Studies suggest that a combination of progesterone and calcitriol might help in preventing or inhibiting the development of endometrial cancer. For the first time, researchers show that progesterone upregulates vitamin D receptor expression in endometrial cancer.1
Advanced-stage or high histological grade endometrial cancer indicates poor prognosis and results in increased death rates.2,3 Progestins have been shown to decrease the risk of endometrial cancer in both premenopausal and menopausal women, at increased or potent doses.4 However, the high-dose progestin is associated with side effects. Researchers have, therefore, been seeking an agent with an acceptable safety profile to enhance the potency of the progestin, even at low doses.
In the report published in Cancer Prevention Research, Viqar Syed, PhD, Assistant Professor at the Uniformed Services University of the Health Sciences Department of Obstetrics and Gynecology (Bethesda, MD) and colleagues investigated the mechanisms underlying the effects of progesterone, calcitriol and their combination on immortalized human endometrial epithelial cells and endometrial cancer cells.1
Calcitriol, or 1,25(OH)(2)D(3), is the dihydroxylated form of Vitamin D3, which is produced in the epidermis or obtained from the diet. Evidence suggests that calcitriol prevents cancers of prostate, breast, colon, skin and leukemic cells by inducing cell cycle arrest, programmed cell death and differentiation.5 ,6 It enhances paclitaxel anti-tumour activity in vitro and in vivo and accelerates paclitaxel-induced apoptosis.7 Calcitriol has dose-limiting side effects, however, including harmful hypercalcemia. Evidence shows that combining calcitriol with other chemotherapeutic agents results in tumour growth inhibition, even at low dosages.8
Researchers therefore sought to investigate the underlying mechanisms of progesterone’s synergism with calcitriol, in the hope of finding better chemopreventive interventions without the related adverse effects. The immortalized epithelial endometrial and endometrial cancer cell lines were treated with progesterone, calcitriol or both. They were then subjected to cell viability assays and cell cycle analysis. The cell lysates were resolved electrophoretically. After tryptic digestion, the samples were subjected to recursive nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses (LTQ-Orbitrap Velos, Thermo Scientific). The goal was to identify differentially expressed proteins in endometrial cancer cells exposed to progesterone, calcitriol or both.
Progesterone-treated cancer cell lines showed enhanced expression of vitamin D receptors. The results showed that the combination of progesterone and calcitriol resulted in a definitive dose-dependent decrease in cell numbers through caspase-3 activation (as compared to either agent used alone), in all the cell lines tested. Caspase-3 is a marker for apoptotic death. The results are consistent with previous preclinical studies showing that calcitriol has additive effects when used in combination with chemotherapeutic agents.8 The combination of progesterone and calcitriol induced G0/G1 cell cycle arrest and modulated cell cycle regulators in endometrial cancer cells. Treatment with progesterone, calcitriol or the combination led to a significant decrease in cyclins D1 and D3 in endometrial cancer cell lines with a simultaneous elevation of p27 protein expression, as compared with untreated cells.
Using MS-based proteomics, the researchers identified additional mediators of progesterone and calcitriol signalling in endometrial cancer cells. Treatment with progesterone, calcitriol or the combination over a 24-hour period resulted in alterations in the levels of 609, 387 and 519 proteins, respectively. After the three treatments, the investigators found only 117 proteins in common that showed differential expression relative to the untreated cells. The differentially altered proteins include oncogene products, tumour suppressors, binding proteins, membrane proteins, structural proteins, transport proteins, ribosomal proteins, receptors and transcription factors, and proteins involved in apoptosis and cell cycle regulation. Of these, the expression of the apoptosis-related proteins histone H1.4 (HIST1H1E), histidine triad nucleotide-binding protein 2 (HINT2), interferon-induced, double-stranded RNA-activated protein kinase (EIF2AK2), and Bcl-2-associated X protein (BAX) was decreased in the three endometrial cancer cell lines, as compared with the immortalized endometrial epithelial cell line. Exposure to progesterone, calcitriol or the combination significantly elevated the expression of these proteins.
The researchers claim that this is the first report showing that HIST1H1E, HINT2, EIF2AK2 and BAX act as tumour suppressors of endometrial cancer. Additional BAX analysis using gain-or-loss-of-function experiments demonstrated that boosting BAX expression decreased cell proliferation by changing the BAX:BCL-2 ratio. Indeed, the investigators used BAX knockdown cells to confirm the finding that the BAX protein mediated the combined progesterone and calcitriol-induced inhibition of endometrial cancer cell growth. BAX induces cytochrome c release and caspase activation during apoptosis.
“Our results showed that progesterone and calcitriol upregulate the expression of BAX along with other apoptosis-related proteins, which induce inhibition of endometrial cancer cell growth by apoptosis and cell cycle arrest,” the investigators conclude. “The identification of these new progesterone and calcitriol molecular targets…offers unique opportunities for future development of novel strategies for endometrial cancer therapies.”
- Lee, L.R., et al. (2013) “Progesterone Enhances Calcitriol Antitumor Activity by Upregulating Vitamin D Receptor Expression and Promoting Apoptosis in Endometrial Cancer Cells,” Cancer Prevention Research (Philadelphia), doi: 10.1158/1940-6207.CAPR-12-0493.
- Di Cristofano, A., and Ellenson, L.H. (2007) “Endometrial carcinoma,” Annual Review of Pathology, 2 (pp. 57–85), doi: 10.1146/annurev.pathol.2.010506.091905.
- Dizon, D.S. (2010) “Treatment options for advanced endometrial carcinoma,” Gynecologic Oncology, 117 (pp. 373–81), doi: http://dx.doi.org/10.1016/j.ygyno.2010.02.007.
- Ehrlich, C.E., et al. (1988) “Steroid receptors and clinical outcome in patients with adenocarcinoma of the endometrium,” American Journal of Obstetrics and Gynecology, 158 (pp. 796–807).
- Getzenberg, R.H., et al. (1997) “Vitamin D inhibition of prostate adenocarcinoma growth and metastasis in the Dunning rat prostate model system,” Urology, 50 (pp. 999–1006), doi:10.1016/S0090-4295(97)00408-1.
- Hershberger, P.A., et al. (2001) “Calcitriol (1,25-dihydroxycholecalciferol) enhances paclitaxel antitumor activity in vitro and in vivo and accelerates paclitaxel-induced apoptosis,” Clinical Cancer Research, 7(4) (pp. 1043–51).
- Wigington, D.P., et al. (2004) “Combination study of 1,24(S)-dihydroxyvitamin D2 and chemotherapeutic agents on human breast and prostate cancer cell lines,” Anticancer Research, 24(5A) (pp. 2905-12).
- Ma, Y., et al. (2008) “1alpha,25-Dihydroxyvitamin D3 potentiates cisplatin antitumor activity by p73 induction in a squamous cell carcinoma model,” Molecular Cancer Therapeutics, 7 (pp. 3047-55), doi: 10.1158/1535-7163.MCT-08-0243.