Določanje estrogenih učinkov izbranih selektivnih modulatorjev estrogenskih receptorjev na celični liniji HeLa9903

Bedek, Naja (author), Peterlin Mašič, Lucija (mentor), Gramec Skledar, Darja (comentor)

Selektivni modulatorji estrogenskih receptorjev so uveljavljene zdravilne učinkovine v terapiji, a je o njih še vedno veliko neznank. Posebnost teh spojin je, da lahko na estrogenske receptorje v enem tkivu delujejo kot agonisti, v drugem pa kot antagonisti. V to skupino učinkovin med drugim spadajo tamoksifen, bazedoksifen, raloksifen, 4-hidroksitamoksifen, ospemifen ter toremifen. Uporabljajo se za zdravljenje raka dojke, osteoporoze ter atrofije vagine in vulve. Uporabni bi lahko bili tudi za lajšanje težav v menopavzi, kot so navali vročine in prekomerno potenje. V okviru magistrske naloge smo želeli z uporabo validiranega in vitro testa na HeLa9903 celični liniji (OECD 455) preučiti tako agonistično kot antagonistično delovanje izbranih modulatorjev estrogenskih receptorjev na estrogenski receptor α. Najprej smo za izbrane spojine določili najvišjo necitotoksično koncentracijo. Najvišje necitotoksične koncentracije, ki smo jih v nadaljevanju uporabili pri luciferaznem testu so bile: 25 µM za bazedoksifen, raloksifen in ospemifen, 10 µM za tamoksifen in toremifen ter 5 µM za 4-hidroksitamoksifen. V naslednjem koraku smo z luciferaznim testom ovrednotili delovanje spojin na estrogenski receptor. Vse testirane spojine z izjemo bazedoksifena delujejo kot agonisti estrogenskega receptorja α z EC50 vrednostmi v pikomolarnem (raloksifen, 4-hidroksitamoksifen) ali nanomolarnem območju (tamoksifen, toremifen, ospemifen). Kljub nizkim EC50 vrednostim, pa je maksimalni odziv raloksifena, ospemifena in toremifena zelo nizek (10-20% odziva pozitivne kontrole), medtem ko tamoksifen in 4-hidroksi tamoksifen dosežeta tudi do 50% odziva glede na pozitivno kontrolo (1 nM E2). Pri testiranju antagonizma so vse testirane spojine razen toremifena znižale odziv 25 pM E2 vsaj za 30%, zato jih lahko uvrstimo med antagoniste. Najnižji IC50 smo določili pri raloksifenu, sledil mu je bazedoksifen, nato 4-hidroksi tamoksifen, ospemifen in tamoksifen z najnižjo jakostjo. Če želimo popolnoma razumeti delovanje teh spojin, jih bo v prihodnosti treba še bolje raziskati. Naša magistrska naloga pokriva le del neodkritega področja, še vedno pa ostaja veliko neznank o njihovemu mehanizmu delovanja.

• Language: Slovenian
• Keywords: estrogenski receptor, selektivni modulatorji estrogenskih receptorjev, SERM, HeLa9903
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-113165
• Publication date in RUL: 09.12.2019

Secondary language

• Language: English
• Title: Determination of estrogenic effects of several selective estrogen receptor modulators on the HeLa9903 cell line

Abstract

Selective estrogen receptor modulators are already widely used active ingredients in therapy, but there is still a lot we do not know about them. Selective estrogen receptor modulators are unusal because they can act as agonists on estrogen receptors in one tissue and as antagonists in another tissue. Tamoxifen, bazedoxifene, raloxifene, 4-hydroxytamoxifen, ospemifene and toremifene are all examples of selective estrogen receptor modulators. They are used for treating breast cancer, osteoporosis and vulvovaginal atrophy. They could also be useful for alleviating postmenopausal symptoms, such as hot flashes and sweating. In our master’s thesis we implemented a validated in vitro test using HeLa9903 cell line (OECD 455) to determine agonistic and antagonistic effects of selected estrogen receptor modulators on estrogen receptor α. First we had to determine the highest non-cytotoxic concentration of our compounds. The results showed that we can use the concentrations up to 25 µM for bazedoxifene, raloxifene and ospemifene; 10 µM for tamoxifen and toremifene and up to 5 µM for 4-hydroxytamoxifen. The next step was determining the action of our compounds on the estrogen receptor α, using luciferase test. Excluding bazedoxifene, all of the other compounds acted as agonists on the estrogen receptor α with EC50 values either picomolar (raloxifene, 4-hydroxytamoxifen) or nanomolar (tamoxifen, toremifene, ospemifene). Despite the low EC50 values the maximum response of raloxifene, ospemifene and toremifene was quite low (10-20% of response given by positive control), while tamoxifen and 4-hydroxytamoxifen reached up to 50% of response given by positive control (1 nM E2). When testing for antiestrogenic properties all of the compounds except toremifene showed at least a 30% reduction in activity compared to spike in control, therefore we can classify all of these compounds as antagonists on the estrogen receptor α. Raloxifene showed the lowest IC50, followed by bazedoxifene, then 4-hydroxytamoxifen, ospemifene and lastly, tamoxifen with the lowest potency. If we wish to understand selective estrogen receptor modulators fully, then more research will need to be done in this area. Our thesis covers only a portion of the undiscovered area. There are still more facts to be gathered surrounding their mechanism of action.

• Keywords: estrogen receptor, selective estrogen receptor modulators, SERM, HeLa9903