63 results on '"Mlinarič-Raščan I"'
Search Results
2. P13-10: In vitro evaluation of immunotoxicity of bisphenol A analogues using human lymphoblastoid cell lines
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Markovič, T., primary, Mlinarič-Raščan, I., additional, Škratek, M., additional, Kodila, A., additional, and Sollner Dolenc, M., additional
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- 2023
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3. Medicines associated with folate–homocysteine–methionine pathway disruption
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Vidmar, M., Grželj, J., Mlinarič-Raščan, I., Geršak, K., and Dolenc, M. Sollner
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- 2019
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4. Personalization of thiopurine therapy: Current recommendations and future perspectives
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Urbančič Dunja, Pasha Flaka, Šmid Alenka, and Mlinarič-Raščan Irena
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thiopurines ,tpmt ,nudt15 ,personalized medicine ,therapeutic drug monitoring ,Pharmaceutical industry ,HD9665-9675 - Abstract
Despite great therapeutic advances in the field of biologics, small synthetic molecules such as thiopurines, including azathioprine, mercaptopurine, and thioguanine, remain an important therapeutic pillar in the treatment of inflammatory bowel disease, other autoimmune disorders, and cancer. This review presents the latest guidelines for thiopurine administration, highlighting the importance of individualized therapy guided by pharmacogenomics. It emphasizes dose adjustment based on nudix hydrolase 15 (NUDT15) and thiopurine S-methyltransferase (TPMT) genotype, along side thiopurine S-methyltransferase activity and thiopurine metabolic profile. In addition, the article takes a critical look at emerging research in the field of thiopurine pharmaco genomics featuring novel genetic markers and technological developments in genetic testing. Finally, the potential of integrated approaches that combine genetic, meta bolic, and clinical factors to further individualize thiopurine therapy is highlighted.
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- 2024
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5. PHARMACOGENETICS OF TPMT: SCREENING FOR NEW BIOCHEMICAL FACTORS INFLUENCING TPMT ACTIVITY
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Šmid, A., Milek, M., Karas-Kuželički, N., Tamm, R., Metspalu, A., and Mlinarič-Raščan, I.
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- 2011
6. Medicines associated with folate–homocysteine–methionine pathway disruption
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Vidmar, M., primary, Grželj, J., additional, Mlinarič-Raščan, I., additional, Geršak, K., additional, and Dolenc, M. Sollner, additional
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- 2018
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7. Oxidatively altered IgG with increased immunoreactivity to β2-glycoprotein I and its peptide clusters influence human coronary artery endothelial cells
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Artenjak, A, primary, Omersel, J, additional, Ahlin Grabnar, P, additional, Mlinarič-Raščan, I, additional, Shoenfeld, Y, additional, Sodin-Semrl, S, additional, Božič, B, additional, and Čučnik, S, additional
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- 2015
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8. A Myc-regulated transcriptional network controls B-cell fate in response to BCR triggering
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Vaigot Pierre, Mlinaric-Rascan Irena, Murn Jernej, Alibert Olivier, Frouin Vincent, and Gidrol Xavier
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Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background The B cell antigen receptor (BCR) is a signaling complex that mediates the differentiation of stage-specific cell fate decisions in B lymphocytes. While several studies have shown differences in signal transduction components as being key to contrasting phenotypic outcomes, little is known about the differential BCR-triggered gene transcription downstream of the signaling cascades. Results Here we define the transcriptional changes that underlie BCR-induced apoptosis and proliferation of immature and mature B cells, respectively. Comparative genome-wide expression profiling identified 24 genes that discriminated between the early responses of the two cell types to BCR stimulation. Using mice with a conditional Myc-deletion, we validated the microarray data by demonstrating that Myc is critical to promoting BCR-triggered B-cell proliferation. We further investigated the Myc-dependent molecular mechanisms and found that Myc promotes a BCR-dependent clonal expansion of mature B cells by inducing proliferation and inhibiting differentiation. Conclusion This work provides the first comprehensive analysis of the early transcriptional events that lead to either deletion or clonal expansion of B cells upon antigen recognition, and demonstrates that Myc functions as the hub of a transcriptional network that control B-cell fate in the periphery.
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- 2009
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9. Exploration and optimisation of structure-activity relationships of new triazole-based C-terminal Hsp90 inhibitors towards in vivo anticancer potency.
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Dernovšek J, Zajec Ž, Poje G, Urbančič D, Sturtzel C, Goričan T, Grissenberger S, Ciura K, Woziński M, Gedgaudas M, Zubrienė A, Grdadolnik SG, Mlinarič-Raščan I, Rajić Z, Cotman AE, Zidar N, Distel M, and Tomašič T
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- Humans, Structure-Activity Relationship, Animals, Cell Line, Tumor, MCF-7 Cells, Cell Proliferation drug effects, HSP90 Heat-Shock Proteins antagonists & inhibitors, HSP90 Heat-Shock Proteins metabolism, Triazoles pharmacology, Triazoles chemistry, Triazoles chemical synthesis, Antineoplastic Agents pharmacology, Antineoplastic Agents chemistry, Zebrafish, Apoptosis drug effects
- Abstract
The development of new anticancer agents is one of the most urgent topics in drug discovery. Inhibition of molecular chaperone Hsp90 stands out as an approach that affects various oncogenic proteins in different types of cancer. These proteins rely on Hsp90 to obtain their functional structure, and thus Hsp90 is indirectly involved in the pathophysiology of cancer. However, the most studied ATP-competitive inhibition of Hsp90 at the N-terminal domain has proven to be largely unsuccessful clinically. Therefore, research has shifted towards Hsp90 C-terminal domain (CTD) inhibitors, which are also the focus of this study. Our recent discovery of compound C has provided us with a starting point for exploring the structure-activity relationship and optimising this new class of triazole-based Hsp90 inhibitors. This investigation has ultimately led to a library of 33 analogues of C that have suitable physicochemical properties and several inhibit the growth of different cancer types in the low micromolar range. Inhibition of Hsp90 was confirmed by biophysical and cellular assays and the binding epitopes of selected inhibitors were studied by STD NMR. Furthermore, the most promising Hsp90 CTD inhibitor 5x was shown to induce apoptosis in breast cancer (MCF-7) and Ewing sarcoma (SK-N-MC) cells while inducing cause cell cycle arrest in MCF-7 cells. In MCF-7 cells, it caused a decrease in the levels of ERα and IGF1R, known Hsp90 client proteins. Finally, 5x was tested in zebrafish larvae xenografted with SK-N-MC tumour cells, where it limited tumour growth with no obvious adverse effects on normal zebrafish development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)
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- 2024
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10. Prevalence and Treatment Outcomes of Childhood Acute Lymphoblastic Leukemia in Kosovo.
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Pasha F, Urbančič D, Maxhuni R, Krasniqi S, Grajçevci Uka V, and Mlinarič-Raščan I
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Advances in research, including novel biomarker identification and patient stratification, have significantly improved the therapy for childhood acute lymphoblastic leukemia (ALL), though access to improved healthcare services varies across geographical regions. In an effort to evaluate the advances in therapeutic approaches, we performed a retrospective analysis of childhood ALL in Kosovo. Our retrospective analysis included 225 cases diagnosed between 2008 and 2023, representing 52% of 429 diagnosed childhood cancers. The average annual incidence was 14, with a median age diagnosis of seven years, and a male predominance (59.54%). Patients were categorized into risk groups, with the majority (43%) in the standard-risk category. We identified five different treatment protocols for this study period. Over 61% of patients achieved remission after the first chemotherapy cycle and we observed a 20% mortality rate. Survival analysis showed that 55% and 40% of patients achieved 2-year and 5-year event-free survival (EFS), respectively, with significant differences across risk groups. Treatment advancements significantly correlated with improved survival rates, achieving a 5-year overall survival (OS) of 88% in the currently used standardized AIEOP-BFM-2009 protocol. Our study emphasizes the need for continued research and customized care strategies to enhance clinical outcomes.
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- 2024
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11. Transcriptome analysis reveals involvement of thiopurine S-methyltransferase in oxidation-reduction processes.
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Šmid A, Štajdohar M, Milek M, Urbančič D, Karas Kuželički N, Tamm R, Metspalu A, and Mlinarič-Raščan I
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- Adult, Female, Humans, Male, Gene Expression Profiling, Mercaptopurine metabolism, Oxidation-Reduction, S-Adenosylmethionine metabolism, Methyltransferases genetics, Methyltransferases metabolism, Purines
- Abstract
Thiopurine S-methyltransferase (TPMT) is an important enzyme involved in the deactivation of thiopurines and represents a major determinant of thiopurine-related toxicities. Despite its well-known importance in thiopurine metabolism, the understanding of its endogenous role is lacking. In the present study, we aimed to gain insight into the molecular processes involving TPMT by applying a data fusion approach to analyze whole-genome expression data. The RNA profiling was done on whole blood samples from 1017 adult male and female donors to the Estonian biobank using Illumina HTv3 arrays. Our results suggest that TPMT is closely related to genes involved in oxidoreductive processes. The in vitro experiments on different cell models confirmed that TPMT influences redox capacity of the cell by altering S-adenosylmethionine (SAM) consumption and consequently glutathione (GSH) synthesis. Furthermore, by comparing gene networks of subgroups of individuals, we identified genes, which could have a role in regulating TPMT activity. The biological relevance of identified genes and pathways will have to be further evaluated in molecular studies., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest that are directly relevant to the content of this article., (Copyright © 2023. Published by Elsevier B.V.)
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- 2024
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12. Nanofibers with genotyped Bacillus strains exhibiting antibacterial and immunomodulatory activity.
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Grilc NK, Zidar A, Kocbek P, Rijavec T, Colja T, Lapanje A, Jeras M, Gobec M, Mlinarič-Raščan I, Gašperlin M, Kristl J, and Zupančič Š
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- Humans, Leukocytes, Mononuclear, Anti-Bacterial Agents pharmacology, Polyethylene Glycols, Alginates, Nanofibers, Bacillus genetics
- Abstract
Biofilm-associated diseases such as periodontitis are widespread and challenging to treat which calls for new strategies for their effective management. Probiotics represent a promising approach for targeted treatment of dysbiosis in biofilm and modulation of host immune response. In this interdisciplinary study, nanofibers with two autochthonous Bacillus strains 27.3.Z and 25.2.M were developed. The strains were isolated from the oral microbiota of healthy individuals, and their genomes were sequenced and screened for genes associated with antimicrobial and immunomodulatory activities, virulence factors, and transferability of resistance to antibiotics. Spores of two Bacillus strains were incorporated individually or in combination into hydrophilic poly(ethylene oxide) (PEO) and composite PEO/alginate nanofibers. The nanofiber mats were characterised by a high loading of viable spores (> 7 log CFU/mg) and they maintained viability during electrospinning and 6 months of storage at room temperature. Spores were rapidly released from PEO nanofibers, while presence of alginate in the nanofibers prolonged their release. All formulations exhibited swelling, followed by transformation of the nanofiber mat into a hydrogel and polymer erosion mediating spore release kinetics. The investigated Bacillus strains released metabolites, which were not cytotoxic to peripheral blood mononuclear cells (PBMCs) in vitro. Moreover, their metabolites exhibited antibacterial activity against two periodontopathogens, an antiproliferative effect on PBMCs, and inhibition of PBMC expression of proinflammatory cytokines. In summary, the developed nanofiber-based delivery system represents a promising therapeutic approach to combat biofilm-associated disease on two fronts, namely via modulation of the local microbiota with probiotic bacteria and host immune response with their metabolites., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)
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- 2023
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13. A Comprehensive Genetic Analysis of Slovenian Families with Multiple Cases of Orofacial Clefts Reveals Novel Variants in the Genes IRF6 , GRHL3 , and TBX22 .
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Slavec L, Geršak K, Eberlinc A, Hovnik T, Lovrečić L, Mlinarič-Raščan I, and Karas Kuželički N
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- Humans, Comparative Genomic Hybridization, DNA-Binding Proteins metabolism, Interferon Regulatory Factors genetics, Mutation, Pedigree, Transcription Factors metabolism, Cleft Lip genetics, Cleft Palate genetics
- Abstract
Although the aetiology of non-syndromic orofacial clefts (nsOFCs) is usually multifactorial, syndromic OFCs (syOFCs) are often caused by single mutations in known genes. Some syndromes, e.g., Van der Woude syndrome (VWS1; VWS2) and X-linked cleft palate with or without ankyloglossia (CPX), show only minor clinical signs in addition to OFC and are sometimes difficult to differentiate from nsOFCs. We recruited 34 Slovenian multi-case families with apparent nsOFCs (isolated OFCs or OFCs with minor additional facial signs). First, we examined IRF6 , GRHL3 , and TBX22 by Sanger or whole exome sequencing to identify VWS and CPX families. Next, we examined 72 additional nsOFC genes in the remaining families. Variant validation and co-segregation analysis were performed for each identified variant using Sanger sequencing, real-time quantitative PCR and microarray-based comparative genomic hybridization. We identified six disease-causing variants (three novel) in IRF6 , GRHL3 , and TBX22 in 21% of families with apparent nsOFCs, suggesting that our sequencing approach is useful for distinguishing syOFCs from nsOFCs. The novel variants, a frameshift variant in exon 7 of IRF6 , a splice-altering variant in GRHL3 , and a deletion of the coding exons of TBX22 , indicate VWS1, VWS2, and CPX, respectively. We also identified five rare variants in nsOFC genes in families without VWS or CPX, but they could not be conclusively linked to nsOFC.
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- 2023
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14. Inhibition of p38 MAPK or immunoproteasome overcomes resistance of chronic lymphocytic leukemia cells to Bcl-2 antagonist venetoclax.
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Avsec D, Škrlj Miklavčič M, Burnik T, Kandušer M, Bizjak M, Podgornik H, and Mlinarič-Raščan I
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- Bridged Bicyclo Compounds, Heterocyclic pharmacology, Bridged Bicyclo Compounds, Heterocyclic therapeutic use, Drug Resistance, Neoplasm, Humans, Ionomycin pharmacology, Ionomycin therapeutic use, Myeloid Cell Leukemia Sequence 1 Protein, Proto-Oncogene Proteins c-bcl-2 metabolism, Sulfonamides, bcl-2-Associated X Protein, p38 Mitogen-Activated Protein Kinases metabolism, Antineoplastic Agents pharmacology, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Leukemia, Lymphocytic, Chronic, B-Cell metabolism
- Abstract
Chronic lymphocytic leukemia (CLL) is a hematological neoplasm of CD19-positive mature-appearing B lymphocytes. Despite the clinical success of targeted therapies in CLL, the development of resistance diminishes their therapeutic activity. This is also true for the Bcl-2 antagonist venetoclax. We investigated the molecular mechanisms that drive venetoclax resistance in CLL, with a clear focus to provide new strategies to successfully combat it. Activation of CLL cells with IFNγ, PMA/ionomycin, and sCD40L diminished the cytotoxicity of venetoclax. We demonstrated that the metabolic activity of cells treated with 1 nM venetoclax alone was 48% of untreated cells, and was higher for cells co-treated with IFNγ (110%), PMA/ionomycin (78%), and sCD40L (62%). As of molecular mechanism, we showed that PMA/ionomycin and sCD40L triggered translocation of NFκB in primary CLL cells, while IFNγ activated p38 MAPK, suppressed spontaneous and venetoclax-induced apoptosis and induced formation of the immunoproteasome. Inhibition of immunoproteasome with ONX-0914 suppressed activity of immunoproteasome and synergized with venetoclax against primary CLL cells. On the other hand, inhibition of p38 MAPK abolished cytoprotective effects of IFNγ. We demonstrated that venetoclax-resistant (MEC-1 VER) cells overexpressed p38 MAPK and p-Bcl-2 (Ser70), and underexpressed Mcl-1, Bax, and Bak. Inhibition of p38 MAPK or immunoproteasome triggered apoptosis in CLL cells and overcame the resistance to venetoclax of MEC-1 VER cells and venetoclax-insensitive primary CLL cells. In conclusion, the p38 MAPK pathway and immunoproteasome represent novel targets to combat venetoclax resistance in CLL., (© 2022. The Author(s).)
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- 2022
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15. A Common Polymorphism in the MTHFD1 Gene Is a Modulator of Risk of Congenital Heart Disease.
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Karas Kuželički N, Šmid A, Vidmar Golja M, Kek T, Geršak B, Mazič U, Mlinarič-Raščan I, and Geršak K
- Abstract
Several environmental and genetic factors may influence the risk of congenital heart defects (CHDs), which can have a substantial impact on pediatric morbidity and mortality. We investigated the association of polymorphisms in the genes of the folate and methionine pathways with CHDs using different strategies: a case-control, mother-child pair design, and a family-based association study. The polymorphism rs2236225 in the MTHFD1 was confirmed as an important modulator of CHD risk in both, whereas polymorphisms in MTRR , FPGS , and SLC19A1 were identified as risk factors in only one of the models. A strong synergistic effect on the development of CHDs was detected for MTHFD1 polymorphism and a lack of maternal folate supplementation during early pregnancy. A common polymorphism in the MTHFD1 is a genetic risk factor for the development of CHD, especially in the absence of folate supplementation in early pregnancy.
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- 2022
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16. The Enhanced Cytotoxic Effects in B-Cell Leukemia and Lymphoma Following Activation of Prostaglandin EP4 Receptor and Targeting of CD20 Antigen by Monoclonal Antibodies.
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Markovič T, Podgornik H, Avsec D, Nabergoj S, and Mlinarič-Raščan I
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- Antibodies, Monoclonal therapeutic use, Antibodies, Monoclonal, Humanized pharmacology, Antibodies, Monoclonal, Humanized therapeutic use, Cell Line, Tumor, Cell Proliferation drug effects, Cell Survival drug effects, Drug Synergism, Gene Expression Regulation, Neoplastic drug effects, Heptanoic Acids pharmacology, Humans, Leukemia, B-Cell drug therapy, Lymphoma, B-Cell drug therapy, Pyrrolidinones pharmacology, Receptors, Prostaglandin E, EP4 Subtype metabolism, Rituximab pharmacology, Rituximab therapeutic use, Tetrazoles pharmacology, Antibodies, Monoclonal pharmacology, Antigens, CD20 immunology, Leukemia, B-Cell metabolism, Lymphoma, B-Cell metabolism, Receptors, Prostaglandin E, EP4 Subtype agonists
- Abstract
Anti-CD20 monoclonal antibodies (MAbs) have revolutionized the treatment of B-cell leukemia and lymphoma. However, many patients do not respond to such treatment due to either deficiency of the complementary immune response or resistance to apoptosis. Other currently available treatments are often inadequate or induce major side effects. Therefore, there is a constant need for improved therapies. The prostaglandin E2 receptor 4 (EP4) receptor has been identified as a promising therapeutic target for hematologic B-cell malignancies. Herein, we report that EP4 receptor agonists PgE1-OH and L-902688 have exhibited enhanced cytotoxicity when applied together with anti-CD20 MAbs rituximab, ofatumumab and obinutuzumab in vitro in Burkitt lymphoma cells Ramos, as well as in p53-deficient chronic lymphocytic leukemia (CLL) cells MEC-1. Moreover, the enhanced cytotoxic effects of EP4 receptor agonists and MAbs targeting CD20 have been identified ex vivo on primary lymphocytes B obtained from patients diagnosed with CLL. Incubation of cells with PgE1-OH and L-902688 preserved the expression of CD20 molecules, further confirming the anti-leukemic potential of EP4 receptor agonists in combination with anti-CD20 MAbs. Additionally, we demonstrated that the EP4 receptor agonist PgE-1-OH induced apoptosis and inhibited proliferation via the EP4 receptor triggering in CLL. This work has revealed very important findings leading towards the elucidation of the anticancer potential of PgE1-OH and L-902688, either alone or in combination with MAbs. This may contribute to the development of potential therapeutic alternatives for patients with B-cell malignancies.
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- 2022
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17. Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring.
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Rajh E, Šket T, Praznik A, Sušjan P, Šmid A, Urbančič D, Mlinarič-Raščan I, Kogovšek P, Demšar T, Milavec M, Prosenc Trilar K, Jensterle Ž, Zidarn M, Tomič V, Turel G, Lejko-Zupanc T, Jerala R, and Benčina M
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- COVID-19 Testing methods, Humans, Mass Screening methods, Nucleic Acid Amplification Techniques methods, RNA isolation & purification, RNA, Viral genetics, SARS-CoV-2 genetics, SARS-CoV-2 pathogenicity, Saliva chemistry, Sensitivity and Specificity, Specimen Handling methods, COVID-19 diagnosis, COVID-19 genetics, Reverse Transcriptase Polymerase Chain Reaction methods
- Abstract
Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.
- Published
- 2021
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18. Polymorphism in Gene for ABCC2 Transporter Predicts Methotrexate Drug Survival in Patients with Psoriasis.
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Grželj J, Marovt M, Marko PB, Mlinarič-Raščan I, Gmeiner T, and Šmid A
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- Adult, Cohort Studies, Female, Humans, Kaplan-Meier Estimate, Methotrexate therapeutic use, Multidrug Resistance-Associated Protein 2, Multidrug Resistance-Associated Proteins, Treatment Outcome, Pharmaceutical Preparations, Psoriasis drug therapy, Psoriasis genetics
- Abstract
Background and Objectives : Methotrexate is widely prescribed for the treatment of moderate-to-severe psoriasis. As drug survival encompasses efficacy, safety, and treatment satisfaction, such studies provide insights into successful drug treatments in the real-life scenario. The objective was to define methotrexate drug survival and reasons for discontinuation, along with factors associated with drug survival, in a cohort of adult patients with moderate-to-severe plaque psoriasis. Materials and Methods : Data on methotrexate treatment were extracted from our institutional registry. Drug survival was estimated by Kaplan-Meier analysis, and predictors of drug survival were analyzed by Cox proportional hazards regression. Results : We included 133 patients treated with methotrexate. Due to significant effects of the year of treatment initiation, drug survival analysis was performed for 117 patients who started methotrexate in 2010 or later. Median methotrexate drug survival was 11.0 months. Overall, 89% of patients discontinued treatment, with over half of these (51%) due to lack of efficacy. Significantly longer drug survival was seen for patients who discontinued treatment due to lack of efficacy versus drug safety ( p = 0.049); when stratified by sex, this remained significant only for women ( p = 0.002). The patient ABCC2 rs717620 genotype was significantly associated with drug survival in both univariate log-rank and multivariate Cox regression analyses, with variant T allele associated with longer drug survival (hazard ratio, 0.606; 95% confidence interval, 0.380-0.967; p = 0.036). Conclusions : We have identified the novel association of patient ABCC2 rs717620 genotype with methotrexate drug survival. This pharmacogenetic marker might thus help in the management of psoriasis patients in daily practice.
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- 2021
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19. Targeting Autophagy Triggers Apoptosis and Complements the Action of Venetoclax in Chronic Lymphocytic Leukemia Cells.
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Avsec D, Jakoš Djordjevič AT, Kandušer M, Podgornik H, Škerget M, and Mlinarič-Raščan I
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Continuous treatment of patients with chronic lymphocytic leukemia (CLL) with venetoclax, an antagonist of the anti-apoptotic protein Bcl-2, can result in resistance, which highlights the need for novel targets to trigger cell death in CLL. Venetoclax also induces autophagy by perturbing the Bcl-2/Beclin-1 complex, so autophagy might represent a target in CLL. Diverse autophagy inhibitors were assessed for cytotoxic activities against patient-derived CLL cells. The AMPK inhibitor dorsomorphin, the ULK1/2 inhibitor MRT68921, and the autophagosome-lysosome fusion inhibitor chloroquine demonstrated concentration-dependent and time-dependent cytotoxicity against CLL cells, even in those from hard-to-treat patients who carried del(11q) and del(17p). Dorsomorphin and MRT68921 but not chloroquine triggered caspase-dependent cell death. According to the metabolic activities of CLL cells and PBMCs following treatments with 10 µM dorsomorphin (13% vs. 84%), 10 µM MRT68921 (7% vs. 78%), and 25 µM chloroquine (41% vs. 107%), these autophagy inhibitors are selective toward CLL cells. In these CLL cells, venetoclax induced autophagy, and addition of dorsomorphin, MRT68921, or chloroquine showed potent synergistic cytotoxicities. Additionally, MRT68921 alone induced G2 arrest, but when combined with venetoclax, it triggered caspase-dependent cytotoxicity. These data provide the rationale to target autophagy and for autophagy inhibitors as potential treatments for patients with CLL.
- Published
- 2021
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20. Structural Fine-Tuning of Desmuramylpeptide NOD2 Agonists Defines Their In Vivo Adjuvant Activity.
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Guzelj S, Nabergoj S, Gobec M, Pajk S, Klančič V, Slütter B, Frkanec R, Štimac A, Šket P, Plavec J, Mlinarič-Raščan I, and Jakopin Ž
- Subjects
- Acetylmuramyl-Alanyl-Isoglutamine metabolism, Acetylmuramyl-Alanyl-Isoglutamine pharmacology, Adjuvants, Immunologic metabolism, Adjuvants, Immunologic pharmacology, Animals, Antibody Formation drug effects, Cell Line, Drug Design, Humans, Immunoglobulin G metabolism, Interleukin-1beta metabolism, Interleukin-6 metabolism, Leukocytes, Mononuclear cytology, Leukocytes, Mononuclear drug effects, Leukocytes, Mononuclear metabolism, Liposomes chemistry, Lymphocyte Activation drug effects, Mice, Mice, Inbred C57BL, Nod2 Signaling Adaptor Protein metabolism, Ovalbumin immunology, Structure-Activity Relationship, Th1 Cells cytology, Th1 Cells immunology, Th1 Cells metabolism, Th2 Cells cytology, Th2 Cells immunology, Th2 Cells metabolism, Acetylmuramyl-Alanyl-Isoglutamine chemistry, Adjuvants, Immunologic chemistry, Nod2 Signaling Adaptor Protein agonists
- Abstract
We report on the design, synthesis, and biological evaluation of a series of nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) desmuramylpeptide agonists with improved in vitro and in vivo adjuvant properties. We identified two promising compounds: 68 , a potent nanomolar in vitro NOD2 agonist, and the more lipophilic 75 , which shows superior adjuvant activity in vivo . Both compounds had immunostimulatory effects on peripheral blood mononuclear cells at the protein and transcriptional levels, and augmented dendritic-cell-mediated activation of T cells, while 75 additionally enhanced the cytotoxic activity of peripheral blood mononuclear cells against malignant cells. The C
18 lipophilic tail of 75 is identified as a pivotal structural element that confers in vivo adjuvant activity in conjunction with a liposomal delivery system. Accordingly, liposome-encapsulated 75 showed promising adjuvant activity in mice, surpassing that of muramyl dipeptide, while achieving a more balanced Th1/Th2 immune response, thus highlighting its potential as a vaccine adjuvant.- Published
- 2021
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21. Polymorphisms in GNMT and DNMT3b are associated with methotrexate treatment outcome in plaque psoriasis.
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Grželj J, Mlinarič-Raščan I, Marko PB, Marovt M, Gmeiner T, and Šmid A
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- Adolescent, Adult, Aged, Child, Child, Preschool, Dermatologic Agents administration & dosage, Female, Humans, Infant, Infant, Newborn, Male, Middle Aged, Multidrug Resistance-Associated Protein 2, Pharmacogenomic Testing methods, Psoriasis diagnosis, Registries, Retrospective Studies, Treatment Outcome, Young Adult, DNA Methyltransferase 3B, DNA (Cytosine-5-)-Methyltransferases genetics, Glycine N-Methyltransferase genetics, Methotrexate administration & dosage, Polymorphism, Single Nucleotide genetics, Psoriasis drug therapy, Psoriasis genetics
- Abstract
Methotrexate is used as first-line treatment of moderate to severe psoriasis. Despite the marked variability in treatment outcomes, no pharmacogenetic markers are currently used for personalised management of therapy. In this retrospective study, we investigated the effects of genetic predisposition on efficacy and toxicity of low-dose methotrexate in a cohort of 137 patients with moderate to severe plaque psoriasis. We genotyped 16 polymorphisms in genes for enzymes involved in the folate-methionine pathway and in methotrexate transport, and analysed their association with treatment efficacy and toxicity using classification and regression tree analysis and logistic regression. The most pronounced effect observed in this study was for GNMT rs10948059, which was identified as a risk factor for inadequate efficacy leading to treatment discontinuation. Patients carrying at least one variant allele had ~7-fold increased risk of treatment failure compared to patients with the wild-type genotype, as shown by the classification and regression tree analysis and logistic regression (odds ratio [OR], 6.94; p = 0.0004). Another risk factor associated with insufficient treatment responses was DNMT3b rs2424913, where patients carrying at least one variant allele had a 4-fold increased risk of treatment failure compared to patients with the wild-type genotype (OR, 4.10; p = 0.005). Using classification and regression tree analysis, we show that DNMT3b rs2424913 has a more pronounced role in patients with the variant GNMT genotype, and hence we suggest an interaction between these two genes. Further, we show that patients with the BHMT rs3733890 variant allele had increased risk of hepatotoxicity (OR, 3.17; p = 0.022), which is the most prominent reason for methotrexate discontinuation. We also show that variants in the genes for methotrexate transporters OATP1B1 (rs2306283/rs4149056 SLCO1B1 haplotypes) and ABCC2 (rs717620) are associated with increased risk of treatment failure. The associations identified have not been reported previously. These data suggest that polymorphisms in genes for enzymes of the methionine cycle (which affect cell methylation potential) might have significant roles in treatment responses to methotrexate of patients with psoriasis. Further studies are warranted to validate the potential of the pharmacogenetic markers identified., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)
- Published
- 2021
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22. A Putative Serine Protease is Required to Initiate the RIPK3-MLKL-Mediated Necroptotic Death Pathway in Neutrophils.
- Author
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Wang X, Avsec D, Obreza A, Yousefi S, Mlinarič-Raščan I, and Simon HU
- Abstract
Adhesion receptors, such as CD44, have been shown to activate receptor interacting protein kinase-3 (RIPK3)-mixed lineage kinase-like (MLKL) signaling, leading to a non-apoptotic cell death in human granulocyte/macrophage colony-stimulating factor (GM-CSF) - primed neutrophils. The signaling events of this necroptotic pathway, however, remain to be investigated. In the present study, we report the design, synthesis, and characterization of a series of novel serine protease inhibitors. Two of these inhibitors, compounds 1 and 3, were able to block CD44-triggered necroptosis in GM-CSF-primed neutrophils. Both inhibitors prevented the activation of MLKL, p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'-kinase (PI3K), hence blocking the increased levels of reactive oxygen species (ROS) required for cell death. Although compounds one and three partially inhibited isolated human neutrophil elastase (HNE) activity, we obtained no pharmacological evidence that HNE is involved in the initiation of this death pathway within a cellular context. Interestingly, neither serine protease inhibitor had any effect on FAS receptor-mediated apoptosis. Taken together, these results suggest that a serine protease is involved in non-apoptotic CD44-triggered RIPK3-MLKL-dependent neutrophil cell death, but not FAS receptor-mediated caspase-dependent apoptosis. Thus, a pharmacological block on serine proteases might be beneficial for preventing exacerbation of disease in neutrophilic inflammatory responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Simon, Wang, Avsec, Obreza, Yousefi and Mlinaric-Rascan.)
- Published
- 2021
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23. EP4 receptor agonist L-902688 augments cytotoxic activities of ibrutinib, idelalisib, and venetoclax against chronic lymphocytic leukemia cells.
- Author
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Nabergoj S, Markovič T, Avsec D, Gobec M, Podgornik H, Jakopin Ž, and Mlinarič-Raščan I
- Subjects
- Adenine administration & dosage, Adult, Antineoplastic Agents administration & dosage, Antineoplastic Combined Chemotherapy Protocols administration & dosage, Apoptosis drug effects, Apoptosis physiology, Dose-Response Relationship, Drug, Drug Synergism, Humans, Jurkat Cells, Leukocytes, Mononuclear drug effects, Leukocytes, Mononuclear metabolism, Receptors, Prostaglandin E, EP4 Subtype metabolism, U937 Cells, Adenine analogs & derivatives, Bridged Bicyclo Compounds, Heterocyclic administration & dosage, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Leukemia, Lymphocytic, Chronic, B-Cell metabolism, Piperidines administration & dosage, Purines administration & dosage, Pyrrolidinones administration & dosage, Quinazolinones administration & dosage, Receptors, Prostaglandin E, EP4 Subtype agonists, Sulfonamides administration & dosage, Tetrazoles administration & dosage
- Abstract
Treatment of patients with relapsed or refractory chronic lymphocytic leukemia (CLL) has significantly improved more recently with the approval of several new agents, including ibrutinib, idelalisib, and venetoclax. Despite the outstanding efficacies observed with these agents, these treatments are sometimes discontinued due to toxicity, unresponsiveness, transformation of the disease and/or resistance. Constitutive NF-κB activation that protects CLL cells from apoptotic stimuli represents one of molecular mechanisms that underlie the emergence of drug resistance. As prostaglandin E (EP)4 receptor agonists have been shown to successfully inhibit the NF-κB pathway in B-cell lymphoma cells, we investigated the potential of the highly specific EP4 receptor agonist L-902688 for the potential treatment of patients with CLL. We show here that low micromolar concentrations of L-902688 can indeed induce selective cytotoxicity towards several B-cell malignancies, including CLL. Moreover, L-902688-mediated activation of the EP4 receptor in patient derived CLL cells resulted in inhibition of the NF-κB pathway, cell proliferation, and induction of apoptosis. Most importantly, we show for the first time that in combination with ibrutinib, idelalisib, or venetoclax, L-902688 induces synergistic cytotoxic activity against patient derived CLL cells. To conclude, the modulation of NF-κB activity by EP4 receptor agonists represents an innovative approach to improve the treatment of patients with CLL. In particular, EP4 receptor agonists appear to represent promising adjuncts to the already existing therapies for patients with CLL due to these promising synergistic activities., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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24. Simultaneous quantification of intracellular concentrations of clinically important metabolites of folate-homocysteine cycle by LC-MS/MS.
- Author
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Vidmar Golja M, Trontelj J, Geršak K, Mlinarič-Raščan I, and Šmid A
- Subjects
- Cell Line, Chromatography, High Pressure Liquid, Humans, Tandem Mass Spectrometry, Folic Acid analysis, Homocysteine analysis, Tetrahydrofolates analysis
- Abstract
Inadequate folate status is detrimental to human development. Deficiency has been implicated in congenital birth defects and cancer, whereas excess has been linked to various negative neurocognitive development outcomes. We developed a method for translational studies involving lymphoblastoid cell models for studying role of folates in vital cell processes. We describe a simple, sensitive, and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of intracellular concentrations of clinically important metabolites of folate-homocysteine cycle; namely, folic acid (FA), 5-methyltetrahydrofolate (5-Me-THF), and homocysteine (Hcy). The method was validated for specificity, linearity, limits of quantification, repeatability, reproducibility, matrix effects, and stability. Method had a wide linear range between 0.341 and 71.053 ng Hcy/mg protein for Hcy, 0.004-0.526 ng FA/mg protein for FA and 0.003-0.526 ng 5-Me-THF/mg protein for 5-Me-THF. The method overcomes challenges associated with the quantification of endogenous molecules, poor stability, and extremely small amounts of the analytes. The method was successfully applied to evaluate the effects of FA and 5-Me-THF treatment of cells in vitro mimicking supplement therapy with various metabolically active species, and showed that 5-Me-THF is more effective than FA in increasing intracellular levels of the biologically active form of folate., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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25. Folate Insufficiency Due to MTHFR Deficiency Is Bypassed by 5-Methyltetrahydrofolate.
- Author
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Vidmar Golja M, Šmid A, Karas Kuželički N, Trontelj J, Geršak K, and Mlinarič-Raščan I
- Abstract
Adequate levels of folates are essential for homeostasis of the organism, prevention of congenital malformations, and the salvage of predisposed disease states. They depend on genetic predisposition, and therefore, a pharmacogenetic approach to individualized supplementation or therapeutic intervention is necessary for an optimal outcome. The role of folates in vital cell processes was investigated by translational pharmacogenetics employing lymphoblastoid cell lines (LCLs). Depriving cells of folates led to reversible S-phase arrest. Since 5,10-methylenetetrahydrofolate reductase (MTHFR) is the key enzyme in the biosynthesis of an active folate form, we evaluated the relevance of polymorphisms in the MTHFR gene on intracellular levels of bioactive metabolite, the 5-methyltetrahydrofolate (5-Me-THF). LCLs ( n = 35) were divided into low- and normal-MTHFR activity groups based on their genotype. They were cultured in the presence of folic acid (FA) or 5-Me-THF. Based on the cells' metabolic activity and intracellular 5-Me-THF levels, we conclude supplementation of FA is sufficient to maintain adequate folate level in the normal MTHFR activity group, while low MTHFR activity cells require 5-Me-THF to overcome the metabolic defects caused by polymorphisms in their MTHFR genes. This finding was supported by the determination of intracellular levels of 5-Me-THF in cell lysates by LC-MS/MS. FA supplementation resulted in a 2.5-fold increase in 5-Me-THF in cells with normal MTHFR activity, but there was no increase after FA supplementation in low MTHFR activity cells. However, when LCLs were exposed to 5-Me-THF, a 10-fold increase in intracellular levels of this metabolite was determined. These findings indicate that patients undergoing folate supplementation to counteract anti-folate therapies, or patients with increased folate demand, would benefit from pharmacogenetics-based therapy choices.
- Published
- 2020
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26. ATG12 deficiency leads to tumor cell oncosis owing to diminished mitochondrial biogenesis and reduced cellular bioenergetics.
- Author
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Liu H, He Z, Germič N, Ademi H, Frangež Ž, Felser A, Peng S, Riether C, Djonov V, Nuoffer JM, Bovet C, Mlinarič-Raščan I, Zlobec I, Fiedler M, Perren A, and Simon HU
- Subjects
- Animals, Cell Line, Tumor, Energy Metabolism, Glycolysis, Humans, Mice, Mice, Inbred NOD, Mice, SCID, Autophagy-Related Protein 12 physiology, Mitochondria metabolism, Neoplasms metabolism
- Abstract
In contrast to the "Warburg effect" or aerobic glycolysis earlier generalized as a phenomenon in cancer cells, more and more recent evidence indicates that functional mitochondria are pivotal for ensuring the energy supply of cancer cells. Here, we report that cancer cells with reduced autophagy-related protein 12 (ATG12) expression undergo an oncotic cell death, a phenotype distinct from that seen in ATG5-deficient cells described before. In addition, using untargeted metabolomics with ATG12-deficient cancer cells, we observed a global reduction in cellular bioenergetic pathways, such as β-oxidation (FAO), glycolysis, and tricarboxylic acid cycle activity, as well as a decrease in mitochondrial respiration as monitored with Seahorse experiments. Analyzing the biogenesis of mitochondria by quantifying mitochondrial DNA content together with several mitochondrion-localizing proteins indicated a reduction in mitochondrial biogenesis in ATG12-deficient cancer cells, which also showed reduced hexokinase II expression and the upregulation of uncoupling protein 2. ATG12, which we observed in normal cells to be partially localized in mitochondria, is upregulated in multiple types of solid tumors in comparison with normal tissues. Strikingly, mouse xenografts of ATG12-deficient cells grew significantly slower as compared with vector control cells. Collectively, our work has revealed a previously unreported role for ATG12 in regulating mitochondrial biogenesis and cellular energy metabolism and points up an essential role for mitochondria as a failsafe mechanism in the growth and survival of glycolysis-dependent cancer cells. Inducing oncosis by imposing an ATG12 deficiency in solid tumors might represent an anticancer therapy preferable to conventional caspase-dependent apoptosis that often leads to undesirable consequences, such as incomplete cancer cell killing and a silencing of the host immune system.
- Published
- 2020
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27. Structural features and functional activities of benzimidazoles as NOD2 antagonists.
- Author
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Guzelj S, Gobec M, Urbančič D, Mlinarič-Raščan I, Corsini E, and Jakopin Ž
- Subjects
- Anti-Inflammatory Agents chemical synthesis, Anti-Inflammatory Agents toxicity, Benzimidazoles chemical synthesis, Benzimidazoles toxicity, Drug Design, HEK293 Cells, Humans, Molecular Structure, Nod1 Signaling Adaptor Protein antagonists & inhibitors, Structure-Activity Relationship, Anti-Inflammatory Agents pharmacology, Benzimidazoles pharmacology, Nod2 Signaling Adaptor Protein antagonists & inhibitors
- Abstract
NOD1 and NOD2 are pattern recognition receptors that have important roles in innate immune responses. Although their overactivation has been linked to a number of diseases, NOD2 in particular remains a virtually unexploited target in this respect, with only one structural class of antagonist reported. To gain insight into the structure-activity relationships of NOD2 antagonists, a series of novel analogs was designed and synthesized, and then screened for antagonist activity versus NOD2, and counter-screened versus NOD1. Compounds 32 and 38 were identified as potent and moderately selective NOD2 antagonists, and 33 and 42 as dual NOD1/NOD2 antagonists, with balanced activities against both targets in the low micromolar range. These data enable in-depth exploration of their structure-activity relationships and provide deeper understanding of the structural features required for NOD2 antagonism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)
- Published
- 2020
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28. Harnessing the untapped potential of nucleotide-binding oligomerization domain ligands for cancer immunotherapy.
- Author
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Nabergoj S, Mlinarič-Raščan I, and Jakopin Ž
- Subjects
- Antineoplastic Agents therapeutic use, Humans, Ligands, Neoplasms metabolism, Nod1 Signaling Adaptor Protein chemistry, Nod1 Signaling Adaptor Protein metabolism, Nod2 Signaling Adaptor Protein chemistry, Nod2 Signaling Adaptor Protein metabolism, Polymerization, Protein Conformation, Signal Transduction, Neoplasms therapy, Nucleotides metabolism
- Abstract
In the last decade, cancer immunotherapy has emerged as an effective alternative to traditional therapies such as chemotherapy and radiation. In contrast to the latter, cancer immunotherapy has the potential to distinguish between cancer and healthy cells, and thus to avoid severe and intolerable side-effects, since the cancer cells are effectively eliminated by stimulated immune cells. The cytosolic nucleotide-binding oligomerization domains 1 and 2 receptors (NOD1 and NOD2) are important components of the innate immune system and constitute interesting targets in terms of strengthening the immune response against cancer cells. Many NOD ligands have been synthesized, in particular NOD2 agonists that exhibit favorable immunostimulatory and anticancer activity. Among them, mifamurtide has already been approved in Europe by the European Medicine Agency for treating patients with osteosarcoma in combination with chemotherapy after complete surgical removal of the primary tumor. This review is focused on NOD receptors as promising targets in cancer immunotherapy as well as summarizing current knowledge of the various NOD ligands exhibiting antitumor and even antimetastatic activity in vitro and in vivo., (© 2018 The Authors. Medicinal Research Reviews Published by Wiley Periodicals, Inc.)
- Published
- 2019
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29. Methylation of selenocysteine catalysed by thiopurine S-methyltransferase.
- Author
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Urbančič D, Kotar A, Šmid A, Jukič M, Gobec S, Mårtensson LG, Plavec J, and Mlinarič-Raščan I
- Subjects
- Catalysis, Catalytic Domain, Humans, Kinetics, Methylation, Molecular Conformation, Molecular Docking Simulation, Protein Binding, Protein Processing, Post-Translational, Recombinant Proteins chemistry, Selenocysteine analogs & derivatives, Magnetic Resonance Spectroscopy, Methyltransferases chemistry, Selenium chemistry, Selenocysteine chemistry
- Abstract
Background: Methylation driven by thiopurine S-methylatransferase (TPMT) is crucial for deactivation of cytostatic and immunosuppressant thiopurines. Despite its remarkable integration into clinical practice, the endogenous function of TPMT is unknown., Methods: To address the role of TPMT in methylation of selenium compounds, we established the research on saturation transfer difference (STD) and
77 Se NMR spectroscopy, fluorescence measurements, as well as computational molecular docking simulations., Results: Using STD NMR spectroscopy and fluorescence measurements of tryptophan residues in TPMT, we determined the binding of selenocysteine (Sec) to human recombinant TPMT. By comparing binding characteristics of Sec in the absence and in the presence of methyl donor, we confirmed S-adenosylmethionine (SAM)-induced conformational changes in TPMT. Molecular docking analysis positioned Sec into the active site of TPMT with orientation relevant for methylation reaction. Se-methylselenocysteine (MeSec), produced in the enzymatic reaction, was detected by77 Se NMR spectroscopy. A direct interaction between Sec and SAM in the active site of rTPMT and the formation of both products, MeSec and S-adenosylhomocysteine, was demonstrated using NMR spectroscopy., Conclusions: The present study provides evidence on in vitro methylation of Sec by rTPMT in a SAM-dependant manner., General Significance: Our results suggest novel role of TPMT and demonstrate new insights into enzymatic modifications of the 21st amino acid., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2019
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30. Novel motif of variable number of tandem repeats in TPMT promoter region and evolutionary association of variable number of tandem repeats with TPMT*3 alleles.
- Author
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Urbančič D, Šmid A, Stocco G, Decorti G, Mlinarič-Raščan I, and Karas Kuželički N
- Subjects
- Alleles, Cell Line, Genotype, Heterozygote, Humans, Linkage Disequilibrium genetics, Pharmacogenetics methods, Phenotype, White People genetics, Methyltransferases genetics, Polymorphism, Single Nucleotide genetics, Promoter Regions, Genetic genetics, Tandem Repeat Sequences genetics
- Abstract
Aim: SNPs in the gene for TPMT exemplify one of the most successful translations of pharmacogenomics into clinical practice. This study explains the correlation between common SNPs and variable number of tandem repeats (VNTR) in promoter of the gene., Materials & Methods: We determined VNTR polymorphisms, as well as TPMT*2 and TPMT*3 SNPs and TPMT activity in Slovenian and Italian individuals and lymphoblastoid cell lines., Results: We observed a previously unreported VNTR allele, AB7C, in a TPMT*3A heterozygous individual. VNTRs with two (AB2C) and three or more (ABnC, n ≥ 3) B motifs were statistically significant in complete linkage disequilibrium (D' = 1, r
2 = 1, p < 0.0001) with the TPMT*3C and TPMT*3A alleles, respectively., Conclusion: The study provides insights into the stepwise evolution of TPMT*3 alleles from *3C to *3A, with increasing number of B motifs in the VNTR region.- Published
- 2018
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31. Endocrine disrupting activities and immunomodulatory effects in lymphoblastoid cell lines of diclofenac, 4-hydroxydiclofenac and paracetamol.
- Author
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Klopčič I, Markovič T, Mlinarič-Raščan I, and Sollner Dolenc M
- Subjects
- Acetaminophen chemistry, Acetaminophen metabolism, Analgesics, Non-Narcotic chemistry, Analgesics, Non-Narcotic metabolism, Androgen Receptor Antagonists adverse effects, Androgen Receptor Antagonists chemistry, Androgen Receptor Antagonists metabolism, Androgens adverse effects, Androgens chemistry, Androgens metabolism, Anti-Inflammatory Agents, Non-Steroidal chemistry, Anti-Inflammatory Agents, Non-Steroidal metabolism, Binding, Competitive, Cell Line, Cell Survival drug effects, Cells, Cultured, Cytokines agonists, Cytokines metabolism, Diclofenac analogs & derivatives, Diclofenac chemistry, Diclofenac metabolism, Endocrine Disruptors chemistry, Endocrine Disruptors metabolism, Estrogens adverse effects, Estrogens chemistry, Estrogens metabolism, Genes, Reporter drug effects, Humans, Immunologic Factors chemistry, Immunologic Factors metabolism, Lymphocytes cytology, Lymphocytes immunology, Lymphocytes metabolism, Receptors, Androgen chemistry, Receptors, Androgen genetics, Receptors, Androgen metabolism, Receptors, Estrogen chemistry, Receptors, Estrogen genetics, Receptors, Estrogen metabolism, Receptors, Glucocorticoid agonists, Receptors, Glucocorticoid antagonists & inhibitors, Receptors, Glucocorticoid genetics, Receptors, Glucocorticoid metabolism, Receptors, Thyroid Hormone agonists, Receptors, Thyroid Hormone antagonists & inhibitors, Receptors, Thyroid Hormone genetics, Receptors, Thyroid Hormone metabolism, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Structure-Activity Relationship, Acetaminophen adverse effects, Analgesics, Non-Narcotic adverse effects, Anti-Inflammatory Agents, Non-Steroidal adverse effects, Diclofenac adverse effects, Endocrine Disruptors adverse effects, Immunologic Factors adverse effects, Lymphocytes drug effects
- Abstract
A critical literature review reveals that knowledge of side effects of pharmaceuticals diclofenac and paracetamol is extremely important because of their widespread use and occurrence in the environment. In order to delineate whether these compounds have endocrine activity and influence on the immune system, we assessed the potential endocrine disrupting and immunomodulatory activities of: diclofenac (DIC), its metabolite 4-hydroxydiclofenac (4-HD) and paracetamol (PAR). Herein, we report on their impact on estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR) and thyroid hormone receptor (TR). The endocrine disrupting effects were assessed in vitro in MDA-kb2 and GH3.TRE-Luc cell lines and by the XenoScreen YES/YAS assay. Moreover, binding affinity to nuclear receptors (GR and AR) was also measured. Immunomodulatory properties of the compounds were evaluated in lymphoblastoid cell lines. All the tested compounds showed endocrine disrupting and immunomodulatory activities. The results revealed that both DIC and its metabolite 4-HD exhibited significant estrogenic, anti-androgenic (in YAS assay), (anti)-androgenic, (anti)-glucocorticoid and anti-thyroid hormonal activities (in luciferase reporter gene assays). DIC showed direct binding to the GR, while its metabolite 4-HD to the GR and AR. Only metabolite 4-HD showed estrogenic, androgenic (in YAS assay) and thyroid-hormonal activities. PAR had anti-androgenic activity and anti-thyroid hormonal activity. PAR displayed GR agonist activity with competition to its receptor and agonistic activity to AR. All of the compounds significantly modulated pro-inflammatory and immunoregulatory cytokine production in lymphoblastoid cell lines and were thus proven immunomodulatory. The study is useful in determining toxicological effects and contributes to the knowledge of possible side effects of diclofenac, its metabolite and paracetamol., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2018
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32. Discovery of Nanomolar Desmuramylpeptide Agonists of the Innate Immune Receptor Nucleotide-Binding Oligomerization Domain-Containing Protein 2 (NOD2) Possessing Immunostimulatory Properties.
- Author
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Gobec M, Tomašič T, Štimac A, Frkanec R, Trontelj J, Anderluh M, Mlinarič-Raščan I, and Jakopin Ž
- Subjects
- Animals, Antineoplastic Agents chemical synthesis, Antineoplastic Agents pharmacology, Cell Line, Tumor, Cytokines metabolism, Drug Design, Drug Discovery, Female, Humans, Immunoglobulin G biosynthesis, Lipopolysaccharides pharmacology, Mice, Models, Molecular, Molecular Conformation, Monocytes drug effects, Monocytes metabolism, Nod2 Signaling Adaptor Protein metabolism, Structure-Activity Relationship, Acetylmuramyl-Alanyl-Isoglutamine agonists, Adjuvants, Immunologic chemical synthesis, Adjuvants, Immunologic pharmacology, Immunity, Innate drug effects, Nod2 Signaling Adaptor Protein drug effects
- Abstract
Muramyl dipeptide (MDP), a fragment of bacterial peptidoglycan, has long been known as the smallest fragment possessing adjuvant activity, on the basis of its agonistic action on the nucleotide-binding oligomerization domain-containing protein 2 (NOD2). There is a pressing need for novel adjuvants, and NOD2 agonists provide an untapped source of potential candidates. Here, we report the design, synthesis, and characterization of a series of novel acyl tripeptides. A pivotal structural element for molecular recognition by NOD2 has been identified, culminating in the discovery of compound 9, the most potent desmuramylpeptide NOD2 agonist to date. Compound 9 augmented pro-inflammatory cytokine release from human peripheral blood mononuclear cells in synergy with lipopolysaccharide. Furthermore, it was able to induce ovalbumin-specific IgG titers in a mouse model of adjuvancy. These findings provide deeper insights into the structural requirements of desmuramylpeptides for NOD2-activation and highlight the potential use of NOD2 agonists as adjuvants for vaccines.
- Published
- 2018
- Full Text
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33. Polymorphic variation in TPMT is the principal determinant of TPMT phenotype: A meta-analysis of three genome-wide association studies.
- Author
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Tamm R, Mägi R, Tremmel R, Winter S, Mihailov E, Smid A, Möricke A, Klein K, Schrappe M, Stanulla M, Houlston R, Weinshilboum R, Mlinarič Raščan I, Metspalu A, Milani L, Schwab M, and Schaeffeler E
- Subjects
- Alleles, Estonia, Humans, Phenotype, Genome-Wide Association Study, Methyltransferases genetics, Polymorphism, Genetic genetics
- Abstract
Thiopurine-related hematotoxicity in pediatric acute lymphoblastic leukemia (ALL) and inflammatory bowel diseases has been linked to genetically defined variability in thiopurine S-methyltransferase (TPMT) activity. While gene testing of TPMT is being clinically implemented, it is unclear if additional genetic variation influences TPMT activity with consequences for thiopurine-related toxicity. To examine this possibility, we performed a genome-wide association study (GWAS) of red blood cell TPMT activity in 844 Estonian individuals and 245 pediatric ALL cases. Additionally, we correlated genome-wide genotypes to human hepatic TPMT activity in 123 samples. Only genetic variants mapping to chromosome 6, including the TPMT gene region, were significantly associated with TPMT activity (P < 5.0 × 10
-8 ) in each of the three GWAS and a joint meta-analysis of 1,212 cases (top hit P = 1.2 × 10-72 ). This finding is consistent with TPMT genotype being the primary determinant of TPMT activity, reinforcing the rationale for genetic testing of TPMT alleles in routine clinical practice to individualize mercaptopurine dosage., (© 2016 American Society for Clinical Pharmacology and Therapeutics.)- Published
- 2017
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34. Structural features of subtype-selective EP receptor modulators.
- Author
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Markovič T, Jakopin Ž, Dolenc MS, and Mlinarič-Raščan I
- Subjects
- Animals, Dinoprostone chemistry, Drug Design, Humans, Ligands, Molecular Structure, Molecular Targeted Therapy, Protein Binding, Pharmaceutical Preparations chemistry, Receptors, Prostaglandin E, EP1 Subtype agonists, Receptors, Prostaglandin E, EP1 Subtype antagonists & inhibitors, Receptors, Prostaglandin E, EP2 Subtype agonists, Receptors, Prostaglandin E, EP2 Subtype antagonists & inhibitors, Receptors, Prostaglandin E, EP3 Subtype agonists, Receptors, Prostaglandin E, EP3 Subtype antagonists & inhibitors, Receptors, Prostaglandin E, EP4 Subtype agonists, Receptors, Prostaglandin E, EP4 Subtype antagonists & inhibitors
- Abstract
Prostaglandin E2 is a potent endogenous molecule that binds to four different G-protein-coupled receptors: EP1-4. Each of these receptors is a valuable drug target, with distinct tissue localisation and signalling pathways. We review the structural features of EP modulators required for subtype-selective activity, as well as the structural requirements for improved pharmacokinetic parameters. Novel EP receptor subtype selective agonists and antagonists appear to be valuable drug candidates in the therapy of many pathophysiological states, including ulcerative colitis, glaucoma, bone healing, B cell lymphoma, neurological diseases, among others, which have been studied in vitro, in vivo and in early phase clinical trials., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2017
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35. Identification of indole scaffold-based dual inhibitors of NOD1 and NOD2.
- Author
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Keček Plešec K, Urbančič D, Gobec M, Pekošak A, Tomašič T, Anderluh M, Mlinarič-Raščan I, and Jakopin Ž
- Subjects
- Cell Proliferation drug effects, Cells, Cultured, Dose-Response Relationship, Drug, Drug Design, Humans, Indoles chemical synthesis, Indoles chemistry, Molecular Structure, NF-kappa B antagonists & inhibitors, NF-kappa B metabolism, Nod1 Signaling Adaptor Protein genetics, Nod2 Signaling Adaptor Protein genetics, Structure-Activity Relationship, Indoles pharmacology, Nod1 Signaling Adaptor Protein antagonists & inhibitors, Nod2 Signaling Adaptor Protein antagonists & inhibitors
- Abstract
NOD1 and NOD2 are important members of the pattern recognition receptor family and play a crucial role within the context of innate immunity. However, overactivation of NODs, especially of NOD1, has also been implicated in a number of diseases. Surprisingly, NOD1 remains a virtually unexploited target in this respect. To gain additional insight into the structure-activity relationships of NOD1 inhibitors, a series of novel analogs has been designed and synthesized and then screened for their NOD1-inhibitory activity. Selected compounds were also investigated for their NOD2-inhibitory activity. Two compounds 4 and 15, were identified as potent mixed inhibitors of NOD1 and NOD2, displaying a balanced inhibitory activity on both targets in the low micromolar range. The results obtained have enabled a deeper understanding of the structural requirements for NOD1 and NOD2 inhibition., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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36. Risk factors for symptomatic osteonecrosis in childhood ALL: A retrospective study of a Slovenian pediatric ALL population between 1970 and 2004.
- Author
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Karas-Kuželički N, Mencej-Bedrač S, Jazbec J, Marc J, and Mlinarič-Raščan I
- Abstract
Treatment induced non-traumatic osteonecrosis (ON) has been reported increasingly in children treated for acute lymphoblastic leukemia (ALL). Several risk factors for ON have been identified in childhood cancer patients; however, their diagnostic and prognostic power is limited and the etiology of the disease remains unclear. Therefore, a continuous effort is focused on the identification of additional ON risk factors. We performed a retrospective study of 313 childhood ALL patients to test the association between the ON occurrence in children receiving ALL therapy and common polymorphisms in potential target genes: Thiopurine S-methyltransferase ( TPMT ; 460G>A, 719A>G), 5,10-methylenetetrahydrofolate reductase ( MTHFR ; 677C>T, 1298A>C), estrogen receptor alpha 1 ( ESR1 ; XbaI) and collagen type I, α1 ( COL1A1 ; Sp1). In the present cohort, higher age and more recently developed treatment protocols were independent risk factors for ON. In children >14.5 years old, TPMT genotype modulated the risk of ON. Additionally, in children <12.9 years old ESR1 genotypes were also implicated in the pathogenesis of ON. Besides greater age and more recent treatment protocols, genetic factors (polymorphisms in ESR1 and TPMT genes) were suggested to be implicated in the pathogenesis of ON and could be potentially used as genetic prognostic markers for ON.
- Published
- 2016
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37. Structural requirements of acylated Gly-l-Ala-d-Glu analogs for activation of the innate immune receptor NOD2.
- Author
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Gobec M, Mlinarič-Raščan I, Dolenc MS, and Jakopin Ž
- Subjects
- Cell Line, Drug Design, Humans, Immunity, Innate drug effects, Indoles chemistry, Indoles pharmacology, Nod2 Signaling Adaptor Protein metabolism, Oligopeptides chemistry, Oligopeptides pharmacology
- Abstract
The fragment of bacterial peptidoglycan muramyl dipeptide (MDP) has long been known for its adjuvant activity, however the underlying mechanism of this action has only recently been elucidated. It is ascribed to its agonist action on the nucleotide-binding oligomerization domain-containing protein 2 (NOD2). In spite of the pressing need for novel adjuvants for human use, this discovery is hampered, by not knowing the structural requirements underlying the immunostimulatory activity. We have investigated how minor modifications of hit compound acyl Gly-L-Ala-D-Glu derivative I modulate the molecular recognition by NOD2. A series of novel desmuramyldipeptides has been designed and synthesized leading to the identification of compound 16, in which the sugar moiety is replaced by a 6-phenylindole moiety, that exhibits the strongest NOD2 activation to date sans the carbohydrate moiety. The results have enabled a deeper understanding of the structural requirements of desmuramylpeptides for NOD2 activation., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)
- Published
- 2016
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38. Nonpeptidic Selective Inhibitors of the Chymotrypsin-Like (β5 i) Subunit of the Immunoproteasome.
- Author
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Sosič I, Gobec M, Brus B, Knez D, Živec M, Konc J, Lešnik S, Ogrizek M, Obreza A, Žigon D, Janežič D, Mlinarič-Raščan I, and Gobec S
- Subjects
- Cell Line, Cell Survival drug effects, HeLa Cells, Humans, Inhibitory Concentration 50, Kinetics, Molecular Docking Simulation, Oligopeptides chemistry, Oligopeptides metabolism, Oligopeptides toxicity, Proteasome Endopeptidase Complex chemistry, Proteasome Inhibitors chemistry, Proteasome Inhibitors toxicity, Protein Subunits antagonists & inhibitors, Protein Subunits metabolism, Structure-Activity Relationship, Proteasome Endopeptidase Complex metabolism, Proteasome Inhibitors metabolism
- Abstract
Elevated expression of the immunoproteasome has been associated with autoimmune diseases, inflammatory diseases, and various types of cancer. Selective inhibitors of the immunoproteasome are not only scarce, but also almost entirely restricted to peptide-based compounds. Herein, we describe nonpeptidic reversible inhibitors that selectively block the chymotrypsin-like (β5i) subunit of the human immunoproteasome in the low micromolar range. The most potent of the reversibly acting compounds were then converted into covalent, irreversible, nonpeptidic inhibitors that retained selectivity for the β5i subunit. In addition, these inhibitors discriminate between the immunoproteasome and the constitutive proteasome in cell-based assays. Along with their lack of cytotoxicity, these data point to these nonpeptidic compounds being suitable for further investigation as β5i-selective probes for possible application in noncancer diseases related to the immunoproteasome., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
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39. Structure-Activity Relationships of Novel Tryptamine-Based Inhibitors of Bacterial Transglycosylase.
- Author
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Sosič I, Anderluh M, Sova M, Gobec M, Mlinarič Raščan I, Derouaux A, Amoroso A, Terrak M, Breukink E, and Gobec S
- Subjects
- Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents toxicity, Enterococcus faecium drug effects, Escherichia coli drug effects, HEK293 Cells, Humans, Methicillin Resistance, Microbial Sensitivity Tests, Protein Binding, Staphylococcus aureus drug effects, Structure-Activity Relationship, Tryptamines pharmacology, Tryptamines toxicity, Uridine Diphosphate N-Acetylmuramic Acid analogs & derivatives, Uridine Diphosphate N-Acetylmuramic Acid metabolism, Anti-Bacterial Agents chemistry, Escherichia coli Proteins antagonists & inhibitors, Penicillin-Binding Proteins antagonists & inhibitors, Peptidoglycan Glycosyltransferase antagonists & inhibitors, Serine-Type D-Ala-D-Ala Carboxypeptidase antagonists & inhibitors, Tryptamines chemistry
- Abstract
Penicillin-binding proteins represent well-established, validated, and still very promising targets for the design and development of new antibacterial agents. The transglycosylase domain of penicillin-binding proteins is especially important, as it catalyzes polymerization of glycan chains, using the peptidoglycan precursor lipid II as a substrate. On the basis of the previous discovery of a noncovalent small-molecule inhibitor of transglycosylase activity, we systematically explored the structure-activity relationships of these tryptamine-based inhibitors. The main aim was to reduce the nonspecific cytotoxic properties of the initial hit compound and concurrently to retain the mode of its inhibition. A focused library of tryptamine-based compounds was synthesized, characterized, and evaluated biochemically. The results presented here show the successful reduction of the nonspecific cytotoxicity, and the retention of the inhibition of transglycosylase enzymatic activity, as well as the ability of these compounds to bind to lipid II and to have antibacterial actions.
- Published
- 2015
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40. Antioxidant and anti-inflammatory properties of 1,2,4-oxadiazole analogs of resveratrol.
- Author
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Gobec M, Tomašič T, Markovič T, Mlinarič-Raščan I, Dolenc MS, and Jakopin Ž
- Subjects
- Anti-Inflammatory Agents chemistry, Anti-Inflammatory Agents pharmacology, Antioxidants chemistry, Antioxidants pharmacology, Blotting, Western, Cell Line, Cell Survival drug effects, Enzyme Activation drug effects, Models, Molecular, Oxadiazoles chemistry, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein Serine-Threonine Kinases metabolism, Reactive Oxygen Species metabolism, Resveratrol, Stilbenes chemistry, NF-kappaB-Inducing Kinase, Oxadiazoles pharmacology, Stilbenes pharmacology
- Abstract
The chemopreventive properties of resveratrol are ascribed mostly to its antioxidant activity, in particular its scavenging ability for reactive oxygen species (ROS), and to the inhibition of NF-κB pathway which has also been suggested as an important underlying mechanism of its reported properties. In present study, a small library of nine 1,2,4-oxadiazole-based structural analogs of resveratrol were assayed for their antioxidant and anti-inflammatory activities. Several compounds showed significant inhibitory activities against NF-κB and/or ROS production. Compound 2, incorporating two para-hydroxyphenyl moieties connected by the 1,2,4-oxadiazole ring, was the most active, its potency in inhibiting activation of NF-κB and ROS scavenging abilities surpassing that of resveratrol. Additionally, we elucidated the mechanisms underlying the NF-κB inhibitory activity of compound 2. Finally, in contrast to resveratrol, compound 2 significantly reduced the LPS-induced release of pro-inflammatory cytokines, indicating its prominent anti-inflammatory potential., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2015
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41. Characterization of human lymphoblastoid cell lines as a novel in vitro test system to predict the immunotoxicity of xenobiotics.
- Author
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Markovič T, Gobec M, Gurwitz D, and Mlinarič-Raščan I
- Subjects
- Animals, Benzo(a)pyrene toxicity, Cell Line, Cyclosporine toxicity, Furosemide toxicity, Humans, Inhibitory Concentration 50, Interferon-gamma metabolism, Interleukin-10 metabolism, Interleukin-2 metabolism, Interleukin-4 metabolism, Interleukin-6 metabolism, Leukocytes, Mononuclear drug effects, Leukocytes, Mononuclear metabolism, Mannitol toxicity, Trialkyltin Compounds toxicity, Tumor Necrosis Factor-alpha metabolism, Urethane toxicity, Verapamil toxicity, Lymphocytes drug effects, Lymphocytes metabolism, Toxicity Tests methods, Xenobiotics toxicity
- Abstract
Evaluating immunomodulatory effects of xenobiotics is an important component of the toxicity studies. Herein we report on the establishment of a novel invitro test system for the immunotoxicity screening of xenobiotics based on human lymphoblastoid cell lines (LCLs). Four immunotoxic compounds; tributyltin chloride, cyclosporine A, benzo(a)pyrene and verapamil hydrochloride, as well as three immune-inert compounds; urethane, furosemide and mannitol were selected for characterization. The treatment of LCLs with immunosuppressive compounds resulted in reduced viability. The IC50 values determined in human LCLs were in agreement with the data obtained for human peripheral mononuclear cells. Since cytokine production reflects lymphocytes responses to external stimuli, we evaluated the functional responses of LCLs by monitoring their pro-inflammatory and immunoregulatory cytokine production. Our findings prove that LCLs allowed for reliable differentiation between immunomodulatory and immune-inert compounds. Hence, pre-treatment with immunomodulatory compounds led to a decrease in the production of pro-inflammatory TNFα, IL-6 and immunoregulatory IL-2, IL-4, IL-10 and IFNγ cytokines, when compared to untreated ionomycin/PMA stimulated cells. Moreover, testing a panel of ten LCLs derived from unrelated healthy individuals reflects inter-individual variability in response to immunomodulatory xenobiotics. In conclusion, LCLs provide a novel alternative method for the testing of the immunotoxic effects of xenobiotics., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2015
- Full Text
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42. Screening of bisphenol A, triclosan and paraben analogues as modulators of the glucocorticoid and androgen receptor activities.
- Author
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Kolšek K, Gobec M, Mlinarič Raščan I, and Sollner Dolenc M
- Subjects
- Drug Evaluation, Preclinical, Humans, Structure-Activity Relationship, Benzhydryl Compounds pharmacology, Parabens pharmacology, Phenols pharmacology, Receptors, Androgen drug effects, Receptors, Glucocorticoid drug effects, Triclosan analogs & derivatives
- Abstract
A homeostasis of the glucocorticoid and androgen endocrine system is essential to human health. Their disturbance can lead to various diseases, for example cardiovascular, inflammatory and autoimmune diseases, infertility, cancer. Fifteen widely used industrial chemicals that disrupt endocrine activity were selected for evaluation of potential (anti)glucocorticoid and (anti)androgenic activities. The human breast carcinoma MDA-kb2 cell line was utilized for reporter gene assays, since it expresses both the androgen and the glucocorticoid-responsive reporter. Two new antiandrogens, 4,4'-sulfonylbis(2-methylphenol) (dBPS) and 4,4'-thiodiphenol (THIO), and two new antiglucocorticoids, bisphenol Z and its analog bis[4-(2-hydroxyethoxy)phenyl] sulfone (BHEPS) were identified. Moreover, four new glucocorticoid agonists (methyl paraben, ethyl paraben, propyl paraben and bisphenol F) were found. To elucidate the structure-activity relationship of bisphenols, we performed molecular docking experiments with androgen and glucocorticoid receptor. These docking experiments had shown that bulky structures such as BHEPS and bisphenol Z act as antiglucocorticoid, because they are positioned toward helix H12 in the antagonist conformation and could therefore be responsible for H12 conformational change and the switch between agonistic and antagonistic conformation of receptor. On the other hand smaller structures cannot interact with H12. The results of in vitro screening of fifteen industrial chemicals as modulators of the glucocorticoid and androgen receptor activities demand additional in vivo testing of these chemicals for formulating any relevant hazard identification to human health., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2015
- Full Text
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43. Novel N-amidinopiperidine-based proteasome inhibitor preserves dendritic cell functionality and rescues their Th1-polarizing capacity in Ramos-conditioned tumor environment.
- Author
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Švajger U, Gobec M, Obreza A, and Mlinarič-Raščan I
- Subjects
- Antineoplastic Agents pharmacology, Apoptosis drug effects, Apoptosis immunology, Blotting, Western, Burkitt Lymphoma drug therapy, Burkitt Lymphoma pathology, Cell Differentiation drug effects, Cell Differentiation immunology, Cell Proliferation drug effects, Cells, Cultured, Cytokines metabolism, Dendritic Cells physiology, Flow Cytometry, Humans, Lymphocyte Activation drug effects, Proteasome Endopeptidase Complex metabolism, Amidines pharmacology, Burkitt Lymphoma immunology, Dendritic Cells drug effects, Piperidines chemistry, Piperidines pharmacology, Proteasome Endopeptidase Complex chemistry, Proteasome Inhibitors pharmacology, Th1 Cells immunology, Tumor Microenvironment drug effects
- Abstract
The tumor microenvironment represents a burden that hampers the proper activation of immune cells, including the dendritic cells (DCs). It is, therefore, desired that the important characteristics of a given anticancer drug candidate be seen as consisting not solely of its antitumor properties, but that it also lacks potential side effects that could additionally constrain the development and function of immune cells associated with tumor immunity. We have previously identified compounds with a N-amidinopiperidine scaffold that selectively induce apoptosis in Burkitt's lymphoma cells through proteasome inhibition. Here, we demonstrate that SPI-15 affected neither the viability of DCs nor their differentiation. In addition, the compound had no significant effect on their cytokine secretion or allostimulatory capacity. Moreover, DC functionality in the context of tumor microenvironment was also unaffected, as demonstrated by experiments performed on DCs differentiated in Ramos-conditioned media in the presence or absence of SPI-15. The cytokine profile and functional assays revealed that SPI-15 rescues DC differentiation from the immunosuppressive environment produced by Ramos cells; this was seen by their reacquired ability to induce IFN-γ-secretion from naïve CD4(+)CD45RA(+) T cells and the consequently induced Th1-effector differentiation. Herein, we present novel characteristics of an N-amidinopiperidine-based protease inhibitor whose anticancer properties are not associated with the immunosuppression of DCs. We propose future studies toward the design of structurally similar compounds with the aim of developing potent anticancer drugs with minimal negative effects on crucial factors involved in tumor immunity.
- Published
- 2015
- Full Text
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44. Discovery of novel small-molecule compounds with selective cytotoxicity for Burkitt's lymphoma cells using 3D ligand-based virtual screening.
- Author
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Gobec M, Sosič I, Brus B, Obreza A, Gobec S, and Mlinarič-Raščan I
- Subjects
- Burkitt Lymphoma metabolism, Cell Line, Tumor, Humans, Ligands, NF-kappa B metabolism, Proteasome Endopeptidase Complex metabolism, Burkitt Lymphoma drug therapy, Small Molecule Libraries pharmacology
- Abstract
We describe a ligand-based approach towards compounds with more specific targeting for Burkitt's lymphoma. Using three-dimensional ligand-based similarity searches and a previously described hit compound, we have identified six compounds that are chemically different but with similar spatial conformations. Biological evaluation revealed that one compound has better growth inhibition and improved selectivity towards Burkitt's lymphoma cells than the query compound. However, initial mechanism-of-action studies show a different target profile in comparison with the previous hit compound, which does not involve the inhibition of the proteasome or the NFκB pathway. The data from this study provide a solid basis for further efforts in the search for selective agents against Burkitt's lymphoma.
- Published
- 2014
- Full Text
- View/download PDF
45. Chemo-sensitizing effects of EP4 receptor-induced inactivation of nuclear factor-κB.
- Author
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Gobec M, Prijatelj M, Delić J, Markovič T, and Mlinarič-Raščan I
- Subjects
- Alprostadil analogs & derivatives, Alprostadil pharmacology, Antineoplastic Agents pharmacology, Apoptosis drug effects, Boronic Acids pharmacology, Bortezomib, Burkitt Lymphoma drug therapy, Burkitt Lymphoma metabolism, Burkitt Lymphoma pathology, Caspases metabolism, Cell Line, Tumor, Doxorubicin pharmacology, Humans, Jurkat Cells, Leukemia, B-Cell drug therapy, Leukemia, B-Cell metabolism, Leukemia, B-Cell pathology, Lymphoma, B-Cell drug therapy, Lymphoma, B-Cell metabolism, Lymphoma, B-Cell pathology, Pyrazines pharmacology, Signal Transduction drug effects, U937 Cells, bcl-X Protein metabolism, NF-kappa B antagonists & inhibitors, NF-kappa B metabolism, Receptors, Prostaglandin E, EP4 Subtype agonists, Receptors, Prostaglandin E, EP4 Subtype metabolism
- Abstract
The EP4 receptor conveys growth-inhibitory effects in mature and immature B cells via NF-κB. Herein, the EP4 receptor was evaluated as a potential therapeutic target for leukemia and lymphoma, whose survival depends on the constitutive activity of NF-κB. Utilizing a pharmacological approach, we proved that the EP4 receptor induces caspase-mediated apoptosis in malignantly transformed B cells, with the most prominent effect being on Burkitt׳s lymphoma cells. Since the increased activation of NF-κB underlies multi-drug resistance phenomena, we modulated this signaling pathway via EP4 receptor triggering. Pge1-OH, a specific EP4 receptor agonist, led to decreased NF-κB activity and a consequent decrease in levels of the antiapoptotic gene Bcl-xL in Ramos cells, resulting in an elevated sensitivity of cells towards bortezomib- and doxorubicin-induced chemotherapeutic effects. Our study identifies the as yet unrecognized potential of EP4 receptor agonists as chemo-sensitizing agents in B-cell lymphoma. The specific downregulation of NF-κB-dependent pathways in B-cell malignancies opens new possibilities for treatment and current therapy optimization using specific EP4 receptor agonists., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
46. From pharmacogenetics to pharmacometabolomics: SAM modulates TPMT activity.
- Author
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Karas-Kuželički N, Šmid A, Tamm R, Metspalu A, and Mlinarič-Raščan I
- Subjects
- Adult, Estonia, Female, Genotype, Heterozygote, Humans, Male, Pharmacogenetics methods, White People genetics, Genetic Variation genetics, Methyltransferases genetics, S-Adenosylmethionine genetics
- Abstract
Aim: In the present study, the influence of SAM on TPMT activity in vivo on human subjects was investigated., Subjects & Methods: A total of 1017 donors from the Estonian Genome Center of the University of Tartu (Estonia) were genotyped for common TPMT variants, evaluated for TPMT activity, SAM levels, a set of 19 biochemical and ten hematological parameters and demographic data., Results: After adjustment in multiple regression models and correction for multiple testing, from the 43 factors that were tested, only TPMT genotype (p = 1 × 10(-13)) and SAM levels (p = 1 × 10(-13)) were found to significantly influence TPMT activity. The influence of SAM on TPMT activity was more pronounced in TPMT-heterozygous than wild-type individuals., Conclusion: SAM represents a potential pharmacometabolomic marker and therapeutic agent in TPMT-heterozygous subjects.
- Published
- 2014
- Full Text
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47. Structural characterization and biological evaluation of a clioquinol-ruthenium complex with copper-independent antileukaemic activity.
- Author
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Gobec M, Kljun J, Sosič I, Mlinarič-Raščan I, Uršič M, Gobec S, and Turel I
- Subjects
- Apoptosis, Cell Cycle, Cell Line, Tumor, Cell Proliferation, Dimethyl Sulfoxide chemistry, Dose-Response Relationship, Drug, Drug Design, Drug Screening Assays, Antitumor, Humans, Inhibitory Concentration 50, Jurkat Cells, Leukemia drug therapy, MCF-7 Cells, NF-kappa B metabolism, Oxyquinoline chemistry, Proteasome Endopeptidase Complex metabolism, Antineoplastic Agents chemistry, Clioquinol chemistry, Copper chemistry, Ruthenium chemistry
- Abstract
In this study, we present the synthesis, biological characterization, and first crystal structure of an organometallic-clioquinol complex. Combining ruthenium with the established apoptotic agent and 8-hydroxyquinoline derivative, clioquinol, resulted in a complex that induces caspase-dependent cell death in leukaemia cells. This activity is copper independent and is improved compared to the parent compound, clioquinol. The study of the mode of action reveals that this clioquinol-ruthenium complex does not intercalate between DNA base pairs. Additionally, this clioquinol-ruthenium complex shows proteasome-independent inhibition of the NFκB signalling pathway, with no effects on cell-cycle distribution. These data suggest a mechanism of action that involves a target profile that is different from that for clioquinol alone.
- Published
- 2014
- Full Text
- View/download PDF
48. Molecular docking revealed potential disruptors of glucocorticoid receptor-dependent reporter gene expression.
- Author
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Kolšek K, Gobec M, Mlinarič Raščan I, and Sollner Dolenc M
- Subjects
- Binding Sites, Binding, Competitive, Cell Line, Databases, Chemical, Dose-Response Relationship, Drug, Endocrine Disruptors chemistry, Endocrine Disruptors metabolism, Hormone Antagonists chemistry, Hormone Antagonists metabolism, Humans, Luciferases genetics, Molecular Structure, Protein Conformation, Receptors, Glucocorticoid chemistry, Receptors, Glucocorticoid metabolism, Transfection, Endocrine Disruptors toxicity, Gene Expression Regulation drug effects, Genes, Reporter, Hormone Antagonists toxicity, Luciferases biosynthesis, Molecular Docking Simulation, Receptors, Glucocorticoid drug effects
- Abstract
Glucocorticoids are an essential part of the endocrine system that is responsible for a variety of functions such as regulation of immune activity, appropriate brain function, and fetal development. Disturbance of glucocorticoid signaling can lead to various cardiovascular, inflammatory, and autoimmune diseases, so the identification of chemicals that can modulate activity of the glucocorticoid receptor (GR) is crucial. In this study, molecular docking was utilized to find new agonists and antagonists of the GR. The best hits were further tested on the in vitro model of MDA-kb2 cells expressing luciferase activity in a GR-dependent manner. Nine new potential modulators of the receptor, belonging to six structurally diverse classes, were identified. Six of them, tetramethrin and cypermethrin, diethyl hexyl phthalate and diphenyl isophthalate, naphthol AS-OL and dicumyl peroxide, induced luciferase activity; while the other three, bisphenol P, bisphenol M, and Antioxidant 425, suppressed luciferase activity. Of the nine potential GR modulators, only bisphenol M displayed appreciable binding affinity for the receptor., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
49. Synthesis of conformationally constrained γ-D-glutamyl-meso-diaminopimelic acid derivatives as ligands of nucleotide-binding oligomerization domain protein 1 (Nod1).
- Author
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Jakopin Ž, Gobec M, Kodela J, Hazdovac T, Mlinarič-Raščan I, and Sollner Dolenc M
- Subjects
- Cells, Cultured, Cytokines metabolism, Diaminopimelic Acid chemical synthesis, Diaminopimelic Acid chemistry, Diaminopimelic Acid pharmacology, Dose-Response Relationship, Drug, Humans, Leukocytes, Mononuclear metabolism, Ligands, Molecular Conformation, NF-kappa B metabolism, Nod1 Signaling Adaptor Protein chemistry, Nod1 Signaling Adaptor Protein genetics, Structure-Activity Relationship, Diaminopimelic Acid analogs & derivatives, Nod1 Signaling Adaptor Protein agonists, Nod1 Signaling Adaptor Protein metabolism
- Abstract
Nod1, an important member of the pattern recognition receptor family, remains a virtually unexploited target. Harnessing its innate immune stimulatory properties still remains an unfulfilled goal of medicinal chemistry. Nucleotide-binding oligomerization domain protein 1 (Nod1) agonists have been shown to boost the inflammatory responses against pathogenic microbes and could thus constitute a new class of broad spectrum antimicrobial agents. To gain additional insight into the structure/activity relationships of Nod1 agonistic compounds, a series of novel, conformationally constrained γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) analogs have been designed and synthesized. Ramos-Blue cells expressing Nod1 were used to screen and validate our compounds for their Nod1-agonist activity. Their immunomodulatory properties were subsequently determined in vitro, by evaluating their capacity to induce pro-inflammatory cytokine and chemokine production from human peripheral blood mononuclear cells (PBMC), by themselves and in synergy with lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand. The synthesized iE-DAP analogs were shown to possess immuno-enhancing properties as a result of their potent and specific Nod1-agonistic effect. The activity of the compound exhibiting the greatest capacity to induce pro-inflammatory cytokine release from PBMC surpassed that of lauroyl-γ-D-glutamyl-meso-diaminopimelic acid (C12-iE-DAP)., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
50. Cinnamic acid derivatives induce cell cycle arrest in carcinoma cell lines.
- Author
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Sova M, Žižak Ž, Stanković JA, Prijatelj M, Turk S, Juranić ZD, Mlinarič-Raščan I, and Gobec S
- Subjects
- Antineoplastic Agents chemical synthesis, Antineoplastic Agents chemistry, Cell Proliferation drug effects, Cell Survival drug effects, Dose-Response Relationship, Drug, Drug Screening Assays, Antitumor, HeLa Cells, Humans, K562 Cells, MCF-7 Cells, Models, Molecular, Molecular Structure, Structure-Activity Relationship, Tumor Cells, Cultured, Antineoplastic Agents pharmacology, Cell Cycle drug effects, Cinnamates chemistry, Cinnamates pharmacology, Neoplasms pathology
- Abstract
Cinnamic acid derivatives can be found in plant material, and they possess a remarkable variety of biological effects. In the present study, we have investigated the cytotoxic effects of representative cinnamic acid esters and amides. The cytotoxicity was determined by MTT test on human cervix adenocarcinoma (HeLa), myelogenous leukemia (K562), malignant melanoma (Fem-x), and estrogen-receptor-positive breast cancer (MCF-7) cells, versus peripheral blood mononuclear cells (PBMCs) without or with the addition of the plant lectin phytohemaglutinin (PHA). The compounds tested showed significant cytotoxicity (IC50s between 42 and 166 µM) and furthermore selectivity of these cytotoxic effects on the malignant cell lines versus the PBMCs was also seen, especially when electron-withdrawing groups, such as a cyano group (compound 5), were present on the aromatic rings of the alcohol or amine parts of the cinnamic acid derivatives. The additional study on cell cycle phase distribution indicated that novel cinnamic acid derivatives inhibit cell growth by induction of cell death. Thus, cinnamic acids derivatives represent important lead compounds for further development of antineoplastic agents.
- Published
- 2013
- Full Text
- View/download PDF
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