RESUMO
Colon cancer cells contain high levels of cystathionine-beta-synthase (CBS). Its product, hydrogen sulfide (H2S) promotes the growth and proliferation of colorectal tumor cells. In order to improve the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle, AOAA (1, 3 or 9 mg/kg/day) or YD0171 (0.1, 0.5 or 1 mg/kg/day) for 3 weeks. Tumor growth was significantly reduced by 9 mg/kg/day AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/day. Thus, the in vivo efficacy of YD0171 is 9-times higher than that of AOAA. YD0171 (1 mg/kg/day) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/day) also reduced tumor growth in patient-derived tumor xenograft (PDTX) bearing athymic mice. YD0171 (3 mg/kg/day) induced the regression of established HCT116 tumors in vivo. A 5-day safety study in mice demonstrated that YD0171 at 20 mg/kg/day (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory anticancer prodrugs. By targeting colorectal cancer bioenergetics, an emerging important hallmark of cancer, the approach exemplified herein may offer direct translational opportunities.
RESUMO
Cystathionine-ß-synthase (CBS) has been recently identified as a drug target for several forms of cancer. Currently no potent and selective CBS inhibitors are available. Using a composite collection of 8871 clinically used drugs and well-annotated pharmacological compounds (including the LOPAC library, the FDA Approved Drug Library, the NIH Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the 'Killer Plates' collection and a small custom collection of PLP-dependent enzyme inhibitors), we conducted an in vitro screen in order to identify inhibitors for CBS using a primary 7-azido-4-methylcoumarin (AzMc) screen to detect CBS-derived hydrogen sulfide (H2S) production. Initial hits were subjected to counterscreens using the methylene blue assay (a secondary assay to measure H2S production) and were assessed for their ability to quench the H2S signal produced by the H2S donor compound GYY4137. Four compounds, hexachlorophene, tannic acid, aurintricarboxylic acid and benserazide showed concentration-dependent CBS inhibitory actions without scavenging H2S released from GYY4137, identifying them as direct CBS inhibitors. Hexachlorophene (IC50: â¼60µM), tannic acid (IC50: â¼40µM) and benserazide (IC50: â¼30µM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: â¼3µM) and NSC67078 (IC50: â¼1µM), while aurintricarboxylic acid (IC50: â¼3µM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: â¼1µM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50 of â¼6µM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: â¼6µM), tannic acid (IC50: â¼20µM), benserazide (IC50: â¼20µM), and NSC67078 (IC50: â¼0.3µM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: â¼300µM). In contrast, although a CBS inhibitor in the cell-free assay, aurintricarboxylic acid failed to inhibit HCT116 proliferation at lower concentrations, and stimulated cell proliferation at 300µM. Copper-containing compounds present in the libraries, were also found to be potent inhibitors of recombinant CBS; however this activity was due to the CBS inhibitory effect of copper ions themselves. However, copper ions, up to 300µM, did not inhibit HCT116 cell proliferation. Benserazide was only a weak inhibitor of the activity of the other H2S-generating enzymes CSE and 3-MST activity (16% and 35% inhibition at 100µM, respectively) in vitro. Benserazide suppressed HCT116 mitochondrial function and inhibited proliferation of the high CBS-expressing colon cancer cell line HT29, but not the low CBS-expressing line, LoVo. The major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity and suppressed HCT116 cell proliferation in vitro. In an in vivo study of nude mice bearing human colon cancer cell xenografts, benserazide (50mg/kg/days.q.) prevented tumor growth. In silico docking simulations showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.
Assuntos
Benserazida/farmacologia , Neoplasias do Colo/tratamento farmacológico , Cistationina beta-Sintase/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cumarínicos/farmacologia , Reposicionamento de Medicamentos/métodos , Metabolismo Energético/efeitos dos fármacos , Feminino , Células HCT116 , Células HT29 , Humanos , Hidrazinas/farmacologia , Sulfeto de Hidrogênio/metabolismo , Masculino , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Morfolinas/farmacologia , Compostos Organotiofosforados/farmacologia , Terapias em Estudo/métodosRESUMO
The physiological functions of hydrogen sulfide (H2S) include vasorelaxation, stimulation of cellular bioenergetics, and promotion of angiogenesis. Analysis of human colon cancer biopsies and patient-matched normal margin mucosa revealed the selective up-regulation of the H2S-producing enzyme cystathionine-ß-synthase (CBS) in colon cancer, resulting in an increased rate of H2S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H2S production, compared with the nonmalignant colonic mucosa cells, NCM356. CBS localized to the cytosol, as well as the mitochondrial outer membrane. ShRNA-mediated silencing of CBS or its pharmacological inhibition with aminooxyacetic acid reduced HCT116 cell proliferation, migration, and invasion; reduced endothelial cell migration in tumor/endothelial cell cocultures; and suppressed mitochondrial function (oxygen consumption, ATP turnover, and respiratory reserve capacity), as well as glycolysis. Treatment of nude mice with aminooxyacetic acid attenuated the growth of patient-derived colon cancer xenografts and reduced tumor blood flow. Similarly, CBS silencing of the tumor cells decreased xenograft growth and suppressed neovessel density, suggesting a role for endogenous H2S in tumor angiogenesis. In contrast to CBS, silencing of cystathionine-γ-lyase (the expression of which was unchanged in colon cancer) did not affect tumor growth or bioenergetics. In conclusion, H2S produced from CBS serves to (i) maintain colon cancer cellular bioenergetics, thereby supporting tumor growth and proliferation, and (ii) promote angiogenesis and vasorelaxation, consequently providing the tumor with blood and nutritients. The current findings identify CBS-derived H2S as a tumor growth factor and anticancer drug target.
Assuntos
Proliferação de Células , Neoplasias do Colo/metabolismo , Cistationina beta-Sintase/metabolismo , Metabolismo Energético , Sulfeto de Hidrogênio/metabolismo , Neovascularização Patológica , Animais , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/patologia , Feminino , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
cAMP plays a critical role in regulating migration of various cancers. This role is context dependent and is determined by which of the two main cAMP sensors is at play: cAMP-dependent protein kinase or exchange protein directly activated by cAMP (EPAC). Recently, we have shown that the cAMP sensor protein EPAC1 promotes invasion/migration of pancreatic ductal adenocarcinoma (PDA) in vitro. In this study, we investigated the role of EPAC1 in invasion and metastasis of PDA in vivo, and evaluated the therapeutic potential of EPAC inhibitors as antimetastasis agents for this neoplasm. We employed an orthotopic metastatic mouse model in which the PDA cells MIA PaCa-2 were injected into the pancreas of athymic nude mice, and their local and distant spread was monitored by in vivo imaging and histologic evaluation of the number of metastatic foci in the liver. Either genetic suppression of EPAC1 or its pharmacologic inhibition with 3-(5-tert-butyl-isoxazol-3-yl)-2-[(3-chloro-phenyl)-hydrazono]-3-oxo-propionitrile, an EPAC-specific antagonist recently identified in our laboratory, decreased invasion and metastasis of the PDA cells. Mechanistically, EPAC1 promotes activation and trafficking of integrin ß1, which plays an essential role in PDA migration and metastasis. Our data show that EPAC1 facilitates metastasis of PDA cells and EPAC1 might be a potential novel therapeutic target for developing antimetastasis agents for PDA.
Assuntos
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Animais , Carcinoma Ductal Pancreático/metabolismo , Feminino , Técnicas de Silenciamento de Genes/métodos , Fatores de Troca do Nucleotídeo Guanina/deficiência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias Pancreáticas/metabolismo , Piperidinas/farmacologiaRESUMO
BACKGROUND: Chronic pancreatitis (CP) is characterized by recurrent pancreatic injury, resulting in inflammation, necrosis, and fibrosis. There are currently no drugs limiting pancreatic fibrosis associated with CP, and there is a definite need to fill this void in patient care. MATERIALS AND METHODS: Pancreatitis was induced in C57/BL6 mice using supraphysiologic doses of cerulein, and apigenin treatment (once daily, 50 µg per mouse by oral gavage) was initiated 1 wk into the recurrent acute pancreatitis (RAP) protocol. Pancreata were harvested after 4 wk of RAP. Immunostaining with fibronectin antibody was used to quantify the extent of pancreatic fibrosis. To assess how apigenin may decrease organ fibrosis, we evaluated the effect of apigenin on the proliferation and apoptosis of human pancreatic stellate cells (PSCs) in vitro. Finally, we assessed apigenin's effect on the gene expression in PSCs stimulated with parathyroid hormone-related protein, a profibrotic and proinflammatory mediator of pancreatitis, using reverse transcription-polymerase chain reaction. RESULTS: After 4 wk of RAP, apigenin significantly reduced the fibrotic response to injury while preserving acinar units. Apigenin inhibited viability and induced apoptosis of PSCs in a time- and dose-dependent manner. Finally, apigenin reduced parathyroid hormone-related protein-stimulated increases in the PSC messenger RNA expression levels of extracellular matrix proteins collagen 1A1 and fibronectin, proliferating cell nuclear antigen, transforming growth factor-beta, and interleukin-6. CONCLUSIONS: These in vivo and in vitro studies provide novel insights regarding apigenin's mechanism(s) of action in reducing the severity of RAP. Additional preclinical testing of apigenin analogs is warranted to develop a therapeutic agent for patients at risk for CP.
Assuntos
Apigenina/uso terapêutico , Células Estreladas do Pâncreas/efeitos dos fármacos , Pancreatite Crônica/tratamento farmacológico , Animais , Apigenina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Estreladas do Pâncreas/patologia , Pancreatite Crônica/metabolismo , Pancreatite Crônica/patologia , Proteína Relacionada ao Hormônio Paratireóideo/farmacologiaRESUMO
Recent studies revealed increased expression of various hydrogen sulfide (H2S)-producing enzymes in cancer cells of various tissue types, and new roles of H2S in the pathophysiology of cancer have emerged. This is particularly evident in cancers of the colon and ovaries, where the malignant cells both overexpress cystathionine-ß-synthase (CBS) and produce increased amounts of H2S, which enhances tumor growth and spread by (a) stimulating cellular bioenergetics, (b) activating proliferative, migratory, and invasive signaling pathways, and (c) enhancing tumor angiogenesis. Importantly, in preclinical models of these cancers, either pharmacological inhibition or genetic silencing of CBS was shown to be sufficient to suppress cancer cell bioenergetics in vitro, inhibit tumor growth and metastasis in vivo, and enhance the antitumor efficacy of frontline chemotherapeutic agents, providing a strong rationale for the development of CBS-targeted inhibitors as anticancer therapies. However, the observation that inhibition of H2S biosynthesis exerts anticancer effects is contradicted by other studies showing that increasing H2S with exogenous donors also exerts antitumor actions. Herein, we present a brief review of the scientific literature documenting the function of H2S, H2S donors, and transsulfuration enzymes in cancers from various tissue types, and propose that the paradoxical actions of H2S can be resolved by considering the bell-shaped pharmacology of H2S, whereby lower (endogenous) H2S production tends to promote, while higher (generated from exogenously added H2S donors) tends to inhibit cancer cell proliferation. Finally, we suggest areas for future investigations to expand our knowledge of this nascent field.
Assuntos
Sulfeto de Hidrogênio/metabolismo , Neoplasias/patologia , Proliferação de Células , HumanosRESUMO
Recent data show that colon cancer cells selectively overexpress cystathionine-ß-synthase (CBS), which produces hydrogen sulfide (H2S), to maintain cellular bioenergetics, support tumor growth and stimulate angiogenesis and vasorelaxation in the tumor microenvironment. The purpose of the current study was to investigate the effect of the allosteric CBS activator S-adenosyl-L-methionine (SAM) on the proliferation and bioenergetics of the CBS-expressing colon cancer cell line HCT116. The non-transformed, non-tumorigenic colon epithelial cell line NCM356 was used as control. For assessment of cell proliferation, the xCELLigence system was used. Bioenergetic function was measured by Extracellular Flux Analysis. Experiments using human recombinant CBS or HCT116 homogenates complemented the cell-based studies. SAM markedly enhanced CBS-mediated H2S production in vitro, especially when a combination of cysteine and homocysteine was used as substrates. Addition of SAM (0.1-3 mM) to HCT116 cells induced a concentration-dependent increase H2S production. SAM exerted time- and concentration-dependent modulatory effects on cell proliferation. At 0.1-1 mM SAM increased HCT116 proliferation between 0 and 12 h, while the highest SAM concentration (3 mM) inhibited proliferation. Over a longer time period (12-24 h), only the lowest concentration of SAM used (0.1 mM) stimulated cell proliferation; higher SAM concentrations produced a concentration-dependent inhibition. The short-term stimulatory effects of SAM were attenuated by the CBS inhibitor aminooxyacetic acid (AOAA) or by stable silencing of CBS. In contrast, the inhibitory effects of SAM on cell proliferation was unaffected by CBS inhibition or CBS silencing. In contrast to HCT116 cells, the lower rate of proliferation of the low-CBS expressor NCM356 cells was unaffected by SAM. Short-term (1 h) exposure of HCT116 cells to SAM induced a concentration-dependent increase in oxygen consumption and bioenergetic function at 0.1-1 mM, while 3 mM was inhibitory. Longer-term (72 h) exposure of HCT116 cells to all concentrations of SAM tested suppressed mitochondrial oxygen consumption rate, cellular ATP content and cell viability. The stimulatory effect of SAM on bioenergetics was attenuated in cells with stable CBS silencing, while the inhibitory effects were unaffected. In NCM356 cells SAM exerted smaller effects on cellular bioenergetics than in HCT116 cells. We have also observed a downregulation of CBS in response to prolonged exposure of SAM both in HCT116 and NCM356 cells. Taken together, the results demonstrate that H2S production in HCT116 cells is stimulated by the allosteric CBS activator, SAM. At low-to intermediate levels and early time periods the resulting H2S serves as an endogenous cancer cell growth and bioenergetic factor. In contrast, the inhibition of cell proliferation and bioenergetic function by SAM does not appear to relate to adverse autocrine effects of H2S resulting from CBS over-stimulation but, rather to CBS-independent pharmacological effects.
Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Cistationina beta-Sintase/metabolismo , Metabolismo Energético/efeitos dos fármacos , S-Adenosilmetionina/farmacologia , Linhagem Celular , Cistationina beta-Sintase/efeitos dos fármacos , Cistationina beta-Sintase/genética , Relação Dose-Resposta a Droga , Inativação Gênica , Células HCT116 , Humanos , Sulfeto de Hidrogênio/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , RNA Interferente PequenoRESUMO
Accumulating evidence suggests that activated pancreatic stellate cells (PSC) play an important role in chronic pancreatitis (CP), and inhibition of the activated PSC is considered as a potential strategy for the treatment and prevention of CP. Herein, we disclose our findings that apigenin and its novel analogues suppress the proliferation and induce apoptosis in PSC, which reduce the PSC-mediated fibrosis in CP. Chemical modifications of apigenin have been directed to build a focused library of O-alkylamino-tethered apigenin derivatives at 4'-O position of the ring C with the attempt to enhance the potency and drug-like properties including aqueous solubility. A number of compounds such as 14, 16, and 24 exhibited potent antiproliferative effects as well as improved aqueous solubility. Intriguingly, apigenin, new analogues 23 and 24 displayed significant efficacy to reduce pancreatic fibrosis even at a low dose of 0.5mg/kg in our proof-of-concept study using a preclinical in vivo mouse model of CP.
Assuntos
Apigenina/farmacologia , Desenho de Fármacos , Fibrose/tratamento farmacológico , Células Estreladas do Pâncreas/efeitos dos fármacos , Pancreatite Crônica/tratamento farmacológico , Apigenina/síntese química , Apigenina/química , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fibrose/patologia , Humanos , Estrutura Molecular , Células Estreladas do Pâncreas/citologia , Pancreatite Crônica/patologia , Solubilidade , Relação Estrutura-AtividadeRESUMO
Excessive alcohol consumption is associated with most cases of chronic pancreatitis, a progressive necrotizing inflammatory disease that can result in pancreatic insufficiency due to acinar atrophy and fibrosis and an increased risk of pancreatic cancer. At a cellular level acute alcohol exposure can sensitize pancreatic acinar cells to secretagogue stimulation, resulting in dysregulation of intracellular Ca(2+) homeostasis and premature digestive enzyme activation; however, the molecular mechanisms by which ethanol exerts these toxic effects have remained undefined. In this study we identify Raf-1 kinase inhibitory protein as an essential mediator of ethanol-induced sensitization of cholecystokinin- and carbachol-regulated Ca(2+) signaling in pancreatic acinar cells. We show that exposure of rodent acinar cells to ethanol induces protein kinase C-dependent Raf-1 kinase inhibitory protein phosphorylation, sensitization of cholecystokinin-stimulated Ca(2+) signaling, and potentiation of both basal and cholecystokinin-stimulated extracellular signal-regulated kinase activation. Furthermore, we show that either suppression of Raf-1 kinase inhibitory protein expression using short hairpin RNA or gene ablation prevented the sensitizing effects of ethanol on cholecystokinin- and carbachol-stimulated Ca(2+) signaling and intracellular chymotrypsin activation in pancreatic acinar cells, suggesting that the modulation of Raf-1 inhibitory protein expression may have future therapeutic utility in the prevention or treatment of alcohol-associated pancreatitis.
Assuntos
Proteína de Ligação a Fosfatidiletanolamina/fisiologia , Células Acinares/citologia , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular , Depressores do Sistema Nervoso Central/farmacologia , Quimotripsina/química , Etanol/farmacologia , Matriz Extracelular/metabolismo , Camundongos , Pâncreas/metabolismo , Pancreatite/induzido quimicamente , Pancreatite/prevenção & controle , Proteína de Ligação a Fosfatidiletanolamina/genética , Fosforilação , Proteína Quinase C/metabolismo , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Risco , Transdução de SinaisRESUMO
Activation of pancreatic stellate cells (PSCs) by transforming growth factor (TGF)-ß is the key step in the development of pancreatic fibrosis, a common pathological feature of chronic pancreatitis (CP). Bone morphogenetic proteins (BMPs), members of the TGF-ß superfamily, have anti-fibrogenic functions, in contrast to TGF-ß, in the kidney, lung, and liver. However, it is not known whether BMPs have an anti-fibrogenic role in the pancreas. The current study was designed to investigate the potential anti-fibrogenic role of BMPs in the pancreas using an in vivo CP model and an in vitro PSC model. CP was induced by repetitive intraperitoneal injections of cerulein in adult Swiss Webster mice. The control mice received saline injections. Compared with the control, cerulein injections induced a time-dependent increase in acinar injury and progression of fibrosis and a steady increase in inflammation. Cerulein injections also induced increases of the extracellular matrix (ECM) protein fibronectin and of α-smooth muscle actin (α-SMA)-positive stellate cells (PSCs). The mice receiving cerulein injections showed increased BMP2 protein levels and phosphorylated Smad1 levels up to 4 wk and then declined at 8 wk to similar levels as the control. In vitro, the isolated mouse and human PSCs were cultured and pretreated with BMP2 followed by TGF-ß treatment. BMP2 pretreatment inhibited TGF-ß-induced α-SMA, fibronectin, and collagen type Ia expression. Knocking down Smad1 with small-interfering RNA reversed the inhibitory effect of BMP2 on TGF-ß-induced α-SMA and fibronectin expression. Thus, BMP2 opposes the fibrogenic function of TGF-ß in PSCs through the Smad1 signaling pathway.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Células Estreladas do Pâncreas/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Actinas/metabolismo , Animais , Ceruletídeo/farmacologia , Matriz Extracelular/metabolismo , Feminino , Fibronectinas/biossíntese , Fibrose , Humanos , Camundongos , Pâncreas/patologia , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/patologia , Transdução de Sinais , Proteína Smad1/fisiologia , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
Human colorectal cancer (CRC) cells are resistant to the anti-proliferative effect of transforming growth factor-ß (TGF-ß), suggesting that disruption of TGF-ß signaling plays an important role in colorectal carcinogenesis. Ecotropic virus integration site-1 (Evi-1) oncoprotein represses TGF-ß signaling by interacting with Smads, but its role in CRC has not been established. The purpose of this study is to determine whether Evi-1 plays role(s) in CRCs and to characterize Evi-1 transcript(s) in CRCs. Evi-1 was overexpressed in 53% of human CRC samples, 100% of colon adenoma samples, and 100% of human colon cancer cell lines tested. Using 5' RACE, we cloned a novel Evi-1 transcript (Evi-1e) from a human CRC tissue and found that this novel transcript was expressed at a higher level in CRC tissues than in normal tissues and was the major Evi-1 transcript in CRCs. Transient Evi-1 transfection inhibited TGF-ß-induced transcriptional activity and reversed the growth inhibitory effect of TGF-ß in MC-26 mouse colon cancer cells. In conclusion, we have identified overexpression of Evi-1 oncoprotein as a novel mechanism by which a subset of human CRCs may escape TGF-ß regulation. We have also identified a novel Evi-1 transcript, Evi-1e, as the major Evi-1 transcript expressed in human CRCs.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a DNA/genética , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/metabolismo , Éxons , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Regiões Promotoras Genéticas , Reto/metabolismo , Reto/patologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Experimental data indicate that colorectal cancer cells with CD133 expression exhibit enhanced tumorigenicity over CD133-negative (CD133-) cells. We hypothesized that CD133-positive (CD133+) cells, compared with CD133-, are more tumorigenic because they are more interactive with and responsive to their stromal microenvironment. Freshly dissected and dissociated cells from a primary colon cancer were separated into carcinoma-associated fibroblasts (CAF) and the epithelial cells; the latter were further separated into CD133+ and CD133- cells using fluorescence-activated cell sorter. The CD133+ cells formed large tumors in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice, demonstrating the phenotypic cellular diversity of the original tumor, whereas CD133- cells were unable to sustain significant growth. Affymetrix gene array analyses using t-test, fold-change and multiple test correction identified candidate genes that were differentially expressed between the CD133+ vs CD133- cells. RT-PCR verified differences in expression for 30 of the 46 genes selected. Genes upregulated (+ vs - cells) included CD133 (9.3-fold) and CXCR4 (4-fold), integrin ß8 and fibroblast growth factor receptor 2. The CAF highly express the respective ligands: stromal-derived factor-1 (SDF-1), vitronectin and FGF family members, suggesting a reciprocal relationship between the CD133+ and CAF cells. SDF-1 caused an increase in intracellular calcium in cells expressing both CD133 and CXCR4, confirming functional CXCR4. The CD133+/CXCR4+ phenotype is increased to 32% when the cells are grown in suspension compared with only 9% when the cells were allowed to attach. In Matrigel 3-D culture, the CD133+/CXCR4+ group treated with SDF-1 grew more colonies compared with vehicle, as well as significantly larger colony sizes of tumor spheres. These data demonstrate proof of principle that the enhanced tumorigenic potential of CD133+, compared with CD133-, cells is due to their increased ability to interact with their neighboring CAF.
Assuntos
Antígenos CD/metabolismo , Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Glicoproteínas/metabolismo , Peptídeos/metabolismo , Antígeno AC133 , Idoso , Animais , Técnicas de Cultura de Células , Transformação Celular Neoplásica , Células Cultivadas , Quimiocina CXCL12/metabolismo , Feminino , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Comunicação Parácrina , Fenótipo , Receptores CXCR4/metabolismoRESUMO
The Interprofessional Research Design course uses authentic learning pedagogy to bring together students from different education tracks (PhD, MD, MD/PhD training) to engage in interprofessional collaborative skills toward completion of a capstone project, a National Health Institutes (NIH) R21-style grant proposal. The course, underpinned by principles of team science, begins with a leadership training workshop to introduce students to effective leadership and teamwork strategies for interprofessional team environments. We used several assessments during the course to monitor leadership and team dynamics. We analyzed three assessments (leadership self-efficacy testing, iterative team contracts and reflective essays) to better understand students' learning experiences. Self-efficacy testing was administered before leadership training (pre) and at the end of the course (post-then); scores were analyzed using a repeated repeated measures ANOVA. Iterative team contracts were analyzed qualitatively using both deductive and descriptive methods. Reflective essays were analyzed using a general inductive approach. Nine teams of 32 students (23 MD; 9 PhD) participated in the class over 2017-2018. Self-efficacy testing using post-then timing to control for response shift showed a statistically significant increase in self-efficacy across all measures. Deductive qualitative analysis of iterative contracting showed evidence of team processes which support successful team performance; and descriptive analysis mapped productive behaviors. In reflective essays, seven of the nine teams collectively described their experiences positively; e.g., themes included empathizing with group members, sophisticated communication and collaborative workflow/styles. For negative experiences, themes were related to basic communication, poor integration and the theory-practice gap of leadership training. These findings demonstrate the usefulness of an authentic learning pedagogy focused on teaching the practice of team science.
RESUMO
Ulcerative colitis (UC) is characterized by widespread relapsing inflammation of the colonic mucosa. Colitis-associated cancer (CAC) is one of the most serious complications of a prolonged history of UC. Hydrogen sulfide (H2S) has emerged as an important physiological mediator of gastrointestinal homeostasis, limiting mucosal inflammation and promoting tissue healing in response to injury. Inhibition of cystathionine-γ-lyase (CSE)-dependent H2S production in animal models of UC has been shown to exacerbate colitis and delay tissue repair. It is unknown whether CSE plays a role in CAC, or the downregulation of CSE expression and/or activity promotes CAC development. In humans, we observed a significant decrease in CSE expression in colonic biopsies from patients with UC. Using the dextran sodium sulfate (DSS) model of epithelium injury-induced colitis and global CSE KO mouse strain, we demonstrated that CSE is critical in limiting mucosal inflammation and stimulating epithelial cell proliferation in response to injury. In vitro studies showed that CSE activity stimulates epithelial cell proliferation, basal and cytokine-stimulated cell migration, as well as cytokine regulation of transepithelial permeability. In the azoxymethane (AOM)/DSS model of CAC, the loss of CSE expression accelerated both the development and progression of CAC. The increased tumor multiplicity and severity of CAC observed in CSE-KO mice were associated with reduced levels of mucosal IL-10 expression and increased levels of IL-6. Restoring CSE expression in bone marrow (BM) cells of CSE-KO mice through reciprocal BM transplantation raised mucosal IL-10 expression, decreased IL-6 level, and reduced the number of aberrant crypt foci and tumors in AOM/DSS-treated mice. These studies demonstrate that CSE expression in BM cells plays a critical role in suppressing CAC in mice. Furthermore, the data suggest that the inhibitory effects of CSE on the development of CAC are due, in part, to the modulation of mucosal pro-and anti-inflammatory cytokine expression.
RESUMO
BACKGROUND: Cyclooxygenase-2 (COX-2) and the bombesin (BBS)-like peptide, gastrin-releasing peptide (GRP), have been implicated in the progression of hormone-refractory prostate cancer; however, a mechanistic link between the bioactive peptide and COX-2 expression in prostate cells has not been made. RESULTS: We report that BBS stimulates COX-2 mRNA and protein expression, and the release of prostaglandin E2 from the GRP receptor (GRPR)-positive, androgen-insensitive prostate cancer cell line, PC-3. BBS-stimulated COX-2 expression is mediated, in part, by p38MAPK and PI3 kinase (PI3K)/Akt pathways, and blocked by a GRPR antagonist. The PI3K/Akt pathway couples GRPR to the transcription factor, activator protein-1 (AP-1), and enhanced COX-2 promoter activity. Although BBS stimulates nuclear factor-kappaB (NF-κB) in PC-3, NF-κB does not regulate GRPR-mediated COX-2 expression. The p38MAPK pathway increases BBS-stimulated COX-2 expression by slowing the degradation of COX-2 mRNA. Expression of recombinant GRPR in the androgen-sensitive cell line LNCaP is sufficient to confer BBS-stimulated COX-2 expression via the p38MAPK and PI3K/Akt pathways. CONCLUSIONS: Our study establishes a mechanistic link between GRPR activation and enhanced COX-2 expression in prostate cancer cell lines, and suggests that inhibiting GRPR may, in the future, provide an effective therapeutic alternative to non-steroidal anti-inflammatory drugs for inhibiting COX-2 in patients with recurrent prostate cancer.
Assuntos
Bombesina/metabolismo , Ciclo-Oxigenase 2/genética , Dinoprostona/metabolismo , Regulação Neoplásica da Expressão Gênica , Neurotransmissores/metabolismo , Neoplasias da Próstata/metabolismo , Receptores da Bombesina/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Cromonas/farmacologia , Ciclo-Oxigenase 2/metabolismo , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , MAP Quinase Quinase Quinases/metabolismo , Masculino , Morfolinas/farmacologia , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/genética , RNA Mensageiro/genética , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Upregulation of hydrogen sulfide (H2S) biosynthesis, at least in part related to the upregulation of cystathionine ß-synthetase (CBS) in cancer cells, serves as a tumor-promoting factor and has emerged as a possible molecular target for antitumor drug development. To facilitate future clinical translation, we have synthesized a variety of novel CBS-targeting, esterase-cleavable prodrugs based on the structure of the prototypical CBS inhibitor aminooxyacetic acid (AOAA). The pharmacological properties of these compounds were evaluated in cell-free assays with recombinant human CBS protein, the human colon cancer cell line HCT116, and in vivo using various tumor-bearing mice models. The prodrug YD0251 (the isopropyl ester derivative of AOAA) was selected for detailed characterization. YD0251 exhibits improved antiproliferative efficacy in cell culture models when compared to AOAA. It is up to 18 times more potent than AOAA at suppressing HCT116 tumor growth in vivo and is effective when administered to tumor-bearing mice either via subcutaneous injection or oral gavage. Patient-derived xenografts (PDTXs) with higher levels of CBS protein grew significantly larger than tumors with lower levels, and YD0251 treatment inhibited the growth of PDTXs with elevated CBS, whereas it had no significant effect on PDTXs with low CBS protein levels. The toxicity of YD0251 was assessed in mice subjected to subchronic administration of supratherapeutic doses the inhibitor; no significant alteration in circulating markers of organ injury or histopathological alterations were noted, up to 60 mg/kg/day × 5 days. In preparation to a future theranostic concept (to match CBS inhibitor therapy to high-CBS expressors), we identified a potential plasma marker of CBS-expressing tumors. Colon cancer cells produced significant levels of lanthionine, a rare metabolic intermediate of CBS-mediated H2S biosynthesis; forced expression of CBS into non-transformed epithelial cells increased lanthionine biogenesis in vitro and in vivo (measured in the urine of tumor-bearing mice). These current results may be useful to facilitate the translation of a CBS inhibition-based antitumor concept into the clinical space.
Assuntos
Ácido Amino-Oxiacético/farmacologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Cistationina beta-Sintase/antagonistas & inibidores , Pró-Fármacos/farmacologia , Animais , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
Expression of gastrin and cholecystokinin 2 (CCK(2)) receptor splice variants (CCK(2)R and CCK(2i4sv)R) are upregulated in human colonic adenomas where they are thought to contribute to tumor growth and progression. To determine the effects of ectopic CCK(2) receptor variant expression on colonic epithelial cell growth in vitro and in vivo, we employed the non-tumorigenic colonic epithelial cell line, NCM356. Receptor expression was induced using a retroviral expression vector containing cDNAs for either CCK(2i4sv)R or CCK(2)R. RT-PCR and intracellular Ca(2+) ([Ca(2+)](i)) imaging of RIE/CCK(2)R cells treated with conditioned media (CM) from NCM356 revealed that NCM356 cells express gastrin mRNA and secrete endogenous, biologically active peptide. NCM356 cells expressing either CCK(2)R or CCK(2i4sv)R (71 and 81 fmol/mg, respectively) grew faster in vitro, and exhibited an increase in basal levels of phosphorylated ERK (pERK), compared with vector. CCK(2) receptor selective antagonist, YM022, partially inhibited the growth of both receptor-expressing NCM356 cells, but not the control cells. Inhibitors of mitogen activated protein kinase pathway (MEK/ERK) or protein kinase C (PKC) isozymes partially inhibited the elevated levels of basal pERK and in vitro growth of receptor-expressing cells. Vector-NCM356 cells did not form tumors in nude mice, whereas, either CCK(2) receptor-expressing cells formed large tumors. Autocrine activation CCK(2) receptor variants are sufficient to increase in vitro growth and tumorigenicity of non-transformed NCM356 colon epithelial cells through a pathway involving PKC and the MEK/ERK axis. These findings support the hypothesis that expression of gastrin and its receptors in human colonic adenomas contributes to tumor growth and progression.
Assuntos
Colo/fisiologia , Neoplasias Colorretais/patologia , Mucosa Intestinal/fisiologia , Receptor de Colecistocinina B/genética , Adenoma/patologia , Animais , Cálcio/metabolismo , Carcinoma/genética , Técnicas de Cultura de Células/métodos , Divisão Celular/genética , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/genética , Primers do DNA , Progressão da Doença , Gastrinas/genética , Gastrinas/metabolismo , Variação Genética , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Mutação , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND AND PURPOSE: During angiogenesis, quiescent endothelial cells (ECs) are activated by various stimuli to form new blood vessels from pre-existing ones in physiological and pathological conditions. Many research groups have shown that hydrogen sulfide (H2 S), the newest member of the gasotransmitter family, acts as a proangiogenic factor. To date, very little is known about the regulatory role of 3-mercaptopyruvate sulfurtransferase (3-MST), an important H2 S-producing enzyme in ECs. The aim of our study was to explore the potential role of 3-MST in human EC bioenergetics, metabolism, and angiogenesis. EXPERIMENTAL APPROACH: To assess in vitro angiogenic responses, we used EA.hy926 human vascular ECs subjected to shRNA-mediated 3-MST attenuation and pharmacological inhibition of proliferation, migration, and tube-like network formation. To evaluate bioenergetic parameters, cell respiration, glycolysis, glucose uptake, and mitochondrial/glycolytic ATP production were measured. Finally, global metabolomic profiling was performed to determine the level of 669 metabolic compounds. KEY RESULTS: 3-MST-attenuated ECs subjected to shRNA or pharmacological inhibition of 3-MST significantly reduced EC proliferation, migration, and tube-like network formation. 3-MST silencing also suppressed VEGF-induced EC migration. From bioenergetic and metabolic standpoints, 3-MST attenuation decreased mitochondrial respiration and mitochondrial ATP production, increased glucose uptake, and perturbed the entire EC metabolome. CONCLUSION AND IMPLICATIONS: 3-MST regulates bioenergetics and morphological angiogenic functions in human ECs. The data presented in the current report support the view that 3-MST pathway may be a potential candidate for therapeutic modulation of angiogenesis. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.
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Células Endoteliais , Sulfeto de Hidrogênio , Sulfurtransferases/metabolismo , Células Endoteliais/metabolismo , Metabolismo Energético , HumanosRESUMO
BACKGROUND: Breast cancers aberrantly express gastrin-releasing peptide (GRP) hormone and its cognate receptor, gastrin-releasing peptide receptor (GRP-R). Experimental evidence suggests that bombesin (BBS), the pharmacological homologue of GRP, promotes breast cancer growth and progression. The contribution of GRP-R to other poor prognostic indicators in breast cancer, such as the expression of the EGF-R family of growth factors and hormone insensitivity, is unknown. MATERIALS AND METHODS: Two estrogen receptor (ER)-negative breast cancer cell lines were used. MDA-MB-231 overexpress both EGFR and GRPR, whereas SK-BR-3 cells express EGF-R but lack GRP-R. Cellular proliferation was assessed by Coulter counter. Chemotactic migration was performed using Transwell chambers, and the migrated cells were quantified. Northern blot and real-time PCR were used to evaluate proangiogenic factor interleukin-8 (IL-8) mRNA expression. RESULTS: In MDA-MB-231 cells, GRP-R and EGF-R synergize to regulate cell migration, IL-8 expression, but not cell proliferation. In SK-BR-3 cells, ectopic expression of GRP-R was sufficient to increase migration and IL-8 mRNA. CONCLUSIONS: These data suggest relevant roles for GRP-R in ER-negative breast cancer progression. Future mechanistic studies to define the molecular role of GRP-R in breast cancer metastasis provide novel targets for the treatment of ER-negative breast cancers.
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Neoplasias da Mama/metabolismo , Movimento Celular , Receptores ErbB/metabolismo , Interleucina-8/metabolismo , Receptores da Bombesina/metabolismo , Bombesina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Fator de Crescimento Epidérmico/farmacologia , Feminino , Humanos , Neurotransmissores/farmacologia , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: Chronic pancreatitis is the consequence of multiple episodes of recurrent acute pancreatitis (RAP). We hypothesized that apigenin can minimize the sequelae of RAP by limiting acinar cells' proinflammatory signaling pathways. METHODS: AR42J acinar cells were treated in vitro with transforming growth factor ß (TGF-ß), apigenin, and other inhibitors. Dual luciferase reporter assay measured parathyroid hormone-related protein (PTHrP) promoter activity. MAPK/ERK pathway activity was assessed by immunoblotting and in vivo by immunohistochemistry with a cerulein-induced RAP mouse model. Nuclear factor κ B nuclear localization was analyzed in vitro in cells stimulated with tumor necrosis factor α. Primary acini were isolated and treated with cerulein; interleukin 6 messenger RNA was measured comparing PTHrP wild-type and knockout mice. RESULTS: Apigenin and PD98059 each downregulated TGF-ß stimulation of PTHrP P3 promoter activity. In a RAP mouse model, apigenin reduced pERK nuclear localization in acinar cells and preserved acinar cell architecture. Apigenin suppressed tumor necrosis factor α-mediated signaling by decreasing nuclear factor κ B nuclear localization and decreased interleukin 6 messenger RNA levels via a PTHrP-dependent mechanism. CONCLUSIONS: Apigenin reduced inflammatory responses in experimental models of RAP. The mechanisms mediating the actions of apigenin, in part, are owing to attenuation of PTHrP and TGF-ß proinflammatory signaling.