Zinc Oxide Nanoparticle-Poly I:C RNA Complexes: Implication as Therapeutics against Experimental Melanoma.

There is current interest in harnessing the combined anticancer and immunological effect of nanoparticles (NPs) and RNA. Here, we evaluate the bioactivity of poly I:C (pIC) RNA, bound to anticancer zinc oxide NP (ZnO-NP) against melanoma. Direct RNA association to unfunctionalized ZnO-NP is shown by observing change in size, zeta potential, and absorption/fluorescence spectra upon complexation. RNA corona was visualized by transmission electron microscopy (TEM) for the first time. Binding constant (Kb = 1.6-2.8 g-1 L) was determined by modified Stern-Volmer, absorption, and biological surface activity index analysis. The pIC-ZnO-NP complex increased cell death for both human (A375) and mouse (B16F10) cell lines and suppressed tumor cell growth in BALB/C-B16F10 mouse melanoma model. Ex vivo tumor analysis indicated significant molecular activity such as changes in the level of phosphoproteins JNK, Akt, and inflammation markers IL-6 and IFN-γ. High throughput proteomics analysis revealed zinc oxide and poly I:C-specific and combinational patterns that suggested possible utility as an anticancer and immunotherapeutic strategy against melanoma.

Enzyme and Cancer Cell Selectivity of Nanoparticles: Inhibition of 3D Metastatic Phenotype and Experimental Melanoma by Zinc Oxide.

Biomedical applications for metal and metal oxide nanoparticles are rapidly increasing. Here their functional impact on two well-characterized model enzymes, Luciferase (Luc) or ß-galactosidase (ß-Gal) was quantitatively compared. Nickel oxide nanoparticle (NiO-NP) activated ß-Gal (>400% control) and boron carbide nanoparticle (B4C-NP) inhibited Luc(<10% control), whereas zinc oxide (ZnO-NP) and cobalt oxide (Co3O4-NP) activated ß-Gal to a lesser extent and magnesium oxide (MgO) moderately inhibited both enzymes. Melanoma specific killing was in the order; ZnO > B4C ≥ Cu > MgO > Co3O4 > Fe2O3 > NiO, ZnO-NP inhibiting B16F10 and A375 cells as well as ERK enzyme (>90%) and several other cancer-associated kinases (AKT, CREB, p70S6K). ZnO-NP or nanobelt (NB) serve as photoluminescence (PL) cell labels and inhibit 3-D multi-cellular tumor spheroid (MCTS) growth and were tested in a mouse melanoma model. These results demonstrate nanoparticle and enzyme specific biochemical activity and suggest their utility as new tools to explore the important model metastatic foci 3-D environment and their chemotherapeutic potential.

Disassociation of muscle insulin signaling and insulin-stimulated glucose uptake during endotoxemia.

Lipopolysaccharide (LPS) elicits a strong immune response, which leads to the release of inflammatory cytokines. Increased cytokine production has been shown to impair insulin-mediated glucose disposal. LPS can alter other factors, such as muscle blood flow and insulin signaling in the myocyte, that can influence glucose disposal. We hypothesize that LPS induced impairments in cardiovascular function contribute to the associated impairments in insulin action in vivo. Male wild-type C57BL/6J mice had a catheter implanted in the jugular vein for infusions and the carotid artery for sampling 5 days prior to the hyperinsulinemic-euglycemic clamp. Mice were treated with vehicle, low- (1 ug/gBW) or high-dose (10 ug/gBW) LPS 4 hours prior to the clamp. Muscle glucose uptake (MGU) was assessed using [2-(14)C] deoxyglucose. While both low- and high-dose LPS inhibited insulin-stimulated MGU compared to vehicle-treated mice, the impairment was more significant with the high-dose treatment (∼25% in soleus and ∼70% in both gastrocnemius and vastus lateralis). Interestingly, insulin signaling through the PI3-kinase pathway in the muscle was not affected by this treatment suggesting that the decrease in MGU is not directly due to impairments in muscle insulin action. Echocardiography demonstrated that high-dose LPS treatment significantly decreased stroke volume (∼30%), heart rate (∼35%), and cardiac output (∼50%). These observations were not seen with vehicle or low-dose LPS treatment. High-dose LPS treatment also significantly decreased muscle blood flow (∼70%) and whole body oxygen consumption (∼50%). Thus, in vivo acute endotoxemia does not impair insulin signaling through the PI3-kinase pathway in skeletal muscle and decreased tissue blood flow likely plays a central role in the impairment of glucose uptake in the muscle.

Cessation of daily exercise dramatically alters precursors of hepatic steatosis in Otsuka Long-Evans Tokushima Fatty (OLETF) rats.

The purpose of this study was to delineate potential mechanisms initiating the onset of hepatic steatosis following the cessation of daily physical activity. Four-week-old, hyperphagic/obese Otsuka Long-Evans Tokushima Fatty rats were given access to voluntary running wheels for 16 weeks to prevent the development of hepatic steatosis. The animals were then suddenly transitioned to a sedentary condition as wheels were locked (wheel lock; WL) for 5 h (WL5), 53 h (WL53) or 173 h (WL173). Importantly after the cessation of daily exercise (5-173 h), no changes occurred in body weight, fat pad mass (omental and retroperitoneal), food intake, serum insulin, hepatic triglycerides or in the exercise-suppressed hepatic stearoyl-CoA desaturase-1 and peroxisome proliferator-activated receptor-gamma protein content. However, complete hepatic fatty acid oxidation and mitochondrial enzyme activities were highest at WL5 and WL53 and dropped significantly to SED levels by WL173. In addition, cessation of daily exercise quickly increased the hepatic protein contents of fatty acid synthase and acetyl-coenzyme A carboxylase (ACC), reduced ACC phosphorylation status, and dramatically increased hepatic malonyl-CoA concentration. This study is the first to show that the sudden cessation of daily exercise in a hyperphagic/obese model activates a subgroup of precursors and processes known to initiate hepatic steatosis, including decreased hepatic mitochondrial oxidative capacity, increased hepatic expression of de novo lipogenesis proteins, and increased hepatic malonyl CoA levels; each probably increasing the susceptibility to non-alcoholic fatty liver disease.

Daily exercise increases hepatic fatty acid oxidation and prevents steatosis in Otsuka Long-Evans Tokushima Fatty rats.

Exercise training is commonly prescribed for treatment of nonalcoholic fatty liver disease (NAFLD). We sought to determine whether exercise training prevents the development of NAFLD in Otsuka Long-Evans Tokushima Fatty (OLETF) rats and to elucidate the molecular mechanisms underlying the effects of exercise on hepatic steatosis. Four-week-old OLETF rats were randomly assigned to either a sedentary control group (Sed) or a group given access to voluntary running wheels for 16 wk (Ex). Wheels were locked 2 days before euthanasia in the Ex animals, and both groups were euthanized at 20 wk old. Voluntary wheel running attenuated weight gain and reduced serum glucose, insulin, free fatty acids, and triglycerides in Ex animals compared with Sed (P < 0.001). Ex animals exhibited significantly reduced hepatic triglyceride levels and displayed fewer lipid droplets (Oil Red O staining) and reduced lipid droplet size compared with Sed. Wheel running increased by threefold the percent of palmitate oxidized completely to CO(2) in the Ex animals but did not alter AMP-activated protein kinase-alpha (AMPKalpha) or AMPK phosphorylation status. However, fatty acid synthase and acetyl-coenzyme A carboxylase (ACC) content were significantly reduced (approximately 70 and approximately 35%, respectively), and ACC phosphorylation and cytochrome c content were significantly elevated (approximately 35 and approximately 30%, respectively) in the Ex animals. These results unequivocally demonstrate that daily physical activity attenuates hepatic steatosis and NAFLD in an obese rodent model and suggest that this effect is likely mediated, in part, through enhancement of hepatic fatty acid oxidation and reductions in key protein intermediates of fatty acid synthesis.

Exercise-induced attenuation of obesity, hyperinsulinemia, and skeletal muscle lipid peroxidation in the OLETF rat.

The Otsuka Long-Evans Tokushima fatty (OLETF) rat is a model of hyperphagic obesity in which the animals retain the desire to run voluntarily. Running wheels were provided for 4-wk-old OLETF rats for 16 wk before they were killed 5 h (WL5), 53 h (WL53), or 173 h (WL173) after the wheels were locked. Sedentary (SED) OLETF rats that were not given access to running wheels served as age-matched cohorts. Epididymal fat pad mass, adipocyte volume, and adipocyte number were 58%, 39%, and 47% less, respectively, in WL5 than SED rats. Contrary to cessation of daily running in Fischer 344 x Brown Norway rats, epididymal fat did not increase during the first 173 h of running cessation in the OLETF runners. Serum insulin and glucose levels were 77% and 29% less, respectively, in WL5 than SED rats. Oil red O staining for intramyocellular lipid accumulation was not statistically different among groups. However, lipid peroxidation levels, as determined by total trans-4-hydroxy-2-nonenal (4-HNE) and 4-HNE normalized to oil red O, was higher in epitrochlearis muscles of SED than WL5, WL53, and WL173 rats. mRNA levels of glutathione S-transferase-alpha type 4, an enzyme involved in cellular defense against electrophilic compounds such as 4-HNE, were higher in epitrochlearis muscle of WL53 than WL173 and SED rats. In contrast, 4-HNE levels in omental fat were unaltered. Epitrochlearis muscle palmitate oxidation and relative transcript levels for peroxisome proliferator-activated receptor-delta and peroxisome proliferator-activated receptor-gamma coactivator type 1 were surprisingly not different between runners and SED rats. In summary, voluntary running was associated with lower levels of lipid peroxidation in skeletal muscle without significant changes in intramyocellular lipids or mitochondrial markers in OLETF rats at 20 wk of age. Therefore, even in a genetic animal model of extreme overeating, daily physical activity promotes improved health of skeletal muscle.

Exercise training prevents development of cardiac contractile dysfunction in hypertensive TG (mREN-2)27 rats.

BACKGROUND: Angiotensin-II (Ang-II) contributes to cardiac remodeling and left ventricular dysfunction. In contrast, exercise may have beneficial effects on left ventricular structure and function. METHODS AND RESULTS: We investigated the effects of low-intensity exercise training (ET) on in vivo cardiac function in hypertensive TG (mREN-2)27 rats (Ren-2) which develop left ventricular hypertrophy and dysfunction. Ren-2 rats and Sprague Dawley (SD) controls (4-5 weeks) began treadmill exercise every day for 5-6 weeks. Cardiac function was evaluated by echocardiography. Cardiac output and stroke volume were increased by ET in both 8-wk-old SD and Ren-2. Slope of mitral deceleration time, a non-invasive measure of diastolic function, was lower in the Ren-2 rats, but not changed by ET. LV collagen deposition, as assessed by hydroxyproline assay, was not affected by rat strain or ET at 10-11 weeks of age. Left ventricular B-type natriuretic peptide mRNA levels were higher in the Ren-2 rats (100%), but not affected by ET. Both alpha (~14.5 fold) and beta (~2.5 fold) myosin heavy chain mRNA were higher in the LV of Ren-2 rats (p < 0.05), but were not changed by ET. CONCLUSION: Low-intensity exercise training in Ren-2 rats, a model of Ang-II-mediated hypertension, maintains cardiac index and stroke volume in the presence of impaired diastolic function at 8 wks of age.

Responsiveness of cell signaling pathways during the failed 15-day regrowth of aged skeletal muscle.

Various cellular signaling pathways, such as phosphatidylinositol 3-kinase, calcineurin, Janus kinase 2/signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase (MAPK) have been suggested to play an important role in skeletal muscle growth. Old muscle, compared with young muscle, lacks the ability to completely regrow its muscle mass after an atrophy-induced stimulus. it is hypothesized that defects and/or delays in the activation of specific cell signaling pathways of aged soleus muscle limit the potential for growth. To test this, 42 male Fischer 344 x Brown Norway rats, 30 mo old, were hindlimb immobilized for 10 days, and their muscle samples were compared with muscle samples analyzed from 3- to 4-mo-old rats in a previous report (Childs TE, Spangenburg EE, Vyas DR, and Booth FW. Am J Physiol Cell Physiol: 285: C391-C398, 2003). After 10 days, the immobilization was removed and rats were allowed to ambulate for a series of days. Alterations in the activation or deactivation status of specific signaling pathways were determined by comparing the phosphorylation (phos) and total concentration of specific signaling proteins (pan) through Western blotting with the 10-day immobilization group. Various cell signals and their respective time groups of the old rats were shown to be significantly different compared with the 10-day immobilization group. For example, peak increases during recovery from the immobilization were observed at 1) the third recovery day for calcineurin B-pan and 2) the sixth recovery day for glycogen synthase kinase-3beta-phos, p70 S6 kinase (p70S6k) -phos and -pan, calcineurin A-pan, STAT3-phos and -pan, p44 MAPK-pan, and p42 MAPK-pan. In contrast, Akt-pan, c-Jun NH2-terminal kinase-phos, and p38 MAPK-phos were observed to decrease from 10-day immobilization values to control levels. Also, Aktphos was unchanged among all groups. In a follow-up experiment in which muscle samples from both the present study and a previous study (Childs TE, Spangenburg EE, Vyas DR, and Booth FW. Am J Physiol Cell Physiol: 285: C391-C398, 2003) were reanalyzed together, the recovery-induced increase in p70S6k-phos from immobilization-atrophy was significantly attenuated in soleus muscles of the old group.