Colchicine as a novel drug for the treatment of osteosarcoma through drug repositioning based on an FDA drug library.

Background: Colchicine is a traditional medication that is currently approved to treat gout and familial Mediterranean fever (FMF). However, colchicine has a wide range of anti-inflammatory activities, and several studies have indicated that it may be useful in a variety of other conditions, such as rheumatic disease, cardiac disease, and cancer. Osteosarcoma, the most common type of bone sarcoma, is derived from primitive bone-forming mesenchymal cells. In this study, we investigated whether colchicine could be used to treat osteosarcoma through the regulation of cell cycle signaling. Methods: Two human osteosarcoma cell lines, U2OS and Saos-2, were used. A clonogenic assay was used to determine the antiproliferative effects of colchicine on osteosarcoma cells. Reactive oxygen species (ROS) production and apoptosis were measured by flow cytometry. Migration and invasion assays were performed to investigate the inhibitory effects of colchicine. The signaling pathways related to colchicine treatment were verified by GO biological process (GOBP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Results: Colchicine was selected as the lead compound based on the results of initial screening and cell viability assays conducted in Saos-2 and U2Os cells. Colchicine reduced the viability of Saos-2 and U2OS cells in a concentration-dependent manner. It also significantly inhibited colony-forming ability and induced ROS production and apoptosis. It also inhibited the migration and invasion of both Saos-2 and U2OS cells. GOBP and KEGG enrichment analyses indicated the involvement of microtubule-based processes and cancer-related pathways. Conclusions: These findings suggest that colchicine has therapeutic potential in osteosarcoma.

Withanolides against TLR4-Activated Innate Inflammatory Signalling Pathways: A Comparative Computational and Experimental Study.

Innate inflammations are dominant causes of poor health and high mortality. The pathogen-associated molecular pattern and lipopolysaccharide (LPS) are sensed by immune cells through activation of toll-like receptor 4 leading to mitogen-activated protein kinases (MAPKs) and NF-κB activations. Controlled MAPK and Nf-κB inhibitors have been proposed as potential antiinflammatory drugs. Withania somnifera is an important medicinal herb with known antiinflammatory activity. In this study, the selected Withania somnifera extracts and withanolides were analysed on LPS-induced macrophages comparatively. Molecular docking analysis revealed withaferin A, withanone and withanolide A as effective withanolides against inflammatory target molecules. In experiments, withaferin A and withanone treatment had prominent suppressions on LPS-induced expression of pro-inflammatory cytokines in bone marrow-derived macrophages. Withaferin A regulated all the major four pathways (MAPKs and NF-κB) involved in innate inflammations. Similarly among the Withania extracts analysed, the in vitro propagated leaf and field grown root extracts containing high withaferin A content suppressed the inflammatory molecules through NF-κB and MAPK pathways. Withaferin A was found to be best in suppressing the activated inflammatory pathways among all the analysed withanolides. Therefore, withaferin A and extracts with high withaferin A content can be used as promising drug candidates against innate inflammations. Copyright © 2016 John Wiley & Sons, Ltd.

Testosterone production by a Leydig tumor cell line is suppressed by hyperthermia-induced endoplasmic reticulum stress in mice.

AIMS: Leydig cells are characterized by their ability to produce testosterone. When the Leydig cells are unable to produce enough testosterone, spermatogenesis fails completely. Considering this, it is of great interest to investigate whether the expressions of steroidogenic enzymes are affected by testicular heat stress. This study aimed to demonstrate that heat induced ER-stress significantly influences steroidogenic enzyme expression and testosterone production in the Leydig cells. MAIN METHODS: C57BL/6 mice were subjected to repetitive testicular heat-treatment at 42 °C for 15 min per day, and heat-treated mLTC-1 cells following hCG treatment for 1h. The protein and RNA expressions were measured by Western blot, RT-PCR. The testosterone and progesterone levels were detected by EIA. The histological and pathological characteristics using hematoxylin and eosin (H&E) and antibody stains. KEY FINDINGS: The 3ß-HSD expression was decreased by heat-stress and hCG treatment. While the GRP78/BiP and CHOP levels were increased by ER-stress inducers, those of the steroidogenic enzyme and progesterone were decreased. In contrast, an ER-stress inhibitor rescued the testosterone levels, even under heat-stress conditions. Moreover, the Leydig cells were randomly scattered, and severely damaged upon repetitive testicular heat-treatment. Additionally, immunohistochemical analyses revealed that cleaved caspase-3 was elevated in the testicular Leydig cells, and rescued by TUDCA. Thus, repetitive testicular heat-treatment in mice promotes excessive ER-stress, thereby leading to apoptosis of the Leydig cells and thus, decreased testosterone production. SIGNIFICANCE: Our findings help to provide an ER-stress mediate mechanistic explanation to the impairment of spermatogenesis upon elevation of the testicular temperature.

Determination of Milk Fat Adulteration with Vegetable Oils and Animal Fats by Gas Chromatographic Analysis.

This study assessed the potential application of gas chromatography (GC) in detecting milk fat (MF) adulteration with vegetable oils and animal fats and of characterizing samples by fat source. One hundred percent pure MF was adulterated with different vegetable oils and animal fats at various concentrations (0%, 10%, 30%, 50%, 70%, and 90%). GC was used to obtain the fatty acid (FA) profiles, triacylglycerol (TG) contents, and cholesterol contents. The pure MF and the adulterated MF samples were discriminated based on the total concentrations of saturated FAs and on the 2 major FAs (oleic acid [C18:1n9c] and linoleic acid [C18:2n6c], TGs [C52 and C54], and cholesterol contents using statistical analysis to compared difference. These bio-markers enabled the detection of as low as 10% adulteration of non-MF into 100% pure MF. The study demonstrated the high potential of GC to rapidly detect MF adulteration with vegetable and animal fats, and discriminate among commercial butter and milk products according to the fat source. These data can be potentially useful in detecting foreign fats in these butter products. Furthermore, it is important to consider that several individual samples should be analyzed before coming to a conclusion about MF authenticity.

Beta-lapachone, a modulator of NAD metabolism, prevents health declines in aged mice.

NADH-quinone oxidoreductase 1 (NQO1) modulates cellular NAD(+)/NADH ratio which has been associated with the aging and anti-aging mechanisms of calorie restriction (CR). Here, we demonstrate that the facilitation of NQO1 activity by feeding ß-lapachone (ßL), an exogenous NQO1 co-substrate, prevented age-dependent decline of motor and cognitive function in aged mice. ßL-fed mice did not alter their food-intake or locomotor activity but did increase their energy expenditure as measured by oxygen consumption and heat generation. Mitochondrial structure and numbers were disorganized and decreased in the muscles of control diet group but those defects were less severe in ßL-fed aged mice. Furthermore, for a subset of genes associated with energy metabolism, mice fed the ßL-diet showed similar changes in gene expression to the CR group (fed 70% of the control diet). These results support the potentiation of NQO1 activity by a ßL diet and could be an option for preventing age-related decline of muscle and brain functions.

Fermentation of Korean red ginseng by Lactobacillus plantarum M-2 and its immunological activities.

We investigated ginsenoside transformation by fermentation of red ginseng with Lactobacillus plantarum M-2. We also examined the anti-metastasis and immune-stimulating activities of EtOH extracts of fermented red ginseng (FRG-E) in animal and human subjects. Total sugar decreased from 85.5 mg mL(-1) to 44.1 mg mL(-1) with increasing culture time during the fermentation with L. plantarum M-2. Uronic acid content reached a maximum level (534.3 µg mL(-1)) at 3 days of fermentation and decreased thereafter. Ginsenoside metabolites increased from 4,637.0 to 7,581.1 µg mL(-1) after 4 days. The prophylactic intraperitoneal injection of FRG-E (500 µg mouse(-1)) inhibited lung metastasis about 81.1%, while the inhibitory effect against tumor metastasis by treatment of EtOH extract from non-fermented red ginseng (NFRG-E) was 66.9%. Immunoglobulin A (IgA) and G (IgG) levels in the serum of healthy subjects were higher after FRG-E administration than at baseline, whereas NFRG-E induced reductions of these variables related to immunity. At 1 week, the change in IgA level by FRG-E (5.14 mg mL(-1)) was significantly higher than that by NFRG-E (-14.50 mg mL(-1); p < 0.05). It was concluded that the immunological activities of FRG-E were higher than those of NFRG-E, indicating that fermentation helped enhance the immunological activities of red ginseng.

Stimulation of osteoblastic differentiation and mineralization in MC3T3-E1 cells by yeast hydrolysate.

In a previous study, it was reported that yeast hydrolysate (YH) was effective in promoting bone growth in Sprague-Dawley (SD) rats. To further clarify the mechanism of YH, the effects of YH on proliferation, differentiation and gene expression in vitro were investigated using osteoblastic cell lines (MC3T3-E1). Cell proliferation increased significantly as much as 110% of the basal value when cells were treated with 100 µg/mL of YH. Alkaline phosphatase (ALP) activity increased significantly with a YH concentration of 25-100 µg/mL, and the activity increased 152% that of the control at 100 µg/mL. The calcium content increased as much as 129% at 100 µg/mL YH. The gene expression levels of ALP and collagen type II (COL II) significantly increased approximately 1.3-fold and 1.7-fold of control, respectively, at 100 µg/mL. YH increased significantly the mRNA level of bone sialoprotein (BSP) but not in a dose-dependent manner. The mRNA levels of bone morphogenetic proteins (BMP)-2, BMP-4, collagen type I (COL I) and osteonectin (ON) did not increase. In summary, YH increased the proliferation of osteoblasts and directly stimulated ALP and bone matrix proteins (e.g. BSP, COL II), and these increases trigger osteoblastic differentiation (e.g. mineralized nodule formation).

Influence of steeping solution and storage temperature on the color change of garlic.

UNLABELLED: The objective of this study was to investigate the browning of garlic under different steeping conditions and storage temperatures. The brown indices of steeped garlics showed lowest values (7.3 and 7) in 25% and 50% EtOH at 7 d of storage. The degree of browning of steeped garlics was lowest (10.2 in 25% EtOH and 10.4 in 50% EtOH) in the samples soaked for 8 h at 13 d of storage. As the storage temperature was increased from 10 to 40 degrees C, the brown indices of garlics revealed an increasing trend relative to storage time regardless of steeping treatment. Overall, the kinetic parameters showed relatively low R(2) and irregular reaction constants, but the k(o) values showed an increasing trend with temperature under a zero-order model. The highest polyphenol content within the garlic bulbs was seen in controls (without steeping treatment, 588.9 microg/g), than 0% EtOH (water, 392.5 microg/g), than 25% EtOH (211.3 microg/g), and finally 50% EtOH (155.6 microg/g). The polyphenol oxidase activity of garlic showed a similar trend to that of polyphenol content. However, the texture properties of garlics steeped with 25% and 50% did not change. PRACTICAL APPLICATION: The garlic color preferred by consumers is a creamy-white, but this is susceptible to enzymatic browning when pre-peeled and chopped. When garlic was steeped in the 25% and 50% alcohol, the browning of garlic was prevented during storage.

Blood lipid-lowering and antioxidant effects of a structured lipid containing monoacylglyceride enriched with monounsaturated fatty acids in C57BL/6 mice.

We recently reported that a synthetic edible oil-containing monoacylglyceride (MAG) and diacylglyceride (DAG) exerted anti-atherosclerotic effects. In order to further investigate the activities and individual effects of MAG and DAG on the atherosclerotic process, we prepared a structured oil with various MAG and DAG contents and tested them both in vitro and in vivo, using C57BL/6 mice. The structured oil to be tested was mixed (final concentration 5%, wt/wt) with a high-cholesterol high-fat diet (1.2% cholesterol/15% fat/0.5% sodium cholate) and provided to the mice for 7 weeks. After administration, the mice consuming MAG97%-oil and DAG50%/MAG10%-oil evidenced 17% and 24% decreases in plasma total cholesterol (TC) level, respectively, as compared to a group of mice fed on lard. The experimental mice also had reduced plasma triglyceride concentrations and elevated high-density lipoprotein-cholesterol to TC ratios, by up to 31% in the case of the DAG50%/MAG10%-oil fed mice. The mice fed on MAG97%-oil exhibited elevated plasma antioxidant activity and lecithin:cholesterol acyltransferase activity. Histological assessments of the livers of the mice showed that the consumption of MAG-containing oil attenuated the adhesion of inflammatory cells and also ameliorated fatty liver changes, as compared to what was observed in the case of DAG85%-oil consumption. In conclusion, the MAG-containing oil exhibited anti-inflammatory and antioxidant activities in vivo, as well as in vitro inhibitory activity against human cholesteryl ester transfer protein. These results provide us with new insights into MAG-containing oil in terms of hypocholesterolemic effects and antioxidant activities.

A murine model for human sepiapterin-reductase deficiency.

Tetrahydrobiopterin (BH(4)) is an essential cofactor for several enzymes, including all three forms of nitric oxide synthases, the three aromatic hydroxylases, and glyceryl-ether mono-oxygenase. A proper level of BH(4) is, therefore, necessary for the metabolism of phenylalanine and the production of nitric oxide, catecholamines, and serotonin. BH(4) deficiency has been shown to be closely associated with diverse neurological psychiatric disorders. Sepiapterin reductase (SPR) is an enzyme that catalyzes the final step of BH(4) biosynthesis. Whereas the number of cases of neuropsychological disorders resulting from deficiencies of other catalytic enzymes involved in BH(4) biosynthesis and metabolism has been increasing, only a handful of cases of SPR deficiency have been reported, and the role of SPR in BH(4) biosynthesis in vivo has been poorly understood. Here, we report that mice deficient in the Spr gene (Spr(-/-)) display disturbed pterin profiles and greatly diminished levels of dopamine, norepinephrine, and serotonin, indicating that SPR is essential for homeostasis of BH(4) and for the normal functions of BH(4)-dependent enzymes. The Spr(-/-) mice exhibit phenylketonuria, dwarfism, and impaired body movement. Oral supplementation of BH(4) and neurotransmitter precursors completely rescued dwarfism and phenylalanine metabolism. The biochemical and behavioral characteristics of Spr(-/-) mice share striking similarities with the symptoms observed in SPR-deficient patients. This Spr mutant strain of mice will be an invaluable resource to elucidate many important issues regarding SPR and BH(4) deficiencies.

Delayed occurrence of H-ras12V-induced hepatocellular carcinoma with long-term treatment with cinnamaldehydes.

Cinnamaldehyde from the bark of Cinnamomum cassia has been reported to have antitumor activity mediated by the inhibition of farnesyl transferase. We assessed in vivo the chemo-preventive effect of cinnamaldehydes on H-ras12V-induced hepatocellular carcinoma formation. A mouse model of hepatocellular carcinoma was established by using the transgene of mutated H-ras12V under the regulation of albumin enhancer/promoter. When treated with cinnamaldehyde for 10 weeks, hepatic tumor development was delayed with 2'-benzoyloxycinnamaldehyde (BCA) compared with control hepatocellular carcinoma formation. The effect of 2'-hydroxycinnamaldehyde (HCA) was comparable. The number of lesions and the size of each lesion were significantly reduced by BCA. Cell proliferation in the lesion was detected by incorporation of 5-bromo-2'-deoxyuridine (BrdU). BCA increased the number of splenocytes, concanavalin A-stimulated splenocyte proliferation and the infiltration of lymphocytes into liver. Data suggest that the delayed hepatic tumor development observed with BCA could be mediated by a long-term immunostimulating effect on T cells.

Antifatigue and antistress effect of the hot-water fraction from mycelia of Cordyceps sinensis.

This study was conducted to investigate the chemical component of the hot water (HW) fraction of mycelia of Cordyceps sinensis and its antifatigue and antistress effect against a stimulus in vivo using rats and mice. The growth of mycelia reached a maximum level of 31.6 g/l after 120 h of incubation. The main chemical composition of the HW fraction of mycelia of C. sinensis was found to be carbohydrate (78.9%) with 5% moisture. The swimming endurance capacity of mice orally administered with the HW fraction (150 and 300 mg/kg/d, respectively) was significantly prolonged from 75 to 90 min with a lessening of fatigue. When the HW fraction (150 mg/kg/d) was given to rats for 8 d including a 48 h stress period, the weight changes of the adrenal gland, spleen, thymus, and thyroid, which is an index of stress, were suppressed. The HW fraction also significantly inhibited the increase in total cholesterol and the decrease in alkaline phosphatase levels as biochemical parameters of immobilization stress in rats.

Thermal kinetics of color degradation of mulberry fruit extract.

The effects of temperature and pH on color degradation kinetics of the mulberry fruit extract were investigated. The absorbance at 510 nm was decreased with increase of heating time, but that at 420 nm was increased with the increase of heating time at 100 degrees C. The change of the browning index (A510/A420) was increased with increase of pH and was lower at pH 2.0 than that at pH 5.0. The browning index variation was adequately described by both the first-order and the zero-order kinetic. However, the zero-order kinetic model was proposed because of the better fit. According to the Arrhenius model, the activation energies for the browning index in the range of 80-100 degrees C for the four different pH values were 30.68 kJ/mol for pH 2.0, 35.87 kJ/mol for pH 3.0, 42.67 kJ/ mol for pH 4.0, and 43.49 kJ/mol for pH 5.0.