Epigallocatechin-3-gallate (EGCG) inhibits the migratory behavior of tumor bronchial epithelial cells

Background

Many studies associated the main polyphenolic constituent of green tea, (-)-Epigallocatechin-3-gallate (EGCG), with inhibition of cancers, invasion and metastasis. To date, most of the studies have focused on the effect of EGCG on cell proliferation or death. Since cell migration is an important mechanism involved in tumor invasion, the aim of the present work was to target another approach of the therapeutic effect of EGCG, by investigating its effect on the cell migratory behavior.

Methods

The effect of EGCG (at concentrations lower than 10 μg/ml) on the migration speed of invasive cells was assessed by using 2D and 3D models of cell culture. We also studied the effects of EGCG on proteinases expression by RT-PCR analysis. By immunocytochemistry, we analyzed alterations of vimentin organization in presence of different concentrations of EGCG.

Results

We observed that EGCG had an inhibitory effect of cell migration in 2D and 3D cell culture models. EGCG also inhibited MMP-2 mRNA and protein expression and altered the intermediate filaments of vimentin.

Conclusion

Taken together, our results demonstrate that EGCG is able to inhibit the migration of bronchial tumor cells and could therefore be an attractive candidate to treat tumor invasion and cell migration.     

CPT I overexpression protects L6E9 muscle cells from fatty acid-induced insulin resistance

Oversupply of lipids to skeletal muscle causes insulin resistance by promoting the accumulation of lipid-derived metabolites that inhibit insulin signaling. In this study, we tested the hypothesis that overexpression of carnitine palmitoyltransferase I (CPT I) could protect myotubes from fatty acid-induced insulin resistance by reducing lipid accumulation in the muscle cell. Incubation of L6E9 myotubes with palmitate caused accumulation of triglycerides, diacylgycerol, and ceramide, produced an activation of PKCtheta and PKCzeta, and blocked insulin-stimulated glucose metabolism, reducing insulin-stimulated PKB activity by 60%. Transduction of L6E9 myotubes with adenoviruses encoding for liver CPT I (LCPT I) wild-type (WT), or a mutant form of LCPT I (LCPT I M593S), which is insensitive to malonyl-CoA, produced a twofold increase in palmitate oxidation when LCPT I activity was increased threefold. LCPT I WT and LCPT I M593S-overexpressing L6E9 myotubes showed normal insulin-stimulated glucose metabolism and an improvement in PKB activity when pretreated with palmitate. Moreover, LCPT I WT- and LCPT I M593S-transduced L6E9 myotubes were protected against the palmitate-induced accumulation of diacylglycerol and ceramide and PKCtheta and -zeta activation. These results suggest that LCPT I overexpression protects L6E9 myotubes from fatty acid-induced insulin resistance by inhibiting both the accumulation of lipid metabolites and the activation of PKCtheta and PKCzeta.     

Pioglitazone has direct effects on insulin sensitivity and intracellular lipid content in L6 skeletal muscle cells

Using L6 skeletal muscle cell line, rendered insulin resistant by incubation with triglyceride-rich lipoproteins (TGRLs), we sought to answer the question whether pioglitazone has direct effects on this cell line. Incubation of L6 cells with TGRLs led to an increase in the intramyocellular triglyceride content. Moreover, TGRLs led to a reduction in insulin-stimulated glycogen content and GSK-3 phosphorylation. All these changes induced by TGRLs could be antagonized by incubation of L6 cells with pioglitazone. The PPAR-γ antagonist GW9662 reversed the pioglitazone effects. We conclude that pioglitazone has direct insulin-sensitizing effects on the L6 skeletal muscle cell line, which are paralleled by a reduction in intramyocellular triglyceride accumulation.     

Protein arginine methylation regulates insulin signaling in L6 skeletal muscle cells

Protein N-arginine methyltransferase (PRMT)1 catalyzes arginine methylation in a variety of substrates, although the potential role of PRMT1 in insulin action has not been defined. We therefore investigated the effect of PRMT1-mediated methylation on insulin signaling and glucose uptake in skeletal L6 myotubes. Exposure of L6 myotubes to insulin rapidly induced translocation of PRMT1 and increased its catalytic activity in membrane fraction. Several proteins in the membrane fraction were arginine-methylated after insulin treatment, which were inhibited by pretreatment with an inhibitor of methyltransferase, 5′-deoxy-5′-(methylthio)adenosine (MTA), or a small interfering RNA against PRMT1 (PRMT1-siRNA). Inhibition of arginine methylation with MTA or PRMT1-siRNA diminished later phase of insulin-stimulated tyrosine phosphorylation of insulin receptor (IR) β and IRS-1, association of IRS-1 with p85α subunit of PI3-K, and glucose uptake. Our results suggest that PRMT1-mediated methylation serves as a positive modulator of IR/IRS-1/PI3-K pathway and subsequent glucose uptake in skeletal muscle cells.     

Epigallocatechin-3-gallate (EGCG) as a pro-osteogenic agent to enhance osteogenic differentiation of mesenchymal stem cells from human bone marrow: an in vitro study

The proliferation and osteogenic capacity of mesenchymal stem cells (MSCs) needs to be improved for their use in cell-based therapy for osteoporosis. (?)-Epigallocatechin-3-gallate (EGCG), one of the green tea catechins, has been widely investigated in studies of osteoblasts and osteoclasts. However, no consensus on its role as an osteogenic inducer has been reached, possibly because of the various types of cell lines examined and the range of concentrations of EGCG used. In this study, the osteogenic effects of EGCG are studied in primary human bone-marrow-derived MSCs (hBMSCs) by detecting cell proliferation, alkaline phosphatase (ALP) activity and the expression of relevant osteogenic markers. Our results show that EGCG has a strong stimulatory effect on hBMSCs developing towards the osteogenic lineage, especially at a concentration of 5 μM, as evidenced by an increased ALP activity, the up-regulated expression of osteogenic genes and the formation of bone-like nodules. Further exploration has indicated that EGCG directes osteogenic differentiation via the continuous up-regulation of Runx2. The underlying mechanism might involve EGCG affects on osteogenic differentiation through the modulation of bone morphogenetic protein-2 expression. EGCG has also been found to promote the proliferation of hBMSCs in a dose-dependent manner. This might be associated with its antioxidative effect leading to favorable amounts of reactive oxygen species in the cellular environment. Our study thus indicates that EGCG can be used as a pro-osteogenic agent for the stem-cell-based therapy of osteoporosis.     

Characteristics of insulin receptors in the heart muscle Binding of insulin to isolated muscle cells from adult rat heart

Adult rat heart muscle cells obtained by perfusion of the heart with collagenase have been used to characterize the insulin receptors by equilibrium binding and kinetic measurements. Binding of ¹²⁵I-labelled insulin to heart cells exhibited a high degree of specificity; it was dependent on pH and temperature, binding at steady increased with decreasing temperatures. About 70% of the radioactivity bound at equilibrium at 25°C could be dissociated by addition of an excess of unlabelled insulin. 54 and 40% of ¹²⁵I-labelled insulin was degraded by isolated heart cells after 2 h at 37°C and 4 h at 25°C, respectively. This degrading activity was effectively inhibited by high concentration of albumin.Equilibrium binding studies were conducted at 25°C using insulin concentrations ranging from 2.5 · 10^?11 mol/l to 10^?6 mol/l. Scatchard analysis of the binding data resulted in a curvilinear plot (concave upward), which was further analyzed using the average affinity profile. The empty site affinity constant was calculated to be 9.5 · 10⁷ l/mol with a total receptor concentration of 3.4 · 10⁶ sites per cell.The presence of site-site interactions of the negative cooperative type among the insulin receptors has been confirmed by kinetic experiments. The rate of dilution induced dissociation was enhanced in the presence of native insulin (5 · 10^?9 mol/l), both, under conditions of low and high fractional saturation of receptors.     

Deteriorating insulin resistance due to WL15 peptide from cysteine and glycine-rich protein 2 in high glucose-induced rat skeletal muscle L6 cells

This study investigates the antioxidant and antidiabetic activity of the WL15 peptide derived from Channa striatus on regulating the antioxidant property in the rat skeletal muscle cell line (L6) and enhancing glucose uptake via glucose metabolism. Increased oxidative stress plays a major role in the development of diabetes and its complications. Strategies are needed to mitigate the oxidative stress that can reduce these pathogenic processes. Our results showed that with treatment with WL15 peptide, the reactive oxygen species significantly decreased in L6 myotubes in a dose-dependent manner, and increased antioxidant enzymes help to prevent the formation of lipid peroxidation in L6 myotubes. The cytotoxicity of WL15 is evaluated in the L6 cells and found to be non-cytotoxic at the tested concentration. Also, for the analysis of glucose uptake activity in L6 cells, the 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxy- d -glucose assay was performed in the presence of wortmannin and genistein inhibitors. WL15 demonstrated antidiabetic activities through a dose-dependent increase in glucose uptake (64%) and glycogen storage (7.8 mM). The optimal concentration for the maximum activity was found to be 50 µM. In addition, studies of gene expression in L6 myotubes demonstrated upregulation of antioxidant genes and genes involved in the pathway of insulin signaling. In cell-based assays, WL15 peptide decreased intracellular reactive oxygen species levels and demonstrated insulin mimic activity by enhancing the primary genes involved in the insulin signaling pathway by increased glucose uptake indicating that glucose transporter type 4 (GLUT4) is regulated from the intracellular pool to the plasma membrane.     

Detection of the GLUT3 facilitative glucose transporter in rat L6 muscle cells: regulation by cellular differentiation, insulin and insulin-like growth factor-I.

The GLUT3 facilitative glucose transporter protein was found to be expressed in rat L6 muscle cells. It was detected at both the myoblast and myotube stage. GLUT3 protein content per mg of total membrane protein increased significantly during L6 cell differentiation. Subcellular fractionation demonstrated that the GLUT3 protein was predominantly localized in plasma membrane-enriched fractions of either myoblasts or myotubes. Short-term exposure of L6 myotubes to IGF-I or insulin caused a redistribution of GLUT3 protein from an intracellular membrane fraction to the plasma membrane, without affecting total membrane GLUT3 protein content. Long-term exposure of L6 myotubes to IGF-I produced an increase of GLUT3 protein in total membranes and all subcellular membrane fractions, especially the plasma membrane. We propose that the GLUT3 glucose transporter may play an important role in glucose metabolism in developing muscle.     

Shifts in phospholipid composition of the nuclear matrix in rat liver cells exposed to insulin

It was shown that the total content of phospholipids of rat liver nuclear matrix increased after in vivo action of insulin (2 units per 100 g of animal weight). At the same time the sphyngomyelin content was increased and the content of phosphatydilcholine and phoshpatydilinositol was decreased significantly under the hormone action. The decreasing of monophosphoinositides amount was accompanied by the increasing of triphosphoinositides content which indicates the redistribution among the phosphoinositides fractions under the hormone action and may be the result of insulin initiation of phosphoinositide pathway in nuclear membrances.     

Epigallocatechin-3-gallate inhibits interleukin-6- and angiotensin II-induced production of C-reactive protein in vascular smooth muscle cells

AimsExtensive research suggests that atherosclerosis is an inflammatory disease and that epigallocatechin-3-gallate (EGCG) is able to inhibit the formation and development of atherosclerosis. However, the mechanisms of action of EGCG against atherosclerosis are still unclear. Therefore, the effect of EGCG on interleukin-6 (IL-6)- and angiotensin II (Ang II)-induced CRP production in vascular smooth muscle cells (VSMCs) was studied to provide experimental evidence for its anti-inflammatory and anti-atherosclerotic actions.Main methodsRat VSMCs were cultured, and IL-6 (10^? 7 M) and Ang II (10^? 7 M) were used as stimulants for CRP generation. The CRP concentration in the supernatant was measured with ELISA, and mRNA and protein expression of CRP was assayed with RT-qPCR and immunocytochemistry, respectively. The production of reactive oxygen species (ROS) and superoxide anion (O₂^?) was detected with ROS and O₂^? assay kits, respectively.Key findingsThe results showed that both IL-6 and Ang II increased CRP levels in the supernatant of VSMCs and induced mRNA and protein expression of CRP in VSMCs, whereas pretreatment of the cells with EGCG (1 × 10^? 6 M, 3 × 10^? 6 M, 10 × 10^? 6 M) significantly inhibited IL-6- and Ang II-induced production and expression of CRP in VSMCs in a concentration-dependent manner. Additionally, Ang II stimulated O₂^? and ROS generations in VSMCs, and EGCG decreased the Ang II-induced increase of O₂^? and ROS in a concentration-dependent fashion.SignificanceThese results suggest that EGCG plays an anti-inflammatory role via inhibiting IL-6- and Ang II-induced CRP secretion, as well as the Ang II-induced generation of O₂^? and ROS in VSMCs, which contributes to its anti-atherosclerotic action.     

Ganoderma lucidum extract stimulates glucose uptake in L6 rat skeletal muscle cells

The effect of Ganoderma lucidum extract on glucose uptake was studied in L6 rat skeletal muscle cells. G. lucidum extract increased glucose uptake about 2-fold compared to control. The extract stimulated the activity of phosphatidylinositol (PI) 3-kinase which is a major regulatory molecule in the glucose uptake pathway. About 7-fold increased activity of a PI 3-kinase was observed after treatment with G. lucidum extract, whereas PI 3-kinase inhibitor, LY294002, blocked the G. lucidum extract-stimulated PI 3-kinase activity in L6 skeletal muscle cells. Protein kinase B, a downstream mediator of PI 3-kinase, was also activated by G. lucidum extract. We then assessed the activity of AMP-activated protein kinase (AMPK), another regulatory molecule in the glucose uptake pathway. G. lucidum extract increased the phosphorylation level of both AMPK alpha1 and alpha2. Activity of p38 MAPK, a downstream mediator of AMPK, was also increased by G. lucidum extract. Taken together, these results suggest that G. lucidum extract may stimulate glucose uptake, through both PI 3-kinase and AMPK in L6 skeletal muscle cells thereby contributing to glucose homeostasis.     

The effect of insulin on delta5 desaturation in hepG2 human hepatoma cells and L6 rat muscle myoblasts.

In humans there is a correlation between the ratio of arachidonic acid (20:4n-6) to cis 8,11,14 eicosatrienoic acid (20:3n-6) in skeletal muscle phospholipids and insulin sensitivity. This has been interpreted as indicating a link between the activity of the delta5 desaturase enzyme and muscle insulin sensitivity. The present study addressed the possibility that insulin regulates delta5 desaturase activity using L6 rat myoblasts and hepG2 human hepatoma cells. Both cell lines responded to insulin by increasing the amount of D-[U-14C] glucose incorporated into glycogen. In L6 cells, insulin stimulated cis 8,11,14 eicosatrienoic acid uptake and arachidonic acid production but had no effect on the percentage conversion of cis 8,11,14 eicosatrienoic acid to arachidonic acid. In hepG2 cells, insulin had no effect on cis 8,11,14 eicosatrienoic acid uptake or arachidonic acid production. These results suggest that insulin has no direct effect on delta5 desaturase activity in the liver but can alter arachidonic acid production in muscle by altering substrate availability.     

The PPARdelta agonist, GW501516, promotes fatty acid oxidation but has no direct effect on glucose utilisation or insulin sensitivity in rat L6 skeletal muscle cells

Peroxisome proliferator-activated receptor-delta (PPARdelta) activation enhances skeletal muscle fatty acid oxidation and improves whole body glucose homeostasis and insulin sensitivity. Recently, GW501516, a selective PPARdelta agonist, was reported to increase glucose uptake in human skeletal myotubes by an AMPK-dependent mechanism that may contribute to the improved glucose tolerance. Here, we demonstrate that whilst GW501516 increases expression of PGC-1alpha and CPT-1 and stimulates fatty-acid oxidation in L6 myotubes, it fails to enhance insulin sensitivity, AMPK activity or glucose uptake and storage. Our findings exclude sarcolemmal glucose transport as a potential target for the therapeutic action of PPARdelta agonists in skeletal muscle.     

Low-dose dexamethasone in the rat: a model to study insulin resistance

The main aim of this study was to set up a new animal model to study insulin resistance. Wistar rats (6 or 7 per group) received the following for 4 wk in experiment 1: 1) vehicle, 2) 2 microg/day subcutaneous dexamethasone, 3) metformin (400 mg x kg(-1) x day(-1) os), and 4) dexamethasone plus metformin. In experiment 2 the rats received the following: 1) vehicle, 2) dexamethasone, 3) dexamethasone plus arginine (2%; as substrate of the nitric oxide synthase for nitric oxide production) in tap water, and 4) dexamethasone plus isosorbide dinitrate (70 mg/kg; as direct nitric oxide donor) in tap water. Insulin sensitivity was significantly reduced by dexamethasone already at week 1, before the increase in blood pressure (day 15) and without significant changes in body weight compared with vehicle. Dexamethasone-treated rats had significantly higher triglycerides, hematocrit, and insulin, whereas serum total nitrates/ nitrites were lower compared with vehicle. The concomitant treatment with metformin minimized all the described effects of dexamethasone. In experiment 2, only isosorbide dinitrate was able to prevent the observed dexamethasone-induced metabolic, hemodynamic, and insulin sensitivity changes. Chronic low-dose subcutaneous dexamethasone (2 microg/day) is a useful model to study the relationships between insulin resistance and blood pressure in the rat, and dexamethasone might decrease insulin sensitivity and increase blood pressure through an endothelium-mediated mechanism.     

Increased response to insulin of glucose metabolism in the 6-day unloaded rat soleus muscle

Hind leg muscles of female rats (85-99 g) were unloaded by tail cast suspension for 6 days. In the fresh-frozen unloaded soleus, the significantly greater concentration of glycogen correlated with a lower activity ratio of glycogen phosphorylase (p less than 0.02). The activity ratio of glycogen synthase also was lower (p less than 0.001), possibly due to the higher concentration of glycogen. In isolated unloaded soleus, insulin (0.1 milliunit/ml) increased the oxidation of D-[U-14C]glucose, release of lactate and pyruvate, incorporation of D-[U-14C]glucose into glycogen, and the concentration of glucose 6-phosphate more (p less than 0.05) than in the weight-bearing soleus. At physiological doses of insulin, the percent of maximal uptake of 2-deoxy-D-[1,2-3H]glucose/muscle also was greater in the unloaded soleus. Unloading of the soleus increased by 50% the concentration of insulin receptors, due to no decrease in total receptor number during muscle atrophy. This increase may account for the greater response of glucose metabolism to insulin in this muscle. The extensor digitorum longus, which generally shows little response to unloading, displayed no differential response of glucose metabolism to insulin.