8-oxo-dGTP curbs tumor development via S phase arrest and AIF-mediated apoptosis.

Oxidative stress can attack precursor nucleotides, resulting in nucleic acid damage in cells. It remains unclear how 8-oxo-dGTP and 8-oxoGTP, oxidized forms of dGTP and GTP, respectively, could affect DNA or RNA oxidation levels and tumor development. To address this, we intravenously administered 8-oxo-dGTP and 8-oxoGTP to wild-type and MTH1-knockout mice. 8-oxoGTP administration increased frequency of tumor incidence, which is more prominent in MTH1-knockout mice. However, 8-oxo-dGTP treatment rather reduced tumor development regardless of the mouse genotype. The tumor suppressive effects of 8-oxo-dGTP were further confirmed using xenograft and C57/6J-ApcMin/Nju mouse models. Mechanistically, 8-oxo-dGTP increased the 8-oxo-dG contents in DNA and DNA strand breakage, induced cell cycle arrest in S phase and apoptosis mediated by AIF, eventually leading to reduced tumor incidence. These results suggest distinct roles of 8-oxo-dGTP and 8-oxoGTP in tumor development.

Faciogenital Dysplasia 5 supports cancer stem cell traits in basal-like breast cancer by enhancing EGFR stability.

Most basal-like breast cancers (BLBCs) are triple-negative breast cancers (TNBCs), which have the worst prognosis and distant metastasis-free survival among breast cancer subtypes. Now, no targeted therapies are available for patients with BLBC due to the lack of reliable and effective molecular targets. Here, we performed the BLBC tissue microarray-based immunohistochemical analysis and showed that Faciogenital Dysplasia 5 (FGD5) abundance is associated with poor prognosis in BLBCs. FGD5 deletion decreased the proliferation, invasion, and tumorsphere formation capacity of BLBC cells. Furthermore, genetic inhibition of Fgd5 in mouse mammary epithelial cells attenuated BLBC initiation and progression by reducing the self-renewal ability of tumor-initiating cells. In addition, FGD5 abundance was positively correlated with the abundance of epidermal growth factor receptor (EGFR) in BLBCs. FGD5 ablation decreased EGFR abundance by reducing EGFR stability in TNBC cells in 2D and 3D culture conditions. Mechanistically, FGD5 binds to EGFR and interferes with basal EGFR ubiquitination and degradation induced by the E3 ligase ITCH. Impaired EGFR degradation caused BLBC cell proliferation and promoted invasive properties and self-renewal. To verify the role of the FGD5-EGFR interaction in the regulation of EGFR stability, we screened a cell-penetrating α-helical peptide PER3 binding with FGD5 to disrupt the interaction. Treatment of BLBC patient-derived xenograft-bearing mice with the peptide PER3 disrupting the FGD5-EGFR interaction either with or without chemotherapy reduced BLBC progression. Our study identified FGD5 as a positive modulator of tumor-initiating cells and suggests a potential therapeutic option for the BLBC subtype of breast cancer.

TRIB3 supports breast cancer stemness by suppressing FOXO1 degradation and enhancing SOX2 transcription.

The existence of breast cancer stem cells (BCSCs) is a major reason underlying cancer metastasis and recurrence after chemotherapy and radiotherapy. Targeting BCSCs may ameliorate breast cancer relapse and therapy resistance. Here we report that expression of the pseudokinase Tribble 3 (TRIB3) positively associates with breast cancer stemness and progression. Elevated TRIB3 expression supports BCSCs by interacting with AKT to interfere with the FOXO1-AKT interaction and suppress FOXO1 phosphorylation, ubiquitination, and degradation by E3 ligases SKP2 and NEDD4L. The accumulated FOXO1 promotes transcriptional expression of SOX2, a transcriptional factor for cancer stemness, which in turn, activates FOXO1 transcription and forms a positive regulatory loop. Disturbing the TRIB3-AKT interaction suppresses BCSCs by accelerating FOXO1 degradation and reducing SOX2 expression in mouse models of breast cancer. Our study provides insights into breast cancer development and confers a potential therapeutic strategy against TRIB3-overexpressed breast cancer.

Oxidative DNA and RNA damage and their prognostic values during Salmonella enteritidis-induced intestinal infection in rats.

Emerging evidence suggests that microbial pathogens may induce oxidative stress in infected hosts. The aim of the present study was to investigate the relationship between changes in oxidative stress and intestinal infection with and without antibiotic treatment in animal models. Sprague-Dawley (SD) rats were divided into three groups: rats infected with Salmonella enterica serovar Enteritidis (S. enteritidis), rats infected with S. enteritidis followed by norfloxacin treatment, and the control group. To evaluate oxidative stress changes, levels of 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dGsn), which represented oxidative damage to RNA and DNA, respectively, were analysed in urine and tissue samples. In urine, the level of 8-oxo-Gsn increased significantly after oral exposure to S. enteritidis (p ≤ 0.001) and returned to baseline after recovery. Notably, norfloxacin treatment decreased the level of 8-oxo-Gsn in urine significantly (p = 0.001). Changes of 8-oxo-Gsn measured in tissues from the small intestine, colon, liver and spleen were consistent with 8-oxo-Gsn measured in urine. Our study suggested that 8-oxo-Gsn in urine may serve as a highly sensitive biomarker for evaluating the severity of S. enteritidis infection and the effectiveness of antibiotic treatment against infection.

Adipose-specific deletion of Kif5b exacerbates obesity and insulin resistance in a mouse model of diet-induced obesity.

Recent studies have shown that KIF5B (conventional kinesin heavy chain) mediates glucose transporter type 4 translocation and adiponectin secretion in 3T3-L1 adipocytes, suggesting an involvement of KIF5B in the homeostasis of metabolism. However, the in vivo physiologic function of KIF5B in adipose tissue remains to be determined. In this study, adipose-specific Kif5b knockout (F-K5bKO) mice were generated using the Cre-LoxP strategy. F-K5bKO mice had similar body weights to controls fed on a standard chow diet. However, F-K5bKO mice had hyperlipidemia and significant glucose intolerance and insulin resistance. Deletion of Kif5b aggravated the deleterious impact of a high-fat diet (HFD) on body weight gain, hepatosteatosis, glucose tolerance, and systematic insulin sensitivity. These changes were accompanied by impaired insulin signaling, decreased secretion of adiponectin, and increased serum levels of leptin and proinflammatory adipokines. F-K5bKO mice fed on an HFD exhibited lower energy expenditure and thermogenic dysfunction as a result of whitening of brown adipose due to decreased mitochondria biogenesis and down-regulation of key thermogenic gene expression. In conclusion, selective deletion of Kif5b in adipose tissue exacerbates HFD-induced obesity and its associated metabolic disorders, partly through a decrease in energy expenditure, dysregulation of adipokine secretion, and insulin signaling.-Cui, J., Pang, J., Lin, Y.-J., Gong, H., Wang, Z.-H., Li, Y.-X., Li, J., Wang, Z., Jiang, P., Dai, D.-P., Li, J., Cai, J.-P., Huang, J.-D., Zhang, T.-M. Adipose-specific deletion of Kif5b exacerbates obesity and insulin resistance in a mouse model of diet-induced obesity.

Effect of CYP2D6 variants on venlafaxine metabolism in vitro.

1. CYP2D6 is an important member of the cytochrome P450 (CYP450) enzyme superfamily, we recently identified 22 CYP2D6 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of venlafaxine in vitro. 2. The wild-type and 24 CYP2D6 variants were expressed in insect cells, and each variant was characterized using venlafaxine as the substrate. Reactions were performed at 37 °C with 5-500 µM substrate (three variants was adjusted to 1000 µM) for 50 min. By using high-performance liquid chromatography to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of O-desmethylvenlafaxine were determined. 3. Among the 22 CYP2D6 variants, the intrinsic clearance (Vmax/Km) values of all variants were significantly decreased (from 0.2% to 84.5%) compared with wild-type CYP2D6*1. In addition, the kinetic parameters of two CYP2D6 variants could not be detected because they have no detectable enzyme activity. 4. The comprehensive in vitro assessment of CYP2D6 variants provides significant insights into allele-specific activity towards venlafaxine in vivo.

Effects of 24 CYP2D6 Variants Found in the Chinese Population on the Metabolism of Risperidone.

AIMS: Cytochrome P450 (CYP450) 2D6 is an important member of the P450 enzyme superfamily and responsible for clearing 25% of clinically important drugs. The aim of this study was to assess the catalytic characteristics of 24 CYP2D6 allelic isoforms found in the Chinese population and their effects on the metabolism of risperidone in vitro. METHODS: Insect microsomes expressing wild-type CYP2D6 and 24 CYP2D6 allelic variants were incubated with 20-1,000 µmol/l risperidone for 40 min at 37°C. After termination, risperidone and 9-OH risperidone, the metabolite of risperidone, were precipitated and used for signal collection by ultra-performance liquid-chromatography tandem mass spectrometry. RESULTS: Among 24 CYP2D6 variants tested, 2 variants (CYP2D6*92 and CYP2D6*96) were found to be with no detectable activity. Two variants (E215K and R440C) exhibited higher intrinsic clearance values than the wild-type protein, while the remaining 20 CYP2D6 allelic variants exhibited significantly decreased clearance values (2.01-87.56%) compared to CYP2D6*1. CONCLUSION: These findings suggest that more attention should be directed to subjects carrying these infrequent CYP2D6 alleles when administering risperidone in the clinic. This is the first report of all these novel alleles for risperidone metabolism, providing fundamental data for further clinical studies on CYP2D6 alleles.

Characterization of a novel CYP2C9 mutation (1009C>A) detected in a warfarin-sensitive patient.

Warfarin is the most frequently prescribed anticoagulant for the long-term treatment in the clinic. Recent studies have shown that polymorphic alleles within the CYP2C9, VKORC1, and CYP4F2 genes are related to the warfarin dosage requirement. In this study, a novel non-synonymous mutation (1009C>A) in CYP2C9 was detected in a warfarin-hypersensitive patient, while the other two candidate genes were both found to be homozygous for the wild-type alleles. The newly identified point mutation results in an amino acid substitution at position 337 of the CYP2C9 protein (P337T) and has been designated as the novel allele CYP2C9*58. When expressed in insect cell microsomes, the relative intrinsic clearance values of the CYP2C9.58 variant for tolbutamide and losartan were quite similar to those of the typical defective variant CYP2C9.3, whereas the clearance value of CYP2C9.58 for diclofenac was slightly higher than that of another typical defective variant CYP2C9.2. These data suggested that when compared with wild-type CYP2C9.1, the enzymatic activity of the novel allelic variant has been greatly reduced by the 1009C>A mutation. If patients carrying this allele take drugs metabolized by CYP2C9, their metabolic rate might be slower than that of wild-type allele carriers and thus much more attention should be paid to their clinical care.

Soluble epoxide hydrolase inhibitors and cardiovascular diseases.

BACKGROUND: Epoxyeicosatrienoic acids (EETs) have been shown to play a role in cardiovascular protection by reducing ischemia reperfusion injury, producing anti-inflammatory effects, and promoting angiogenesis. EETs are regulated through conversion to less active corresponding diols by soluble epoxide hydrolase (sEH). Inhibition of sEH enhances the beneficial properties of EETs and has been investigated as a possible treatment for cardiovascular diseases. CONTENT: sEH inhibitors (sEHIs) have anti-inflammatory effects by stabilizing anti-inflammatory EETs. Additionally, sEHIs strongly inhibit and reverse cardiac hypertrophy. sEHIs have been shown to protect myocardial cells from ischemiareperfusion injury, treat atherosclerosis and prevent the development of hypertension. sEHIs promote blood vessels to release bradykinin via an EET-mediated STAT3 signaling pathway to elicit tolerance to ischemia. SUMMARY: Inhibition of sEH has been shown to improve several aspects of cardiovascular diseases, including inflammation, hypertension, cardiac hypertrophy and atherosclerosis. For this reason, sEHIs are promising new pharmaceutical for the treatment of cardiovascular diseases.

A potent soluble epoxide hydrolase inhibitor, t-AUCB, acts through PPARγ to modulate the function of endothelial progenitor cells from patients with acute myocardial infarction.

BACKGROUND: Epoxyeicosatrienoic acids (EETs) are natural angiogenic mediators regulated by soluble epoxide hydrolase (sEH). Inhibitors of sEH can stabilize EETs levels and were reported to reduce atherosclerosis and inhibit myocardial infarction in animal models. In this work, we investigated whether increasing EETs with the sEH inhibitor t-AUCB would increase angiogenesis related function in endothelial progenitor cells (EPCs) from patients with acute myocardial infarction (AMI). METHODS AND RESULTS: EPCs were isolated from 50 AMI patients and 50 healthy subjects (control). EPCs were treated with different concentrations of t-AUCB for 24h with or without peroxisome proliferator activated receptor γ (PPARγ) inhibitor GW9662. Migration of EPCs was assayed in trans-well chambers. Angiogenesis assays were performed using a Matrigel-Matrix in vitro model. The expression of vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1α (HIF-1α) mRNA and protein in EPCs was measured by real-time PCR or Western blot, respectively. Also, the concentration of EETs in the culture supernatant was detected by ELISA. The activity of EPCs in the AMI patient group was reduced compared to healthy controls. Whereas increasing EET levels with t-AUCB promoted a dose dependent angiogenesis and migration in EPCs from AMI patients. Additionally, the t-AUCB dose dependently increased the expression of the angiogenic factors VEGF and HIF-α. Lastly, we provide evidence that these effects were PPARγ dependent. CONCLUSION: The results demonstrate that the sEH inhibitor positively modulated the functions of EPCs in patients with AMI through the EETs-PPARγ pathway. The present study suggests the potential utility of sEHi in the therapy of ischemic heart disease.