ABSTRACT
Hepatocellular carcinoma (HCC) is still one of the major causes of cancer-related death. Kinetochore-associated protein 2 (KNTC2) is specifically upregulated in tumor tissues of HCC patients and recognized as a potential candidate target for the treatment of HCC. However, the relationship between KNTC2 and in vivo tumor growth of HCC is not yet fully understood. Here we encapsulated KNTC2 siRNAs into a lipid nanoparticle (LNP) and investigated their knockdown activity, target engagement marker, anti-tumor activity and hepatotoxicity in an orthotopic HCC model mice of Hep3B-luc cells. Single i.v. administration of KNTC2 siRNA-LNP specifically suppressed the expression levels of both human KNTC2 mRNA and mouse Kntc2 mRNA in tumor tissues. Phosphorylation levels of histone H3 (HH3) at serine 10 in tumor tissues were increased by KNTC2 siRNA-LNP. Repeated administration of KNTC2 siRNA-LNP (twice a week) specifically inhibited the growth of tumor tissues without increasing the plasma AST and ALT levels. Their growth inhibitory activities were consistent with knockdown activities. These data strongly indicated that KNTC2 is a promising target for the treatment of HCC and that phosphorylated HH3 at serine 10 is one of the target engagement markers for KNTC2.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/therapy , Genetic Therapy/methods , Liver Neoplasms/genetics , Liver Neoplasms/therapy , Nuclear Proteins/genetics , RNA, Small Interfering/administration & dosage , Animals , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Apoptosis/genetics , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cytoskeletal Proteins , Gene Knockdown Techniques/methods , Humans , Liver Neoplasms/pathology , Male , Mice , Mice, SCID , Molecular Targeted Therapy/methods , Treatment OutcomeABSTRACT
Cholinergic neurons in the CNS are involved in synaptic plasticity and cognition. Both muscarinic and nicotinic acetylcholine receptors (nAChRs) influence plasticity and cognitive function. The mechanism underlying nAChR-induced plasticity, however, has remained elusive. Here, we demonstrate morphological changes in dendritic spines following activation of α4ß2* nAChRs, which are expressed on glutamatergic pre-synaptic termini of cultured hippocampal neurons. Exposure of the neurons to nicotine resulted in a lateral enlargement of spine heads. This was abolished by dihydro-ß-erythroidine, an antagonist of α4ß2* nAChRs, but not by α-bungarotoxin, an antagonist of α7 nAChRs. Tetanus toxin or a mixture of 2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, antagonists of NMDA- and AMPA-type glutamate receptors, blocked the nicotine-induced spine remodeling. In addition, nicotine exerted full spine-enlarging response in the post-synaptic neuron whose ß2 nAChR expression was knocked down. Finally, pre-treatment with nicotine enhanced the Ca(2+)-response of the neurons to glutamate. These data suggest that nicotine influences the activity of glutamatergic neurotransmission through the activation of pre-synaptic α4ß2 nAChRs, resulting in the modulation of spinal architecture and responsiveness. The present findings may represent one of the cellular mechanisms underlying cholinergic tuning of brain function. Activation of nicotinic acetylcholine receptors (nAChRs) in brain influences plasticity and cognition. Here, activation of α4ß2* nAChRs, which are expressed on glutamatergic presynaptic termini, results in the enlargement of dendritic spines through the modulation of the glutamatergic neurotransmission. The remodeled spinal architecture might be responsible for the change in responsiveness of neural circuitry, leading to cholinergic tuning of brain function.
Subject(s)
Dendritic Spines/drug effects , Hippocampus/cytology , Neurons/drug effects , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Receptors, Nicotinic/metabolism , Animals , Cells, Cultured , Dendritic Spines/ultrastructure , Glutamates/metabolism , Neurons/ultrastructure , Rats , Rats, Sprague-DawleyABSTRACT
Recent studies have revealed that Argonaute proteins are crucial components of the RNA-induced silencing complexes (RISCs) that direct both small interfering RNA (siRNA)- and microRNA (miRNA)-mediated gene silencing. Full complementarity between the small RNA and its target messenger RNA (mRNA) results in RISC-mediated cleavage ("Slicing") of the target mRNA. A subset of Argonaute proteins directly contributes to the target cleavage ("Slicer") activity of the RISC. We describe (in vitro) Slicer assays using endogenous Argonaute protein immunopurified from animal cells and recombinant Argonaute protein produced in and purified from Escherichia coli.
Subject(s)
Drosophila Proteins/metabolism , Eukaryotic Initiation Factor-2/metabolism , RNA/metabolism , Animals , Argonaute Proteins , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/isolation & purification , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/isolation & purification , Eukaryotic Initiation Factors , Gene Silencing , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , RNA/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Induced Silencing Complex/chemistry , RNA-Induced Silencing Complex/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Diabetes, a disease in which carbohydrate and lipid metabolism are regulated improperly by insulin, is a serious worldwide health issue. Insulin is secreted from pancreatic beta cells in response to elevated plasma glucose, with various factors modifying its secretion. Free fatty acids (FFAs) provide an important energy source as nutrients, and they also act as signalling molecules in various cellular processes, including insulin secretion. Although FFAs are thought to promote insulin secretion in an acute phase, this mechanism is not clearly understood. Here we show that a G-protein-coupled receptor, GPR40, which is abundantly expressed in the pancreas, functions as a receptor for long-chain FFAs. Furthermore, we show that long-chain FFAs amplify glucose-stimulated insulin secretion from pancreatic beta cells by activating GPR40. Our results indicate that GPR40 agonists and/or antagonists show potential for the development of new anti-diabetic drugs.
Subject(s)
Fatty Acids, Nonesterified/pharmacology , Insulin/metabolism , Pancreas/drug effects , Pancreas/metabolism , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled , Animals , CHO Cells , Calcium/metabolism , Calcium Signaling/drug effects , Cricetinae , Enzyme Activation/drug effects , Glucose/pharmacology , Haplorhini , Humans , Insulin Secretion , MAP Kinase Signaling System/drug effects , Male , Mice , Mitogen-Activated Protein Kinases/metabolism , Molecular Sequence Data , Pancreas/cytology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Rats, Wistar , Receptors, Cell Surface/agonists , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cell Surface/genetics , TransfectionABSTRACT
Imprinting is an epigenetic modification leading to monoallelic expression of some genes, and disrupted imprinting is believed to be a barrier to human stem cell transplantation, based on studies that suggest that epigenetic marks are unstable in mouse embryonic germ (EG) and embryonic stem (ES) cells. However, stem cell imprinting has not previously been examined directly in humans. We found that three imprinted genes, TSSC5, H19, and SNRPN, show monoallelic expression in in vitro differentiated human EG-derived cells, and a fourth gene, IGF2, shows partially relaxed imprinting at a ratio from 4:1 to 5:1, comparable to that found in normal somatic cells. In addition, we found normal methylation of an imprinting control region (ICR) that regulates H19 and IGF2 imprinting, suggesting that imprinting may not be a significant epigenetic barrier to human EG cell transplantation. Finally, we were able to construct an in vitro mouse model of genomic imprinting, by generating EG cells from 8.5-day embryos of an interspecific cross, in which undifferentiated cells show biallelic expression and acquire preferential parental allele expression after differentiation. This model should allow experimental manipulation of epigenetic modifications of cultured EG cells that may not be possible in human stem cell studies.
