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The hypothalamic-pituitary-adrenal (HPA) axis controls the release of glucocorticoids, which regulate immune and inflammatory function by modulating cytokines, white blood cells and oxidative stress via glucocorticoid receptor (GR) signaling. Although the response to HPA activation is well characterized in many species, little is known about the impacts of HPA activation during extreme physiological conditions. Hence, we challenged 18 simultaneously fasting and developing elephant seal pups with daily intramuscular injections of adrenocorticotropin (ACTH), a GR antagonist (RU486), or a combination of the two (ACTH+RU486) for 4 days. We collected blood at baseline, 2â h and 4 days after the beginning of treatment. ACTH and ACTH+RU486 elevated serum aldosterone and cortisol at 2â h, with effects diminishing at 4 days. RU486 alone induced a compensatory increase in aldosterone, but not cortisol, at 4â days. ACTH decreased neutrophils at 2â h, while decreasing lymphocytes and increasing the neutrophil:lymphocyte ratio at 4 days. These effects were abolished by RU486. Despite alterations in white blood cells, there was no effect of ACTH or RU486 on transforming growth factor-ß or interleukin-6 levels; however, both cytokines decreased with the 4 day fasting progression. Similarly, ACTH did not impact protein oxidation, lipid peroxidation or antioxidant enzymes, but plasma isoprostanes and catalase activity decreased while glutathione peroxidase increased with fasting progression. These data demonstrate differential acute (2â h) and chronic (4â days) modulatory effects of HPA activation on white blood cells and that the chronic effect is mediated, at least in part, by GR. These results also underscore elephant seals' extraordinary resistance to oxidative stress derived from repeated HPA activation.
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Sistema Hipófiso-Suprarrenal , Phocidae , Animales , Citocinas , Ayuno , Hidrocortisona , Sistema Hipotálamo-Hipofisario , Recuento de Leucocitos , Estrés OxidativoRESUMEN
Homeobox D10 (HoxD10 ) gene plays a critical role in cell differentiation and morphogenesis during development. However, the function of HoxD10 in tumor progression remains largely unknown. We demonstrate that the expression of HoxD10 is commonly downregulated in gastric cancer tissues (n = 33) and cell lines (n = 8) relative to normal stomach tissues. Functionally, reexpression of HoxD10 results in significant inhibition of cell survival, induction of cell apoptosis, and impairment of cell migration and invasion. Moreover, ectopic expression of HoxD10 suppresses gastric tumor growth in a mouse xenograft model. To identify target candidates of HoxD10, we performed cDNA microarray and showed that HoxD10 regulates multiple downstream genes including IGFBP3. Reintroduction of HoxD10 transcriptionally upregulates IGFBP3, activates caspase 3 and caspase 8, and subsequently induces cell apoptosis. Methylation specific PCR revealed that HoxD10 promoter DNA was hypermethylated in gastric cancer cell lines. Additionally, 5-aza demethylation treatment could transiently reactivate the expression of HoxD10 in gastric cancer cells. HoxD10 promoter methylation frequently was detected in gastric cancer tissues obtained from endoscopic biopsies (85.7%, 24/28) and surgically resected samples (82.6%, 57/69). Intestinal metaplasia tissues showed a 60% methylation rate (18/30), but no detectable methylation in normal stomach tissues (0%, 0/10). Taken together, our results suggest that HoxD10 functions as a candidate tumor suppressor in gastric cancer, which is inactivated through promoter hypermethylation.
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Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Regiones Promotoras Genéticas , Neoplasias Gástricas/metabolismo , Factores de Transcripción/genética , Animales , Apoptosis , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación hacia Abajo , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Metilación , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Carga TumoralRESUMEN
Thermal extremes alter population processes, which can result in part from temperature-induced movement at different spatial and temporal scales. Thermal thresholds for animal movement likely change based on underlying thermal physiology and life-history stage, a topic that requires greater study. The intertidal porcelain crab Petrolisthes cinctipes currently experiences temperatures that can reach near-lethal levels in the high-intertidal zone at low tide. However, the thermal thresholds that trigger migration to cooler microhabitats, and the extent to which crabs move in response to temperature, remain unknown. Moreover, the influence of reproductive status on these thresholds is rarely investigated. We integrated demographic, molecular, behavioral, and physiological measurements to determine if behavioral thermal limits varied due to reproductive state. Demographic data showed a trend for gravid, egg bearing, crabs to appear more often under rocks in the cooler intertidal zone where crab density is highest. In situ expression of 31 genes related to stress, metabolism, and growth in the field differed significantly based on intertidal elevation, with mid-intertidal crabs expressing the gene for the reproductive yolk protein vitellogenin (vg) earlier in the season. Furthermore, VG protein levels were shown to increase with density for female hemolymph. Testing for temperatures that elicit movement revealed that gravid females engage in heat avoidance behavior at lower temperatures (i.e., have a lower voluntary thermal maximum, VTmax) than non-gravid females. VTmax was positively correlated with the temperature of peak firing rate for distal afferent nerve fibers in the walking leg, a physiological relationship that could correspond to the mechanistic underpinning for temperature dependent movement. The vulnerability of marine organisms to global change is predicated by their ability to utilize and integrate physiological and behavioral strategies in response to temperature to maximize survival and reproduction. Interactions between fine-scale temperature variation and reproductive biology can have important consequences for the ecology of species, and is likely to influence how populations respond to ongoing climate change.
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Regular surveillance testing of asymptomatic individuals for SARS-CoV-2 has been center to SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants' experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.
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COVID-19/diagnóstico , Evaluación de Programas y Proyectos de Salud , Saliva/virología , Adulto , Anciano , COVID-19/epidemiología , COVID-19/virología , Prueba de COVID-19/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , ARN Viral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Normas Sociales , Encuestas y Cuestionarios , Universidades , Adulto JovenRESUMEN
Marine mammals exhibit some of the most dramatic physiological adaptations in their clade and offer unparalleled insights into the mechanisms driving convergent evolution on relatively short time scales. Some of these adaptations, such as extreme tolerance to hypoxia and prolonged food deprivation, are uncommon among most terrestrial mammals and challenge established metabolic principles of supply and demand balance. Non-targeted omics studies are starting to uncover the genetic foundations of such adaptations, but tools for testing functional significance in these animals are currently lacking. Cellular modeling with primary cells represents a powerful approach for elucidating the molecular etiology of physiological adaptation, a critical step in accelerating genome-to-phenome studies in organisms in which transgenesis is impossible (e.g., large-bodied, long-lived, fully aquatic, federally protected species). Gene perturbation studies in primary cells can directly evaluate whether specific mutations, gene loss, or duplication confer functional advantages such as hypoxia or stress tolerance in marine mammals. Here, we summarize how genetic and pharmacological manipulation approaches in primary cells have advanced mechanistic investigations in other non-traditional mammalian species, and highlight the need for such investigations in marine mammals. We also provide key considerations for isolating, culturing, and conducting experiments with marine mammal cells under conditions that mimic in vivo states. We propose that primary cell culture is a critical tool for conducting functional mechanistic studies (e.g., gene knockdown, over-expression, or editing) that can provide the missing link between genome- and organismal-level understanding of physiological adaptations in marine mammals.
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Cetáceos/genética , Genoma , Fenotipo , AnimalesRESUMEN
Aberrant regulation of APC/beta-catenin signaling pathway is common in the pathogenesis of colorectal and other cancers. Targets regulated by APC/beta-catenin signaling pathway play crucial roles in cancer development. In the current study, we aimed to illustrate the influence of APC/beta-catenin signaling pathway on expression of microRNAs, one new group of players important to carcinogenesis. Restoration of APC function in colorectal cancer cells led to the deregulation of several cancer-related microRNAs, such as miR-122a which was recognized as the liver-specific microRNA. MiR-122a was down-regulated in gastrointestinal cancer cell lines as well as primary carcinoma tissues. Inhibition of miR-122a could reverse wild-type APC-induced growth inhibition of gastrointestinal cancer cells while miR-122a mimic inhibited cell growth. In summary, we identified some cancer-related microRNAs regulated by APC/beta-catenin signaling pathway. The down-regulation of miR-122a mediated by aberrant APC/beta-catenin signaling is important to the pathogenesis of gastrointestinal cancers.
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Poliposis Adenomatosa del Colon/genética , Carcinoma/genética , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , MicroARNs/genética , beta Catenina/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Perfilación de la Expresión Génica , HumanosRESUMEN
As one of major epigenetic changes responsible for tumor suppressor gene inactivation in the development of cancer, promoter hypermethylation was proposed as a marker to define novel tumor suppressor genes. In the current study we identified ZIC1 (Zic family member 1, odd-paired Drosophila homolog) as a novel tumor suppressor gene silenced through promoter hypermethylation in gastric cancer, the second leading cause of cancer death worldwide. In all of gastric cancer cells lines examined, ZIC1 expression was downregulated and such downregulation was accompanied with the hypermethylation of ZIC1 promoter. Demethylation treatment with 5-aza-2'-deoxycytidine (Aza) reversed ZIC1 downregulation, highlighting the importance of promoter methylation to ZIC1 downregulation in gastric cancer cells. Notably, ZIC1 expression was significantly downregulated in primary gastric carcinoma tissues in comparison with non-tumor adjacent gastric tissues (p<0.01). Accordingly, promoter methylation of ZIC1 was frequently detected in primary gastric carcinoma tissues (94.6%, 35/37) but not normal gastric tissues, indicating that promoter hypermethylation mediated ZIC1 downregulation may play an important role in gastric carcinogenesis. Indeed, ectopic expression of ZIC1 led to the growth inhibition of gastric cancer cells through the induction of S-phase cell cycle arrest (p<0.01). Our results revealed ZIC1 as a novel candidate tumor suppressor gene downregulated through promoter hypermethylation in gastric cancer.
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Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Neoplasias Gástricas/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Regiones Promotoras GenéticasRESUMEN
As an important way to inactivate tumor suppressor genes (TSGs) during cancer development, promoter hypermethylation can be used to define novel TSGs and identify biomarkers for cancer diagnosis. SLC19A3 (solute carrier family 19, member 3) was found to be such a biomarker. SLC19A3 expression was downregulated in gastric cancer cell lines (71%, 5/7) and restored after pharmacological demethylation. Notably, hypermethylation of SLC19A3 promoter was detected in gastric cancer cell lines (57%, 4/7), primary gastric carcinoma tissues (51%, 52/101) and precancerous lesion (intestinal metaplasia) tissues (32%, 8/25). Exogenous SLC19A3 expression caused growth inhibition of gastric cancer cells. In summary, SLC19A3 was epigenetically downregulated in gastric cancer. Methylation of SLC19A3 promoter could be a novel biomarker for early gastric cancer development.
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Metilación de ADN , Proteínas de Transporte de Membrana/genética , Regiones Promotoras Genéticas/genética , Neoplasias Gástricas/genética , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Supervivencia Celular/genética , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/patología , Análisis de Supervivencia , Factores de TiempoRESUMEN
Mucus forms the physical barrier along the gastrointestinal tract. It plays an important role to prevent mucosal damage and inflammation. Our animal study showed that antibacterial peptide 'cathelicidin' increased mucus thickness and prevented inflammation in the colon. In the current study, we examined the direct effect and mechanisms by which the peptide increased mucus synthesis in a human colonic cell line (HT-29). Human cathelicidin (LL-37) dose-dependently (10-40 microg/ml) and significantly stimulated mucus synthesis by increasing the D-[6-(3)H] glucosamine incorporation in the cells. Real-time PCR data showed that addition of LL-37 induced more than 50% increase in MUC1 and MUC2 mRNA levels. Treatment with MUC1 and MUC2 siRNAs normalized the stimulatory action of LL-37 on mucus synthesis. LL-37 also activated the phosphorylation of mitogen-activated protein (MAP) kinase in the cells. A specific inhibitor of the MAP kinase pathway, U0126, completely blocked the increase of MUC1 and MUC2 expression as well as mucus synthesis by LL-37. Taken together, LL-37 can directly stimulate mucus synthesis through activation of MUC1 and MUC2 expression and MAP kinase pathway in human colonic cells.
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Péptidos Catiónicos Antimicrobianos/farmacología , Colon/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mucina-1/genética , Mucinas/genética , Moco/metabolismo , Transducción de Señal , Línea Celular , Colon/citología , Humanos , Mucina 2 , ARN Interferente Pequeño/farmacología , Regulación hacia Arriba/genética , CatelicidinasRESUMEN
Probiotics are widely used as functional foods which have been advocated for the maintenance of gastrointestinal microflora equilibrium and treatment of gastrointestinal disorders. However, studying the role of probiotics in peptic ulcer disease is limited. The aim of the present study is to investigate the effect of a probiotic strain Lactobacillus rhamnosus GG on gastric ulcer and to elucidate the mechanisms involved. Gastric kissing ulcers were induced in rats by acetic acid (60% v/v). L. rhamnosus GG was given intragastrically at 10(8) cfu/day or 10(9) cfu/day for three consecutive days after ulcer induction. L. rhamnosus GG successfully colonized in the gastric mucosa especially at the ulcer margin. It also significantly and dose-dependently reduced gastric ulcer area. Cell apoptosis to cell proliferation ratio was strongly decreased and accompanied by significant up-regulation of ornithine decarboxylase (ODC) and B-cell lymphoma 2 (Bcl-2) protein expression at the ulcer margin. Angiogenesis was also significantly stimulated together with the induction of vascular endothelial growth factor (VEGF) expression. Furthermore, L. rhamnosus GG up-regulated the phosphorylation level of epidermal growth factor receptor (EGF receptor) without altering the total EGF receptor expression. These findings suggested that L. rhamnosus GG enhanced gastric ulcer healing via the attenuation of cell apoptosis to cell proliferation ratio and increase in angiogenesis. Regulators of these processes such as ODC, Bcl-2, VEGF and EGF receptor are likely to be involved in the healing action of L. rhamnosus GG for gastric ulcer.
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Lacticaseibacillus rhamnosus , Probióticos/uso terapéutico , Úlcera Gástrica/tratamiento farmacológico , Cicatrización de Heridas/efectos de los fármacos , Animales , Apoptosis , Proliferación Celular , Receptores ErbB/metabolismo , Masculino , Neovascularización Fisiológica/efectos de los fármacos , Ornitina Descarboxilasa/biosíntesis , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Ratas , Ratas Sprague-Dawley , Estómago/microbiología , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
Cathelicidin, an antimicrobial peptide of the innate immune system, modulates microbial growth, wound healing, and inflammation. However, its association with inflammatory bowel diseases (IBDs) is unknown. Our objective was to determine whether cathelicidin would exert a modulatory effect on the progression of IBD and, if so, investigate the mechanism of action through which this effect occurred. We evaluated the potential for a synthetic cathelicidin, the mouse cathelin-related antimicrobial peptide (mCRAMP), to prevent the initiation and promote the healing of lesions from inflammatory colitis that was experimentally induced in mice with dextran sulfate sodium (DSS). During the experiment, mCRAMP was given: (i) as a parallel treatment starting together with 3% DSS feeding, and (ii) as a posttreatment starting 7 days after 3% DSS feeding. The body weight, fecal microflora populations, clinical symptoms, and histologic findings of colonic tissues were measured. Relative gene expression of mucins (MUC1, MUC2, MUC3, and MUC4) in colonic tissues was determined by real-time polymerase chain reaction. Intrarectal administration of mCRAMP ameliorated DSS-induced colitis with negligible effects on mucosal healing. The peptide also significantly reduced the increased number of fecal microflora in colitis animals. It reversed the decline of colonic mucus thickness during colitis through upregulation of the expression of mucin genes. Treatment with mCRAMP also prevented colitis development by suppressing the induction of apoptosis by DSS. The current study demonstrates for the first time that intrarectal administration of cathelicidin may be a novel therapeutic option for IBDs.
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Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Colitis Ulcerosa/prevención & control , Colon/efectos de los fármacos , Administración Rectal , Animales , Apoptosis/efectos de los fármacos , Catelicidinas , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Colon/metabolismo , Colon/patología , Sulfato de Dextran , Heces/microbiología , Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Mucinas/genética , Moco/metabolismo , Peroxidasa/metabolismoRESUMEN
The gastric mucosa is frequently exposed to different exogenous and endogenous ulcerative agents. Alcoholism is one of the risk factors for the development of mucosal damage in the stomach. This study aimed to assess if a probiotic strain Lactobacillus rhamnosus GG (LGG) is capable of protecting the gastric mucosa from acute damage induced by intragastric administration of ethanol. Pre-treatment of rats with LGG at 10(9) cfu/ml twice daily for three consecutive days markedly reduced ethanol-induced mucosal lesion area by 45%. LGG pre-treatment also significantly increased the basal mucosal prostaglandin E(2) (PGE(2)) level. In addition, LGG attenuated the suppressive actions of ethanol on mucus-secreting layer and transmucosal resistance and reduced cellular apoptosis in the gastric mucosa. It is suggested that the protective action of LGG on ethanol-induced gastric mucosal lesions is likely attributed to the up-regulation of PGE(2), which could stimulate the mucus secretion and increase the transmucosal resistance in the gastric mucosa. All these would protect mucosal cells from apoptosis in the stomach.
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Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Lacticaseibacillus rhamnosus/metabolismo , Animales , Apoptosis , Dinoprostona/metabolismo , Etanol/química , Etanol/farmacología , Masculino , Mucina 6 , Mucinas/biosíntesis , Membrana Mucosa/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
Cigarette smoking is a risk factor for colorectal cancer. It is suggested that 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, mediates the carcinogenic action of cigarette smoking by promoting cancer growth. In the present study, the proliferative response of a cultured colon cancer cell line HT-29 to NNK was determined. It was found that NNK dose-dependently stimulated HT-29 cell proliferation. In this regard, the stimulatory action of NNK was abolished by atenolol and ICI 118,551, a beta1- and beta2-selective antagonist, respectively. In addition, cell growth was stimulated by the nonselective adrenergic agonist, noradrenaline, and more effectively by the beta-selective agonist, isoproterenol. The second message cyclic AMP level for beta-adrenoceptor activation was elevated by isoproterenol and NNK treatment. These agents also up-regulated cyclooxygenase-2 expression, cytosolic phospholipase A2 expression, and prostaglandin E2 release. Beta2-adrenoceptor blockade with ICI 118,551, in contrast, significantly decreased cyclooxygenase-2 expression, cytosolic phospholipase A2 expression and prostaglandin E2 release induced by NNK and isoproterenol. To conclude, it is proposed that NNK stimulates HT-29 cell proliferation through beta-adrenoceptors, preferentially beta2 receptors. Activation of the beta-adrenoceptors, and the consequent cyclic AMP elevation coupled with the downstream arachidonic acid pathway, is perhaps an important mechanistic cascade in the promotion of colon cancer growth. These findings partly elucidate the carcinogenic actions of cigarette smoke and shed new light on the novel modulatory role of beta-adrenoceptors in the development of colon cancer.
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Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Carcinógenos/toxicidad , Neoplasias del Colon/patología , Nitrosaminas/toxicidad , Receptores Adrenérgicos beta/fisiología , Atenolol/farmacología , Carcinógenos/antagonistas & inhibidores , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/etiología , AMP Cíclico/metabolismo , Ciclooxigenasa 2 , Dinoprostona/metabolismo , Interacciones Farmacológicas , Células HT29 , Humanos , Isoproterenol/farmacología , Proteínas de la Membrana , Nitrosaminas/antagonistas & inhibidores , Norepinefrina/farmacología , Fosfolipasas A/biosíntesis , Fosfolipasas A/genética , Fosfolipasas A2 , Propanolaminas/farmacología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , Humo/efectos adversos , Nicotiana/efectos adversos , Nicotiana/química , Regulación hacia ArribaRESUMEN
Blockade of angiogenesis is a promising strategy to suppress tumor growth, invasion, and metastasis. Vascular endothelial growth factor (VEGF), which binds to tyrosine kinase receptors [VEGF receptors (VEGFR) 1 and 2], is the mediator of angiogenesis and mitogen for endothelial cells. Cyclooxygenase-2 (COX-2) plays an important role in the promoting action of nicotine on gastric cancer growth. However, the action of nicotine and the relationship between COX-2 and VEGF/VEGFR system in tumorigenesis remain undefined. In this study, the effects of nicotine in tumor angiogenesis, invasiveness, and metastasis were studied with sponge implantation and Matrigel membrane models. Nicotine (200 microg/mL) stimulated gastric cancer cell proliferation, which was blocked by SC-236 (a highly selective COX-2 inhibitor) and CBO-P11 (a VEGFR inhibitor). This was associated with decreased VEGF levels as well as VEGFR-2 but not VEGFR-1 expression. Topical injection of nicotine enhanced tumor-associated vascularization, with a concomitant increase in VEGF levels in sponge implants. Again, application of SC-236 (2 mg/kg) and CBO-P11 (0.4 mg/kg) partially attenuated vascularization by approximately 30%. Furthermore, nicotine enhanced tumor cell invasion through the Matrigel membrane by 4-fold and promoted migration of human umbilical vein endothelial cells in a cocultured system with gastric cancer cells. The activity of matrix metalloproteinases 2 and 9 and protein expressions of plasminogen activators (urokinase-type plasminogen activator and its receptor), which are the indicators of invasion and migration processes, were increased by nicotine but blocked by COX-2 and VEGFR inhibitors. Taken together, our results reveal that the promoting action of nicotine on angiogenesis, tumor invasion, and metastasis is COX-2/VEGF/VEGFR dependent.
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Adenocarcinoma/patología , Ciclooxigenasa 2/metabolismo , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Neoplasias Gástricas/patología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Adenocarcinoma/metabolismo , Animales , División Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Colágeno , Combinación de Medicamentos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Laminina , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Desnudos , Invasividad Neoplásica/patología , Trasplante de Neoplasias , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Proteoglicanos , Receptores de Superficie Celular/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Neoplasias Gástricas/metabolismo , Tapones Quirúrgicos de Gaza , Venas Umbilicales/citología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
The transcription factor, Zinc finger of the cerebellum (ZIC1), plays a crucial role in vertebrate development. Recently, ZIC1 has also been found to participate in the progression of human cancers, including medulloblastomas, endometrial cancers, and mesenchymal neoplasms. However, the function of ZIC1 in colon cancer progression has not been defined. In this study, we demonstrate ZIC1 to be silenced or significantly downregulated in colon cancer cell lines. These effects were reversed by demethylation treatment with 5-aza-2'-deoxycytidine (Aza). ZIC1 expression is also significantly downregulated in primary colorectal cancer tissues relative to adjacent non-tumor tissues (pâ=â0.0001). Furthermore, methylation of ZIC1 gene promoter is frequently detected in primary tumor tissues (85%, 34/40), but not in adjacent non-tumor tissues. Ectopic expression of ZIC1 suppresses cell proliferation and induces apoptosis, which is associated with MAPK and PI(3)K/Akt pathways, as well as the Bcl-xl/Bad/Caspase3 cascade. To identify target candidates of ZIC1, we employed cDNA microarray and found that 337 genes are downregulated and 95 genes upregulated by ectopic expression of ZIC1, which were verified by 10 selected gene expressions by qRT-PCR. Taken together, our results suggest that ZIC1 may potentially function as a tumor suppressor gene, which is downregulated through promoter hypermethylation in colorectal cancers.
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Neoplasias Colorrectales/genética , Metilación de ADN/genética , Regulación hacia Abajo/genética , Genes Supresores de Tumor , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/patología , Femenino , Silenciador del Gen , Humanos , Masculino , Persona de Mediana Edad , Factores de Transcripción/metabolismo , Transcriptoma/genéticaRESUMEN
AIMS: Stress has been implicated in the development of cancers. Adrenaline levels are increased in response to stress. The effects of adrenaline on colon cancer are largely unknown. The aims of the study are to determine the effects of adrenaline in human colon adenocarcinoma HT-29 cells and the possible underlying mechanisms involved. MAIN METHODS: The effect of adrenaline on HT-29 cell proliferation was determined by [(3)H] thymidine incorporation assay. Expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) were detected by Western blot. Matrix metalloproteinase-9 (MMP-9) activity and prostaglandin E(2) (PGE(2)) release were determined by zymography and enzyme immunoassay, respectively. KEY FINDINGS: Adrenaline stimulated HT-29 cell proliferation. This was accompanied by the enhanced expression of COX-2 and VEGF in HT-29 cells. Adrenaline also upregulated MMP-9 activity and PGE(2) release. Adrenaline stimulated HT-29 cell proliferation which was reversed by COX-2 inhibitor sc-236. COX-2 inhibitor also reverted the action of adrenaline on VEGF expression and MMP-9 activity. Further study was performed to determine the involvement of ß-adrenoceptors. The stimulatory action of adrenaline on colon cancer growth was blocked by atenolol and ICI 118,551, a ß(1)- and ß(2)-selective antagonist, respectively. This signified the role of ß-adrenoceptors in this process. In addition, both antagonists also abrogated the stimulating actions of adrenaline on COX-2, VEGF expression, MMP-9 activity and PGE(2) release in HT-29 cells. SIGNIFICANCE: These results suggest that adrenaline stimulates cell proliferation of HT-29 cells via both ß(1)- and ß(2)-adrenoceptors by a COX-2 dependent pathway.
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Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Epinefrina/farmacología , Antagonistas Adrenérgicos beta/farmacología , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/enzimología , Neoplasias del Colon/etiología , Ciclooxigenasa 2/biosíntesis , Inhibidores de la Ciclooxigenasa 2/farmacología , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Epinefrina/metabolismo , Células HT29 , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Estrés Psicológico/complicaciones , Estrés Psicológico/enzimología , Estrés Psicológico/metabolismo , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
Wound healing in the gastrointestinal tract is an orderly process involving orchestrated responses of various cell types. Lipopolysaccharides (LPS) are major components of the outer membrane of Gram-negative bacteria, which are known to impair gastric ulcer healing in animals. The influence of LPS on intercellular communication in wound healing, however, is unknown. We examined the effects of LPS-induced macrophage activation on the proliferative response in cultured rat gastric epithelial cells (RGM-1) and fibroblasts JHU-25. Rat peritoneal resident macrophages were activated with increasing doses of LPS. The supernatant from the activated macrophage preparation, designated as macrophage-conditioned medium, was then used to treat RGM-1 or JHU-25 cells. Cell proliferation and migration were determined by [(3)H]-thymidine incorporation and a monolayer wound-healing assay, respectively. Macrophage-conditioned medium significantly suppressed RGM-1 cell proliferation but had no effect on cell migration. The same medium, however, increased JHU-25 cell proliferation. LPS treatment alone suppressed JHU-25 cell proliferation while it had no effect on RGM-1 cell proliferation, indicating that the differential effects of the macrophage-conditioned medium on cell proliferation were elicited by the factors derived from macrophages. In this regard, tumor necrosis factor (TNF)-alpha stimulated while interleukin (IL)-1beta suppressed RGM-1 cell proliferation, suggesting that IL-1beta but not TNF-alpha may play a part in the mediation of the antiproliferative effect of macrophage-conditioned medium on gastric epithelial cells. In contrast, IL-1beta suppressed while TNF-alpha had no effect on JHU-25 cell proliferation. Collectively, LPS-activated macrophages delay gastric mucosal regeneration but promote fibroblast proliferation in vitro. Such changes may partly elucidate the detrimental effect of bacterial infection on tissue repair in the stomach.
Asunto(s)
Fibroblastos/fisiología , Mucosa Gástrica/fisiología , Homeostasis/fisiología , Cicatrización de Heridas/fisiología , Animales , Movimiento Celular , Proliferación Celular , Células Cultivadas , Medios de Cultivo Condicionados , Lipopolisacáridos/farmacología , Macrófagos/fisiología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Cathelicidin, a cationic host defense peptide, has been shown to promote cutaneous wound repair and reaches high levels in the gastric mucosa during infection and inflammation. Therefore, we investigated whether this peptide contributes to gastric ulcer healing in rats. Ulcer induction increased the expression of rat cathelicidin rCRAMP in the gastric mucosa. Further increase in expression of rCRAMP by local injection of rCRAMP-encoding plasmid promoted ulcer healing by enhancing cell proliferation and angiogenesis. rCRAMP directly stimulated proliferation of cultured rat gastric epithelial cells (RGM-1), which was abolished by inhibitors of matrix metalloproteinase (MMP), epidermal growth factor receptors (EGFR) tyrosine kinase, or mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase. rCRAMP also increased EGFR and ERK1/2 phosphorylation via an MMP-dependent mechanism. Knockdown of transforming growth factor alpha (TGFalpha), which is a ligand of EGFR, by small interfering RNA completely nullified the mitogenic signals evoked by rCRAMP in RGM-1 cells. These findings suggest that rCRAMP exhibits prohealing activity in stomachs through TGFalpha-dependent transactivation of EGFR and its related signaling pathway to induce proliferation of gastric epithelial cells.
Asunto(s)
Antiulcerosos , Péptidos Catiónicos Antimicrobianos/farmacología , Terapia Genética , Úlcera Gástrica/terapia , Ácido Acético , Actinas/genética , Actinas/fisiología , Animales , Western Blotting , Proliferación Celular , Supervivencia Celular/genética , Células Cultivadas , Células Epiteliales/fisiología , Receptores ErbB/metabolismo , Mucosa Gástrica/citología , Mucosa Gástrica/efectos de los fármacos , Inmunohistoquímica , Inmunoprecipitación , Masculino , Mitosis/efectos de los fármacos , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , Fosforilación , Plásmidos/genética , ARN Interferente Pequeño , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Úlcera Gástrica/inducido químicamente , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/fisiología , CatelicidinasRESUMEN
To further explore the antiobesity effect of freeze-dried bitter melon (BM) juice, activities of mitochondrial lipid oxidative enzymes as well as the expression of uncoupling proteins and their transcription coactivator peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1alpha) were determined in diet-induced obese (DIO) rats. Rats were fed high-fat (HF) diets to induce obesity, and the effect of BM was assessed at doses of 0.75, 1.0, or 1.25% (wt:wt). In a dose-response experiment, BM-supplemented rats had lower energy efficiency (g weight gained/kJ consumed), visceral fat mass, serum glucose, and insulin resistance index, but higher plasma norepinephrine than unsupplemented rats (P < 0.05). Hepatic and skeletal muscle triglyceride concentrations were lower in supplemented HF diet-fed rats than in unsupplemented HF diet-fed rats (P < 0.05). An HF diet supplemented with BM elevated activities of hepatic and muscle mitochondrial carnitine palmitoyl transferase-I (CPT-I) and acyl-CoA dehydrogenase (AD) (P < 0.05). In another experiment, BM (1.0 g/100 g) lowered visceral fat mass but increased serum adiponectin concentration in HF diet-fed rats (P < 0.05). In the final study, rats were fed the HF diet with 0, 1.0 or 1.25% BM. Both groups of BM-supplemented rats had higher uncoupling protein 1 in brown adipose tissue (P < 0.05) and uncoupling protein 3 in red gastrocnemius muscle (P < 0.05), measured by Western blotting and RT-PCR, than the controls. The expression of the transcription coactivator PGC-1alpha in both tissues was also significantly elevated in the BM-supplemented rats (P < 0.05). The present results suggest that decreased adiposity in BM-supplemented rats may result from lower metabolic efficiency, a consequence of increased lipid oxidation and mitochondrial uncoupling.