Publications by authors named "Ana Damjanovic"

45 Publications

White blood cell subsets in HER2-positive breast cancer patients treated with trastuzumab in relation to clinical outcome.

Pathol Res Pract 2021 Jul 4;224:153543. Epub 2021 Jul 4.

Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia.

To examine whether HER2+ breast cancer patients who have decreased immune effector cells could respond well to trastuzumab, we evaluated the alterations in circulating immune system cell subsets: CD16+ and/or CD56+ lymphocytes, lymphocytes and granulocytes in these patients before and after treatment with trastuzumab-based regimens in relation to clinical response to therapy. The study involved 55 patients with HER2+ breast cancer before and 2 months after the initiation of the therapy. Progressive disease was confirmed in nine out of 55 patients (non-responders), while other patients achieved complete or partial response, or stable disease (responders). Control group consisted of up to 52 healthy individuals. Significantly lower percentages of total lymphocytes, CD16+, CD56+, and CD16+CD56+ lymphocytes as well as higher percentage of granulocytes and a higher ratio of granulocyte to lymphocyte percentages were found in patients before therapy and 2 months after the initiation of the therapy, compared with those in healthy individuals. Responder subgroup showed significantly lower percentages of CD16+, CD56+, and CD16+CD56+ lymphocytes before therapy, compared with those in healthy controls. Two months after the initiation of the therapy, the percentages of immune cell subsets remained significantly lower in responders in comparison with those in the healthy donors, while a significantly decreased percentages of CD56+ and CD16+CD56+ lymphocytes were observed in non-responders, in comparison with those in healthy controls. Our study demonstrated that HER2+ breast cancer patients who have decreased percentages of CD16+, CD56+, and CD16+CD56+ lymphocytes may achieve response to trastuzumab-containing treatment.
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http://dx.doi.org/10.1016/j.prp.2021.153543DOI Listing
July 2021

Determinants of conductance of a bacterial voltage-gated sodium channel.

Biophys J 2021 Jun 30. Epub 2021 Jun 30.

Laboratory of Computational Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; Department of Biophysics, Johns Hopkins University, Baltimore, Maryland. Electronic address:

Through molecular dynamics (MD) and free energy simulations in electric fields, we examine the factors influencing conductance of bacterial voltage-gated sodium channel NaMs. The channel utilizes four glutamic acid residues in the selectivity filter (SF). Previously, we have shown, through constant pH and free energy calculations of pKa values, that fully deprotonated, singly protonated, and doubly protonated states are all feasible at physiological pH, depending on how many ions are bound in the SF. With 173 MD simulations of 450 or 500 ns and additional free energy simulations, we determine that the conductance is highest for the deprotonated state and decreases with each additional proton bound. We also determine that the pKa value of the four glutamic residues for the transition between deprotonated and singly protonated states is close to the physiological pH and that there is a small voltage dependence. The pKa value and conductance trends are in agreement with experimental work on bacterial Na channels, which show a decrease in maximal conductance with lowering of pH, with pKa in the physiological range. We examine binding sites for Na in the SF, compare with previous work, and note a dependence on starting structures. We find that narrowing of the gate backbone to values lower than the crystal structure's backbone radius reduces the conductance, whereas increasing the gate radius further does not affect the conductance. Simulations with some amount of negatively charged lipids as opposed to purely neutral lipids increases the conductance, as do simulations at higher voltages.
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http://dx.doi.org/10.1016/j.bpj.2021.06.013DOI Listing
June 2021

3D HeLa spheroids as a model for investigating the anticancer activity of Biginelli-hybrids.

Chem Biol Interact 2021 Aug 20;345:109565. Epub 2021 Jun 20.

Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000, Belgrade, Serbia. Electronic address:

In previous study, we examined the anticancer effects of novel Biginelli-hybrids against HeLa cell line on 2D monolayer culture. The five most effective compounds were chosen for further analysis of their anticancer activity against HeLa spheroids. Using the 3D models implies the possible differences in anticancer effects and mechanisms of activity of tested compounds. The compounds 4c and 4d exerted the strongest activity against 3D HeLa spheroids and induced to some extent loosened cell-cell contacts in spheroids, leading to the largest reduction in the diameter of the spheroids. Additionally, the highest accumulation of the cells in the subG1 phase of the cell cycle was observed after the treatment with compounds 4d and 4c, while the compound 4f led to the G2/M arrest. The invasion potential of treated HeLa cells in spheroids was monitored by imaging of spheroids embedded in a matrix made of matrigel and collagen and by determination of MMP2, MMP9, and VEGF gene expression levels. The compound 4l did not show invasion-suppressive activity, while the compounds 4c and 4d exerted the strongest anti-invasive activity.
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http://dx.doi.org/10.1016/j.cbi.2021.109565DOI Listing
August 2021

Cytotoxic activities of L. extracts against 2D and 3D cancer cell models.

Cytotechnology 2021 Jun 1;73(3):373-389. Epub 2021 Apr 1.

Institute of Oncology and Radiology of Serbia, Pasterova 14, Belgrade, Serbia.

Six extracts were obtained from plant species L., collected at Samsun in Turkey. The aim of this study was to examine the mechanisms of the anticancer activity of these extracts. Methanol, ethyl-acetate and hexane were used as a solvents for extraction from both branch-body part of the plant (extracts 1, 2 and 3) and from plant flowers (extracts 4, 5 and 6). The cytotoxic effects of the extracts were determined against 2D and 3D cancer cell models. Cell cycle changes of treated HeLa cells were analyzed by flow cytometry. Measurements of gene and microRNA expression levels in treated HeLa cells were done by quantitative real time PCR. Five examined extracts (2-6) exerted selective concentration-dependent cytotoxic effects on HeLa, K562, and A549 cancer cells, while the extract 1 exhibited very weak cytotoxicity. The extract 6 showed the highest intensity of cytotoxic activity. All tested extracts (2-6) demonstrated the ability to induce apoptosis in HeLa cells through activation of caspase-3. These extracts remarkably decreased gene expression levels of , , , and in HeLa cells. Flower extracts might have stronger effects on miR128/193a-5p/335 level changes than branch-body extracts. extracts exerted weaker cytotoxic effects on 3D HeLa spheroids when compared with their effects on 2D monolayer HeLa cells. Taken together, results of our research may suggest the promising anticancer properties of the extracts.
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http://dx.doi.org/10.1007/s10616-021-00464-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167025PMC
June 2021

Influence of selective acid-etching on functionality of halloysite-chitosan nanocontainers for sustained drug release.

Mater Sci Eng C Mater Biol Appl 2021 Apr 10;123:112029. Epub 2021 Mar 10.

University of Belgrade, Faculty of Pharmacy, Vojvode Stepe 450, 11 221 Belgrade, Serbia.

The functionality of halloysite (Hal) nanotubes as drug carriers can be improved by lumen enlargement and polymer modification. This study investigates the influence of selective acid etching on Hal functionalization with cationic biopolymer chitosan. Hal was subjected to lumen etching under mild conditions, loaded under vacuum with nonsteroidal antiinflammatory drug aceclofenac, and incubated in an acidic solution of chitosan. The functionality of pristine and etched Hal before and upon polymer functionalization was assessed by ζ-potential measurements, structural characterization (FT-IR, DSC and XRPD analysis), cell viability assay, drug loading and drug release studies. Acid etching increased specific surface area, pore volume and pore size of Hal, decreased ζ-potential and facilitated binding of the cationic polymer. XRPD and DSC analysis revealed crystalline structure of etched Hal. Successful chitosan binding and drug entrapment were further confirmed by FT-IR and DSC studies. XRPD showed surface polymer binding. DSC and FT-IR analyses confirmed the presence of the entrapped drug in its crystalline form. Drug loading was increased for ≈81% by selective lumen etching. Slight decrease of drug content occurred during chitosan functionalization due to aceclofenac diffusion in the polymer solution. The drug release was more sustained from etched Hal nanocomposites (up to ≈87% for 12 h) than from pristine Hal (up to ≈97% for 12 h) due to more intensive chitosan binding. High human fibroblast survival rates upon exposure to pristine and etched Hal before and after chitosan functionalization (>90% in the concentration of 1000 μg/mL) confirmed that both lumen etching under mild conditions and polymer functionalization had no significant effect on cytocompatibility. Based on these findings, selective lumen etching in combination with polycation modification appears to be a promising approach for improvement of Hal nanotubes functionality by increasing payload, polymer binding capacity, and sustained release properties with no significant effect on their cytocompatibility.
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http://dx.doi.org/10.1016/j.msec.2021.112029DOI Listing
April 2021

EGFR mutation testing from liquid biopsy of non-small cell lung cancer at the Institute for Oncology and Radiology of Serbia.

J BUON 2020 Nov-Dec;25(6):2635-2642

Department of Experimental Oncology, Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia.

Purpose: Resistance to tyrosine kinase inhibitors (TKIs) in lung cancer often occurs, so mutation testing from liquid biopsy is the method of choice as a minimally invasive approach that quickly provides information for additional therapeutic options. The purpose of this study was to assess the success rate and usefulness of EGFR testing from liquid biopsy at the Institute for Oncology and Radiology of Serbia (IORS).

Methods: EGFR mutation testing was performed by real-time qPCR in 4750 tumor samples using the Cobas® EGFR Mutation Test v2. EGFR testing from 104 liquid biopsy samples was used to track the resistance on first-line EGFR-TKIs as well as for initial testing of 124 patients without tissue biopsies.

Results: Liquid biopsy samples were tested in cases with inadequate material for DNA isolation or without tissue biopsy at diagnosis. Nine mutated samples were detected (7.3 %) with a 99.2 % testing success rate. Testing liquid biopsy samples of patients who progressed on EGFR-TKIs showed an accordance rate of 67% with driver mutations, and 49% of mutated patients had the T790M mutation which rendered them eligible for third-generation EGFR-TKIs. An additional 5 patients tested EGFR wild type from plasma after progression were rebiopsied and 3 of them had the T790M mutation.

Conclusions: EGFR mutation testing from liquid biopsy has been successfully implemented in Serbia and has proven invaluable for detecting molecular resistance mechanisms to EGFR-TKIs and as an alternative sample source for patients with scarce biopsy material or without any at all.
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January 2021

Exploring the real-world effect of the SARS-CoV-2 pandemic on the molecular diagnostics for cancer patients and high-risk individuals.

Expert Rev Mol Diagn 2021 01 21;21(1):101-107. Epub 2020 Dec 21.

Department of Experimental Oncology, Institute for Oncology and Radiology of Serbia, Belgrade, Serbia.

: The SARS-CoV-2 pandemic introduced a global distraction effect in cancer patients' care. The aim of this study was to explore the effect of the pandemic on the largest molecular diagnostics center for cancer patients and high-risk individuals in Serbia.: and mutation testing were performed by qPCR and NGS. NGS was used for panel testing of hereditary breast/ovarian cancer and cancers associated with Lynch syndrome. The analytical output during the state of emergency (SoE) was compared to the period before and after the outbreak using one-way ANOVA. Statistical significance was set at p < 0.05.: A 38% reduction in the number of analysis was detected during the SoE. After the SoE, a 19% reduction was noted compared to SoE and 50% compared to the period before the SoE (p = 0.038). Three of the 48 scheduled appointments for pretest genetic counseling were carried out during the SoE, but the number of NGS tests increased by 50%.: The SARS-CoV-2 pandemic had a profound negative effect on the diagnostic output of our centralized molecular diagnostics center. The only positive effect was shortening of waiting lists for hereditary cancer patients and high-risk individuals.
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http://dx.doi.org/10.1080/14737159.2021.1860760DOI Listing
January 2021

Extracts Promote Doxorubicin Effects against Lung Adenocarcinoma Cells In Vitro.

Molecules 2020 Nov 10;25(22). Epub 2020 Nov 10.

Department for Experimental Oncology, Institute of Oncology and Radiology of Serbia, 11 000 Belgrade, Serbia.

and its secondary metabolites have been shown to have anticancer potential. We performed MTT, scratch, and colony formation assays; analyzed cell cycle phase distribution and doxorubicin uptake and retention with flow cytometry; and detected alterations in the expression of genes involved in the formation of cell-cell interactions and migration using quantitative real-time PCR following treatment of lung adenocarcinoma cells with doxorubicin, extracts, or their combination. MTT assay results suggested strong synergistic effects of the combined treatments, and their application led to an increase in cell numbers in the subG1 phase of the cell cycle. Both extracts were shown to prolong doxorubicin retention time in cancer cells, while the application of doxorubicin/extract combination led to a decrease in expression. Furthermore, cells treated with doxorubicin/extract combinations were shown to have lower migratory and colony formation potentials than untreated cells or cells treated with doxorubicin alone. The obtained results suggest that nontoxic extracts can enhance the activity of doxorubicin, thus potentially allowing the application of lower doxorubicin doses in vivo, which may decrease its toxic effects in normal tissues.
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http://dx.doi.org/10.3390/molecules25225233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697947PMC
November 2020

Protonation state of the selectivity filter of bacterial voltage-gated sodium channels is modulated by ions.

Proteins 2020 03 12;88(3):527-539. Epub 2019 Nov 12.

Laboratory of Computational Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland.

The selectivity filter (SF) of bacterial voltage-gated sodium channels consists of four glutamate residues arranged in a C symmetry. The protonation state population of this tetrad is unclear. To address this question, we simulate the pore domain of bacterial voltage-gated sodium channel of Magnetococcus sp. (Na Ms) through constant pH methodology in explicit solvent and free energy perturbation calculations. We find that at physiological pH the fully deprotonated as well as singly and doubly protonated states of the SF appear feasible, and that the calculated pKa decreases with each additional bound ion, suggesting that a decrease in the number of ions in the pore can lead to protonation of the SF. Previous molecular dynamics simulations have suggested that protonation can lead to a decrease in the conductance, but no pKa calculations were performed. We confirm a decreased ionic population of the pore with protonation, and also observe structural symmetry breaking triggered by protonation; the SF of the deprotonated channel is closest to the C symmetry observed in crystal structures of the open state, while the SF of protonated states display greater levels of asymmetry which could lead to transition to the inactivated state which possesses a C symmetry in the crystal structure. We speculate that the decrease in the number of ions near the mouth of the channel, due to either random fluctuations or ion depletion due to conduction, could be a self-regulatory mechanism resulting in a nonconducting state that functionally resembles inactivated states.
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http://dx.doi.org/10.1002/prot.25831DOI Listing
March 2020

Integration of dry-column flash chromatography with NMR and FTIR metabolomics to reveal cytotoxic metabolites from Amphoricarpos autariatus.

Talanta 2020 Jan 10;206:120248. Epub 2019 Aug 10.

Institute of Chemistry, Technology and Metallurgy, National Institute, University of Belgrade, Studentskitrg12-16, 11000, Belgrade, Serbia. Electronic address:

Metabolomics generate a profile of small molecules from plant extracts, which could be directly responsible for bioactivity effects. Using dry-column flash chromatography enabled a rapid and inexpensive method for the very efficient separation of plant extract with a high resolution. This separation method coupled to NMR and FTIR-based metabolomics is applied to identify bioactive natural products. OPLS multivariate analysis method, was used for correlation the chemical composition of the plant extracts, Amphoricarpos autariatus, with the results of cytotoxic activity against Human cervical adenocarcinoma cell line (HeLa) and epithelial lung cancer cell line (A549). In this way, the highest contribution to the cytotoxic activity was recorded for the guaianolide sesquiterpene lactones named amphoricarpolides. The compounds indicated as bioactive after metabolomics analysis were tested, and their cytotoxic activity were confirmed.
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http://dx.doi.org/10.1016/j.talanta.2019.120248DOI Listing
January 2020

Mechanisms of Chemotherapy Resistance in Triple-Negative Breast Cancer-How We Can Rise to the Challenge.

Cells 2019 08 22;8(9). Epub 2019 Aug 22.

Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia.

Triple-negative (TNBC) is the most lethal subtype of breast cancer owing to high heterogeneity, aggressive nature, and lack of treatment options. Chemotherapy remains the standard of care for TNBC treatment, but unfortunately, patients frequently develop resistance. Accordingly, in recent years, tremendous effort has been made into elucidating the mechanisms of TNBC chemoresistance with the goal of identifying new molecular targets. It has become evident that the development of TNBC chemoresistance is multifaceted and based on the elaborate interplay of the tumor microenvironment, drug efflux, cancer stem cells, and bulk tumor cells. Alterations of multiple signaling pathways govern these interactions. Moreover, TNBC's high heterogeneity, highlighted in the existence of several molecular signatures, presents a significant obstacle to successful treatment. In the present, in-depth review, we explore the contribution of key mechanisms to TNBC chemoresistance as well as emerging strategies to overcome them. We discuss novel anti-tumor agents that target the components of these mechanisms and pay special attention to their current clinical development while emphasizing the challenges still ahead of successful TNBC management. The evidence presented in this review outlines the role of crucial pathways in TNBC survival following chemotherapy treatment and highlights the importance of using combinatorial drug strategies and incorporating biomarkers in clinical studies.
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http://dx.doi.org/10.3390/cells8090957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770896PMC
August 2019

Biological Potential of Novel Methoxy and Hydroxy Substituted Heteroaromatic Amides Designed as Promising Antioxidative Agents: Synthesis, 3D-QSAR Analysis, and Biological Activity.

Chem Res Toxicol 2019 09 19;32(9):1880-1892. Epub 2019 Aug 19.

Department of Chemistry, Faculty of Science , University of Zagreb , Horvatovac 102a , HR 10000 Zagreb , Croatia.

This paper discusses antioxidative and biological activities of 25 novel amidino substituted benzamides with a variety of heteroaromatic nuclei attached to the benzamide moiety and with a variable number of methoxy or hydroxy substituents. Targeted compounds, bearing either amidino or 2-imidazolinyl substituent, were obtained in the Pinner reaction from cyano precursors. 3D-QSAR models were generated to predict antioxidative activity of the 25 novel aromatic and heteroaromatic benzamide derivatives. The compounds were tested for antioxidative activity using spectrophotometric assays. Direct validation of 3D-QSAR approach for predicting activities of novel benzamide derivatives was carried out by comparing experimental and computationally predicted antioxidative activity. Experimentally determined activities for all novel compounds were found to be within a standard deviation of error of the models. Following this, structure-activity relationships among the synthesized compounds are discussed. Furthermore, antiproliferative activity against HeLa cells as well as antibacterial and antifungal activity was tested to confirm the other biological activities of the prepared compounds.
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http://dx.doi.org/10.1021/acs.chemrestox.9b00256DOI Listing
September 2019

Association of uPA and PAI-1 tumor levels and 4G/5G variants of PAI-1 gene with disease outcome in luminal HER2-negative node-negative breast cancer patients treated with adjuvant endocrine therapy.

BMC Cancer 2019 Jan 15;19(1):71. Epub 2019 Jan 15.

Institute of Oncology and Radiology of Serbia, Pasterova 14, Belgrade, 11000, Serbia.

Background: The aim of this study was to evaluate the prognostic potential of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) tumor tissue levels and examine the association between these biomarkers and classical prognostic factors in early node-negative luminal breast cancer patients. The clinical value of 4G/5G variants of PAI-1 gene was evaluated.

Patients And Methods: This study involved 81 node-negative, estrogen receptor-positive and/or progesterone receptor-positive and human epidermal growth factor receptor 2-negative operable breast cancer patients who underwent radical surgical resection and received adjuvant endocrine therapy. Determination of uPA and PAI-1 concentrations in the breast cancer tissue extracts was performed using FEMTELLE® uPA/PAI-1 ELISA. An insertion (5G)/deletion (4G) polymorphism at position - 675 of the PAI-1 gene was detected by PCR-RFLP analysis.

Results: Our research showed that patients with uPA tumor tissue levels higher than 3 ng/mg of protein had significantly reduced disease-free survival (DFS) and overall survival (OS) when compared to patients with uPA tumor tissue levels lower or equal to 3 ng/mg of protein. Patients with PAI-1 tumor tissue levels higher than 14 ng/mg of protein had significantly decreased OS in comparison with patients with PAI-1 tumor tissue levels lower or equal to 14 ng/mg of protein. ROC analysis confirmed the uPA and PAI-1 discriminative potential for the presence/absence of relevant events in these patients and resulted in higher cut-off values (5.65 ng/mg of protein for uPA and 27.10 ng/mg of protein for PAI-1) than standard reference cut-off values for both biomarkers. The prognostic importance of uPA and PAI-1 ROC cut-off values was confirmed by the impact of uPA higher than 5.65 ng/mg of protein and PAI-1 higher than 27.10 ng/mg of protein on poorer DFS, OS and event-free survival (EFS). We observed that patients with dominant allele in PAI-1 genotype (heterozygote and dominant homozygote, - 675 4G/5G and - 675 5G/5G) had significantly increased DFS, OS and EFS when compared with patients with recessive homozygote genotype (- 675 4G/4G).

Conclusion: Our study indicates that uPA and PAI-1 tumor tissue levels and 4G/5G variants of PAI-1 gene might be of prognostic significance in early node-negative luminal HER2-negative breast cancer patients treated with adjuvant endocrine therapy.
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http://dx.doi.org/10.1186/s12885-018-5255-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6332605PMC
January 2019

Comparative in vitro studies of the biological potential and chemical composition of stems, leaves and berries Aronia melanocarpa's extracts obtained by subcritical water extraction.

Food Chem Toxicol 2018 Nov 21;121:458-466. Epub 2018 Sep 21.

Department of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska 33, 18000, Niš, Serbia.

Preparation of functional products as well as natural-based products requires non-toxic but effective extraction techniques. In this study, subcritical water extraction was used for the extraction of different aronia parts in order to explore their potential. Stems, leaves and berries of Aronia melanocarpa were extracted under the following conditions: temperature 130 °C; pressure 35 bar; time 20 min. The total phenols and flavonoid contents of the produced extracts were evaluated by conventional spectrophotometric methods. Additionally, the main phenolic compounds were also identified and quantified by high performance liquid chromatography with diode array detection (HPLC-DAD). The biological potential of the extracts was evaluated by determining their antioxidant (DPPH, ABTS and lipid peroxidation assays), antimicrobial, enzyme inhibitory (cholinesterase and elastase) and cytotoxic effects (HeLa, A-549, LS-174T, MRC-5 cell lines). The results indicate that leaves and berries extracts exhibited stronger antioxidant action when compared with stems. The strongest cholinesterase and elastase inhibitory activity was also found in berries extract. Similarly, the extracts obtained from leaves and berries showed considerable cytotoxic effects against tested cell lines. A moderate antimicrobial effects was observed too. Demonstrated biological potential of all three aronia parts can trace a new road map for developing newly designed functional products.
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http://dx.doi.org/10.1016/j.fct.2018.09.045DOI Listing
November 2018

Reservoir pH replica exchange.

J Chem Phys 2018 Aug;149(7):072321

Laboratory of Computational Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-5690, USA.

We present the reservoir pH replica exchange (R-pH-REM) method for constant pH simulations. The R-pH-REM method consists of a two-step procedure; the first step involves generation of one or more reservoirs of conformations. Each reservoir is obtained from a standard or enhanced molecular dynamics simulation with a constrained (fixed) protonation state. In the second step, fixed charge constraints are relaxed, as the structures from one or more reservoirs are periodically injected into a constant pH or a pH-replica exchange (pH-REM) simulation. The benefit of this two-step process is that the computationally intensive part of conformational search can be decoupled from constant pH simulations, and various techniques for enhanced conformational sampling can be applied without the need to integrate such techniques into the pH-REM framework. Simulations on blocked Lys, KK, and KAAE peptides were used to demonstrate an agreement between pH-REM and R-pH-REM simulations. While the reservoir simulations are not needed for these small test systems, the real need arises in cases when ionizable molecules can sample two or more conformations separated by a large energy barrier, such that adequate sampling is not achieved on a time scale of standard constant pH simulations. Such problems might be encountered in protein systems that exploit conformational transitions for function. A hypothetical case is studied, a small molecule with a large torsional barrier; while results of pH-REM simulations depend on the starting structure, R-pH-REM calculations on this model system are in excellent agreement with a theoretical model.
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http://dx.doi.org/10.1063/1.5027413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6005788PMC
August 2018

Synthesis, characterization and biological evaluation of Pd(ii), Cu(ii), Re(i) and Tc(i) thiazole-based complexes.

Medchemcomm 2018 May 13;9(5):831-842. Epub 2018 Apr 13.

Department of Pharmaceutical Chemistry , School of Pharmacy , Faculty of Health Sciences , Aristotle University of Thessaloniki , 54124 University Campus , Thessaloniki , Greece . Email: ; ; Tel: +30 2310 998680.

A new thiazole-containing multidentate ligand 2-((2-phenylthiazol-4-yl)methylthio)ethanamine, L, was synthesized and used to prepare new complexes of the formula PdLCl (Pd-L), CuLCl (Cu-L) and -[Re/Tc(CO)(L)] (Re/Tc-L). The ligand L and the metal complexes were characterized spectroscopically. Furthermore, the structures of Re-L and Cu-L were elucidated by X-ray crystallography. Ligand L acts as a bidentate (N, S) chelator in Pd-L, as a bidentate (N, S) chelator in Cu-L and as a tridentate (N, S, N) chelator in Re-L. The radiotracer Tc-L was synthesized in high yield and characterised by HPLC comparison with the Re-L analog. The synthesized compounds were evaluated for their anti-inflammatory and cytotoxic properties. The compounds exhibited low anti-inflammatory activity with Pd-L showing the highest activity among them. The cytotoxic activity of the ligand and the complexes against several human cancer cell lines (cervical adenocarcinoma HeLa, colorectal adenocarcinoma LS-174T, lung adenocarcinoma A549, breast adenocarcinoma MDA-MB-231 and normal human lung fibroblast cell line MRC-5) was examined using the MTT assay. The complex Cu-L exhibited the highest cytotoxicity and the complex Pd-L showed the best tumor selectivity. The changes in the cell cycle phase distribution were determined by flow cytometry and it was found that ligand L shows the highest apoptotic activity. The biodistribution studies of Tc-L in mice showed fast tissue clearance. Of all the thiazole-containing compounds, the palladium complex appears to be more promising for future efforts.
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http://dx.doi.org/10.1039/c8md00067kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072317PMC
May 2018

Peripheral White Blood Cell Subsets in Metastatic Colorectal Cancer Patients Treated with Cetuximab: The Potential Clinical Relevance.

Front Immunol 2017 4;8:1886. Epub 2018 Jan 4.

Institute of Oncology and Radiology of Serbia, Belgrade, Serbia.

It was demonstrated that cetuximab-induced tumor regression is based on the effects exerted by immune cells included mainly in the innate immune response. Therefore, the focus of this study was to explore the alterations in the percentages of CD16+, and/or CD56+ lymphocytes, which are comprised of NK cells, and minority of CD56+CD3+ cells, in patients with metastatic colorectal cancer before or 2 months after the treatment with cetuximab-based regimens associated with the response to therapy. The changes in the percentages of lymphocytes and granulocytes in these patients were evaluated as well. We enrolled 50 patients with metastatic colorectal cancer. Disease progression was observed in 11/50 patients (non-responders), while other patients achieved partial response or stable disease (responders). Control groups included up to 72 healthy individuals. A significant decrease in the percentages of CD56+ and CD16+CD56+ lymphocytes together with a significant decrease in the percentage of lymphocytes and an increase in the ratio of granulocyte to lymphocyte percentages were observed in patients with metastatic colorectal cancer before therapy, compared with those in the healthy individuals. In contrast to those in the responders, the percentage of CD16+ lymphocytes in the overall white blood cell pool was shown to be significantly decreased in the non-responders, together with a significantly decreased percentage of lymphocytes, a significantly increased percentage of granulocytes, and an increased ratio of granulocyte to lymphocyte percentages before treatment compared with those in the healthy controls. Two months after the initiation of the treatment, significantly decreased percentages of CD16+, CD56+, and CD16+CD56+ lymphocytes were observed in patients, compared with those determined in the healthy controls. The same changes in the amounts of circulating immune cells were also observed in the responder subgroup, but the percentages of CD16+, CD56+, and CD16+CD56+ lymphocytes 2 months after treatment in the non-responder group did not differ significantly in comparison with healthy individuals. Considerable alterations of immune cell percentages observed in patients with metastatic colorectal cancer with disease progression indicate that the assessment of peripheral white blood cell architecture before treatment initiation may be clinically relevant.
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http://dx.doi.org/10.3389/fimmu.2017.01886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758541PMC
January 2018

Identification of cytotoxic metabolites from Mahonia aquifolium using H NMR-based metabolomics approach.

J Pharm Biomed Anal 2018 Feb 1;150:9-14. Epub 2017 Dec 1.

Institute for Medicinal Plant Research "Dr. Josif Pančić", Tadeuša Košćuška 1, 11000 Belgrade, Serbia.

Herein, we propose a H NMR-based metabolomics method to reveal cytotoxic metabolites from Mahonia aquifolium stem-bark. Primary and secondary metabolites in the Mahonia aquifolium extracts were identified by thorough analysis of H and 2D NMR spectra, without prior isolation. An OPLS multivariate analysis method was used to correlate the chemical composition of the plant extracts with the results of cytotoxic activity against Human cervical adenocarcinoma cell line. Protoberberine alkaloids berberinе and palmatine, along with bisbenzylisoquinoline alkaloid berbamine were identified as the most influential in the OPLS model, with the highest cytotoxic activity.
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http://dx.doi.org/10.1016/j.jpba.2017.11.075DOI Listing
February 2018

Absolute binding free energy calculations of CBClip host-guest systems in the SAMPL5 blind challenge.

J Comput Aided Mol Des 2017 01 27;31(1):71-85. Epub 2016 Sep 27.

Laboratory of Computational Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, 20852, USA.

Herein, we report the absolute binding free energy calculations of CBClip complexes in the SAMPL5 blind challenge. Initial conformations of CBClip complexes were obtained using docking and molecular dynamics simulations. Free energy calculations were performed using thermodynamic integration (TI) with soft-core potentials and Bennett's acceptance ratio (BAR) method based on a serial insertion scheme. We compared the results obtained with TI simulations with soft-core potentials and Hamiltonian replica exchange simulations with the serial insertion method combined with the BAR method. The results show that the difference between the two methods can be mainly attributed to the van der Waals free energies, suggesting that either the simulations used for TI or the simulations used for BAR, or both are not fully converged and the two sets of simulations may have sampled difference phase space regions. The penalty scores of force field parameters of the 10 guest molecules provided by CHARMM Generalized Force Field can be an indicator of the accuracy of binding free energy calculations. Among our submissions, the combination of docking and TI performed best, which yielded the root mean square deviation of 2.94 kcal/mol and an average unsigned error of 3.41 kcal/mol for the ten guest molecules. These values were best overall among all participants. However, our submissions had little correlation with experiments.
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http://dx.doi.org/10.1007/s10822-016-9968-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5241186PMC
January 2017

Enhancing constant-pH simulation in explicit solvent with a two-dimensional replica exchange method.

J Chem Theory Comput 2015 Jun 26;11(6):2560-74. Epub 2015 May 26.

Laboratory of Computational Biology, National Heart, Lung, and Blood Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.

We present a new method for enhanced sampling for constant-pH simulations in explicit water based on a two-dimensional (2D) replica exchange scheme. The new method is a significant extension of our previously developed constant-pH simulation method, which is based on enveloping distribution sampling (EDS) coupled with a one-dimensional (1D) Hamiltonian exchange method (HREM). EDS constructs a hybrid Hamiltonian from multiple discrete end state Hamiltonians that, in this case, represent different protonation states of the system. The ruggedness and heights of the hybrid Hamiltonian's energy barriers can be tuned by the smoothness parameter. Within the context of the 1D EDS-HREM method, exchanges are performed between replicas with different smoothness parameters, allowing frequent protonation-state transitions and sampling of conformations that are favored by the end-state Hamiltonians. In this work, the 1D method is extended to 2D with an additional dimension, external pH. Within the context of the 2D method (2D EDS-HREM), exchanges are performed on a lattice of Hamiltonians with different pH conditions and smoothness parameters. We demonstrate that both the 1D and 2D methods exactly reproduce the thermodynamic properties of the semigrand canonical (SGC) ensemble of a system at a given pH. We have tested our new 2D method on aspartic acid, glutamic acid, lysine, a four residue peptide (sequence KAAE), and snake cardiotoxin. In all cases, the 2D method converges faster and without loss of precision; the only limitation is a loss of flexibility in how CPU time is employed. The results for snake cardiotoxin demonstrate that the 2D method enhances protonation-state transitions, samples a wider conformational space with the same amount of computational resources, and converges significantly faster overall than the original 1D method.
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http://dx.doi.org/10.1021/ct501101fDOI Listing
June 2015

Metformin effects on malignant cells and healthy PBMC; the influence of metformin on the phenotype of breast cancer cells.

Pathol Oncol Res 2015 Jul 29;21(3):605-12. Epub 2014 Oct 29.

Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000, Belgrade, Serbia.

The aim of research was to determine the effects of maximally therapeutically achievable concentrations of metformin on malignant cells and healthy peripheral blood mononuclear cells (PBMC). Eight patients with T2D or hyperglycemia and nine healthy volunteers were included in the study. For determination of the influence of metformin on the phenotype of breast carcinoma, 1,410 patients with surgically removed tumors were included. From this group 37 breast cancer patients had DM type 2 or hyperglycemia and were pretreated with metformin alone or sometimes in combination with other antidiabetic drugs. Our results proved that metformin at low concentrations induced mild decrease in survival of malignant cells and PBMC stimulated for proliferation, but it didn't affect survival of resting PBMC. The effects of plasma of hyperglycemic patients who were under metformin therapy on autologous PBMC-induced decrease in survival of MDA-MB-361 cells, was noticeable in some patients. Metformin pretreatment for 24 h of HER2+ MDA-MB-361 cells, which were subsequently treated for 48 h with Herceptin, induced additional decline in cell survival. The analysis of influence of metformin on phenotype of breast cancer cells revealed significantly lower number of diabetic cancer patients treated with metformin with overexpressed HER2+ tumors (p < 0.013), while the number of patients with ER+PR+ tumors was not significantly changed (p < 0.832). In conclusion, therapeutically used concentrations of metformin exhibit mild cytotoxic action on malignant and dividing normal cells pointing to its preferred role in malignant and autoimmune diseases. The use of metformin was associated with pronounced decrease in HER2 overexpressing tumors.
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http://dx.doi.org/10.1007/s12253-014-9864-9DOI Listing
July 2015

Computational and experimental investigation of constitutive behavior in AraC.

Proteins 2014 Dec 21;82(12):3385-96. Epub 2014 Oct 21.

Physics Department, Loyola University Maryland, Baltimore, Maryland.

Many mutations in the N-terminal arm of AraC result in constitutive behavior in which transcription of the araBAD genes occurs even in the absence of arabinose. To begin to understand the mechanism underlying this class of mutations, we used molecular dynamics with self-guided Langevin dynamics to simulate (1) wild-type (WT) AraC, (2) known constitutive mutants resulting from alterations in the regulatory arm, particularly alanine and glycine substitutions at residue 8 because P8G is constitutive, whereas P8A behaves like wild type, and (3) selected variant AraC proteins containing alterations in the dimerization core. In all of the constitutive arm mutants, but not the WT protein, residues 37-42, which are located in the core of the dimerization domain, became restructured. This raised the question of whether or not these structural changes are an obligatory component of constitutivity. Using molecular dynamics, we identified alterations in the core that produced a similar restructuring. The corresponding mutants were constructed and their ara constitutivity status was determined experimentally. Because the core mutants were not found to be constitutive, we conclude that restructuring of core residues 37-42 does not, itself, lead to constitutivity of AraC. The available data lead to the hypothesis that the interaction of the N-terminal arm with something other than the front lip is the primary determinant of the inducing versus repressing state of AraC.
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http://dx.doi.org/10.1002/prot.24693DOI Listing
December 2014

Constant pH Molecular Dynamics in Explicit Solvent with Enveloping Distribution Sampling and Hamiltonian Exchange.

J Chem Theory Comput 2014 Jul 3;10(7):2738-2750. Epub 2014 Jun 3.

Laboratory of Computational Biology, National Heart, Lung, and Blood Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.

We present a new computational approach for constant pH simulations in explicit solvent based on the combination of the enveloping distribution sampling (EDS) and Hamiltonian replica exchange (HREX) methods. Unlike constant pH methods based on variable and continuous charge models, our method is based on discrete protonation states. EDS generates a hybrid Hamiltonian of different protonation states. A smoothness parameter is used to control the heights of energy barriers of the hybrid-state energy landscape. A small value facilitates state transitions by lowering energy barriers. Replica exchange between EDS potentials with different values allows us to readily obtain a thermodynamically accurate ensemble of multiple protonation states with frequent state transitions. The analysis is performed with an ensemble obtained from an EDS Hamiltonian without smoothing, = ∞, which strictly follows the minimum energy surface of the end states. The accuracy and efficiency of this method is tested on aspartic acid, lysine, and glutamic acid, which have two protonation states, a histidine with three states, a four-residue peptide with four states, and snake cardiotoxin with eight states. The p values estimated with the EDS-HREX method agree well with the experimental p values. The mean absolute errors of small benchmark systems range from 0.03 to 0.17 p units, and those of three titratable groups of snake cardiotoxin range from 0.2 to 1.6 p units. This study demonstrates that EDS-HREX is a potent theoretical framework, which gives the correct description of multiple protonation states and good calculated p values.
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http://dx.doi.org/10.1021/ct500175mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4095908PMC
July 2014

Immunoreactivity to food antigens in patients with chronic urticaria.

Immunol Invest 2014 24;43(5):504-16. Epub 2014 Mar 24.

Institute of Immunology and Allergology, Clinical Center of Serbia , 11000 Belgrade , Serbia .

The goal of study was better understanding of complex immune mechanisms that can help to evaluate patients with chronic urticaria (CU), especially those with unknown etiology. The study involved 55 patients with CU. Control group consisted of up to 90 healthy persons. The presence and intensity of serum IgG, IgA, IgM and IgE antibodies to common food antigens: cow's milk proteins (CMP), gliadin and phytohemagglutinin were determined by ELISA. Determination of subpopulations of immunocompetent cells was performed by flow cytometry. Significantly enhanced IgE, but also IgA immunity to CMP was found in patients with CU in comparison to healthy controls: (p < 0.000004) and (p < 0.002), respectively. Notably, in 40 out of 55 CU patients, the increased levels of some type of immunoglobulin reactivity to CMP were found. Regarding gliadin, only the levels of serum IgE anti-gliadin antibodies were significantly enhanced in patients with CU (p < 0.04). Significantly enhanced percentage of CD89+ cells accompanied with significantly lower percentage of lymphocytes and significantly higher mean fluorescence intensity of CD26 expression on lymphocytes were found in patients with CU in comparison to healthy controls (p < 0.04), (p < 0.02) and (p < 0.003), respectively. Results of this study may help in better understanding the complex immune disturbances in patients with CU.
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http://dx.doi.org/10.3109/08820139.2014.892509DOI Listing
January 2015

Gold(III) complexes with esters of cyclohexyl-functionalized ethylenediamine-N,N'-diacetate.

J Inorg Biochem 2013 Nov 13;128:146-53. Epub 2013 Aug 13.

Faculty of Agriculture, University of Belgrade, Nemanjina 6, Belgrade-Zemun, Serbia.

Six novel gold(III) complexes containing O,O'-dialkyl-(S,S)-ethylenediamine-N,N'-di-2-(3-cyclohexyl)propanoate ([AuCl2{(S,S)-R2eddch}]PF6, R=Me, Et, n-Pr, n-Bu, i-Bu, i-Am; 1-6, respectively) were synthesized and characterized by elemental analysis, UV/Visible, IR and NMR spectroscopy, mass spectrometry and differential pulse voltammetry. Density functional theory (DFT) calculations confirmed that diastereoisomer with the N,N' atoms configured (S,S) was the most stable. In vitro antiproliferative activity was determined against human cervix adenocarcinoma HeLa and human myelogenous leukemia K562 tumor cell lines, as well as against rested and stimulated normal immunocompetent human peripheral blood mononuclear cells (PBMC) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Complex 6 expressed the highest activity against K562 cells (IC50 = 3.8 ± 0.5 μM). Apoptosis, seen as condensation of HeLa cell nuclei was the mode of cell death induced by complexes 2-6. Complexes 3-6 induced death of K562 cells inhibiting cell entry in mitosis.
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http://dx.doi.org/10.1016/j.jinorgbio.2013.08.002DOI Listing
November 2013

Dipeptidyl peptidase IV: serum activity and expression on lymphocytes in different hematological malignancies.

Leuk Lymphoma 2013 Dec 19;54(12):2701-6. Epub 2013 Apr 19.

Institute of Oncology and Radiology of Serbia , Belgrade , Serbia.

The aim of this research was to determine the serum dipeptidyl peptidase IV (DPPIV) activity as well as the percentages of CD26 + lymphocytes and CD26 + overall white blood cells in patients with hematological malignancies: non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), leukemia, plasmacytoma and multiple myeloma, and in healthy individuals. Data from our study showed significantly decreased serum DPPIV activity and a significant decrease in the percentage of: CD26 + lymphocytes, CD26 + overall white blood cells and lymphocytes in patients with NHL in comparison to healthy controls. Patients with leukemia had a statistically significant lower activity of DPPIV in serum and significant decrease in the percentage of CD26 + lymphocytes in relation to healthy controls. Furthermore, significantly decreased DPPIV serum activity associated with a significantly reduced percentage of CD26 + overall white blood cells and percentage of lymphocytes was found in patients with multiple myeloma when compared to the healthy control group. The obtained results indicate that immune disturbances that can occur in hematological malignancies might be related to the decreased expression and activity of CD26/DPPIV that we observed.
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http://dx.doi.org/10.3109/10428194.2013.782611DOI Listing
December 2013

Good tolerance to goat's milk in patients with recurrent aphthous ulcers with increased immunoreactivity to cow's milk proteins.

J Oral Pathol Med 2013 Aug 25;42(7):523-7. Epub 2013 Feb 25.

Institute of Oncology and Radiology of Serbia, Belgrade, Serbia.

Background: Recurrent aphthous ulcers (RAU) represent a very common, but poorly understood mucosal disorder. The connection between immunity to cow's milk proteins (CMP) and oral diseases was noted earlier. The goal of this study was to determine the prevalence of the increased levels of serum antibodies to goat's milk proteins (GMP), by enzyme-linked immunosorbent assay (ELISA) test, in subjects who have RAU and proven increased immunity to CMP.

Methods: Fifty subjects with RAU (36 with proven increased immunity to CMP and 14 without this increased immunity) were included in this research. Levels of serum IgA, IgG, and IgE antibodies to the same quantity of the examined antigens were determined by ELISA. The statistical analysis of data was performed by Wilcoxon rank-sum test and Mann-Whitney test.

Results: The levels of serum antifresh cow's milk IgA, IgG, and IgE antibodies were significantly higher than the levels of serum antifresh goat's milk, in subjects with RAU with proven increased immunoreactivity to CMP (P = 0.0003; P < 0.0001; P < 0.0001).

Conclusions: These results indicate that patients with RAU with increased immunity to CMP could consider the use of goat's milk as the alternative protein source.
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http://dx.doi.org/10.1111/jop.12052DOI Listing
August 2013

Understanding the basis of a class of paradoxical mutations in AraC through simulations.

Proteins 2013 Mar 24;81(3):490-8. Epub 2012 Dec 24.

Department of Biophysics, Johns Hopkins University, Baltimore, Maryland 21218, USA.

Most mutations at position 15 in the N-terminal arm of the regulatory protein AraC leave the protein incapable of responding to arabinose and inducing the proteins required for arabinose catabolism. Mutations at other positions of the arm do not have this behavior. Simple energetic analysis of the interactions between the arm and bound arabinose do not explain the uninducibility of AraC with mutations at position 15. Extensive molecular dynamics (MD) simulations, carried out largely on the Open Science Grid, were done of the wild-type protein with and without bound arabinose and of all possible mutations at position 15, many of which were constructed and measured for this work. Good correlation was found for deviation of arm position during the simulations and inducibility as measured in vivo of the same mutant proteins. Analysis of the MD trajectories revealed that preservation of the shape of the arm is critical to inducibility. To maintain the correct shape of the arm, the strengths of three interactions observed to be strong in simulations of the wild-type AraC protein need to be preserved. These interactions are between arabinose and residue 15, arabinose and residues 8-9, and residue 13 and residue 15. The latter interaction is notable because residues L9, Y13, F15, W95, and Y97 form a hydrophobic cluster which needs to be preserved for retention of the correct shape.
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http://dx.doi.org/10.1002/prot.24207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3557760PMC
March 2013

Serum activity of DPPIV and its expression on lymphocytes in patients with melanoma and in people with vitiligo.

BMC Immunol 2012 Aug 21;13:48. Epub 2012 Aug 21.

Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000, Belgrade, Serbia.

Background: Dipeptidyl peptidase IV, a multifunctional serine protease, is implicated in regulation of malignant transformation, promotion and further progression of cancer, exerting tumor-suppressing or even completely opposite - tumor-promoting activities. The aim of present research was to determine the serum DPPIV activity, as well as the percentages of CD26+ lymphocytes, CD26+ overall white blood cells and the mean fluorescence intensity of CD26 expression on lymphocytes in patients with melanoma, people with vitiligo and in healthy controls.

Methods: The activity of DPPIV in serum was determined by colorimetric test. Expression of DPPIV (as CD26) on immunocompetent peripheral white blood cells was done using flow cytometry analysis.

Results: Data from our study show for the first time statistically significant decrease: in the serum DPPIV activity, in the percentage of CD26+ overall white blood cells and in the percentage of lymphocytes in patients with melanoma in comparison to healthy control people. In addition, significantly lower serum DPPIV activity was found in the group of patients with melanoma in relation to people with vitiligo too.

Conclusion: This study indicates the need for exploring the cause and the importance of the disturbances in the serum DPPIV activity and in the CD26 expression on immunocompetent cells in complex molecular mechanisms underlying the development and progression of melanoma.
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http://dx.doi.org/10.1186/1471-2172-13-48DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464610PMC
August 2012

Immunity to melanin and to tyrosinase in melanoma patients, and in people with vitiligo.

BMC Complement Altern Med 2012 Jul 26;12:109. Epub 2012 Jul 26.

Institute of Oncology and Radiology of Serbia, Pasterova 14, Belgrade, Serbia.

Background: The aim of this study was to determine the presence and the intensity of humoral immunity to melanoma-associated antigens: tyrosinase and melanin, in patients with melanoma, in persons with vitiligo and in control healthy people.

Methods: The study involved 63 patients with melanoma and 19 persons with vitiligo. Control group consisted up to 41 healthy volunteers. Mushroom tyrosinase and synthetic melanin were used as the antigens.

Results: ELISA test showed significantly (p < 0.0000004 and p < 0.04) lower levels of IgM anti-tyrosinase autoantibodies, in melanoma and vitiligo patients respectively, compared to controls.Although there was no significant difference between the levels of IgA anti-melanin autoantibodies in melanoma or vitiligo patients in comparison with controls, the enhanced concentrations of anti-melanin IgA autoantibodies were preferentially found in melanoma patients with metastatic disease. Significantly high percentage in the Fc alphaRI (CD89) positive cells was determined in melanoma patients (p < 0.002 and p < 0.008) in comparison to that found in healthy people or in patients with vitiligo, in the already mentioned order, pointing that IgA dependent cellular cytotoxicity is not important for the immune action against melanoma, even more that it is included in some immune suppression.Levels of IgG autoantibodies to mentioned antigens in melanoma patients although low were not significantly lower from controls. These findings analyzed together with the statistically significant low percentage of FcgammaRIII, (CD16) positive immunocompetent cells (p < 0.0007 and p < 0.003), which was found in patients with melanoma compared with healthy or vitiligo people respectively, and statistically significant low percentage of (CD16 + CD56+) natural killer (NK) cells (p < 0.005) found in melanoma patients in comparison to healthy controls pointed to the low probability for anti-melanoma IgG mediated, antibody mediated cellular cytotoxicity, (ADCC) and NK cytotoxicity. Moreover the ratio of the percentages of granulocytes and percentage of lymphocytes was statistically higher in patients with melanoma in relation to healthy people as well as to people with vitiligo (p < 0.0007 and p < 0.05 respectively).

Conclusion: Autoantibodies to tyrosinase and to melanin which are found even in healthy people, point that consummation of edible mushrooms that carry the antigen tyrosinase and melanin, could influence the humoral anti-melanoma immune response.Levels of different immunoglobulin classes of anti-melanin and anti-tyrosinase antibodies varied depending on the presence and the stage of studied diseases. Besides, the statistically enhanced ratio of the percentages of granulocytes and percentage of lymphocytes, together with statistically decreased percentage of NK cells is found in analyzed melanoma patients.
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http://dx.doi.org/10.1186/1472-6882-12-109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457868PMC
July 2012
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