查看更多>>摘要：Poly (ADP-ribose) polymerase (PARP) inhibitors provide a promising therapeutic strategy for triple-negative breast cancers (TNBCs) with BRCA1/2 mutation. However, acquire resistance mechanisms and genetic alterations limit the clinical efficacy of PARP inhibitors. The aberrant activation of phosphatidylinositol 3-kinase (PI3K) is a significant problem for cancer development and thus the inhibition of PI3K by PI3K inhibitors is a novel targeted therapy in advanced breast cancer. Here, we, for the first time, investigated that the combined inhibition of PARP by Talazoparib (TAL) and PI3K by LY294002 synergistically inhibited proliferation of BRCA1 mutant HCC1937 TNBC cells through apoptosis, G0/G1 arrest, oxidative stress and increased DNA damage compared to drug alone. Additionally, TAL and LY294002 combination could be a promising strategy for overcoming TAL resistance. Co-treatment of TAL with LY294002 considerably suppressed the activation of PI3K, Akt1 and mTOR expression and phosphorylated protein levels in TNBC cells and caused changes in the multiple kinase phosphorylation. Our findings revealed that the dual inhibition of PARP and PI3K might represent an effective therapeutic strategy for TNBC and potentially overcome TAL resistance.
Hafiane, AnouarGianopoulos, IoannaSorci-Thomas, Mary G.Daskalopoulou, Stella S....
3页
查看更多>>摘要：Adiponectin exerts its atheroprotection by stimulating adenosine triphosphate binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux to apolipoprotein A-I (apoA-I). However, involvement of the apoA-I resi-dues in this process have not been studied. In Tamm-Horsfall 1 (THP-1) macrophages and baby hamster kidney (BHK) cells we assessed adiponectin's potential to restore cholesterol efflux in the presence of apoA-I and ABCA1 mutants, respectively. Adiponectin was unable to restore efflux from THP-1 macrophages in the presence of apoA-I carboxy-terminal domain (CTD) successive mutants from residues 187-243 versus apoA-I mutants alone. Furthermore, adiponectin did not significantly influence cholesterol efflux to apoA-I from BHK-ABCA1 mutant cells. Adiponectin appears to require functional apoA-I CTD residues 187-243 and wild-type ABCA1 to mediate efficient cholesterol efflux from THP-1 macrophages and BHK cells, respectively. Therefore, adiponectin cannot rescue defective cholesterol efflux in apoA-I-or ABCA1-mutant conditions, but rather increases cholesterol efflux in wild-type apoA-I conditions compared to apoA-I exposure alone.
查看更多>>摘要：Activation of a specific protein kinase C (PKC) isoform during stimulation of Gq protein-coupled receptors (GqPCRs) is determined by homologous receptor desensitization that controls the spatiotemporal formation of downstream Gq signalling molecules. Furthermore, GqPCR-activated PKC isoforms specifically regulate receptor activity via a negative feedback mechanism. In the present study, we investigated the contribution of several phosphorylation sites in the alpha 1B-adrenergic receptor (alpha 1B-AR) for PKC and G protein coupled receptor kinase 2 (GRK2) to homologous receptor desensitization and effector modulation. We analyzed signalling events downstream to human wildtype alpha 1B-ARs and alpha 1BARs lacking PKC or GRK2 phosphorylation sites (Delta 391-401, alpha 1B-Delta PKC-AR and Delta 402-520, alpha 1B-Delta GRK-AR) by means of FRET-based biosensors in HEK293 that served as online-assays of receptor activity. K+ currents through KCNQ1/KCNE1 channels (IKs), which are regulated by both phosphatidylinositol 4,5-bisphosphate (PIP2)depletion and/or phosphorylation by PKC, were measured as a functional readout of wildtype and mutant alpha 1B-AR receptor activity. As a novel finding, we provide evidence that deletion of PKC and GRK2 phosphorylation sites in alpha 1B-ARs abrogates the contribution of PKC alpha to homologous receptor desensitization. Instead, the time course of mutant receptor activity was specifically modulated by PKC beta. Mutant alpha 1B-ARs displayed pronounced homologous receptor desensitization that was abolished by PKC beta-specific pharmacological inhibitors. IKs modulation during stimulation of wildtype and mutant alpha 1B-ARs displayed transient inhibition and current facilitation after agonist withdrawal with reduced capability of mutant alpha 1B-ARs to induce IKs inhibition. Pharmacological inhibition of the PKC beta isoform did not augment IKs reduction by mutant alpha 1B-ARs, but shifted IKs modulation towards current facilitation. Coexpression of an inactive (dominant-negative) PKC delta isoform (DNPKC delta) abolished IKs facilitation in alpha 1B-Delta GRK-AR-expressing cells, but not in alpha 1B-Delta PKC-AR-expressing cells. The data indicate that the differential modulation of IKs activity by alpha 1B-Delta GRK- and alpha 1B-Delta PKC-receptors is attributed to the activation of entirely distinct novel PKC isoforms. To summarize, specific phosphorylation sites within the wildtype and mutant alpha 1B-adrenergic receptors are targeted by different PKC isoforms, resulting in differential regulation of receptor desensitization and effector function.
查看更多>>摘要：Hepatic regeneration after hepatectomy is a great concern in clinical practice. Recently, the neuronal guidance protein netrin-1 has been reported to enhance regeneration after nerve injury. The goal of this study was to preliminarily investigate whether netrin-1 stimulates vagus nerve regeneration to promote liver regeneration after partial hepatectomy in mice. The expression of netrin-1 in murine remnant livers after partial hepatectomy (PHx) was evaluated in initial studies. C57BL/6 mice that received exogenous netrin-1 after PHx were used to examine liver regeneration. PHx was performed in wild-type mice after adeno-associated virus injection (Ntn1 gene silencing) to detect the impact of endogenous netrin-1. After PHx and hepatic branch vagotomy (HV), the mice were injected with or without netrin-1 to evaluate the effects on hepatic regeneration and vagal nerve recovery. Significant reductions in netrin-1 at the transcript and protein levels in murine liver tissue after hepatectomy were observed. Subsequent studies of netrin-1 administration revealed the promotion of hepatocyte proliferation and specific growth factors contributing to liver repair and a decrease in hepatic-specific injury enzymes. Furthermore, the opposite results were observed in the netrin-1 knockdown group. HV delayed liver regeneration after PHx. However, this retardation was reversed by exogenous netrin-1 supplementation. In addition, the results of nerve growth and vagal nerve repair in the remnant liver suggested that netrin-1 promoted vagal nerve regeneration after hepatectomy. Netrin-1 accelerates liver regeneration after partial hepatectomy in mice, and the potential mechanism is related to the promotion of vagus nerve repair and regeneration.
查看更多>>摘要：Adaptation of gene expression is one of the most fundamental response of cardiomyocytes to hypertrophic stimuli. G3bp1, an RNA binding protein with site-specific endoribonuclease activity regulates the processing of pre-miR-1 stem-loop, and thus levels of cardiomyocyte-enriched mature miR-1. Here, we examine the role of G3bp1 in regulating gene expression in quiescent cardiomyocytes and those undergoing growth-factor induced hypertrophy. Further, we determine if these changes are facilitated through G3bp1-mediated regulation of miR-1 in these cardiomyocytes. Using isolated cardiomyocytes with knockdown of endogenous G3bp1, we performed high throughput RNA sequencing to determine the change in cardiac transcriptome. Then, using gain and loss of function approach for both, G3bp1 and miR-1, alone or in combination we examine the G3bp1-miR-1 signaling in regulating gene expression and Endothelin (ET-1)-induced cardiomyocyte hypertrophy. We show that knockdown of endogenous G3bp1 results in inhibition of genes involved in calcium handling, cardiac muscle contraction, action potential and sarcomeric structure. In addition, there is inhibition of genes that contribute to hypertrophic and dilated cardiomyopathy development. Conversely, an increase is seen in genes that negatively regulate the Hippo signaling, like Rassf1 and Arrdc3, along with inflammatory genes of TGF-beta and TNF pathways. Knockdown of G3bp1 restricts ET-1 induced cardiomyocyte hypertrophy. Interestingly, concurrent silencing of G3bp1 and miR-1 rescues the change in gene expression and inhibition of hypertrophy seen with knockdown of G3bp1 alone. Similarly, expression of exogenous G3bp1 reverses the miR-1 induced inhibition of gene expression. Intriguingly, expression of Gfp tagged G3bp1 results in perinuclear accumulations of G3bp1Gfp, resembling Stress Granules. Based on our results, we conclude that G3bp1 through its regulation of mature miR-1 levels plays a critical role in regulating the expression of essential cardiac-enriched genes and those involved in development of cardiomyocyte hypertrophy.
Jones-Tabah, JaceMartin, Ryan D.Chen, Jennifer J.Tanny, Jason C....
16页
查看更多>>摘要：The activity of striatal medium-spiny projection neurons is regulated by D1 and D2 dopamine receptors. The D1 receptor (D1R) is a G alpha s/olf-coupled GPCR which activates a cAMP/PKA/DARPP-32 signalling cascade that increases excitability and facilitates plasticity, partly through the regulation of transcription. Upon activation via D1R, PKA can translocate to the nucleus to regulate transcription through the phosphorylation of various targets. One candidate effector of PKA-dependent transcriptional regulation is the BET protein Brd4. It is known that when Brd4 is activated by phosphorylation, it binds more readily to acetylated histones at promoters and enhancers; moreover, in non-neuronal cells, PKA signalling has been shown to increase recruitment of Brd4 to chromatin. However, it is unknown whether BET proteins, or Brd4 specifically, are involved in transcriptional activation by cAMP/PKA in neurons. Here, we demonstrate that in adult rats, inhibition of BET proteins with the bromodomain inhibitor JQ1 suppressed the expression of ~25% of D1R-upregulated genes, while also increasing the expression of a subset of immediate-early genes. We further found that cAMP/PKA signalling promotes Brd4 recruitment to dopamine-induced genes in striatal neurons, and that knockdown of Brd4 attenuates D1R-induced gene expression. Finally, we report that JQ1 treatment downregulated expression of many GPCRs and also impaired ERK1/2 signalling in striatal neurons. Our findings identify the BET protein family, and Brd4 in particular, as novel regulators of basal and D1R-dependent transcription in rat striatal neurons, and delineate complex bi-directional effects of bromodomain inhibitors on neuronal transcription.
Natani, SirishaSruthi, K. K.Asha, Sakkarai MohamedKhilar, Priyanka...
11页
查看更多>>摘要：Neuroendocrine prostate cancer (NEPC) is an aggressive, androgen independent PCa and it is detected in patients undergoing androgen deprivation therapy (ADT). Interleukin-6 (IL-6) is a pleiotropic cytokine elevated in PCa patients promotes neuroendocrine differentiation (NED). In this study, PCa cells were differentiated with IL-6 in in-vitro to identify novel targets or signaling pathways associated with emergence of NEPC on deprivation of androgens. From the results, we observed an activation of TGF-beta signaling pathway is altered through multiple proteins in differentiated LNCaP cells. Hence, we investigated the role of TGF-beta axis in PCa cells differentiation. LNCaP cells treated with IL-6 in androgens deprived media release excess TGF-beta ligand and this as conditioned media added to cells stimulated NED of PCa cells. TGF-beta released by IL-6 stimulated cells activate p38MAPK through SMAD2 thereby promote NED. Inhibition of TGF-beta RI and TGF-beta RII signaling activation in LNCaP cells treated with IL-6 did not reversed the NED of cells, possibly due to the reason that the inhibition of TGF-beta axis is further activating p38MAPK through SMAD independent manner in PCa cells. However, siRNA mediated knock down or inhibition p38MAPK inactivated TGF-beta - SMAD axis in differentiating cells and attenuated NED of LNCaP cells. This result suggests that p38MAPK is the central node for receiving IL-6 signals and promotes NED of LNCaP cells in androgens free media. Remarkably, downregulation or inhibition of p38MAPK in NCI-H660 reversed NED characteristics as well as markers along with inactivation of SMAD2 whereas no effect observed in WPMY-1 normal prostate cells. Taken together these findings unveil that p38MAPK and its upstream regulators are potential targets to overcome the progression of NED of PCa and develop novel therapeutic measures along ADT for effective treatment of PCa.
查看更多>>摘要：Intracellular calcium ion (Ca2+) in cytoplasm as an intracellular second messenger is involved in almost all important cellular activities of organisms. Generally its concentration ([Ca2+](i)) is tested by live imaging followed image and data processes, in which much tedious and subjective manual work is involved. Here we show a computational approach of Deep Calcium following the principles of deep learning to predict the cytoplasmic Ca2+ ranges and calcium peaks in calcium curve of objective cells. To validate Deep Calcium, chondrocytes, bone marrow stromal cells (BMSCs) and osteoblastic like cells (MC3T3-E1) from both the tissue and cell samples as well as from spontaneous and mechanical stimulated calcium response patterns are used. The good performance comparing with other relative machine learning models, as well as consistency biological results with human experts are demonstrated. Deep Calcium provides references for other image and data processes of intracellular range determination and curve peak identification.
查看更多>>摘要：Microfibril-associated glycoprotein 2 (MAGP2) plays an important role in regulating cell signaling and acts as a biomarker to predict the prognostic effect of tumor therapy. However, research on MAGP2 mostly focuses on its extracellular signal transmission features, and its potential intracellular function is rarely reported. Here, we reported that intracellular MAGP2 increased the stability of urokinase-type plasminogen activator receptor (uPAR) in the cell by direct interaction which inhibits the lysosomal-mediated degradation of uPAR. Further-more, with the detection of protein content changes and proteomics analysis, we found that highly expressed MAGP2 promoted the proliferation of tumor cells through uPAR-mediated p38-NF-kappa B signaling axis activation, enhancement of DNA damage repair and reduction of cell stagnation in the S phase of the cell cycle. In the nude mouse xenograft model of colorectal cancer, the upregulation of MAGP2 in tumor cells significantly promoted tumor progression, while the downregulation of uPAR significantly attenuated tumor progression. These studies elucidate the role of MAGP2 inside the cell and provide a new explanation for why patients with higher MAGP2 expression in tumors are associated with a worse prognosis. In addition, we also determined a mechanism for the stable existence of uPAR in the cell, providing information for the development of tumor drugs targeting uPAR.
查看更多>>摘要：Background: Growing evidence has showed long noncoding RNAs (lncRNAs) play critical roles in bladder cancer (BC) progression. LncRNA taurine upregulated gene 1 (TUG1) was involved in the development of human malignancies. However, the intrinsic and concrete molecular mechanisms of TUG1 in BC remain largely unknown.Methods: Expression patterns of TUG1, miR-320a and FOXQ1 in BC tissues and cell lines were measured using qRT-PCR and western blot, respectively. Cell proliferation was detected by CCK-8 and colony formation assays. The capacity of cell migration and invasion was evaluated using wound healing and transwell assay. Tumor xenograft assay was performed to further validate the role of TUG1 in BC progression. Dual luciferase reporter assay and FISH analysis were employed to verify the TUG1/miR-320a/FOXQ1 regulatory network.Results: TUG1 was significantly higher expression in BC specimens and cell lines. TUG1 knockdown suppressed BC cells malignant behaviors in vitro and inhibited tumor growth and metastasis in vivo, while TUG1 over expression promoted BC cells malignant behaviors in vitro. However, the function of miR-320a was opposite to that of TUG1, and miR-320a inhibitor partially eliminated the inhibitory effect of TUG1 knockdown on the malignant behavior of BC cells. As a microRNA sponge, TUG1 actively elevated FOXQ1 expression to sponge miR-320a and subsequently promoted BC cells malignant phenotypes.Conclusion: TUG1 may have great potential as therapeutic target for BC, since TUG1 silencing inhibited cell proliferation, migration and invasion in BC, while promoted cell apoptosis, by regulating the miR-320a/FOXQ1 axis.
NSTL
Elsevier