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1.
Talanta ; 235: 122814, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517670

RESUMO

Simple and sensitive detection of telomerase activity is of vital importance for both early diagnosis and therapy of malignant tumors. Inspired by DNA-biobarcode amplification reported by Chad A. Mirkin, we developed a facile DNA-biobarcode-like SERS-based copper-mediated signal amplification strategy for sensitive detection of telomerase activity. In this strategy, a duplex DNA constructed by hybridization of a copper oxide nanoparticle (CuO NP)-labeled reporting sequence (RS) with the telomerase primer sequence (TS) is ingeniously designed, and anchored on the magnetic bead (MB) to build the CuO NPs-encoded magnetic bead (MB-CuO NPs) detection probe. Upon selective sensing of telomerase, telomerase elongation reaction and structure change of TS products make the CuO NP-RS displace and separate from MB. The separated CuO NPs are dissolved into a mass of Cu2+, which prompt monodisperse dopamine-functionalized AgNPs (D-AgNPs) signal probe into aggregation, resulting in color changes and significantly enhancing of SERS signal. The SERS signal increases with the increase of Cu2+, which is directly proportional to the telomerase. Benefiting from the transformation of CuO NP to Cu2+ with a high amplification effect, this strategy could realize the telomerase activity measurement down to 3 HeLa cells and a dynamic range of 10-10000 cells. It shows a significant improvement of sensitivity without need for other enzymes and elaborate design, which escapes from the complicated manipulations and design in polymerase chain reaction (PCR) and DNA amplification techniques. Moreover, with this strategy, telomerase activities of different cell lines and telomerase inhibitors screening were successfully performed. Significantly, it can also be utilized for visual detection of telomerase, which validates the potential on-site application and its application as point-of-care testing (POCT) for efficient monitoring. Given the high-performance for telomerase analysis, the strategy has a promising application in biological detection and clinical diagnosis, as well as point-of-care tests.


Assuntos
Técnicas Biossensoriais , Telomerase , Cobre , DNA , Células HeLa , Humanos , Técnicas de Amplificação de Ácido Nucleico , Telomerase/genética , Telomerase/metabolismo
2.
Reprod Biomed Online ; 38(5): 761-767, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30885666

RESUMO

RESEARCH QUESTION: The aim of this study was to compare expression of hypoxia-inducible factor 1-alpha (HIF-1α), angiogenesis and apoptosis in endometrial tissue near the implantation window of women with recurrent implantation failure (RIF) and in fertile control women, and to describe possible mechanisms of endometrial injury. DESIGN: A controlled clinical study was conducted. Endometrial tissue specimens were obtained from 20 women undergoing IVF who had had at least three previous failed treatment cycles; normal endometrial specimens were obtained from 10 fertile control women. RESULTS: HIF-1α expression was down-regulated in the endometrium of women with RIF compared with that of control women. In addition, micro-vessel density (MVD) was much lower in the endometrium of women with RIF than in that of the control women. Apoptosis was significantly reduced in the endometrium of the RIF group compared with the control group. Endometrial injury increased HIF-1α expression and MVD in endometrial samples of the RIF group, but apoptosis was not significantly altered. CONCLUSIONS: HIF-1α expression, MVD and endometrial apoptosis were reduced in the peri-implantation endometrium of women with RIF. This suggests that altered endometrial HIF-1α expression and angiogenesis may contribute to implantation failure.


Assuntos
Implantação do Embrião , Transferência Embrionária , Endométrio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Fisiológica , Adulto , Apoptose , Endométrio/irrigação sanguínea , Feminino , Humanos , Falha de Tratamento
3.
Cell Oncol (Dordr) ; 37(6): 429-37, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25404385

RESUMO

PURPOSE: The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, mediates a broad spectrum of biological processes, including ovarian growth and ovulation. Recently, we found that an endogenous AhR ligand (ITE) can inhibit ovarian cancer proliferation and migration via the AhR. Here, we tested whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, an exogenous AhR ligand) may exert similar anti-ovarian cancer activities using human ovarian cancer and non-cancerous human ovarian surface epithelial cells. METHODS: Two human ovarian cancer cell lines (SKOV-3 and OVCAR-3) and one human ovarian surface epithelial cell line (IOSE-385) were used. Cell proliferation and migration activities were determined using crystal violet and FluoroBlok insert system assays, respectively. AhR protein expression was assessed by Western blotting. Expression of cytochrome P450, family 1, member A1 (CYP1A1) and member B1 (CYP1B1) mRNA was assessed by qPCR. Small interfering RNAs (siRNAs) were used to knock down AhR expression. RESULTS: We found that TCDD dose-dependently suppressed OVCAR-3 cell proliferation, with a maximum effect (~70% reduction) at 100 nM. However, TCDD did not affect SKOV-3 and IOSE-385 cell proliferation and migration. The estimated IC50 of TCDD for inhibiting OVCAR-3 cell proliferation was 4.6 nM. At 10 nM, TCDD time-dependently decreased AhR protein levels, while it significantly increased CYP1A1 and CYP1B1 mRNA levels in SKOV-3, OVCAR-3 and IOSE-385 cells, indicating activation of AhR signaling. siRNA-mediated AhR knockdown readily blocked TCDD-mediated suppression of OVCAR-3 cell proliferation. CONCLUSION: Our data indicate that TCDD can suppress human ovarian cancer cell proliferation via the AhR signaling pathway and that TCDD exhibits an anti-proliferative activity in at least a subset of human ovarian cancer cells.


Assuntos
Neoplasias Ovarianas/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Antineoplásicos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/metabolismo
4.
Mol Cell Endocrinol ; 391(1-2): 60-7, 2014 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-24796659

RESUMO

Fetoplacental endothelial cells reside under physiological normoxic conditions (∼2-8% O2) in vivo. Under such conditions, cells are believed to sense O2 changes primarily via hypoxia inducible factor 1 α (HIF1A). However, little is known regarding the role of HIF1A in fetoplacental endothelial function under physiological normoxia. We recently reported that physiological chronic normoxia (PCN; 20-25 day, 3% O2) enhanced FGF2- and VEGFA-stimulated proliferation and migration of human umbilical vein endothelial cells (HUVECs) via the MEK/ERK1/2 and PI3K/AKT1 pathways compared to standard cell culture normoxia (SCN; ambient O2: ∼21% O2). Here, we investigated the action of HIF1A in regulating these cellular responses in HUVECs. HIF1A adenovirus infection in SCN-cells increased HIF1A protein expression, enhanced FGF2- and VEGFA-stimulated cell proliferation by 2.4 and 2.0-fold respectively, and promoted VEGFA-stimulated cell migration by 1.4-fold. HIF1A adenovirus infection in SCN-cells did not affect either basal or FGF2- and VEGFA-induced ERK1/2 activation, but it decreased basal AKT1 phosphorylation. Interestingly, HIF1A knockdown in PCN-cells via specific HIF1A siRNA transfection did not alter FGF2- and VEGFA-stimulated cell proliferation and migration, or ERK1/2 activation; however, it inhibited FGF2-induced AKT1 activation by ∼50%. These data indicate that HIF1A differentially regulates cell proliferation and migration, and ERK1/2 and AKT1 activation in PCN- and SCN-HUVECs. These data also suggest that HIF1A critically regulates cell proliferation and migration in SCN-, but not in PCN-HUVECs.


Assuntos
Adaptação Fisiológica/genética , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Oxigênio/farmacologia , Adenoviridae/genética , Hipóxia Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica , Vetores Genéticos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos
5.
Reprod Sci ; 21(1): 102-11, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23757313

RESUMO

Estradiol 17ß (E2ß) and ascorbic acid (AA) have been implicated in cancer progression. However, little is known about the actions of biologically active metabolites of E2ß, 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 2-methoxyestradiol (2ME2), and 4-methoxyestradiol (4ME2) synthesized sequentially by cytochrome P450, family 1, subfamily A (CYP1A1) and B (CYP1B1), polypeptide 1, and catechol-O-methyltransferase (COMT) on ovarian cancer. Herein, we examined the expression of CYP1A1, CYP1B1, COMT, and estrogen receptor α (ERα) and ß (ERß) in human ovarian surface epithelial (IOSE-385) and cancer cell lines (OVCAR-3, SKOV-3, and OVCA-432). We also investigated the roles of E2ß, 2OHE2, 4OHE2, 2ME2, and 4ME2 in cell proliferation, and their interactive effects with AA on ovarian cells. We found the expression of CYP1A1, CYP1B1, COMT, ERα, and ERß in most cell lines tested. Treating cells with physiological concentrations of E2ß and its metabolites promoted (13%-42% of the control) IOSE-385 and OVCAR-3 proliferation. The ER blockade inhibited IOSE-385 (∼76%) and OVCAR-3 (∼87%) proliferative response to E2ß but not to its metabolites. The ERα blockade inhibited (∼85%) E2ß-stimulated OVCAR-3 proliferation, whereas ERß blockade attenuated (∼83%) E2ß-stimulated IOSE-385 proliferation. The AA at ≥250 µmol/L completely inhibited serum-stimulated cell proliferation in all cell lines tested; however, such inhibition in IOSE-385, OVCAR-3, and OVCA-432 was partially (∼10%-20%) countered by E2ß and its metabolites. Thus, our findings indicate that E2ß and its metabolites promote cell proliferation and antagonize the AA-suppressed cell proliferation in a subset of ovarian cancer cells, suggesting that blocking the actions of E2ß and its metabolites may enhance AA's antiovarian cancer activity.


Assuntos
Ácido Ascórbico/farmacologia , Proliferação de Células/efeitos dos fármacos , Estradiol/farmacologia , Neoplasias Ovarianas/patologia , 2-Metoxiestradiol , Hidrocarboneto de Aril Hidroxilases/metabolismo , Catecol O-Metiltransferase/metabolismo , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1 , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estradiol/análogos & derivados , Estradiol/metabolismo , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/efeitos dos fármacos , Receptor beta de Estrogênio/metabolismo , Estrogênios de Catecol/metabolismo , Estrogênios de Catecol/farmacologia , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Fatores de Tempo
6.
Biol Reprod ; 89(6): 133, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24152727

RESUMO

Fetoplacental endothelial cells are exposed to oxygen levels ranging from 2% to 8% in vivo. However, little is known regarding endothelial function within this range of oxygen because most laboratories use ambient air (21% O2) as a standard culture condition (SCN). We asked whether human umbilical artery endothelial cells (HUAECs) that were steadily exposed to the physiological chronic normoxia (PCN, 3% O2) for ∼20-25 days differed in their proliferative and migratory responses to FGF2 and VEGFA as well as in their global gene expression compared with those in the SCN. We observed that PCN enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. In oxygen reversal experiments (i.e., when PCN cells were exposed to SCN for 24 h and vice versa), we found that preexposure to 21% O2 decreased the migratory ability, but not the proliferative ability, of the PCN-HUAECs in response to FGF2 and VEGFA. These PCN-enhanced cellular responses were associated with increased protein levels of HIF1A and NOS3, but not FGFR1, VEGFR1, and VEGFR2. Microarray analysis demonstrated that PCN up-regulated 74 genes and down-regulated 86, 14 of which were directly regulated by hypoxia-inducible factors as evaluated using in silico analysis. Gene function analysis further indicated that the PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from our functional assays. Given that PCN significantly alters cellular responses to FGF2 and VEGFA as well as transcription in HUAECs, it is likely that we may need to reexamine the current cellular and molecular mechanisms controlling fetoplacental endothelial functions, which were largely derived from endothelial models established under ambient O2.


Assuntos
Células Endoteliais/metabolismo , Circulação Placentária/genética , Transcriptoma , Artérias Umbilicais/metabolismo , Hipóxia Celular/genética , Movimento Celular/genética , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Análise em Microsséries , Oxigênio/farmacologia , Gravidez , Estresse Fisiológico/genética , Fatores de Tempo , Artérias Umbilicais/citologia
7.
Cancer Lett ; 340(1): 63-71, 2013 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-23851185

RESUMO

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor mediates many biological processes. Herein, we investigated if 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE, an endogenous AhR ligand) regulated proliferation and migration of human ovarian cancer cells via AhR. We found that AhR was widely present in many histotypes of ovarian cancer tissues. ITE suppressed OVCAR-3 cell proliferation and SKOV-3 cell migration in vitro, which were blocked by AhR knockdown. ITE also suppressed OVCAR-3 cell growth in mice. These data suggest that the ITE might potentially be used for therapeutic intervention for at least a subset of human ovarian cancer.


Assuntos
Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Indóis/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Receptores de Hidrocarboneto Arílico/metabolismo , Tiazóis/farmacologia , Animais , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Receptores de Hidrocarboneto Arílico/genética , Análise Serial de Tecidos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Biol Reprod ; 88(5): 114, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23536375

RESUMO

Endothelial cells chronically reside in low-O2 environments in vivo (2%-13% O2), which are believed to be critical for cell homeostasis. To elucidate the roles of this physiological chronic normoxia in human endothelial cells, we examined transcriptomes of human umbilical vein endothelial cells (HUVECs), proliferation and migration of HUVECs in response to fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA), and underlying signaling mechanisms under physiological chronic normoxia. Immediately after isolation, HUVECs were cultured steadily under standard cell culture normoxia (SCN; 21% O2) or physiological chronic normoxia (PCN; 3% O2) up to 25 days. We found that PCN up-regulated 41 genes and down-regulated 21 genes, 90% of which differed from those previously reported from HUVECs cultured under SCN and exposed to acute low O2. Gene ontology analysis indicated that PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from benchtop assays that showed that PCN significantly enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. Interestingly, preexposing the PCN cells to 21% O2 up to 5 days did not completely diminish PCN-enhanced cell proliferation and migration. These PCN-enhanced cell proliferations and migrations were mediated via augmented activation of MEK1/MEK2/ERK1/ERK2 and/or PI3K/AKT1. Importantly, these PCN-enhanced cellular responses were associated with an increase in activation of VEGFR2 but not FGFR1, without altering their expression. Thus, PCN programs endothelial cells to undergo dramatic changes in transcriptomes and sensitizes cellular proliferative and migratory responses to FGF2 and VEGFA. These PCN cells may offer a unique endothelial model, more closely mimicking the in vivo states.


Assuntos
Adaptação Fisiológica/fisiologia , Hipóxia Celular/fisiologia , Movimento Celular/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/farmacologia
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