Alpha-1-Antitrypsin in Pathogenesis of Hepatocellular Carcinoma

Context: Alpha-1-antitrypsin (A1AT) is the most abundant liver-derived, highly polymorphic, glycoprotein in plasma. Hereditary deficiency of alpha-1-antitrypsin in plasma (A1ATD) is a consequence of accumulation of polymers of A1AT mutants in endoplasmic reticulum of hepatocytes and other A1AT-producing cells. One of the clinical manifestations of A1ATD is liver disease in childhood and cirrhosis and/or hepatocellular carcinoma (HCC) in adulthood. Epidemiology and pathophysiology of liver failure in early childhood caused by A1ATD are well known, but the association with hepatocellular carcinoma is not clarified. The aim of this article is to review different aspects of association between A1AT variants and hepatocellular carcinoma, with emphasis on the epidemiology and molecular pathogenesis. The significance of A1AT as a biomarker in the diagnosis of HCC is also discussed. Evidence Acquisitions: Search for relevant articles were performed through Pub Med, HighWire, and Science Direct using the keywords "alpha-1-antitrypsin", "liver diseases", "hepatocellular carcinoma", "SERPINA1". Articles published until 2011 were reviewed. Results: Epidemiology studies revealed that severe A1ATD is a significant risk factor for cirrhosis and HCC unrelated to the presence of HBV or HCV infections. However, predisposition to HCC in moderate A1ATD is rare, and probably happens in combination with HBV and/or HCV infections or other unknown risk factors. It is assumed that accumulation of polymers of A1ATD variants in endoplasmic reticulum of hepatocytes leads to damage of hepatocytes by gain-of-function mechanism. Also, increased level of A1AT was recognized as diagnostic and prognostic marker of HCC. Conclusions: Clarification of a carcinogenic role for A1ATD and identification of pro-inflammatory or some still unknown factors that lead to increased susceptibility to HCC associated with A1ATD may contribute to a better understanding of hepatic carcinogenesis and to the development of new drugs. Published by Kowsar Corp, 2012. cc 3.0

Structural and functional analysis of alpha-1-antitrypsin gene in human lung diseases

Alfa-1-antitripsin je inhibitor serin proteaza čija je osnovna biološka funkcija inhibicija elastaze neutrofila – enzima koji učestvuje u degradaciji elastina i koji dovodi do oštećenja tkiva pluća. Deficijencija i disfunkcija alfa-1-antitripsina leže u osnovi više oboljenja od kojih su najčešći emfizem pluća i oboljenja jetre, a mogu predstavljati i faktor rizika za nastanak karcinoma pluća. Ovaj rad je imao za cilj da ispita postojanje strukturnih promena u kodirajućim egzonima gena za alfa-1-antitripsin kod ispitanika sa emfizemom pluća i kod ispitanika sa karcinomom pluća, kao i da istraži funkcionalne posledice otkrivenih promena i njihov eventualni značaj u nastanku i razvoju bolesti. Strukturnom analizom gena za alfa-1-antitripsin DGGE metodom i DNK sekvenciranjem detektovano je prisustvo deficijentnih varijanti Z, S i Mmalton, kao i dve nove varijante označene kao G320R i V321F. Elektroforezom u denaturišućem poliakrilamidnom gelu uočeno je da varijante G320R i V321F migriraju brže u odnosu na WT varijantu, a analizom u denaturišućem poliakrilamidnom gelu sa 5M ureom pokazano je da ove varijante verovatno imaju smanjenu hidrofobnost u odnosu na WT varijantu. Analizom formiranih kompleksa između alfa-1-antitripsina i elastaze pankreasa u SDS gelovima, utvrđeno je da se inhibitorna aktivnost ispitivanih varijanti ne razlikuje od inhibitorne aktivnosti WT varijante proteina. Nativna elektroforeza preparata alfa-1-antitripsina inkubiranih na različitim temperaturama pokazala je da mutirane varijante proteina imaju blago smanjenu termostabilnost u odnosu na WT protein, a elektroforeza proteina eksprimiranih u COS7 ćelijskoj liniji pokazala je da varijante G320R i V321F pod fiziološkim uslovima ne formiraju polimere. Metodom imunofluorescencije na tranzijentno transfekovanim COS7 ćelijama pokazano je da su varijante G320R i V321F, kao i WT protein, lokalizovane u Goldžijevom aparatu i da se ne zadržavaju u endoplazmatičnom retikulumu...Alpha-1-antitrypsin is a serine protease inhibitor whose main biological function is inhibition of neutrophil elastase – enzyme capable of digestion of elastin and involved in lung tissue destruction. Deficiency and disfunction of alpha-1-antitrypsin is associated with emphysema and liver disease, and can represent a risk factor for development of lung cancer. The objective of this work was to perform structural analysis of alpha-1-antitrypsin gene in subjects with emphysema and lung cancer, as well as to perform functional analysis of discovered variants in order to estimate their role in disease development. Structural analysis of alpha-1-antitrypsin gene, using DGGE and DNA sequencing, revealed presence of deficient variants Z, S and Mmalton, as well as two novel variants – G320R and V321F. Denaturing PAGE analysis showed that variants G320R and V321F exibited increased gel mobility compared to WT variant, and electrophoresis in the presence of 5M urea showed that these variants effected the protein structure, probably by changing its hydrophobicity. Nondenaturing electrophoresis of heat treated proteins showed that both variants had slightly decreased thermostability when compared to WT variant and non-denaturing PAGE of COS7 expressed proteins showed that these variants did not form polymers under physiological conditions. Inibitory activity of variants G320R and V321F, determined by analysis of SDS-PAGE resistant complexes between alpha-1-antitrypsin and porcine pancreas elastase, was same as in WT variant. Immunofluorescence of transiently transfected COS7 cell line showed that variants G320R and V321F, as well as WT protein, were predominantly localized at Golgi apparatus indicating that they are not retained in endoplasmic reticulum..

Functional analysis of novel alpha-1 antitrypsin variants G320R and V321F

Alpha-1 antitrypsin (AAT) gene is highly polymorphic, with a large number of rare variants whose phenotypic consequences often remain inconclusive. Studies addressing functional characteristics of AAT variants are of significant biomedical importance since deficiency and dysfunctionality of AAT are associated with liver and lung diseases. We report the results of the functional analysis of two naturally occurring AAT variants, G320R and V321F, previously identified in patients with lung disease. Neither of variants has been fully functionally characterized. In order to perform their functional analysis both variants were expressed in prokaryotic and eukaryotic systems and their intracellular localization, activity, stability, and polymerization were determined. The results of this study demonstrated that variants G320R and V321F have neither impaired activity against porcine pancreatic elastase nor propensity to form polymers. However, both variants had altered electrophoretic mobility and reduced thermostability when compared to M variant of the protein, indicating a slightly impaired secondary or tertiary structure

Targeting autophagy to modulate cell survival: a comparative analysis in cancer, normal and embryonic cells

Autophagy is linked to multiple cancer-related signaling pathways, and represents a defense mechanism for cancer cells under therapeutic stress. The crosstalk between apoptosis and autophagy is essential for both tumorigenesis and embryonic development. We studied the influence of autophagy on cell survival in pro-apoptotic conditions induced by anticancer drugs in three model systems: human cancer cells (NCI-H460, COR-L23 and U87), human normal cells (HaCaT and MRC-5) and zebrafish embryos (Danio rerio). Autophagy induction with AZD2014 and tamoxifen antagonized the pro-apoptotic effect of chemotherapeutics doxorubicin and cisplatin in cell lines, while autophagy inhibition by wortmannin and chloroquine synergized the action of both anticancer agents. This effect was further verified by assessing cleaved caspase-3 and PARP-1 levels. Autophagy inhibitors significantly increased both apoptotic markers when applied in combination with doxorubicin while autophagy inducers had the opposite effect. In a similar manner, autophagy induction in zebrafish embryos prevented cisplatin-induced apoptosis in the tail region while autophagy inhibition increased cell death in the tail and retina of cisplatin-treated animals. Autophagy modulation with direct inhibitors of the PI3kinase/Akt/mTOR pathway (AZD2014 and wortmannin) triggered the cellular response to anticancer drugs more effectively in NCI-H460 and zebrafish embryonic models compared to HaCaT suggesting that these modulators are selective towards rapidly proliferating cells. Therefore, evaluating the autophagic properties of chemotherapeutics could help determine more accurately the fate of different cell types under treatment. Our study underlines the importance of testing autophagic activity of potential anticancer agents in a comparative approach to develop more rational anticancer therapeutic strategies.Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1004]This is the peer reviewed version of the paper: Divac Rankov, A., Ljujić, M., Petrić, M., Radojković, D., Pesić, M., & Dinić, J. (2017). Targeting autophagy to modulate cell survival: A comparative analysis in cancer, normal and embryonic cells. Histochemistry and Cell Biology, 148(5), 529–544. [https://doi.org/10.1007/s00418-017-1590-4

Analysis of CFTR Gene Variants in Idiopathic Bronchiectasis in Serbian Children

This study has investigated a potential role of common Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene variants in the etiology of noncystic fibrosis bronchiectasis in Serbian children. The study has encompassed 48 patients (19 male and 29 female, aged between 5 and 18 years, median age 10.6 +/- 3.3), diagnosed with idiopathic bronchiectasis based on high-resolution computed tomography of thorax and pathologic examination of lobectomy materials. The CFTR gene analysis was performed on genomic DNA extracted from peripheral blood samples of patients by polymerase chain reaction (PCR)-Mediated Site-Directed Mutagenesis method, Denaturing Gradient Gel Electrophoresis method, and DNA sequencing. Mutation c.1521_1523delCTT (F508del) was detected with an allelic frequency of 1.0%, and c.224G gt A (R75Q) variant. Carriers of c.1210-12T[5] (IVS8-5T) allele were significantly more common than in the general population (10.4% vs. 5.0%, P = 0.0302). The frequency of homozygotes for Met 470 allele was higher in patients than in the general population (33% vs. 20%), while heterozygotes for p. Met470Val were less frequent (31% vs. 50%), and this difference was statistically significant (P = 0.0222). The results obtained in this study indicate involvement of 2 common CFTR variants, c. 1210-12T[5] and c. 1408A, in idiopathic bronchiectasis in children, but this observation should be further confirmed by more extensive analysis of the CFTR gene in a larger group of patients

Combined effect of GSTM1 gene deletion, GSTT1 gene deletion and MTHFR C677T mutation in male infertility

The aim of the study was to investigate the association between the GSTM1 and GSTT1 gene deletion and MTHFR C677T mutation and male infertility. The study has encompassed 52 infertile and 56 fertile males. Genotype distributions of GSTM1 and GSTT1 gene deletions and the MTHFR C677T mutation did not differ significantly among the analyzed groups, however, a difference in distribution of certain genotype combinations was observed. The obtained results indicate that carriers of double GSTM1/GSTT1 deletion and the MTHFR 677CC genotype are at higher risk of infertility than carriers of any other combination of genotypes (OR 3.5, 95%CI 0.68-18.30)

E-cigarette liquid and condensate leads to impaired embryonic development of zebrafish

Introduction: E-cigarettes are advertised as safer alternative to tradicional cigarettes. However, they contain chemicals that can exhibit toxic effects on the organism. Notably, effects of e-cigarettes on in utero development are not well studied. We wanted to compare potential toxic effects of e-cigarette liquid and vapor condensate on development of zebrafish embryos. Methods: Six hour old zebrafish embryos were exposed to different concentrations of e-cigarette liquid or vapour condensate – 0.1% and 1%. Untreated embryos were used as control. Each treatment and control were set up in triplicate, with at least 20 embryos per treatment. The effects on survival, hatching and developmental malformations were monitored using light microscopy, at 3 timepoints - 24, 48 and 72 hours post fertilization (hpf). Results: No noticeable differences between control and treated groups were observed 24 hpf. Hatched larvae (35%) treated with 0.1% condensate had scoliosis and malformations- yolk sac and pericardial edema at 48 hpf. In groups treated with 1% of condensate or liquid, hatching was delayed and did not start 48 hpf. At 72 hpf timepoint, in wells with 1% condensate, less than 30% of larvae hatched in total, which was comparable to wells with e-cigarette liquid (25%). Malformations that were observed in all treatements are hemagglutionation, pericardial or yolk sac edema, and scoliosis. In groups with 0.1% condensate these maformations were observed in lower number of embryos, but the percentage of hatched larvae was higher (approximately 80%) at 72 hpf. Conclusions: Chronic exposure to e-cigarette vapor condensate and liquid leads to severe disorders of zebrafish embryonic development

Алфа-1 антитрипсин подстиче регенерцију репног пераја ембриона зебрице (Danio rerio)

Komplikacije sa zarastanjem rana i formiranje ožiljnog tkiva predstavljaju globalni zdravstveni i ekonomski problem. Postoji evidentna potreba za razvijanjem novih, efikasnih terapija koje bi poboljšale proces zarastanja rana i transformisale ga u regenerativni proces. Alfa-1 antitripsin (AAT) je multifunkcionalni protein koji učestvuje u različitim procesima značajnim za zarastanje rana i regeneraciju – proliferaciji, migraciji, apoptozi i inflamaciji. Zebrica (Danio rerio) predstavlja značajan model za istraživanja procesa zarastanja rana, jer ima sposobnost potpune regeneracije amputiranog repnog peraja. Cilj ovog rada je bio da se ispita uticaj proteina AAT na regeneraciju repnog peraja embriona zebrice. Embrionima zebrice starim 48 sati amputirano je repno peraje i zatim merena dužina repa posle 48 h pod tretmanom (2 mg/ml AAT) i bez njega. Analizirana je ekspresija fibronektina 1a i 1b, dok je formiranje kolagena analizirano korišćenjem drugog harmonika, nelinearnom mikroskopijom. Pokazano je da je dužina regenerisanog dela repa veća kada su embrioni tretirani AAT-om. Ekspresija fibronektina 1a je povećana pod tretmanom. Primećen je trend povećanja dužine i debljine kolagenskih vlakana pri tretmanu, što dalje podržava rezultate o podsticanju regeneracije pod delovanjem AAT. Dobijeni rezultati ukazuju na značajan uticaj alfa-1 antitripsina na brzinu regeneracije repa embriona zebrice. S obzorom da je AAT odobren kao lek postoji i potencijal da se njegova primena proširi i na lečenje rana kod ljudi.Компликације са зарастањем рана и формирање ожиљног ткива представљају глобални здравствени и економски проблем. Постоји евидентна потреба за развијањем нових, ефикасних терапија које би побољшале процес зарастања рана и трансформисале га у регенеративни процес. Алфа-1 антитрипсин (ААТ) је мултифункционални протеин који учествује у различитим процесима значајним за зарастањe рана и регенерацију – пролиферацији, миграцији, апоптози и инфламацији. Зебрица (Danio rerio) представља значајан модел за истраживања процеса зарастања рана, јер има способност потпуне регенерације ампутираног репног пераја. Циљ овог рада је био да се испита утицај протеина ААТ на регенерацију репног пераја ембриона зебрице. Ембрионима зебрице старим 48 сати ампутирано је репно пераје и затим мерена дужина репа после 48 h под третманом (2 мг/мл ААТ) и без њега. Aнализирана је експресија фибронектина 1а и 1b, док је формирање колагена анализирано коришћењем другог хармоника, нелинеарном микроскопијом. Показано је да је дужина регенерисаног дела репа већа када су ембриони третирани ААТ-ом. Експресија фибронектина 1а је повећана под третманом. Примећен је тренд повећања дужине и дебљине колагенских влакана при третману, што даље подржава резултате о подстицању регенерације под деловањем ААТ. Добијени резултати указују на значајан утицај алфа-1 антитрипсина на брзину регенерације репа ембриона зебрице. С обзором да је ААТ одобрен као лек постоји и потенцијал да се његова примена прошири и на лечење рана код људи.Knjiga sažetaka: Treći Kongres biologa Srbije, Zlatibor, Srbija 21 - 25. 9. 2022.Autori D. P. i M. R. se zahvaljuju na finansiranju koje im je obezbedio Institut za fiziku u Beogradu, kroz ugovor sa Ministarstvom prosvete, nauke i tehnološkog razvoja Republike Srbij

E-cigarette vapor condensate affects mitochondrial function in A549 cells

Introduction: E-cigarettes are becoming increasingly popular, but their potentially harmful effects are not well studied. Although they are used to aid smoking cessation, there is evidence that the chemicals they contain can affect lung cells, and the effects may also be important for lung cancer cells. Our objective wasto investigate the effects of e-cigarette vapor condensate on mitochondrial function of A549 cells. Methods: We used the Agilent Seahorse Mito Stress Test, according to the manufacturer’s protocol to evaluate the mitochondrial function of A549 cells under the treatment with different concentrations of e-cigarette vapor condensates. Vapor condensates were prepared from e-cigarette liquid base (PG/VG), base with nicotine (PG/VG+N), with flavoring (PG/VG+F), or with nicotine and flavoring (PG /VG+F+N). The cells were exposed to 2% or 3% PG /VG, PG /VG+F, PG/VG+N, and PG/VG+F+N for 24 hours before testing. Results: All treatments with 2% vapor condensates affected mitochondrial function of A549 cells. Basal and maximal respiration were decreased, indicating mitochondrial dysfunction. Higher concentrations of vapor condensate (3%) significantly increased proton leak and decreased mitochondrial coupling efficiency, indicating mitochondrial damage. However, the increased spare respiratory capacity, in 3% vapor condensate treated cells, may indicate activation of a compensatory response in mitochondria. Conclusion: Our resultssuggest that e-cigarette vapor condensate may have deleterious effects on mitochondria. Further analysis of mitochondrial function and morphology would further elucidate the effects of e-cigarette vapor condensate

Decreased TSPAN14 Expression Contributes to NSCLC Progression

Tspan14 is a transmembrane protein of the tetraspanin (Tspan) protein family. Different members of the Tspan family can promote or suppress tumor progression. The exact role of Tspan14 in tumor cells is unknown. Earlier, mutational inactivation of the TSPAN14 gene has been proposed to coincide with a low survival rate in NSCLC patients. This study aimed to investigate the correlation of TSPAN14 lack of function with clinicopathological features of NSCLC patients, and to elucidate the role TSPAN14 might have in NSCLC progression. TSPAN14 expression was lower in tumor cells than non-tumor cells in NSCLC patients' samples. The decreased gene expression was correlated with a low survival rate of patients and was more frequent in patients with aggressive, invasive tumor types. Additionally, the role of decreased TSPAN14 expression in the metastatic potential of cancer cells was confirmed in NSCLC cell lines. The highly invasive NSCLC cell line (NCI-H661) had the lowest TSPAN14 gene and protein expression, whereas the NSCLC cell line with the highest TSPAN14 expression (NCI-H460) had no significant metastatic potential. Finally, silencing of TSPAN14 in these non-metastatic cancer cells caused an increased expression of matrix-degrading enzymes MMP-2 and MMP-9, followed by an elevated capacity of cancer cells to degrade gelatin. The results of this study propose TSPAN14 expression as an indicator of NSCLC metastatic potential and progression