Abstract
Human studies suggest that progesterone and calcitriol may prove beneficial in preventing or inhibiting oncogenesis, but the underlying mechanism is not fully understood. The current study investigates the effects of progesterone, calcitriol, and their combination on immortalized human endometrial epithelial cells and endometrial cancer cells and identifies their targets of action. Combination treatment with both agents enhanced vitamin D receptor expression and inhibited cell proliferation through caspase-3 activation and induction of G0-G1 cell-cycle arrest with associated downregulation of cyclins D1 and D3 and p27 induction. We used mass spectrometry-based proteomics to measure protein abundance differences between calcitriol-, progesterone-, or combination-exposed endometrial cells. A total of 117 proteins showed differential expression among these three treatments. Four proteins were then selected for validation studies: histone H1.4 (HIST1H1E), histidine triad nucleotide-binding protein 2 (HINT2), IFN-induced, double-stranded RNA-activated protein kinase (EIF2AK2), and Bcl-2-associated X protein (BAX). Abundance levels of selected candidates were low in endometrial cancer cell lines versus the immortalized endometrial epithelial cell line. All four proteins displayed elevated expression in cancer cells upon exposure to calcitriol, progesterone, or the combination. Further BAX analysis through gain- or loss-of-function experiments revealed that upregulation of BAX decreased cell proliferation by changing the BAX:BCL-2 ratio. Knockdown of BAX attenuated progesterone- and calcitriol-induced cell growth inhibition. 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.
Highlights
Endometrial cancer is the most common gynecologic malignancy in the United States
Progesterone receptors were not affected by progesterone or calcitriol treatment The protein expression levels of progesterone receptor (PR)-A and PR-B were studied in EM-E6/E7-TERT and 3 endometrial cancer (Ishikawa, HEC-1B, and RL95-2) cell lines by quantification of Western blot analyses (Fig. 1), using a mouse anti-human PR antibody that recognizes both human PR isoforms: PR-A (85–94 kDa) and PR-B (116–120 kDa)
We studied the effects of progesterone, calcitriol, or both on PR protein expression in the same normal and endometrial cancer cell lines
Summary
Endometrial cancer is the most common gynecologic malignancy in the United States. In 2012, 47,130 new cases are anticipated, resulting in 8,010 deaths [1]. Endometrial cancer is usually treated with surgical removal of the uterus. Adjuvant therapy is administered in selected cases based on surgico-pathologic factors predictive of recurrence risk. For early-stage disease, outcomes are quite favorable with 5year survival rates more than 90% [2]. The number of women presenting with endometrial cancer in an advanced-stage or a high histologic grade, which is indicative of a poor prognosis, is increasing and mortality rates are rising [3, 4]. Discovery of novel molecular targets for the diagnosis, prognosis, and treatment of endometrial cancer is imperative to improve the management and outcome of this disease
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