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1.
Biol Reprod ; 101(5): 1001-1017, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31350850

RESUMO

Yes-associated protein 1 (YAP1) is a major component of the Hippo signaling pathway. Although the exact extracellular signals that control the Hippo pathway are currently unknown, increasing evidence supports a critical role for the Hippo pathway in embryonic development, regulation of organ size, and carcinogenesis. Granulosa cells (GCs) within the ovarian follicle proliferate and produce steroids and growth factors, which facilitate the growth of follicle and maturation of the oocyte. We hypothesize that YAP1 plays a role in proliferation and estrogen secretion of GCs. In the current study, we examined the expression of the Hippo signaling pathway in bovine ovaries and determined whether it was important for GC proliferation and estrogen production. Mammalian STE20-like protein kinase 1 (MST1) and large tumor suppressor kinase 2 (LATS2) were identified as prominent upstream components of the Hippo pathway expressed in granulosa and theca cells of the follicle and large and small cells of the corpus luteum. Immunohistochemistry revealed that YAP1 was localized to the nucleus of growing follicles. In vitro, nuclear localization of the downstream Hippo signaling effector proteins YAP1 and transcriptional co-activator with PDZ-binding motif (TAZ) was inversely correlated with GC density, with greater nuclear localization under conditions of low cell density. Treatment with verteporfin and siRNA targeting YAP1 or TAZ revealed a critical role for these transcriptional co-activators in GC proliferation. Furthermore, knockdown of YAP1 in GCs inhibited follicle-stimulating hormone (FSH)-induced estradiol biosynthesis. The data indicate that Hippo pathway transcription co-activators YAP1/TAZ play an important role in GC proliferation and estradiol synthesis, two processes necessary for maintaining normal follicle development.

2.
Molecules ; 24(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30978940

RESUMO

Saikosaponin d (SSd) is one of the main active ingredients in Radix Bupleuri. In our study, network pharmacology databases and metabolomics were used in combination to explore the new targets and reveal the in-depth mechanism of SSd. A total of 35 potential targets were chosen through database searching (HIT and TCMID), literature mining, or chemical similarity predicting (Pubchem). Out of these obtained targets, Neuropilin-1 (NRP-1) was selected for further research based on the degree of molecular docking scores and novelty. Cell viability and wound healing assays demonstrated that SSd combined with NRP-1 knockdown could significantly enhance the damage of HepG2. Metabolomics analysis was then performed to explore the underlying mechanism. The overall difference between groups was quantitatively evaluated by the metabolite deregulation score (MDS). Results showed that NRP-1 knockdown exhibited the lowest MDS, which demonstrated that the metabolic profile experienced the slightest interference. However, SSd alone, or NRP-1 knockdown in combination with SSd, were both significantly influenced. Differential metabolites mainly involved short- or long-chain carnitines and phospholipids. Further metabolic pathway analysis revealed that disturbed lipid transportation and phospholipid metabolism probably contributed to the enhanced anti-hepatoma effect by NRP-1 knockdown in combination with SSd. Taken together, in this study, we provided possible interaction mechanisms between SSd and its predicted target NRP-1.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Neuropilina-1/genética , Ácido Oleanólico/análogos & derivados , Saponinas/farmacologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Redes e Vias Metabólicas/efeitos dos fármacos , Metabolômica/métodos , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Neuropilina-1/antagonistas & inibidores , Ácido Oleanólico/farmacologia
3.
J Cell Biochem ; 2019 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-30697802

RESUMO

Previous studies have reported that microRNAs function as key regulators in tumor development and progression. This study aims to investigate the functional effects of miR-503 expression in cervical cancer (CC) progression. We detected the expression of miR-503 in CC tissues and cell lines using quantitative real-time polymerase chain reaction. Synthesized miR-503 mimics or inhibitors were used to upregulate or downregulate the expression of miR-503 in HeLa or SiHa cells. Cell Counting Kit-8 and colony formation assay were used to detect the ability of cell proliferation. Furthermore, luciferase assay and Western blot were applied to confirm the target of miR-503 in CC cells. Here, we demonstrated that miR-503 expression was significantly downregulated in CC tissues, compared with adjacent normal tissues. miR-503 expression was significantly associated with tumor size and International Federation of Gynecology and Obstetrics stage. Furthermore, increasing miR-503 expression in CC cells dramatically inhibited cell proliferation, colony formation ability of CC. However, reducing miR-503 had reverse effects on these malignant behaviors. Moreover, we demonstrated that miR-503 inhibited cell proliferation by targeting AKT2 3'-untranslated region and affected its expression. Overexpression of AKT2 rescued the effects induced by miR-503 on cell proliferation. Therefore, our results indicated that miR-503 may serve as a tumor suppressor in CC and provide a potential value for CC treatment.

4.
Phytomedicine ; 54: 120-131, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30668361

RESUMO

BACKGROUND: Emerging hallmark of cancer is reprogrammed cellular metabolism, increased glycolytic metabolism is physiological characteristic of human malignant neoplasms. Saponin monomer 13 of the dwarf lilyturf tuber (DT-13) is the main steroidal saponin from Liriopes Radix, which has been reported to exert anti-inflammation and anti-tumor activities but low toxicity to normal tissue. However, the effect of DT-13 on metabolism process is still unclear. PURPOSE: This study aims to characterize the role of DT-13 in glucose metabolism in colorectal cancer cells, and investigate whether the metabolism process is involved in the anti-cancer response of DT-13. METHODS: Colony formation assay was employed to determine anti-proliferative effect induced by DT-13 at 2.5, 5, 10 µM. Apoptosis and cell cycle arrest were detected by Annexin V/PI staining and PI staining, respectively. Genetic inhibition of glycolytic metabolism was carried out by knockdown of GLUT1. Orthotopic implantation mouse model of colorectal cancer was used to assess in vivo antitumor effect of DT-13 (0.625, 1.25, 2.5 mg/kg). The chemoprevention effect of DT-13 (10mg/kg) was evaluated by using C57BL/6J APCmin mice model. Glycolytic-related key enzymes and AMPK pathway were detected by using quantitative real-time PCR, western blotting, and immunohistochemical staining. RESULTS: Our results showed that cell proliferation was significantly inhibited by DT-13 in a dose-dependent manner. DT-13 inhibited glucose uptake, ATP generation, and reduced lactate production. Furthermore, DT-13 remarkably inhibited GLUT1 expression in both mRNA and protein levels. Knocking down of GLUT1 led to reduced inhibition of glucose uptake after DT-13 treatment. Moreover, deletion of GLUT1 decreased inhibitory ratio of DT-13 on cancer growth. Orthotopic implantation mouse model of colorectal cancer further confirmed that DT-13 inhibited colorectal cancer growth via blocking GLUT1 in vivo. In addition, C57BL/6J APCmin mice model revealed that DT-13 dramatically reduced the total number of spontaneous adenomas in intestinal, which further confirmed the anti-tumor activity of DT-13 in colorectal cancer. Furthermore, the mechanistically investigation showed DT-13 activated AMPK and inhibited m-TOR to block cancer growth in vitro. CONCLUSION: DT-13 is a potent anticancer agent for colorectal cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Saponinas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Glicólise , Humanos , Liriope (Planta)/química , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
5.
J Org Chem ; 83(19): 11905-11916, 2018 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-30199250

RESUMO

With the aid of density functional theory (DFT) calculations, mechanistic investigations have been carried out for the nickel-catalyzed dehydrogenative cross-coupling reaction of benzaldehyde with benzyl alcohol in the presence of N-heterocyclic carbene (NHC) ligand. The overall Ni(0)/Ni(II) catalytic cycle consists of four basic steps: ligand exchange, oxidative addition, hydrogen transfer, and reductive elimination. Considerable interests are paid on detecting the transition state of the rate-determining step, with particular emphasis on the structural and electronic properties, together with clarifying the important roles of external oxidant and hydrogen acceptor. The hydrogen transfer process in the oxidative addition step is rate-determining in the whole catalytic cycle, which is accomplished by C-Ha (active Ha) activation without generating the high energy nickel hydride intermediate. Such process could be understood as the direct hydrogen transfer, instead of general concerted oxidative addition to low valent transition metal. The analysis of the bond distances, electron distributions, and orbital interactions highlights the direct hydrogen transfer mechanism. Furthermore, by exploring the influences from the electronic effect of different substrates on the reaction energy barriers, the  a,a,a-trifluoroacetophenone could accelerate the direct hydrogen transfer with low activate energy.

6.
Eur J Pharm Sci ; 123: 546-559, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30118848

RESUMO

At present, chemotherapy is still to be the preferred and most significant therapeutic strategy for cancer patients in clinical practice. Although Camptothecin (CPT) has been discovered for over half century, a series of CPT derivatives such as Topotecan (TPT) and irinotecan (CPT-11) have been approved and are still to be the first-line medicines for clinical application. Up to now, the topoisomerase 1 inhibitor continues to be a significant drug development research field. Based on previous study of the structure-activity relationship, we consider that the introduction of lipophilic group at C7 position can prolong the retention time and the hydroxyl esterification at C20 can eliminate the hydrogen bond interaction, stabilize the E-lactone form and promote the anti-cancer effect. In this study, we carried out an optimization at C7 and C20 positions to afford two CPT derivatives 3g and 3j. Firstly, we predicted the possibly binding sites of two compounds with topoisomerase 1 by molecular docking. Then we evaluated the anti-proliferation effect of the two novel derivatives and compared the IC50 with CPT-11. Furthermore, the induction of cell cycle arrest and apoptosis was explored through karyomorphology, flow cytometry (FCM) and Western blot analysis. At last, we evaluated the anti-cancer effect and detected the mechanism in colorectal cancer xenograft model. In brief, all the data showed that the novel CPT derivatives (3g and 3j) could inhibit colorectal cancer proliferation via induction of cell cycle arrest and apoptosis in vitro and in vivo. It suggested that the two agents may be a new potential therapeutic strategy in the future.


Assuntos
Apoptose/efeitos dos fármacos , Camptotecina/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Inibidores da Topoisomerase I/farmacologia , Animais , Sítios de Ligação , Camptotecina/análogos & derivados , Camptotecina/química , Camptotecina/metabolismo , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Projeto Auxiliado por Computador , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , Desenho de Drogas , Feminino , Células HCT116 , Células HT29 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Int J Mol Med ; 41(5): 2793-2801, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29484370

RESUMO

Chemotherapy is the preferred and most common treatment for cancer in clinical practice. An increasing number of researchers all over the world are focusing on natural medicines to find new antitumor drugs, and several reports have shown that Camellia nitidissima (C. nitidissima) Chi could reduce blood-lipid, decrease blood pressure, resist oxidation, prevent carcinogenesis and inhibit tumors. Therefore, the pharmacodynamics of the chemical constituents in C. nitidissima need to be investigated further. In the present study, 16 chemical constituents were isolated from the leaves of C. nitidissima, of which 6 compounds are reported to be found in this plant for the first time. Furthermore, all these phytochemicals were screened for antitumor activity on 4 common cancer cell lines, while compound 3, one oleanane-type triterpene, exhibited the most potential antitumor effects. Interestingly, to our knowledge, this was the first report that compound 3 inhibits cancer cells. Compound 3 inhibited EGFR-mutant lung cancer cell line, NCI-H1975 via apoptosis effect, with an IC50 of 13.37±2.05 µM at 48 h. Based on the data, compound 3 showed potential for antitumor drug development, suggesting the scientific basis for the antitumor activity of C. nitidissima.


Assuntos
Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Camellia/química , Neoplasias/tratamento farmacológico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Extratos Vegetais/química , Extratos Vegetais/farmacologia
8.
Front Pharmacol ; 9: 21, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29472856

RESUMO

Tumor metastasis is the most lethal and debilitating process that threatens cancer patients. Among the regulators involved in tumor metastasis, lysyl oxidase (LOX) is an important contributor for tumor invasion, migration and the formation of the pre-metastatic niche. Although the relationship between LOX and poor prognosis of lung patients has been preliminary reported, the mechanism remains poorly understood. Here, we found that LOX overexpression is closely related to the survival of lung adenocarcinoma patients but not squamous cell carcinoma patients. Moreover, we confirmed that LOX expression is regulated by the activation of epidermal growth factor receptor (EGFR) via the PI3K/AKT, MEK/ERK, and SAPK/JNK signaling pathways in non-small cell lung cancer (NSCLC). Meanwhile, the study also suggested that the traditional anti-fibrosis drug silibinin inhibited NSCLC cell migration in an EGFR/LOX dependent manner. In addition, an orthotopic implantation metastasis model also confirmed that the EGFR inhibitor WZ4002 and silibinin decreased tumor metastasis through the EGFR/LOX pathway. Altogether, this study revealed that LOX expression is regulated by the EGFR pathway and this may account for the anti-cancer metastasis effects of silibinin, indicating LOX as a potentially therapeutic target for NSCLC treatment.

9.
Org Lett ; 20(3): 510-513, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29355325

RESUMO

A convenient copper-catalyzed intra-/intermolecular diamination of ß,γ-unsaturated hydrazones has been developed with simple amines as external amine sources. The protocol enables efficient access to various nitrogen-containing pyrazolines under mild reaction conditions.

10.
Eur J Pharmacol ; 818: 124-131, 2018 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-29037767

RESUMO

Natural medicine has multi-levels, multi-paths and multi-targets, and an increasing number of reports have confirmed that the combination of natural medicine with chemotherapy drugs exhibit a significant synergistic effect. It is necessary to find drug combination strategies to enhance efficacy and reduce toxicity, which can relieve the restrictions on the use of several chemotherapy drugs that have serious toxicity. Our previous reports showed that DT-13 inhibits cancer proliferation, invasion, migration, metastasis, and angiogenesis and induces autophagy. In this study, we evaluated the anti-proliferation effect of DT-13 on a panel of 40 different cancer cell lines for the first time. Moreover, it is also the first time that the combination of DT-13 with 5 different chemotherapy drugs on 3 common cancer cells has been examined. We further confirmed that DT-13 enhanced the sensitivity of gastric cancer cells to topotecan (TPT) via cell cycle arrest in vitro and in vivo. Considering that TPT has been subjected to restriction because of its serious toxicity, DT-13 showed the ability to enhance its effect and reduce its toxicity, which could provide a strategy to reduce the toxic and clinical side effects of TPT.


Assuntos
Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Saponinas/farmacologia , Neoplasias Gástricas/patologia , Topotecan/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Humanos
11.
Cell Death Dis ; 8(10): e3143, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29072684

RESUMO

In multiple types of tumors, fibrotic collagen is regarded as the 'highway' for cancer cell migration, which is mainly modified by lysyl hydroxylase 2 (PLOD2). The previous findings have demonstrated that the expression of PLOD2 was regulated by multiple factors, including HIF-1α, TGF-ß and microRNA-26a/b. Although PLOD2 was confirmed to be related to poor prognosis in lung adenocarcinoma, the regulatory mechanism and function of PLOD2 in human lung adenocarcinoma is poorly understood. On the other hand, upregulation or hyperactivation of epidermal growth factor receptor is considered as a prognostic marker in many cancers, especially in non-small-cell lung cancer (NSCLC). In this study, we found that PLOD2 was elevated in NSCLC specimens and positively links to NSCLC poor prognosis. Gain- and loss-of-function studies and orthotopic implantation metastasis model pinpointed that PLOD2 promotes NSCLC metastasis directly by enhancing migration and indirectly by inducing collagen reorganization. In addition, we revealed that PLOD2 was regulated by PI3K/AKT-FOXA1 axis. The transcription factor FOXA1 directly bound to the PLOD2 promoter, and turned on PLOD2 transcription. In summary, our findings revealed a regulatory mechanism of NSCLC metastasis through EGFR-PI3K/AKT-FOXA1-PLOD2 pathway, and provided PLOD2 as a therapeutic target for NSCLC treatment.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Neoplasias Pulmonares/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Análise de Sobrevida , Transfecção
12.
Data Brief ; 14: 695-706, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28932774

RESUMO

RNA expression analysis was performed on the corpus luteum tissue at five time points after prostaglandin F2 alpha treatment of midcycle cows using an Affymetrix Bovine Gene v1 Array. The normalized linear microarray data was uploaded to the NCBI GEO repository (GSE94069). Subsequent statistical analysis determined differentially expressed transcripts ± 1.5-fold change from saline control with P ≤ 0.05. Gene ontology of differentially expressed transcripts was annotated by DAVID and Panther. Physiological characteristics of the study animals are presented in a figure. Bioinformatic analysis by Ingenuity Pathway Analysis was curated, compiled, and presented in tables. A dataset comparison with similar microarray analyses was performed and bioinformatics analysis by Ingenuity Pathway Analysis, DAVID, Panther, and String of differentially expressed genes from each dataset as well as the differentially expressed genes common to all three datasets were curated, compiled, and presented in tables. Finally, a table comparing four bioinformatics tools' predictions of functions associated with genes common to all three datasets is presented. These data have been further analyzed and interpreted in the companion article "Early transcriptome responses of the bovine mid-cycle corpus luteum to prostaglandin F2 alpha includes cytokine signaling" [1].

13.
Mol Cell Endocrinol ; 452: 93-109, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28549990

RESUMO

In ruminants, prostaglandin F2alpha (PGF2α)-mediated luteolysis is essential prior to estrous cycle resumption, and is a target for improving fertility. To deduce early PGF2α-provoked changes in the corpus luteum a short time-course (0.5-4 h) was performed on cows at midcycle. A microarray-determined transcriptome was established and examined by bioinformatic pathway analysis. Classic PGF2α effects were evident by changes in early response genes (FOS, JUN, ATF3) and prediction of active pathways (PKC, MAPK). Several cytokine transcripts were elevated and NF-κB and STAT activation were predicted by pathway analysis. Self-organizing map analysis grouped differentially expressed transcripts into ten mRNA expression patterns indicative of temporal signaling cascades. Comparison with two analogous datasets revealed a conserved group of 124 transcripts similarly altered by PGF2α treatment, which both, directly and indirectly, indicated cytokine activation. Elevated levels of cytokine transcripts after PGF2α and predicted activation of cytokine pathways implicate inflammatory reactions early in PGF2α-mediated luteolysis.


Assuntos
Corpo Lúteo/metabolismo , Citocinas/metabolismo , Dinoprosta/metabolismo , Ciclo Estral/metabolismo , Luteólise/genética , Transcriptoma , Animais , Bovinos , Colesterol/genética , Colesterol/metabolismo , Corpo Lúteo/efeitos dos fármacos , Citocinas/genética , Dinoprosta/farmacologia , Feminino , Perfilação da Expressão Gênica/veterinária , Inflamação , Modelos Lineares , Células Lúteas/metabolismo , Ovariectomia , Cultura Primária de Células , Progesterona/sangue , Progesterona/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Biomed Pharmacother ; 90: 670-676, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28415047

RESUMO

Collagen is not only the most abundant protein providing the scaffold for assembly of the extracellular matrix (ECM), but also considered to be the "highway" for cancer cell migration and invasion depending on the different collagen organizations. The accumulation of stabilized collagen is enhanced by different covalent collagen cross-links, lysyl hydroxylases 2 (encoded by the PLOD2 gene) is the key enzyme mediating the formation of the stabilized collagen cross-link. Interestingly, PLOD2 is overexpressed in different cancers and closely related to a poor prognosis. To the best of our knowledge, only the mechanisms of PLOD2 regulated by HIF-1α, TGF-ß and microRNA-26a/b have been elaborated. In addition, several pharmacologic inhibitors of PLOD2 have been confirmed to have an anti-metastasis effect. However, there have been no reviews about PLOD2 in cancer research published thus far. In brief, this review about PLOD2 will describe the function, regulatory mechanism, and the inhibitors of PLOD2 in cancer, suggesting the credible clinical evaluation of a prognostic signature in pathological examination and the possible development of therapeutic strategies targeting PLOD2 in the future.


Assuntos
Neoplasias/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Movimento Celular/fisiologia , Colágeno/metabolismo , Humanos , Metástase Neoplásica/patologia , Neoplasias/patologia , Prognóstico
15.
Mol Cell Endocrinol ; 439: 379-394, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-27693538

RESUMO

After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these four cell types. Analysis of the RNA present in each bovine cell type using Affymetrix microarrays yielded new cell-specific genetic markers, functional insight into the behavior of each cell type via Gene Ontology Annotations and Ingenuity Pathway Analysis, and evidence of small and large luteal cell lineages using Principle Component Analysis. Enriched expression of select genes for each cell type was validated by qPCR. This expression analysis offers insight into cell-specific behaviors and the differentiation process that transforms somatic follicular cells into luteal cells.


Assuntos
Perfilação da Expressão Gênica , Células Lúteas/metabolismo , Folículo Ovariano/metabolismo , Animais , Biomarcadores/metabolismo , Bovinos , Linhagem da Célula/genética , Análise por Conglomerados , Feminino , Regulação da Expressão Gênica , Células da Granulosa/metabolismo , Luteinização/genética , Análise em Microsséries , Análise de Componente Principal , Reprodutibilidade dos Testes , Células Tecais/metabolismo , Transcriptoma/genética
16.
Data Brief ; 10: 335-339, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28004024

RESUMO

Affymetrix Bovine GeneChip® Gene 1.0 ST Array RNA expression analysis was performed on four somatic ovarian cell types: the granulosa cells (GCs) and theca cells (TCs) of the dominant follicle and the large luteal cells (LLCs) and small luteal cells (SLCs) of the corpus luteum. The normalized linear microarray data was deposited to the NCBI GEO repository (GSE83524). Subsequent ANOVA determined genes that were enriched (≥2 fold more) or decreased (≤-2 fold less) in one cell type compared to all three other cell types, and these analyzed and filtered datasets are presented as tables. Genes that were shared in enriched expression in both follicular cell types (GCs and TCs) or in both luteal cells types (LLCs and SLCs) are also reported in tables. The standard deviation of the analyzed array data in relation to the log of the expression values is shown as a figure. These data have been further analyzed and interpreted in the companion article "Gene expression profiling of ovarian follicular and luteal cells provides insight into cellular identities and functions" (Romereim et al., 2017) [1].

17.
J Ovarian Res ; 9: 8, 2016 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-26911253

RESUMO

BACKGROUND: Granulosa cell tumors (GCT) are a rare ovarian neoplasm but prognosis is poor following recurrence. Keratin intermediate filaments expressed in these tumors are a diagnostic marker, yet paradoxically, may also constitute a target for therapeutic intervention. In the current study, we evaluated keratin 8/18 (K8/18) filament expression as a mechanism of resistance to apoptosis in GCT, specifically focusing on regulation of the cell surface death receptor, Fas (FAS). METHODS: The GCT cell line, KGN, was transiently transfected with siRNA to KRT8 and KRT18 to reduce K8/18 filament expression. Expression of K8/18, FAS, and apoptotic proteins (PARP, cleaved PARP) were evaluated by fluorescence microscopy, flow cytometric analysis, and immunoblotting, respectively. The incidence of FAS-mediated apoptosis in KGN cells was measured by caspase 3/7 activity. All experiments were performed independently three to six times, using a fresh aliquot of KGN cells for each experiment. Quantitative data were analyzed by one- or two-way analysis of variance (ANOVA), followed by a Tukey's post-test for multiple comparisons; differences among means were considered statistically significant at P < 0.05. RESULTS: Control cultures of KGN cells exhibited abundant K8/18 filament expression (~90 % of cells), and minimal expression of FAS (<25 % of cells). These cells were resistant to FAS-activating antibody (FasAb)-induced apoptosis, as determined by detection of cleaved PARP and measurement of caspase 3/7 activity. Conversely, siRNA-mediated knock-down of K8/18 filament expression enhanced FAS expression (> 70 % of cells) and facilitated FasAb-induced apoptosis, evident by increased caspase 3/7 activity (P < 0.05). Additional experiments revealed that inhibition of protein synthesis, but not MEK1/2 or PI3K signaling, also prompted FasAb-induced apoptosis. CONCLUSIONS: The results demonstrated that K8/18 filaments provide resistance to apoptosis in GCT by impairing FAS expression. The abundance of keratin filaments in these cells and their role in apoptotic resistance provides a greater mechanistic understanding of ovarian tumorgenicity, specifically GCT, as well as a clinically-relevant target for potential therapeutic intervention.


Assuntos
Tumor de Células da Granulosa/metabolismo , Queratina-18/metabolismo , Queratina-8/metabolismo , Neoplasias Ovarianas/metabolismo , Receptor fas/fisiologia , Apoptose , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Tumor de Células da Granulosa/genética , Tumor de Células da Granulosa/patologia , Humanos , Queratina-18/genética , Queratina-8/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Transdução de Sinais , Receptor fas/antagonistas & inibidores
18.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi ; 32(3): 542-7, 2015 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-26485975

RESUMO

Multivariate time series problems widely exist in production and life in the society. Anomaly detection has provided people with a lot of valuable information in financial, hydrological, meteorological fields, and the research areas of earthquake, video surveillance, medicine and others. In order to quickly and efficiently find exceptions in time sequence so that it can be presented in front of people in an intuitive way, we in this study combined the Riemannian manifold with statistical process control charts, based on sliding window, with a description of the covariance matrix as the time sequence, to achieve the multivariate time series of anomaly detection and its visualization. We made MA analog data flow and abnormal electrocardiogram data from MIT-BIH as experimental objects, and verified the anomaly detection method. The results showed that the method was reasonable and effective.


Assuntos
Interpretação Estatística de Dados , Interpretação de Imagem Assistida por Computador , Reconhecimento Automatizado de Padrão , Eletrocardiografia , Humanos
19.
J Clin Endocrinol Metab ; 100(6): E852-60, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25915568

RESUMO

CONTEXT: Previous studies suggest that aging in women is associated with a reduction in hypoglycosylated forms of FSH. OBJECTIVE: Experiments were performed to determine whether glycosylation of the FSHß subunit modulates the biological activity of FSH in human granulosa cells. DESIGN AND SETTING: Recombinant human FSH (hFSH) derived from GH3 pituitary cells was purified into fractions containing hypoglycosylated hFSH(21/18) and fully glycosylated hFSH(24). The response to FSH glycoforms was evaluated using the well-characterized, FSH-responsive human granulosa cell line, KGN at an academic medical center. INTERVENTIONS: Granulosa cells were treated with increasing concentrations of fully- or hypoglycosylated FSH glycoforms for periods up to 48 hours. MAIN OUTCOME MEASURE(S): The main outcomes were indices of cAMP-dependent cell signaling and estrogen and progesterone synthesis. RESULTS: We observed that hypoglycosylated FSH(21/18) was significantly more effective than fully glycosylated FSH(24) at stimulating cAMP accumulation, protein kinase A (PKA) activity, and cAMP response element binding protein (CREB) (S133) phosphorylation. FSH(21/18) was also much more effective than hFSH(24) on the stimulation CREB-response element-mediated transcription, expression of aromatase and STAR proteins, and synthesis of estrogen and progesterone. Adenoviral-mediated expression of the endogenous inhibitor of PKA, inhibited FSH(21/18)- and FSH(24)-stimulated CREB phosphorylation, and steroidogenesis. CONCLUSIONS: Hypoglycosylated FSH(21/18) has greater bioactivity than fully glycosylated hFSH(24), suggesting that age-dependent decreases in hypoglycosylated hFSH contribute to reduced ovarian responsiveness. Hypoglycosylated FSH may be useful in follicle stimulation protocols for older patients using assisted reproduction technologies.


Assuntos
Hormônio Foliculoestimulante Humano/metabolismo , Hormônio Foliculoestimulante Humano/farmacologia , Células da Granulosa/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Sequência de Carboidratos , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Glicosilação , Células da Granulosa/metabolismo , Humanos , Fosforilação , Isoformas de Proteínas
20.
Reproduction ; 148(1): 21-31, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24686456

RESUMO

Recent studies have suggested that chemokines may mediate the luteolytic action of prostaglandin F2α (PGF). Our objective was to identify chemokines induced by PGF in vivo and to determine the effects of interleukin 8 (IL8) on specific luteal cell types in vitro. Mid-cycle cows were injected with saline or PGF, ovaries were removed after 0.5-4 h, and expression of chemokine was analyzed by qPCR. In vitro expression of IL8 was analyzed after PGF administration and with cell signaling inhibitors to determine the mechanism of PGF-induced chemokine expression. Purified neutrophils were analyzed for migration and activation in response to IL8 and PGF. Purified luteal cell types (steroidogenic, endothelial, and fibroblast cells) were used to identify which cells respond to chemokines. Neutrophils and peripheral blood mononuclear cells (PBMCs) were cocultured with steroidogenic cells to determine their effect on progesterone production. IL8, CXCL2, CCL2, and CCL8 transcripts were rapidly increased following PGF treatment in vivo. The stimulatory action of PGF on IL8 mRNA expression in vitro was prevented by inhibition of p38 and JNK signaling. IL8, but not PGF, TNF, or TGFB1, stimulated neutrophil migration. IL8 had no apparent action in purified luteal steroidogenic, endothelial, or fibroblast cells, but stimulated ERK phosphorylation in neutrophils. In coculture experiments neither IL8 nor activated neutrophils altered basal or LH-stimulated luteal cell progesterone synthesis. In contrast, activated PBMCs inhibited LH-stimulated progesterone synthesis from cultured luteal cells. These data implicate a complex cascade of events during luteolysis, involving chemokine signaling, neutrophil recruitment, and immune cell action within the corpus luteum.


Assuntos
Corpo Lúteo/metabolismo , Interleucina-8/metabolismo , Luteólise , Progesterona/metabolismo , Animais , Bovinos , Comunicação Celular , Células Cultivadas , Quimiotaxia , Técnicas de Cocultura , Corpo Lúteo/citologia , Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/imunologia , Dinoprosta/farmacologia , Ciclo Estral , Feminino , Regulação da Expressão Gênica , Humanos , Interleucina-8/genética , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Luteólise/efeitos dos fármacos , Ativação de Neutrófilo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Tempo
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