The endothelin-1 (ET-1)/endothelin A receptor (ETAR, a G protein-coupled receptor) axis confers pleiotropic effects on both tumor cells and the tumor microenvironment, modulating chemo-resistance and other tumor-associated processes by activating G?q- and ?-arrestin-mediated pathways. While the precise mechanisms by which these effects occur remain to be elucidated, interference with ETAR signaling has emerged as a promising antitumor strategy in many cancers including ovarian cancer (OC). However, current clinical approaches using ETAR antagonists in the absence of a detailed knowledge of downstream signaling have resulted in multiple adverse side effects and limited therapeutic efficacy. To maximize the safety and efficacy of ETAR-targeted OC therapy, we investigated the role of other G protein subunits such as G?s in the ETAR-mediated ovarian oncogenic signaling. In HEY (human metastatic OC) cells where the ET-1/ETAR axis is well-characterized, G?s signaling inhibits ETAR-mediated OC cell migration, wound healing, proliferation and colony formation on soft agar while inducing OC cell apoptosis. Mechanistically, ET-1/ETAR is coupled to G?s/cAMP signaling in the same ovarian carcinoma-derived cell line. G?s/cAMP/PKA activation inhibits ETAR-mediated ?-arrestin activation of angiogenic/metastatic Calcrl and Icam2 expression. Consistent with our findings, G?s overexpression is associated with improved survival in OC patients in the analysis of the Cancer Genome Atlas data. In conclusion, our results indicate a novel function for G?s signaling in ET-1/ETAR-mediated OC oncogenesis and may provide a rationale for a biased signaling mechanism, which selectively activates G?s-coupled tumor suppressive pathways while blocking G?q-/?-arrestin-mediated oncogenic pathways, to improve the targeting of the ETAR axis in OC.
Chondrocyte proliferation and differentiation is a fundamental process during hard palatogenesis. Excessive retinoic acid (RA), the biologically most active metabolite of vitamin A, has been reported to adversely affect chondrogenesis. The aim of this study was to investigate the mechanisms underlying RA-induced chondrocyte differentiation by using human fetal palatal chondrocytes (hFPCs) aging about 9 weeks of amenorrhea. RA treatment inhibited proliferation and induced apoptosis in hFPCs. Alkaline phosphatase activity assay, quantitative alcian blue staining, and real-time PCR analysis revealed that RA treatment stimulated hFPCs to undergo maturation and terminal differentiation, as demonstrated by decreased chondrogenic markers and increased osteogenic markers. Further studies demonstrated that RA treatment increased Wnt/?-catenin signaling, as demonstrated by Wnt/?-catenin target gene expression analysis and a luciferase-based ?-catenin-activated reporter assay. To address the role of Wnt/?-catenin signaling, we treated hFPCs with Dickkopf-related protein 1, an extracellular inhibitor of Wnt/?-catenin signaling, and the observed all-trans retinoic acid-mediated increases in nuclear accumulation of ?-catenin, alkaline phosphatase activity, and type I collagen mRNA were attenuated, suggesting that RA modulated Wnt signaling at ligand-receptor level. In summary, excessive all-trans retinoic acid inhibited proliferation and promoted ossification of hFPCs by upregulation of Wnt/?-catenin signaling.
Ovarian cancer (OC) is the second most common and the most fatal gynecologic cancer in the United States. Over the last decade, various targeted therapeutics have been introduced but there has been no corresponding improvement in patient survival mainly because of the lack of effective early detection methods. microRNAs (miRs) are small, non-coding RNAs that regulate gene expression post-transcriptionally. Accumulating data suggest central regulatory roles of miRs in modulating OC initiation, progression, and metastasis. More recently, aberrant miR expression has been also associated with cancer stem cell (CSC) phenotypes and development of CSC chemo-resistance. Here, we review recent advances on miRs and OC metastasis and discuss the concept that miRs are involved in both CSC transformation and subsequent OC metastasis. Finally, we describe the prevalence of circulating miRs and assess their potential utilities as biomarkers for OC diagnosis, prognosis, and therapeutics.
MicroRNAs (miRs) are small, non-coding RNAs that function to post-transcriptionally regulate gene expression. First transcribed as long primary miR transcripts (pri-miRs), they are enzymatically processed in the nucleus by Drosha into hairpin intermediate miRs (pre-miRs) and further processed in the cytoplasm by Dicer into mature miRs where they regulate cellular processes following activation by a variety of signals such as those stimulated by ?-adrenergic receptors (?ARs). Initially discovered to desensitize ?AR signaling, ?-arrestins are now appreciated to transduce multiple effector pathways independent of G protein-mediated second messenger accumulation, a concept known as biased signaling. We previously showed that the ?-arrestin-biased ?AR agonist carvedilol activates cellular pathways in the heart.
Apolipoprotein F is a component protein mainly secreted by liver and resides on several lipoprotein classes. It can inhibit lipids transfer between different lipoproteins. FXR is a member of the nuclear receptor superfamily which is also highly expressed in the liver. It modulates bile acids synthesis and lipids metabolism by transcriptional regulation. We aimed to determine whether apoF can be regulated by FXR. The FXR agonist Chenodeoxycholic acid (CDCA) and GW4064 both can activate the expression of apoF in liver cell lines and in C57/BL6 mouse liver. This is dependent on the binding of FXR to the FXR element ER1 (-2904 to -2892bp) in the apoF gene promoter. Taken together, we have identified apoF as likely another target gene of FXR.
Traumatic brain injury (TBI) is a leading cause of mortality and disability in children and young adults worldwide. Neurologic impairment is caused by both immediate brain tissue disruption and post-injury cellular and molecular events that worsen the primary neurologic insult. The ?-lactam antibiotic ceftriaxone (CTX) has been reported to induce neuroprotection in animal models of diverse neurologic diseases via up-regulation of GLT-1. However, no studies have addressed the neuroprotective role of CTX in the setting of TBI, and whether the mechanism is involved in the modulation of neuronal autophagy remains totally unclear. The present study was designed to determine the hypothesis that administration of CTX could significantly enhance functional recovery in a rat model of TBI and whether CTX treatment could up-regulate GLT-1 expression and suppress post-TBI neuronal autophagy. The results demonstrated that daily treatment with CTX attenuated TBI-induced brain edema and cognitive function deficits in rats. GLT-1 is down-regulated following TBI and this phenomenon can be reversed by treatment of CTX. In addition, we also found that CTX significantly reduced autophagy marker protein, LC3 II, in hippocampus compared to the TBI group. These results suggest that CTX might provide a new therapeutic strategy for TBI and this protection might be associated with up-regulation of GLT-1 and suppression of neuronal autophagy.
Oxidative stress is a recognized factor in nephrotoxicity induced by chronic exposure to inorganic arsenic (As). Grape seed extract (GSE) possesses antioxidant properties. The present study was designed to evaluate the beneficial effects of GSE against arsenic-induced renal injury. Healthy, male Sprague-Dawley rats were exposed to As in drinking water (30 ppm) with or without GSE (100 mg/kg) for 12 months. The serum proinflammatory cytokine levels and mRNA expression levels of fibrogenic markers in the renal tissues were evaluated using enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively. The protein expression levels of nicotinamide adenine dinucleotide phosphate (NADPH) subunits, transforming growth factor-?1 (TGF-?1) and phosphorylated Smad2/3 (pSmad2/3) were assessed using western blot analysis. The results demonstrated that cotreatment with GSE significantly improved renal function, as demonstrated by the reductions in relative kidney weight (% of body weight) and blood urea nitrogen, and the increase in the creatinine clearance capacity. GSE attenuated the As-induced changes in the serum levels of tumor necrosis factor-? (TNF-?), interleukin-6 (IL-6) and IL-1? and the mRNA levels of TGF-?1, ?-smooth muscle actin (?-SMA), connective tissue growth factor (CTGF) and fibronectin (FN) in renal tissue. Furthermore, administration of GSE markedly reduced As-stimulated reactive oxygen species (ROS) production and Nox activity, as well as the protein expression levels of the NADPH subunits (Nox2, p47phox and Nox4). In addition, GSE cotreatment was correlated with a significant reduction in TGF-?/Smad signaling, as demonstrated by the decreased protein levels of TGF-?1 and pSmad2/3 in renal tissue. This study indicated that GSE may be a useful agent for the prevention of nephrotoxicity induced by chronic exposure to As. GSE may exert its effects through the suppression of Nox and inhibition of TGF-?/Smad signaling activation.
A concise and efficient route for the synthesis of highly substituted imidazopyrroloquinoline derivatives by simply refluxing a reaction mixture of different types of isatins and heterocyclic ketene aminals (HKAs) by acetic acid was developed. This method is suitable for combinatorial and parallel syntheses in drug discovery; consequently, a library of highly substituted imidazopyrroloquinoline derivatives was rapidly constructed using the present protocol.
Retrotransposons including endogenous retroviruses and their solitary long terminal repeats (LTRs) compose >40% of the human genome. Many of them are located in intergenic regions far from genes. Whether these intergenic retrotransposons serve beneficial host functions is not known. Here we show that an LTR retrotransposon of ERV-9 human endogenous retrovirus located 40-70 kb upstream of the human fetal gamma- and adult beta-globin genes serves a long-range, host function. The ERV-9 LTR contains multiple CCAAT and GATA motifs and competitively recruits a high concentration of NF-Y and GATA-2 present in low abundance in adult erythroid cells to assemble an LTR/RNA polymerase II complex. The LTR complex transcribes intergenic RNAs unidirectionally through the intervening DNA to loop with and modulate transcription factor occupancies at the far downstream globin promoters, thereby modulating globin gene switching by a competitive mechanism.
The mutant JAK2V617F tyrosine kinase (TK) is present in the majority of patients with BCR-ABL-negative myeloproliferative neoplasms (MPNs). JAK2V617F activates downstream signaling through the signal transducers and activators of transcription (STAT), RAS/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3 (PI3)/AKT pathways, conferring proliferative and survival advantages in the MPN hematopoietic progenitor cells (HPCs). Treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) is known to inhibit the chaperone function of heat shock protein 90, as well as induce growth arrest and apoptosis of transformed HPCs. Here, we demonstrate that PS treatment depletes the autophosphorylation, expression, and downstream signaling of JAK2V617F. Treatment with PS also disrupted the chaperone association of JAK2V617F with hsp90, promoting proteasomal degradation of JAK2V617F. PS also induced apoptosis of the cultured JAK2V617F-expressing human erythroleukemia HEL92.1.7 and Ba/F3-JAK2V617F cells. Treatment with the JAK2 TK inhibitor TG101209 attenuated JAK2V617F autophosphorylation and induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with PS and TG101209 further depleted JAK/STAT signaling and synergistically induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with TG101209 and PS exerted greater cytotoxicity against primary CD34(+) MPN cells than normal CD34(+) HPCs. These in vitro findings suggest combination therapy with HDAC and JAK2V617F inhibitors is of potential value for the treatment of JAK2V617F-positive MPN.
The polycomb repressive complex (PRC) 2 contains 3 core proteins, EZH2, SUZ12, and EED, in which the SET (suppressor of variegation-enhancer of zeste-trithorax) domain of EZH2 mediates the histone methyltransferase activity. This induces trimethylation of lysine 27 on histone H3, regulates the expression of HOX genes, and promotes proliferation and aggressiveness of neoplastic cells. In this study, we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) depletes EZH2 levels, and inhibits trimethylation of lysine 27 on histone H3 in the cultured human acute myeloid leukemia (AML) HL-60 and OCI-AML3 cells and in primary AML cells. DZNep treatment induced p16, p21, p27, and FBXO32 while depleting cyclin E and HOXA9 levels. Similar findings were observed after treatment with small interfering RNA to EZH2. In addition, DZNep treatment induced apoptosis in cultured and primary AML cells. Furthermore, compared with treatment with each agent alone, cotreatment with DZNep and the pan-histone deacetylase inhibitor panobinostat caused more depletion of EZH2, induced more apoptosis of AML, but not normal CD34(+) bone marrow progenitor cells, and significantly improved survival of nonobese diabetic/severe combined immunodeficiency mice with HL-60 leukemia. These findings indicate that the combination of DZNep and panobinostat is effective and relatively selective epigenetic therapy against AML cells.
The PRC2 complex protein EZH2 is a histone methyltransferase that is known to bind and recruit DNMT1 to the DNA to modulate DNA methylation. Here, we determined that the pan-HDAC inhibitor panobinostat (LBH589) treatment depletes DNMT1 and EZH2 protein levels, disrupts the interaction of DNMT1 with EZH2, as well as de-represses JunB in human acute leukemia cells. Similar to treatment with the hsp90 inhibitor 17-DMAG, treatment with panobinostat also inhibited the chaperone association of heat shock protein 90 with DNMT1 and EZH2, which promoted the proteasomal degradation of DNMT1 and EZH2. Unlike treatment with the DNA methyltransferase inhibitor decitabine, which demethylates JunB promoter DNA, panobinostat treatment mediated chromatin alterations in the JunB promoter. Combined treatment with panobinostat and decitabine caused greater attenuation of DNMT1 and EZH2 levels than either agent alone, which was accompanied by more JunB de-repression and loss of clonogenic survival of K562 cells. Co-treatment with panobinostat and decitabine also caused more loss of viability of primary AML but not normal CD34(+) bone marrow progenitor cells. Collectively, these findings indicate that co-treatment with panobinostat and decitabine targets multiple epigenetic mechanisms to de-repress JunB and exerts antileukemia activity against human acute myeloid leukemia cells.
Heat shock protein (hsp) 90 inhibitors promote proteasomal degradation of pro-growth and pro-survival hsp90 client proteins, including CDK4, c-RAF and AKT, and induce apoptosis of human lymphoma cells. The pan-histone deacetylase inhibitor vorinostat has also been shown to induce growth arrest and apoptosis of lymphoma cells. Here, we determined the effects of the more soluble, orally bio-available, geldanamycin analogue 17-NN-dimethyl ethylenediamine geldanamycin (DMAG, Kosan Biosciences Inc.) and/or vorinostat in cultured and primary human MCL cells. While vorinostat induced accumulation in the G(1) phase, treatment with DMAG arrested MCL cells in the G(2)/M phase of the cell cycle. Both agents dose-dependently induced apoptosis of MCL cells. Vorinostat also induced hyperacetylation of hsp90 and disrupted the association of hsp90 with its co-chaperones p23 and cdc37, as well as with its client proteins CDK4 and c-RAF. Treatment of MCL cells with vorinostat or 17-DMAG was associated with the inductionof p21 and p27, as well as with depletion of c-Myc, c-RAF, AKT and CDK4. Compared to treatment with either agent alone, co-treatment with DMAG and vorinostat markedly attenuated the levels of cyclin D1 and CDK4, as well as of c-Myc, c-RAF and AKT. Combined treatment with DMAG and vorinostat synergistically induced apoptosis of the cultured MCL cells, as well as induced more apoptosis of primary MCL cells than either agent alone. Therefore, these findings support the rationale to determine the in vivo efficacy of co-treatment with vorinostat and DMAG against human MCL cells.
Pan-histone deacetylase inhibitors, for example, vorinostat and panobinostat (LBH589; Novartis Pharmaceuticals, East Hanover, NJ), have shown clinical efficacy against advanced cutaneous T-cell lymphoma (CTCL). However, the molecular basis of this activity remains unclear. HDAC7, a class IIA histone deacetylase (HDAC), is overexpressed in thymocytes, where it represses expression of the proapoptotic nuclear orphan receptor Nur77. Here, we demonstrate that treatment with panobinostat rapidly inhibits the in vitro and intracellular activity, as well as the mRNA and protein levels of HDAC7, and induces expression and translocation of Nur77 to the mitochondria. There, Nur77 converts death resistance protein Bcl-2 into a killer protein, promoting cell death of cultured and patient-derived human CTCL cells. Treatment with panobinostat improved survival of athymic nude mice implanted with human CTCL cells. Ectopic expression of Nur77 induced apoptosis and sensitized HH cells to panobinostat, whereas combined knockdown of Nur77 and its family member Nor1 was necessary to inhibit panobinostat-induced apoptosis of CTCL cells. Cotreatment with the Bcl-2/Bcl-x(L) antagonist ABT-737 decreased resistance and synergistically induced apoptosis of human CTCL cells. These findings mechanistically implicate HDAC7 and Nur77 in sensitizing human CTCL cells to panobinostat as well as suggest that cotreatment with an anti-Bcl-2 agent would augment the anti-CTCL activity of panobinostat.
A novel protein that associates with interphase nucleus and mitotic apparatus (INMAP) was identified by screening HeLa cDNA expression library with an autoimmune serum followed by tandem mass spectrometry. Its complete cDNA sequence of 1.818 kb encodes 343 amino acids with predicted molecular mass of 38.2 kDa and numerous phosphorylation sites. The sequence is identical with nucleotides 1-1800 bp of an unnamed gene (GenBank accession no. 7022388) and highly homologous with the 3-terminal sequence of POLR3B. A monoclonal antibody against INMAP reacted with similar proteins in S. cerevisiae, Mel and HeLa cells, suggesting that it is a conserved protein. Confocal microscopy using either GFP-INMAP fusion protein or labeling with the monoclonal antibody revealed that the protein localizes as distinct dots in the interphase nucleus, but during mitosis associates closely with the spindle. Double immunolabeling using specific antibodies showed that the INMAP co-localizes with alpha-tubulin, gamma-tubulin, and NuMA. INMAP also co-immunoprecipitated with these proteins in their native state. Stable overexpression of INMAP in HeLa cell lines leads to defects in the spindle, mitotic arrest, formation of polycentrosomal and multinuclear cells, inhibition of growth, and apoptosis. We propose that INMAP is a novel protein that plays essential role in spindle formation and cell-cycle progression.
A series of N-sulfonyl-3,7-dioxo-5?-cholan-24-amides, ursodeoxycholic acid derivatives, have been designed and synthesized in nine steps starting from ursodeoxycholic acid. The in vitro antitumor activity of the target compounds has been evaluated against HCT-116, MCF-7, K562, and SGC-7901 cell lines. The pharmacological results showed that most of the prepared compounds display excellent selective cytotoxicity toward HCT-116, MCF-7, and K562 cell lines. Particularly, compounds 10c, 10f and 10g show high inhibitory activity on these human cancer cell lines (IC50: 2.39-9.34 ?M). Conversely, all compounds are generally inactive against SGC-7901, with only 10b having IC?? below 50 ?M.
The human embryonic, fetal and adult ?-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal ?-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal ?-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal ?-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed ?-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of ?-globin gene.
To investigate the effectiveness of reconstructing medial patellofemoral ligament with hamstring tendon autografts for the treatment of recurrent patellar dislocation under arthroscopy.
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