Calcitriol stimulates gene expression of cathelicidin antimicrobial peptide in breast cancer cells with different phenotype

Abstract

Background: In normal and neoplastic cells, growth-promoting, proangiogenic, cytotoxic and pro-apoptotic effects have all been attributed to cathelicidin antimicrobial peptide (CAMP). Nevertheless, little is known about the factors regulating this peptide expression in breast cancer. Herein we asked if the well-known antineoplastic hormone calcitriol could differentially modulate CAMP gene expression in human breast cancer cells depending on the cell phenotype in terms of efficacy and potency.

Methods: The established breast cancer cell lines MCF7, BT-474, HCC1806, HCC1937, SUM-229PE and a primary cell culture generated from invasive ductal breast carcinoma were used in this study. Calcitriol regulation of cathelicidin gene expression in vitro and in human breast cancer xenografts was studied by real time PCR. Tumorigenicity was evaluated for each cell line in athymic mice.

Results: Estrogen receptor (ER)α + breast cancer cells showed the highest basal CAMP gene expression. When incubated with calcitriol, CAMP gene expression was stimulated in a dose-dependent and cell phenotype-independent manner. Efficacy of calcitriol was lower in ERα + cells when compared to ERα- cells (<10 vs. >70 folds over control, respectively). Conversely, calcitriol lowest potency upon CAMP gene expression was observed in the ERα-/EGFR+ SUM-229PE cell line (EC₅₀ = 70.8 nM), while the highest was in the basal-type/triple-negative cells HCC1806 (EC₅₀ = 2.13 nM) followed by ERα + cells MCF7 and BT-474 (EC₅₀ = 4.42 nM and 14.6 nM, respectively). In vivo, lower basal CAMP gene expression was related to increased tumorigenicity and lack of ERα expression. Xenografted triple-negative breast tumors of calcitriol-treated mice showed increased CAMP gene expression compared to vehicle-treated animals.

Conclusions: Independently of the cell phenotype, calcitriol provoked a concentration-dependent stimulation on CAMP gene expression, showing greater potency in the triple negative HCC1806 cell line. Efficacy of calcitriol was lower in ERα + cells when compared to ERα- cells in terms of stimulating CAMP gene expression. Lower basal CAMP and lack of ERα gene expression was related to increased tumorigenicity. Our results suggest that calcitriol anti-cancer therapy is more likely to induce higher levels of CAMP in ERα- breast cancer cells, when compared to ERα + breast cancer cells.

Keywords: Breast cancer; Calcitriol; Cathelicidin; LL-37; Vitamin D.

Fig. 1

Basal CAMP gene expression. Basal CAMP gene expression was evaluated in several breast cancer cell lines with different phenotype. Data are depicted as the mean ± SD. N = 3. Results were normalized against ACTB mRNA expression

Fig. 2

CAMP is transcriptionally upregulated by calcitriol in different human breast cancer cell lines. Cells were incubated in the presence of different calcitriol concentrations during 24 h. Afterwards cells were processed for qPCR. CAMP mRNA levels were obtained by normalizing against ACTB mRNA expression. Vehicle values were set to one. N = 3, *P < 0.05 vs. control

Fig. 3

CAMP gene expression is stimulated by calcitriol in xenografted tumors. IDC (white bars) and HCC1806 (black bars) cells were inoculated in athymic mice. After tumor onset calcitriol was administered once per week during 3 weeks. Mice were sacrificed and tumors were collected to evaluate CAMP gene expression by qPCR. Results are depicted as the mean ± SEM. Controls were set to one N ≥ 5; *P < 0.05 vs. control

Fig. 4

Calcitriol reduces tumor growth in mice xenografted with human breast cancer cells. HCC1806 cells (triangles) and IDC cells (circles) were subcutaneously injected in athymic mice, which were treated without (black) or with 12.5 μg/kg calcitriol (white) during three weeks. Relative tumor volume is shown as the mean ± SEM. N ≥ 5