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N-methyl-glycine (sarcosine) is known to promote metastatic potential in some cancers; however, its effects on bladder cancer are unclear. T24 cells derived from invasive cancer highly expressed GNMT, and S-adenosyl methionine (SAM) treatment increased sarcosine production, promoting proliferation, invasion, anti-apoptotic survival, sphere formation, and drug resistance. In contrast, RT4 cells derived from non-invasive cancers expressed low GNMT, and SAM treatment did not produce sarcosine and did not promote malignant phenotypes. In T24 cells, the expression of miR-873-5p, which suppresses GNMT expression, was suppressed, and the expression of ERVK13-1, which sponges miR-873-5p, was increased. The growth of subcutaneous tumors, lung metastasis, and intratumoral GNMT expression in SAM-treated nude mice was suppressed in T24 cells with ERVK13-1 knockdown but promoted in RT4 cells treated with miR-873-5p inhibitor. An increase in mouse urinary sarcosine levels was observed to correlate with tumor weight. Immunostaining of 86 human bladder cancer cases showed that GNMT expression was higher in cases with muscle invasion and metastasis. Additionally, urinary sarcosine concentrations increased in cases of muscle invasion. Notably, urinary sarcosine concentration may serve as a marker for muscle invasion in bladder cancer; however, further investigation is necessitated.
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MicroARNs , Neoplasias de la Vejiga Urinaria , Humanos , Animales , Ratones , Sarcosina/farmacología , Ratones Desnudos , S-Adenosilmetionina/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Movimiento CelularRESUMEN
INTRODUCTION: The aim of this study was to investigate the association of the HtrA1 rs11200638 polymorphism with neovascular age-related macular degeneration (nAMD) in Indonesia. METHODS: This case-control study included 80 patients with nAMD and 85 controls. Demographic parameters and whole blood were collected from each participant. Genomic DNA was extracted and used to assess the rs11200638 genotype by PCR and restriction enzyme digestion. Associations between the HtrA1 rs11200638 polymorphism and other risk factors for susceptibility to nAMD were assessed using the logistic regression model. RESULTS: Significant allelic associations between the HtrA1 polymorphism and nAMD were detected (odds ratio [OR] 8.67; 95% confidence interval [CI] 4.88-15.41; P < 0.001). Genotype analysis showed a statistical difference between the nAMD group and the control group (P < 0.001). In the multiple adjusted logistic regression model, people with the AA genotype were more likely to have nAMD although there was a wide confidence interval (OR 19.65; 95% CI 4.52-85.38; P < 0.001). CONCLUSION: Our findings show that the risk of nAMD increased in the presence of risk alleles of HtrA1 rs11200638.
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Purpose: The current study aims to evaluate the in vitro cytotoxic and cell migration effects of synthetic curcumin and its analogues on HER2 and nuclear factor kappa B (NFκB) pathways, as well as the in vivo inhibitory effect on cancer growth of metastatic breast cancer. Methods: Cell viability, protein expression, and protein localization were determined in vitro using MTT assay, western blotting, and immunofluorescence, respectively. Meanwhile, scratch wound healing assay and gelatin zymography were conducted to investigate the metastasis inhibitory effect. The in vivo anti-tumor ability was evaluated in xenograft mouse model using triple-negative breast cancer (TNBC) cells. Results: Curcumin, PGV-0, and PGV-1 exhibited cytotoxic effect against HER2-overexpressing breast cancer cells. Although PGV-1 showed the best activity in the single cytotoxic assay, curcumin showed the strongest synergism with doxorubicin. Curcumin and PGV-0 inhibited membrane localization of HER2. In contrast, PGV-1 neither inhibited localization nor decreased the expression of HER2, nonetheless showed the most potent inhibition against nuclear localization of p65 indicating the different mechanisms of curcumin, PGV-0, and PGV-1. Regarding cancer metastasis, curcumin and PGV-1 showed inhibitory activities against cell migration and inhibited MMP-2 and MMP-9 protein expression. Lastly, PGV-1 was more potent compared to curcumin to suppress the tumor formation of metastatic breast cancer xenograft model in nude mice. Conclusion: Overall, our study strengthens the potency of curcumin analogue, PGV-1, for treating several types of cancer, including metastatic breast cancer.
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PURPOSE: This study aimed to determine the association of ARMS2 A69S, ARMS2 del443ins54, and CFH Y402H polymorphisms with neovascular age-related macular degeneration (nAMD) for the first time in an Indonesian population. PATIENTS AND METHODS: Our case-control study involved 104 nAMD and 100 control subjects. AMD diagnosis was evaluated by retinal specialists based on color fundus photography and optical coherence tomography. The polymorphisms on CFH Y402H and ARMS2 A69S were analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP), whereas ARMS2 del443ins54 was evaluated by PCR-based assay. RESULTS: Significant allelic associations with nAMD were detected on all polymorphisms (P<0.05), with stronger association with the ARMS2 A69S (OR 3.13; 95% CI 2.08-4.71; P<0.001) and ARMS2 del443ins54 (OR 3.28; 95% CI 2.17-4.95; P<0.001) polymorphisms than with CFH Y402H (OR 2.08; 95% CI 1.08-3.99; P=0.028). Genotype analysis showed a statistical difference between nAMD and the control group for all polymorphisms (P<0.05). However, the association with nAMD was weaker for CFH Y402H (P=0.043) than for ARMS2 A69S and ARMS2 del443ins54 (P<0.001). A significant interaction between ARMS2 A69S and hypertension was documented (OR 9.53; 95% CI 3.61-25.1; P<0.001). CONCLUSION: Our findings indicate that ARMS2 A69S and ARMS2 del443ins54 polymorphisms are strongly associated with the risk of nAMD for the first time in an Indonesian population. The risk of nAMD increased when the presence of risk alleles from ARMS2 A69S was combined with the presence of hypertension.
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Random gene trapping is the application of insertional mutagenesis techniques that are conventionally used to inactivate protein-coding genes in mouse embryonic stem (ES) cells. Transcriptionally silent genes are not effectively targeted by conventional random gene trapping techniques, thus we herein developed an unbiased poly (A) trap (UPATrap) method using a Tol2 transposon, which preferentially integrated into active genes rather than silent genes in ES cells. To achieve efficient trapping at transcriptionally silent genes using random insertional mutagenesis in ES cells, we generated a new diphtheria toxin (DT)-mediated trapping vector, DTrap that removed cells, through the expression of DT that was induced by the promoter activity of the trapped genes, and selected trapped clones using the neomycin-resistance gene of the vector. We found that a double-DT, the dDT vector, dominantly induced the disruption of silent genes, but not active genes, and showed more stable integration in ES cells than the UPATrap vector. The dDT vector disrupted differentiated cell lineage genes, which were silent in ES cells, and labeled trapped clone cells by the expression of EGFP upon differentiation. Thus, the dDT vector provides a systematic approach to disrupt silent genes and examine the cellular functions of trapped genes in the differentiation of target cells and development.
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Elementos Transponibles de ADN , Toxina Diftérica/genética , Marcación de Gen/métodos , Células Madre Embrionarias de Ratones/metabolismo , Animales , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Mutagénesis , Mutagénesis InsercionalRESUMEN
Purpose: Pentagamavunon-1 (PGV-1) is a curcumin analogue that shows cytotoxic activity in various cancer cells. In this study, we evaluated the effect of PGV-1 on a highly metastatic breast cancer cell line, the 4T1 cells, as an anti-metastatic and anti-proliferative agent. Methods: Cell viability was evaluated using MTT assay; while cell cycle profile, apoptosis incidence, and ROS intracellular level were determined by flow cytometry. Cell senescence was observed under senescence-associated-ß-galactosidase (SA-ß-gal) staining assay. The expression of matrixmetalloproteinase-9 (MMP-9) was determined using immunoreaction based-ELISA, while other proteins expression were detected using immunoblotting. Results: Curcumin and PGV-1 showed cytotoxic effects on 4T1 cells with IC50 value of 50 and 4 µM, respectively. The cytotoxic activity of PGV-1 was correlated to the induction of G2/M cell cycle arrest and cell senescence. Furthermore, PGV-1 increased the accumulation of intracellular ROS level. We also revealed that PGV-1 bound to several ROS-metabolizing enzymes, including glyoxalase I (GLO1), peroxiredoxin 1 (PRDX1), N-ribosyldihydronicotinamide: quinone reductase 2 (NQO2), aldo-keto reductase family 1 member c1 (AKR1C1). As an antimetastatic agent, PGV-1 showed less inhibitory effect on cell migration compared to curcumin. However, PGV-1 significantly decreased MMP-9 protein expression in a dose-dependent manner suggesting it still potent to inhibit metastatic cells. Conclusion: Overall, our findings suggest that PGV-1 is potential to be developed as an antiproliferative and anti-metastatic agent.
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Homozygous mutations of human HTRA1 cause cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). HtrA1-/- mice were examined for arterial abnormalities. Although their cerebral arteries were normal, the thoracic aorta was affected in HtrA1-/- mice. The number of vascular smooth muscle cells (VSMCs) in the aorta was increased in HtrA1-/- mice of 40 weeks or younger, but decreased thereafter. The cross-sectional area of the aorta was increased in HtrA1-/- mice of 40 weeks or older. Aortic VSMCs isolated from HtrA1-/- mice rapidly proliferated and migrated, produced high MMP9 activity, and were prone to oxidative stress-induced cell death. HtrA1-/- VSMCs expressed less smooth muscle α-actin, and more vimentin and osteopontin, and responded to PDGF-BB more strongly than wild type VSMCs, indicating that HtrA1-/- VSMCs were in the synthetic phenotype. The elastic lamina was disrupted, and collagens were decreased in the aortic media. Calponin in the media was decreased, whereas vimentin and osteopontin were increased, suggesting a synthetic shift of VSMCs in vivo. Loss of HtrA1 therefore skews VSMCs toward the synthetic phenotype, induces MMP9 expression, and expedites cell death. We propose that the synthetic modulation is the primary event that leads to the vascular abnormalities caused by HtrA1 deficiency.
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Serina Peptidasa A1 que Requiere Temperaturas Altas/fisiología , Músculo Liso Vascular/metabolismo , Animales , Aorta/enzimología , Aorta/metabolismo , Aorta/patología , Apoptosis , Supervivencia Celular , Células Cultivadas , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , Estrés OxidativoRESUMEN
Cell cycle regulation and the NF-κB pathway in cancer cells are important in mediating resistance to 5-Fluorouracil (5-FU). Pentagamavunon-1 (PGV-1), a curcumin analog, is known to exhibit stronger growth inhibitory effects than curcumin itself in several cancer cells. In this study, we evaluated the potency of PGV-1 in combination with 5-FU in WiDr colon cancer cells. In MTT assays, PGV-1 did not only exhibit stronger growth inhibitory effects than both 5-FU and curcumin, but also enhanced the cytotoxicity of 5-FU. Flow cytometry demonstrated that single treatments with PGV-1 and 5-FU resulted in different effects on cell cycle profiles. PGV-1 induced G2/M arrest while 5-FU caused S-phase arrest at low concentration (1 µM) and G1-phase arrest at high concentration (100 µM). Interestingly, the combination of 5-FU and PGV-1 enhanced cell accumulation in S-phase. Although a single treatment with either 5-FU or PGV-1 increased cyclin D1 at the protein level, the combination treatment resulted in significant suppression. In addition, PGV-1 inhibited activation of NF-κB and suppressed the expression of cyclooxygenase-2, an NF-κB downstream protein. In conclusion, PGV-1 increased the cytotoxic effect of 5-FU on WiDr cells through inhibition of NF-κB activation.
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A pathogen of giant freshwater prawn, Macrobrachium rosenbergii, was recently recorded in a hatchery in Yogyakarta. The clinical symptom in post-larvae (PL) was a whitish appearance of the muscles in the tail. Histological examination revealed myonecrosis with massive infiltration of myonuclei and hemocytes. RT-PCR products of 850 bp were obtained when using RNA from diseased PL as a template. The clinical signs and RT-PCR amplicon were reproduced in M. rosenbergii inoculated with bacteria-free inocula. Electron microscopy demonstrated that the M. rosenbergii nodavirus (MrNV) was icosahedral in shape and 28.12 ± 2.31 nm in diameter. RT-PCR products of the RNA-dependent RNA polymerase gene (RNA-1) and capsid protein gene (RNA-2) of MrNV were obtained using designed primer pairs, cloned into pBluescript-KS, and sequenced. The 1312 nucleotide (nt) sequence of MrNV RNA-1 revealed 98.0 % identity with isolates from China and India. Additionally, the 1112 nt sequence of MrNV RNA-2 displayed 98.0 % identity with isolates from China and Taiwan. Disease control efforts involving disinfection of PL, broodstocks, water media, tanks, equipment and ponds successfully eradicated white tail disease from the hatchery. This study is the first report on white tail disease and the isolation and characterization of MrNV in Indonesia.
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The molecular mechanisms underlying mesodermal and cardiac specification from embryonic stem cells (ESCs) are not fully understood. Here, we showed that the BTB domain-containing zinc finger protein CIBZ is expressed in mouse ESCs but is dramatically downregulated during ESC differentiation. CIBZ deletion in ESCs induced specification toward mesoderm phenotypes and their differentiation into cardiomyocytes, whereas overexpression of CIBZ delayed these processes. During ESC differentiation, CIBZ loss-and-gain-of-function data indicate that CIBZ negatively regulates the expressions of Brachyury (T) and Mesp1, the key transcriptional factors responsible for the specification of mammalian mesoderm and cardiac progenitors, respectively. Chromatin immunoprecipitation assays showed that CIBZ binds to T and Mesp1 promoters in undifferentiated ESCs, and luciferase assays indicate that CIBZ suppresses T and Mesp1 promoters. These findings demonstrate that CIBZ is a novel regulator of mesodermal and cardiac differentiation of ESCs, and suggest that CIBZ-mediated cardiac differentiation depends on the regulation of these two genes.
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BACKGROUND: Increased numbers and improperly positioned centrosomes, aneuploidy or polyploidy, and chromosomal instability are frequently observed characteristics of cancer cells. While some aspects of these events and the checkpoint mechanisms are well studied, not all players have yet been identified. As the role of proteases other than the proteasome in tumorigenesis is an insufficiently addressed question, we investigated the epigenetic control of the widely conserved protease HTRA1 and the phenotypes of deregulation. METHODS: Mouse embryonal fibroblasts and HCT116 and SW480 cells were used to study the mechanism of epigenetic silencing of HTRA1. In addition, using cell biological and genetic methods, the phenotypes of downregulation of HTRA1 expression were investigated. RESULTS: HTRA1 is epigenetically silenced in HCT116 colon carcinoma cells via the epigenetic adaptor protein MBD2. On the cellular level, HTRA1 depletion causes multiple phenotypes including acceleration of cell growth, centrosome amplification and polyploidy in SW480 colon adenocarcinoma cells as well as in primary mouse embryonic fibroblasts (MEFs). CONCLUSIONS: Downregulation of HTRA1 causes a number of phenotypes that are hallmarks of cancer cells suggesting that the methylation state of the HtrA1 promoter may be used as a biomarker for tumour cells or cells at risk of transformation.
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Neoplasias del Colon/genética , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Fibroblastos/citología , Serina Endopeptidasas/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Centrosoma/metabolismo , Neoplasias del Colon/patología , Regulación hacia Abajo , Epigénesis Genética , Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Ratones , Trasplante de Neoplasias , Poliploidía , Regiones Promotoras GenéticasRESUMEN
trans-Bisthioglycosylated tetrakis(fluorophenyl)chlorin (7) was designed as a powerful photodynamic therapy (PDT) photosensitizer based on the findings of our systematic studies. We show here that the trans-bisthioglycosylated structure of 7 enhanced its uptake by HeLa cells and that the chlorin ring of 7 increased the efficiency of reactive oxygen species generation under the standard condition of our photocytotoxicity test. The versatility of 7 in PDT treatment was established using weakly metastatic B16F1 melanoma cells, metastatic 4T1 breast cancer cells, the RGK-1 gastric carcinoma mucosal cell line, and three human glioblastoma cell lines (U87, U251, and T98G). The pharmacokinetics of 7 in mice bearing 4T1 breast cancer cells showed a high tumor-to-skin concentration ratio (approximately 60) at 24 h after intraperitoneal injection. The PDT efficacy of 7 in vivo was approximately 250-times higher than that of mono-l-aspartyl chlorin e6 (9) in mice bearing 4T1 breast cancer cells.
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Neoplasias de la Mama/tratamiento farmacológico , Hidrocarburos Clorados/química , Hidrocarburos Clorados/uso terapéutico , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/uso terapéutico , Animales , Mama/efectos de los fármacos , Mama/patología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Halogenación , Humanos , Hidrocarburos Clorados/síntesis química , Hidrocarburos Clorados/farmacocinética , Ratones , Ratones Endogámicos BALB C , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Fotoquimioterapia , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/farmacocinéticaRESUMEN
Abnormal levels of High temperature requirement A1 (HtrA1) protein have been repeatedly observed in sera and placentas of preeclampsia patients. To understand the functions of HtrA1 in placentation and in the etiology of preeclampsia, we established HtrA1(-/-) mice. HtrA1(-/-) mice show intrauterine growth retardation, and their placentas are small due to a reduced size of the junctional zone and aberrant vascularization in the labyrinth at the mid-gestation stage. HtrA1 is expressed by Tpbpa-positive trophoblast precursors in the outer ectoplacental cone and junctional zone from embryonic day 7.5 to 10.5. In the HtrA1(-/-) placenta, Tpbpa-positive cell precursors are decreased in the early stage. Spongiotrophoblasts and glycogen trophoblast cells, both of which differentiate from Tpbpa-positive precursors, are consequently decreased in the junctional zone. Fewer spiral artery-associated trophoblast giant cells, another cell type derived from Tpbpa-positive precursors, invade the decidua and associate with maternal arteries in the HtrA1(-/-) placenta than in the wild type placenta. Maternal arteries in the HtrA1(-/-) decidua have narrower lumens, thicker arterial walls, and more vascular smooth muscle cells remaining in the walls than those in the wild type decidua, indicating impaired remodeling of maternal arteries. These results indicate that HtrA1 plays important roles in the differentiation of trophoblasts from Tpbpa-positive precursors in the ectoplacental cone. Insufficient levels of HtrA1 cause poor placental development and intrauterine growth retardation, due to aberrant trophoblast differentiation and consequent defects in maternal artery remodeling, and may contribute to the onset of preeclampsia.
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Placentación , Serina Endopeptidasas/genética , Serina Endopeptidasas/fisiología , Animales , Diferenciación Celular , Linaje de la Célula , Decidua/fisiología , Modelos Animales de Enfermedad , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas , Ratones , Ratones Noqueados , Fenotipo , Preeclampsia , Embarazo , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismo , Trofoblastos/metabolismoRESUMEN
BNIP-2 shows high homology with the Cayman ataxia protein, caytaxin, which functions as a kinesin-1 adapter bridging cargos and kinesin light chains (KLCs). BNIP-2 is known to induce cell shape changes when over-expressed in culture cells, but its physiological functions are mostly unknown. BNIP-2 interacts with KLC through the conserved WED motif in the N-terminal region of BNIP-2. Interaction with KLC and transportation by kinesin-1 are essential for over-expressed BNIP-2 to elongate cells and induce cellular processes. Endogenous BNIP-2 localizes to the Golgi apparatus, early and recycling endosomes and mitochondria, aligned with microtubules, and moves at a speed compatible with kinesin-1 transportation. The CRAL-TRIO domain of BNIP-2 specifically interacts with phosphatidylserine, and the vesicular localization of BNIP-2 requires interaction with this phospholipid. BNIP-2 mutants which do not bind phosphatidylserine do not induce morphological changes in cells. These data show that similar to caytaxin, BNIP-2 is a kinesin-1 adapter involved in vesicular transportation in the cytoplasm and that association with cargos depends on interaction of the CRAL-TRIO domain with membrane phosphatidylserine.
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Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfatidilserinas/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Cinesinas , Unión ProteicaRESUMEN
Chemoresistance of breast cancer to doxorubicin is mediated mainly through activation of NF-kB and over expression of HER2. Curcumin and its analogues (PGV-0 and PGV-1) exert cytotoxic effects on T47D breast cancer cells. Suppression of NF-kB activation is suggested to contribute to this activity. The present study aimed to explore the effects of curcumin, PGV-0, and PGV-1 singly and in combination with doxorubicin on MCF-7/Dox cells featuring over-expression of HER2. In MTT assays, curcumin, PGV-0, and PGV-1 showed cytotoxicity effects against MCF-7/Dox with IC50 values of 80 µM, 21 µM, and 82 µM respectively. These compounds increased MCF-7/Dox sensitivity to doxorubicin. Cell cycle distribution analysis exhibited that the combination of curcumin and its analogues with Dox increased sub G-1 cell populations. Curcumin and PGV-1 but not PGV-0 decreased localization of p65 into the nucleus induced by Dox, indicating that activation of NF- kB was inhibited. Molecular docking of curcumin, PGV-0, and PGV-1 demonstrated high affinity to HER2 at ATP binding site. This interaction were directly comparable with those of ATP and lapatinib. These findings suggested that curcumin, PGV-0 and PGV-1 enhance the Dox cytotoxicity to MCF-7 cells through inhibition of HER2 activity and NF-kB activation.
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Antineoplásicos/farmacología , Curcumina/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , FN-kappa B/metabolismo , Receptor ErbB-2/metabolismo , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Curcumina/análogos & derivados , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Células MCF-7 , FN-kappa B/antagonistas & inhibidores , Receptor ErbB-2/antagonistas & inhibidores , Factor de Transcripción ReIA/metabolismoRESUMEN
Genetic predisposition and senescence of retinal pigment epithelium induced by oxidative stress are major contributors to age-related macular degeneration (AMD). Single-nucleotide polymorphisms in HTRA1 are strongly linked to the onset of AMD. In this study, we examine the role of HtrA1 in premature senescence and cell death induced by oxidative stress. HtrA1 mRNA and protein were up-regulated during premature senescence induced by H2O2 in both mouse embryonic fibroblasts (MEFs) and ARPE-19 cells. Expression of the senescence markers p21(CIP1/WAF1) and p16(INK4a), and SA-ß-galactosidase activity, were higher in HtrA1+/- MEFs than in HtrA1-/- MEFs. HtrA1+/+ and HtrA1+/- MEFs were more resistant than HtrA1-/- MEFs to H2O2-induced cell death. Activation of p38 MAPK by oxidative stress was quicker in HtrA1+/- MEFs than in HtrA1-/- MEFs. The effects of excess HtrA1 were examined by transient transfection of cells with HtrA1 expression vectors or by addition of recombinant proteins. Excess wild type HtrA1 accelerated premature senescence of MEFs and ARPE-19 cells, while the protease-inactive HtrA1 S328A did not. HtrA1-induced senescence was abrogated by inhibition of p38 MAPK. We conclude that HtrA1 is induced by oxidative stress and promotes premature cell senescence through p38 MAPK in a protease activity-dependent manner.
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Apoptosis/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Peróxido de Hidrógeno/toxicidad , Estrés Oxidativo , Serina Endopeptidasas/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Western Blotting , Femenino , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH/efectos de los fármacos , Células 3T3 NIH/metabolismo , Plásmidos , ARN Mensajero/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/metabolismo , Transfección , Regulación hacia ArribaRESUMEN
OBJECTIVE: To evaluate the effects of n-hexane insoluble fraction (HIF) of Ficus septica leaves in combination with doxorubicin on cytotoxicity, cell cycle and apoptosis induction of breast cancer T47D cell lines. METHODS: The in vitro drugs-stimulated cytotoxic effects were determined using MTT assay. Analysis of cell cycle distribution was performed using flowcytometer and the data was analyzed using ModFit LT 3.0 program. Apoptosis assay was carried out by double staining method using ethydium bromide-acridin orange. The expression of cleaved-poly (ADP-ribose) polymerase (PARP) on T47D cell lines was identified using immunocytochemistry. RESULTS: The combination exhibited higher inhibitory effect on cell growth than the single treatment of doxorubicin in T47D cells. In addition, combination of doxorubicin and HIF increased the incidence of cells undergoing apoptosis. HIF could improve doxorubicin cytotoxic effect by changing the accumulation of cell cycle phase from G2/M to G1 phase. The combination also exhibited upregulation of cleaved-PARP in T47D cells. CONCLUSIONS: Based on this results, HIF is potential to be developed as co-chemotherapeutic agent for breast cancer by inducing apoptosis and cell cycle arrest. However, the molecular mechanism need to be explored further.
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Antineoplásicos/farmacología , Doxorrubicina/farmacología , Ficus/química , Hexanos/química , Hexanos/farmacología , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Neoplasias de la Mama , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , SolubilidadRESUMEN
Palladium(II) complexes of glycoconjugated porphyrin and pyrrolidine-fused chlorin were prepared to examine sugar and heavy atom effects on in vitro photocytotoxicity. Cellular uptake into HeLa cells was enhanced by introducing sugar units regardless of other features, such as the central ion (free base or palladium(II) ion) and the ring structure (porphyrin or chlorin). The palladium(II) complex of glycoconjugated pyrrolidine-fused chlorin (PdPC2) exerted an excellent degree of photocytotoxicity not only on HeLa cells, but also on metastatic B16-BL6 cells, weakly metastatic B16F1 cells, and metastatic 4T1 cells. However, free-base glycoconjugated pyrrolidine-fused chlorin (PC2) also exerted similar or much higher photocytotoxicity rather than PdPC2. Therefore, the palladium(II) ion did not improve the in vitro photocytotoxicity of PC2. The enhanced singlet oxygen generation of palladium(II) complexes (i.e., the heavy atom effect) was confirmed at least in O(2)-saturated D(2)O. In addition, the formation of hydrogen peroxide and hydroxyl radical were also detected in O(2)-saturated phosphate buffered saline. However, the reactive oxygen species (ROS) generation efficiency, which is the product of the (relative) quantum yield of each ROS and the light absorbing ability, did not fit the trends of photocytotoxicity seen for the photosensitizers. In our glycoconjugated photosensitizers tested, the best indicator of the photocytotoxicity was found to be the light absorbing ability (namely, the oscillator strength in the wavelength region applied in the photocytotoxicity test). These results indicated that photochemical characteristics of glycoconjugated photosensitizers were notably susceptible to the microenvironment. The biological characteristics, such as the sugar effect, were a much more reliable approach to improving the photocytotoxicity of photosensitizers.
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Carbohidratos/farmacología , Supervivencia Celular/efectos de los fármacos , Glicoconjugados/farmacología , Paladio/química , Porfirinas/química , Línea Celular Tumoral , Glicoconjugados/química , Humanos , Espectroscopía de Resonancia Magnética , Especies Reactivas de Oxígeno/metabolismo , Espectrofotometría UltravioletaRESUMEN
Upf1 is a highly conserved RNA helicase essential for nonsense-mediated mRNA decay (NMD), an mRNA quality-control mechanism that degrades aberrant mRNAs harboring premature termination codons (PTCs). For the activation of NMD, UPF1 interacts first with a translation-terminating ribosome and then with a downstream exon-junction complex (EJC), which is deposited at exon-exon junctions during splicing. Although the helicase activity of Upf1 is indispensable for NMD, its roles and substrates have yet to be fully elucidated. Here we show that stable RNA secondary structures between a PTC and a downstream exon-exon junction increase the levels of potential NMD substrates. We also demonstrate that a stable secondary structure within the 3'-untranslated region (UTR) induces the binding of Upf1 to mRNA in a translation-dependent manner and that the Upf1-related molecules are accumulated at the 5'-side of such a structure. Furthermore, we present evidence that the helicase activity of Upf1 is used to bridge the spatial gap between a translation-termination codon and a downstream exon-exon junction for the activation of NMD. Based on these findings, we propose a model that the Upf1-related molecular motor scans the 3'-UTR in the 5'-to-3' direction for the mRNA-binding factors including EJCs to ensure mRNA integrity.