ABSTRACT
Since impaired mitochondrial ATP production in cardiomyocytes is thought to lead to heart failure, a drug that protects mitochondria and improves ATP production under disease conditions would be an attractive treatment option. In this study, we identified small-molecule drugs, including the anti-parasitic agent, ivermectin, that maintain mitochondrial ATP levels under hypoxia in cardiomyocytes. Mechanistically, transcriptomic analysis and gene silencing experiments revealed that ivermectin increased mitochondrial ATP production by inducing Cox6a2, a subunit of the mitochondrial respiratory chain. Furthermore, ivermectin inhibited the hypertrophic response of human induced pluripotent stem cell-derived cardiomyocytes. Pharmacological inhibition of importin ß, one of the targets of ivermectin, exhibited protection against mitochondrial ATP decline and cardiomyocyte hypertrophy. These findings indicate that maintaining mitochondrial ATP under hypoxia may prevent hypertrophy and improve cardiac function, providing therapeutic options for mitochondrial dysfunction.
Subject(s)
Adenosine Triphosphate/metabolism , Cardiotonic Agents/pharmacology , Cell Hypoxia/drug effects , Mitochondria/drug effects , Myocytes, Cardiac/cytology , Small Molecule Libraries/pharmacology , Animals , Cells, Cultured , Electron Transport Complex IV/genetics , Gene Expression Profiling , Gene Expression Regulation/drug effects , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Ivermectin/pharmacology , Mice , Mitochondria/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , beta Karyopherins/metabolismABSTRACT
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
Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. Expanding the application outside the liver is required to increase the value of siRNAs. Here we report on a novel platform targeted to muscular organs by conjugation of siRNAs with anti-CD71 Fab' fragment. This conjugate showed durable gene-silencing in the heart and skeletal muscle for one month after intravenous administration in normal mice. In particular, 1µg siRNA conjugate showed significant gene-silencing in the gastrocnemius when injected intramuscularly. In a mouse model of peripheral artery disease, the treatment with myostatin-targeting siRNA conjugate by intramuscular injection resulted in significant silencing of myostatin and hypertrophy of the gastrocnemius, which was translated into the recovery of running performance. These data demonstrate the utility of antibody conjugation for siRNA delivery and the therapeutic potential for muscular diseases.
Subject(s)
Immunoconjugates/therapeutic use , Muscle, Skeletal/metabolism , Myocardium/metabolism , Myostatin/genetics , Peripheral Arterial Disease/therapy , RNA, Small Interfering/therapeutic use , Animals , Antigens, CD/immunology , Cells, Cultured , Female , Immunoconjugates/genetics , Immunoconjugates/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Peripheral Arterial Disease/genetics , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/immunology , RNAi Therapeutics , Rats , Receptors, Transferrin/immunologyABSTRACT
Lysophosphatidyl-L-serine (lysoPS) is thought to be an immunological regulator because it dramatically augments the degranulation of rat peritoneal mast cells (RPMCs). This stimulatory effect may be mediated by a lysoPS receptor, but its molecule has not been identified yet. During a ligand fishing study for the orphan G-protein-coupled receptor 34 (GPR34), we found that lysoPS caused a dose-dependent inhibition of forskolin-stimulated cAMP accumulation in human GPR34-expressing Chinese hamster ovary (CHO/hGPR34) cells. The CHO/hGPR34 cells were unresponsive to other structurally related phospholipids examined. Quantitative real-time-PCR demonstrated that mRNAs of GPR34 are particularly abundant in mast cells. The effective lysoPS concentration for RPMC degranulation was similar to that required for GPR34 activation, and the structural requirement of lysoPS for RPMC degranulation was in good agreement with that observed in CHO/hGPR34 cells. These results suggest that GPR34 is the functional mast cell lysoPS receptor.
