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Evaluation of nanobodies against the selected protein biomarkers of glioblastoma and attempt of their delivery with exosomes
Get PDFGlioblastom (GBM) je najpogostejši primarni možganski tumor, ki se pojavlja s pogostnostjo 3,2 primera na 100 000 prebivalcev. Kljub uveljavljenem zdravljenju, ki obsega kirurško odstranitev tumorja, kemoterapijo s temozolomidom in radioterapijo, večina bolnikov ne preživi več kot 18 mesecev po postavljeni diagnozi. Eden izmed sodobnih možnih načinov zdravljenja GBM je uporaba nanoteles, antigen-prepoznavnih delov težkoverižnih protiteles, ki jih proizvajajo le nekatere živali, npr. lame. Nanotelesa imajo namreč v primerjavi s klasičnimi protiteles precej prednosti, kot so visoka stabilnost, možnost proizvodnje z bakterijo E. coli in hitrejše prehajanje v tumor.
V okviru doktorskega dela smo proučili vpliv nanoteles na preživetje celic, proti osmim možnim označevalcem glioblastoma, njihovo migracijo in tvorjenje kolonij. Najprej smo z uporabo imunohistokemije ugotovili, da na osnovi navzočnosti biooznačevalec oziroma razlik v izražanju, vimentin lahko razlikuje med glioblastomom, gliomi nižje stopnje in normalno možganovino, medtem ko biooznačevalci TUFM, DPYSL1 in CRMP1 razlikujejo med glioblastomom in normalno možganovino. Rezultati proučevanja citotoksičnega delovanja nanoteles kažejo, da nanotelesa Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) in Nb314 (anti-DPYSL2) delujejo citotoksično na glioblastomske celice. Posebno velik vpliv na citotoksičnost glioblastomskih matičnih celic ima nanotelo anti-TUFM (Nb225). Na migracijo glioblastomskih celic pa najbolj vpliva nanotelo anti-vimentin (Nb79), ki je popolnoma inhibiralo migracijo celic glioblastomske celične linije U87MG.
V drugem delu raziskave smo razvili dostavni sistem, ki temelji na zunajceličnih veziklih eksosomih, v katere smo zapakirali nanotelesa, da bi izboljšali njihovo dostavo in povečali učinkovitost. Eksosomi so najmanjši zunajcelični vezikli, ki jih izločajo celice, in naj bi, v primerjavi s primerljivimi dostavljalci, liposomi, hitreje prehajali v tarčne celice, obenem pa naj bi tudi imeli daljši razpolovni čas. Eksosome smo izolirali iz glioblastomske celične linije U251MG in jih opredelili z uporabo prenosa western za detekcijo eksosomalnih označevalcev, z uporabo metode sledenja nanodelcem za določitev števila in velikosti eksosomov, ter z elektronsko mikroskopijo za ugotavljanje njihove oblike. V eksosome smo nanotelesa uspešno zapakirali z metodama inkubacije z 0,4 % saponinom in sonikacije, ki sta bili približno enako učinkoviti, medtem ko posredno pakiranje nanoteles v eksosome preko inkubacije celic z nanotelesi ni bilo uspešno.
Eksosomi so poleg tega, da so potencialni dostavljalci oz. komponente sistemov za dostavo zdravil do tkiv in celic, tudi možen vir biooznačevalcev. V naši študiji smo z metodo qPCR analizirali izražanje izbranih mRNA, miRNA in proteinov v eksosomih celic glioblastomskih celičnih linij. Ugotovili smo, da so miR-9-5p, miR-124-3p, mRNA TUFM in mRNA CRMP1 možni označevalci eksosomov glioblastomskih matičnih celic, mRNA VIM pa primeren označevalec eksosomov diferenciranih glioblastomskih celic. Za razliko od omenjenih molekul RNA so bili proteini v eksosomih proučevanih celic slabše zastopani, v njih smo lahko detektirali samo proteinska biooznačevalca ALYREF in DPYSL2.
V doktorski nalogi smo pokazali, da so nanotelesa primerno sredstvo za doseganje citotoksičnega učinka in zmanjševanje migracije glioblastomskih celic. Uspešno smo razvili dostavno sredstvo, eksosome, ki vsebujejo nanotelesa, in te bi lahko v prihodnosti uporabili za povečanje učinkovitosti samih nanoteles. Naši rezultati kažejo, da so citotoksična nanotelesa in eksosomi, kot dostavni sistem, obetavna učinkovita in specifična oblika terapije za zdravljenje GBM. Vzporedno smo v naši študiji tudi določili možne eksosomalne označevalce glioblastomskih celic, za katere predlagamo ovrednotenje pri nadaljnjem proučevanju eksosomov, izoliranih iz telesnih tekočin bolnikov. Ti bi v prihodosti lahko služili kot potencialni biomarkerji GBM iz krvi ali cerebrospinalne tekočine bolnikov z GBM.Glioblastoma is the most common primary brain tumor, occurring with a frequency of 3.2 cases per 100 000 population. Despite established treatment, which includes surgical removal of the tumor, chemotherapy with temozolomide, and radiotherapy, most patients do not survive more than 18 months after diagnosis. One of the modern possible ways of treating GBM is the use of nanobodies, antigen-recognizing parts of heavy chain-only antibodies produced by only some animals, e.g. llamas. Namely, nanobodies have many advantages over classical antibodies, such as high stability, possibility of production with E. coli and faster transition and penetration to the tumor.
In the doctoral thesis, we studied the influence of nanobodies against eight possible biomarkers of glioblastoma on cell survival, migration and colony formation. First, using immunohistochemistry, we found that, based on differences in expression or presence, the biomarker vimentin could distinguish between glioblastoma, lower-grade gliomas, and normal brain, while biomarkers TUFM, DPYSL1, and CRMP1 distinguished between glioblastoma and normal brain. The results of the study of the cytotoxic action of nanobodies showed that nanobodies Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) and Nb314 (anti-DPYSL2) have a cytotoxic effect on glioblastoma cells. The anti-TUFM nanobody (Nb225) has a particularly large effect on the cytotoxicity of glioblastoma stem cells. The migration of glioblastoma cells is mostly affected by the anti-vimentin nanobody (Nb79), which completely inhibited the migration of cells of the glioblastoma cell line U87MG.
In the second part of the study, we developed a delivery system based on extracellular vesicles, exosomes, into which we packaged nanobodies to improve their delivery and increase efficiency. Exosomes are the smallest extracellular vesicles secreted by cells and are thought to pass more rapidly into target cells than comparable delivery systems, while also having a longer half-life. Exosomes were isolated from the U251MG glioblastoma cell line cells and characterized by detection of exosomal markers using Western blot, a nanoparticle tracking analysis method to determine their number and size, and by electron microscopy to determine their shape. Nanobodies were successfully packaged into exosomes by 0.4% saponin incubation and sonication methods, which were approximately equally effective, while indirect packaging by incubating cells with nanobodies was not successful.
In addition to being carriers of a potential delivery system, exosomes are also a possible source of biomarkers. In our study, we analyzed the expression of selected mRNAs, miRNAs, and proteins in the exosomes of glioblastoma cell lines using the qPCR method. We found that miR-9-5p, miR-124-3p, TUFM mRNA, and CRMP1 mRNA are possible markers of glioblastoma stem cell exosomes, and VIM mRNA is a suitable marker of differentiated glioblastoma cell exosomes. In contrast to the mentioned RNA molecules, proteins were less represented in the exosomes of the studied cells, in which only the protein biomarkers ALYREF and DPYSL2 could be detected.
In our doctoral dissertation, we showed that nanobodies are a suitable means of achieving a cytotoxic effect and reducing the migration of glioblastoma cells. We have successfully developed a delivery vehicle, exosomes that contain nanobodies, and these could be used in the future to increase the efficiency of the nanobodies themselves. Our results suggest that cytotoxic nanobodies and exosomes, as a delivery system, are a promising form of efficient and specific therapy for the treatment of GBM. In parallel, in this study we also identified possible exosomal markers of glioblastoma cells, for which we propose evaluation in the further study of exosomes isolated from patients’ body fluids. These could in the future serve as potential biomarkers of GBM from the blood or cerebrospinal fluid of patients with GBM
Implementation of nanobodies for the design of glioblastoma targeting therapy
Get PDFGlioblastom multiforme (GBM) je gliom, tumor osrednjega živčnega sistema, najpogostejša oblika možganskega tumorja, za katerim zboli 3-5 bolnikov na 100.000 ljudi. Povprečni čas preživetja bolnikov z GBM je od časa diagnosticiranja 12 do 18 mesecev, ob kombinirani postoperativni terapiji z uporabo temozolomida. Genetska heterogenost GBM je posledica glioblastomskih matičnih celic (GMC). GMC zaradi svoje odpornosti proti kemoterapevtikom in radioterapiji uspešno napadajo zdravo okoljsko tkivo. Zdravljenje je dodatno oteženo zaradi krvno-možganske pregrade (KMP), saj je prehod kemoterapevtikov skozi njo zelo otežen. Za izboljšanje zdravljenja GBM in izid za bolnika je potrebna stalna dostava zdravil v celice glioma, obenem pa je potrebno zmanjšati učinek zdravil na sosednje, zdrave nevrone in celice glije. Novi pristopi zdravljenja GBM so zato zelo zaželeni in potrebni. Za razvoj usmerjenega zdravljenja pa še vedno potrebujemo odkritje bolj specifičnih biomolekularnih označevalcev GMC, kot tudi njihovih tarčnih zdravil, ki bi prehajala KMP. To lahko dosežemo s proteomskim pristopom, ki temelji na nano-protitelesih, ki so eno-domenski antigen-vezavni fragmenti, pridobljeni iz kemelidnih težkoverižnih protiteles, ki zaradi svoje majhnosti lahko prehajajo KMP.
V doktorski nalogi smo izdelali knjižnico nano-protiteles in opravili njihovo imunoafinitetno obogatitev, izvedeno na celotnih GMC, in tako pridobili nano-protitelo, specifično za bio-označevalec GMC. Z masno spektrometrijo smo ugotovili, da je nov bio-označevalec mitohondrijski translacijsko-elongacijski dejavnik TUFM. Diferenčno izražanje TUFM smo proučevali na ravni proteinov in mRNA, in sicer v celičnih linijah GBM (U87MG in U251MG), GMC in tkivih GBM, v primerjavi z izražanjem v nevralnih matičnih celicah (NSC) in normalnih možganskih tkivih. S prenosom western smo na ravni proteinov ter qPCR na ravni mRNA potrdili nadizražanje TUFM v GMC. Z imunohistokemijo, na tkivnih rezinah, vklopljenih v parafin, smo potrdili nadizražanje TUFM v tkivih GBM, v normalnem možganskem tkivu pa se TUFM ni izražal. Z imunocitokemijo smo potrdili vstop nano-protitelesa anti-TUFM v GBM celice U87MG, U251MG in GMC ter njegovo vezavo v območje mitohondrijev. Citotoksičnost vezave nano-protitelesa anti-TUFM na antigen smo preverili z metabolnimi testi na celičnih linijah GBM (U87MG in U251MG), GMC ter kontrolnih celičnih linijah – astrocitih, nevralnih matičnih celicah in človeških nesmrtnih keratinocitih. Nano-protitelo anti-TUFM je imelo citotoksičen učinek na vse celične linije GBM, medtem ko toksičnosti anti-TUFM na kontrolnih celičnih linijah nismo opazili.
Z vklapljanjem nano-protiteles anti-TUFM v arheosome smo preverili ali obstaja boljši oz. bolj učinkovit dostavni sistem za vnos nano-protitelesa anti-TUFM v celice. Arheosomi niso citotoksični in vivo in imajo edinstvene strukturne lastnosti, ki so osnova za razvoj novih dostavnih sistemov zdravil. Te lastnosti so stabilnost pri visokih temperaturah, nizkem oz. visokem pH, odpornost proti fosolipazam in solem žolčnih kislin ter manjša membranska prepustnost. Nano-protitelesa anti-TUFM smo uspešno vklopili v arheosome in ugotovili, da le-ti vstopajo v celice GBM, U251MG in U87MG. Ugotovili smo, da ima nano-protitelo anti-TUFM, vklopljeno v arheosom, na celice GBM manjši citotoksični učinek kot ga ima samo nano-protitelo anti-TUFM.
V doktorski nalogi smo dokazali specifičnost in izrazit zaviralni učinek nano-protitelesa anti-TUFM na rast GMC, kar bi lahko v prihodnje pripomoglo k razvoju specifičnega pristopa za zdravljenje glioblastoma.Glioblastoma multiforme (GBM) is a glioma, a tumor found in central nervous system. It is the most common form of brain tumors, which affects 3-5 patients per 100,000 people. The average survival period of patients with GBM is 12 to 18 months, which includes resection and combination of postoperative therapy with temozolomide. Glioblastoma stem cells (GMC) are responsible for high genetic heterogeneity of GBM, and due to their resistance to chemotherapy and radiotherapy they successfully invade healthy tissue. The treatment is further aggravated, due to difficult transition of chemotherapeutics through blood-brain barrier (BBB). To improve GBM treatment and the outcome of patients, it is necessary to continuously deliver drugs to the glioma cells while reducing the effect of drugs on adjacent, healthy neurons and glial cells. New GBM treatment approaches are therefore much needed. For the development of targeted GBM treatment, we still need the discovery of more specific GMC biomarkers and the corresponding targeting drugs that would pass BBB. This can be achieved by a proteomic approach based on nanobodies, single-domain antigen-binding fragments, derived from camelid heavy chain antibodies that can, due to their small size, pass BBB.
In the doctoral thesis, we constructed a nanobody library and biopannings were made on the whole GMCs. We obtained a nanobody specific for a GMC antigen. Mass spectrometry determined that the new biomarker of GMC is the mitochondrial translational-elongation factor TUFM. Differential expression of TUFM was studied at the protein and mRNA levels in the GBM cell lines (U87MG, and U251MG), GMC and GBM tissue, compared to its expression in neural stem cells (NSC) and normal brain tissue. Western blot and qPCR confirmed the TUFM overexpression in GMC. With immunohistochemistry, on paraffin-embedded GBM tissue, we confirmed the TUFM overexpression, whereas the normal brain tissue was negative for TUFM. Immunocytochemistry confirmed the entry of anti-TUFM nanobodies to the U87MG, U251MG and GMC cells and its binding to mitochondria. The cytotoxic effect of anti-TUFM nanobodies on GBM-related cell lines (U87MG, U251MG and GMC), and on control cell lines (astrocytes, NSC and human immortal keratinocytes) was measured through metabolic assays. Anti-TUFM nanobody had cytotoxic effect on all GBM cell lines, while on the other hand no toxicity of anti-TUFM on control cell lines was observed.
Anti-TUFM nanobodies were encapsulated to archeosomes and used to verify their delivery in cells. Aerheosomes are not cytotoxic in vivo and have unique structural properties for the development of new drug delivery systems. These properties are stability at high temperatures, extreme pH values, resistance to phospholipases and bile salts and lower membrane permeability. Successfully, anti-TUFM nanobodies were encapsulated in the archeosomes and were found that they could enter the U251MG cells. Therefore, an encapsulated anti-TUFM nanobody exhibited lower cytotoxic effect on GBM cells than the anti-TUFM nanobody itself.
In the doctoral thesis, we showed the specificity and pronounced inhibitory effect of anti-TUFM nanobody on GMC growth, which could in the future contribute to the development of a specific approach for the treatment of glioblastoma
Developing methods for in vitro studies of tissue protective effects of erythropoietin in a cell model
Get PDFAttempt of enrichment of plasma membrane proteins from gastric tissue, and a computer search of potential biomarkers in gastric cancer
Get PDF-actin, CD9, FTL, S100A9 and TRIM28 proteins as potential candidates for glioblastoma stem cell markers
Get PDFGlioblastom (GBM) je najbolj agresivna maligna bolezen na možganih. Čeprav so že identificirali nekatere kandidate za označevalce glioblastoma, trenutno še ne poznamo dovolj kandidatov za označevalce, ki bi se izražali na celični površini ter bi bili sposobni ločevati med nemalignim možganskim tkivom in glioblastomskimi tumorskimi celicami. Znotraj glioblastomskega tumorja se med običajnimi tumorskimi celicami nahajajo glioblastomske matične celice (GMC), ki so vpletene v proces ponovnega nastanka tumorja po kirurški odstranitvi.
V doktorski nalogi smo želeli najti nove kandidate za označevalce glioblastoma ali glioblastomskih matičnih celic. Za osnovo smo vzeli izhodiščne kandidate (IK) CD9, FTL, S100A9, beta-aktin in TRIM28, ki so jih pred kratkih predlagali kot kandidate za označevalce GMC s povišanim izražanjem v GMC. V literaturi smo poiskali maligne bolezni, v katerih so IK prav tako povišano izraženi kot v GBM, in interakcijske partnerje, s katerimi se povezujejo v patologiji teh bolezni. Za vsak par IK-interakcijski partner smo naredili model interakcijskega omrežja na genski in proteinski ravni s pomočjo podatkovnih zbirk Biomine Explorer (genska raven) in String (proteinska raven). V teh omrežjih smo poiskali skupna vozlišča med IK in interakcijskim partnerjem, ki so predstavljala morebitne nove kandidate za označevalce GBM/GMC. Za kandidate iz skupnih vozlišč smo preverili, če so jih že povezali s karcinogenezo GBM. Tiste kandidate, ki jih še niso povezali z GBM, smo bioinformatsko validirali s primerjavo njihovega izražanja v različnih celičnih/tkivnih tipih z izražanjem uveljavljenih kandidatov za označevalce GBM/GMC. S tem namenom smo naredili meta-analizo podatkov o izražanju mRNA na ravni celotnega genoma, ki smo jih našli v treh podatkovnih zbirkah (GEO, ArrayExpress in GLIOMASdb). V omenjenih podatkovnih zbirkah smo našli deset podatkovnih nizov, ki smo jih uporabili za validacijo kandidatov iz skupnih vozlišč.
Našli smo 16 interakcijskih partnerjev, ki so se povezovali z našimi IK pri 10 različnih malignih patologijah. Z analizo vseh možnih omrežij IK-interakcijski partner smo prišli do 82 skupnih vozlišč, med katerimi je bilo 23 še nepovezanih s karcinogenezo GBM. Te kandidate smo bioinformatsko validirali in prišli do treh morebitnih novih kandidatov za označevalce GBM/GMC s povišanim izražanjem v malignih celicah (CCT2, RUVBL1, BST1). Podrobnejša analiza rezultatov je pokazala, da se vsi trije kandidati neenakomerno izražajo v različnih za GBM/GMC relevantnih celičnih/tkivnih tipih (povišano izražanje v tkivnih vzorcih GBM, ne pa tudi v matičnih celičnih linijah in nevrosferah).
Zato smo poiskali kandidate, ki so v bioinformatskih testih dosegli najboljše rezultate in so se torej najbolj konsistentno izražali v vseh relevantnih celičnih/tkivnih tipih. V tej fazi smo se osredotočili na kandidate, ki se na proteinski ravni povišano izražajo na celični površini tumorskih celic. Za izbiro najboljših kandidatov smo uporabili podatkovne nize GSE4290/GDS1962, GSE23806/GDS3885 in GLIOMASdb, preostalih sedem nizov, ki smo jih našli v podatkovnih zbirkah (GSE4412/GDS1975, GSE4412/GDS1976, E-GEOD-52009, E-GEOD-68848, E-GEOD-16011, E-GEOD-4536 in E-GEOD-74571), pa smo uporabili za validacijo izbranih kandidatov.
V fazi selekcije najboljših kandidatov smo identificirali štiri gene, ki kodirajo površinske proteine (CD276, FREM2, SPRY1 in SLC47A1), in z bioinformatsko validacijo potrdili njihovo povišano izraženost v GBM/GMC. Pregled literature je razkril, da so CD276 že povezali s karcinogenezo glioblastoma, medtem ko je SLC47A1 v bioinformatski validaciji dosegel najslabši rezultat med štirimi novimi kandidati, zato ga nismo vključili v fazo eksperimentalne validacije.
V tej fazi – v katero smo vključili kandidata FREM2 in SPRY1 – smo ugotovili, da je izražanje FREM2 – ne pa tudi SPRY1 – višje v glioblastomskih celičnih linijah kot v nemalignih astrocitih. Poleg tega se je FREM2 tako na genski kot na proteinski ravni višje izražal v glioblastomskim matičnim celicam podobnih celičnih linijah kot v konvencionalnih glioblastomskih celičnih linijah. Zato predlagamo FREM2 kot novega kandidata za označevalca glioblastomskih celic in tudi kot potencialnega kandidata za označevalca GMC. Tako FREM2 kot SPRY1 sta izražena na površini glioblastomskih celic, medtem ko smo povišano izraženost v citosolu nemalignih astrocitov opazili samo pri SPRY1. Tudi SPRY1 bi lahko bil zanimiva terapevtska tarča, saj se v malignih glioblastomskih celicah izraža na površini, v nemalignih astrocitih pa v citosolu.Glioblastoma (GBM) is the most aggressive brain malignancy. Although some potential glioblastoma biomarkers have already been identified, there is a lack of cell membrane-bound biomarkers capable of distinguishing brain tissue from glioblastoma and/or glioblastoma stem cells (GSC), which are responsible for the rapid post-operative tumor reoccurrence.
Our aim was to find new candidates for glioblastoma or glioblastoma stem cell markers. Our startig point were the candidates CD9, FTL, S100A9, beta-actin and TRIM28 (starting point candidates – SPC). These candidates have ben recenty proposed as novel GSC biomarkers with elevated expression in GSC. We conducted a literature search in order to find all malignant pathologies in which SPC likewise have elevated expression – as in GBM. In each such pathology, we identified interaction partners for our SPC. For each pair SPC-interaction partner an interaction network was created at the gene and protein levels of expression, using the databases Biomine Explorer (gene level) and String (protein level). In each of these networks we identified common nodes between SPC and its interaction partner, and these nodes represented potential novel GBM/GSC marker candidates. For each candidate represented in the common nodes, a check was made whether it had already been linked to GBM carcinogenesis. Only candidates with no such links were bioinformatically validated by comparing their expression in various GBM/GSC-relevant cell/tissue types with that of established GBM/GSC marker candidates. This validation consisted of meta-analysis of genome-scale mRNA expression data from three data repositories (GEO, ArrayExpress and GLIOMASdb). The search yielded ten appropriate datasets, which were used for validation of the candidates from the common nodes.
16 interaction partners that were linked to our SPC in 10 different malignant pathologies were identified. Analysis of all possible networks SPC-interaction partner yielded 82 common nodes, of which only 23 had not yet been linked to GBM carcinogenesis. Bioinformatic validation of these candidates revealed three potential new GBM/GSC marker candidates with elevated expression in malignant cells (CCT2, RUVBL1, BST1). However, detailed results analysis highlighted that all these three candidates have uneven expression in various GBM/GSC-relevant cell/tissue types (e.g., elevated expression in GBM tissue samples, but not in stem-like cell lines and neurospheres).
Thus, we decided to identify candidates that achieved the best results in the bioinformatic validation tests and had the most consistent expression pattern in all relevant cell/tissue types. In this stage the search was focused on candidates with elevated expression at the protein level on the tumor cells surface. Three datasets (GSE4290/GDS1962, GSE23806/GDS3885, and GLIOMASdb) were used for selection of new GBM/GSC marker candidates, while the other seven (GSE4412/GDS1975, GSE4412/GDS1976, E-GEOD-52009, E-GEOD-68848, E-GEOD-16011, E-GEOD-4536, and E-GEOD-74571) were used for bioinformatic validation of the selected candidates.
The selection identified four new CSP-encoding candidate genes—CD276, FREM2, SPRY1, and SLC47A1—and the bioinformatic validation confirmed their elevated expression in GBM/GSC. A review of the literature revealed that CD276 is not a novel candidate, while SLC47A1 had lower validation test scores than the other new candidates and was therefore not considered for experimental validation.
This validation revealed that the expression of FREM2—but not SPRY1—is higher in glioblastoma cell lines when compared to non-malignant astrocytes. In addition, FREM2 gene and protein expression levels are higher in glioblastoma stem-like cell lines than in conventional glioblastoma cell lines. FREM2 is thus proposed as a novel GBM marker candidate, as well as a putative candidate for GSC biomarker. Both FREM2 and SPRY1 are expressed on the surface of the GBM cells, while SPRY1 alone was found over-expressed in the cytosol of non-malignant astrocytes. SPRY could also be useful as a therapeutic target since it is expressed on the surface of malignant GBM cells but in the cytosol of non-malignant astrocytes
VPLIV POLIMORFIZMOV V SEGREGACIJSKIH GENIH NA RAZVOJ RAKA ŽELODCA
Get PDFINTRODUCTION. Gastric cancer represents the fifth most frequently diagnosed cancer in the world. Despite numerous research studies, mechanisms leading to disease are poorly known and unclear. At the molecular level, many changes are involved in the development of gastric cancer, including malfunction of chromosome segregation genes. These abnormalities can lead to chromosomal instability (CIN). Segregation gene function can be affected by the low penetrance errors which include polymorphisms.
AIM. The aim of this study is to examine the effect of selected polymorphisms in specific segregation genes on gastric cancer development.
HYPOTHESIS. The study focused on exploring genotypes of selected polymorphisms in specific mitotic segregation genes. Those that differ significantly between the subjects and the healthy control population, may be associated with higher risk for developing gastric cancer or with certain clinical and histopathological characteristics, and may have effect on the survival of gastric cancer patients.
METHODS. 30 polymorphisms in genes BUB1B, CASC5, ESPL1, PTTG1, SMC1A, TPX2, TTK and ZWINT were included in the study. Subjects were compared with the control group. Polymorphisms were determined using quantitative real-time PCR (qPCR), restriction fragment length polymorphism (RFLP) and DNA sequencing.
RESULTS. The association between polymorphisms rs2277559 (BUB1B), rs2241666 (ZWINT), rs11858113 (CASC5) and rs11855334 (CASC5) and increased risk of developing gastric cancer in male population was determined. As concerning rs11855334, statistically significant difference was also observed in the genotype distribution between the whole population of subjects and controls. The association between the genotypes of polymorphisms (in gene BUB1B) rs2277559, rs2290551, rs1801376, rs1047130, rs1565866, rs2277560 and Lauren classification was recognized. Genotypes of polymorphisms rs1801376 (BUB1B), rs11855334 (CASC5), rs2241666 (ZWINT), rs2910101 (PTTG1) and rs1047130 (BUB1B) are linked to different tumour differentiation grades. Survival analysis revealed association between the lymph node involvement and perineural invasion. Statistically higher frequencies of haplotypes G-A-G-T-G-G-A, G-G-A-G-A-A-G and A-G-G-T-A-G-A in gene BUB1B and of haplotypes A-A-A-C and C-C-G-T in gene ESPL1 were observed in gastric cancer patients, whereas haplotypes A-C-A-T and C-A-G-T in gene ESPL1 were significantly more frequent in the control group. Association with gastric cancer was not noted with other polymorphisms.
CONCLUSIONS. The association between specific polymorphisms of selected chromosome segregation genes and gastric cancer was recognized. Findings could provide guidelines for further research and polymorphisms linked to gastric cancer could serve as potential diagnostic and prognostic biomarkers.IZHODIŠČE. Rak želodca je peta najpogosteje diagnosticirana oblika raka v svetovnem merilu. Kljub številnim raziskavam so mehanizmi, ki vodijo do nastanka bolezni, še vedno slabo poznani in nejasni. Na molekulski ravni so v razvoj raka na želodcu vpletene številne spremembe, med drugim tudi napake v delovanju segregacijskih genov. Omenjene nepravilnosti lahko vodijo v kromosomsko nestabilnost (CIN). Na spremembe v delovanju segregacijskih genov lahko vplivajo tudi napake z nizko penetranco, med katere uvrščamo polimorfizme.
NAMEN. Z raziskavo smo želeli preveriti vpliv izbranih polimorfizmov v posameznih segregacijskih genih na razvoj raka želodca.
HIPOTEZE. Želeli smo preveriti ali se genotipi izbranih polimorfizmov značilno razlikujejo med preiskovanci in kontrolami. Tisti, pri katerih ugotovimo značilno razliko, lahko vplivajo na višje tveganje za razvoj raka želodca in so povezani s patohistološkimi značilnostmi preiskovancev. Preveriti želimo, kakšen je njihov vpliv na preživetje bolnikov z rakom želodca.
METODE. V študijo smo vključili 30 polimorfizmov v genih BUB1B, CASC5, ESPL1, PTTG1, SMC1A, TPX2, TTK in ZWINT. Primerjali smo preiskovance s kontrolno skupino. Za določanje polimorfizmov smo uporabili metode kvantitativni PCR v realnem času (qPCR), polimorfizem dolžin restrikcijskih fragmentov (RFLP) in določanje zaporedja nukleotidov (sekvenciranje DNA).
REZULTATI. Ugotovili smo povezavo med polimorfizmi rs2277559 (BUB1B), rs2241666 (ZWINT), rs11858113 (CASC5) ter rs11855334 (CASC5) in višjim tveganjem za razvoj raka želodca pri moških. Pri rs11855334 se je značilna razlika v porazdelitvi genotipov pokazala tudi pri primerjavi celotne populacije preiskovancev in kontrol. Dokazali smo povezavo med genotipi polimorfizmov (v genu BUB1B) rs2277559, rs2290551, rs1801376, rs1047130, rs1565866, rs2277560 in Laurenovo klasifikacijo. Z različno stopnjo diferenciranosti tumorjev so povezani genotipi polimorfizmov rs1801376 (BUB1B), rs11855334 (CASC5), rs2241666 (ZWINT), rs2910101 (PTTG1) in rs1047130 (BUB1B). Analiza preživetja je pokazala, da je bilo preživetje preiskovancev povezano s prizadetostjo bezgavk in perinevralno invazijo. Bolniki z rakom želodca so imeli statistično značilno višje frekvence haplotipov G-A-G-T-G-G-A, G-G-A-G-A-A-G in A-G-G-T-A-G-A v genu BUB1B in haplotipov A-A-A-C in C-C-G-T v genu ESPL1. Haplotipa A-C-A-T in C-A-G-T v genu ESPL1 pa sta bila značilno bolj pogosta v kontrolni populaciji. Analize ostalih polimorfizmov niso pokazale povezave z rakom želodca.
ZAKLJUČEK. Dokazali smo povezavo med posameznimi polimorfizmi izbranih mitoznih segregacijskih genov in rakom želodca. Ti polimorfizmi bi lahko služili kot smernice za nadaljnje raziskave, uporabni pa bi bili lahko tudi kot potencialni diagnostični in prognostični bio-označevalci
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