2-Methoxymethylestradiol: a new 2-methoxy estrogen analog that exhibits antiproliferative activity and alters tubulin dynamics

An estradiol metabolite, 2-methoxyestradiol (2-MeOE(2)), has shown antiproliferative effects in both hormone-dependent and hormone-independent breast cancer cells. Previously, a series of 2-hydroxyalkyl estradiol analogs had been synthesized in our laboratories as potential probes for comparison of estrogen receptor (ER)-mediated versus non-ER-mediated effects in breast cancer cells. A methoxy derivative of 2-hydroxymethyl estradiol was prepared for biological evaluation and comparison with 2-MeOE(2). Estrogenic activity of the synthetic analogs was evaluated in two ways, one by examining affinity of the analogs for the estrogen receptor in MCF-7 cells and the other by examining the ability of the analogs to induce estrogen-responsive gene expression. The analog, 2-methoxymethyl estradiol (2-MeOMeE(2)), demonstrated weak affinity for the estrogen receptor (0.9% of estradiol) and weak ability to stimulate estrogen-induced expression of the pS2 gene (0.02% of estradiol). Antitumor activity was evaluated both in vitro and in vivo. The steroidal nucleus seems to be an attractive target for developing novel tubulin polymerization inhibitors. Additionally, such steroidal compounds may have low toxicity compared to the natural products known to interact with tubulin. Interestingly, 2-MeOMeE(2) inhibited tubulin polymerization in vitro at concentrations of 1 and 3 microM and was more effective than 2-MeOE(2). In cells, 2-MeOMeE(2) was effective in suppressing growth and inducing cytotoxicity in MCF-7 and MDA-MB-231 breast cancer cells. The cytotoxic effects of 2-MeOMeE(2) are associated with alterations in tubulin dynamics, with the frequent appearance of misaligned chromosomes, a significant mitotic delay, and the formation of multinucleated cells. In comparison, 2-MeOE(2) was more effective than 2-MeOMeE(2) in producing cytotoxicity and altering tubulin dynamics in intact cells. Assessment of in vivo antitumor activity was performed in athymic mice containing human breast tumor xenografts. Nude mice bearing MDA-MB-435 tumor xenografts were treated i.p. with 50 mg/kg per day of 2-MeOMeE(2) or vehicle control for 45 days. Treatment with 2-MeOMeE(2) resulted in an approximate 50% reduction in mean tumor volume at treatment day 45 when compared to control animals and had no effect on animal weight. Thus, 2-MeOMeE(2) is an estrogen analog with minimal estrogenic properties that demonstrates antiproliferative effects both in vitro and in the human xenograft animal model of human breast cancer.     

Mitochondrial DNA Released by Trauma Induces Neutrophil Extracellular Traps

Neutrophil extracellular traps (NETs) are critical for anti-bacterial activity of the innate immune system. We have previously shown that mitochondrial damage-associated molecular patterns (mtDAMPs), including mitochondrial DNA (mtDNA), are released into the circulation after injury. We therefore questioned whether mtDNA is involved in trauma-induced NET formation. Treatment of human polymorphoneutrophils (PMN) with mtDNA induced robust NET formation, though in contrast to phorbol myristate acetate (PMA) stimulation, no NADPH-oxidase involvement was required. Moreover, formation of mtDNA-induced NETs was completely blocked by TLR9 antagonist, ODN-TTAGGG. Knowing that infective outcomes of trauma in elderly people are more severe than in young people, we measured plasma mtDNA and NET formation in elderly and young trauma patients and control subjects. MtDNA levels were significantly higher in the plasma of elderly trauma patients than young patients, despite lower injury severity scores in the elderly group. NETs were not visible in circulating PMN isolated from either young or old control subjects. NETs were however, detected in PMN isolated from young trauma patients and to a lesser extent from elderly patients. Stimulation by PMA induced widespread NET formation in PMN from both young volunteers and young trauma patients. NET response to PMA was much less pronounced in both elderly volunteers’ PMN and in trauma patients’ PMN. We conclude that mtDNA is a potent inducer of NETs that activates PMN via TLR9 without NADPH-oxidase involvement. We suggest that decreased NET formation in the elderly regardless of higher mtDNA levels in their plasma may result from decreased levels of TLR9 and/or other molecules, such as neutrophil elastase and myeloperoxidase that are involved in NET generation. Further study of the links between circulating mtDNA and NET formation may elucidate the mechanisms of trauma-related organ failure as well as the greater susceptibility to secondary infection in elderly trauma patients.     

Enhanced G2/M Arrest,Caspase Related Apoptosis and Reduced E-Cadherin Dependent Intercellular Adhesion by Trabectedin in Prostate Cancer Stem Cells

Trabectedin (Yondelis, ET-743) is a marine-derived tetrahydroisoquinoline alkaloid. It is originally derived from the Caribbean marine tunicate Ecteinascidia turbinata and currently produced synthetically. Trabectedin is active against a variety of tumor cell lines growing in culture. The present study focused on the effect of trabectedin in cell proliferation, cell cycle progression, apoptosis and spheroid formation in prostate cancer stem cells (CSCs). Cluster of differentiation (CD) 133^+high/CD44^+high prostate CSCs were isolated from the DU145 and PC-3 human prostate cancer cell line through flow cytometry. We studied the growth-inhibitory effects of trabectedin and its molecular mechanisms on human prostate CSCs and non-CSCs. DU-145 and PC-3 CSCs were treated with 0.1, 1, 10 and 100 nM trabectedin for 24, 48 and 72 h and the growth inhibition rates were examined using the sphere-forming assay. Annexin-V assay and immunofluorescence analyses were performed for the detection of the cell death. Concentration-dependent effects of trabectedin on the cell cycle were also evaluated. The cells were exposed to the different doses of trabectedin for 24, 48 and 72 h to evaluate the effect of trabectedin on the number and diameter of spheroids. According to the results, trabectedin induced cytotoxicity and apoptosis at the IC₅₀ dose, resulting in a significant increase expression of caspase-3, caspase-8, caspase-9, p53 and decrease expression of bcl-2 in dose-dependent manner. Cell cycle analyses revealed that trabectedin induces dose-dependent G2/M-phase cell cycle arrest, particularly at high-dose treatments. Three-dimensional culture studies showed that trabectedin reduced the number and diameter of spheroids of DU145 and PC3 CSCs. Furthermore, we have found that trabectedin disrupted cell-cell interactions via E-cadherin in prostasphere of DU-145 and PC-3 CSCs. Our results showed that trabectedin inhibits cellular proliferation and accelerates apoptotic events in prostate CSCs; and may be a potential effective therapeutic agent against prostate cancer.     

Nucleotide binding interactions modulate dNTP selectivity and facilitate 8-oxo-dGTP incorporation by DNA polymerase lambda

8-Oxo-7,8,-dihydro-2′-deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol λ) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol λ. We have solved high resolution crystal structures showing how Pol λ accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn-conformation, and is stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala⁵¹⁰ and Asn⁵¹³, play differential roles in dNTP selectivity. Specifically, Ala⁵¹⁰ and Asn⁵¹³ facilitate incorporation of 8-oxo-dGMP opposite dA and dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol λ and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases.     

A new modified concomitant therapy for Helicobacter pylori eradication in Turkey

Background: Helicobacter pylori eradication rates have tended to decrease recently, mostly due to antibiotic resistance. In the present study, our aim was to determine Hp eradication rate with the LAC plus tid metronidazole regimen and the secondary objective of this study was to identify an effective regimen for our population. Methods: Eighty‐four Hp‐positive patients with non‐ulcer dyspepsia were assigned into the same group. Patients were administered the classical LAC protocole (lansoprazole 30 mg bid, amoxicillin 1 g bid and claritromycin 500 mg bid for 14 days) plus metronidazole 500 mg tid for 14 days. Gastroscopy and histopathological assessment were performed before enrollment and C¹⁴ urea breath test and stool antigen test were performed 6 weeks after treatment. Results: All 84 patients completed the study. No patient left the study because of drug side effect. Total eradication rate was 75% (63/84). Conclusion: Although LAC plus tid metronidazole regimen achieved a much better eradication rate compared with the standard LAC regimen; this is the first study that has a relatively low success with a concomitant therapy. So in areas of high resistance like Turkey, one cannot expect a high success with any clarithromycin containing regimen and those should be avoided.     

Detection of arbuscular mycorrhizal fungi within colonised roots of the Gramineae family members by nested-polymerase chain reaction (PCR)

In this study, it is aimed to asses the association of arbuscular mycorrhizal (AM) fungi within colonised rhizosphere of Gramineae family members through a survey by using nested- polymerase chain reaction method in Van province (Turkey). From 24 agro-ecological fields, a total of 82 samples belonging to Gramineae family were tested by molecular methods. The presence of Glomus intraradices and Glomus mosseae was ascertained in 10 plants belonging to eight different species by using fungus specific primers. Root colonisation ranged from 6 to 37% within rhizosphere of Gramineae family members and the average root colonisation by AM fungi was 22%.     

Effects of protonation state of Asp181 and position of active site water molecules on the conformation of PTP1B

In protein tyrosine phosphatase 1B (PTP1B), the flexible WPD loop adopts a closed conformation (WPD_closed) in the active state of PTP1B, bringing the catalytic Asp181 close to the active site pocket, while WPD loop is in an open conformation (WPD_open) in the inactive state. Previous studies showed that Asp181 may be protonated at physiological pH, and ordered water molecules exist in the active site. In the current study, molecular dynamics simulations are employed at different Asp181 protonation states and initial positions of active site water molecules, and compared with the existing crystallographic data of PTP1B. In WPD_closed conformation, the active site is found to maintain its conformation only in the protonated state of Asp181 in both free and liganded states, while Asp181 is likely to be deprotonated in WPD_open conformation. When the active site water molecule network that is a part of the free WPD_closed crystal structure is disrupted, intermediate WPD loop conformations, similar to that in the PTPRR crystal structure, are sampled in the MD simulations. In liganded PTP1B, one active site water molecule is found to be important for facilitating the orientation of Cys215 and the phosphate ion, thus may play a role in the reaction. In conclusion, conformational stability of WPD loop, and possibly catalytic activity of PTP1B, is significantly affected by the protonation state of Asp181 and position of active site water molecules, showing that these aspects should be taken into consideration both in MD simulations and inhibitor design. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Life cycle sustainability assessment of autonomous heavy‐duty trucks

Connected and automated vehicles (CAVs) are emerging technologies expected to bring important environmental, social, and economic improvements in transportation systems. Given their implications in terms of air quality and sustainable and safer movement of goods, heavy‐duty trucks (HDTs), carrying the majority of U.S. freight, are considered an ideal domain for the application of CAV technology. An input–output (IO) model is developed based on the Eora database—a detailed IO database that consists of national IO tables, covering almost the entire global economy. Using the Eora‐based IO model, this study quantifies and assesses the environmental, economic, and social impacts of automated diesel and battery electric HDTs based on 20 macro‐level indicators. The life cycle sustainability performances of these HDTs are then compared to that of a conventional diesel HDT. The study finds an automated diesel HDT to cause 18% more fatalities than an automated electric HDT. The global warming potential (GWP) of automated diesel HDTs is estimated to be 4.7 thousand metric tons CO₂‐eq. higher than that of automated electric HDTs. The health impact costs resulting from an automated diesel HDT are two times higher than that of an automated electric HDT. Overall, the results also show that automation brings important improvements to the selected sustainability indicators of HDTs such as global warming potential, life cycle cost, GDP, decrease in import, and increase in income. The findings also show that there are significant trade‐offs particularly between mineral and fossil resource losses and environmental gains, which are likely to complicate decision‐making processes regarding the further development and commercialization of the technology.