Aktivacija Hh-Gli signalnog puta u zloćudnim epitelnim tumorima jajnika

Kalafatić, Držislav (2011) Aktivacija Hh-Gli signalnog puta u zloćudnim epitelnim tumorima jajnika. PhD thesis, Sveučilište u Zagrebu.

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Abstract

Introduction: Ovarian cancer is the fourth most common cancer in women, and survival of ovarian cancer has not significantly changed over the past few decades. There is still no effective method of primary screening of patients, as well as sensitive clinical and laboratory diagnosis of early stage disease. Hedgehog-Gli (Hh-Gli) pathway is involved in the occurrence of at least one third of all cancers, but its importance in the genesis of ovarian cancer has not yet been explored. ----- Patients and Methods: We analyzed the biological material (tumor tissue, the tissue not affected by tumor and blood specimens) of 18 patients, of which 12 had ovarian cancer, and the remaining 6 atypical proliferative ovarian tumor. We performed the analysis of gene expression of Hh-Gli pathway and β-catenin, their mutual correlation and correlation with clinical parameters of disease, immunohistochemical analysis of protein expression and analysis of genetic polymorphisms and the mutation of Ptch and Smo. Finally, the blocking of the signaling pathway with cyclopamine and curcumin on primary cell cultures was investigated. ----- Results: The gene expression of Hh-Gli pathway was found in most of the analyzed tumor samples (63-79%), but also in the majority of tissue samples not affected by tumor (68-74%). All atypical proliferative tumor samples showed increased expression of the ligand, Shh gene (47%), which could classify them into ligand-induced tumor. Most tumors have increased expression of genes Ptch, Smo and β-catenin (63%). At the protein level increased protein expression of Ptch (64%) and Smo (78%) was found, and we showed that expression of Ptch protein was significantly associated with the anatomical extension of disease (p<0,05). In 6 samples a polymorphism in exon 6 (31,6%) was found, of which 3 had loss of heterozygosity (LOH) in the Ptch gene promotor region, showing significantly higher incidence compared to control group (p<0,01). Primary cell cultures showed inhibition of expression of Hh-Gli genes and β-catenin with cyclopamine, a dose-dependent effect 96 hours after treatment. For the first time we revealed that curcumin caused cell death in ovarian cancer primary cell culture. ----- Conclusion: The ovarian cancers are very heterogeneous in terms of Hh-Gli pathway and β-catenin gene expression. Increased expression of Ptch protein is significantly associated with extension of disease. The analysis of genetic polymorphisms could serve as a molecular marker of the ovarian cancer. The blocking of Hh-Gli signaling pathway is dose-dependent after the treatment with cyclopamine and the ability of curcumin to cause cell death opens up new perspectives in the study of therapeutic modalities of ovarian cancer.

Abstract in Croatian

Uvod: Rak jajnika je četvrto najčešće sijelo raka u žena, a preživljenje od raka jajnika nije se značajnije mijenjalo tijekom zadnjih nekoliko desetljeća. Još uvijek ne postoji učinkovita metoda primarnoga probira bolesnica, kao ni osjetljiva klinička ili laboratorijska dijagnostika ranoga stadija bolesti. Hedgehog-Gli (Hh-Gli) put sudjeluje u nastanku barem jedne trećine svih zloćudnih tumora, ali njegov značaj u genezi karcinoma jajnika još uvijek nije istražen. ----- Bolesnice i metode: Analizirali smo biološki materijal (tumorsko tkivo, tkivo koje nije zahvaćeno tumorom i punu krv) 18 bolesnica, od kojih su 12 imale karcinom jajnika, a preostalih 6 atipični epitelni proliferativni tumor jajnika. Provedena je analiza ekspresije gena signalnog puta Hh-Gli i β-katenina, njihova međusobna korelacija, korelacija s kliničkim parametrima bolesti, imunohistokemijska analiza ekspresije proteina signalnoga puta te analiza genskih polimorfizama i mutacija gena Ptch i Smo. U konačnici je ispitana mogućnost blokiranja signalnoga puta ciklopaminom i kurkuminom na primarnim staničnim kulturama. ----- Rezultati: Ekspresija gena Hh-Gli signalnog puta pronađena je u većini analiziranih uzoraka tumora (63-79%), ali i u većini pratećih uzoraka tkiva (68-74%), koja nisu zahvaćena tumorom. Svi uzorci atipičnih proliferativnih tumora pokazali su povećanu ekspresiju liganda, gena Shh (47%), zbog čega bi se mogli svrstati u ligandom inducirani tip tumora. Većina tumora ima povećanu ekspresiju gena Ptch i Smo te β–katenina (63%). Na proteinskoj razini nađena je povećana ekspresija proteina Ptch (64%) i Smo (78%), a pokazali smo i da je ekspresija Ptch proteina značajno povezana s anatomskom proširenošću bolesti (p<0,05). U 6 uzoraka nađen je polimorfizam u eksonu 6 (31,6%), od kojih 3 imaju gubitak heterozigotnosti u promotorskoj regiji gena Ptch, što je značajno češće u odnosu na kontrolnu skupinu (p<0,01). Primarne kulture su pokazale inhibiciju ekspresije Hh-Gli gena i β-katenina ciklopaminom, koja je ovisna o dozi, 96 sati nakon tretmana. Po prvi puta smo na primarnoj staničnoj kulturi pokazali i da kurkumin izaziva staničnu smrt u karcinomima jajnika. ----- Zaključak: Karcinomi jajnika vrlo su heterogeni s aspekta ekspresije gena signalnog puta Hh-Gli i β-katenina. Pojačana ekspresija Ptch proteina značajno je povezana s proširenošću bolesti, a analiza genskih polimorfizama mogla bi poslužiti kao molekularni biljeg sklonosti razvoju zloćudnih tumora jajnika. Budućoj terapiji karcinom jajnika mogla bi doprinijeti činjenica kako je blokada signalnoga puta ovisna o dozi i ekspoziciji ciklopaminom, a sposobnost izazivanja stanične smrti kurkuminom otvara nove perspektive u istraživanju terapijskih modaliteta karcinoma jajnika.

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