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Abstract

Vitamin D and its analogs are known for their role in the development of breast cancer and in immunomodulation. Our previous studies have shown the pro-metastatic effect of calcitriol and tacalcitol (PRI-2191) in young mice bearing 4T1 breast cancer and the anti-metastatic effect in aged ovariectomized (OVX) mice. Therefore, the aim of our work was to characterize Th17 cell population in young and aged OVX mice bearing 4T1 tumors treated with calcitriol and PRI-2191. The expression of genes typical for Th17 cells was examined in splenocytes, as well as splenocytes differentiated with IL-6 and TGF-β to Th17 cells (iTh17). Expression of genes encoding vitamin D receptor (Vdr) and osteopontin (Spp1) as well as the secretion of IL-17A were evaluated in iTh17 cells. PRI-2191 treatment increased the expression of Rora and Rorc transcription factors, Il17a, Il17re and Il21 in iTh17 cells from young mice. In aged OVX mice this effect was not observed. Increased expression was observed in the case of Vdr and Spp1 genes in iTh17 cells from young mice treated with PRI-2191. What is more, in young mice treated with PRI-2191 the secretion of IL-17A to the culture media by iTh17 cells was increased, whereas in aged OVX mice a significant decrease was noted. Increased expression of Spp1 in young mice treated with PRI-2191 may enhance the differentiation of Th17 cells.

Splenocytes and lymph nodes used in the studies

The splenocytes and lymph nodes utilized in this study were collected from female BALB/c mice used in experiments described in our previous work [,]. The course of experiment on 6-8-week-old mice weighing 20-25 g was described in [] and on 60-week-old mice weighing 24-30 g in [] (mice obtained from the Center of Experimental Medicine of the Medical University of Bialystok, Poland). All methods used in those experiments were compliant with the EU Directive 2010/63/EU on the protection of animals used for scientific purposes and was approved by the First Local Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland (no. of permission: 40/2014). Calcitriol (1,25(OH)2D3) and PRI-2191 (1,24(OH)2D3, tacalcitol) were obtained from the Pharmaceutical Research Institute, Warsaw, Poland. PRI-2191 was described as compound with lowered calcemic activity and increased anticancer activity as compared to calcitriol [,].

Splenocytes and lymph nodes used in the studies

Briefly, 1×104 4T1 mammary gland cancer cells (American Type Culture Collection, Rockville, MD, USA) per mouse were injected orthotopically into young mice [] - as a premenopausal model or into old mice four weeks after they had been ovariectomized [] - as a postmenopausal model. Starting from day 7 after cells inoculation, mice were subcutaneously (s.c.) injected with 80% propylene glycol (vehicle), calcitriol (0.5 µg/kg) and PRI-2191 (1.0 µg/kg) three times a week. On days 0, 7, 14, 21, 28, and 33 after transplantation, spleens and lymph nodes were harvested after euthanasia of mice.

Splenocytes and lymph nodes used in the studies

The experiment on young mice was repeated twice, whereas the experiment on aged OVX mice was conducted once.

PRI-2191 increased the Th17 genes expression in iTh17 splenocytes from young mice bearing 4T1 mouse mammary gland cancer

CD4+ splenocytes stimulated by IL-6 and TGF-β to Th17 cells had different expression level of selected genes when harvested from young and old OVX mice. The induced iTh17 cells from splenocytes of young mice treated with PRI-2191 expressed significantly higher levels of Il17a mRNA as compared to iTh17 splenocytes from control tumor bearing mice (). The expression of transcription factors Rora and Rorc and IL-17 receptor (Il17re) as well as IL-21 (Il21) mRNA was higher in iTh17 cells from mice treated with PRI-2191 (). Vitamin D receptor mRNA (Vdr) as well as osteopontin (Spp1) significantly increased in iTh17 cells from young mice treated with PRI-2191 (). Calcitriol did not affect in statistically significant manner the expression of genes in young mice (). Aged OVX mice did not reveal any significant changes in the expression levels of analyzed genes (). The purity of isolated CD4+ splenocytes from young () and aged OVX () were controlled by flow cytometry after staining of isolated cells with anti-CD4 antibody.