GT-11 impairs insulin signaling through modulation of sphingolipid metabolism in C2C12 myotubes.

AIMS: Sphingolipids are involved in the regulation of insulin signaling, which is linked to the development of insulin resistance, leading to diabetes mellitus. We aimed to study whether modulation of sphingolipid levels by GT-11 may regulate insulin signaling in C2C12 myotubes. MAIN METHODS: We investigated the effects of sphingolipid metabolism on Akt phosphorylation and glucose uptake using C2C12 myotubes. Either GT-11, an inhibitor of dihydroceramide desaturase 1 and S1P lyase, or siRNA targeting Sgpl1, the gene encoding the enzyme, was employed to determine the effect of sphingolipid metabolism modulation on insulin signaling. Western blotting and glucose uptake assays were used to evaluate the effect of treatments on insulin signaling. Sphingolipid metabolites were analyzed by high performance liquid chromatography (HPLC). KEY FINDINGS: Treatment with GT-11 resulted in decreased Akt phosphorylation and reduced glucose uptake. Silencing the Sgpl1 gene, which encodes S1P lyase, mimicked these findings, suggesting the potential for regulating insulin signaling through S1P lyase modulation. GT-11 modulated sphingolipid metabolism, inducing the accumulation of sphingolipids. Using PF-543 and ARN14974 to inhibit sphingosine kinases and acid ceramidase, respectively, we identified a significant interplay between sphingosine, S1P lyase, and insulin signaling. Treatment with either exogenous sphingosine or palmitic acid inhibited Akt phosphorylation, and reduced S1P lyase activity. SIGNIFICANCE: Our findings highlight the importance of close relationship between sphingolipid metabolism and insulin signaling in C2C12 myotubes, pointing to its potential therapeutic relevance for diabetes mellitus.

Ginseng Saponin Enriched in Rh1 and Rg2 Ameliorates Nonalcoholic Fatty Liver Disease by Inhibiting Inflammasome Activation.

Nonalcoholic fatty liver disease (NAFLD) is becoming one of the most common chronic liver diseases in the world. One of the features of NAFLD is hepatic fat accumulation, which further causes hepatic steatosis, fibrosis, and inflammation. Saponins, the major pharmacologically active ingredients isolated from Panax notoginseng, contain several ginsenosides, which have various pharmacological and therapeutic functions. However, the ginsenoside-specific molecular mechanism of saponins in NAFLD remains unknown. This study aimed to elucidate the effects of ginseng saponin extract and its ginsenosides on hepatic steatosis, fibrosis, and inflammation and their underlying action mechanism in NAFLD. Mice were fed a fast food diet (FFD) for 16 weeks to induce NAFLD and then treated with saponin extract (50 or 150 mg/kg) for the remaining nine weeks to determine the effects of saponin on NAFLD. Saponin extract administration significantly alleviated FFD-induced hepatic steatosis, fibrosis, and inflammation. Particularly, saponin extract, compared with conventional red ginseng, contained significantly increased amounts of ginsenosides (Rh1 (10.34-fold) and Rg2 (7.1-fold)). In vitro Rh1 and Rg2 treatments exerted an anti-steatotic effect in primary hepatocytes, an antifibrotic effect in hepatic stellate cells, and anti-inflammatory and pro-mitophagy effects in immortalized mouse Kupffer cells. Mechanistically, saponin extract alleviated lipopolysaccharide-induced NLRP3 inflammasome activation by promoting mitophagy. In conclusion, saponin extract inhibited inflammation-mediated pathological inflammasome activation in macrophages, thereby preventing NAFLD development. Thus, saponin extract administration may be an alternative method for NAFLD prevention.

Fumonisin B1-Induced Toxicity Was Not Exacerbated in Glutathione Peroxidase-1/Catalase Double Knock Out Mice.

Fumonisin B1 (FB1) structurally resembles sphingolipids and interferes with their metabolism leading to sphingolipid dysregulation. We questioned if FB1 could exacerbate liver or kidney toxicities in glutathione peroxidase 1 (Gpx1) and catalase (Cat) knockout mice. While higher serum levels of thiobarbituric acid reactive substances (TBARS) and sphinganine (Sa) were measured in Gpx1/Cat knockout mice (Gpx1/Cat KO) than wild type mice after 5 days of FB1 treatment, serum levels of alanine aminotransferase (ALT), sphingosine-1 phosphate (So-1-P), and sphinganine-1 phosphate (Sa-1-P) were found to be relatively low. Although Sa was highly elevated in Gpx1/Cat KO mice and wild mice, lower levels of So and Sa were found in both the kidney and liver tissues of Gpx/Cat KO mice than wild type mice after FB1 treatment. Paradoxically, FB1-induced cellular apoptosis and necrosis were hastened under oxidative stress in Gpx1/Cat KO mice.

Fermented Korean Red Ginseng Extract Enriched in Rd and Rg3 Protects against Non-Alcoholic Fatty Liver Disease through Regulation of mTORC1.

The fermentation of Korean red ginseng (RG) increases the bioavailability and efficacy of RG, which has a protective role in various diseases. However, the ginsenoside-specific molecular mechanism of the fermented RG with Cordyceps militaris (CRG) has not been elucidated in non-alcoholic fatty liver disease (NAFLD). A mouse model of NAFLD was induced by a fast-food diet (FFD) and treated with CRG (100 or 300 mg/kg) for the last 8 weeks. CRG-mediated signaling was assessed in the liver cells isolated from mice. CRG administration significantly reduced the FFD-induced steatosis, liver injury, and inflammation, indicating that CRG confers protective effects against NAFLD. Of note, an extract of CRG contains a significantly increased amount of ginsenosides (Rd and Rg3) after bioconversion compared with that of conventional RG. Moreover, in vitro treatment with Rd or Rg3 produced anti-steatotic effects in primary hepatocytes. Mechanistically, CRG protected palmitate-induced activation of mTORC1 and subsequent inhibition of mitophagy and PPARα signaling. Similar to that noted in hepatocytes, CRG exerted anti-inflammatory activity through mTORC1 inhibition-mediated M2 polarization. In conclusion, CRG inhibits lipid-mediated pathologic activation of mTORC1 in hepatocytes and macrophages, which in turn prevents NAFLD development. Thus, the administration of CRG may be an alternative for the prevention of NAFLD.

Amelioration of Hyperglycemia-Induced Nephropathy by 3,3'-Diindolylmethane in Diabetic Mice.

Type 1 diabetes mellitus (insulin-dependent diabetes) is characterized by hyperglycemia caused by an insulin deficiency. Diabetic nephropathy is a major complication of hyperglycemia. 3,3'-diindolylmethane (DIM)-a natural compound produced from indole-3-carbinol, found in cruciferous vegetables-enhances glucose uptake by increasing the activation of the insulin signaling pathway in 3T3-L1 adipocytes. In this study, we investigated whether DIM could improve insulin-dependent diabetes and nephropathy in streptozotocin (STZ)-induced diabetic mice. In mice, STZ induced hyperglycemia, hunger, thirst, and abnormally increased kidney weight and serum creatinine, which is a renal functional parameter. DIM decreased STZ-increased high blood glucose levels and food and water intake in diabetic mice. DIM also improved diabetic nephropathy by inhibiting the expression of PKC-α, the marker of albuminuria, and TGF-ß1, an indicator of renal hypertrophy, in diabetic mice. Our findings suggest that DIM may ameliorate hyperglycemia and diabetic nephropathy through the inhibition of PKC-α and TGF-ß1 signaling.

3,3'-Diindolylmethane Enhances Glucose Uptake Through Activation of Insulin Signaling in 3T3-L1 Adipocytes.

OBJECTIVE: Indole-3-carbinol (I3C), a naturally occurring compound found in cruciferous vegetables, and its metabolite 3,3'-diindolylmethane (DIM) reduce body mass and serum glucose levels in high-fat-diet-induced obese mice. This study aimed to determine whether I3C or DIM could increase glucose uptake via enhanced insulin sensitivity in 3T3-L1 adipocytes, as well as the mechanism involved. METHODS: 3T3-L1 preadipocytes were differentiated by using a mixture of adipogenic inducers, including a suboptimal concentration of insulin. RESULTS: DIM, but not I3C, increased adipocyte differentiation through upregulation of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α. DIM also enhanced glucose uptake by increasing expression of glucose transporter 4 in adipocytes. This was associated with DIM-enhanced phosphorylation of the signaling intermediates Akt, insulin receptor substrate-1, and insulin receptor early in differentiation. CONCLUSIONS: Our findings suggest that DIM may improve insulin sensitivity through the activation of the insulin signaling pathway, leading to enhanced glucose uptake.

Enhancement of Glucose Uptake by Meso-Dihydroguaiaretic Acid through GLUT4 Up-Regulation in 3T3-L1 Adipocytes.

Type 2 diabetes is characterized by insulin resistance, which leads to increased blood glucose levels. Adipocytes are involved in the development of insulin resistance, resulting from the dysfunction of the insulin signaling pathway. In this study, we investigated whether meso-dihydroguaiaretic acid (MDGA) may modulate glucose uptake in adipocytes, and examined its mechanism of action. MDGA enhanced adipogenesis through up-regulation of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α in 3T3-L1 adipocytes partially differentiated with sub-optimal concentrations of insulin. MDGA also increased glucose uptake by stimulating expression and translocation of glucose transporter 4 (GLUT4) in adipocytes. These results suggest that MDGA may increase GLUT4 expression and its translocation by promoting insulin sensitivity, leading to enhanced glucose uptake.

Activated Natural Killer Cells Mediate the Suppressive Effect of Interleukin-4 on Tumor Development via STAT6 Activation in an Atopic Condition Melanoma Model.

A protective effect of allergy for cancer has been suggested, but the results are somewhat conflicting, and the mechanism remains elusive. Interleukin-4 (IL-4) signaling has been identified as a potentially important pathway in the development of allergies and the suppression of cancer development. To evaluate the allergy responses in IL-4-mediated tumor development, we compared the growth of B16F10 melanoma cells in 4% phthalic anhydride (PA)-treated IL-4/Luc/CNS-1 transgenic mice (IL-4 mice) and acetone-olive oil (AOO)-treated IL-4 mice as a control for 3 weeks. Much higher allergic responses and natural killer (NK) and STAT6 activation were found in PA-treated IL-4 mice compared with AOO-treated IL-4 control mice. Tumor volume and weight showed an inverse association with the higher allergic response and were significantly reduced in the PA-treated IL-4 mice when compared with those of AOO-treated IL-4 control mice. Significantly higher activation of STAT6, as well as IL-4 and NK cell activation, was found in the tumor tissues of PA-treated IL-4 mice. Infiltration of immune cells and cytokine levels were also higher in the tumor tissues of PA-treated IL-4 mice. We further found that IL-4-activated NK-92MI cells showed increased anticancer effects in human melanoma cells. Overall, these results showed that allergy responses further accelerated the IL-4-induced inhibition of tumor development through the activation of STAT6 pathways.

Inhibition of Ceramide Decreased the Expression of ATP-Binding Cassette Transporter G5/8 mRNA in an Animal Model of Cholesterol Gallstone.

BACKGROUND: The increased risk of gallstone has been reported in patients with ATP-binding cassette (ABC) transporter polymorphism. The half-transporters ABCG5 and ABCG8 mediate the efflux of cholesterol in hepatocytes and the intestine. We investigated whether ceramide plays a role in cholesterol efflux through the ABC transporters. METHODS: Six-week-old C57BL/6J mice were assigned to 3 groups. The normal group (n = 5) was fed a normal chow diet, the cholesterol group (n = 10) was fed a lithogenic diet, and the myriocin group (n = 15) was fed the lithogenic diet and myriocin, a specific inhibitor of serine-palmitoyl transferase. After 6 weeks, the ABCG5 and ABCG8 transporters were analyzed. RESULTS: The rate of cholesterol gallstone formation in cholesterol group was also higher than that in normal and myriocin groups (0, 70, and 40%, respectively). ABCG5 and ABCG8 mRNA levels were significantly increased in cholesterol group and less increased in myriocin group, relative to that in normal group (p < 0.05). CONCLUSIONS: The inhibition of ceramide biosynthesis by myriocin suppressed gallstone formation and ABCG5/8 mRNA expression. We expect that ceramide's role as a regulator of the ABCG5/8 transporter might be linked to cholesterol gallstone formation.

Dodeca-2(E),4(E)-dienoic acid isobutylamide enhances glucose uptake in 3T3-L1 cells via activation of Akt signaling.

Dodeca-2(E),4(E)-dienoic acid isobutylamide (DDI), an alkamide derived from the plant Echinacea purpurea, promotes adipocyte differentiation and activates peroxisome proliferator-activated receptor γ, which is associated with enhanced insulin sensitivity. In the present study, we investigated whether DDI may increase glucose uptake through activation of the insulin signaling pathway in 3T3-L1 adipocytes. DDI increased insulin-stimulated glucose uptake, and expression and translocation of glucose transporter 4 in adipocytes treated with sub-optimal levels of insulin. Additionally, DDI enhanced Akt phosphorylation, whereas phosphoinositide 3-kinase/Akt inhibitors suppressed DDI-induced glucose uptake. These results suggest that DDI may improve insulin sensitivity through the activation of Akt signaling, which leads to enhanced glucose uptake.

(-)-Epigallocatechin-3-O-gallate (EGCG) attenuates the hemodynamics stimulated by caffeine through decrease of catecholamines release.

A human study of the effects on hemodynamics of caffeine and epigallocatechin-3-O-gallate (EGCG) was performed. Caffeine tablets (200 mg) were orally administered to healthy males aged between 25 and 35 years 30 min after oral administration of EGCG tablets (100 and 200 mg). The increase in BP induced by caffeine was inhibited when co-administrated with EGCG. We found that caffeine slightly decreased heart rate (HR) in the volunteers. Although EGCG enhanced HR reduction, the effect was not significant. In addition, caffeine increased blood catecholamine levels, but EGCG inhibited the increase in noradrenaline, adrenaline and dopamine levels induced by caffeine. Whether EGCG decreases the elevated HR and systolic perfusion pressure, and ventricular contractility induced by adrenergic agonists in the isolated rat heart was investigated. The modified Krebs-Henseleit solution was perfused through a Langendorff apparatus to the isolated hearts of rats. HR, systolic perfusion pressure, and developed maximal rates of contraction (+dP/dtmax) and relaxation (-dP/dtmax) were increased by epinephrine (EP) and isoproterenol (IP). In contrast, EGCG decreased the elevated HR, systolic perfusion pressure, and left ventricular ±dp/dtmax induced by EP and/or IP. In conclusion, EGCG could attenuate the hemodynamics stimulated by caffeine through decreasing catecholamine release.

Anti-cancer effect of N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxamide, a novel synthetic compound.

Naphthofuran compounds have been known to regulate HNF 4α which is associated with proliferation, progression and metastasis of HCC. In this study, we investigated whether N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxamide (NHDC), a novel synthetic naphthofuran compound inhibits liver tumor growth through activation of HNF 4α. Treatment with different concentrations (1-10.8 µM) of NHDC for various periods (0-72 h) inhibited liver cancer cells (HepG2, Hep3B) growth as well as colony formation followed by induction of apoptosis in a concentration dependent manner. NHDC also induced expression of the apoptosis regulating genes as well as inhibiting the action of STAT3. These inhibitory effects were associated with enhancement of expression and DNA binding activity of HNF 4α. In vivo study confirmed that liver tumor growth was prevented with NHDC (5 mg/kg), and its effect was also related with inhibition of STAT3 pathway through enhancement of expression and DNA binding activity of HNF 4α. Moreover, siRNA of HNF 4α abolished NHDC-induced cell growth inhibition as well as DNA binding activity and phosphorylation of STAT3. Pull down assay docking prediction analysis proved that NHDC directly binds to hydrophobic fatty acid ligand binding site of HNF 4α. A novel naphthofuran compound, NHDC inhibited liver tumor growth by inactivating of STAT3 through direct biding to HNF 4α.

Anti-Obesity Effect of 6,8-Diprenylgenistein, an Isoflavonoid of Cudrania tricuspidata Fruits in High-Fat Diet-Induced Obese Mice.

Obesity, which is characterized by excessive fat accumulation, is associated with several pathological disorders, including metabolic diseases. In this study, the anti-obesity effect of 6,8-diprenylgenistein (DPG), a major isoflavonoid of Cudrania tricuspidata fruits was investigated using high fat-diet (HFD)-induced obese mice at the doses of 10 and 30 mg/kg for six week. The body weight of the DPG-treated groups was significantly lower compared to the HFD-treated group. In addition, fat accumulation in epididymal adipose tissue and liver was dramatically decreased in the HFD + DPG groups. The food efficiency ratios of the HFD + DPG groups were also lower compared to the HFD group with the same food intake. Metabolic parameters that had increased in the HFD group were decreased in the HFD + DPG groups. Further studies demonstrate that DPG efficiently reduces lipogenic genes by regulation of transcription factors, such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), and hormones, such as leptin and adiponection. DPG also regulates acetyl-CoA carboxylase (ACC) and hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) by AMP-activated protein kinase (AMPK) activation. Taken together, DPG is beneficial for the regulation of obesity, especially resulting from high fat intake.

Bee venom inhibits growth of human cervical tumors in mice.

We studied whether bee venom (BV) inhibits cervical tumor growth through enhancement of death receptor (DR) expressions and inactivation of nuclear factor kappa B (NF-κB) in mice. In vivo study showed that BV (1 mg/kg) inhibited tumor growth. Similar inhibitory effects of BV on cancer growth in primary human cervical cancer cells were also found. BV (1-5 µg/ml) also inhibited the growth of cancer cells, Ca Ski and C33Aby the induction of apoptotic cell death in a dose dependent manner. Agreed with cancer cell growth inhibition, expression of death receptors; FAS, DR3 and DR6, and DR downstream pro-apoptotic proteins including caspase-3 and Bax was concomitantly increased, but the NF-κB activity and the expression of Bcl-2 were inhibited by treatment with BV in tumor mice, human cancer cell and human tumor samples as well as cultured cancer cells. In addition, deletion of FAS, DR3 and DR6 by small interfering RNA significantly reversed BV-induced cell growth inhibitory effects as well as NF-κB inactivation. These results suggest that BV inhibits cervical tumor growth through enhancement of FAS, DR3 and DR6 expression via inhibition of NF-κB pathway.

Interleukin-32γ attenuates ethanol-induced liver injury by the inhibition of cytochrome P450 2E1 expression and inflammatory responses.

Alcohol abuse and alcoholism lead to alcoholic liver disease (ALD), which is a major type of chronic liver disease worldwide. Interleukin-32 (IL-32) is a novel cytokine involved in inflammation and cancer development. However, the role of IL-32 in chronic liver disease has not been reported. In the present paper, we tested the effect of IL-32γ on ethanol-induced liver injury in IL-32γ-overexpressing transgenic mice (IL-32γ mice) after chronic ethanol feeding. Male C57BL/6 and IL-32γ mice (10-12 weeks old) were fed on a Lieber-DeCarli diet containing 6.6% ethanol for 6 weeks. IL-32γ-transfected HepG2 and Huh7 cells, as well as primary hepatocytes from IL-32γ mice, were treated with or without ethanol. The hepatic steatosis and damage induced by ethanol administration were attenuated in IL-32γ mice. Ethanol-induced cytochrome P450 2E1 expression and hydrogen peroxide levels were decreased in the livers of IL-32γ mice, primary hepatocytes from IL-32γ mice and IL-32γ-overexpressing human hepatic cells. The ethanol-induced expression levels of cyclo-oxygenase-2 (COX-2) and IL-6 were reduced in the livers of IL-32γ mice. Because nuclear transcription factor κB (NF-κB) is a key redox transcription factor of inflammatory responses, we examined NF-κB activity. Ethanol-induced NF-κB activities were significantly lower in the livers of IL-32γ mice than in wild-type (WT) mice. Furthermore, reduced infiltration of natural killer cells, cytotoxic T-cells and macrophages in the liver after ethanol administration was observed in IL-32γ mice. These data suggest that IL-32γ prevents ethanol-induced hepatic injury via the inhibition of oxidative damage and inflammatory responses.

Memory Impairment in Estrogen Receptor α Knockout Mice Through Accumulation of Amyloid-ß Peptides.

Estrogen has been known to reduce the development of Alzheimer's disease (AD). However, exact mechanisms are not clear. We investigated whether estrogen can increase amyloid-beta (Aß) degradation and affects Aß-induced memory impairment in an estrogen deficiency model. Estrogen receptor alpha (ERα) knockout mice and wild-type mice were intracerebroventricular (ICV) infused with Aß (300 pmol) for 2 weeks. Cognitive function was then assessed by the Morris water maze test and passive avoidance test. In addition, Western blot analysis, immunostaining, immunofluorescence staining, ELISA, and enzyme activity assays were used to examine the degree of Aß deposition in the brains of ERα knockout mice. In our present study, Aß was accumulated more in the ERα knockout mice brain and greatly worsened memory impairment and glial activation as well as neurogenic inflammation. These results suggest that estrogen may protect memory impairment by stimulating the degradation of Aß and down-regulate neurogenic inflammation as well as amyloidogenesis.

Quantification of 4-methylimidazole in carbonated beverages by ultra-performance liquid chromatography-tandem mass spectrometry.

2- and 4-methylimidazoles (2-MI and 4-MI) are undesired byproducts produced during the manufacture of caramel color used to darken food products such as carbonated beverages. The Office of Environmental Health Hazard Assessment in California listed 4-MI as carcinogen in January 2011 with a proposed no significant risk level at 29 µg per person per day. Thus, a quantitative analytical measurement for 2-MI and 4-MI is desired for reliable risk assessments for exposure. An ultra-performance liquid chromatography (UPLC) coupled tandem mass spectrometric (MS/MS) method was developed for the quantification of 4-MI in beverage samples. Chromatographic separation of 2-MI and 4-MI were achieved by using a PFP reversed-phase column and a stepwise gradient of methanol and distilled water containing 0.1 % formic acid. Identification and quantification of 2-MI and 4-MI were performed using electrospray ionization-tandem mass monitoring the precursor to product ion transitions for 2-MI at m/z 83.1 → 42.2 and 4-MI at m/z 83.1 → 56.1 with melamine at m/z 127.1 → 85.1 as the internal standard. The performance of the method was evaluated against validation parameters such as specificity, carryover, linearity and calibration, correlation of determination (r(2)), detection limit, precision, accuracy, and recovery. Calibration curves at 10-400 ng/mL were constructed by plotting concentration versus peak-area ratio (analyte/internal standard) and fitting the data with a weighted 1/x. The accuracy of the assay ranged from 93.58 to 110.53 % for all analytes. Intra-assay precision for 2-MI and 4-MI were below 7.28 (relative standard deviation/RSD %) at QC samples. Here we present a new and improved method using UPLC-MS/MS to significantly simplify sample preparation and decrease chromatographic run time. This method allows accurate and reproducible quantification of 4-MI in carbonated beverages as low as sub ng/mL (ppb) levels.

Ginsenoside compound K inhibits angiogenesis via regulation of sphingosine kinase-1 in human umbilical vein endothelial cells.

Ginsenoside compound K (CK) is a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer (Araliaceae), has long been used to treat against the development of cancer, inflammation, allergies, and diabetes. This study examined the anti-angiogenic properties of CK against sphingosine 1-phosphate (S1P)-induced cell migration via regulation of sphingosine kinase 1 (SPHK1) in human umbilical vein endothelial cells (HUVEC). Studies on S1P-induced cell migration, expression of SPHK1 and MMPs and analysis of sphingolipid metabolites by LC-MS/MS were examined after the treatment of CK (2.5, 5, 10 µg/mL) in HUVEC. S1P produced by SPHK1 is also involved in cell growth, migration, and protection of apoptosis; therefore, we sought to investigate whether ginsenosides are able to regulate SPHK1. For this purpose, we developed an inhibitory assay of SPHK1 activity and an analytical method for detection of S1P and other sphingolipid metabolites in HUVEC. Ginsenoside CK inhibited 100 nM S1P-induced cell migrations in a dose-dependent manner. Among tested ginsenosides, CK exclusively inhibited S1P production, SPHK1 activity and SPHK1 expression in HUVEC, whereas expression of the pro-apoptotic sphingolipids, sphingosine and ceramide, was increased in response to CK. The major subspecies of the increased ceramide was C24:0-ceramide. CK also disrupted the sphingolipid rheostat, which ultimately influences cell fate, and dose-dependently inhibited HUVEC migration by reducing expression of metalloproteinases (MMPs). Ginsenoside CK acts as a unique HUVEC migration inhibitor by regulating MMP expression, as well as the activity of SPHK1 and its related sphingolipid metabolites.

Anti-cancer effect of thiacremonone through down regulation of peroxiredoxin 6.

Thiacremonone (2, 4-dihydroxy-2, 5-dimethyl-thiophene-3-one) is an antioxidant substance as a novel sulfur compound generated from High-Temperature-High-Pressure-treated garlic. Peroxiredoxin 6 (PRDX6) is a member of peroxidases, and has glutathione peroxidase and calcium-independent phospholipase A2 (iPLA2) activities. Several studies have demonstrated that PRDX6 stimulates lung cancer cell growth via an increase of glutathione peroxidase activity. A docking model study and pull down assay showed that thiacremonone completely fits on the active site (cys-47) of glutathione peroxidase of PRDX6 and interacts with PRDX6. Thus, we investigated whether thiacremonone inhibits cell growth by blocking glutathione peroxidase of PRDX6 in the human lung cancer cells, A549 and NCI-H460. Thiacremonone (0-50 µg/ml) inhibited lung cancer cell growth in a concentration dependent manner through induction of apoptotic cell death accompanied by induction of cleaved caspase-3, -8, -9, Bax, p21 and p53, but decrease of xIAP, cIAP and Bcl2 expression. Thiacremonone further inhibited glutathione peroxidase activity in lung cancer cells. However, the cell growth inhibitory effect of thiacremonone was not observed in the lung cancer cells transfected with mutant PRDX6 (C47S) and in the presence of dithiothreitol and glutathione. In an allograft in vivo model, thiacremonone (30 mg/kg) also inhibited tumor growth accompanied with the reduction of PRDX6 expression and glutathione peroxidase activity, but increased expression of cleaved caspase-3, -8, -9, Bax, p21 and p53. These data indicate that thiacremonone inhibits tumor growth via inhibition of glutathione peroxidase activity of PRDX6 through interaction. These data suggest that thiacremonone may have potentially beneficial effects in lung cancer.

Myriocin induces apoptotic lung cancer cell death via activation of DR4 pathway.

It has been known that myriocin inhibits melanoma growth. However, the effects and action mechanisms of myriocin on lung cancer cell growth have not been reported. In this study, we examined whether myriocin isolated from Mycelia sterilia inhibits cell growth of lung cancer cells (A549 and NCI-H460) as well as possible signaling pathways involved in cell growth inhibition. Different concentrations of myriocin inhibited the growth of lung cancer cells through the induction of apoptotic cell death. Consistent with cancer cell growth inhibition, myriocin induced the expression of death receptors (DRs) as well as p-JNK and p-p38 in both cell lines. Moreover, the combination of myriocin with DR4 ligand TRAIL, and other well known anti-tumor drugs (docetaxel and cisplatin) synergistically inhibited cancer cell growth, and induced DR4 expression. These results showed that myriocin inhibits lung cancer cells growth through apoptosis via the activation of DR4 pathways, and enhanced anti-cancer effects with well known drugs. Thus, our study indicates that myriocin could be effective for lung cancer cells as an anti-cancer drug and/or a conjunction agent with well known anti-cancers.