Glioblastoma cells express crucial enzymes involved in androgen synthesis: 3β‐hydroxysteroid dehydrogenase, 17‐20α‐hydroxylase, 17β‐hydroxysteroid dehydrogenase and 5α‐reductase

Abstract Glioblastoma (GB) is the most common and aggressive primary brain tumour in adult humans. Therapeutic resistance and tumour recurrence after surgical removal contribute to poor prognosis for glioblastoma patients. Men are known to be more likely than women to develop an aggressive form of GB, and differences in sex steroids have emerged as a leading explanation for this finding. Studies indicate that the metabolism and proliferation of GB‐derived cells are increased by sex steroids, the expression of androgen receptors (ARs) and the synthesis of androgens and oestrogens, suggesting that these hormones have a role in the tumour pathogenesis. The expression of aromatase, the enzyme that converts androgens to oestrogens, has been reported in glial cells and GB cell lines. Thus, it was necessary to test whether the steroidogenic enzymes involved in androgen synthesis are expressed in GB cells. Therefore, here, we investigated the expression of four key enzymes involved in androgen synthesis in human‐derived GB cells. U87 cells were cultured in Dulbecco's modified Eagle medium plus foetal bovine serum and antibiotics on slides for immunocytochemistry or immunofluorescence. U87, LN229 and C6 cells were also cultured in multi‐well chambers to obtain proteins for Western blotting. We used primary antibodies against 3β‐hydroxysteroid dehydrogenase (3β‐HSD), 17α‐hydroxilase/17,20‐lyase (P450c17), 17β‐hydroxysteroid dehydrogenase (17β‐HSD) and 5α‐reductase. Immunocytochemistry, and immunofluorescence results revealed that glioblastoma cells express 3β‐HSD, P450c17, 17β‐HSD and 5α‐reductase proteins in their cytoplasm. Moreover, Western blot analyses revealed bands corresponding to the molecular weight of these four enzymes in the three GB cell lines. Thus, glioblastoma cells have the key enzymatic machinery necessary to synthesize androgens, and these enzymes might be useful targets for new therapeutic approaches.

gens and oestrogens, suggesting that these hormones have a role in the tumour pathogenesis. The expression of aromatase, the enzyme that converts androgens to oestrogens, has been reported in glial cells and GB cell lines. Thus, it was necessary to test whether the steroidogenic enzymes involved in androgen synthesis are expressed in GB cells. Therefore, here, we investigated the expression of four key enzymes involved in androgen synthesis in human-derived GB cells. U87 cells were cultured in Dulbecco's modified Eagle medium plus foetal bovine serum and antibiotics on slides for immunocytochemistry or immunofluorescence. U87, LN229 and C6 cells were also cultured in multi-well chambers to obtain proteins for Western blotting. We used primary antibodies against 3β-hydroxysteroid dehydrogenase (3β-HSD), 17α-hydroxilase/17,20-lyase (P450c17), 17β-hydroxysteroid dehydrogenase (17β-HSD) and 5α-reductase. Immunocytochemistry, and immunofluorescence results revealed that glioblastoma cells express 3β-HSD, P450c17, 17β-HSD and 5αreductase proteins in their cytoplasm. Moreover, Western blot analyses revealed bands corresponding to the molecular weight of these four enzymes in the three GB cell lines. Thus, glioblastoma cells have the key enzymatic machinery necessary to synthesize androgens, and these enzymes might be useful targets for new therapeutic approaches.

| INTRODUC TI ON
Among local brain tumour types, glioma is the most aggressive. The World Health Organization (WHO) has proposed classifying gliomas into four grades based on a set of morphological criteria that determine the cell lineage. 1 More recently, a molecular-based classification (WHO, 2016) for GB was proposed, where isocitrate dehydrogenase wild-type IDH that accounts for about 90% of cases, frequently corresponds with the clinically defined primary or de novo glioblastoma, and predominates in patients over 55 years old, and GB IDH-mutant (accounting for 10% of cases), socalled secondary glioblastoma with a history of prior lower grade diffuse glioma, occurs preferentially in younger patients. 2 The treatments for those types of neoplasms consist mainly of surgery for tumour ablation, radiotherapy and chemotherapy, mainly with temozolomide (TMZ). Unfortunately, patient survival is poor because of frequent recurrences.
Several studies have proposed that steroid hormones play an important role in developing gliomas. 3 Preliminary evidence was provided by statistics from the United States of America, where the incidence in the adult population is 50% higher in men than in women 4,5 ). Likewise, the expression of receptors to oestrogen, progesterone and androgens in glioma cell lines and tumour biopsies in GB cell lines has been reported (Gandhari et al., 2006, 6,7,40 ,Chang et al. 2014 The synthesis of neurosteroids by the central nervous system (CNS) comprises several enzymes. Steroidogenesis in the CNS begins with cholesterol entry into the mitochondria, which is regulated by the protein StAR (steroidogenic acute regulatory protein), which captures cholesterol in the outer mitochondrial membrane and transfers it to the peripheral receptor to benzodiazepines (PBR) located in the mitochondrial membrane. This forms a channel that directs the cholesterol to the inner mitochondrial membrane, where the first steroidogenic enzyme is found, cholesterol desmolase, cytochrome P450scc (side-chain cleavage), which synthesizes pregnenolone (P 5 ) in the endoplasmic reticulum. Starting from cholesterol, the first step of the pathway is to synthesize pregnenolone (P5), which is then metabolized to various hormones. The 3β-hydroxysteroid dehydrogenase delta 4-5 isomerase (3β-HSD) is a key enzyme in these metabolic pathways and is necessary for producing the 3-oxo-group present in most steroid hormones. This enzyme is also expressed in SH-SY5Y cells 8 [9]. CYP17 A1 (also termed 17α-hydroxylase/C17-20-lyase) catalyses the hydroxylation of C21 steroids to C19 steroids. CYP17 A1 is also required to form dehydroepiandrosterone (DHEA) and androstenedione, weak androgens that are converted to testosterone (T) and estrone (E 1 ).
The expression of CYP17A1 mRNAs in the nervous system is controversial, but has been reported in neurons and astrocytes of the rat brain and SH-SY5Y cells. 8,9 The multifunctional enzyme 17βhydroxysteroid dehydrogenase catalyses the oxidation of neuroactive steroids in the brain, which are involved in brain development, neurogenesis, neural plasticity and neuroprotection and has an impact on brain diseases. The originally described functions of 17βhydroxysteroid dehydrogenases were to maintain the balance between the less potent (17-Keto) and more potent (17β-hydroxy) forms of oestrogen and androgens, also, they are involved in the metabolism of fatty acids and sterols (for a review, please see. 10 The expression of the steroidogenic enzymes P450 cholesterol side-chain-cleavage and P450-aromatase (P450arom) in glioma cell The ability of GB-derived cells to synthesize steroids has been reported by Papadopolous et al. 11 , who demonstrated that rat C6 cells transformed 22-hydroxy cholesterol to pregnenolone, and by Melcangi et al. 14 , who reported that C6 and 1321 N1 human astrocytoma-derived cells converted testosterone and progesterone into 5α-reduced derivatives DHT and dihydroprogesterone (DHP). We have recently shown that U87 cells synthesize several androgens, and that the production can be inhibited by the 5α-reductase inhibitors finasteride and dutasteride. 15 Those results strongly suggest the presence of a panoply of enzymes involved in androgen synthesis.
The final products of steroidogenesis in each tissue depend on the enzymes that synthesize these hormones, such as P450cytochromes, steroid dehydrogenases and reductases. 16 Baulieu 17 described the CNS as a steroidogenic tissue and showed that steroids (eg pregnenolone (P 5 ) and DHEA) and their sulphated forms (P 4 S and DHEAS) were found in higher concentrations in the brain than in the peripheral circulation of rodents Robel and

Baulieu. 18
The central nervous system (CNS) expresses most of the synthesizing enzymes or modifiers of steroids; most enzymes involved in the steroid synthesis have been documented in the rat brain and regions of the human brain. [19][20][21][22] The enzymatic pathways involved in neurosteroid synthesis and regulation have been extensively studied. 23 Oligodendrocytes, astrocytes and neurons present steroidogenic capacity. However, it is important to note that these enzymes have distinct expression profiles and activities in neurons and glial cells. 9 The conversion of androgens like testosterone K E Y W O R D S cancer, glioblastoma, neurosteroids, steroidogenic enzymes to DHT, the potent AR agonist, results from 5α-reductase activities.
Therefore, these enzymes are essential for androgen synthesis in several classic steroidogenic tissues. 24 Three types of this enzyme have been described, they are expressed in the normal prostate and are associated with prostate cancer initiation and progression 25 ,the prostate 5α-reductase mRNA levels of types 1 and 2 are regulated by testosterone and DHT. 25 The 5α-reductase isoforms 1 and 2 are express in the human brain where they participate in synthesis of neurosteroids. 22 The 5α-reductase type 1 is constitutively expressed in the rat brain,its expression is negatively regulated by T and DHT, while gene expression of 5α-reductase type 2 is under the positive control of T and DHT. 26 The physiological importance of 5α-reductases in the brain derives from its ability to convert testosterone (T) to the potent androgen, dihydrotestosterone (DHT) and progesterone and deoxycorticosterone (DOC) to their respective 5α-reduced derivatives, precursors of allopregnanolone and tetrahydro DOC, which are both strong allosteric modulators of the aminobutyric acid receptor (GABA A R). 27 The implication of 5α-reductases and aromatase in the growth of androgen and oestrogen-sensitive tumours, such as prostate, breast and GB cancers, suggested that these steroidogenic enzymes are expressed in GB-derived cells. Using RT-qPCR, Zamora-Sanchez et al. 28 have shown 5α-reductase mRNA expression in U87

cells.
We have recently shown that the androgens T 4 and DHT, but not androstenedione, significantly increase GB metabolism, and that the effect of these androgens is inhibited by using the 5α-reductase inhibitor dutasteride or the androgen receptor blocker finasteride. 29 Since androgens have a role in GB development, it is important to have a deeper insight into the tumour steroidogenic ability, focusing on androgen synthesis. Here, we aimed to describe the expression of key steroidogenic enzymes involved in the androgen synthesis by GB-derived cells.

| Immunocytochemistry
The cultured cells were fixed in 4% paraformaldehyde and ethanol dehydrated. An antigenic unmask was carried using a 0.1 M citrate buffer solution, pH 6.0 (DECLERE, Cell Mark, Rocklin, CA, USA), followed by the inhibition of endoperoxidase activity with 5% H 2 O 2 .
After washing the slides, they were incubated with specific primary antibodies for CYP17 (Rabbit Polyclonal CYP17A1 antibody, GeneTex), for 5α-reductase Type2 (rabbit monoclonal [EPR6281(B)] to SRD5A2, Abcam) and antisera against 17β HSD type I (Raised in rabbit using purified type 1 from the human placenta) and 3β-HSD were taken with a digital camera (Infinity 1-Lumenera) with a 40× objective, using an Infinity 1-Lumenera camera equipped with a 10× objective, aided by an Olympus microscope.

| Immunofluorescence
For immunofluorescence observations, we fixed the slides as described for immunocytochemistry and exposed them to the same

| Western Blotting
Cells were scrapped with a cell homogenizer and mixed for 30 s at Nitrocellulose membranes with the transferred proteins were placed in a blocking solution (Millipore Chemiluminescent Blocker) at 50% for 30 min. The membranes were incubated with the same primary antibodies specific for 3β-HSD, CYP17, 17β HSD and 5α reductase used for immunocytochemistry described above, at a dilution of 1:1000 in the same blocking solution. On the following day, membranes were incubated with a secondary anti-rabbit antibody (BioRad) at a dilution of 1:5000 in blocking solution. Membranes were revealed with chemiluminescence (Millipore, corporation, Billerica, U.S.A) and exposed to a film (Hiperfilm). The internal control used was glyceraldehyde-3-phosphate dehydrogenase (GADPH) or tubulin. The bands obtained were analysed and quantified by densitometry using Image Studio Software (LI-COR Biosciences). The expression of U87 protein (42 KDa MW) corresponding to 3β-HSD can be observed in the Western blot analysis in Figure 1D. This figure also shows the expression of the 3β-HSD protein in another GB cell lines, LN229 and C6.

| RE SULTS
By immunocytochemistry, we detected the expression of 17αhydroxylase (CYP17), the enzyme that converts progesterone to 17OH-progesterone and androstenedione in U87 cells ( Figure 2B).
Confocal images confirmed the cytoplasmic distribution of the enzyme in U87 cells ( Figure 2C). Western blot analysis showed a band corresponding to the molecular weight of the of CYP17 protein (57 KDa MW, Figure 2D). This figure also shows the expression of the 3β-HSD protein in another GB cell lines, LN229 and C6.
The 17β-hydroxyesteroid dehydrogenase expression was found in the cytoplasm of U87 cells exposed to a specific antibody by immunocytochemistry ( Figure 3B). Figure 3C Figure 3D). This figure also shows that the expression of the 3β-HSD protein is also present in another GB cell lines, LN229 and C6. The expression of 5α-reductase type 2, an enzyme that converts T to the potent androgen DHT, is present in the cytoplasm of U 87 cells, as shown by immunocytochemistry ( Figure 4B). The enzyme was also observed in confocal microscopy images ( Figure 4C).
Western blot analysis detected the expression of the 5α-reductase protein (25 KDa MW) that can be observed in Figure 4D. This figure also shows that L229 and C6 cells express the protein. The activity of 17beta-HSD has been suspected based on brain tissue capacity to synthesize androgens and oestrogens 18,23 . We have previously found the transformation of A 4 to testosterone, and DHEA to androstenediol by U87-derived GB cells 15 ,therefore, the enzyme should be necessarily present in these cells, as we show in this study using immunohistochemical procedures. The expression of the enzyme found by Western blot further confirms the presence of the protein in GB cells.
Type 1 17beta-HSD also participates in the synthesis of oestrogens, transforming estrone to estradiol and vice-versa. 36 Our study showed that the type 1 17beta-HSD antibody recognized the GB enzyme in the immunocytochemical and Western blot studies presented here, confirming that type 1 is present in the cells. This type facilitates the reduction in DHEA to androstenediol, and DHT to 3α and 3β-diol, and reactions that other authors and we have shown to occur in GB cells. 37 We and others have previously shown that the potent androgens testosterone and DHT increased the metabolism and proliferation of GB-derived cells 29,38 , and these steroids positively control the gene expression of 5α-reductase type 2. 26 The 5α-reductase protein was  shown that testosterone, promotes cell proliferation, migration and invasion of GB-derived cells through the androgen receptors.
Therefore, the expression of 5α-reductase would be essential to provide the cells with potent androgens necessary for cell metabolism. We recently showed that dutasteride, or the combination of this 5α-reductase inhibitor with the AR blockers, cyproterone and flutamide, significantly reduced the metabolism, cell proliferation and migration capacity of U87, 29 findings that strongly support the presence of 5α-reductases found in these cells.
The results presented here indicate that U87 cells maintain the steroidogenic enzymes present in glial cells. Therefore, U87 cells have the machinery necessary to synthesize androgens and other neurosteroids that might have a role in the GB development. These findings were also shown in LN229 and C6 cells. These enzymes might be considered as therapeutic targets for the treatment of human glioblastomas. Romano.

CO N FLI C T O F I NTE R E S T
We declare that there is notconflict of interest

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available