Vitamin D Compounds PRI-2191 and PRI-2205 Enhance Anastrozole Activity in Human Breast Cancer Models

Abstract

1,25-Dihydroxycholecalciferol, the hormonally active vitamin D₃ metabolite, is known to exhibit therapeutic effects against breast cancer, mainly by lowering the expression of estrogen receptors and aromatase activity. Previously, the safety of the vitamin D active metabolite (24R)-1,24-dihydroxycholecalciferol (PRI-2191) and 1,25(OH)₂D₃ analog PRI-2205 was tested, and the in vitro activity of these analogs against different cancer cell lines was studied. We determined the effect of the two vitamin D compounds on anastrozole (An) activity against breast cancer based on antiproliferative activity, ELISA, flow cytometry, enzyme inhibition potency, PCR, and xenograft study. Both the vitamin D active metabolite and synthetic analog regulated the growth of not only estrogen receptor-positive cells (T47D and MCF-7, in vitro and in vivo), but also hormone-independent cancer cells such as SKBR-3 (HER-2-positive) and MDA-MB-231 (triple-negative), despite their relatively low VDR expression. Combined with An, PRI-2191 and PRI-2205 significantly inhibited the tumor growth of MCF-7 cells. Potentiation of the antitumor activity in combined treatment of MCF-7 tumor-bearing mice is related to the reduced activity of aromatase by both An (enzyme inhibition) and vitamin D compounds (switched off/decreased aromatase gene expression, decreased expression of other genes related to estrogen signaling) and by regulation of the expression of the estrogen receptor ERα and VDR.

Keywords: anastrozole; aromatase; breast cancer; estrogen receptor; mice; vitamin D analog.

Figure 1

Structures of vitamin D compounds.

Figure 2

The proliferation inhibition (A–D) of human breast cancer cell lines after treatment with analogs of 1,25(OH)₂D₃ alone or in combination with anastrozole. As indicated (A) MCF-7 cell line, (B) T47D cell line, (C) SKBR-3 cell line, and (D) MDA-MB-231 cell line after 120 h of treatment with calcitriol or PRI-2191 and PRI-2205 and anastrozole. Calcitriol and PRI-2191 and PRI-2005 were used at the dose of 100 nM (A,C,D) or 10 nM (B). Anastrozole was used at the dose of 0.1 mg/mL. The tests were performed 3 to 6 times, each in triplicate. The % (mean ± SD) of proliferation inhibition of cancer cells is shown on the graphs. The nonparametric Kruskal-Wallis ANOVA for multiple comparisons was performed. p < 0.05 was considered to be statistically significant; a—compared to anastrozole, b—compared to PRI-2205 alone.

Figure 3

Aromatase inhibition by calcitriol and vitamin D compounds PRI-2191 and PRI-2205 (Corning CYP19 inhibition test). The concentrations of the compounds were used according to the manufacturers’ protocol by referring to the concentrations of the positive control used (ketoconazole). Ethanol was used as a solvent for the compounds. The test was performed three times, each time in triplicates. Statistical analysis was performed using the nonparametric Kruskal-Wallis test. p < 0.05 was considered to be statistically significant; * compared to calcitriol.

Figure 4

Expression of the estrogen receptors ERα and ERβ in the MCF-7 breast cancer cell line after 72 h of treatment with calcitriol, PRI-2191 and PRI-2205, and anastrozole as measured by flow cytometry. Calcitriol, PRI-2191, and PRI-2205 were used at the concentration of 100 nM and anastrozole at the concentration of 0.1 mg/mL. The test was performed three times, each time in duplicates.

Figure 5

Secretion of 17-β estradiol by MCF-7 cells after in vitro treatment with vitamin D analogs and anastrozole Calcitriol, PRI-2191, and PRI-2205 were tested at 100 nM, while anastrozole was tested at 0.1 mg/mL concentration. (A) After 48 h of treatment, the culture medium was replaced with fresh, colorless DMEM medium without the tested compounds. The culture media were collected after additional 24 h. Total time of cell culture = 72 h. The test was performed two times, each time in triplicates. (B) After 96 h of treatment, the medium was replaced with fresh, colorless DMEM medium without the tested compounds. The culture media were collected after additional 24 h. Total time of cell culture = 120 h. The test was performed three times, each time in triplicates. The nonparametric Kruskal-Wallis ANOVA was used for multiple comparisons. p < 0.05 was considered to be statistically significant; * compared to untreated control.

Figure 6

Changes in the mRNA profile of MCF-7 cells after treatment with calcitriol or vitamin D analogs with/without anastrozole. The figures presented above represent genes encoding molecules involved in estrogen metabolism. (A) BCAR3—breast cancer antiestrogen resistance protein 3; (B,C) ERα, ERβ—estrogen receptors α and β, respectively; (D) CYP19—aromatase; (E,F) ESRRA, ESRRG—Estrogen Related Receptor Alpha and Gamma, respectively; (G) GNRH1—Gonadotropin-Releasing Hormone 1; (H) SULT1E1—sulfotransferase family 1E, estrogen-preferring member 1; (I) HSD17B2—hydroxysteroid dehydrogenase 17 beta 2. PCR analysis was performed for two independent samples (each sample was tested in triplicate).

Figure 7

The antitumor effect of vitamin D analogs when used alone or in combination with anastrozole in MCF-7 tumor-bearing mice. Five days after tumor inoculation (tumor volume was measured), the mice received subcutaneous (s.c.) injections of PRI-2191 (1 μg/kg body weight (b.w.) in each injection) or PRI-2205 (10 μg/kg b.w. in each injection) and/or anastrozole (200 μg/mouse in each injection). PRI-2191 and PRI-2205 were administered three times a week, and the total dose was 17 μg/kg and 170 μg/kg respectively. Anastrozole was administered five times a week, at the total dose of 5.2 mg/mouse. (A,B) tumor growth kinetics after treatment with PRI-2191 (A) or PRI-2205 (B) alone or combined with anastrozole. (C,D) present the tumor growth inhibition (TGI) in % calculated according to the formula given in this article. (C) Red bars indicate TGI for combined PRI-2191 and anastrozole treatment. □—PRI-2191 alone, ▼—anastrozole alone treatment. (D) Green bars indicate TGI for combined PRI-2205 and anastrozole treatment, ○—PRI-2205 alone, ▼—anastrozole alone treatment. (E,F) body weight (b.w.) changes during the treatment in all the experimental groups. (G) The serum calcium level at the end of the experiment. The serum calcium levels were measured at the end of the experiment by using a Roche Diagnostic Kit. Data presented in mmol/L are expressed as mean with SD for each group, except for calcitriol, which was administered subcutaneously to a single NOD/SCID mouse at the dose of 1 µg/kg b.w. (sacrificed after 48 h). To demonstrated the safety of using both vitamin D analogs in comparison to calcitriol, the serum calcium level was evaluated in one mouse. (H) The 17β-estradiol serum levels after treatment. The 17-β estradiol levels were measured using the IBL ELISA kit, which is used for in vitro diagnostic quantitative determination of 17β-estradiol. Statistical analysis was performed using Kruskal–Wallis ANOVA, For calcium and 17β-estradiol level in serum * p < 0.05 compared to the control group.

Figure 8

The expression of selected proteins in MCF-7 tumor tissue. NOD/SCID mice were treated with PRI-2191, PRI-2205, and/or anastrozole. Primary antibodies were used in the following concentrations: anti-VDR (sc-1008) 1:400, anti-CYP27B1 (sc-67261) 1:500, and anti-ERα (sc-542) and anti-ERβ (sc-8974) 1:500 (all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and anti-β-actin (Sigma-Aldrich, Poznań, Poland) 1:500. ECF Western Blotting Reagent Pack (Amersham, GE Healthcare, Little Chalfont, Buckinghamshire, UK) was used for the analysis. Densitometric analysis was performed using ImageJ 1.43u (Wayne Rasband, National Institutes of Health, USA, https://imagej.nih.gov/ij/, accessed on 8 March 2021).