro. Silencing LTbetaR using LTbetaR-specific short hairpin RNA reduced the ability of pfp CTLs to induce tumor rejection in vivo. CONCLUSION: LTbetaR directly mediates CTL-directed tumor rejection in vivo. Targeting LTbetaR with tumor-specific CTLs is a potential therapeutic approach.
Assuntos
Imunoterapia Adotiva , Linfotoxina-beta/imunologia , Neoplasias/terapia , Linfócitos T Citotóxicos/imunologia , Animais , Citotoxicidade Imunológica , Feminino , Rejeição de Enxerto , Interferon gama/farmacologia , Receptor beta de Linfotoxina/análise , Receptor beta de Linfotoxina/fisiologia , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Perforina , Proteínas Citotóxicas Formadoras de Poros/fisiologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Receptor fas/fisiologiaRESUMO
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.
RESUMO
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.
Assuntos
Carcinogênese/metabolismo , Colite/complicações , Neoplasias do Colo/etiologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Inativação Gênica , Fatores Reguladores de Interferon/genética , Interleucina-10/biossíntese , Células Supressoras Mieloides/metabolismo , Animais , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Inflamação/patologia , Fatores Reguladores de Interferon/metabolismo , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Fator de Transcrição STAT3/metabolismo , Regulação para Cima , DNA Metiltransferase 3BRESUMO
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.
Assuntos
Polipose Adenomatosa do Colo/prevenção & controle , Transformação Celular Neoplásica/efeitos dos fármacos , GMP Cíclico/metabolismo , Agonistas da Guanilil Ciclase C/farmacologia , Neoplasias Intestinais/prevenção & controle , Lesões Pré-Cancerosas/prevenção & controle , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Feminino , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/farmacologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Citrato de Sildenafila/farmacologiaRESUMO
Anoikis is an essential process in which a loss of adhesion to the substratum alters intracellular signaling pathways that lead to apoptosis. Using phosphorylation of vasodilator stimulated phosphoprotein (VASP) as an indicator of cGMP-dependent protein kinase (PKG) activity in vivo, it was found that suspension of the colon epithelial cell line (CCD841) leads to rapid and transient activation of PKG that lasted several hours. The colon carcinoma lines SW480 and SW620 do not express endogenous PKG, but exogenously expressed PKG was similarly activated upon cell suspension. To determine whether PKG has a role in apoptosis following cell suspension, poly-ADP ribose polymerase (PARP) cleavage and propidium iodide staining were measured. After 24 h in suspension it was found that approximately 50% of CCD841 cells exhibited apoptosis, whereas apoptosis was not detected in either of the colon carcinoma cell lines. Inhibition of type 1 PKG by expression of a dominant negative PKG construct (G1alphaR-GFP), or by incubation with the PKG inhibitor peptide DT-2, blocked apoptosis in suspended CCD841 cells by approximately 50%. Furthermore, expression of exogenous PKG in SW620 and SW480 cells conferred partial sensitivity anoikis. Taken together these findings indicate that PKG has an important role in the induction of apoptosis following suspension of normal colon epithelial cells, and loss of PKG expression in colon tumor cells may contribute to resistance to anoikis.
Assuntos
Anoikis/fisiologia , Carcinoma/enzimologia , Neoplasias do Colo/enzimologia , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Células Epiteliais/enzimologia , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Células Cultivadas , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/genética , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Técnicas de Transferência de Genes , Humanos , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Células Tumorais CultivadasRESUMO
The biological functions of heterotrimeric G proteins and small GTPases are modulated by both extracellular stimuli and intracellular regulatory proteins. Using Saccharomyces cerevisiae two-hybrid screening, we identified tetratricopeptide repeat 1 (TPR1), a 292-amino-acid protein with three TPR motifs, as a Galpha16-binding protein. The interaction was confirmed both in vitro and in transfected mammalian cells, where TPR1 also binds to several other Galpha proteins. TPR1 was found to interact with Ha-Ras preferentially in its active form. Overexpression of TPR1 promotes accumulation of active Ras. TPR1 was found to compete with the Ras-binding domain (RBD) of Raf-1 for binding to the active Ras, suggesting that it may also compete with Ras GTPase-activating protein, thus contributing to the accumulation of GTP-bound Ras. Expression of Galpha16 strongly enhances the interaction between TPR1 and Ras. Removal of the TPR1 N-terminal 112 residues abolishes potentiation by Galpha16 while maintaining the interaction with Galpha16 and the ability to discriminate active Ras from wild-type Ras. We have also observed that LGN, a Galphai-interacting protein with seven TPR motifs, binds Ha-Ras. Thus, TPR1 is a novel adaptor protein for Ras and selected Galpha proteins that may be involved in protein-protein interaction relating to G-protein signaling.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Sequências Repetitivas de Ácido Nucleico , Proteínas ras/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Motivos de Aminoácidos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/fisiologia , Técnicas do Sistema de Duplo-Híbrido , Proteínas ras/genéticaRESUMO
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.
Assuntos
Constipação Intestinal/tratamento farmacológico , Trânsito Gastrointestinal/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/uso terapêutico , Citrato de Sildenafila/uso terapêutico , Administração Oral , Animais , Colite/induzido quimicamente , Colite/tratamento farmacológico , GMP Cíclico/metabolismo , Sulfato de Dextrana , Avaliação Pré-Clínica de Medicamentos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/administração & dosagem , Peptídeos/uso terapêutico , Inibidores da Fosfodiesterase 5/administração & dosagem , Citrato de Sildenafila/administração & dosagemRESUMO
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.
Assuntos
Antiparkinsonianos/farmacologia , Autoimunidade/efeitos dos fármacos , Carbidopa/farmacologia , Dopaminérgicos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Mediadores da Inflamação/metabolismo , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismoRESUMO
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.