The effects of selective estrogen receptor modulators on the death of breast cancer cells and osteoblastic cells

Selektiivisten estrogeenireseptorin muuntelijoiden (serm) vaikutus rintasyöpäsolujen ja luun solujen kuolemaan Selektiiviset estrogeenireseptorin muuntelijat (SERMit) ovat ryhmä kemialliselta rakenteeltaan erilaisia yhdisteitä jotka sitoutuvat solunsisäisiin estrogeenireseptoreihin toimien joko estrogeenin kaltaisina yhdisteinä tai estrogeenin vastavaikuttajina. Tamoksifeeni on SERM –yhdiste, jota on jo pitkään käytetty estrogeenireseptoreita (ER) ilmentävän rintasyövän lääkehoidossa. Tamoksifeeni sekä estää rintasyöpäsolujen jakaantumista että toisaalta aikaansaa niiden apoptoosin eli ohjelmoidun solukuoleman muuntelemalla ER-välitteisesti kohdesolun geenien ilmentymistä. Viimeaikaiset tutkimustulokset ovat kuitenkin osoittaneet tamoksifeenilla olevan myös nopeampia, nongenomisia vaikutusmekanismeja. Tässä väitöskirjatyössä tutkimme niitä nopeita vaikutusmekanismeja joiden avulla tamoksifeeni vaikuttaa rintasyöpäsolujen elinkykyyn. Osoitamme että tamoksifeeni farmakologisina pitoisuuksina aikaansaa nopean mitokondriaalisen solukuolemaan johtavan signallointireitin aktivoitumisen rintasyöpäsoluissa. Tämän lisäksi tutkimme myös tamoksifeenin aiheuttamaan mitokondriovaurioon johtavia tekijöitä. Tutkimustuloksemme osoittavat että ER-positiivisissa rintasyöpäsoluissa tamoksifeeni indusoi pitkäkestoisen ERK-kinaasiaktivaation, joka voidaan estää 17-beta-estradiolilla. Tamoksifeenin aikaansaama nopea solukuolema on pääosin ER:sta riippumaton tapahtuma, mutta siihen voidaan vaikuttaa myös ER-välitteisin mekanismein. Sen sijaan epidermaalisen kasvutekijäreseptorin (EGFR) voitiin osoittaa osallistuvan tamoksifeenin nopeiden vaikutusten välittämiseen. Tämän lisäksi vertailimme myös estradiolin ja eri SERM-yhdisteiden kykyä suojata apoptoosilta käyttämällä osteoblastiperäisiä soluja. Pytyäksemme vertailemaan ER-isotyyppien roolia eri yhdisteiden suojavaikutuksissa, transfektoimme U2OS osteosarkoomasolulinjan ilmentämään pysyvästi joko ERalfaa tai ERbetaa. Tulostemme mukaan sekä estradioli että uusi SERM-yhdiste ospemifeeni suojaavat osteoblastin kaltaisia soluja etoposidi-indusoidulta apoptoosilta. Sekä ERalfa että ERbeta pystyivät välittämään suojavaikutusta, joskin vaikutukset erosivat toisistaan. Lisäksi havaitsimme edellä mainitun suojavaikutuksen olevan yhteydessä muutoksiin solujen sytokiiniekspressiossa. Tietoa SERM-yhdisteiden anti-ja proapoptoottisten vaikutusmekanismeista eri kohdekudoksissa voidaan mahdollisesti hyödyntää kehiteltäessä uusia kudosspesifisiä SERM-yhdisteitä.

The Effects of Estradiol, Androgens and the Selective Estrogen Modulators: Ospemifene, Raloxifene and Tamoxifen on Explant Cultures of Human Breast Tissue

The impact of menopausal hormone therapy (MHT) on increasing the risk for breast cancer (BC) remains controversial. To understand MHT-elicited cellular breast effects and the potential risks, included with using this therapy, a further investigation into this controversy is the subject of this thesis. In this thesis, to study the effects of estrogen, progestin, androgens and selective estrogen receptor modulators (SERMs), a modified tissue explant culture system was used. The different types of human breast tissues (HBTs) used in this study were normal HBTs, obtained from reduction mammoplasties of premenopausal women (prem-HBTs) or postmenopausal (postm-HBTs) women and peritumoral HBTs (peritum-HBTs) which were obtained from surgeries on postmenopausal BC patients. The explants were cultured up to three weeks in the presence or absence of estradiol (E2), medroxyprogesterone acetate (MPA), testosterone (T), dihydrotestosterone (DHT) and SERMs - ospemifene (OSP), raloxifene (RAL) and tamoxifen (TAM). The cultured HBTs maintained morphological integrity and responded to hormonal treatment in vitro. E2, MPA or E2/MPA increased proliferative activity and was associated with increased cyclin-D1 and caused changes in the cell cycle inhibitors p21 and p27, whereas the androgens T and DHT inhibited proliferation and increased apoptosis in HBT epithelia and opposed E2-stimulated proliferation and cell survival. The postm-HBTs were more sensitive to E2 than prem-HBTs. The effects of OSP, RAL and TAM on HBT epithelium were antiproliferative. E2, androgens and SERMs were associated with marked changes in the proportions of epithelial cells expressing steroid hormone receptors: E2 increased ERα expressing cells and decreased androgen receptor (AR) positive cells, whereas T and DHT had opposite effects. The OSP, RAL and TAM, also decreased a proportion of ERα positive cells in HBT epithelium. At 100 nM, these compounds maintained the relative number of AR positive cells, present at control level, which may partly explain proliferative inhibition. In conclusion, the proliferative activity of E2, in the epithelium of postm-HBTs, is opposed by T and DHT, which suggests that the inclusion of androgens in MHT may decrease the risk for developing BC.

Selective inhibition of human tankyrases

Tankyrases belong to the Diphtheria toxin-like ADP-ribosyltransferase (ARTD) enzyme superfamily, also known as poly(ADP-ribose) polymerases (PARPs). They catalyze a covalent post-translational modification reaction where they transfer ADP-ribose units from NAD+ to target proteins. Tankyrases are involved in many cellular processes and their roles in telomere homeostasis, Wnt signaling and in several diseases including cancers have made them interesting drug targets. In this thesis project, selective inhibition of human tankyrases was studied. A homogeneous fluorescence-based assay was developed to screen the compound libraries. The assay is inexpensive, operationally easy, and performs well according to the statistical analysis. Assay suitability was confirmed by screening a natural product library. Flavone was identified as the most potent inhibitor in the library and this motivated us to screen a larger flavonoid library. Results showed that flavones were indeed the best inhibitor of tankyrases among flavonoids. To further study the structure-activity relationship, a small library of flavones containing single substitution was screened and potency measurements allowed us to generate structure-activity relationship. Compounds containing substitutions at 4´-position were more potent in comparison to other substitutions, and importantly, hydrophobic groups improved isoenzyme selectivity as well as the potency. A flavone derivative containing a hydrophobic isopropyl group (compound 22), displayed 6 nM potency against TNKS1, excellent isoenzyme selectivity and Wnt signaling inhibition. Protein interactions with compounds were studied by solving complex crystal structures of the compounds with TNKS2 catalytic domain. A novel tankyrase inhibitor (IWR-1) was also crystallized in complex with TNKS2 catalytic domain. The crystal structure of TNKS2 in complex with IWR-1 showed that the compound binds to adenosine site and it was the first known ARTD inhibitor of this kind. To date, there is no structural information available about the substrate binding with any of the ARTD family members; therefore NAD+ was soaked with TNKS2 catalytic domain crystals. However, analysis of crystal structure showed that NAD+ was hydrolyzed to nicotinamide. Also, a co-crystal structure of NAD+ mimic compound, EB-47, was solved which was used to deduce some insights about the substrate interactions with the enzyme. Like EB-47, other ARTD1 inhibitors were also shown to inhibit tankyrases. It indicated that selectivity of the ARTD1 inhibitors should be considered as some of the effects in cells could come from tankyrase inhibition. In conclusion, the study provides novel information on tankyrase inhibition and presents new insight into the selectivity and potency of compounds.