Statins inhibit expression of thioredoxin reductase 1 in rat and human liver and reduce tumour development

BackgroundStatins have been reported to have anti-carcinogenic properties in addition to their cholesterol-lowering effects, but the mechanism is unknown. Thioredoxin reductases (TrxR) are selenium-containing enzymes of great importance for carcinogenesis and their levels are increased in neoplastic cells. The aim of the present study was to investigate if statin treatment is associated with alterations in the hepatic expression of TrxR.MethodsHuman liver biopsies from a study where patients had been randomised to statin treatment or placebo were analysed. In addition we used liver tissue from a human liver bank where statin treated subjects were compared with non-treated. We also used tissue from a rat liver cancer model in which we have previously shown anti-carcinogenic effects of statins. Real-time PCR and activity assay were used to determine TrxR-levels and activity in tissue extracts.ResultsIn humans 80 mg atorvastatin treatment for 4 weeks (n = 6) was associated with 85% lower levels of TrxR1 and TrxR2 compared to placebo-treated patients (n = 8) (p = 0.03). In liver biopsies from a human donor liver bank 3 statin treated subjects had 90% lower expression of TrxR1 than 15 non-treated subjects (p = 0.04). Statin treatment was associated with 45% lower expression and activity of TrxR1 in a rat model for liver cancer (p = 0.03). There was a clear correlation between inhibition of carcinogenesis and decreased TrxR1-levels (p = 0.003).ConclusionStatin treatment decreases the hepatic expression of TrxR1 in humans and rats. Suppression of TrxR1 expression could explain possible anti-carcinogenic effects of statins. In addition, decreased levels of TrxR1 during statin treatment may shed light on the mechanism of other side-effects of statins.     

Exonuclease 1 (EXO1) gene variation and melanoma risk

ObjectiveDNA repair pathway genes play an important role in maintaining genomic integrity and protecting against cancer development. This study aimed to identify novel SNPs in the DNA repair-related genes associated with melanoma risk from a genome-wide association study (GWAS).MethodsA total of 8422 SNPs from the 165 DNA repair-related genes were extracted from a GWAS of melanoma risk, including 494 cases and 5628 controls from the Nurses’ Health Study (NHS) and the Health Professionals Follow-up Study (HPFS). We further replicated the top SNPs in a GWAS of melanoma risk from the MD Anderson Cancer Center (1804 cases and 1026 controls).ResultsA total of 3 SNPs with P value <0.001 were selected for in silico replication. One SNP was replicated: rs3902093 [A] in EXO1 promoter region (P_discovery = 6.6 × 10^?4, P_replication = 0.039, P_joint = 2.5 × 10^?4; OR_joint = 0.80, 95% CI: 0.71, 0.90). This SNP was associated with the expression of the EXO1; carriers of the A allele showed lower expression (P = 0.002).ConclusionOur study found that a promoter region SNP in the editing and processing nucleases gene EXO1 was associated with decreased expression of EXO1 and decreased melanoma risk. Further studies are warranted to validate this association and to investigate the potential mechanisms.     

Codon optimization through a two-step gene synthesis leads to a high-level expression of Aspergillus niger lip2 gene in Pichia pastoris

Aspergillus niger lipases are important biocatalysts for a broad range of industrial applications. To enhance the expression level of a newly cloned lipase gene lip2 of A. niger in Pichia pastoris, we applied codon optimization and synthesized the full length codon-optimized gene by a two-step gene synthesis strategy. This strategy consists of an assembly PCR for several small DNA fragments and enzymatic digestion and ligation steps to ligate these fragments into the full-length gene. First, the full-length lip2 gene was divided into three fragments F1 (237 bp), F2 (238 bp) and F3 (422 bp) with the additions of proper restriction sites, and separately amplified by assembly PCR reactions. Second, three PCR amplified fragments were digested and ligated into the full-length lip2 gene. In the two-step gene synthesis, synthesis of smaller DNA fragments resulted in a significant lower level of nonspecific mismatching among oligonucleotides and a very low mutational rate of the PCR products, demonstrating the superiority of the method. When compared with the originally cloned lip2 gene of A. niger, the new codon optimized lip2 gene expressed at a significantly higher level in yeasts after methanol induction for 72 h, and both the enzyme activity and protein content reached maximal levels of 191 U/ml and 154 mg/1, with 11.6- and 5.3-fold increases, respectively.     

TNF-α -308 genotypes are associated with TNF-α and TGF-β₁ mRNA expression in blood leucocytes of humans

AimTumor necrosis factor α (TNF-α) influences the pathogenesis of lung-fibrosis and carcinogenesis in normal cells. Polymorphisms of this gene are suggested to be associated with susceptibility to lung-diseases. Additionally TNF-α is postulated to play a significant role in regulating. Transforming growth factor (TGF-β₁) expression Therefore we investigated if the TNF-α or TGF-β₁ gene expression level is different within the ?308 TNF-α genotypes.MethodsQuantitative Real-time PCR of TNF-α and TGF-β₁ was performed in 178 Germans. Calculations of expression were made with the 2^?ΔΔCT method. Detection of the ?308 promoter polymorphism of the TNF-α gene was performed by rapid capillary PCR with melting curve analysis.ResultsThe relative TNF-α mRNA expression revealed significant differences between the TNF-α ?308 homozygote wild-type G/G (0.00079 ± 0.00011; n = 113) and the heterozygote genotype G/A (0.0005 ± 0.00008; n = 52; p = 0.030) as well as between homozygote wild-type G/G and the homozygote mutant A/A (0.00029 ± 0.00009; n = 5; p = 0.004). The relative TGF-β mRNA expression showed, similar to TNF-α, the highest mRNA expression was seen within the TNF-α ?308 homozygote wild-types, while the lowest mRNA expression lay within the homozygote mutant-types.ConclusionOur findings suggest that the G-allele of TNF-α ?308 is associated with a significantly higher TNF-α mRNA expression compared to the A-allele and that this also reflects in TGF-β expression. Therefore we support the thesis that TGF-β is regulated by TNF-α.     

Adenovirus-mediated tissue factor pathway inhibitor gene transfer induces apoptosis by blocking the phosphorylation of JAK-2/STAT-3 pathway in vascular smooth muscle cells

ObjectiveIn our previous study, we have demonstrated that tissue factor pathway inhibitor (TFPI) gene could induce vascular smooth muscle cell (VSMC) apoptosis. This study was conducted to investigate whether the overexpression of the TFPI gene can induce VSMC apoptosis by inhibiting JAK-2/STAT-3 pathway phosphorylation and thereby inhibiting the expression of such downstream targets as the apoptotic protein Bcl-2 and cell cycle protein cyclin D1. The effect of TFPI on the expression of survivin, a central molecule in cell survival, was also investigated.MethodsRat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro. TFPI expression was detected by ELISA. TUNEL staining and electron microscope were carried out to determine the apoptosis of VSMCs. The expression levels of JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1, Bcl-2 and survivin were examined by western blot analysis.ResultsTFPI protein was detected in the TFPI group after gene transfer and the peak expression was at the 3rd day. At the 3rd, 5th and 7th days after gene transfer, the apoptotic rates by TUNEL assay in the TFPI group were 10.91 ± 1.66%, 13.46 ± 1.28% and 17.04 ± 1.95%, respectively, whereas those in the LacZ group were 3.28 ± 0.89%, 4.01 ± 0.72% and 4.89 ± 1.17%, respectively. We observed cell contraction, slight mitochondrial swelling, nuclear pyknosis and apoptotic body formation in TFPI-treated VSMCs using electron microscopy. JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1 and Bcl-2, which are all involved in the JAK-2/STAT-3 pathway, were detected in the VSMCs on the 3rd, 5th and 7th days after gene transfer, which is consistent with previously demonstrated time points when VSMCs apoptosis occurred. The expression levels of p-JAK-2, p-STAT-3, cyclin D1 and Bcl-2 were significantly decreased over time in the TFPI group (each P < 0.05) but not in the Ad-LacZ and DMEM groups. However, this attenuation of expression was not observed for JAK-2 and STAT-3 in any of the groups at any time points after gene transfer (each P > 0.05). The expression level of survivin in the TFPI group also weakened significantly over time compared with the levels in the Ad-LacZ and DMEM groups (each P < 0.05) at the 3rd, 5th and 7th days after gene transfer.ConclusionThe results demonstrated that TFPI played an apoptosis-inducing role in VSMCs in a manner that involves both the suppression of JAK-2/STAT-3 pathway phosphorylation and the down-regulation of survivin. Our data show for the first time that targeting the JAK-2/STAT-3 pathway and survivin by overexpressing TFPI may be a new avenue for the treatment of restenosis.     

Induced Pluripotent Stem Cells Are Sensitive to DNA Damage

Induced pluripotent stem cells(iPSCs)resemble embryonic stem cells(ESCs)in morphology,gene expression and in vitro differentiation,raising new hope for personalized clinical therapy.While many efforts have been made to improve reprogramming effciency,signifcant problems such as genomic instability of iPSCs need to be addressed before clinical therapy.In this study,we try to fgure out the real genomic state of iPSCs and their DNA damage response to ionizing radiation(IR).We found that iPSC line 3FB4-1 had lower DNA damage repair ability than mouse embryonic fbroblast(MEF)cells,from which 3FB4-1line was derived.After the introduction of DNA damage by IR,the number of c-H2AX foci in 3FB4-1 increased modestly compared to a large increase seen in MEF,albeit both signifcantly(P<0.01).In addition,whole-genome sequencing analysis showed that after IR,3FB4-1 possessed more point mutations than MEF and the point mutations spread all over chromosomes.These observations provide evidence that iPSCs are more sensitive to ionizing radiation and their relatively low DNA damage repair capacity may account for their high radiosensitivity.The compromised DNA damage repair capacity of iPSCs should be considered when used in clinical therapy.     

Metabolism of the synthetic progestogen norethynodrel by human ketosteroid reductases of the aldo-keto reductase superfamily

Menopause is associated with changes in lipid levels resulting in increased risk of atherosclerosis and cardiovascular events. Hormone therapy (HT) and atorvastatin have been used to improve lipid profile in postmenopausal women.Effects of HT, atorvastatin and APOE polymorphisms on serum lipids and APOE and LXRA expression were evaluated in 87 hypercholesterolemic postmenopausal women, randomly selected for treatment with atorvastatin (AT, n = 17), estrogen or estrogen plus progestagen (HT, n = 34) and estrogen or estrogen plus progestagen associated with atorvastatin (HT + AT, n = 36). RNA was extracted from peripheral blood mononuclear cells (PBMC) and mRNA expression was measured by TaqMan^® PCR. APOE ?2/?3/?4 genotyping was performed using PCR-RFLP.Total cholesterol (TC), LDL-c and apoB were reduced after each treatment (p < 0.001). Triglycerides, VLDL-c and apoAI were reduced only after atorvastatin (p < 0.05), whereas triglycerides and VLDL-c were increased after HT (p = 0.01). HT women had lower reduction on TC, LDL-c and apoB than AT and HT + AT groups (p < 0.05). APOE mRNA expression was reduced after atorvastatin treatment (p = 0.03). Although LXRA gene expression was not modified by atorvastatin, it was correlated with APOE mRNA before and after treatments. Basal APOE mRNA expression was not influenced by gene polymorphisms, however the reduction on APOE expression was more pronounced in ?3?3 than in ?3?4 carriers.Atorvastatin down-regulates APOE mRNA expression and it is modified by APOE genotypes in PBMC from postmenopausal women.     

Role of damage-specific DNA polymerases in M13 phage mutagenesis induced by a major lipid peroxidation product trans-4-hydroxy-2-nonenal

One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G > C ? A > T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZα gene of M13mp18 phage, whose DNA was treated in vitro with HNE, and which was grown in uvrA^? Escherichia coli strains, carrying one, two or all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II.Mutation spectrum established for strains expressing only Pol V, showed that in uvrA^? bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:T → C:G, A:T → G:C, G:C → A:T and G:C → T:A prevailed.The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent.     

BRCA1 promoter methylation in peripheral blood DNA was identified in sporadic breast cancer and controls

Objective Epigenetics, particularly DNA methylation, has recently been shown to be important in breast cancer initiation. We investigated the clinical and prognostic importance of whole blood breast cancer early onset gene 1 (BRCA1) DNA methylation in sporadic breast cancer. Methods Genomic DNA was extracted from the peripheral blood cells (PBCs) of 902 breast cancer patients at diagnosis, with no BRCA1 mutation, and 990 control women. DNA methylation was measured by quantitative analysis of methylated alleles (QAMA) to estimate the extent of methylation of 2 CpG sites in the promoter region of BRCA1 oncosuppressor. Results BRCA1 promoter methylation rate in PBCs was 47.1% with a 95% confidence interval [46.1; 48.1] in breast cancer patients, and 45.9% with a 95% confidence interval [45.0; 46.8] in controls. We found a trend toward BRCA1 promoter hypermethylation in PBCs of sporadic breast cancer patients compared with controls. Association between methylation and clinicopathological features was evaluated using statistical tests. BRCA1 promoter methylation in PBCs increased significantly in breast cancer patients compared with controls, for age over 70 years (p = 0.022), in post-menopausal status (p = 0.013), for a body mass index (BMI) <20 (p = 0.0095) or a waist-to-hip ratio (WHR) ≤76.8 (p = 0.0027). We also found an association of increased BRCA1 promoter methylation in PBCs with ACA/ACA genotype for the SNP Thr594Thr in ESR (estrogen receptor gene), known to be associated with breast cancer risk (p = 0.092), reflecting the reduced presence of this genotype in this breast cancer case-control study. Conclusion Analysis of site-specific DNA methylation in PBCs by QAMA provides quantitative DNA methylation values that may serve as important prognostic indicators.     

Investigation of porcine circovirus contamination in human vaccines

DNA from porcine circovirus type 1 (PCV1) and 2 (PCV2) has recently been detected in two vaccines against rotaviral gastroenteritis from manufacturers A and B. We investigated if PCV1 sequences are present in other viral vaccines. We screened seeds, bulks and final vaccine preparations from ten manufacturers using qRT-PCR. We detected 3.8 × 10³ to 1.9 × 10⁷ PCV1 DNA copies/milliliter in live poliovirus seeds for inactivated polio vaccine (IPV) from manufacturer A, however, following inactivation and purification, the finished IPV was PCV1-negative. PCV1 DNA was not detectable in live polio preparations from other vaccine producers. There was no detectable PCV1 DNA in the measles, mumps, rubella and influenza vaccines analysed including material supplied by manufacturer A. We confirmed that the PCV1 genome in the rotavirus vaccine from manufacturer A is near full-length. It contains two mutations in the PCV cap gene, which may result from viral adaptation to Vero cells. Bulks of this vaccine contained 9.8 × 10¹⁰ to 1.8 × 10¹¹ PCV1 DNA copies/millilitre and between 4.1 × 10⁷ and 5.5 × 10⁸ DNA copies were in the final doses. We found traces of PCV1 and PCV2 DNA in the rotavirus vaccine from manufacturer B. This highlights the issue of vaccine contamination and may impact on vaccine quality control.