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OBJECTIVE: Stable luteal cell function is an important prerequisite for reproductive ability and embryonic development. However, luteal insufficiency seriously harms couples who have the desire to have a pregnancy, and the most important thing is that there is no complete solution. In addition, Vaspin has been shown to have regulatory effects on luteal cells, but the complex mechanisms involved have not been fully elucidated. Therefore, this study aimed to explore the effect of Vaspin on rat luteal cells and its mechanism. METHODS: Granulosa lutein cells separated from the ovary of female rats were incubated for 24h with gradient concentrations of Vaspin, and granulosa lutein cells incubated with 0.5% bovine serum albumin were used as controls. The proliferation, apoptosis, angiogenesis, progesterone (P4) and estradiol (E2) were detected by CCK-8, Anneixn-FITC/PI staining, angiogenesis experiment and ELISA. Western blot was applied to observe the expression levels of proteins related to cell proliferation, apoptosis, angiogenesis and MEK/MAPK signaling pathway. RESULTS: Compared with the Control group, Vaspin could significantly up-regulate the proliferation of granulosa lutein cells and reduce the apoptosis. Moreover, Vaspin promoted the angiogenesis of granulosa lutein cells and the production of P4 and E2 in a concentration-dependent manner. Furthermore, Vaspin up-regulated the CyclinD1, CyclinB1, Bcl2, VEGFA and FGF-2 expression in granulosa lutein cells, and down-regulated the level of Bax. Also, Vaspin increased the p-MEK1 and p-p38 levels. CONCLUSION: Vaspin can up-regulate the proliferation and steroidogenesis of rat luteal cells and reduce apoptosis, which may be related to the influence of MEK/MAPK activity.
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Apoptose , Proliferação de Células , Células Lúteas , Progesterona , Serpinas , Animais , Feminino , Proliferação de Células/efeitos dos fármacos , Serpinas/metabolismo , Serpinas/farmacologia , Ratos , Células Lúteas/efeitos dos fármacos , Células Lúteas/metabolismo , Apoptose/efeitos dos fármacos , Progesterona/farmacologia , Estradiol/farmacologia , Células Cultivadas , Ratos Sprague-Dawley , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacosRESUMO
Participation of major endocrine and/or local autocrine/paracrine factors and potential interplay between apparently disparate intra-oocyte signalling events during maintenance and withdrawal of meiotic prophase arrest has been an area of active research in recent years. Studies on oocyte maturation have contributed substantially in the discovery of some of the most important biochemical and cellular events like functional significance of novel membrane-associated steroid receptors, elucidation of maturation promoting factor (MPF), cytostatic factor (CSF) and other signalling cascades that entrain the cell cycle clock to hormonal stimuli. While follicular estrogen has largely been implicated in maintenance of prophase arrest, involvement of maturational steroid and membrane progestin receptor in resumption of meiotic G2-M1 transition in piscine oocytes has been shown earlier. Moreover, detection of ovarian IGF system, maturational gonadotropin stimulation of IGF ligands and potential synergism between maturational steroid and IGF1 in zebrafish oocytes are most recent advancements. Though endocrine/paracrine regulation of cyclic nucleotide-mediated signalling events in meiotic cell cycle progression is well established, involvement of PI3K/Akt signalling cascade has also been reported in fish, amphibian and mammalian oocytes. The major objective of this overview is to describe how fish oocytes maintain high cAMP/PKA activity and how steroid- and/or growth factor-mediated signalling cascade regulate this pathway for the withdrawal of meiotic arrest. Moreover, special emphasis is placed on some recent findings on interaction of PKA with some of the MPF-regulating components (e.g., synthesis of cyclin B or MEK/MAPK signalling cascade) for the maintenance of prophase arrest.
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AMP Cíclico/metabolismo , Hormônios/farmacologia , Meiose/fisiologia , Oócitos/fisiologia , Oogênese/genética , Peixe-Zebra , Animais , Feminino , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Peixe-Zebra/fisiologiaRESUMO
Generating cellular heterogeneity is crucial to the development of complex organs. Organ-fate selector genes and signalling pathways generate cellular diversity by subdividing and patterning naïve tissues to assign them regional identities. The Drosophila eye-antennal imaginal disc is a well-characterised system in which to study regional specification; it is first divided into antennal and eye fates and subsequently retinal differentiation occurs within only the eye field. During development, signalling pathways and selector genes compete with and mutually antagonise each other to subdivide the tissue. Wingless (Wg) signalling is the main inhibitor of retinal differentiation; it does so by promoting antennal/head-fate via selector factors and by antagonising Hedgehog (Hh), the principal differentiation-initiating signal. Wg signalling must be suppressed by JAK/STAT at the disc posterior in order to initiate retinal differentiation. Ras/MEK/MAPK signalling has also been implicated in initiating retinal differentiation but its mode of action is not known. We find that compromising Ras/MEK/MAPK signalling in the early larval disc results in expanded antennal/head cuticle at the expense of the compound eye. These phenotypes correspond both to perturbations in selector factor expression, and to de-repressed wg. Indeed, STAT activity is reduced due to decreased mobility of the ligand Unpaired (Upd) along with a corresponding loss in Dally-like protein (Dlp), a heparan sulphate proteoglycan (HSPG) that aids Upd diffusion. Strikingly, blocking HSPG biogenesis phenocopies compromised Ras/MEK/MAPK, while restoring HSPG expression rescues the adult phenotype significantly. This study identifies a novel mode by which the Ras/MEK/MAPK pathway regulates regional-fate specification via HSPGs during development.
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Proteoglicanas de Heparan Sulfato/química , MAP Quinase Quinase 1/metabolismo , Retina/embriologia , Proteínas ras/metabolismo , Animais , Linhagem da Célula , Cruzamentos Genéticos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Larva/metabolismo , Ligantes , Sistema de Sinalização das MAP Quinases , Fenótipo , Proteínas Quinases/metabolismo , Interferência de RNA , Transdução de Sinais , Proteína Wnt1/metabolismoRESUMO
Psoriasis and atopic dermatitis are chronic inflammatory skin diseases characterized by keratinocyte (KC) hyperproliferation and epidermal acanthosis (hyperplasia). The milieu of disease-associated cytokines and soluble factors is considered a mitogenic factor; however, pinpointing the exact mitogens in this complex microenvironment is challenging. We employed organotypic human epidermal equivalents, faithfully mimicking native epidermal proliferation and stratification, to evaluate the proliferative effects of a broad panel of (literature-based) potential mitogens. The KC GF molecule, the T-helper 2 cytokines IL-4 and IL-13, and the psoriasis-associated cytokine IL-17A caused acanthosis by hyperplasia through a doubling in the number of proliferating KCs. In contrast, IFN-γ lowered proliferation, whereas IL-6, IL-20, IL-22, and oncostatin M induced acanthosis not by hyperproliferation but by hypertrophy. The T-helper 2âcytokineâmediated hyperproliferation was Jak/signal transducer and activator of transcription 3 dependent, whereas IL-17A and KC GF induced MAPK/extracellular signalâregulated kinase kinase/extracellular signalâregulated kinaseâdependent proliferation. This discovery that key regulators in atopic dermatitis and psoriasis are direct KC mitogens not only adds evidence to their crucial role in the pathophysiological processes but also highlights an additional therapeutic pillar for the mode of action of targeting biologicals (e.g., dupilumab) or small-molecule drugs (e.g., tofacitinib) by the normalization of KC turnover within the epidermal compartment.
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BACKGROUND: Long non-coding RNAs (lncRNAs) have recently become the vital gene regulators in diverse cancers. In our study, we purposed to inquiry into the mechanisms of lncRNA PRNCR1 in breast cancer via microRNA-377 (miR-377)/CCND2/MEK/MAPK axis. METHODS: PRNCR1 expression in breast cancer tissues was detected, and the correlation between PRNCR1 expression and prognostic survival was analyzed. The expressions of PRNCR1 and miR-377 in breast cancer cell lines were detected. Relationships among PRNCR1, miR-377 and CCND2 were confirmed by luciferase activity, RNA pull-down or RIP assays. Breast cancer cells were introduced with silenced PRNCR1 or restored miR-377 to explore their functions in malignant phenotype of breast cancer cells. The expression of MEK/MAPK pathway-related proteins was determined by western blot analysis. RESULTS: PRNCR1 was highly expressed and miR-377 was poorly expressed in patients with breast cancer, and patients with high expression of PRNCR1 had a poor prognosis. PRNCR1 silencing or miR-377 overexpression resulted in suppressed breast cancer cell proliferation ability, blocked cell cycle process and induced apoptosis. PRNCR1 regulated CCND2 expression by competitively binding to miR-377. CCND2 activated the MEK/MAPK pathway, and after treatment with Mirdametinib, the MEK/MAPK pathway was inhibited, which was found to retard breast cancer growth. CONCLUSION: Our study highlights that lncRNA PRNCR1 may competitively bind to miR-377, leading to upregulated CCND2, which in turn activated MEK/MAPK pathway to promote breast cancer growth.
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Neoplasias da Mama , MicroRNAs , RNA Longo não Codificante , Apoptose , Neoplasias da Mama/genética , Proliferação de Células , Ciclina D2 , Feminino , Humanos , MicroRNAs/genética , Quinases de Proteína Quinase Ativadas por Mitógeno , RNA Longo não Codificante/genéticaRESUMO
BACKGROUND/AIM: The aim of this study was to examine clonal heterogeneity, to test the utility of liquid biopsy in monitoring disease progression and to evaluate the usefulness of ex vivo drug screening in a BRAF L597Q-mutated colorectal cancer (CRC) patient developing metastases during adjuvant therapy. MATERIALS AND METHODS: Next generation sequencing (NGS) and droplet digital PCR (ddPCR) were performed in samples from tumor tissues and liquid biopsies. Live cancer cells from a metastatic lesion were used in ex vivo drug sensitivity assays. RESULTS: We found evidence of continued dependence of MEK/MAPK pathway activation, but different activating mutations in primary tumor and metastases. Liquid biopsy based BRAF L597Q ddPCR testing was a sensitive personalized biomarker predicting the rise of clinically aggressive metastatic disease. Ex vivo drug sensitivity assays with BRAF L597Q mutated cells showed response to MEK/MAPK targeted therapies. CONCLUSION: The rare BRAF L597Q mutation may be associated with aggressive tumor behavior in CRC. Liquid biopsy can be used to capture clinically relevant tumor features.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/secundário , MAP Quinase Quinase 1/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Capecitabina/administração & dosagem , Evolução Clonal , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , MAP Quinase Quinase 1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxaliplatina/administração & dosagem , PrognósticoRESUMO
HER3 belongs to the human epidermal growth factor receptor (HER) family which also includes HER1/EGFR/erbB1, HER2/erbB2, and HER4/erbB4. As a unique member of the HER family, HER3 lacks or has little intrinsic tyrosine kinase activity. It frequently co-expresses and forms heterodimers with other receptor tyrosine kinases (RTKs) in cancer cells to activate oncogenic signaling, especially the PI-3K/Akt pathway and Src kinase. Elevated expression of HER3 has been observed in a wide variety of human cancers and associates with a worse survival in cancer patients with solid tumors. Studies on the underlying mechanism implicate HER3 expression as a major cause of treatment failure in cancer therapy. Activation of HER3 signaling has also been shown to promote cancer metastasis. These data strongly support the notion that therapeutic inactivation of HER3 and/or its downstream signaling is required to overcome treatment resistance and improve the outcomes of cancer patients.
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Definition of cell cycle control proteins that modify tumor cell resistance to estrogen (E2) signaling antagonists could inform clinical choice for estrogen receptor positive (ER+) breast cancer (BC) therapy. Cyclin G2 (CycG2) is upregulated during cell cycle arrest responses to cellular stresses and growth inhibitory signals and its gene, CCNG2, is directly repressed by E2-bound ER complexes. Our previous studies showed that blockade of HER2, PI3K and mTOR signaling upregulates CycG2 expression in HER2+ BC cells, and that CycG2 overexpression induces cell cycle arrest. Moreover, insulin and insulin-like growth factor-1 (IGF-1) receptor signaling strongly represses CycG2. Here we show that blockade of ER-signaling in MCF7 and T47D BC cell lines enhances the expression and nuclear localization of CycG2. Knockdown of CycG2 attenuated the cell cycle arrest response of E2-depleted and fulvestrant treated MCF7 cells. These muted responses were accompanied by sustained inhibitory phosphorylation of retinoblastoma (RB) protein, expression of cyclin D1, phospho-activation of ERK1/2 and MEK1/2 and expression of cRaf. Our work indicates that CycG2 can form complexes with CDK10, a CDK linked to modulation of RAF/MEK/MAPK signaling and tamoxifen resistance. We determined that metformin upregulates CycG2 and potentiates fulvestrant-induced CycG2 expression and cell cycle arrest. CycG2 knockdown blunts the enhanced anti-proliferative effect of metformin on fulvestrant treated cells. Meta-analysis of BC tumor microarrays indicates that CCNG2 expression is low in aggressive, poor-prognosis BC and that high CCNG2 expression correlates with longer periods of patient survival. Together these findings indicate that CycG2 contributes to signaling networks that limit BC.
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Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Ciclina G2/metabolismo , Estradiol/análogos & derivados , Metformina/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Intervalo Livre de Doença , Estradiol/farmacologia , Estrogênios/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fulvestranto , Técnicas de Silenciamento de Genes , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Recidiva Local de Neoplasia/patologia , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/metabolismo , Interferência de RNA , Receptores de Estrogênio/metabolismo , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Regulação para Cima/efeitos dos fármacosRESUMO
We previously demonstrated that blocking hepatocyte growth factor (HGF) receptor/c-Met signaling inhibited arthritis and articular bone destruction in mouse models of rheumatoid arthritis (RA). In the present study, we investigated the role of c-Met signaling in osteoblast differentiation using the C2C12 myoblast cell line derived from murine satellite cells and the MC3T3-E1 murine pre-osteoblast cell line. Osteoblast differentiation was induced by treatment with bone morphogenetic protein (BMP)-2 or osteoblast-inducer reagent in the presence or absence of either HGF antagonist (NK4) or c-Met inhibitor (SU11274). Osteoblast differentiation was confirmed by Runx2 expression, and alkaline phosphatase (ALP) and osteocalcin production by the cells. Production of ALP, osteocalcin and HGF was verified by enzyme-linked immunosorbent assay. Runx2 expression was confirmed by reverse transcription-PCR analysis. The phosphorylation status of ERK1/2, AKT, and Smads was determined by Western blot analysis. Both NK4 and SU11274 enhanced Runx2 expression, and ALP and osteocalcin production but suppressed HGF production in BMP-2-stimulated C2C12 cells. SU11274 also enhanced ALP and osteocalcin production in osteoblast-inducer reagent-stimulated MC3T3-E1 cells. SU11274 inhibited ERK1/2 and AKT phosphorylation in HGF-stimulated C2C12 cells. This result suggested that ERK and AKT were functional downstream of the c-Met signaling pathway. However, both mitogen-activated protein kinase/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitor suppressed osteocalcin and HGF production in BMP-2-stimulated C2C12 cells. Furthermore, SU11274, MEK, and PI3K inhibitor suppressed Smad phosphorylation in BMP-2-stimulated C2C12 cells. These results indicate that although the c-Met-MEK-ERK-Smad and c-Met-PI3K-AKT-Smad signaling pathways positively regulate osteoblast differentiation, c-Met signaling negatively regulates osteoblast differentiation, independent of the MEK-ERK-Smad and PI3K-AKT-Smad pathways. Therefore, blocking c-Met signaling might serve as a therapeutic strategy for the repair of destructed bone in patients with RA.
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In cultured cells, not many mitochondria are degraded by mitophagy induced by physiological cellular stress. We observed mitophagy in HeLa cells using a method that relies on the pH-sensitive fluorescent protein Keima. With this approach, we found that mitophagy was barely induced by carbonyl cyanide m-chlorophenyl hydrazone treatment, which is widely used as an inducer of PARK2/Parkin-related mitophagy, whereas a small but modest amount of mitochondria were degraded by mitophagy under conditions of starvation or hypoxia. Mitophagy induced by starvation or hypoxia was marginally suppressed by knockdown of ATG7 and ATG12, or MAP1LC3B, which are essential for conventional macroautophagy. In addition, mitophagy was efficiently induced in Atg5 knockout mouse embryonic fibroblasts. However, knockdown of RAB9A and RAB9B, which are essential for alternative autophagy, but not conventional macroautophagy, severely suppressed mitophagy. Finally, we found that the MAPKs MAPK1/ERK2 and MAPK14/p38 were required for mitophagy. Based on these findings, we conclude that mitophagy in mammalian cells predominantly occurs through an alternative autophagy pathway, requiring the MAPK1 and MAPK14 signaling pathways.
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Autofagia/fisiologia , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mitofagia/fisiologia , Transdução de Sinais/fisiologia , Animais , Autofagia/efeitos dos fármacos , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Células HeLa , Humanos , Lisossomos/metabolismo , Camundongos , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Hypoxia-inducible factor-1 (HIF-1) has been recognized as an important cancer drug target. Many recent studies have provided convincing evidences of strong correlation between elevated levels of HIF-1 and tumor metastasis, angiogenesis, poor patient prognosis as well as tumor resistance therapy. It was found that hypoxia (low O2 levels) is a common character in many types of solid tumors. As an adaptive response to hypoxic stress, hypoxic tumor cells activate several survival pathways to carry out their essential biological processes in different ways compared with normal cells. Recent advances in cancer biology at the cellular and molecular levels highlighted the HIF-1α pathway as a crucial survival pathway for which novel strategies of cancer therapy could be developed. However, targeting the HIF-1α pathway has been a challenging but promising progresses have been made in the past twenty years. This review summarizes the role and regulation of the HIF-1α in cancer, and recent therapeutic approaches targeting this important pathway.
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The kinin-B2 receptor (B2BKR) activated by its endogenous ligand bradykinin participates in various metabolic processes including the control of arterial pressure and inflammation. Recently, functions for this receptor in brain development and protection against glutamate-provoked excitotoxicity have been proposed. Here, we report neuroprotective properties for bradykinin against organophosphate poisoning using acute hippocampal slices as an in vitro model. Following slice perfusion for 10min with diisopropylfluorophosphate (DFP) to initiate the noxious stimulus, responses of pyramidal neurons upon an electric impulse were reduced to less than 30% of control amplitudes. Effects on synaptic-elicited population spikes were reverted when preparations had been exposed to bradykinin 30min after challenging with DFP. Accordingly, bradykinin-induced population spike recovery was abolished by HOE-140, a B2BKR antagonist. However, the kinin-B1 receptor (B1BKR) agonist Lys-des-Arg(9)-bradykinin, inducing the phosphorylation of mitogen-activated protein kinase (MEK/MAPK) and cell death, abolished bradykinin-mediated neuroprotection, an effect, which was reverted by the ERK inhibitor PD98059. In agreement with pivotal B1BKR functions in this process, antagonism of endogenous B1BKR activity alone was enough for restoring population spike activity. On the other hand pralidoxime, an oxime, reactivating acetylcholinesterase (AChE) after organophosphate poisoning, induced population spike recovery after DFP exposure in the presence of bradykinin and Lys-des-Arg(9)-bradykinin. Lys-des-Arg(9)-bradykinin did not revert protection exerted by pralidoxime, however when instead bradykinin and Ly-des-Arg(9)-bradykinin were superfused together, recovery of population spikes diminished. These findings again confirm the neuroprotective feature of bradykinin, which is, diminished by its endogenous metabolites, stimulating the B1BKR, providing a novel understanding of the physiological roles of these receptors.