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

Study of element contents in bivalve molluscs: health benefit and risk

Bivalve molluscs, which include mussels, oysters and clams, have high nutritional value. They are regarded as a good source for proteins, lipids, carbohydrates and minerals [1]. On the other hand, seafood may also contain harmful contaminants and other undesirable substances such as mercury and persistent halogenated compounds, which has resulted in a number of risk-benefit assessments during the last decade [2]. Four species of bivalve molluscs Ruditapes philippinarum (Manila clam, MC), Yesso scallop (YS), Tegillarca granosa (TG) and Anadara broughtonii (AB) were bought in two fish markets in Incheon, Korea, in order to determine content of As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Se, Zn, and Fe and consequently, to estimate the health hazards associated to dietary intake. The samples were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) after closed-vessel microwave digestion. The analytical accuracy of the method was evaluated by using the SRM (TORT-2, lobster hepatopancreas). Application of principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed a tendency to form three groups between samples belonging to different genus of samples. European food safety authority (EFSA) has established recommended daily intake (RDI) values for Cu, Fe, Mn and Zn of 3, 14, 5 and 10 mg/day, respectively. We calculated the RDI for daily consumption in milligrams per 300 g of sample. Our results showed that these species could serve as a good dietary source of essential elements, especially Fe, Mn and Zn. However, all species showed As content higher than the maximum tolerable limit specified by EFSA. Seafood is the major contributor to As in the diet though As in seafood mostly occurs as organic As species [3]. In addition, content of Mn in Yesso scallop is few times higher than in other species

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

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

Determination of toxic elements (mercury, cadmium, lead and arsenic) in shellfish samples

Morski plodovi imaju visoku nutritivnu vrednost, ali sa druge strane postoji rizik od kontaminacije toksičnim elementima. Sadržaj As, Cd, Hg i Pb je određen u četiri vrste školjki Ruditapes philippinarum (Manila clam, MC), Yesso scallop (YS), Tegillarca granosa (TG) i Anadara broughtonii (AB) kupljene u Incheonu, Koreja. Uzorci su analizirani induktivno spregnutom plazmom - masenom spektrometrijom (ICP-MS) nakon mikrotalasne digestije. Izračunati su dnevni/nedeljni unosi za ove elemente u mg/300 g uzorka. Ukupan sadržaj As (neoganski i organski) u svim vrstama je bio veći od dozvoljenog limita preporučenog od Svetske zdravstvene organizacije (WHO). Procenjeni dnevni unosi ostalih elemenata (Hg, Cd i Pb) su niži od maksimalnih podnošljivih granica (MDI) koje je odredila Evropska agencija za bezbednost hrane (EFSA) što ukazuje da nema rizika za konzumente.Bivalve molluscs, which include mussels, oysters and clams, have high nutritional value. On the other hand, seafood may also contain harmful contaminants and other undesirable substances such as mercury and persistent halogenated compounds. Four species of bivalve molluscs Ruditapes philippinarum (Manila clam, MC), Yesso scallop (YS), Tegillarca granosa (TG) and Anadara broughtonii (AB) were bought in Incheon, Korea, in order to determine content of As, Cd, Hg, and Pb and consequently, to estimate the health hazards associated to dietary intake. The samples were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) after microwave digestion. All species showed As content higher than the maximum tolerable limit specified by EFSA. Estimated daily intake of Hg, Cd and Pb from consumption of 300 g was very low and hence poses no toxicological risk

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

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

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