No abstract

Methylglyoxal (MG) and related alpha-oxoaldehydes react with proteins, lipids and DNA to give rise to covalent adducts known as advanced glycation end-products (AGEs). Elevated levels of AGEs have been implicated in the pathological complications of diabetes, uremia, Alzheimers disease, and possibly cancer. There is therefore widespread interest in developing sensitive methods for the in vivo measurement of AGEs as prognostic biomarkers and for treatment monitoring. The two diastereomeric MG-DNA adducts of N2-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) are the primary glycation products formed in DNA; however, accurate assessment of their distribution in vivo has not been possible since there is no readily available quantitative method for CEdG determination in biological samples. In order to address these issues we have developed a sensitive and quantitative LC-ESI-MS/MS assay using the stable isotope dilution method with an 15N5-CEdG standard. Methods for CEdG determination in urine or tissue extracted DNA are described. Changes in urinary CEdG in diabetic rats in response to oral administration of the AGE inhibitor LR-90 are used to demonstrate the potential utility of the method for treatment monitoring. Both stereoisomeric CEdG adducts were detected in a human breast tumor and normal adjacent tissue at levels of 3–12 adducts/107 dG, suggesting that this lesion may be widely distributed in vivo. Strategies for dealing with artifactual adduct formation due to oxoaldehyde generation during DNA isolation and enzymatic workup procedures are described.

Ligation of advanced glycation end products (AGEs) with their receptor (RAGE) plays an important role in the development of various diabetes complications, including atherosclerosis. Monocyte activation, adhesion, and migration are key events in the pathogenesis of atherosclerosis. Previous studies showed that AGEs and S100b, a specific RAGE ligand, could augment monocyte inflammatory responses via RAGE. In this study, we examined whether LR-90, a compound belonging to a new class of AGE inhibitor, could inhibit inflammatory responses in human monocytes. Human THP-1 cells were pretreated with LR-90 and then stimulated with S100b. LR-90 significantly inhibited S100b-induced expression of RAGE and other proinflammatory genes including monocyte chemoattractant protein-1, interferon-␥-inducible protein-10, and cyclooxygenase-2 in a dose-dependent manner. These inhibitory effects may be exerted via inhibition of nuclear factor-B (NF-B) activation, as LR-90 suppressed both S100b-and tumor necrosis factor-␣-induced IB-␣ degradation as well as NF-B promoter transcriptional activity. LR-90 also prevented oxidative stress in activated monocytes, as demonstrated by its inhibitory effects on S100b-induced expression of NADPH oxidase and intracellular superoxide production. In addition, LR-90 blocked S100b-induced monocyte adhesion to human umbilical vein endothelial cell. These new data show that, in addition to its AGE inhibitory effects, LR-90 has novel anti-inflammatory properties and might therefore have additional protective effects against diabetic vascular complications. Diabetes 56:647-655, 2007

OBJECTIVE—The reactions of carbohydrate- or lipid-derived intermediates with proteins lead to the formation of Maillard reaction products, which subsequently leads to the formation of advanced glycation/lipoxidation end products (AGE/ALEs). Levels of AGE/ALEs are increased in diseases like diabetes. Unlike AGEs, very little is known about ALE effects in vitro. We hypothesized that ALEs can have proinflammatory effects in monocytes. RESEARCH DESIGN AND METHODS—In a profiling approach, conditioned media from THP-1 cells either cultured in normal glucose (5.5 mmol/l) or treated with MDA-Lys or MDA alone were hybridized to arrays containing antibodies to 120 known human cytokines/chemokines. Pathway analyses with bioinformatics software were used to identify signalling networks. RESULTS—Synthetic ALE (malondialdehyde-lysine [MDA-Lys]) (50 μmol/l) could induce oxidant stress and also activate the transcriptional factor nuclear factor-κB (NF-κB) in THP-1 monocytes. MDA-Lys also significantly increased the expression of key candidate proinflammatory genes, interferon-γ–inducible protein-10, β1- and β2-integrins, cyclooxygenase-2 (COX-2), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 and -8, and inducible nitric-oxide synthase, which are also associated with monocyte dysfunction. Several key target proinflammatory proteins were significantly induced by MDA-Lys relative to normal glucose or MDA alone, including MCP-1; tumor necrosis factor ligand superfamily member-14; chemokine CC motif ligand-11 (CCL11); growth-related oncogene-α, -β, and -γ; and chemokine CXC motif ligand-13. Bioinformatics analyses identified a network of chemokine signaling among MDA-Lys–regulated genes. MDA-Lys also increased monocyte binding to vascular smooth muscle and endothelial cells. Furthermore, plasma from diabetic rats showed significantly higher levels of MDA-Lys and CCL11. CONCLUSIONS—These new results suggest that ALEs can promote monocyte activation and vascular complications via induction of inflammatory pathways and networks.

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis.

Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity. Hepatocellular carcinoma (HCC)2 is the most common and severe form of liver cancer, accounting for about 80 -90% of primary liver cancers and 5% of all human cancers. More than 600,000 deaths are attributed to HCC every year with 2:1 ratio for men versus women (1, 2). HCC is a primary cancer of hepatocytes that most typically occurs in the setting of known risk factors, including cirrhosis and chronic hepatitis B virus or hepatitis C virus infections (2, 3), although recently, several lines of evidence suggest that type 2 diabetes is also an independent risk factor for HCC development (4). HCC is an aggressive tumor and represents a major health problem as its incidence is increasing. Systemic chemotherapies have proven ineffective against advanced HCC, so it typically leads to death within 6 -20 months (5). Hepatocarcinogenesis is a multistep process involving inflammation, hyperplasia, and dysplasia that finally leads to malignant transformation. In recent years, mitochondria have been found to provide a novel targeting site for new anticancer drugs (known as "mitocans") that ca...

Abstract. Obesity is a chronic metabolic disorder caused by an imbalance between energy intake and expenditure. It is one of the principal causative factors involved in the development of metabolic syndrome and cancer. Inhibition of adipocyte differentiation has often been a target of anti-obesity strategies since obesity is caused not only by hypertrophy but also by adipocyte hyperplasia. In this study, we investigated the effects of COH-SR4, a novel compound with anticancer properties, on the adipogenesis in 3T3-L1 cells. Treatment with COH-SR4 significantly inhibited adipocyte differentiation in a dose-dependent manner. This inhibitory effect mainly occurred at the early phase of differentiation through inhibition of mitotic clonal expansion and cell cycle arrest at the G1/S phase transition. In differentiating adipocytes, COH-SR4 significantly reduced intracellular lipid accumulation and downregulated the expression of key adipogenesis-related transcription factors and lipogenic proteins. COH-SR4 exhibited no cytotoxic effects in 3T3-L1 cells, but indirectly activated AMP-activated protein kinase (AMPK). AMPK activation by COH-SR4 also resulted in the phosphorylation of raptor and tuberous sclerosis protein 2 (TSC2), two proteins involved in the mammalian target of rapamycin (mTOR) signaling pathways. Additionally, COH-SR4 decreased the phosphorylation of p70 kDa ribosomal protein S6 kinase (S6K) and initiation factor 4E (eIF4E) binding protein 1 (4EB-P1), two downstream effectors of mTOR that regulate protein synthesis. Interestingly, knockdown of AMPKα1/α2 prevented the ability of COH-SR4 to inhibit cell cycle arrest and overall adipogenesis and lipid accumulation in the differentiating 3T3-L1 cells. Taken together, these results suggest that COH-SR4 inhibits 3T3-L1 adipogenesis via AMPK activation. COH-SR4 may be a promising compound for the treatment of obesity and related metabolic disorders.

These findings show that LR-90 can effectively inhibit important lesions of diabetic retinopathy. This agent has potential for preventing retinopathy in patients with diabetes.