Progesterone Enhances Calcitriol Antitumor Activity by Upregulating Vitamin D Receptor Expression and Promoting Apoptosis in Endometrial Cancer Cells

Published OnlineFirst May 16, 2013; DOI: 10.1158/1940-6207.CAPR-12-0493

Cancer Prevention Research Article Research

Laura R. Lee1, Pang-Ning Teng4, Huyen Nguyen1, Brian L. Hood4, Leyla Kavandi1, Guisong Wang4, Jane M. Turbov6, Larry G. Thaete6, Chad A. Hamilton3, George L. Maxwell5, Gustavo C. Rodriguez6, Thomas P. Conrads4, and Viqar Syed1,2

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). Abun- dance 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. Cancer Prev Res; 6(7); 731–43. 2013 AACR.

Introduction and adjuvant therapy is administered in selected cases based Endometrial cancer is the most common gynecologic on surgico-pathologic factors predictive of recurrence risk. malignancy in the United States. In 2012, 47,130 new cases For early-stage disease, outcomes are quite favorable with 5- are anticipated, resulting in 8,010 deaths (1). Endometrial year survival rates more than 90% (2). However, the num- cancer is usually treated with surgical removal of the uterus ber of women presenting with endometrial cancer in an advanced-stage or a high histologic grade, which is indic- ative of a poor prognosis, is increasing and mortality rates are rising (3, 4). Discovery of novel molecular targets for the Authors' Afﬁliations: Departments of 1Obstetrics and Gynecology and 2Molecular and Cell Biology, Uniformed Services University of the Health diagnosis, prognosis, and treatment of endometrial cancer Sciences; 3Division of Gynecologic Oncology, Walter Reed National Mil- is imperative to improve the management and outcome of 4 itary Medical Center, Bethesda, Maryland; Women's Health Integrated this disease. Research Center at Inova Health System, Department of Defense Gyne- cologic Cancer Center of Excellence, Annandale; 5Department of Obstet- A strong body of clinical and epidemiologic evidence rics and Gynecology, Inova Fairfax Hospital, Falls Church, Virginia; and suggests that progestins are highly effective endometrial 6Division of Gynecologic Oncology, North Shore University Health System, University of Chicago, Evanston, Illinois cancer preventive agents. Routine use of progestins lowers endometrial cancer risk and the protective effect increases Note: Supplementary data for this article are available at Cancer Prevention Research Online (http://cancerprevres.aacrjournals.org/). with increasing progestin potency. In premenopausal women, use of progestin containing oral contraceptives Corresponding Author: Viqar Syed, Department of Obstetrics and Gyne- cology, Uniformed Services University of the Health Sciences, Room# confers a signiﬁcant reduction in endometrial cancer risk A-3080, 4301 Jones Bridge Road, Bethesda, MD 20814. Phone: 301-295- (5–7). In addition, progestin-potent oral contraceptives 3128; Fax: 301-295-6774; E-mail: [email protected] have enhanced endometrial cancer protective effects doi: 10.1158/1940-6207.CAPR-12-0493 compared with oral contraceptives containing weak pro- 2013 American Association for Cancer Research. gestins (8, 9). In menopausal women, the addition of a

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progestin to estrogen replacement therapy decreases the rationale for chemoprevention or treatment trials using this risk of precancerous endometrial hyperplasias, suggesting combination. chemopreventive effects of progestins on the endometrium (10, 11). Currently, progestins are often used as Materials and Methods salvage therapy in patients with recurrent endometrial Cell lines and culture conditions cancer and in young patients to preserve childbearing The immortalized human endometrial epithelial cell capacity (12). Progesterone mediates its inhibitory effects line, EM-E6/E7-TERT, established and characterized by on the endometrium and endometrial cancer via the Dr. Satoru Kyo, (Kanazawa University, School of Medi- progesterone receptor (PR), an intracellular steroid cine, Kanazawa, Japan) was kindly provided to us in receptor with A and B isoforms. An increase in the 2008. This line was not authenticated by us once received response rates to progestin therapy and improved survival in our laboratory. Human endometrial cancer cell lines outcomes have been reported in tumors with a higher HEC-1B and RL-95 were obtained from the American percentage of PR (12, 13). Progestin potency can be Type Culture Collection (ATCC) and Ishikawa cell line enhanced by either increasing the dosage of progestin or was obtained from Sigma. These human-derived cell lines by selecting a pharmacologically potent progestin. How- were authenticated by DNA short-tandem repeat analysis ever, both of these approaches are likely to be associated by ATCC and Sigma. All 4 cell lines were initially expand- with an increase in side effects that would be undesirable ed and cryopreserved within 1 month of receipt. Cells for long-term chemoprevention in women. An alternative were typically used for 3 months, at which time a fresh strategy would be to combine a shorter course or lower vial of cryopreserved cells was used. The cells were rou- dose of progestin with a second preventive agent that has tinely tested for Mycoplasma. an excellent safety profile and enhances the potency of the Immortalized epithelial endometrial (EM-E6/E7-TERT) progestin. cells were established and characterized by Kyo and collea- There is a growing body of epidemiologic and laboratory gues (23). Primary endometrial epithelial cells were trans- evidence in support of vitamin D for the prevention of formed by the combination of human papillomavirus-16 a number of malignancies including endometrial cancer E6/E7 expression and telomerase activation by the intro- (14, 15). Vitamin D3, produced in the epidermis or duction of human telomerase reverse transcriptase obtained from the diet, is metabolized to the dihydroxy- (hTERT). The immortalized cells contained no chromo- lated form [calcitriol, 1,25-(OH)2D3] to be biologically somal abnormalities, retained responsiveness to sex-steroid active. Calcitriol is well known for its antiproliferative roles hormones, exhibited glandular structure on 3-dimensional through multiple mechanisms including the induction of culture, and lacked transformed phenotypes on soft agar or cell-cycle arrest, apoptosis, and differentiation in a variety of in nude mice. The cells were grown in Dulbecco’s modified cancer types, including prostate, breast cancer, colon, skin, Eagle medium (DMEM). Ishikawa cells were derived from a and leukemic cells (16–18). The inhibitory role of calcitriol well-differentiated adenocarcinoma of the human endome- in cellular growth and proliferation potentially provides trial epithelium from a 39-year-old woman, and express protection from various types of cancers, as indicated by functional steroid receptors for estrogen, progesterone, and several epidemiologic studies (19, 20). However, as calci- androgen. Cells were grown in DMEM:F12 supplemented triol has been found to be efficacious in several preclinical with insulin (Invitrogen). HEC-1-B cells derived from a and clinical studies, dose-limiting calcemic effects have moderately differentiated adenocarcinoma of endometrial proved a major obstacle for the use of this compound as epithelium from a 71-year-old patient were cultured in a chemotherapeutic or chemopreventive agent (14, 15). Eagle minimum essential medium (Invitrogen). RL95-2 One approach to limit the toxicity is to use lower concen- was derived from a grade 2 moderately differentiated ade- trations of calcitriol in combination with other agents that nosquamous carcinoma of the endometrium. The cells are enhance its anticancer activity. characteristically epithelioid with well-defined junctional A general approach to cancer chemotherapy entails complexes, tonofilaments, filopodia-like extensions, and simultaneous administration of 2 or more chemotherapeu- surface microvilli. Nuclei are large, irregular, and invagi- tic drugs to patients, in anticipation that the drug combi- nated frequently with multiple, prominent, and lamellar nation will be more effective than any single agent. Several nucleoli. The RL95-2 cells were grown in DMEM:F12 medi- studies have reported that calcitriol works synergistically um supplemented with insulin (0.005 mg/mL) and FBS as with chemotherapeutic drugs (21, 22). Combining proges- described earlier (24, 25). The cells were treated with terone with low doses of calcitriol might be a beneficial progesterone (20 mmol/L P4, 99.9% pure; Sigma), calcitriol chemopreventive or therapeutic strategy in endometrial (100 nmol/L; Sigma), or both for 24, 72, or 120 hours and cancer. Therefore, in the present study, we evaluated the collected for protein extraction. The time of treatment and effects of combinations of calcitriol and progesterone on doses of progesterone and calcitriol were based on our endometrial cancer cells. The data show that combinations previous studies showing inhibition of cell growth and achieve significantly greater inhibition of tumor cell growth apoptosis of cancer cells (24). For a set of experiments, and enhanced anticancer activity than individual agents. endometrial cancer cells were transfected with siRNA-tar- Characterizing the underlying mechanisms of progester- geting Bcl-2–associated X protein (BAX) or scrambled one’s synergism with calcitriol will provide an important siRNA and then treated with progesterone, calcitriol, or

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both for 120 hours. Protein extracts were prepared for BAX 6-well plates and transfected the following day with BAX analysis. siRNA (SC29212; Santa Cruz Biotechnology) or scrambled siRNA (SC37007; Santa Cruz Biotechnology) using the Cell growth and death assays Lipofectamine 2000 reagent (Invitrogen). Transfected cells Endometrial cells were plated at 5,000 cells/cm2 and fed were treated with or without progesterone (20 mmol/L), after 24 hours with fresh medium and treated with proges- calcitriol (100 nmol/L), or both for 5 days. Cell extracts were terone, calcitriol, or both. After 120 hours of these treat- used to assess expression of BAX. The overall transfection ments, total cells were collected by brief trypsinization, and efficiency for endometrial cells assessed by luciferase assay washed with PBS. Total cell number was determined by was 76% to 83%. counting each sample in triplicate using a hemocytometer under an inverted microscope. Cell viability was deter- Cell viability assay mined using the Trypan blue exclusion method. Each Cell viability was evaluated after transfection of endo- treatment had 3 independent plates. The data shown in metrial cancer cells with BAX siRNA or scrambled siRNA by this study are the mean of 3 independent experiments. using the CellTiter 96 AQueous One Solution cell viability assay (Promega) according to the instructions of the man- Cell-cycle analysis ufacturer. CellTiter 96 AQueous One Solution reagent (20 Cells were plated onto tissue culture flasks and the medi- mL) was added into each well of the 96-well assay plate um was replaced the next day with medium containing containing the samples in 100 mL of culture medium. progesterone, calcitriol, combined treatment, or vehicle. Absorbance was measured at 490 nm using a microtiter Cell-cycle status was measured after 24 hours of exposure plate reader. Relative cell viability was expressed as percent- to treatments. Cells (1 106) were collected and centrifuged age change of transfected cells over scrambled siRNA– at 250 g for 5 minutes. Supernatant was removed and cells transfected cells. were fixed by slowly adding 1.0 mL of a chilled ethanol (20C) while vortexing the tube at low speed. Cells were Western blot analysis kept at 20C until DNA staining. On the day of DNA Endometrial cancer cell extracts from progesterone- or staining, samples were centrifuged at 250 g for 5 minutes. calcitriol-treated and nontreated control cells as well as Then supernatant was removed. DNA staining buffer (1 mL extracts from BAX-silenced and control siRNA-transfected containing 100 mg propidium iodide; Sigma) was added to cells treated with or without progesterone or calcitriol were cell pellet and briefly vortexed. Cells were kept in the dark analyzed using antibodies against BAX, caspase-3, cyclin for 15 minutes at room temperature. The distribution of D1, cyclin D3, p27 (Cell Signaling Technology), BCL-2 cells between phases of the cell cycle was determined using a (BioLegend), PR (PgR 1294; Dako Corporation), VDR, BD LSR II Flow Cytometer (Becton Dickinson, LSR 2) and histone H1.4 (HIST1H1E), histidine triad nucleotide-bind- ModFit LT software (Verity Software House). Data are ing protein 2 (HINT2), IFN-induced, double-stranded means SD of 3 independent experiments. RNA-activated protein kinase (EIF2AK2; Santa Cruz Bio- technology), and b-actin antibody (Sigma-Aldrich). Equal Liquid chromatography/tandem mass spectrometry amounts of protein were subjected to SDS-PAGE. The To identify differentially expressed proteins in endome- enhanced chemiluminescence system was used to visualize trial cancer cells exposed to progesterone, calcitriol, or both, the protein bands as recommended by the manufacturer 40 mg of each total cell lysate was resolved by one-dimen- (Pierce). Protein bands were quantified using densitometry sional SDS-PAGE. Twenty equivalently sized gel slices were software (Bio-Rad), and normalized using actin as a loading excised and digested using trypsin. Tryptic digests were control. To calculate the relative intensity of each band, resuspended in mobile phase A and each subjected to 5 individual bands were divided by the corresponding load- recursive nanoflow liquid chromatography/tandem mass ing control intensity. spectrometry (LC/MS-MS) analyses (LTQ-Orbitrap Velos; Thermo Fisher Scientific) as previously described (26). Statistical analysis Tandem mass spectrometry (MS-MS) spectra were searched Data are presented as the mean of triplicate determinants against the UniProt human protein database using Mascot with SEM. Experiments carried out in triplicate were repeat- (Matrix Science) with a decoy database to result in a false ed at least 3 times. Statistically significant differences were peptide discovery rate of less than 1%. Differences in determined between control and treatment groups using protein abundance between the samples were derived by two-way ANOVA followed by Tukey post hoc test. A P value of spectral counting (SC), and peptides whose sequence less than 0.05 was considered statistically significant. The mapped to multiple protein isoforms were grouped as per Pearson correlation coefficient test was used to calculate the the principle of parsimony. correlation between BAX expression and cell growth. West- ern blot analysis data were quantified using densitometry. Silencing of BAX in endometrial cancer cells A representative of 3 immunoblots is presented in the To establish that BAX is a mediator of progesterone and figures along with average relative density of the bands calcitriol-induced growth inhibition, endometrial (Ishi- normalized to b-actin. Statistically significant differences kawa, HEC-1B, and RL95-2) cancer cells were seeded in were determined between control and treatment groups by

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quantiﬁcation of 3 immunoblots and P values are provided in the results section. A Results Progesterone receptors were not affected by Control PROGCalcitriolPROG + calcitriol progesterone or calcitriol treatment 1.71 1.60 1.75 1.69 The protein expression levels of PR-A and PR-B were 1.73 1.62 1.64 1.76 studied in EM-E6/E7-TERT and 3 endometrial cancer PR-B (118 kDa) PR-A (90 kDa) (Ishikawa, HEC-1B, and RL95-2) cell lines by quantiﬁcation EM-E6/E7-TERT β of Western blot analyses (Fig. 1), using a mouse anti-human -Actin (42 kDa) PR antibody that recognizes both human PR isoforms: PR-A 1.21 1.31 1.25 1.14 (85–94 kDa) and PR-B (116–120 kDa). Both isoforms were 1.23 1.34 1.15 1.16 expressed in endometrial cancer and in immortalized cell PR-B (118 kDa) HEC-1B PR-A (90 kDa) lines (Fig. 1A). However, expression of both isoforms was β-Actin (42 kDa) reduced in cancer cell lines compared with the immortal- < 1.11 1.23 1.19 1.25 ized cell line (P 0.05). We studied the effects of proges- 1.19 1.32 1.31 1.33 terone, calcitriol, or both on PR protein expression in the PR-B (118 kDa) same normal and endometrial cancer cell lines. No marked Ishikawa PR-A (90 kDa) change was noticed in the expression of PR isoforms in β-Actin (42 kDa) immortalized cells and in endometrial cancer cells follow- 1.12 1.09 1.23 1.16 ing progesterone, calcitriol, or combination treatments 1.15 1.17 1.29 1.28 (Fig. 1A). PR-B (118 kDa) PR-A (90 kDa) RL-95 β Progesterone enhances vitamin D receptors in -Actin (42 kDa) endometrial cancer cells We analyzed the effect of progesterone, calcitriol, or both B on VDR protein expression in normal and malignant endometrial cells. As shown in Fig. 1B, Western blotting of cell

lysates for VDR revealed a doublet at approximately 60 and Control PROGCalcitriolPROG + calcitriol 48 kDa, which likely represents multiple phosphorylated 0.76 0.65 2.53 1.91 forms of the receptor. Interestingly, quantification of VDR VDR (60 kDa) VDR (48 kDa) expression data in progesterone-treated cancer cell lines EM-E6/E7-TERT resulted in a marked (P < 0.05) stimulation of VDR expres- β-Actin (42 kDa) sion. Exposure to the combined treatment showed similar 0.13 1.57 0.41 2.01 expression of VDR versus cells treated with only progester- VDR (60 kDa) VDR (48 kDa) one. Although calcitriol enhanced VDR expression, the HEC-1B β enhancement was not as pronounced as that shown by -Actin (42 kDa) progesterone-treated cells. In EM-E6/E7-TERT cells, calci- 0.17 1.84 0.44 2.11 < VDR (60 kDa) triol, but not progesterone, enhanced (P 0.05) VDR VDR (48 kDa) expression (Fig. 1B). Ishikawa β-Actin (42 kDa) Progesterone and calcitriol inhibit cell proliferation 0.27 1.71 0.52 2.16 VDR (60 kDa) and cause cell death VDR (48 kDa) We examined the dose response effects of these agents RL-95 β alone or in combination on cell growth and death in EM-E6/ -Actin (42 kDa) E7-TERT and 3 endometrial cancer lines (Ishikawa, HEC-1B, and RL95-2). Cells were treated with progesterone (10, 20, m Figure 1. Expression of PR and VDR in immortalized human or 40 mol/L), calcitriol (50, 100, and 200 nmol/L), or the endometrial (EM-E6/E7-TERT) and endometrial cancer (Ishikawa, combination of the 2 for 120 hours. This treatment time was HEC-1B, and RL95-2) cell lines. Whole-cell extracts of progesterone- selected on the basis of our previous studies, which indi- (20 mmol/L), calcitriol- (100 nmol/L), or combination-treated cell lines cated significant inhibition of cell growth after 5 days (25). containing an equal amount of protein (100 mg) for PRs (A) and (20 mg) At the end of treatment time, determination of total cell for VDR (B) were separated by electrophoresis, and immunoblots were probed with 3 mg/mL of mouse monoclonal anti-human PR antibody number as well as dead cells showed that both progesterone from Dako Corporation or VDR antibody at 4C. The immunoblots and calcitriol inhibit cell growth and cause cell death in a shown here are representative of 3 independent experiments with dose-dependent manner (Fig. 2A and B). Combined pro- similar results. The values above the bands represent relative density gesterone and calcitriol treatment showed a pronounced of the bands normalized to b-actin. The upper values represent quantification of PR (B) and lower represent PR (A). The quantitation of dose-dependent decrease in cell numbers compared with VDR includes both the upper and lower bands. either single agent in all the cell lines tested. Using the

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A B 60 40 50 32 40 24 30 EM-E6/E7-TERT 16 20 10 8 Figure 2. Effect of progesterone, 0 0 calcitriol, and combination 60 40 treatment on cell growth and death 50 32 in human immortalized and 4 40 endometrial cancer cells. Cells 24 30 HEC-1B were treated with ethanol 16 20 (control) or 10, 20, and 40 mmol/L 10 8 progesterone or 50, 100, or 200 0 0 nmol/L of calcitriol or the 60 40 combination for 120 hours. At the 50 end of the treatment period, both 32 40 ﬂoaters and attached cells were Dead cells (%) 24 30 Ishikawa collected and processed for (A) 16 20

total cell number determination of cells x 10 number Total 10 8 and (B) dead cell count using 0 0 Trypan blue exclusion method. 60 40 The data shown are mean SEM 50 of 3 samples for each treatment. 32 40 These results were similar in 24 30 RL-95 3 independent experiments. 16 20 Two-way ANOVA and the Tukey 10 8 post hoc test were used to 0 0 determine differences between treatment groups. ,statistically Control significant difference across the PROG entire cell line, calculated by two- Calcitriol way ANOVA (P < 0.05). "a" and "b" show statistically significant difference from the lowest dose within a treatment group at (P < C 0.01) and (P < 0.05) respectively. #, a significant interaction between PROG and calcitriol combination group compared with single ControlPROGCalcitriolPROG + calcitriol agent groups. C, representative 0.14 0.77 0.98 1.44 Western blot analyses and quantification of caspase-3 protein Caspase-3 (19–17 kDa) m EM-E6/E7-TERT in progesterone- (20 mol/L), β-Actin (42 kDa) calcitriol- (100 nmol/L), and combination-treated endometrial 0.18 1.20 1.33 1.71 cells. The relative expression of Caspase-3 (19–17 kDa) caspase-3 protein was analyzed HEC-1B β using b-actin as a control. The -Actin (42 kDa) quantitation of caspase-3 includes 0.11 0.83 0.76 1.46 both the upper and lower bands. Caspase-3 (19–17 kDa) Ishikawa β-Actin (42 kDa) 0.13 0.72 0.78 1.09 Caspase-3 (19–17 kDa) RL-95 β-Actin (42 kDa)

Trypan blue dye exclusion method, we observed that the death caused by each of the 3 treatments, we assessed the decrease in cell number by both the agents was accompa- levels of cleaved caspase-3 in cells. Although treatment of nied by an increase in cell death. However, no treatment cells with progesterone or calcitriol alone showed moderate caused the percentage of cell death to increase beyond 20%, elevation of cleaved caspase-3, their combination substan- suggesting that the overall decrease in cell number was tially increased (P < 0.05) caspase-3 levels (Fig. 2C). Next, partially caused by the cell death–inducing effect of both we investigated the effect of progesterone and calcitriol on the agents. In an attempt to identify the mechanism of cell cell-cycle arrest in endometrial cancer cells to determine

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whether the inhibitory effect on cell proliferation is accom- tural proteins, transport proteins, ribosomal proteins, panied by modulation of cell-cycle progression. receptors, and transcription factors, as well as proteins involved in apoptosis and cell-cycle regulation. Of the Combination of progesterone and calcitriol induces G1 117 proteins, 4 apoptosis-related proteins were selected for cell-cycle arrest and modulates cell-cycle regulators in validation studies, HIST1H1E, HINT2, EIF2AK2, and BAX endometrial cancer cells (Fig. 4A). The expression levels of the selected candidates Many apoptotic stimuli induce cell-cycle arrest before cell were successfully verified by Western blot analysis to be at death, thereby affecting both cell cycle and apoptotic low levels in 3 endometrial cancer cell lines and their machinery. To determine whether inhibition of cell prolif- expression was significantly elevated (P < 0.05) upon expo- eration was associated with cell-cycle arrest, endometrial sure to progesterone, calcitriol, or the combination (Fig. cells EM-E6/E7-TERT, Ishikawa, HEC-1B, and RL95-2 were 4B). We selected BAX for further analysis and investigated treated for 24 hours with vehicle, progesterone, calcitriol, or the time-dependent induction of BAX and BCL-2 in combined treatment and analyzed by flow cytometry. The response to progesterone, calcitriol, or both. A time-depen- initial cell population in the G0–G1 phase was 56.39% dent increase in BAX and concomitant decrease in BCL-2 0.42%, 36.44% 0.35%, 49.69% 0.41%, and 47.14% expression was observed in all cell lines (Fig. 5). Quantifi- 0.14% in EM-E6/E7-TERT, HEC-1B, Ishikawa, and RL95-2 cation of the Western blot analysis images and calculation cells, respectively. However, after 24 hours of incubation of the BAX:BCL2 ratio clearly showed a marked (P < 0.05) with the combined progesterone-calcitriol treatment, a increase of BAX:BCL2 following combined treatment versus significantly greater number of cells were arrested in the either single treatment in cancer cells (Fig. 5). However, in G1 phase (62.86% 0.57%, 60.24% 0.4%, 59.979% immortalized cells no significant changes were noted in 0.48%, and 58.97% 0.48% in EM-E6/E7-TERT, HEC, BAX:BCL2 between vehicle-treated cells versus single or Ishikawa, and RL95-2 cells, respectively) compared with combination treatment cells (Fig. 5). vehicle-treated cells. In RL-95 cells, cell-cycle arrest was more pronounced (P < 0.05) following combination treat- Knockdown of BAX enhances growth of cancer cells ment compared with either single treatment (Fig. 3A–D and restoration of BAX expression by progesterone and Table 1). These results suggest that a combination and calcitriol attenuates proliferation treatment induces G0–G1 phase arrest in endometrial To confirm that the progesterone and calcitriol-induced cancer cells. To identify cell-cycle regulatory molecules, inhibition of endometrial cancer cell growth is mediated via we examined the expression of cyclins and cyclin-depen- BAX expression, we transfected cells with BAX-targeted dent kinases (CDK) inhibitors. Cells were treated with siRNA oligonucleotides. Cells were harvested at 24 hours progesterone, calcitriol, or the combination for 5 days after transfection and BAX expression was analyzed by and cell lysates were prepared for Western blot analysis. Western blot analysis (Fig. 6A). Although BAX-specific Our results showed a marked decrease (P < 0.05) in siRNA suppressed BAX synthesis almost completely, control cyclins D1 and D3 in endometrial cancer cell lines with (scrambled) siRNA had no effect. Next, we assessed the concomitant elevation (P < 0.05) of p27 protein expression effect of BAX-specific siRNA on cell viability. Progesterone-, following treatment with progesterone, calcitriol, or the calcitriol-, and combination-induced growth inhibition combination compared with untreated cells (Fig. 3E). was attenuated in BAX knockdown cells compared with Together, these results suggested that progesterone and scrambled siRNA-transfected cells (Fig. 6B). BAX expression calcitriol treatments in endometrial cancer cells strongly was correlated with cell proliferation using Pearson corre- modulate cyclins and inhibit their normal regulation of cell- lation. Strong correlation (correlation coefficient r ¼ 0.81; P cycle progression. < 0.001, HEC-1B, r ¼ 0.90; P < 0.001, Ishikawa, r ¼ 0.89; P < 0.001, RL-95) was observed between BAX expression and Identification of novel mediators of calcitriol and inhibition of cell growth. progesterone signaling in endometrial cancer cells We used a proteomic approach to identify additional Discussion mediators of progesterone and calcitriol signaling in endo- Although both progesterone and calcitriol may have metrial cancer cells. Comparative analysis of protein expres- potential as therapeutic interventions in selected cancers, sion in HEC-1B cells treated with progesterone, calcitriol, or adverse side effects associated with high dosages may limit the combination for 24 hours resulted in the identification their clinical use, especially for calcitriol, which can cause of alterations in the abundance levels of 609, 387, and 519 harmful hypercalcemia. These limitations could be over- proteins, respectively. Interestingly, between the 3 treat- come if the 2 agents act additively or synergistically or via a ments, only 117 proteins were found in common that different mechanism of action, thereby allowing the use of exhibited altered expression in HEC-1B relative to the lower doses of each. On the basis of this rationale, in the untreated cells (Fig. 4A). The list of 117 proteins is provided present study, we tested the efficacy of progesterone in in the Supplementary Table. The proteins that were differ- combination with calcitriol against endometrial cancer. The entially altered by these 3 treatments in the HEC-1B cells results show that progesterone and calcitriol produced were categorized into different groups: oncogenes, tumor dose-dependent growth inhibition in endometrial cancer suppressors, binding proteins, membrane proteins, struc- and in immortalized endometrial cells, although growth

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ABEM-E6/E7-TERT HEC-1B

Vehicle control PROG Vehicle control PROG

G –G G1–G0 G1–G0 G1–G0 1 0

S S G –M S S 2 G2–M G2–M G2–M

Calcitriol PROG + calcitriol Calcitriol PROG + calcitriol

G –G G1–G0 1 0 G1–G0 G1–G0 S SS G –M S G2–M G2–M 2 G2–M

C Ishikawa D RL-95

Vehicle control PROG Vehicle control PROG

G1–G0 G1–G0 G1–G0 G1–G0

S G –M G –M 2 S G2–M S 2 S G2–M

Calcitriol PROG + calcitriol Calcitriol PROG + calcitriol

G1–G0 G1–G0 G1–G0 G1–G0

S G –M SSG2–M G2–M S 2 G2–M

ControlPROGCalcitriolPROG + ControlcalcitriolPROGCalcitriolPROG + ControlcalcitriolPROGCalcitriolPROG + calcitriolControlPROGCalcitriolPROG + calcitriol

0.46 0.25 0.24 0.21 0.38 0.20 0.26 0.19 0.52 0.14 0.22 0.16 0.42 0.22 0.18 0.12 Cyclin D1 (36 kDa)

0.43 0.17 0.20 0.14 0.45 0.19 0.24 0.19 0.38 0.14 0.22 0.18 0.45 0.22 0.24 0.17 Cyclin D3 (31 kDa)

0.06 0.27 0.29 0.33 0.11 0.29 0.42 0.43 0.05 0.32 0.40 0.48 0.13 0.35 0.31 0.37 P27 (27 kDa)

β-Actin (42 kDa) EM-E6/E7-TERT HEC-1B Ishikawa RL-95

Figure 3. Progesterone and calcitriol induce cell-cycle arrest in endometrial cancer cells. A–D, cells were treated with progesterone (20 mmol/L), calcitriol (100 nmol/L), or a combination for 24 hours and stained with propidium iodide. DNA content was analyzed using ﬂow cytometry. Results are shown as the percentage of cell population in G1, S, and G2–M phases of the cell cycle. E, Cell-cycle regulatory proteins expression in progesterone- and calcitriol-treated cells. The immunoblots shown are representative of 3 independent experiments with similar results. The values above the bands represent relative density of the bands normalized to b-actin.

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Table 1. Effect of progesterone and calcitriol on cell-cycle arrest

EM-E6/E7-TERT HEC-1B

c c G0–G1 SG2–MG0–G1 SG2–M Control 56.39 0.43 24.58 0.56 19.03 0.95 36.44 0.35 45.28 0.84 18.28 0.58 PROG 59.19 0.84 25.70 0.73 15.11 0.17 60.94 0.45 30.54 0.53 8.53 0.16 Calcitriol 63.71 0.37 21.34 0.73 14.95 0.44 38.01 0.57 43.05 0.26 18.94 0.42 Progþcalcitriol 62.86 0.57a 20.04 0.46 17.11 0.11 60.24 0.4b 30.38 0.83 9.38 0.48 Ishikawa RL-95

c c G0–G1 SG2–MG0–G1 SG2–M Control 49.69 0.41 34.58 0.05 15.74 0.37 47.14 0.42 37.15 0.23 15.71 0.63 PROG 55.42 0.74 30.13 0.47 14.45 0.32 57.03 0.76 35.66 0.67 7.33 0.22 Calcitriol 49.51 1.03 35.34 1.05 15.15 0.3 52.47 0.21 32.49 0.37 15.03 0.35 Progþcalcitriol 59.05 0.69a,b 29.02 0.2 11.92 0.71 58.97 0.48b 33.35 0.33 7.68 0.24

Abbreviation: HSD, honest significant difference. aStatiscally significant difference from PROG, calculated by Tukey HSD (P < 0.05). bStatiscally significant difference from calcitriol calculated by Tukey HSD (P < 0.05). cStatiscally significant difference across the entire cell line, calculated by two-way ANOVA (P > 0.05).

inhibition was less pronounced with calcitriol treatment either agent alone, suggesting an additive effect on tumor alone. Of note, cells exposed to a combination of these cell growth. Our results agree with preclinical studies show- agents showed greater inhibition than did cells exposed to ing that calcitriol has additive effects when used in

A Isoform α of apoptosis regulator, BAX, accession no Q07812-1 PROG Calcitriol (609) (387) 88 119 HIST1H1E, accession no P10412 278

117 63 HINT2, accession no Q9BX68 ﬁ 126 Figure 4. Identi cation and EIF2AK2, accession no P19525 validation of new progesterone and 213 calcitriol targets in endometrial cancer cells. A, total number of PROG + calcitriol proteins altered in endometrial (519) cancer cells by progesterone, calcitriol, and combination B treatment. Common proteins differentially regulated by all treatments are shown in the center and 4 proteins selected for ﬁ Control PROGCalcitriolPROG + calcitriolControl PROG CalcitriolPROG +Control calcitriolPROGCalcitriolPROG + calcitriolControl PROGCalcitriolPROG + calcitriol validation are listed. B, veri cation of a subset of progesterone- and 0.67 0.53 0.62 0.67 0.17 0.41 0.45 0.54 0.15 0.54 0.46 0.63 0.12 0.32 0.38 0.46 calcitriol-regulated proteins in endometrial cancer cell lines by BAX Western blotting. Representative 0.56 0.60 0.53 0.55 0.12 0.57 0.54 0.65 0.09 0.48 0.52 0.50 0.13 0.28 0.38 0.42 Western blot analyses of 3 independent experiments with HIST1H1E similar results are shown. The values above the bands represent 0.47 0.51 0.45 0.44 0.12 0.52 0.48 0.46 0.12 0.41 0.56 0.49 0.19 0.40 0.56 0.61 relative density of the bands HINT2 normalized to b-actin.

0.33 0.35 0.37 0.28 0.11 0.42 0.38 0.52 0.17 0.43 0.51 0.53 0.10 0.37 0.43 0.58

EIF2AK2

β-Actin EM-E6/E7-TERT HEC-1B Ishikawa RL-95

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Control PROG Calcitriol PROG + calcitriolControl PROG Calcitriol PROG + calcitriolControl PROG Calcitriol PROG + calcitriol NS NS NS

Figure 5. Upregulation of BAX and Ratio 1.75 ± 0.06 1.71 ± 0.08 2.03 ± 0.17 1.57 ± 0.08 1.56 ± 0.17 1.52 ± 0.25 1.81 ± 0.26 1.88 ± 0.15 2.00 ± 0.39 2.14 ± 0.31 2.16 ± 0.37 downregulation of BCL-2 in 1.80 ± 0.08 endometrial cancer cells by BAX progesterone and calcitriol. EM-E6/E7-TERT Immortalized EM-E6/E7-TERT and BCL2 endometrial cancer cells (Ishikawa, β HEC-1B, and RL95-2) treated with -Actin progesterone (PROG, 20 mmol/L), * calcitriol (100 nmol/L), or a * * combination for 24, 72, and 120 hours were evaluated by Western Ratio

blot analysis for expression of BAX 0.59 ± 0.02 1.56 ± 0.12 1.52 ± 0.15 2.63 ± 0.29 0.52 ± 0.01 2.40 ± 0.41 2.07 ± 0.20 3.37 ± 0.55 0.57 ± 0.11 2.53 ± 0.23 2.42 ± 0.07 3.62 ± 0.79 b and BCL-2. -Actin was used as a BAX loading control. Representative Western blot analyses of 3 HEC-1B BCL2 independent experiments with similar results are shown. The β-Actin values at the top of the blots * represent the ratio of BAX to BCL-2 * * after normalization to b-Actin as determined by densitometric Ratio

scanning. BAX and BCL-2 density 0.54 ± 0.01 2.16 ± 0.07 1.18 ± 0.19 3.33 ± 0.25 0.47 ± 0.07 1.23 ± 0.16 1.68 ± 0.12 3.50 ± 0.42 0.45 ± 0.06 2.27 ± 0.20 2.11 ± 0.34 3.85 ± 0.29 values are the average of the BAX 3 independent immunoblot Ishikawa densities. , a signiﬁcant difference BCL2 between combined PROG and calcitriol treatment and single β-Actin agent groups. NS, P values not statistically signiﬁcant between * * * the 3 treatment groups. Ratio 0.54 ± 0.01 1.55 ± 0.07 1.62 ± 0.19 2.50 ± 0.25 0.43 ± 0.05 2.20 ± 0.16 1.86 ± 0.12 2.50 ± 0.24 0.50 ± 0.06 2.35 ± 0.20 2.40 ± 0.34 3.28 ± 0.23 BAX RL-95 BCL2

β-Actin

24 h 72 h 120 h

combination with chemotherapeutic agents including cis- endometrial, cervical, breast, and colorectal cancer (30– platin and paclitaxel (27, 21). Such combination therapy 36). Herein, we showed for the ﬁrst time that progesterone potentially offers a therapeutic advantage over a standard upregulated VDR expression in endometrial cancer but dose of a single drug by enhancing the effectiveness of the not in immortalized endometrial cells, suggesting that therapy without increasing the dose, thus limiting dose- progesterone is able to improve calcitriol’s biologic effects dependent toxic side effects. in cancer cells. This is corroborated by our results show- Calcitriol has antiproliferative and antineoplastic activ- ing a signiﬁcant decrease in cell proliferation in cells treat- ities, which include activation of apoptosis, induction of ed with a combination of progesterone and calcitriol. cell-cycle arrest, differentiation, inhibition of invasion and Although progesterone-treated cells showed a dramatic motility, and reduction of angiogenesis (28, 29). These increase in VDR expression, calcitriol-treated cells display- activities are exerted through both genomic and nonge- ed a much more subtle increase. This suggests low basal nomic pathways. The classic genomic response is believed VDR expression in the cell lines of interest, which may to be the most responsible for the vitamin D action, and explain the moderate antiproliferative activity caused by it is mediated through the VDR. The VDR is highly expres- calcitriol treatment in comparison with progesterone or sed in a large number of tumor tissues including ovarian, combination treatment.

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Control PROGCalcitriolPROG + calcitriolControl PROGCalcitriolPROG + calcitriol

0.520.47 0.51 0.45 0.110.14 0.18 0.15

EM-E6/E7-TERT BAX β-Actin

0.21 0.640.720.73 0.13 0.140.120.11 BAX Figure 6. Effect of BAX silencing on HEC-1B protein expression and growth of β -Actin normal and malignant cells. Cells were transfected with BAX siRNA 0.240.68 0.59 0.71 0.16 0.150.140.13 (5 mmol/L) or control siRNA m BAX (5 mol/L) using Lipofectamine Ishikawa 2000. After 24 hours, cells were β-Actin treated with progesterone or calcitriol or a combination for 5 0.13 0.610.530.66 0.150.11 0.10 0.13 days. A, expression of BAX was analyzed by Western blot analysis. RL-95 BAX Representative Western blot β-Actin analyses of 3 independent experiments with similar results Control siRNA BAX siRNA are shown. The values above the bands represent relative density of the bands normalized to b-actin. B B, cell proliferation was evaluated by MTS assay. Data shown are mean SEM of values from 3 independent experiments. Control PROG PROG + calcitriolControl PROG PROG + calcitriol Calcitriol Calcitriol , P < 0.05 (statistically signiﬁcant) between the control 120 and the 3 treatment groups. *** *** 80

40 Cell viability (%) 0

Control siRNA BAX siRNA

EM-E6/E7-TERT HEC-1B Ishikawa RL-95

Because apoptotic stimuli often arrest growth before the sensitivity of cell lines to treatment as 48-hour treatment inducing cell death and as significant apoptosis was not (data not shown) significantly increased cell-cycle arrest evident in calcitriol-treated endometrial cells for the first 24 in immortalized endometrial cell line. In EM-E6/E7-TERT hours (results not shown), we opted to investigate the early cells, increased cell-cycle arrest in G0–G1 in response to effects of progesterone, calcitriol, and combined treatment calcitriol can be ascribed to increased VDR expression (Fig. on the cell cycle in the first 24 hours of exposure. Our results 2). In nontumorigenic cells, this is not surprising because showed G0–G1 cell-cycle arrest following progesterone vitamin D3/VDR signaling is involved in maintenance of treatment. The combined treatment resulted in a greater cellular homeostasis and modulation of growth by inter- number of cells undergoing cell-cycle arrest in the Ishikawa actions with other transcriptional regulators and cell sig- cell line. However, combined treatment failed to show an naling systems, including TGF-b and b-catenin, to control additive effect on the cell cycle in EM-E6/E7-TERT, RL-95, cell growth and differentiation. and HEC-1B cell lines. In some cell lines, the delayed effect Cells progress through the various phases of the cell cycle of calcitriol and combined treatment can be attributed to via the interactions of different cyclins with their respective

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CDK subunits (37–39). Quiescent cells enter the cell cycle of BAX gene expression promoted tumorigenesis, whereas after mitogenic stimuli and upregulate cyclins D and E its overexpression showed an inhibitory effect on cell during the G1 phase of the cell cycle (38). Cyclins D1 growth (52). and D3 are known to bind with and activate CDK4, which In the present study, lower basal levels of BAX expression phosphorylates Rb (Rb) protein to release E2F transcription were observed in endometrial cancer cell lines than in factors to transcribe genes needed for the G1 to S transition immortalized endometrial cells. Progesterone and calcitriol (40). Cyclin D1 is considered an oncogene and is over- were shown to upregulate BAX expression in cancer expressed in many cancers including prostate, breast, cells. Cell survival is maintained by a fine balance of the esophagus, lung, head and neck, and colon (41–43). The proapoptotic (e.g., Bad and Bax) and antiapoptotic proteins Cip/Kip family of CDK inhibitors binds to and inhibits (e.g., BCL-2 and BCL-xL), which control the process of the activity of CDK-cyclin complexes that regulates G1–S apoptosis through release of caspases (51). Therefore, the and G2–M phase transitions (38, 40). The Cip/Kip family increase in the ratio of BAX to BCL-2 proteins observed plays a critical role in the cellular response to DNA damage following treatment of endometrial cancer cells with a for cell-cycle arrest (44, 45) and is upregulated in response combination of progesterone and calcitriol could be to antiproliferative signals for cell-cycle arrest (46, 47). responsible for the observed apoptotic effect. Experimental Endometrial cancer cells treated with a combination of alterations of BAX levels disrupt cellular processes. We were progesterone and calcitriol showed a decrease in cyclins able to elevate BAX protein expression via progesterone and D1 and D3, and an increase in p27 expression. Others (48, calcitriol treatment in endometrial cancer cells and subse- 49) have shown an increase in the steady-state levels of p27 quently relate their effects to cellular functions such as protein in prostate and endometrial cancer cells in response proliferation. The highlight of this study is that BAX knock- to calcitriol. Although cyclins are downregulated, we cannot down attenuated progesterone- and calcitriol-induced conclude from our studies that cyclins are direct transcrip- growth inhibition. Interestingly, the upregulation of BAX tional targets of calcitriol and progesterone. Cyclin suppres- by combined treatment of endometrial cancer cells resulted sion may have occurred as a downstream consequence of in a greater decrease in cell proliferation compared with the prior modulation of direct target genes. either progesterone or calcitriol alone. This suggests that the Induction of apoptosis in cancer cells is another approach upregulation of BAX by a combined treatment may have a to limit their uncontrolled proliferation (50, 51). In this cumulative effect on inhibition of cell growth. process, activation of caspases is the central event (50, 51). Our results showed no significant effects of progesterone Once activated, the executioner caspases downstream of the and calcitriol treatment on BAX-BCL2 expression in EM-E6/ caspase cascade act on the key molecules inside the cells to E7-TERT cells. Normal cells maintain critical balance of orchestrate cell death (51). In fact, many cancer preventive proteins promoting or inhibiting cell proliferation. Knock- and chemotherapeutic agents have been shown to activate ing down of BAX perturbs the balance and exposure to the 2 the caspases. Cleavage of caspase-3 is considered to be a agents triggers expression of proteins that may inhibit cell marker for apoptotic death (51). Confirming these reports, growth. our study shows treatment of endometrial cancer cells with These results provide evidence that calcitriol and proges- progesterone, calcitriol, and the combination significantly terone produce cell death in endometrial cells by apoptosis. enhanced levels of cleaved caspase-3. Together these obser- All investigations into vitamin D and cell death to this point vations suggest that progesterone and calcitriol inhibit cell have pointed to an apoptotic mechanism. Although caspase growth through apoptosis. cleavage and the action of other apoptosis-related mole- Many proteins either sensitize cancer cells to apoptosis or cules usually occurs within 24 hours or less, it has been are directly involved in apoptosis. In an effort to identify the shown that vitamin D acts much more slowly to effect molecular targets of progesterone and calcitriol in endo- apoptosis. For example, Zhang and colleagues (53, 54) have metrial cancer cells, we used a proteomic approach. Our shown the strongest apoptotic response at 6 days following data showed 117 proteins differentially regulated by pro- treatment using a vitamin D analog in several cell lines. gesterone, calcitriol, and the combination. The 4 apoptosis- Mathiasen and colleagues (55) also showed peak vitamin related proteins selected for validation were upregulated by D–induced apoptosis at 6 to 7 days posttreatment. The latter all 3 treatments in endometrial cancer cells. To our knowl- group also concluded that vitamin D–induced apoptosis edge, this is the first report showing BAX, HIST1H1E, may not be caspase-dependent. Although we have not HINT2, and EIF2AK2 as tumor suppressors of endometrial conducted caspase inhibition studies, it should be noted cancer and showed their regulation by progesterone and that cleaved caspase-3 increased primarily in response to calcitriol. These proteins have not been reported to be progesterone treatment, particularly in combination with regulated by progesterone or calcitriol in endometrial calcitriol. The cumulative evidence presented herein points cancer. strongly to an apoptotic pathway for cell death in these BAX resides in the cytosol and translocates to mito- endometrial cancer cell lines in response to progesterone chondria upon induction of apoptosis. BAX has been and calcitriol. showntoinducecytochromecreleaseandcaspaseacti- Induction of chromatin and condensation and cleavage vation. Its expression is considerably diminished at the of DNA are characteristic steps of apoptosis, leading to the mRNA and protein level in cancerous tissues. Suppression formation of oligomers of nucleosomes. HIST1H1E is

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Lee et al.

essential for the generation of condensed structures and Disclosure of Potential Conflicts of Interest initiation of the subsequent internucleosomal cleavage of No potential conflicts of interest were disclosed. DNA, which is a hallmark of programmed cell death. During early apoptosis, induction of internucleosomal Disclaimer DNA cleavage and increase in HIST1H1E expression is a The opinions and assertions expressed herein are those of the authors prerequisite for DNA accessibility and/or endonuclease. and should not be construed as reflecting those of the Uniformed Services University of the Health Sciences, Department of the Air Force, or the U.S. Similar to our findings in human leukemic cells, HIST1H1E Department of Defense. is low and expression increases when cells are exposed to an apoptosis-inducing monoclonal anti-CD95 antibody (56). Authors' Contributions HINT proteins are AMP–lysine hydrolases. HINT2 is Conception and design: C.A. Hamilton, G.L. Maxwell, G.C. Rodriguez, T.P. a mammalian-specific, nuclear-encoded mitochondrial Conrads, V. Syed Development of methodology: B.L. Hood, T.P. Conrads, V. Syed HINT hydrolase. We have shown low expression of Acquisition of data (provided animals, acquired and managed patients, HINT2 in endometrial cancer cells compared with control provided facilities, etc.): L.R. Lee, P.-N. Teng, H. Nguyen, B.L. Hood, cells and expression of HINT2 was upregulated by pro- L. Kavandi, J.M. Turbov, L.G. Thaete, T.P. Conrads Analysis and interpretation of data (e.g., statistical analysis, biosta- gesterone and calcitriol treatment. These results are in tistics, computational analysis): L.R. Lee, B.L. Hood, L. Kavandi, G. Wang, agreement with a study suggesting that HINT2 overex- J.M. Turbov, L.G. Thaete, C.A. Hamilton, G.L. Maxwell, G.C. Rodriguez, T.P. Conrads, V. Syed pression sensitizes hepatocellular carcinoma to apoptosis Writing, review, and/or revision of the manuscript: L.R. Lee, B.L. Hood, (57). J.M. Turbov, L.G. Thaete, C.A. Hamilton, G.L. Maxwell, G.C. Rodriguez, T.P. EIF2AK2 is a member of the eukaryotic initiation-factor 2 Conrads, V. Syed a Administrative, technical, or material support (i.e., reporting or orga- kinase family. EIF2AK2 has been shown to play a signif- nizing data, constructing databases): L. Kavandi, T.P. Conrads icant role in signaling pathways involved in other cellular Study supervision: V. Syed processes such as cell proliferation, differentiation, metab- olism, DNA repair, tumor suppression, and apoptosis (58). b Acknowledgments EIF2AK2 regulates TNF- –induced p53-dependent apopto- The authors thank Dr. Satoru Kyo for providing the EM-E6/E7/TERT sis in U937 cells. In addition, it can interact directly with cell line. p53 and phosphorylate it (58). The identification of these new progesterone and calcitriol molecular targets in endo- Grant Support metrial cancer cells offers unique opportunities for future This study was funded by the United States Medical Acquisition Activity award W81XWH-11-2-0131. development of novel strategies for endometrial cancer The costs of publication of this article were defrayed in part by the therapies. payment of page charges. This article must therefore be hereby marked In summary, we conclude that progesterone combined advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. with calcitriol enhances expression of proapoptotic (BAX, HIST1H1E, HINT2, and EIF2AK2) proteins and causes Received December 17, 2012; revised April 29, 2013; accepted May 5, apoptosis and cell-cycle arrest in endometrial cancer cells. 2013; published OnlineFirst May 16, 2013.

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www.aacrjournals.org Cancer Prev Res; 6(7) July 2013 743

Progesterone Enhances Calcitriol Antitumor Activity by Upregulating Vitamin D Receptor Expression and Promoting Apoptosis in Endometrial Cancer Cells

Laura R. Lee, Pang-Ning Teng, Huyen Nguyen, et al.

Cancer Prev Res 2013;6:731-743. Published OnlineFirst May 16, 2013.

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