Oleuropein inhibits migration ability through suppression of epithelial-mesenchymal transition and synergistically enhances doxorubicin-mediated apoptosis in MCF-7 cells

Distinct metastasis is one of the main causes of breast cancer (BC)-related mortality and epithelial-mesenchymal transition (EMT) is a primary step in metastasis dissemination. On the other hand, doxorubicin (DOX) is an effective chemotherapeutic agent against BC; unfortunately, its clinical use is limited by dose-dependent side effects. Therefore, extensive efforts have been dedicated to suppressing metastasis of BC and also to overcome DOX side effects together with keeping its antitumor efficacy. Studies supported the role of oleuropein (OLEU) in reducing DOX-induced side effects besides its antitumor actions. In this study, the antimigratory effect of OLEU was assessed and real-time PCR (RT-PCR) was used to detect OLEU effect on the expression level of EMT markers, in MCF-7 cells. The cytotoxic effect of OLEU and DOX was assessed by MTT assay, whereas the ratio of apoptosis was investigated by flow cytometry. The results showed that migration ability of MCF-7 cells remarkably decreased in OLEU treated group and RT-PCR results showed that OLEU may exert its antimigratory action by suppressing EMT through downregulation of sirtuin1 (SIRT1). Also, the results indicated that both OLEU and DOX were cytotoxic to MCF-7 cells, whereas DOX-OLEU cotreatment led to additive cytotoxicity and apoptosis rate. This study provides evidence regarding the suppressive role of OLEU on MCF-7 cells migration ability through suppression of EMT, and for the first time, it was proposed that SIRT1 downregulation can be involved in the OLEU antimigratory effect. Also, the findings demonstrated that OLEU can reduce DOX-induced side effects by reducing its effective dose.     

Antibody–drug conjugates (ADCs) for cancer therapy: Strategies,challenges, and successes

Targeted delivery of therapeutic molecules into cancer cells is considered as a promising strategy to tackle cancer. Antibody–drug conjugates (ADCs), in which a monoclonal antibody (mAb) is conjugated to biologically active drugs through chemical linkers, have emerged as a promising class of anticancer treatment agents, being one of the fastest growing fields in cancer therapy. The failure of early ADCs led researchers to explore strategies to develop more effective and improved ADCs with lower levels of unconjugated mAbs and more-stable linkers between the drug and the antibody, which show improved pharmacokinetic properties, therapeutic indexes, and safety profiles. Such improvements resulted in the US Food and Drug Administration approvals of brentuximab vedotin, trastuzumab emtansine, and, more recently, inotuzumab ozogamicin. In addition, recent clinical outcomes have sparked additional interest, which leads to the dramatically increased number of ADCs in clinical development. The present review explores ADCs, their main characteristics, and new research developments, as well as discusses strategies for the selection of the most appropriate target antigens, mAbs, cytotoxic drugs, linkers, and conjugation chemistries.     

Radioprotective effect of olanzapine as an anti-psychotic drug against genotoxicity and apoptosis induced by ionizing radiation on human lymphocytes

Molecular Biology Reports - Olanzapine (OLA), is prescribed as an anti-psychotic medicine in schizophrenia patients. In this study, the protective effect of OLA against genotoxicity and...     

Correction to: Effects of Exogenous Spermidine and Elevated CO2 on Physiological and Biochemical Changes in Tomato Plants Under Iso-osmotic Salt Stress

Journal of Plant Growth Regulation - The original version of this article unfortunately contained errors in two authors' names. The given and family names of the authors were incorrectly...     

Initial reaction(s) in biotransformation of CL-20 is catalyzed by salicylate 1-monooxygenase from Pseudomonas sp. strain ATCC 29352

CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) (C(6)H(6)N(12)O(12)), a future-generation high-energy explosive, is biodegradable by Pseudomonas sp. strain FA1 and Agrobacterium sp. strain JS71; however, the nature of the enzyme(s) involved in the process was not understood. In the present study, salicylate 1-monooxygenase, a flavin adenine dinucleotide (FAD)-containing purified enzyme from Pseudomonas sp. strain ATCC 29352, biotransformed CL-20 at rates of 0.256 +/- 0.011 and 0.043 +/- 0.003 nmol min(-1) mg of protein(-1) under anaerobic and aerobic conditions, respectively. The disappearance of CL-20 was accompanied by the release of nitrite ions. Using liquid chromatography/mass spectrometry in the negative electrospray ionization mode, we detected a metabolite with a deprotonated mass ion [M - H](-) at 345 Da, corresponding to an empirical formula of C(6)H(6)N(10)O(8), produced as a result of two sequential N denitration steps on the CL- 20 molecule. We also detected two isomeric metabolites with [M - H](-) at 381 Da corresponding to an empirical formula of C(6)H(10)N(10)O(10). The latter was a hydrated product of the metabolite C(6)H(6)N(10)O(8) with addition of two H(2)O molecules, as confirmed by tests using (18)O-labeled water. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.7 nitrite ions, 3.2 molecules of nitrous oxide, 1.5 molecules of formic acid, and 0.6 ammonium ion. Diphenyliodonium-mediated inhibition of salicylate 1-monooxygenase and a comparative study between native, deflavo, and reconstituted enzyme(s) showed that FAD site of the enzyme was involved in the biotransformation of CL-20 catalyzed by salicylate 1-monooxygenase. The data suggested that salicylate 1-monooxygenase catalyzed two oxygen-sensitive single-electron transfer steps necessary to release two nitrite ions from CL-20 and that this was followed by the secondary decomposition of this energetic chemical.     

Parathyroid hormone regulation of type II sodium-phosphate cotransporters is dependent on an A kinase anchoring protein

Parathyroid hormone inhibits sodium-phosphate cotransport in proximal renal tubule cells through activation of several kinases. We tested the hypothesis that the activity of these kinases was coordinated by an A kinase anchoring protein (AKAP) by demonstrating that the type II sodium-phosphate cotransporter (NaPi-4) physically associated with an AKAP and that this association was necessary for regulation of phosphate transport by parathyroid hormone. Immunoprecipitation with anti-NaPi-4 antiserum and glutathione S-transferase pull-down with GST-NaPi-4 showed that NaPi-4 associated with AKAP79, protein kinase A catalytic and regulatory subunits, and the parathyroid hormone receptor in opossum kidney cells. When the regulatory subunit of protein kinase A was uncoupled from the AKAP by a competing peptide, parathyroid hormone lost the ability to inhibit phosphate transport. This result was confirmed by co-transfecting HEK293 cells with the sodium-phosphate cotransporter and wild type AKAP, a mutant AKAP79, or the empty vector. 8-Bromo-cAMP was able to inhibit phosphate transport in cells expressing the wild type AKAP79 but not empty vector or mutant AKAP79. We conclude that parathyroid hormone inhibits proximal renal tubule sodium-phosphate cotransport through a signaling complex dependent upon an AKAP.     

Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450: insight into the mechanism of RDX biodegradation by Rhodococcus sp. strain DN22

A unique metabolite with a molecular mass of 119 Da (C(2)H(5)N(3)O(3)) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.     

Biotransformation of 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) by denitrifying Pseudomonas sp. strain FA1

The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood. Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment. In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source, Pseudomonas sp. strain FA1, was isolated from a garden soil. Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation. An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells. The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 +/- 0.1 nmol h(-1) mg of cell biomass(-1) and 11.5 +/- 0.4 nmol h(-1) mg of protein(-1), respectively, under anaerobic conditions. In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO(2)(-)), 1.5 molecules of nitrous oxide (N(2)O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule. The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions. A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme. The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20.     

Fate of explosives and their metabolites in bioslurry treatment processes

Microcosm tests simulating bioslurry reactors with 40% soilcontent, containing high concentrations of TNT and/or RDX,and spiked with either [¹⁴C]-TNT or[¹⁴C]-RDX were conducted to investigate the fate ofexplosives and their metabolites in bioslurry treatment processes.RDX is recalcitrant to indigenous microorganisms in soil andactivated sludge under aerobic conditions. However, soilindigenous microorganisms alonewere able to mineralize 15% of RDX to CO₂ underanaerobic condition, and supplementation of municipal anaerobicsludge as an exogenous source of microorganismssignificantly enhanced the RDX mineralization to 60%. RDXmineralizing activity of microorganisms in soil and sludge wassignificantly inhibited by the presence of TNT. TNTmineralization was poor (< 2%) and was not markedlyimproved by the supplement ofaerobic or anaerobic sludge. Partitioning studies of[¹⁴C]-TNT in the microcosmsrevealed that the removal of TNTduring the bioslurry process was due mainly to thetransformation of TNT and irreversiblebinding of TNT metabolites onto soil matrix. In the case ofRDX under anaerobic conditions,a significant portion (35%) of original radioactivity wasalso incorporated into the biomass andbound to the soil matrix.     

Complement Activation in Patients with Focal Segmental Glomerulosclerosis

Background

Recent pre-clinical studies have shown that complement activation contributes to glomerular and tubular injury in experimental FSGS. Although complement proteins are detected in the glomeruli of some patients with FSGS, it is not known whether this is due to complement activation or whether the proteins are simply trapped in sclerotic glomeruli. We measured complement activation fragments in the plasma and urine of patients with primary FSGS to determine whether complement activation is part of the disease process.

Study Design

Plasma and urine samples from patients with biopsy-proven FSGS who participated in the FSGS Clinical Trial were analyzed.

Setting and Participants

We identified 19 patients for whom samples were available from weeks 0, 26, 52 and 78. The results for these FSGS patients were compared to results in samples from 10 healthy controls, 10 patients with chronic kidney disease (CKD), 20 patients with vasculitis, and 23 patients with lupus nephritis.

Outcomes

Longitudinal control of proteinuria and estimated glomerular filtration rate (eGFR).

Measurements

Levels of the complement fragments Ba, Bb, C4a, and sC5b-9 in plasma and urine.

Results

Plasma and urine Ba, C4a, sC5b-9 were significantly higher in FSGS patients at the time of diagnosis than in the control groups. Plasma Ba levels inversely correlated with the eGFR at the time of diagnosis and at the end of the study. Plasma and urine Ba levels at the end of the study positively correlated with the level of proteinuria, the primary outcome of the study.

Limitations

Limited number of patients with samples from all time-points.

Conclusions

The complement system is activated in patients with primary FSGS, and elevated levels of plasma Ba correlate with more severe disease. Measurement of complement fragments may identify a subset of patients in whom the complement system is activated. Further investigations are needed to confirm our findings and to determine the prognostic significance of complement activation in patients with FSGS.