Functional characterisation of gonadotropin-releasing hormone-estrogen conjugates

Abstract

Prostate cancer (PC) is the second most commonly occurring cancer in men, and the fourth most common commonly occurring cancer overall. Almost all PC begins in an androgen-dependent state with androgen deprivation therapy (ADT) an effective treatment at this stage. PC can overtime develop into an androgen independent state at which point it can no longer be treated with ADT. The focus of this research is on androgen dependent PC and ADT. The hypothalamic–pituitary–gonadal (HPG) axis, controlled by gonadotropin releasing hormone (GnRH) is responsible for regulating reproduction, puberty and the production of spermatozoa and androgens in men. GnRH analogues (which downregulate the axis) are the foremost ADT agents. GnRH analogues may also have direct anti-proliferative effects in some cancers. However, ADT has negative side effects including loss of bone mass, hot flushes and loss of libido, due to a concomitant decrease in estrogen which is synthesised from androgen. We hypothesise that a molecule which retains GnRH receptor (GnRHR) activation while replacing estrogen activity which activates the estrogen receptors (ERs) may be beneficial. Conjugates of GnRH analogue (GnRHag) with 17β-Estradiol (E2C) or genistein (GenC) were studied examining GnRH and estrogen activity in vitro to evaluate their potential as novel PC therapeutics.

Synthesis of the conjugates was commissioned from a commercial company. GnRHR activation was tested in HEK 293T cells by determining the generation of inositol phosphate in cells expressing GnRHR. ER activation was determined in MCF-7 cells using an E-screen assay in a cell line expressing ERs. Anti-proliferative effects were assessed in PC cell lines by crystal violet assay. Potential bone-protective capability was measured by ability to inhibit RANKL-induced osteoclast differentiation of Raw 264.7 macrophages.

E2C and GenC elicited potent stimulation of GnRHR. The conjugates also had estrogenic activity similar to the unconjugated estrogen and phytoestrogen in the E-screen assay. Their estrogenic activities were confirmed by their ability to inhibit osteoclast differentiation to the same degree as unconjugated 17β-Estradiol and genistein. No direct antiproliferative effects, by the conjugates or GnRH, on PC cells were observed, indicating that GnRHR may not be expressed in these cell lines. The demonstration that E2C and GenC displayed GnRHR and ER activities similar to their unconjugated counterparts suggests they may be efficacious as ADT agents with reduced side effects of estrogen deprivation.

Key Words: GnRH analogues, Prostate Cancer, Androgen Deprivation Therapy, Estrogen Deficiency, RANKL, Hot Flushes, Libido, Bone Loss