Dimethoxytolyl propylresorcinol induces apoptosis and mitophagy in human leukemia cells through the PI3K/AKT pathway.

Dimethoxytolyl propylresorcinol (UP302), a natural compound extracted from Dianella ensifolia, owing to its tyrosinase inhibitory and strong antioxidant properties, is used in whitening cosmetics. However, the role of UP302 has not been reported in cancer treatment. This study aimed to assess the in vitro antitumor activity of UP302 in different tumor cells. It inhibited the growth of certain cancer cell lines and especially in leukemia cells. Therefore, we investigated the antitumor effect of UP302 in leukemia by examining the cell cycle, apoptosis, reactive oxygen species levels (ROS) production, and changes in mitochondrial membrane potential. Our results demonstrated that UP302 inhibited the growth of leukemia cells both in vivo and in vitro and exerted a proapoptotic effect on MV411 and K562 cells, confirmed by flow cytometry and western blot analysis. Furthermore, UP302 promoted autophagy in MV411 and K562 cells. Transmission electron microscopy and western blot analysis showed that UP302 induced mitophagy in MV411 and K562 cells. In addition, the autophagy inhibitor chloroquine could enhance UP302-induced apoptosis, suggesting that UP302-mediated autophagy may be protective in MV411 and K562 cells. In conclusion, our study is the first to provide evidence for the anti-leukemia properties of UP302 and the potential clinical use of UP302 combined with autophagy inhibitors as a chemotherapeutic strategy for human leukemia.

Hepatic DDAH1 mitigates hepatic steatosis and insulin resistance in obese mice: Involvement of reduced S100A11 expression.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is an important regulator of plasma asymmetric dimethylarginine (ADMA) levels, which are associated with insulin resistance in patients with nonalcoholic fatty liver disease (NAFLD). To elucidate the role of hepatic DDAH1 in the pathogenesis of NAFLD, we used hepatocyte-specific Ddah1-knockout mice (Ddah1HKO) to examine the progress of high-fat diet (HFD)-induced NAFLD. Compared to diet-matched flox/flox littermates (Ddah1f/f), Ddah1HKO mice exhibited higher serum ADMA levels. After HFD feeding for 16 weeks, Ddah1HKO mice developed more severe liver steatosis and worse insulin resistance than Ddah1f/f mice. On the contrary, overexpression of DDAH1 attenuated the NAFLD-like phenotype in HFD-fed mice and ob/ob mice. RNA-seq analysis showed that DDAH1 affects NF-κB signaling, lipid metabolic processes, and immune system processes in fatty livers. Furthermore, DDAH1 reduces S100 calcium-binding protein A11 (S100A11) possibly via NF-κB, JNK and oxidative stress-dependent manner in fatty livers. Knockdown of hepatic S100a11 by an AAV8-shS100a11 vector alleviated hepatic steatosis and insulin resistance in HFD-fed Ddah1HKO mice. In summary, our results suggested that the liver DDAH1/S100A11 axis has a marked effect on liver lipid metabolism in obese mice. Strategies to increase liver DDAH1 activity or decrease S100A11 expression could be a valuable approach for NAFLD therapy.

DDAH1 Protects against Cardiotoxin-Induced Muscle Injury and Regeneration.

Nitric oxide (NO) is an important biological signaling molecule affecting muscle regeneration. The activity of NO synthase (NOS) is regulated by dimethylarginine dimethylaminohydrolase 1 (DDAH1) through degradation of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA). To investigate the role of DDAH1 in muscle injury and regeneration, muscle-specific Ddah1-knockout mice (Ddah1MKO) and their littermates (Ddah1f/f) were used to examine the progress of cardiotoxin (CTX)-induced muscle injury and subsequent muscle regeneration. After CTX injection, Ddah1MKO mice developed more severe muscle injury than Ddah1f/f mice. Muscle regeneration was also delayed in Ddah1MKO mice on Day 5 after CTX injection. These phenomena were associated with higher serum ADMA and LDH levels as well as a great induction of inflammatory response, oxidative stress and cell apoptosis in the gastrocnemius (GA) muscle of Ddah1MKO mice. In the GA muscle of CTX-treated mice, Ddah1 deficiency decreased the protein expression of M-cadherin, myogenin, Bcl-2, peroxiredoxin 3 (PRDX3) and PRDX5, and increased the protein expression of MyoD, TNFα, Il-6, iNOS and Bax. In summary, our data suggest that DDAH1 exerts a protective role in muscle injury and regeneration.

Dimethylarginine dimethylaminohydrolase 1 protects PM2.5 exposure-induced lung injury in mice by repressing inflammation and oxidative stress.

BACKGROUND: Airborne fine particulate matter with aerodynamic diameter ≤ 2.5 µm (PM2.5) pollution is associated with the prevalence of respiratory diseases, including asthma, bronchitis and chronic obstructive pulmonary disease. In patients with those diseases, circulating asymmetric dimethylarginine (ADMA) levels are increased, which contributes to airway nitric oxide deficiency, oxidative stress and inflammation. Overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1), an enzyme degrading ADMA, exerts protective effects in animal models. However, the impact of DDAH1/ADMA on PM2.5-induced lung injury has not been investigated. METHODS: Ddah1-/- and DDAH1-transgenic mice, as well as their respective wild-type (WT) littermates, were exposed to either filtered air or airborne PM2.5 (mean daily concentration ~ 50 µg/m3) for 6 months through a whole-body exposure system. Mice were also acutely exposed to 10 mg/kg PM2.5 and/or exogenous ADMA (2 mg/kg) via intratracheal instillation every other day for 2 weeks. Inflammatory response, oxidative stress and related gene expressions in the lungs were examined. In addition, RAW264.7 cells were exposed to PM2.5 and/or ADMA and the changes in intracellular oxidative stress and inflammatory response were determined. RESULTS: Ddah1-/- mice developed more severe lung injury than WT mice after long-term PM2.5 exposure, which was associated with greater induction of pulmonary oxidative stress and inflammation. In the lungs of PM2.5-exposed mice, Ddah1 deficiency increased protein expression of p-p65, iNOS and Bax, and decreased protein expression of Bcl-2, SOD1 and peroxiredoxin 4. Conversely, DDAH1 overexpression significantly alleviated lung injury, attenuated pulmonary oxidative stress and inflammation, and exerted opposite effects on those proteins in PM2.5-exposed mice. In addition, exogenous ADMA administration could mimic the effect of Ddah1 deficiency on PM2.5-induced lung injury, oxidative stress and inflammation. In PM2.5-exposed macrophages, ADMA aggravated the inflammatory response and oxidative stress in an iNOS-dependent manner. CONCLUSION: Our data revealed that DDAH1 has a marked protective effect on long-term PM2.5 exposure-induced lung injury.

Genetic and Pharmacological Inhibition of GCN2 Ameliorates Hyperglycemia and Insulin Resistance in Type 2 Diabetic Mice.

It is well recognized that there is a strong and complex association between nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D). We previously demonstrated that genetic knockout or pharmacological inhibition of general control nondepressible kinase 2 (GCN2), a well-known amino acid sensor, alleviated hepatic steatosis and insulin resistance in obese mice. However, whether GCN2 affects the development of T2D remains unclear. After a high-fat diet (HFD) plus low-dose streptozotocin (STZ) treatments, Gcn2-/- mice developed less hyperglycemia, insulin resistance, hepatic steatosis, and oxidative stress than wild-type (WT) mice. Inhibition of GCN2 by intraperitoneal injection of 3 mg/kg GCN2iB (a specific inhibitor of GCN2) every other day for 6 weeks also ameliorated hyperglycemia, insulin resistance, hepatic steatosis, and oxidative stress in HFD/STZ- and leptin receptor deletion (db/db)-induced T2D mice. Moreover, depletion of hepatic GCN2 in db/db mice by tail vein injection of an AAV8-shGcn2 vector resulted in similar improvement in those metabolic disorders. The protective mechanism of GCN2 inhibition in T2D mice was associated with regulation of the glucose metabolic pathway, repression of lipogenesis genes, and activation of the Nrf2 pathway. Together, our data provide evidence that strategies to inhibit hepatic GCN2 activity may be novel approaches for T2D therapy.

Inhibition of GCN2 Alleviates Cardiomyopathy in Type 2 Diabetic Mice via Attenuating Lipotoxicity and Oxidative Stress.

Diabetic cardiomyopathy (DCM) is a kind of heart disease that affects diabetic patients and is one of the primary causes of death. We previously demonstrated that deletion of the general control nonderepressible 2 (GCN2) kinase ameliorates cardiac dysfunction in diabetic mice. The aim of this study was to investigate the protective effect of GCN2iB, a GCN2 inhibitor, in type 2 diabetic (T2D) mice induced by a high-fat diet (HFD) plus low-dose streptozotocin (STZ) treatments or deletion of the leptin receptor (db/db). GCN2iB (3 mg/kg/every other day) treatment for 6 weeks resulted in significant decreases in fasting blood glucose levels and body weight and increases in the left ventricular ejection fraction. GCN2iB treatment also attenuated myocardial fibrosis, lipid accumulation and oxidative stress in the hearts of T2D mice, which was associated with decreases in lipid metabolism-related genes and increases in antioxidative genes. Untargeted metabolomics and RNA sequencing analysis revealed that GCN2iB profoundly affected myocardial metabolomic profiles and gene expression profiles. In particular, GCN2iB increased myocardial phosphocreatine and taurine levels and upregulated genes involved in oxidative phosphorylation. In conclusion, the data provide evidence that GCN2iB effectively protects against cardiac dysfunction in T2D mice. Our findings suggest that GCN2iB might be a novel drug candidate for DCM therapy.

Inhibition of GCN2 alleviates hepatic steatosis and oxidative stress in obese mice: Involvement of NRF2 regulation.

The development of nonalcoholic fatty liver disease (NAFLD) is associated with increased reactive oxygen species (ROS) production. Previous observations on the contradictory roles of general control nonderepressible 2 (GCN2) in regulating the hepatic redox state under different nutritional conditions prompted an investigation of the underlying mechanism by which GCN2 regulates ROS homeostasis. In the present study, GCN2 was found to interact with NRF2 and decrease NRF2 expression in a KEAP1-dependent manner. Activation of GCN2 by halofuginone treatment or leucine deprivation decreased NRF2 expression in hepatocytes by increasing GSK-3ß activity. In response to oxidative stress, GCN2 repressed NRF2 transcriptional activity. Knockdown of hepatic GCN2 by tail vein injection of an AAV8-shGcn2 vector attenuated hepatic steatosis and oxidative stress in leptin-deficient (ob/ob) mice in an NRF2-dependent manner. Inhibition of GCN2 by GCN2iB also ameliorated hepatic steatosis and oxidative stress in both ob/ob mice and high fat diet-fed mice, which was associated with significant changes in lipid and amino acid metabolic pathways. Untargeted metabolomics analysis revealed that GCN2iB decreased fatty acid and sphingomyelin levels but increased aliphatic amino acid and phosphatidylcholine levels in fatty livers. Collectively, our results provided the first direct evidence that GCN2 is a novel regulator of NRF2 and that specific GCN2 inhibitors might be potential drugs for NAFLD therapy.

Metformin protects against PM2.5-induced lung injury and cardiac dysfunction independent of AMP-activated protein kinase α2.

Fine particulate matter (PM2.5) airborne pollution increases the risk of respiratory and cardiovascular diseases. Although metformin is a well-known antidiabetic drug, it also confers protection against a series of diseases through the activation of AMP-activated protein kinase (AMPK). However, whether metformin affects PM2.5-induced adverse health effects has not been investigated. In this study, we exposed wild-type (WT) and AMPKα2-/- mice to PM2.5 every other day via intratracheal instillation for 4 weeks. After PM2.5 exposure, the AMPKα2-/- mice developed more severe lung injury and cardiac dysfunction than were developed in the WT mice; however the administration of metformin was effective in attenuating PM2.5-induced lung injury and cardiac dysfunction in both the WT and AMPKα2-/- mice. In the PM2.5-exposed mice, metformin treatment resulted in reduced systemic and pulmonary inflammation, preserved left ventricular ejection fraction, suppressed induction of pulmonary and myocardial fibrosis and oxidative stress, and increased levels of mitochondrial antioxidant enzymes. Moreover, pretreatment with metformin significantly attenuated PM2.5-induced cell death and oxidative stress in control and AMPKα2-depleted BEAS-2B and H9C2 cells, and was associated with preserved expression of mitochondrial antioxidant enzymes. These data support the notion that metformin protects against PM2.5-induced adverse health effects through a pathway that appears independent of AMPKα2. Our findings suggest that metformin may also be a novel drug for therapies that treat air pollution associated disease.

A glutathione peroxidase from Antarctic psychrotrophic bacterium Pseudoalteromonas sp. ANT506: Cloning and heterologous expression of the gene and characterization of recombinant enzyme.

A glutathione peroxidase (GPx) gene, designated as PsGPx, was cloned from Antarctic psychrotrophic bacterium Pseudoalteromonas sp. ANT506 and expressed in Escherichia coli. The full-length PsGPx contained a 585-bp encoding 194 amino acids with predicted molecular masses of approx. 21.7 kDa. Multiple sequence alignments revealed that PsGPx belonged to the thioredoxin-like superfamily. PsGPx was heterologously overexpressed in E. coli, purified and characterized. The maximum catalytic temperature and pH value for recombinant PsGPx (rPsGPx) were 30°C and pH 9.0, respectively. rPsGPx retained 45% of the maximum activity at 0°C and exhibited high thermolability with a half-life of approx. 40 min at 40°C. In addition, the enzymatic activity of rPsGPx was still manifested under 3 M NaCl. The Km and Vmax values of the recombinant enzyme using GSH and H2O2 as substrates were 1.73 mM and 16.28 nmol/mL/min versus 2.46 mM and 21.50 nmol/mL/min, respectively.