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The active hormonal form of vitamin D, 1 alpha,25-dihydroxyvitamin D-3, is reported to have 1000s of biological targets. The growth-suppressive properties of 1 alpha,25-dihydroxyvitamin D-3 and its synthetic analogs have attracted interest for the development of treatment and/or prevention of cancer. We examined effects of 1 alpha,25-dihydroxyvitamin D-3 and the vitamin D analog tacalcitol on signaling pathways and anchorage-independent growth in T98G and U251 glioblastoma cells. Assay of signaling proteins important for cellular growth indi-cated suppression of p70-S6 kinase levels by 1 alpha,25-dihydroxyvitamin D-3 and tacalcitol in T98G cells, whereas the levels of PLC gamma, a target for phospholipid signaling, was slightly increased. Activation of STAT3, an important regulator of malignancy, was suppressed by 1 alpha,25-dihydroxyvitamin D-3 and tacalcitol in T98G and U251 cells. However, despite the close structural similarity of these compounds, suppression was stronger by tacalcitol (1 alpha,24-dihydroxyvitamin D-3), indicating that even minor modifications of a vitamin D analog can impact its effects on signaling. Experiments using soft agar colony formation assay in T98G and U251 cells revealed significant suppression by 1 alpha,25-dihydroxyvitamin D-3 and tacalcitol on anchorage-independent growth, a property for cancer invasion and metastasis known to correlate with tumor-igenicity. These findings indicate that vitamin D and its analogs may be able to counteract the oncogenic transformation, invasion and metastatic potential of glioblastoma and prompt further study of these compounds in the development of improved therapy for brain cancer.

The active form of vitamin D, 1,25-dihydroxyvitamin D₃, is known to act via VDR (vitamin D receptor), affecting several physiological processes. In addition, PDIA3 (protein disulphide-isomerase A3) has been associated with some of the functions of 1,25-dihydroxyvitamin D₃. In the present study we used siRNA-mediated silencing of PDIA3 in osteosarcoma and prostate carcinoma cell lines to examine the role(s) of PDIA3 for 1,25-dihydroxyvitamin D₃-dependent responses. PDIA3 silencing affected VDR target genes and significantly altered the 1,25-dihydroxyvitamin D₃-dependent induction of CYP24A1, essential for elimination of excess 1,25-dihydroxyvitamin D₃. Also, PDIA3 silencing significantly altered migration and proliferation in prostate PC3 cells, independently of 1,25-dihydroxyvitamin D₃. 1,25-Dihydroxyvitamin D₃ increased thermostability of PDIA3 in cellular thermal shift assay, supporting functional interaction between PDIA3 and 1,25-dihydroxyvitamin D₃-dependent pathways. In summary, our data link PDIA3 to 1,25-dihydroxyvitamin D₃-mediated signalling, underline and extend its role in proliferation and reveal a novel function in maintenance of 1,25-dihydroxyvitamin D₃ levels.

This study explored the signaling interplay between the vitamin D receptor (VDR) and receptor tyrosine kinases (RTKs). Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-BB promotes cell proliferation in normal and cancer cells. At the same time, the active form of vitamin D (1,25(OH)₂-vitamin D₃) inhibits proliferation in some cells. Although EGF receptors (EGFR) and PDGF receptors (PDGFR) activate similar downstream pathways, we found that they interact with VDR signaling in distinct ways. We confirmed that 1,25(OH)₂-vitamin D₃ induces CYP24A1 gene expression in U2OS, T98G, and U251 cells. We found this to be potentiated when combined with EGF. In contrast, PDGF-BB did not impact 1,25(OH)₂-vitamin D₃-induced CYP24A1 expression in U2OS cells. The increase in CYP24A1 expression due to the combined action of EGF and 1,25(OH)₂-vitamin D₃ was dependent on AKT and ERK1/2 activation. Another VDR-responsive gene, CYP27B1, was unaffected by the addition of EGF, suggesting that EGF may have gene-specific effects on VDR signaling. While PDGF-BB did not influence CYP24A1 expression, 1,25(OH)₂-vitamin D₃ significantly influenced PDGF-BB-induced receptor phosphorylation and cell proliferation. In summary, we found that EGF, but not PDGF-BB, influenced the expression of the VDR-dependent gene CYP24A1, while 1,25(OH)₂-vitamin D₃ had an inhibitory effect on PDGFR signaling and proliferation. These findings highlight unique crosstalk between 1,25(OH)₂-vitamin D₃ signaling and EGF or PDGF-BB.

Glioblastoma is an incurable brain cancer, known for its rapid growth and invasive properties. The present study reports increased migration and levels of proteins associated with EMT (epithelial-to-mesenchymal transition) in T98G glioblastoma cells treated with 24-hydroxycholesterol, an endogenous cholesterol derivative involved in brain cholesterol homeostasis. In addition to the observed increase in migration, our data show decreased proliferation in the presence of 24-hydroxycholesterol, suggesting that this compound may influence the cells towards favoring migration rather than proliferation. A morphological change was observed in cell cultures exposed to 24-hydroxycholesterol, where many of the cells developed a longer, more outstretched shape, consistent with a migratory phenotype. Cell painting analysis indicated that about 40% of the cells responded to 24-hydroxycholesterol in this way. Also, morphological profiling using cell painting indicate dose-dependent effects by 24-hydroxycholesterol on a large number of different morphological features in T98G cells. Treatment of T98G glioblastoma cells with 24-hydroxycholesterol increased the levels of fibronectin and N-cadherin, proteins that are associated with a mesenchymal migratory phenotype. These effects were decreased or abolished by silencing of LXR (liver X receptor) a and/or b, indicating that LXR(s) are important for the observed 24-hydroxycholesterol-induced responses. In conclusion, the current results support previous data reporting that 24-hydroxycholesterol has multiple effects on cells of the brain and indicate a previously unknown role for 24-hydroxycholesterol in pro-migratory and EMT-like processes of importance for glioblastoma tumor invasion.