Progestins inhibit calcitriol-induced CYP24A1 and synergistically inhibit ovarian cancer cell viability: An opportunity for chemoprevention
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
References (42)
- et al.
Role of ultraviolet B irradiance and vitamin D in prevention of ovarian cancer
Am. J. Prev. Med.
(2006) - et al.
Induction of ovarian cancer cell apoptosis by 1,25-dihydroxyvitamin D3 through the down-regulation of telomerase
J. Biol. Chem.
(2004) - et al.
A local effect of CYP24 inhibition on lung tumor xenograft exposure to 1,25-dihydroxyvitamin D(3) is revealed using a novel LC-MS/MS assay
Steroids
(2012) - et al.
Bcl-2, BAX, and apoptosis in endometrial hyperplasia after high dose gestagen therapy: a comparison of responses in patients treated with intrauterine levonorgestrel and systemic medroxyprogesterone
Gynecol. Oncol.
(2005) - et al.
Phytoestrogens regulate vitamin D metabolism in the mouse colon: relevance for colon tumor prevention and therapy
J. Nutr.
(2002) - et al.
Phytoestrogens and vitamin D metabolism: a new concept for the prevention and therapy of colorectal, prostate, and mammary carcinomas
J. Nutr.
(2004) - et al.
Isoflavonoids inhibit catabolism of vitamin D in prostate cancer cells
J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
(2002) - et al.
Genistein potentiates the growth inhibitory effects of 1,25-dihydroxyvitamin D3 in DU145 human prostate cancer cells: role of the direct inhibition of CYP24 enzyme activity
Mol. Cell. Endocrinol.
(2005) - et al.
Cancer CGoESoO. Ovarian cancer and oral contraceptives: collaborative reanalysis of data from 45 epidemiological studies including 23,257 women with ovarian cancer and 87,303 controls
Lancet
(2008) - et al.
Progestin-induced apoptosis in the macaque ovarian epithelium: differential regulation of transforming growth factor-beta
J. Natl. Cancer Inst.
(2002)
Progestin treatment induces apoptosis and modulates transforming growth factor-beta in the uterine endometrium
Cancer Epidemiol. Biomark. Prev.
Impact of progestin and estrogen potency in oral contraceptives on ovarian cancer risk
J. Natl. Cancer Inst.
Vitamin D: biology, action and clinical applications
Vitamin D deficiency
N. Engl. J. Med.
Sunlight, vitamin D, and ovarian cancer mortality rates in US women
Int. J. Epidemiol.
The regulation of apoptosis by activin and transforming growth factor-beta in early neoplastic and tumorigenic ovarian surface epithelium
J. Clin. Endocrinol. Metab.
Nutritional determinants of epithelial ovarian cancer risk: a case-control study in Mexico
Oncology
Progesterone enhances calcitriol antitumor activity by upregulating vitamin D receptor expression and promoting apoptosis in endometrial cancer cells
Cancer Prev. Res. (Phila)
Sensitive induction of apoptosis in breast cancer cells by a novel 1,25-dihydroxyvitamin D3 analogue shows relation to promoter selectivity
J. Cell. Biochem.
Indian hedgehog is a major mediator of progesterone signaling in the mouse uterus
Nat. Genet.
Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function
Mol. Endocrinol.
Cited by (28)
Sex steroid hormones and DNA repair regulation: Implications on cancer treatment responses
2023, Journal of Steroid Biochemistry and Molecular BiologyCitation Excerpt :In the endometrium progesterone has an antagonistic effect on estrogen-driven proliferation, so that sufficient progesterone action is required to lower endometrial cancer risk [74] and ovarian cancer risk [48]. Progestins (synthetic progesterone) and progesterone have been reported to inhibit cell growth whilst increasing cell differentiation and apoptosis in endometrial cancer [30] and in ovarian cancer cells [121]. Although progesterone is predominantly linked with anti-cancer effects (decreased cancer risk) as evidenced in the use of oral contraceptives and pregnancy [148,164], a recent finding has revealed progesterone can increase the risk of metastatic ovarian cancer.
Calcitriol and cancer therapy: A missed opportunity
2018, Bone ReportsCitation Excerpt :Synergistic antitumor effects of calcitriol and glucocorticoids have been demonstrated in human prostate cancer xenografts, and the dexamethasone does reduce calcitriol-induced hypercalcemia in patients (Mccarthy et al., 1983; Trump et al., 2004). Non-specific (e.g. ketoconazole and liarazole, isoflavones such as genistein, progesterone) and specific inhibitors of CYP24A1 each accentuate the antitumor activity of calcitriol (Trump et al., 2004; Zhang et al., 2012; Peehl et al., 2002; Swami et al., 2005; Muindi et al., 2010; Chiellini et al., 2012; Lechner et al., 2007; Yee & Simons, 2004; Schuster et al., 2003; Ly et al., 1999; Zhao et al., 1996; Rao et al., 2002; Rodriguez et al., 2016; Lee et al., 2013; Lou & Tuohimaa, 2006; Yee et al., 2006). Prostaglandin synthesis inhibitors may potentiate the activity of vitamin D compounds.
Age-associated expression of vitamin D receptor and vitamin D-metabolizing enzymes in the male reproductive tract and sperm of Hu sheep
2018, Animal Reproduction ScienceCitation Excerpt :Vit D3 binds and activates VDR in target cell, until 24-hydroxylase (CYP24A1) inactivates it (Krause et al., 2017). The expression of VDR and Vitamin D-metabolizing enzymes (CYP2R1, CYP27A1, CYP27B1, and CYP24A1) has been widely demonstrated in numerous reproductive tissues, such as testes, epididymis, ovaries, and uterus, as well as in spermatozoa (Jensen et al., 2010; Mahmoudi et al., 2013; Bergada et al., 2014; Jensen, 2014; Brozyna et al., 2015; Rodriguez et al., 2016), which suggests that target-cell responsiveness is affected not only by circulating 25(OH)D3 levels, but also the cellular expression of Vitamin D-metabolizing enzymes. Particularly, CYP27B1 and CYP2R1 are more strongly expressed in the testes than in other organs.
Reevaluating the Role of Progesterone in Ovarian Cancer: Is Progesterone Always Protective?
2023, Endocrine ReviewsVitamin D metabolism in cancer: potential feasibility of vitamin D metabolism blocking therapy
2023, Medical Molecular Morphology