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Glioblastoma is the most aggressive primary brain tumor in adults. Androgens are reported to influence the development of glioblastoma. Dihydrotestosterone (DHT) is formed from testosterone by action of the enzyme steroid 5α-reductase and is the most potent growth-inducing androgen metabolite. 24-Hydroxycholesterol, another steroid in the brain, is pivotal for brain cholesterol homeostasis and has been suggested to influence glioblastoma cells. However, a connection between 24-hydroxycholesterol and androgen metabolism related to glioblastoma has not previously been reported. The present study reports that human T98G glioblastoma cells metabolize testosterone into DHT, 3αandrostanediol and androstenedione. The 5α-reductase pathway converted testosterone to DHT and further to 3α-androstanediol. The 17β-hydroxysteroid dehydrogenase pathway metabolized testosterone to androstenedione. Results indicated that the 5α-reductase pathway is the major pathway for testosterone metabolism in this cell line. 24-Hydroxycholesterol significantly suppressed the conversion of testosterone to DHT and 3α-androstanediol, to a similar degree as the synthetic 5α-reductase inhibitor finasteride. Suppression of DHT formation resulted in increased metabolism to androstenedione. Similar effects on DHT formation were observed with the LXR agonist T0901317 as with 24-hydroxycho-lesterol. In addition, 24-hydroxycholesterol suppressed DHT formation in patient-derived primary GB cell lines U3009 and U3013, indicating that the observed connection between 24-hydroxycholesterol and androgen metabolism is not unique for T98G cells. Furthermore, 24-hydroxycholesterol-mediated suppression of DHT formation was also observed in human neuroblastoma SH-SY5Y cells. To summarize, the present data provide information on androgen metabolism in glioblastoma cells and indicate a previously unknown link between cholesterol homeostasis and growth-inducing androgens in glioblastoma and potentially other cell types.

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.

Vitamin D signaling is important in regulating calcium homeostasis essential for bone health but also displays other functions in cells of several tissues. Disturbed vitamin D signaling is linked to a large number of diseases. The multiple cytochrome P450 (CYP) enzymes catalyzing the different hydroxylations in bioactivation of vitamin D₃ are crucial for vitamin D signaling and function. This review is focused on the progress achieved in identification of the bioactivating enzymes and their genes in production of 1α,25-dihydroxyvitamin D₃ and other active metabolites. Results obtained on species- and tissue-specific expression, catalytic reactions, substrate specificity, enzyme kinetics, and consequences of gene mutations are evaluated. Matters of incomplete understanding regarding the physiological roles of some vitamin D hydroxylases are critically discussed and the authors will give their view of the importance of each enzyme for vitamin D signaling. Roles of different vitamin D receptors and an alternative bioactivation pathway, leading to 20-hydroxylated vitamin D₃ metabolites, are also discussed. Considerable progress has been achieved in knowledge of the vitamin D₃ bioactivating enzymes. Nevertheless, several intriguing areas deserve further attention to understand the pleiotropic and diverse activities elicited by vitamin D signaling and the mechanisms of enzymatic activation necessary for vitamin D-induced responses.

Determination of interactions between native proteins in cells is important for understanding function. Here the authors report MolBoolean as a method to detect interactions between endogenous proteins in subcellular compartments, using antibody-DNA conjugates for identification and signal amplification. Determining the levels of protein-protein interactions is essential for the analysis of signaling within the cell, characterization of mutation effects, protein function and activation in health and disease, among others. Herein, we describe MolBoolean - a method to detect interactions between endogenous proteins in various subcellular compartments, utilizing antibody-DNA conjugates for identification and signal amplification. In contrast to proximity ligation assays, MolBoolean simultaneously indicates the relative abundances of protein A and B not interacting with each other, as well as the pool of A and B proteins that are proximal enough to be considered an AB complex. MolBoolean is applicable both in fixed cells and tissue sections. The specific and quantifiable data that the method generates provide opportunities for both diagnostic use and medical research.

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 vitamin D hormone, 1,25-dihydroxyvitamin D-3, exerts many physiological actions in the body, including effects on the nervous system. Studies of steroidogenesis in cells of the nervous system and elsewhere not only indicate that 1,25-dihydroxyvitamin D-3 affects steroidogenic pathways but also suggest varying responses in different cell types. For example, 1,25-dihydroxyvitamin D-3 stimulates the expression of aromatase in human glioma but not in human neuroblastoma cells or rat astrocytes. However, in astrocytes, 1,25-dihydroxyvitamin D-3 suppresses 3 beta-hydroxysteroid dehydrogenase and steroid 17-hydroxylase/lyase. Other studies indicate cross-talk between vitamin D signalling and signalling of oestrogens, progesterone or glucocorticoids. Reported data indicate synergistic effects of combinations of 1,25-dihydroxyvitamin D-3 and other steroid hormones on neuroinflammation, neurite outgrowth and neuroprotection. Also, dysregulation of steroid pathways affecting brain cells is found in vitamin D deficiency. Thus, several studies suggest that active vitamin D may affect steroid hormone synthesis and/or signalling in the nervous system, although the potential mechanisms for these responses remain unclear. 1,25-Dihydroxyvitamin D-3 suppresses proliferation in several cell types and is therefore of interest in cancer treatment. Also, epidemiological studies associate vitamin D levels with cancer risk or outcomes. Reported data on tumours of the nervous system are mainly on glioma, a common type of brain cancer. Expression of the vitamin D receptor in glioma tumours is associated with improved survival. Several studies show that 1,25-dihydroxyvitamin D-3 and vitamin D analogues (synthetic vitamin D-like compounds) suppress proliferation and migration in human vitamin D receptor-expressing glioma cell lines. Studies on mechanisms for actions of 1,25-dihydroxyvitamin D-3 or its analogues indicate regulation of cell cycle proteins and senescence markers. These compounds also show synergism in combination with other cancer therapies treating glioma. From the data available, vitamin D analogues emerge as interesting candidates for the future improved treatment of human glioma and possibly also other cancers of the nervous system.

Vitamin D is essential for bone function and deficiency in active vitamin D hormone can lead to bone disorders. Long-term treatment with glucocorticoids results in osteoporosis and increased risk of fractures. Much remains unclear regarding the effects of these compounds in bone cells. In the current study, human osteosarcoma Saos-2 cells and primary human osteoblasts were found to express mRNA for the vitamin D receptor as well as activating and deactivating enzymes in vitamin D-3 metabolism. These bone cells exhibited CYP24A1-mediated 24-hydroxylation which is essential for deactivation of the active vitamin form. However, bioactivating vitamin D-3 hydroxylase activities could not be detected in either of these cells. Several glucocorticoids, including prednisolone, down regulated CYP24A1 mRNA and CYP24A1-mediated 24-hydroxylase activity in both Saos-2 and primary human osteoblasts. Also, prednisolone significantly suppressed a human CYP24A1 promoter-luciferase reporter gene in Saos-2 cells co-transfected with the glucocorticoid receptor. Thus, the results of the present study show suppression by glucocorticoids on CYP24A1 mRNA, CYP24A1-mediated metabolism and CYP24A1 promoter activity in human osteoblast-like cells. As part of this study we examined if glucocorticoids are formed locally in Saos-2 cells. The experiments indicate formation of 11-deoxycortisol, a steroid with glucocorticoid activity, which can bind the glucocorticoid receptor. Our data showing suppression by glucocorticoids on CYP24A1 expression in human osteoblasts suggest a previously unknown mechanism for effects of glucocorticoids in human bone, where these compounds may interfere with regulation of active vitamin D levels.

Steroids are reported to have diverse functions in the nervous system. Enzymatic production of steroid hormones has been reported in different cell types, including astrocytes and neurons. However, the information on some of the steroidogenic enzymes involved is insufficient in many respects. Contradictory results have been reported concerning the relative importance of different cell types in the nervous system for expression of CYP17A1 and 3b-hydroxysteroid dehydrogenase (3b-HSD). 3b-HSD is important in all basic steroidogenic pathways and CYP17A1 is required to form sex hormones. In the current investigation we studied the expression of these enzymes in cultured primary rat astrocytes, in neuron-enriched cells from rat cerebral cortex and in human neuroblastoma SH-SY5Y cells, a cell line often used as an in vitro model of neuronal function and differentiation. As part of this study we also examined potential effects on CYP17A1 and 3b-HSD by vitamin D, a compound previously shown to have regulatory effects in steroid hormone-producing cells outside the brain. The results of our study indicate that astrocytes are a major site for expression of 3b-HSD whereas expression of CYP17A1 is found in both astrocytes and neurons. The current data suggest that neurons, contrary to some previous reports, are not involved in 3b-HSD reactions. Previous studies have shown that vitamin D can influence gene expression and hormone production by steroidogenic enzymes in some cells. We found that vitamin D suppressed CYP17A1-mediated activity by 20% in SH-SY5Ycells and astrocytes. Suppression of CYP17A1 mRNA levels was considerably stronger, about 50% in SH-SY5Y cells and 75% in astrocytes. In astrocytes 3b-HSD was also suppressed by vitamin D, about 20% at the enzyme activity level and 60% at the mRNA level. These data suggest that vitamin D-mediated regulation of CYP17A1 and 3b-HSD, particularly on the transcriptional level, may play a role in the nervous system.

The active form of vitamin D (1,25-dihydroxyvitamin D) acts as a steroid hormone and binds to the vitamin D receptor. This receptor is expressed in most cell types including cells in the central nervous system (CNS). Vitamin D has several functions in the body including effects on brain development, neuroprotection and immunological regulation. It has been shown that vitamin D has antiproliferative activities in different cancer cell lines. Tacalcitol and calcipotriol are synthetic analogues of 1,25-dihydroxyvitamin D with reduced effect on calcium metabolism. The aim of this study was to analyse the effects of tacalcitol and calcipotriol on cell viability, proliferation and migration in the human glioblastoma cell line T98G. Glioblastoma is the most lethal type of primary tumours in the CNS. Both analogues decreased cell viability and/or growth, dose-dependently, in concentrations between 1 nM and 10 M. Manual counting indicated suppressive effects by the vitamin D analogues on proliferation. Treatment with tacalcitol strongly suppressed thymidine incorporation, indicating that the vitamin D analogues mainly inhibit proliferation. Also, effects on cell migration were measured with wound-healing assay. Both calcipotriol and tacalcitol reduced the migration rate of T98G cells compared to vehicle-treated cells. However, they had no effect on caspase-3 and -7 activities, suggesting that their mechanism of action does not involve induction of apoptosis. The current results indicate that the vitamin D analogues tacalcitol and calcipotriol strongly reduce proliferation and migration of human glioblastoma T98G cells, suggesting a potential role for this type of compounds in treatment of brain cancer.