Revisiting the expression and function of follicle-stimulation hormone receptor in human umbilical vein endothelial cells

Expression of follicle-stimulation hormone receptor (FSHR) is confined to gonads and at low levels to some extragonadal tissues like human umbilical vein endothelial cells (HUVEC). FSH-FSHR signaling was shown to promote HUVEC angiogenesis and thereafter suggested to have an influential role in pregnancy. We revisited hereby the expression and functionality of FSHR in HUVECs angiogenesis, and were unable to reproduce the FSHR expression in human umbilical cord, HUVECs or immortalized HUVECs (HUV-ST). Positive controls as granulosa cells and HEK293 cells stably transfected with human FSHR cDNA expressed FSHR signal. In contrast to positive control VEGF, FSH treatment showed no effects on tube formation, nitric oxide production, wound healing or cell proliferation in HUVEC/HUV-ST. Thus, it remains open whether the FSH-FSHR activation has a direct regulatory role in the angiogenesis of HUVECs

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model

Background/Aims: The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. Methods: We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7(shPRODH/POX)) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. Results: PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7(shPRODH/POX) cells. All studied compounds decreased cell viability in MCF-7 and MCF-7(shPRODH/POX) cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7(shPRODH/POX) and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7(shPRODH/POX) cells and contributed to the induction of pro-survival mode only in MCF-7(shPRODH/POX) cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. Conclusion: PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7(shPRODH/POX) cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways. (C) 2017 The Author(s) Published by S. Karger AG, Base

Platelet Carbonic Anhydrase II, a Forgotten Enzyme, May Be Responsible for Aspirin Resistance

Background. Thromboembolic events constitute a major health problem, despite the steadily expanding arsenal of antiplatelet drugs. Hence, there is still a need to optimize the antiplatelet therapy. Objectives. The aim of our study was to verify a hypothesis that there are no differences in platelet proteome between two groups of healthy people representing different acetylsalicylic acid (aspirin) responses as assessed by the liquid chromatography/mass spectrometry (LC/MS) technique. Patients/Methods. A total of 61 healthy volunteers were recruited for the study. Physical examination and blood collection were followed by platelet-rich plasma aggregation assays and platelet separation for proteomic LC/MS analysis. Arachidonic acid- (AA-) induced aggregation (in the presence of aspirin) allowed to divide study participants into two groups aspirin-resistant (AR) and aspirin-sensitive (AS) ones. Subsequently, platelet proteome was compared in groups using the LC/MS analysis. Results. The LC/MS analysis of platelet proteome between groups revealed that out of all identified proteins, the only discriminatory protein, affecting aspirin responsiveness, is platelet carbonic anhydrase II (CA II). Conclusions. CA II is a platelet function modulator and should be taken into consideration as a cardiovascular event risk factor or therapeutic target

Constitutively active follicle-stimulating hormone receptor enables androgen-independent spermatogenesis

Spermatogenesis is regulated by the 2 pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This process is considered impossible without the absolute requirement of LH-stimulated testicular testosterone (T) production. The role of FSH remains unclear because men and mice with inactivating FSH receptor (FSHR) mutations are fertile. We revisited the role of FSH in spermatogenesis using transgenic mice expressing a constitutively strongly active FSHR mutant in a LH receptor-null (LHR-null) background. The mutant FSHR reversed the azoospermia and partially restored fertility of Lhr(-/-) mice. The finding was initially ascribed to the residual Leydig cell T production. However, when T action was completely blocked with the potent antiandrogen flutamide, spermatogenesis persisted. Hence, completely T-independent spermatogenesis is possible through strong FSHR activation, and the dogma of T being a sine qua non for spermatogenesis may need modification. The mechanism for the finding appeared to be that FSHR activation maintained the expression of Sertoli cell genes considered androgen dependent. The translational message of our findings is the possibility of developing a new strategy of high-dose FSH treatment for spermatogenic failure. Our findings also provide an explanation of molecular pathogenesis for Pasqualini syndrome (fertile eunuchs; LH/T deficiency with persistent spermatogenesis) and explain how the hormonal regulation of spermatogenesis has shifted from FSH to T dominance during evolution

Luteinizing hormone and GATA4 action in the adrenocortical tumorigenesis of gonadectomized female mice

Background/Aims: Physiological role of luteinizing hormone (LH) and its receptor (LHCGR) in adrenal remains unknown. In inhibin-α/Simian Virus 40 T antigen (SV40Tag) (inhα/Tag) mice, gonadectomy-induced (OVX) elevated LH triggers the growth of transcription factor GATA4 (GATA4)-positive adrenocortical tumors in a hyperplasia-adenoma-adenocarcinoma sequence. Methods: We investigated the role of LHCGR in tumor induction, by crossbreeding inhα/Tag with Lhcgr knockout (LuRKO) mice. By knocking out Lhcgr and Gata4 in Cα1 adrenocortical cells (Lhcgr-ko, Gata4-ko) we tested their role in tumor progression. Results: Adrenal tumors of OVX inhα/Tag mice develop from the hyperplastic cells localized in the topmost layer of zona fasciculata. OVX inhα/Tag/LuRKO only developed SV40Tag positive hyperplastic cells that were GATA4 negative, cleaved caspase-3 positive and did not progress into adenoma. In contrast to Lhcgr-ko, Gata4-ko Cα1 cells presented decreased proliferation, increased apoptosis, decreased expression of Inha, SV40Tag and Lhcgr tumor markers, as well as up-regulated adrenal- and down-regulated sex steroid gene expression. Both Gata4-ko and Lhcgr-ko Cα1 cells had decreased expression of steroidogenic genes resulting in decreased basal progesterone production. Conclusion: Our data indicate that LH/LHCGR signaling is critical for the adrenal cell reprogramming by GATA4 induction prompting adenoma formation and gonadal-like phenotype of the adrenocortical tumors in inhα/Tag mice.</p

A Patient-Derived Cell Atlas Informs Precision Targeting of Glioblastoma

Glioblastoma (GBM) is a malignant brain tumor with few therapeutic options. The disease presents with a complex spectrum of genomic aberrations, but the pharmacological consequences of these aberrations are partly unknown. Here, we report an integrated pharmacogenomic analysis of 100 patient-derived GBM cell cultures from the human glioma cell culture (HGCC) cohort. Exploring 1,544 drugs, we find that GBM has two main pharmacological subgroups, marked by differential response to proteasome inhibitors and mutually exclusive aberrations in TP53 and CDKN2A/B. We confirm this trend in cell and in xenotransplantation models, and identify both Bcl-2 family inhibitors and p53 activators as potentiators of proteasome inhibitors in GBM cells, We can further predict the responses of individual cell cultures to several existing drug classes, presenting opportunities for drug repurposing and design of stratified trials. Our functionally profiled biobank provides a valuable resource for the discovery of new treatments for GBM.Patrik Johansson, Cecilia Krona and Soumi Kundu share first authorship</p

Follicle-stimulating hormone promotes growth of human prostate cancer cell line-derived tumor xenografts

Chemical castration in prostate cancer can be achieved with gonadotropin-releasing hormone (GnRH) agonists or antagonists. Their effects differ by the initial flare of gonadotropin and testosterone secretion with agonists and the immediate pituitary-testicular suppression by antagonists. While both suppress luteinizing hormone (LH) and follicle-stimulating hormone (FSH) initially, a rebound in FSH levels occurs during agonist treatment. This rebound is potentially harmful, taken the expression of FSH receptors (R) in prostate cancer tissue. We herein assessed the role of FSH in promoting the growth of androgen-independent (PC-3, DU145) and androgen-dependent (VCaP) human prostate cancer cell line xenografts in nude mice. Gonadotropins were suppressed with the GnRH antagonist degarelix, and effects of add-back human recombinant FSH were assessed on tumor growth. All tumors expressed GnRHR and FSHR, and degarelix treatment suppressed their growth. FSH supplementation reversed the degarelix-evoked suppression of PC-3 tumors, both in preventive (degarelix and FSH treatment started upon cell inoculation) and therapeutic (treatments initiated 3 weeks after cell inoculation) setting. A less marked, though significant FSH effect occurred in DU145, but not in VCaP xenografts. FSHR expression in the xenografts supports direct FSH stimulation of tumor growth. Testosterone supplementation, to maintain the VCaP xenografts, apparently masked the FSH effect on their growth. Treatment with the LH analogue hCG did not affect PC-3 tumor growth despite their expression of luteinizing hormone/choriongonadotropin receptor. In conclusion, FSH, but not LH, may directly stimulate the growth of androgen-independent prostate cancer, suggesting that persistent FSH suppression upon GnRH antagonist treatment offers a therapeutic advantage over agonist