Elsevier

Gynecologic Oncology

Volume 143, Issue 1, October 2016, Pages 159-167
Gynecologic Oncology

Progestins inhibit calcitriol-induced CYP24A1 and synergistically inhibit ovarian cancer cell viability: An opportunity for chemoprevention

https://doi.org/10.1016/j.ygyno.2016.04.022Get rights and content

Highlights

  • Progestins and vitamin D synergistically reduce viability in ovarian cancer cells.

  • Progestins inhibit CYP24A1, extending vitamin D activity in ovarian cancer cells.

  • Combination progestin and vitamin D may inhibit ovarian carcinogenesis.

Abstract

Objectives

Previously we have shown in endometrial cells that progesterone (P4) and calcitriol (CAL, 1,25(OH)2D3) synergistically promote apoptosis and that progestins induce expression of the vitamin D receptor. In the current study we examined the progestin/vitamin D combination in ovarian cells and searched for other progestin-related effects on vitamin D metabolism that may underlie the novel interaction between progestins and vitamin D, including whether progestins inhibit CYP24A1, the enzyme that renders CAL inactive.

Methods

We investigated the impact of P4 on CAL-induced CYP24A1 expression in cancer cell lines expressing progesterone receptors (PRs), [OVCAR-5, OVCAR-3-PGR (PR-transfected OVCAR-3 ovarian line), and T47D-WT, T47D-A and T47D-B (breast lines expressing PRs or individual PR isoforms)] or lines that do not express PRs (OVCAR-3 and T47D-Y). We examined CYP24A1 expression using RT-PCR and western blotting, and apoptosis by TUNEL. We also investigated P4 inhibition of Cyp24a1 in ovaries from CAL-treated mice.

Results

CAL treatment induced CYP24A1 expression. When co-treated with P4, cell lines expressing PRs showed marked inhibition of CYP24A1 expression (p < 0.001), along with increased apoptosis (p < 0.01); cells not expressing PRs did not. Mouse ovaries showed a significant reduction in CAL-induced Cyp24a1 mRNA (p < 0.001) and protein (p < 0.01) in response to P4.

Conclusions

We show for the first time that progestins and vitamin D synergistically reduce cell viability and induce apoptosis in ovarian cells and that progestins PR-dependently inhibit CAL-induced CYP24A1, thus extending CAL activity. The combination of progestins and vitamin D deserves further consideration as a strategy for inhibiting ovarian carcinogenesis.

Introduction

There is immense potential to decrease ovarian cancer incidence and mortality through prevention. Extensive epidemiological evidence has shown that routine use of the combination estrogen-progestin oral contraceptive pill (OC) confers a remarkable 30–50% reduced risk of ovarian cancer [1]. Based on our research findings, we believe the progestin component of the OC is functioning as a chemopreventive agent by activating potent and well-known molecular pathways such as apoptosis and transforming growth factor-beta (TGF-β) signaling in the genital tract [2], [3]. Our animal research findings are supported by human data demonstrating that progestin-potent OCs confer twice the protective effect against ovarian cancer as OCs containing weak progestins [4]. These human data suggest that enhancing progestin potency will confer enhanced ovarian cancer preventive efficacy.

Progestin potency can be enhanced by either increasing the dosage of progestin or by selecting a pharmacologically potent progestin. Both of these approaches, however, may increase side effects that would be undesirable for long-term chemoprevention. An alternative strategy would combine a progestin with a second preventive agent that is both non-toxic and enhances progestin potency. In this regard, there is epidemiological and laboratory evidence in support of vitamin D, which is non-toxic, for the prevention of malignancy including ovarian cancer, making vitamin D an attractive second agent.

The beneficial effects of vitamin D are due to the activity of its dihydroxylated metabolite, 1,25(OH)2D3 (“calcitriol”, CAL), the active form of the molecule. The human body obtains vitamin D (specifically vitamin D3 or “cholecalciferol”, chole) through synthesis by skin exposed to sunlight or orally through the diet. Of these two sources, however, very little is supplied by the diet as few foods contain appreciable amounts of vitamin D. Through a series of enzymatic reactions, vitamin D3 is converted to 1,25(OH)2D3, which binds to the vitamin D receptor (VDR). Vitamin D-responsive genes then confer a number of chemopreventive effects, including cell cycle arrest, apoptosis and differentiation in a variety of cells, including prostate, breast, colon, and ovarian [5], [6]. Notably, the VDR is expressed ubiquitously throughout most epithelia. Additionally, via expression of the 1-alpha hydroxylase enzyme, many normal tissues convert circulating 25(OH)D3 to 1,25(OH)2D3, which acts in an autocrine or paracrine fashion to regulate cell growth and biology. Once 1,25(OH)2D3 confers its local effect, it induces production of vitamin d-24-hydroyxlase (CYP24A1), which catalyzes its conversion to inactive metabolites.

Worldwide, the geographic distribution of ovarian cancer shows increasing incidence commensurate with distance from the equator [7]. Similarly, in the U.S. a North-South gradient favors a higher ovarian cancer risk in Northern versus Southern latitudes, demonstrating a statistically significant inverse correlation between regional sunlight exposure and ovarian cancer mortality [8]. Given that sunlight induces production of pre-vitamin D3 in the skin, it is interesting to speculate that vitamin D might confer protection against ovarian cancer via direct chemopreventive biologic effects in the non-malignant ovarian epithelium, similar to that induced by progestins. For example through induction of apoptosis and/or TGF-β in the ovarian epithelium, vitamin D may cause the selective removal of non-malignant, but genetically damaged ovarian epithelial cells [9], [10]. A small case-control study supports the notion that vitamin D confers ovarian cancer prevention, at dosages easy to achieve through the diet. Compared to a low dietary intake of vitamin D, a high dietary intake of vitamin D was associated with a 50% reduction in ovarian cancer risk [11].

Recently, we reported that progesterone (P4) and CAL have synergistic inhibitory effects on cell viability in cells derived from the endometrium, characterized by a marked increase in apoptosis [12]. We also demonstrated that progestin increases expression of the vitamin D receptor, thereby providing a potential mechanism underlying the novel interaction between the two agents. In this study, we sought to characterize the effect of progestins on vitamin D metabolism in cells derived from the ovarian epithelium, and we report that progestins inhibit CYP24A1, the enzyme that catalytically degrades vitamin D. We also report that vitamin D and progestins synergistically reduce ovarian cancer cell viability.

Section snippets

Cell lines and treatment

All culture media, sera and reagents were purchased from Invitrogen except insulin, RU486 (mifepristone) and CAL, which were purchased from Sigma. CB1089, a stable analog of CAL with a functional Kd of 3.4 nM (compared with 0.9 nM for CAL) [13], was kindly provided by Cougar Biotechnology, Inc. (now a division of Johnson and Johnson). The parental ovarian cancer cell line OVCAR-3 (purchased from ATCC) was grown in RPMI 1640 medium with glutamine supplemented with 10% heat-inactivated FBS, 10 μg/ml

Impact of vitamin D and progesterone on the viability of parental OVCAR-3 and PR-transfected (OVCAR-3-PGR) cell lines

OVCAR-3 parent cells do not express classical nuclear PRs (Fig. 1A), although they do express membrane progesterone receptors. Treatment with P4 alone only modestly reduced viability of the parental OVCAR-3 cells in a 3-day MTS assay, and only at the highest dose (Fig. 1B). In combination with CAL, viability was reduced dose-dependently at all doses of P4, demonstrating an inhibitory effect of P4 and vitamin D on cell viability of the parental OVCAR-3 cells, but viability remained at 60% or

Discussion

A growing body of human and animal evidence supports a robust cancer preventive role for progestins in the upper gynecologic tract, including a) evidence of a marked reduction in ovarian, fallopian tube and endometrial cancers associated with routine use of progestin-containing contraceptives [1], [17], b) evidence of progestin activation of surrogate endpoint biomarkers relevant to chemoprevention in the ovary and endometrium [2], [3], [18], c) evidence that progestins effectively reverse

Conflict of interest statement

The authors declare no conflicts of interest.

Acknowledgements

The authors are grateful to Dr. Weidong Xu for his expertise with the transfection of OVCAR-3 cells with PR to produce the OVCAR-3-PGR line; and to Ms. Dan Li for her excellent technical assistance with some of the molecular biological procedures reported herein.

This work was supported by Grant #DAMD17-00-1-0570 from the Department of Defense, and also by an award from the United States Army Medical Research and Material Command (PI: Chad A. Hamilton), W81XWH-11-2-0131. Additional support also

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