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

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

Toxic metals in Loggerhead sea turtles (Caretta caretta) stranded freshly dead along Sicilian coasts

Abstract Background: The Loggerhead sea turtle (Caretta caretta) is a marine reptile belonging to a monophyletic group of chelonians. As these animals are long-lived, they have the ability to accumulate pollutants. Aim: To collect epidemiological data on toxic metals in marine Loggerhead sea turtles. Materials and Methods: Forty Loggerhead sea turtles comprising 25 males and 15 females stranded freshly dead between 2013 and 2018 along the coasts of Sicily, Southern Italy, were examined for arsenic, cadmium, and lead accumulation in muscle and adipose tissues by means of a validated ICP-MS method. A modified K index as a growth condition factor, namely Fulton's K index, was used. Samples were tested in duplicate. A Wilcoxon rank sum test was carried out to evaluate metal contents differences between muscle and adipose tissues and between genders. Results: The Fulton's K index suggested a good body condition of the C. caretta recovered with mean values of 5.34±3.40 (n=40; ±SD). Detectable concentrations of lead were found in 70% of the samples analysed with mean values of 0.65±1.67 mg/kg wet weight and 0.51±1.29 mg/kg wet weight in muscle and adipose tissues, respectively. No significant differences in arsenic, cadmium, and lead were detected between genders. In addition, no significant correlation was found between modified K index and concentrations of arsenic, cadmium, and lead. Clinical relevance: Findings on muscle and adipose tissues suggest chronic exposure of Caretta caretta to high concentrations of especially lead which might negatively affect health and welfare of these marine turtles although body condition was good

Histamine in Fish Products Randomly Collected in Southern Italy: A 6-Year Study.

In total, 4,615 fresh and processed fish samples collected from 2010 to 2015 were analyzed for histamine by ultrahigh-performance liquid chromatography with diode array detection. Histamine levels were detected in 352 (7.6%) samples, with a maximum of 4,110 mg kg-1 and mean values of 908.9 ± 1,226.79 and 344.01 ± 451.18 mg kg-1 for fresh and processed fish samples, respectively. No histamine levels were found in canned tuna and smoked fish samples in contrast to most of the data reported in the literature. A low percentage (2.79%) of noncompliant samples was found. The highest mean values were found during 2011 and 2015 for fresh and processed fish samples, respectively, showing a significant (P < 0.05) difference between the sampling years. The histamine contents found in fresh fish samples were significantly higher (P < 0.05) than those of processed samples. Most of the positive samples came from street vendors, suggesting the need to improve inspection measures in these commercial categories to ensure fish product safety