Inhibition of microRNA-128-3p attenuates hypercholesterolemia in mouse model.

AIMS: Hypercholesterolemia remains a critical risk factor for cardiovascular diseases and there is an urgent need to develop effective alternative therapeutics. Herein, we investigated the effects of miR-128-3p inhibition on serum cholesterol levels using a hypercholesterolemic mouse model. MATERIALS AND METHODS: Five injections of anti-miR-128-3p (AM-128) treatment were given, and the cholesterol profile in serum and liver was quantified. We validated the underlying gene network using qRT-PCR, western blotting, ELISA, and dual luciferase assays. KEY FINDINGS: AM-128 treatment inhibits cholesterol biosynthesis by upregulating INSIG1 and downregulating HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase) expression. The serum cholesterol clearance by SR-B1 (scavenger receptor class B member 1) and LDLR (low density lipoprotein receptors) was also increased. Furthermore, the catabolism of cholesterol by CYP7A1 (cytochrome P450 family 7 subfamily A member 1) was increased. SIGNIFICANCE: Our results confirmed a critical role of miR-128-3p inhibition in lowering serum cholesterol and suggest its potential therapeutic implications in reversing hypercholesterolemia.

Radioresistant Sf9 insect cells undergo an atypical form of Bax-dependent apoptosis at very high doses of γ-radiation.

PURPOSE: To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in radioresistant Spodoptera frugiperda-9 (Sf9) insect cells. MATERIALS AND METHODS: Morphology, cell proliferation and DNA-fragmentation analysis was performed at 500-2000 Gy. Changes in intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cardiolipin oxidation and Annexin-V externalization were studied using flow-cytometry. Cytochrome-c release was measured using immunofluorescence microscopy. Inhibitors of apoptosis, i.e., Bongkrekic acid (BKA), Caspase-9 inhibitor (C9i), 5-(4-fluorosulfonylbenzoyl) adenosine hydrochloride (FSBA) and Cyclosporin-A (CsA) were used to dissect apoptotic mechanism at many classical steps. Caspase-3 activity was measured using a caspase-activity assay kit. RESULTS: A dose-dependent induction of typical apoptosis was observed at extremely high doses, marked by extensive apoptotic body formation. However, certain atypical responses such as cellular hypertrophy and the lack of phosphatidylserine-externalization were observed during the initial hours after radiation. Loss of mitochondrial membrane potential observed at 48 h following a 2000 Gy dose was accompanied by an increase in ROS that caused significant cardiolipin oxidation leading to cytochrome-c release, caspase activation and internucleosomal DNA fragmentation. Inhibitors of B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax)-mediated cytochrome-c release, apoptosome formation and caspase-9 effectively prevented radiation-induced apoptosis, strongly suggesting the role of Bax-dependent cell death mechanism. CONCLUSIONS: Our study demonstrates that the Sf9 insect cells display good homology with human cells in the mitochondria-dependent events during radiation-induced apoptosis, although doses eliciting similar responses were 50-200 times higher than human cells. Factors upstream to mitochondrial damage remain pertinent for a thorough understanding of this extreme radioresistance displayed by lepidopteran cells.

Evidence for involvement of cytosolic thioredoxin peroxidase in the excessive resistance of Sf9 Lepidopteran insect cells against radiation-induced apoptosis.

Lepidopteran insect cells display 50-100 times higher radioresistance compared to human cells, and reportedly have more efficient antioxidant system that can significantly reduce radiation-induced oxidative stress and cell death. However, the antioxidant mechanisms that contribute substantially to this excessive resistance still need to be understood thoroughly. In this study, we investigated the role of thioredoxin peroxidase (TPx) in high-dose γ-radiation response of Sf9 cell line derived from Spodoptera frugiperda, the Fall armyworm. We identified a TPx orthologue (Sf-TPx) in Spodoptera system, with primarily cytosolic localization. Gamma-irradiation at 500 Gy dose significantly up-regulated Sf-TPx, while higher doses (1000 Gy-2000 Gy) had no such effect. G2/M checkpoint induced following 500 Gy was associated with transition of Sf-TPx decamer into enzymatically active dimer. Same effect was observed during G2/M block induced by 5 nM okadaic acid or 10 µM CDK1 (cycline dependent kinase-1) inhibitor roscovitine, thus indicating that radiation-induced Sf-TPx activity is mediated by CDKs. Accumulation of TPx dimer form during G2/M checkpoint might favour higher peroxidase activity facilitating efficient survival at this dose. Confirming this, higher lethal doses (1000 Gy-2000 Gy) caused significantly less accumulation of dimer form and induced dose-dependent apoptosis. A ∼50% knock-down of Sf-TPx by siRNA caused remarkable increase in radiation-induced ROS as well as caspase-3 dependent radiation-induced apoptosis, clearly implying TPx role in the radioresistance of Sf9 cells. Quite importantly, our study demonstrates for the first time that thioredoxin peroxidase contributes significantly in the radioresistance of Lepidopteran Sf9 insect cells, especially in their exemplary resistance against radiation-induced apoptosis. This is an important insight into the antioxidant mechanisms existing in this highly stress-resistant model cell system.

Radioresistant Sf9 insect cells display moderate resistance against cumene hydroperoxide.

Lepidopteran insect cells serve as excellent model to study stress responses and are known to display resistance against DNA damaging agents including ionizing radiation; however, limited information is available on the effects of membrane damaging agents in these cells. In this study, we investigated the response of Sf9 cells (derived from ovaries of Spodoptera frugiperda; order Lepidoptera) to cumene hydroperoxide (CHPx), compared to human BMG-1 cells. CHPx treatment at doses lethal for human cells also caused typical necrosis in Sf9. Severe necrosis in human BMG-1 cells was observed at 125 µM, whereas similar effect in Sf9 cells was observed at 250 µM. In Sf9 cells, CHPx (250 µM) induced negligible changes in mitochondrial membrane potential and intracellular reactive oxygen species, while moderate effect was observed on intracellular calcium distribution. Reduced DNA damage and lipid (including cardiolipin) oxidation was observed in Sf9 cells that could be due to moderate total antioxidant status and constitutive/induced glutathione S-transferase activity. This study importantly demonstrates that Lepidopteran insect cells having extensive resistance towards DNA damaging agents show only moderately higher resistance to membrane damaging agents. A stronger reducing environment involving efficient antioxidant system seems to contribute significantly in this response.