The effect of prothrombin, the precursor of thrombin, on the proliferation and migration of colorectal cancer cells

Thrombotic disorders are some of the main comorbidities in cancer patients. So far, research has indicated that thrombin, a key regulator of hemostasis, contributes to cancer progression. However, data on its origin in tumor microenvironments remain elusive. Based on previous research, we analyzed the RNA and protein expression of prothrombin, a precursor of thrombin, in selected colorectal cancer (CRC) cell lines. Since the effect of prothrombin in cancer development has not been previously reported, we treated the cells for 24 h and 48 h with different prothrombin concentrations and assessed the effect on cell proliferation and migration. Our results show that the tested CRC cell lines expressed prothrombin and that prothrombin inhibited proliferation and migration. The presented results suggest that prothrombin may contribute to CRC etiopathology and could serve as a potential diagnostic biomarker and therapeutic target. The mechanisms underlying prothrombin expression in cancer cells, potential prothrombin activation, and the underlying processes driving the described effects warrant further investigation

Prothrombin expression in cancer-derived cell lines

The link between thrombotic disorders and cancer has been known for over 150 years, although the precise mechanism of this relationship has not yet been resolved. Current data show that thrombin has a significant role in cancer metabolism, invasiveness, adhesion and survival. However, data regarding the expression of the thrombin precursor prothrombin in various cancer cell lines are scarce. Therefore, it was our objective to determine whether common cancer-derived cell lines (Caco-2, MCF-7, SK-BR-3, U-87 and U-251) express prothrombin. The prothrombin RNA expression level was assessed by qPCR, and the presence of prothrombin was analyzed by Western blot analysis. Our results show that Caco-2 cells originating from colorectal adenocarcinoma express prothrombin, whereas other analyzed cell lines do not. Our results provide a background for further research into the role of (pro) thrombin in cancer etiopathology

Наследни фактори ризика за тромбофилију – од тачкастих мутација до примене вештачке интелигенције

Trombofilija je patofiziološko stanje povećanog rizika za nastanak hiperkoagulacije, koja može dovesti do začepljenja krvnog suda (tromboze). Faktori rizika za nastanak ove multifaktorijalne bolesti mogu biti sredinski, uzrokovani načinom života, i nasledni (genetski). Do sada je identifikovan veliki broj naslednih faktora rizika, uglavnom tačkastih mutacija u genima za proteine hemostaznog sistema. Iako se ove mutacije analiziraju u okviru rutinskih kliničkih testova, kod značajnog broja bolesnika i nakon sprovedenih dijagnostičkih procedura, uzrok trombotičkog događaja ostaje nepoznat, što implicira postojanje neidentifikovanih naslednih faktora rizika. U cilju njihove identifikacije, vrše se dalje genske analize, asocijativne studije, karakterizacije potencijalnih faktora rizika u in vitro i in vivo studijama na različitim model sistemima. U našem dosadašnjem radu detektovano je više varijanti u kodirajućem i nekodirajućem regionu gena, za koje je karakterizacijom utvrđeno da utiču na ekspresiju i/ili funcionalnost koagulacionih proteina. Primenom sekvenciranja nove generacije i bioinformatičke obrade, omogućena je sveobuhvatnija analiza celokupnog genoma i identifikacija klastera gena koji su povezani sa kompleksnom kliničkom slikom tromboza. Kombinovanjem velikog broja podataka o genetskim i sredinskim faktorima, primenom veštačke inteligencije, otvara se mogućnost kompletnijeg sagledavanja mehanizama trombofilije i multifaktorijalnih bolesti uopšte.Тромбофилија је патофизиолошко стање повећаног ризика за настанак хиперкоагулације, која може довести до зачепљења крвног суда (тромбозе). Фактори ризика за настанак ове мултифакторијалне болести могу бити средински, узроковани начином живота, и наследни (генетски). До сада је идентификован велики број наследних фактора ризика, углавном тачкастих мутација у генима за протеине хемостазног система. Иако се ове мутације анализирају у оквиру рутинских клиничких тестова, код значајног броја болесника и након спроведених дијагностичких процедура, узрок тромботичког догађаја остаје непознат, што имплицира постојање неидентификованих наследних фактора ризика. У циљу њихове идентификације, врше се даље генске анализе, асоцијативне студије, карактеризације потенцијалних фактора ризика у in vitro и in vivo студијама на различитим модел системима. У нашем досадашњем раду детектовано је више варијанти у кодирајућем и некодирајућем региону гена, за које је карактеризацијом утврђено да утичу на експресију и/или фунционалност коагулационих протеина. Применом секвенцирања нове генерације и биоинформатичке обраде, омогућена је свеобухватнија анализа целокупног генома и идентификација кластера гена који су повезани са комплексном клиничком сликом тромбоза. Комбиновањем великог броја података о генетским и срединским факторима, применом вештачке интелигенције, отвара се могућност комплетнијег сагледавања механизама тромбофилије и мултифакторијалних болести уопште.Knjiga sažetaka: Treći Kongres biologa Srbije, Zlatibor, Srbija 21 - 25. 9. 2022

Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22 Exhibit Anti-Inflammatory Effect by Attenuation of NF-kappa B and MAPK Signaling in Human Bronchial Epithelial Cells

Bronchial epithelial cells are exposed to environmental influences, microbiota, and pathogens and also serve as a powerful effector that initiate and propagate inflammation by the release of proinflammatory mediators. Recent studies suggested that lung microbiota differ between inflammatory lung diseases and healthy lungs implicating their contribution in the modulation of lung immunity. Lactic acid bacteria (LAB) are natural inhabitants of healthy human lungs and also possess immunomodulatory effects, but so far, there are no studies investigating their anti-inflammatory potential in respiratory cells. In this study, we investigated immunomodulatory features of 21 natural LAB strains in lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Our results show that several LAB strains reduced the expression of pro-inflammatory cytokine and chemokine genes. We also demonstrated that two LAB strains, Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22, effectively attenuated LPS-induced nuclear factor-kappa B (NF-kappa B) nuclear translocation. Moreover, BGZLS10-17 and BGPKM22 reduced the activation of p38, extracellular signal-related kinase (ERK), and c-Jun amino-terminal kinase (JNK) signaling cascade resulting in a reduction of pro-inflammatory mediator expressions in BEAS-2B cells. Collectively, the LAB strains BGZLS10-17 and BGPKM22 exhibited anti-inflammatory effects in BEAS-2B cells and could be employed to balance immune response in lungs and replenish diminished lung microbiota in chronic lung diseases

Reconstitution of non-carrier, heterozygous and homozygous prothrombin belgrade mutation carrier plasma using recombinant proteins

Introduction: The prothrombin Belgrade variant (c.1787G>A, p.Arg596Gln) is a rare mutation found in Serbia, Japan, China, America, India and leads to antithrombin resistance. Prothrombin Belgrade mutation influencesthrombin-antithrombin interactions and leadsto impaired inactivation of mutated thrombin. Also, it affectssodium binding site in thrombin, which isimportant forswitching from fast thrombin configuration (coagulant properties) to slow configuration (anticoagulant properties). It has only been found in a heterozygous state, which could mean that homozygous carriers are incompatible with life. By using prothrombin (FII) deficient plasma, we could reconstitute plasma of wild type, heterozygous and homozygous carrier, which could give more insight into the mechanism of this mutation. Methods: Recombinant wild type and mutated prothrombin were generated by transient transfection in HEK293T cell line. Western blot analysis was performed to test the efficiency of transfection. Human Prothrombin ELISA (Nordic BioSite, Sweden) was used in order to measure recombinant prothrombin concentration. Overall Hemostasis Potential (OHP) assay was performed to assess recombinant protein activity. Recombinant wild type and mutated prothrombin were added to FII deficient plasma (Siemens, Germany) in order to create reconstituted plasma, in the final concentration of 0.1 mg/mL, as it is approximately the level of prothrombin in human plasma. Results: Reconstituted plasma samples that correspond to non-carrier, heterozygous carrier, and homozygous mutation carrier plasma were reconstructed. Recombinant proteinstested by OHP assay were functional. Conclusion: Reconstituted plasma samples allow us to examine the mechanism of prothrombin Belgrade mutation in various assays and in homozygous form as well

Reconstitution of non-carrier, heterozygous and homozygous prothrombin belgrade mutation carrier plasma using recombinant proteins

Introduction: The prothrombin Belgrade variant (c.1787G>A, p.Arg596Gln) is a rare mutation found in Serbia, Japan, China, America, India and leads to antithrombin resistance. Prothrombin Belgrade mutation influencesthrombin-antithrombin interactions and leadsto impaired inactivation of mutated thrombin. Also, it affectssodium binding site in thrombin, which isimportant forswitching from fast thrombin configuration (coagulant properties) to slow configuration (anticoagulant properties). It has only been found in a heterozygous state, which could mean that homozygous carriers are incompatible with life. By using prothrombin (FII) deficient plasma, we could reconstitute plasma of wild type, heterozygous and homozygous carrier, which could give more insight into the mechanism of this mutation. Methods: Recombinant wild type and mutated prothrombin were generated by transient transfection in HEK293T cell line. Western blot analysis was performed to test the efficiency of transfection. Human Prothrombin ELISA (Nordic BioSite, Sweden) was used in order to measure recombinant prothrombin concentration. Overall Hemostasis Potential (OHP) assay was performed to assess recombinant protein activity. Recombinant wild type and mutated prothrombin were added to FII deficient plasma (Siemens, Germany) in order to create reconstituted plasma, in the final concentration of 0.1 mg/mL, as it is approximately the level of prothrombin in human plasma. Results: Reconstituted plasma samples that correspond to non-carrier, heterozygous carrier, and homozygous mutation carrier plasma were reconstructed. Recombinant proteinstested by OHP assay were functional. Conclusion: Reconstituted plasma samples allow us to examine the mechanism of prothrombin Belgrade mutation in various assays and in homozygous form as well

Prothrombin 3'end Gene Variants in Patients With Sporadic Colon Adenocarcinoma

Background/ Aim: Thrombin plays significant roles in various types of cancer. However, the expression levels of prothrombin, the thrombin precursor, in cancer remain unclear. Variants of the 3'end of the prothrombin gene lead to increased prothrombin expression. This study aimed to analyze prothrombin 3'end gene variants in colon tumor and adjacent normal tissue samples. Materials and Methods: The study group consisted of 93 patients suffering from colon adenocarcinoma. The 3'end of the prothrombin gene was analyzed by DNA sequencing. Results: Three variants, all previously associated with increased prothrombin expression were detected. Frequency of the FII 19911G allele was 46.77% and 47.85% in tumor and normal tissue, respectively. For the FII 20210A allele, the detected frequencies were 2.15% and 1.61%, respectively. The frequency of the FII c.1824T allele was 0.54% in both tissues. Four patients showed different genotypes in tumor and normal tissue. Conclusion: Prothrombin 3' end gene variants may play a role in colorectal cancer

Frequency of FV Leiden and FII G20210A Mutations in Patients with Inherited Antithrombin Deficiency from Serbia

PURPOSE: Thrombosis is multicausal disease in which both acquired and genetic risk factors play important roles. The most frequent genetic risk factors known to date are the Factor V G1691A (FV Leiden) and FII G20210A mutations. On the other hand, inherited antithrombin (AT) deficiency, caused by mutations in the AT gene (SERPINC1) is a very rare disorder, but it is associated with significant risk for thrombotic complications. AT deficiency is classified into two types: type-I is a quantitative disorder characterized by decreased amount and activity of AT, while type-II is a qualitative - functional disorder. Aim of our study was to analyze the frequency of FV Leiden and FII G20210A mutations in patients with inherited AT deficiency from Serbia. METHODOLOGY: A study was carried out in large group of AT deficiency patients from Serbia. Cohort of 42 subjects (15m/27f; 36.7±18.7y) from 18 Serbian families included 24 symptomatic and 18 asymptomatic first-degree relatives. Among them, type-I AT deficiency were detected in 9 families (19 members: 6m/13f; 37.1±19.0y) and type-II in 9 families (23 members: 9m/14f; 36.5±18.8y). FV Leiden and FII G2010A mutations were detected by PCR, followed by digestion with specific restriction enzymes (PCR-RFLP). RESULTS: We have detected 3 FV Leiden heterozygous carriers in 3 different families (1 with type-I and 2 with type-II AT deficiency). All 3 carriers were symptomatic. Regarding FII G20210A mutation, 2 heterozygous carriers, both asymptomatic and from same family with type-I deficiency, were identified. According to our findings in families with AT deficiency from Serbia frequency of FV Leiden and FII G20210A mutation are 16.7% and 5.6%, respectively. CONCLUSION: This is the first study in which frequency of FV Leiden and FII G20210A mutations in patients with inherited AT deficiency from Serbia were examined. Results of our study suggest that these mutations can be relevant for AT deficiency patients’ phenotype, but further studies are required.Synergy in Hemophilia; 16th International Hemophilia Congress of Turkey, April 2019, Antaly

Prothrombin influences proliferation and migration of colon cancer in vitro

Introduction: Thrombin, crucial member of the coagulation cascade, can influence growth and development of different types of cancer. Prothrombin, thrombin precursor, although predominantly secreted from the liver into the bloodstream, can also be expressed in the cancer cells. According to latest data prothrombin can bind in vitro to transmembrane receptors, which have previously been shown to be up-regulated in cancers and activate migration and invasion. Despite the significant amount of data on the effects of thrombin in cancer progression, there are little data of prothrombin´s effect. The aim of this study was to further examine the effects of prothrombin and thrombin in cancer cell lines. Methods: Colon cancer cell lines (Caco2, SW480, SW620, HT29 and HCT116) were treated with prothrombin, thrombin and direct thrombin inhibitor, dabigatran, for 24h and 48h. To assess the effects of treatment on cell viability and proliferation MTT test was used, and wound healing assay was used for cell migration potential. Results: Detected effects of treatment with prothrombin, thrombin and dabigatran varied between cell lines. Trend of lower cell viability, proliferation and migration was observed in cells treated with prothrombin in comparison to untreated controls. Conclusion: Our resultsindicate that prothrombin, although considered an inactive zymogen, can exert an effect on colon cancer cells proliferation and migration in vitro

A phenotype driven integrative framework uncovers molecular mechanisms of a rare hereditary thrombophilia

Antithrombin resistance is a rare subtype of hereditary thrombophilia caused by prothrombin gene variants, leading to thrombotic disorders. Recently, the Prothrombin Belgrade variant has been reported as a specific variant that leads to antithrombin resistance in two Serbian families with thrombosis. However, due to clinical data scarcity and the inapplicability of traditional genome-wide association studies (GWAS), a broader perspective on molecular and phenotypic mechanisms associated with the Prothrombin Belgrade variant is yet to be uncovered. Here, we propose an integrative framework to address the lack of genomic samples and support the genomic signal from the full genome sequences of five heterozygous subjects by integrating it with subjects’ phenotypes and the genes’ molecular interactions. Our goal is to identify candidate thrombophilia-related genes for which our subjects possess germline variants by focusing on the resulting gene clusters of our integrative framework. We applied a Non-negative Matrix Tri-Factorization-based method to simultaneously integrate different data sources, taking into account the observed phenotypes. In other words, our data-integration framework reveals gene clusters involved with this rare disease by fusing different datasets. Our results are in concordance with the current literature about antithrombin resistance. We also found candidate disease-related genes that need to be further investigated. CD320, RTEL1, UCP2, APOA5 and PROZ participate in healthy-specific or disease-specific subnetworks involving thrombophilia-annotated genes and are related to general thrombophilia mechanisms according to the literature. Moreover, the ADRA2A and TBXA2R subnetworks analysis suggested that their variants may have a protective effect due to their connection with decreased platelet activation. The results show that our method can give insights into antithrombin resistance even if a small amount of genetic data is available. Our framework is also customizable, meaning that it applies to any other rare disease