Targeting RNA: A Transformative Therapeutic Strategy.

The therapeutic pathways that modulate transcription mechanisms currently include gene knockdown and splicing modulation. However, additional mechanisms may come into play as more understanding of molecular biology and disease etiology emerge. Building on advances in chemistry and delivery technology, oligonucleotide therapeutics is emerging as an established, validated class of drugs that can modulate a multitude of genetic targets. These targets include over 10,000 proteins in the human genome that have hitherto been considered undruggable by small molecules and protein therapeutics. The approval of five oligonucleotides within the last 2 years elicited unprecedented excitement in the field. However, there are remaining challenges to overcome and significant room for future innovation to fully realize the potential of oligonucleotide therapeutics. In this review, we focus on the translational strategies encompassing preclinical evaluation and clinical development in the context of approved oligonucleotide therapeutics. Translational approaches with respect to pharmacology, pharmacokinetics, cardiac safety evaluation, and dose selection that are specific to this class of drugs are reviewed with examples. The mechanism of action, chemical evolution, and intracellular delivery of oligonucleotide therapies are only briefly reviewed to provide a general background for this class of drugs.

Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat.

Friedreich's Ataxia (FA) is an inherited neurologic disorder caused by an expanded GAA repeat within intron 1 of the frataxin (FXN) gene that reduces expression of FXN protein. Agents that increase expression of FXN have the potential to alleviate the disease. We previously reported that duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) complementary to the GAA repeat could enhance expression of FXN protein. We now explore the potential of a diverse group of chemically modified dsRNAs and ASOs to define the breadth of repeat-targeted synthetic nucleic acids as a platform for therapeutic development for FA. ASOs and dsRNAs can activate FXN protein expression in FA patient-derived cell lines that possess varied numbers of GAA repeats. Increased FXN protein expression was achieved by ASOs incorporating diverse chemical modifications with low nanomolar potencies, suggesting substantial flexibility in choosing compounds for further chemical optimization and animal studies. Our data encourage further development of ASOs as agents to treat FA.

Effects of erythropoietin and methylprednisolone on AQP4 expression in astrocytes.

Methylprednisolone sodium succinate (MPSS) has been suggested as a treatment for spinal cord injury (SCI), but its use has been limited due to its adverse effects. Erythropoietin (EPO) has been suggested as a promising candidate for limiting SCI in mammals. The aim of the present study was to investigate the effects of EPO in combination with MPSS on astrocytes following ischemic injury in vitro. Astrocytes were isolated from the cerebral cortex of postnatal day 3 Sprague­Dawley rats and cultured in vitro. Astrocyte ischemic injury was induced by oxygen and glucose deprivation for 4 h, and reperfusion was simulated by subsequent culture under normoxic conditions. The effects of EPO and MPSS on the expression of aquaporin­4 (AQP4) were investigated. Ischemic astrocytes were treated with EPO (10 U/ml), MPSS (10 µg/ml), or EPO (10 U/ml) in combination with MPSS (10 µg/ml) during reperfusion. The cell viability of astrocytes was assessed using an MTT assay. The mRNA and protein expression levels of AQP4 were determined using reverse transcription­quantitative polymerase chain reaction and western blot analysis, respectively. The role of the protein kinase C (PKC) signaling pathway in the molecular mechanisms underlying the effects of EPO and MPSS was also investigated. The present results demonstrated that following treatment with EPO and MPSS, the mRNA expression levels of AQP4 were upregulated and cell viability was enhanced. EPO and MPSS effectively inhibited the oxygen and glucose deprivation­mediated downregulation of AQP4 following reperfusion. In addition, the combined treatment with EPO and MPSS exhibited higher AQP4 expression levels and cell viability compared with each treatment alone. Finally, the effects of EPO and MPSS on AQP4 expression were partially reversed by pretreatment with the PKC inhibitor Ro 31­8220. The present study indicated that EPO and MPSS had a synergistic effect on AQP4 expression following reperfusion, and suggest that they may be combined in the treatment of SCI.

Cinnamomum osmophloeum Kanehira ethanol extracts prevents human liver-derived HepG2 cell death from oxidation stress by induction of ghrelin gene expression.

Diabetes patients associated with liver disease carry a significant risk of morbidity and mortality. Cinnamon has been reported to reduce fructose-induced oxidative stress in the rat liver. However, the mechanism by which cinnamon protects the liver in a high-saccharide environment remains to be investigated. HepG2 cells were cultured with 30 mM D-ribose to mimic the high-oxidative-stress environment, typical of a liver in a diabetic patient. Three different chemical types of C. osmophloeum ethanol extracts (CEEs) were added in HepG2 culture media and the administration of all three CEEs protected HepG2 cells from D-ribose damage and increased cell survival by approximately 20 percent. Exclusively, the transcript variant 1 of the ghrelin gene, but not variant 3, was 2-3 times induced by the addition of these CEEs. Moreover, the mRNAs of ghrelin processing enzyme, furin, and mboat4 were detected in HepG2 cells. The ghrelin hormones in the culture media were increased 4-9 times by the addition of CEEs. The protective effects of ghrelin on HepG2 cells in D-ribose environment were further confirmed by recombinant ghrelin transfection. We conclude that the CEEs induce ghrelin gene expression and protect HepG2 cells from D-ribose-induced oxidative damage through ghrelin signalling.

Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages.

We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.

5,6-Dehydrokawain from Alpinia zerumbet promotes osteoblastic MC3T3-E1 cell differentiation.

Bone homeostasis is maintained by balancing bone formation and bone resorption, but an imbalance between them is associated with various bone-related diseases such as osteoporosis and rheumatoid arthritis. We found that 5,6-dehydrokawain (DK) and dihydro-5,6-dehydrokawain (DDK), which were isolated as promising compounds from Alpinia zerumbet rhizomes, promote differentiation of osteoblastic MC3T3-E1 cells. DK and DDK increased the alkaline phosphatase activity and matrix mineralization of MC3T3-E1 cells. DK exerts larger effects than DDK. The gene expression of runt-related transcription factor 2 and osterix, which are essential transcription factors in the early period of osteoblast differentiation, was significantly increased by DK treatment. The mRNA level of distal-less homeobox 5 was also enhanced by DK treatment, and DK activated the p38 mitogen-activated protein kinase pathway. Therefore, DK may have clinical potential for preventing osteoporosis, and could be considered as a potential anabolic therapeutic agent.

Estrogenic effects of flavonoid components in Xiaoyao powder.

The objective of this study was to evaluate the estrogenic effects and mechanisms of three flavonoid components in Xiaoyao powder: quercetin, kaempferol, and isorhamnetin. The drugs were used to treat estrogen receptor (ER)-positive human breast cancer MCF-7 cells, and proliferation was measured using the MTT method. The expression of proteins and mRNA of the ER subtype were measured using western blotting and real time polymerase chain reaction. The quercetin (10(-2) µM, 10(-3) µM), kaempferol (100 µM, 10(-2) µM), and isorhamnetin (10(-3) µM) promoted the proliferation of MCF-7 cells, and the expression of ERα and ERß proteins and mRNA were all increased significantly (P < 0.05). These effects were reversed by treatment with 0.1 µM estrogen antagonist ICI182780. Three flavonoid components in Xiaoyao powder increased the expression of proteins and mRNA of ERα and ERß and promoted the proliferation of MCF-7 cells. These estrogenic effects were mediated by the ER.

Berberine inhibits tumor necrosis factor-α-induced expression of inflammatory molecules and activation of nuclear factor-κB via the activation of AMPK in vascular endothelial cells.

Berberine, which is a well­known drug used in traditional medicine, has been demonstrated to exert diverse pharmacological effects, including anti­inflammatory effects. However, whether berberine can affect the production of inflammatory molecules in vascular endothelial cells remains to be elucidated. Therefore, the present study aimed to determine the effects of berberine, and the underlying molecular mechanisms of these effects. The effect of berberine on tumor necrosis factor (TNF)­α­induced inflammatory molecule expression was examined in cultured human aortic endothelial cells (HAECs). The HAECs were stimulated with TNF­α and incubated with or without berberine. The activation of nuclear factor (NF)­κB and adenosine monophosphate­activated protein kinase (AMPK) were analyzed using western blotting, and the protein secretion of intercellular adhesion molecule (ICAM)­1 and monocyte chemoattractant protein (MCP)­1 was measured using ELISA kits. The mRNA expression levels of ICAM­1 and MCP­1 were analyzed using reverse transcription­quantitative polymerase chain reaction. The results of the present study demonstrated that berberine significantly inhibited the TNF­α­induced expression of ICAM­1 and MCP­1, as well as the activation of NF­κB in the HAECs. These effects were attenuated following co­treatment with AMPK inhibitor compound C, or specific small interfering RNAs. In conclusion, the results of the present study indicated that berberine inhibits the TNF­α­induced expression of ICAM­1 and MCP­1, and the activation of NF­κB in HAECs in vitro, possibly through the AMPK­dependent pathway.