Disruptions in reproductive health, sex hormonal profiles, and hypothalamic hormone receptors content in females of the C58/J mouse model of autism.

Autism Spectrum Disorder (ASD) is characterized by differences in social communication and interaction, as well as areas of focused interests and/or repetitive behaviors. Recent studies have highlighted a higher prevalence of endocrine and reproductive disturbances among females on the autism spectrum, hinting at potential disruptions within the hypothalamus-pituitary-ovary (HPO) axis. This research aims to explore the reproductive health disparities in ASD using an animal model of autism, the C58/J inbred mouse strain, with a focus on reproductive performance and hormonal profiles compared to the C57BL/6J control strain. Our findings revealed that the estrous cycle in C58/J females is disrupted, as evidenced by a lower frequency of complete cycles and a lack of cyclical release of estradiol and progesterone compared to control mice. C58/J females also exhibited poor performance in several reproductive parameters, including reproductive lifespan and fertility index. Furthermore, estrogen receptor alpha content showed a marked decrease in the hypothalamus of C58/J mice. These alterations in the estrous cycle, hormonal imbalances, and reduced reproductive function imply dysregulation in the HPO axis. Additionally, our in-silico study identified a group of genes involved in infertility carrying single-nucleotide polymorphisms (SNPs) in the C58/J strain, which also have human orthologs associated with autism. These findings could offer valuable insights into the molecular underpinnings of neuroendocrine axis disruption and reproductive issues observed in ASD.

Progesterone and its metabolite allopregnanolone promote invasion of human glioblastoma cells through metalloproteinase­9 and cSrc kinase.

Glioblastomas are the most aggressive and common primary brain tumors in adults. Glioblastoma cells have a great capacity to migrate and invade the brain parenchyma, often reaching the contralateral hemisphere. Progesterone (P4) and its metabolite, allopregnanolone (3α-THP), promote the migration and invasion of human glioblastoma-derived cells. P4 induces migration in glioblastoma cells by the activation of the proto-oncogene tyrosine-protein kinase Src (cSrc) and focal adhesion kinase (Fak). In breast cancer cells, cSrc and Fak promote invasion by increasing the expression and activation of extracellular matrix metalloproteinases (MMPs). However, the mechanism of action by which P4 and 3a-THP promote invasion in glioblastoma cells remains unclear. The effects of P4 and 3α-THP on the protein expression levels of MMP-2 and -9 and the participation of cSrc in progestin effects in U251 and U87 human glioblastoma-derived cells were evaluated. It was determined by western blotting that the P4 increased the protein expression level of MMP-9 in U251 and U87 cells, and 3α-THP increased the protein expression level of MMP-9 in U87 cells. None of these progestins modified MMP-2 protein expression levels. The increase in MMP-9 expression was reduced when the intracellular progesterone receptor and cSrc expression were blocked with small interfering RNAs. Cell invasion induced by P4 and 3α-THP was also blocked by inhibiting cSrc activity with PP2 or by cSrc gene silencing. These results suggest that P4 and its metabolite 3α-THP induce the invasion of glioblastoma cells by increasing MMP-9 expression through the cSrc kinase family. The results of this study provide information of interest in the context of targeted therapies against molecular pathways involved in glioblastoma invasion.

Rapid Actions of the Nuclear Progesterone Receptor through cSrc in Cancer.

The nuclear progesterone receptor (PR) is mainly known for its role as a ligand-regulated transcription factor. However, in the last ten years, this receptor's extranuclear or rapid actions have gained importance in the context of physiological and pathophysiological conditions such as cancer. The PR's polyproline (PXPP) motif allows protein-protein interaction through SH3 domains of several cytoplasmatic proteins, including the Src family kinases (SFKs). Among members of this family, cSrc is the most well-characterized protein in the scenario of rapid actions of the PR in cancer. Studies in breast cancer have provided the most detailed information on the signaling and effects triggered by the cSrc-PR interaction. Nevertheless, the study of this phenomenon and its consequences has been underestimated in other types of malignancies, especially those not associated with the reproductive system, such as glioblastomas (GBs). This review will provide a detailed analysis of the impact of the PR-cSrc interplay in the progression of some non-reproductive cancers, particularly, in GBs.

Allopregnanolone Promotes Migration and Invasion of Human Glioblastoma Cells through the Protein Tyrosine Kinase c-Src Activation.

Glioblastomas (GBs) are the most aggressive and common primary malignant brain tumors. Steroid hormone progesterone (P4) and its neuroactive metabolites, such as allopregnanolone (3α-THP) are synthesized by neural, glial, and malignant GB cells. P4 promotes cellular proliferation, migration, and invasion of human GB cells at physiological concentrations. It has been reported that 3α-THP promotes GB cell proliferation. Here we investigated the effects of 3α-THP on GB cell migration and invasion, the participation of the enzymes involved in its metabolism (AKR1C1-4), and the role of the c-Src kinase in 3α-THP effects in GBs. 3α-THP 100 nM promoted migration and invasion of U251, U87, and LN229 human-derived GB cell lines. We observed that U251, LN229, and T98G cell lines exhibited a higher protein content of AKR1C1-4 than normal human astrocytes. AKR1C1-4 silencing did not modify 3α-THP effects on migration and invasion. 3α-THP activated c-Src protein at 10 min (U251 cells) and 15 min (U87 and LN229 cells). Interestingly, the pharmacological inhibition of c-Src decreases the promoting effects of 3α-THP on cell migration and invasion. Together, these data indicate that 3α-THP promotes GB migration and invasion through c-Src activation.

Allopregnanolone: Metabolism, Mechanisms of Action, and Its Role in Cancer.

Allopregnanolone (3α-THP) has been one of the most studied progesterone metabolites for decades. 3α-THP and its synthetic analogs have been evaluated as therapeutic agents for pathologies such as anxiety and depression. Enzymes involved in the metabolism of 3α-THP are expressed in classical and nonclassical steroidogenic tissues. Additionally, due to its chemical structure, 3α-THP presents high affinity and agonist activity for nuclear and membrane receptors of neuroactive steroids and neurotransmitters, such as the Pregnane X Receptor (PXR), membrane progesterone receptors (mPR) and the ionotropic GABAA receptor, among others. 3α-THP has immunomodulator and antiapoptotic properties. It also induces cell proliferation and migration, all of which are critical processes involved in cancer progression. Recently the study of 3α-THP has indicated that low physiological concentrations of this metabolite induce the progression of several types of cancer, such as breast, ovarian, and glioblastoma, while high concentrations inhibit it. In this review, we explore current knowledge on the metabolism and mechanisms of action of 3α-THP in normal and tumor cells.

LPA1 Receptor Promotes Progesterone Receptor Phosphorylation through PKCα in Human Glioblastoma Cells.

Lysophosphatidic acid (LPA) induces a wide range of cellular processes and its signaling is increased in several cancers including glioblastoma (GBM), a high-grade astrocytoma, which is the most common malignant brain tumor. LPA1 receptor is expressed in GBM cells and its signaling pathways activate protein kinases C (PKCs). A downstream target of PKC, involved in GBM progression, is the intracellular progesterone receptor (PR), which can be phosphorylated by this enzyme, increasing its transcriptional activity. Interestingly, in GBM cells, PKCα isotype translocates to the nucleus after LPA stimulation, resulting in an increase in PR phosphorylation. In this study, we determined that LPA1 receptor activation induces protein-protein interaction between PKCα and PR in human GBM cells; this interaction increased PR phosphorylation in serine400. Moreover, LPA treatment augmented VEGF transcription, a known PR target. This effect was blocked by the PR selective modulator RU486; also, the activation of LPA1/PR signaling promoted migration of GBM cells. Interestingly, using TCGA data base, we found that mRNA expression of LPAR1 increases according to tumor malignancy and correlates with a lower survival in grade III astrocytomas. These results suggest that LPA1/PR pathway regulates GBM progression.

Estradiol Induces Epithelial to Mesenchymal Transition of Human Glioblastoma Cells.

The mesenchymal phenotype of glioblastoma multiforme (GBM), the most frequent and malignant brain tumor, is associated with the worst prognosis. The epithelial-mesenchymal transition (EMT) is a cell plasticity mechanism involved in GBM malignancy. In this study, we determined 17ß-estradiol (E2)-induced EMT by changes in cell morphology, expression of EMT markers, and cell migration and invasion assays in human GBM-derived cell lines. E2 (10 nM) modified the shape and size of GBM cells due to a reorganization of actin filaments. We evaluated EMT markers expression by RT-qPCR, Western blot, and immunofluorescence.We found that E2 upregulated the expression of the mesenchymal markers, vimentin, and N-cadherin. Scratch and transwell assays showed that E2 increased migration and invasion of GBM cells. The estrogen receptor-α (ER-α)-selective agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT, 10 nM) affected similarly to E2 in terms of the expression of EMT markers and cell migration, and the treatment with the ER-α antagonist methyl-piperidino-pyrazole (MPP, 1 µM) blocked E2 and PPT effects. ER-ß-selective agonist diarylpropionitrile (DNP, 10 nM) and antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazole[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 1 µM) showed no effects on EMT marker expression. These data suggest that E2 induces EMT activation through ER-α in human GBM-derived cells.

Sex hormones and proteins involved in brain plasticity.

It is well known that peripheral sex steroid hormones cross the blood-brain barrier and control a broad spectrum of reproductive behaviors. However, their role in other essential brain functions was investigated since the 1980s, when the accumulation of pregnenolone and dehydroepiandrosterone in the brain of mammalian species was determined. Since then, numerous studies have demonstrated the participation of sex hormones in brain plasticity processes. Sex hormones through both genomic and non-genomic mechanisms of action are capable of inducing gene transcription or activating signaling cascades that result in the promotion of different physiological and pathological events of brain plasticity, such as remodeling or formation of dendritic spines, neurogenesis, synaptogenesis or myelination. In this chapter, we will present the effects of sex hormones and proteins involved in brain plasticity.

5alpha-dihydroprogesterone promotes proliferation and migration of human glioblastoma cells.

Glioblastomas (GBMs) are the most common and deadliest intracranial tumors. Steroid hormones, such as progesterone (P4), at physiological concentrations, promote proliferation, and migration of human GBM cells in vivo and in vitro. Neuronal and glial cells, but also GBMs, metabolize P4 and synthesize different active metabolites such as 5α-dihydroprogesterone (5α-DHP). However, their contribution to GBM malignancy remains unknown. Here, we determined the 5α-DHP effects on the number of cells, proliferation, and migration of the U87 and U251 human GBM-derived cell lines. Of the tested concentrations (1 nM-1 µM), 5α-DHP 10 nM significantly increased the number of U87 and U251 cells from day 2 of treatment, and proliferation (at day 3) in a similar manner as P4 (10 nM). The treatment with the progesterone receptor (PR) antagonist RU486 (mifepristone), blocked the effects of 5α-DHP on the number of cells and proliferation. Besides, in U251 and LN229 GBM cells, 5α-DHP promoted cell migration (from 12 to 24 h). We also determined that GBM cells expressed the 3α-hydroxysteroid oxidoreductases (3α-HSOR), which reversibly reduce 5α-DHP to allopregnanolone (3α-THP). These data indicate that 5α-DHP induces proliferation and migration of human GBM through the activation of PR.

Tibolone Effects on Human Glioblastoma Cell Lines.

BACKGROUND: Ovarian steroid hormones are involved in modulating the growth of glioblastomas (the most common, aggressive, and lethal brain tumor) through the interaction with their intracellular receptors. Activation of sex hormone receptors is involved in glioblastomas progression. Tibolone (TIB) is a selective tissue estrogenic activity regulator widely prescribed to treat menopausal symptoms and to prevent bone lost. The effects of TIB on the growth of glioblastoma are unknown. AIM OF THE STUDY: To evaluate the effects of TIB on cell number, migration, and invasion of two derived human glioblastoma cell lines (U251 MG and U87), as well as the role of this steroid in estrogen and progesterone receptors activity and content. METHODS: U251-MG and U87 human glioblastoma cell lines were grown with different doses of TIB. The number of cells was determined and migration and invasion tests were carried out. Protein expression was performed by Western blot. RESULTS: We observed that TIB (10 nM) increased the number of cells by inducing proliferation with no effects on cell migration or invasion. The increase in cell proliferation induced by TIB was blocked by estrogen (ERs) or progesterone receptor (PRs) antagonists, ICI 182, 780 and RU 486, suggesting that these receptors mediate proliferating actions of TIB; TIB also modified the content of ERs and PRs by increasing ER-α, ER-ß, and PR-B, while decreased PR-A. CONCLUSION: Our results suggest that TIB increases cell number and proliferation of human glioblastoma cells through the regulation of ERs and PRs actions and content.

Allopregnanolone Alters the Gene Expression Profile of Human Glioblastoma Cells.

Glioblastomas (GBM) are the most frequent and aggressive brain tumors. In these malignancies, progesterone (P4) promotes proliferation, migration, and invasion. The P4 metabolite allopregnanolone (3α-THP) similarly promotes cell proliferation in the U87 human GBM cell line. Here, we evaluated global changes in gene expression of U87 cells treated with 3α-THP, P4, and the 5α-reductase inhibitor, finasteride (F). 3α-THP modified the expression of 137 genes, while F changed 90. Besides, both steroids regulated the expression of 69 genes. After performing an over-representation analysis of gene ontology terms, we selected 10 genes whose products are cytoskeleton components, transcription factors, and proteins involved in the maintenance of DNA stability and replication to validate their expression changes by RT-qPCR. 3α-THP up-regulated six genes, two of them were also up-regulated by F. Two genes were up-regulated by P4 alone, however, such an effect was blocked by F when cells were treated with both steroids. The remaining genes were regulated by the combined treatments of 3α-THP + F or P4 + F. An in-silico analysis revealed that promoters of the six up-regulated genes by 3α-THP possess cyclic adenosine monophosphate (cAMP) responsive elements along with CCAAT/Enhancer binding protein alpha (CEBPα) binding sites. These findings suggest that P4 and 3α-THP regulate different sets of genes that participate in the growth of GBMs.

Allopregnanolone promotes proliferation and differential gene expression in human glioblastoma cells.

Allopregnanolone (3α-THP) is one of the main reduced progesterone (P4) metabolites that is recognized as a neuroprotective and myelinating agent. 3α-THP also induces proliferation of different neural cells. It has been shown that P4 favors the progression of glioblastomas (GBM), the most common and aggressive primary brain tumors. However, the role of 3α-THP in the growth of GBMs is unknown. Here, we studied the effects of 3α-THP on the number of cells, proliferation and gene expression in U87 cell line derived from a human GBM. 3α-THP (10, 100nM and 1µM) increased the number of U87 cells, and at 10nM exerted a similar increase in both the number of total and proliferative U87 cells as compared with P4 (10nM). Interestingly, finasteride (F; 100nM), an inhibitor of 5α-reductase (5αR), an enzyme necessary to metabolize P4 and produce 3α-THP, blocked the increase in the number of U87 cells induced by P4. By using RT-qPCR, we determined that U87 cells express 5α-R isoenzymes 1 and 2 (5αR1 and 5αR2), being 5αR1 the predominant one in these cells. 3α-THP (10nM) increased the expression of TGFß1, EGFR, VEGF and cyclin D1 genes. P4 increased TGFß1 and EGFR expression, and this effect was blocked by F. These data provide evidence that P4, through its metabolite 3α-THP, can promote in part cell proliferation of human GBM cells by changing the expression of genes involved in tumor progression.