Proučevanje vloge estrogenov pri neodzivnosti celične linije raka jajčnikov na karboplatin

Kreft, Tinkara (author), Lanišnik Rižner, Tea (mentor)

Serozni rak jajčnikov visokega gradusa (HGSOC) je najbolj smrtonosna oblika raka jajčnikov predvsem zaradi pozne postavitve diagnoze in razvoja na kemoterapevtike odporne oblike bolezni. Natančna vloga estrogenov v patogenezi HGSOC še ni jasna, lahko pa ti spodbujajo celično proliferacijo preko receptorjev za estrogene (ER) ali kažejo genotoksične lastnosti preko svojih metabolitov. Naš namen je bilo raziskati vlogo estrogenov pri neodzivnosti celične linije HGSOC (OVCAR-3) na karboplatin (kemoterapevtik za zdravljenje raka jajčnikov). Najprej smo novovzpostavljeni celični liniji OVCAR-3/Rcarbo potrdili večjo odpornost na karboplatin v primerjavi z osnovno celično linijo OVCAR 3. Z LC-MS/MS smo ocenili, da OVCAR-3 tvori aktivne estrogene iz estron sulfata (E1 S), a šibko. Nato smo izvedli analizo qPCR na obeh celičnih linijah in primerjali izražanje genov, ki sodelujejo pri metabolizmu in delovanju estrogenov. S prenosom western (WB) smo ocenili izražanje dveh ER (ERα in GPER) ter steroid sulfataze (STS), glavnega encima biosintezne poti estradiola. Na koncu smo raziskali še učinke estrogenov na celično proliferacijo in občutljivost celic na karboplatin. Ugotovili smo, da celična linija OVCAR-3 v manjši meri metabolizira E1 S v aktivne estrogene, kar je potrdilo tudi izražanje genov, vključenih v sulfatazno pot. Kljub temu, da nismo potrdili spremenjenega izražanja teh genov na ravni mRNA, smo pokazali večjo raven izražanja izooblik STS z molekulskima masama 100 kDa in 30 kDa v celicah OVCAR-3/R. Potrdili smo tudi izražanje ERα, ERβ in GPER ter ugotovili povečano izražanje gena za ERα v OVCAR 3/R. WB je pokazal, da obe celični liniji izražata izoobliko ERα46, vendar ne osnovne ERα. Estradiol (E2) ni vplival na celično proliferacijo, so pa rezultati potrdili, da višja raven kemorezistence zmanjša z E2 spodbujeno odpornost na karboplatin. Ekvilin in etinilestradiol sta zmanjšala viabilnost celic OVCAR-3/R v primerjavi s celicami OVCAR-3, vendar nista vplivala na občutljivost celic na karboplatin. Z našim delom smo dokazali obstoj razlik v metabolizmu in delovanju estrogenov med celicami OVCAR-3, ki so bolj ter manj odporne na karboplatin, potrebne pa so nadaljnje raziskave za boljše razumevanje vloge estrogenov pri kemorezistenci HGSOC.

• Language: Slovenian
• Keywords: rak jajčnikov, estrogeni, kemorezistenca, OVCAR-3, karboplatin
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Year: 2023
• PID: 20.500.12556/RUL-152563
• COBISS.SI-ID: 174362115
• Publication date in RUL: 29.11.2023

Secondary language

• Language: English
• Title: Study on the influence of estrogens on the unresponsiveness of an ovarian cancer cell line to carboplatin

Abstract

High-grade serous ovarian cancer (HGSOC) is the most lethal form of ovarian cancer, mainly because of late diagnosis and development of resistance to platinum-based chemotherapy. Estrogens can promote cell proliferation via estrogen receptors (ERs) or show genotoxic properties through their metabolites; however, the exact role of estrogens in HGSOC pathogenesis remains unclear. Our aim was to investigate the role of estrogens in the chemoresistance of the HGSOC cell line OVCAR-3. First, we established a carboplatin-resistant cell line OVCAR-3/R by prolonged carboplatin exposure and confirmed its higher resistance compared with the parental cell line by determining IC50 values. With LC MS/MS we assessed the capacity of OVCAR-3 to form active estrogens from the precursor estrone sulfate (E1-S). We then performed qPCR analysis on both cell lines to compare the expression of genes involved in estrogen biosynthesis, metabolism, and action. Western blotting (WB) was used to evaluate the expression of two ERs (ERα and GPER) and steroid sulfatase (STS), the main enzyme of the estrogen biosynthesis pathway. Finally, we investigated the effects of estrogens on cell proliferation and sensitivity to carboplatin. We found that the OVCAR-3 cell line weakly metabolizes E1-S to active estrogens, which was also confirmed by the expression of genes involved in the sulfatase pathway. We did not confirm any differential gene expression at the mRNA level, but we did show higher expression of 100 kDa and 30 kDa STS isoforms in OVCAR-3/R cells. We affirmed the expression of ERα, ERβ and GPER, and found increased expression of ESR1 in OVCAR 3/R vs OVCAR-3. WB showed that both cell lines express ERα46 isoform but not full-length ERα. Estradiol (E2) had no effect on cell proliferation, although our results showed that a higher level of chemoresistance reduced E2-promoted resistance to carboplatin. In contrast, equilin and ethinylestradiol decreased viability of OVCAR 3/R cells compared with that of OVCAR 3 cells, but had no effect on sensitivity to carboplatin. Our work demonstrated some differences in the metabolism and action of estrogens between more and less chemoresistant OVCAR-3 cells. Further research is needed to better understand the role of estrogens in the chemoresistance of HGSOC.

• Keywords: ovarian cancer, estrogens, chemoresistance, OVCAR-3, carboplatin