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Biomedicines Mar 2023Preclinical studies show that the anticancer actions of vitamin D metabolites are mediated by apoptosis, inhibition of cell proliferation and induction of cell cycle...
Preclinical studies show that the anticancer actions of vitamin D metabolites are mediated by apoptosis, inhibition of cell proliferation and induction of cell cycle arrest. Cervical cancer cells express an autocrine vitamin D metabolising system (VDMS) comprised of a vitamin D receptor, vitamin D catabolic enzyme (CYP24A1), and the activating enzyme of 25-hydroxycholecalciferol (25(OH)D3), CYP27B1. We assessed the anticancer effects of 25(OH)D3 at clinically relevant concentrations on a cervical squamous cell cancer cell line, SiHa. We evaluated cell health parameters (cell count, viability, and cell cycle), cell death modes (apoptosis, autophagic-dependent death, and necrosis by flow cytometry and transmission electron microscopy), and autocrine VDMS gene and protein expression by qPCR and Western blot, respectively. Our study demonstrates that physiological and supraphysiological doses of 25(OH)D3 inhibit cell growth and viability and induce biochemical and morphological apoptosis in SiHa cells. These growth effects are mediated by alteration in the VDMS gene and protein expression, with prominent negative feedback at supraphysiological treatment dose. These data identify promising therapeutic potential of 25(OH)D3 in cervical cancer, which warrants further clinical translational investigations.
PubMed: 36979850
DOI: 10.3390/biomedicines11030871 -
Frontiers in Endocrinology 2023The apelin receptor binds two distinct endogenous peptides, apelin and ELA, which act in an autocrine/paracrine manner to regulate the human cardiovascular system. As a...
INTRODUCTION
The apelin receptor binds two distinct endogenous peptides, apelin and ELA, which act in an autocrine/paracrine manner to regulate the human cardiovascular system. As a class A GPCR, targeting the apelin receptor is an attractive therapeutic strategy. With improvements in imaging techniques, and the stability and brightness of dyes, fluorescent ligands are becoming increasingly useful in studying protein targets. Here, we describe the design and validation of four novel fluorescent ligands; two based on [Pyr1]apelin-13 (apelin488 and apelin647), and two based on ELA-14 (ELA488 and ELA647).
METHODS
Fluorescent ligands were pharmacologically assessed using radioligand and functional in vitro assays. Apelin647 was validated in high content imaging and internalisation studies, and in a clinically relevant human embryonic stem cell-derived cardiomyocyte model. Apelin488 and ELA488 were used to visualise apelin receptor binding in human renal tissue.
RESULTS
All four fluorescent ligands retained the ability to bind and activate the apelin receptor and, crucially, triggered receptor internalisation. In high content imaging studies, apelin647 bound specifically to CHO-K1 cells stably expressing apelin receptor, providing proof-of-principle for a platform that could screen novel hits targeting this GPCR. The ligand also bound specifically to endogenous apelin receptor in stem cell-derived cardiomyocytes. Apelin488 and ELA488 bound specifically to apelin receptor, localising to blood vessels and tubules of the renal cortex.
DISCUSSION
Our data indicate that the described novel fluorescent ligands expand the pharmacological toolbox for studying the apelin receptor across multiple platforms to facilitate drug discovery.
Topics: Cricetinae; Animals; Humans; Apelin Receptors; Ligands; Peptide Hormones; Cricetulus; Protein Binding
PubMed: 36967803
DOI: 10.3389/fendo.2023.1139121 -
Journal of Animal Science Jan 20235-Hydroxytryptamine (5-HT) is an amine produced in both the mammary gland and the central nervous system. Tryptophan hydroxylase 1 (TPH1) catalyzes the conversion of...
5-Hydroxytryptamine (5-HT) is an amine produced in both the mammary gland and the central nervous system. Tryptophan hydroxylase 1 (TPH1) catalyzes the conversion of 5-hydroxytryptophan (5-HTP) into l-tryptophan, which is then converted into 5-HT by monoamine-oxidase (MAO-A). In the mammary gland, 5-HT has been shown to have a variety of paracrine-autocrine actions, including suppressing lactation, controlling the destiny of mammary epithelial cells, and maintaining calcium homeostasis throughout the transition from pregnancy to lactation. To examine the effects of 5-HT on the composition of colostrum and milk, a total of 30 transition Guan Zhong dairy goats were intramuscularly injected with 5-HTP (1.0 mg/kg) every morning before feeding from 10 d before the projected parturition date to the day of parturition. The average number of days animals received injections was 8.2 ± 3.2 d. 5-HTP treatment increased serum 5-HT concentration from days 5 to 2 relative to parturition (P < 0.05), and decreased the casein concentration of colostrum (P < 0.05). In the in vitro experiment, mammary epithelial cells isolated from three individual goats' mammary glands were separately treated with 200 μM 5-HTP, 30 μM PCPA (the specific inhibitor of TPH1), or 200 μM 5-HTP + 50 μM SB269970 (the selective antagonist of 5-HTR7). The results showed that 200 μM 5-HTP inhibited the expression of β-casein, downregulated the activity of the JAK2/ STAT5a signaling pathway, and promoted the apoptosis of goat mammary epithelial cells (GMECs) (P < 0.05). When GMECs were treated with 30 μM Four-chloro-dl-phenylalanine (PCPA), a specific inhibitor of 5-HT synthesis, the mRNA expression of STAT5a and the phosphorylated STAT5a protein level were upregulated. The 50 μM SB269970 treatment rescued the effects of 5-HTP on GMECs (P < 0.05). Taken together, the results indicated that 5-HTP exerted an inhibitory effect on β-casein synthesis and a proapoptotic effect in GMECs via HTR7 and the JAK2/STAT5a axis.
Topics: Animals; Female; Pregnancy; 5-Hydroxytryptophan; Apoptosis; Caseins; Epithelial Cells; Goats; Lactation; Mammary Glands, Animal; Serotonin; STAT5 Transcription Factor; Receptors, Serotonin
PubMed: 36964762
DOI: 10.1093/jas/skad089 -
Purinergic Signalling Apr 2024María Teresa Miras Portugal devoted most of her scientific life to the study of purinergic signalling. In an important part of her work, she used a model system: the... (Review)
Review
María Teresa Miras Portugal devoted most of her scientific life to the study of purinergic signalling. In an important part of her work, she used a model system: the chromaffin cells of the adrenal medulla. It was in these cells that she identified diadenosine polyphosphates, from which she proceeded to the study of adrenomedullary purinome: nucleotide synthesis and degradation, adenosine transport, nucleotide uptake into chromaffin granules, exocytotic release of nucleotides and autocrine regulation of chromaffin cell function via purinoceptors. This short review will focus on the current state of knowledge of the purinoceptors of adrenal chromaffin cells, a subject to which María Teresa made seminal contributions and which she continued to study until the end of her scientific life.
Topics: Portugal; Chromaffin Cells; Adrenal Medulla; Receptors, Purinergic; Nucleotides
PubMed: 36941507
DOI: 10.1007/s11302-023-09934-1 -
Communications Biology Mar 2023Prolactin (PRL) is elevated in B-cell-mediated lymphoproliferative diseases and promotes B-cell survival. Whether PRL or PRL receptors drive the evolution of B-cell...
Prolactin (PRL) is elevated in B-cell-mediated lymphoproliferative diseases and promotes B-cell survival. Whether PRL or PRL receptors drive the evolution of B-cell malignancies is unknown. We measure changes in B cells after knocking down the pro-proliferative, anti-apoptotic long isoform of the PRL receptor (LFPRLR) in vivo in systemic lupus erythematosus (SLE)- and B-cell lymphoma-prone mouse models, and the long plus intermediate isoforms (LF/IFPRLR) in human B-cell malignancies. To knockdown LF/IFPRLRs without suppressing expression of the counteractive short PRLR isoforms (SFPRLRs), we employ splice-modulating DNA oligomers. In SLE-prone mice, LFPRLR knockdown reduces numbers and proliferation of pathogenic B-cell subsets and lowers the risk of B-cell transformation by downregulating expression of activation-induced cytidine deaminase. LFPRLR knockdown in lymphoma-prone mice reduces B-cell numbers and their expression of BCL2 and TCL1. In overt human B-cell malignancies, LF/IFPRLR knockdown reduces B-cell viability and their MYC and BCL2 expression. Unlike normal B cells, human B-cell malignancies secrete autocrine PRL and often express no SFPRLRs. Neutralization of secreted PRL reduces the viability of B-cell malignancies. Knockdown of LF/IFPRLR reduces the growth of human B-cell malignancies in vitro and in vivo. Thus, LF/IFPRLR knockdown is a highly specific approach to block the evolution of B-cell neoplasms.
Topics: Mice; Humans; Animals; Receptors, Prolactin; Prolactin; Protein Isoforms; Lymphoma, B-Cell; Lupus Erythematosus, Systemic; Proto-Oncogene Proteins c-bcl-2
PubMed: 36941341
DOI: 10.1038/s42003-023-04667-8 -
Journal For Immunotherapy of Cancer Mar 2023Progression of colorectal cancer (CRC), a leading cause of cancer-related death worldwide, is driven by colorectal cancer stem cells (CR-CSCs), which are regulated by...
BACKGROUND
Progression of colorectal cancer (CRC), a leading cause of cancer-related death worldwide, is driven by colorectal cancer stem cells (CR-CSCs), which are regulated by endogenous and microenvironmental signals. Interleukin (IL)-30 has proven to be crucial for CSC viability and tumor progression. Whether it is involved in CRC tumorigenesis and impacts clinical behavior is unknown.
METHODS
IL30 production and functions, in stem and non-stem CRC cells, were determined by western blot, immunoelectron microscopy, flow cytometry, cell viability and sphere formation assays. CRISPR/Cas9-mediated deletion of the gene, RNA-Seq and implantation of gene transfected or deleted CR-CSCs in NSG mice allowed to investigate IL30's role in CRC oncogenesis. Bioinformatics and immunopathology of CRC samples highlighted the clinical implications.
RESULTS
We demonstrated that both CR-CSCs and CRC cells express membrane-anchored IL30 that regulates their self-renewal, via WNT5A and RAB33A, and/or proliferation and migration, primarily by upregulating CXCR4 STAT3, which are suppressed by IL30 gene deletion, along with WNT and RAS pathways. Deletion of gene downregulates the expression of proteases, such as MMP2 and MMP13, chemokine receptors, mostly CCR7, CCR3 and CXCR4, and growth and inflammatory mediators, including ANGPT2, CXCL10, EPO, IGF1 and EGF. These factors contribute to IL30-driven CR-CSC and CRC cell expansion, which is abrogated by their selective blockade. gene deleted CR-CSCs displayed reduced tumorigenicity and gave rise to slow-growing and low metastatic tumors in 80% of mice, which survived much longer than controls. Bioinformatics and CIBERSORTx of the '' collection, and morphometric assessment of IL30 expression in clinical CRC samples revealed that the lack of IL30 in CRC and infiltrating leucocytes correlates with prolonged overall survival.
CONCLUSIONS
IL30 is a new CRC driver, since its inactivation, which disables oncogenic pathways and multiple autocrine loops, inhibits CR-CSC tumorigenicity and metastatic ability. The development of CRISPR/Cas9-mediated targeting of IL30 could improve the current therapeutic landscape of CRC.
Topics: Mice; Animals; Colonic Neoplasms; Cell Line, Tumor; Gene Editing; CRISPR-Cas Systems; Cell Proliferation; Neoplastic Stem Cells; Colorectal Neoplasms; Cell Transformation, Neoplastic; Carcinogenesis; Interleukins
PubMed: 36927528
DOI: 10.1136/jitc-2022-006056 -
Cancer Cell Mar 2023The mechanisms underlying the multistep process of tumorigenesis can be distilled into a logical framework involving the acquisition of functional capabilities, the... (Review)
Review
The mechanisms underlying the multistep process of tumorigenesis can be distilled into a logical framework involving the acquisition of functional capabilities, the so-called hallmarks of cancer, which are collectively envisaged to be necessary for malignancy. These capabilities, embodied both in transformed cancer cells as well as in the heterotypic accessory cells that together constitute the tumor microenvironment (TME), are conveyed by certain abnormal characteristics of the cancerous phenotype. This perspective discusses the link between the nervous system and the induction of hallmark capabilities, revealing neurons and neuronal projections (axons) as hallmark-inducing constituents of the TME. We also discuss the autocrine and paracrine neuronal regulatory circuits aberrantly activated in cancer cells that may constitute a distinctive "enabling" characteristic contributing to the manifestation of hallmark functions and consequent cancer pathogenesis.
Topics: Humans; Tumor Microenvironment; Neoplasms; Carcinogenesis; Neurons
PubMed: 36917953
DOI: 10.1016/j.ccell.2023.02.012 -
Molecules (Basel, Switzerland) Feb 2023Myocardial remodelling is a molecular, cellular, and interstitial adaptation of the heart in response to altered environmental demands. The heart undergoes reversible... (Review)
Review
Myocardial remodelling is a molecular, cellular, and interstitial adaptation of the heart in response to altered environmental demands. The heart undergoes reversible physiological remodelling in response to changes in mechanical loading or irreversible pathological remodelling induced by neurohumoral factors and chronic stress, leading to heart failure. Adenosine triphosphate (ATP) is one of the potent mediators in cardiovascular signalling that act on the ligand-gated (P2X) and G-protein-coupled (P2Y) purinoceptors via the autocrine or paracrine manners. These activations mediate numerous intracellular communications by modulating the production of other messengers, including calcium, growth factors, cytokines, and nitric oxide. ATP is known to play a pleiotropic role in cardiovascular pathophysiology, making it a reliable biomarker for cardiac protection. This review outlines the sources of ATP released under physiological and pathological stress and its cell-specific mechanism of action. We further highlight a series of cardiovascular cell-to-cell communications of extracellular ATP signalling cascades in cardiac remodelling, which can be seen in hypertension, ischemia/reperfusion injury, fibrosis, hypertrophy, and atrophy. Finally, we summarize current pharmacological intervention using the ATP network as a target for cardiac protection. A better understanding of ATP communication in myocardial remodelling could be worthwhile for future drug development and repurposing and the management of cardiovascular diseases.
Topics: Adenosine Triphosphate; Receptors, Purinergic P2; Signal Transduction; Cell Communication; Myocardium
PubMed: 36903347
DOI: 10.3390/molecules28052102 -
International Journal of Molecular... Feb 2023Neurotensin (NTS) is a peptide discovered in 1973, which has been studied in many fields and mainly in oncology for its action in tumor growth and proliferation. In this... (Review)
Review
Neurotensin (NTS) is a peptide discovered in 1973, which has been studied in many fields and mainly in oncology for its action in tumor growth and proliferation. In this review of the literature, we wanted to focus on its involvement in reproductive functions. NTS participates in an autocrine manner in the mechanisms of ovulation via NTS receptor 3 (NTSR3), present in granulosa cells. Spermatozoa express only its receptors, whereas in the female reproductive system (endometrial and tube epithelia and granulosa cells), we find both NTS secretion and the expression of its receptors. It consistently enhances the acrosome reaction of spermatozoa in mammals in a paracrine manner via its interaction with NTSR1 and NTSR2. Furthermore, previous results on embryonic quality and development are discordant. NTS appears to be involved in the key stages of fertilization and could improve the results of in vitro fertilization, especially through its effect on the acrosomal reaction.
Topics: Animals; Female; Male; Mammals; Neurotensin; Humans
PubMed: 36902025
DOI: 10.3390/ijms24054594 -
Aging Mar 2023Sarcopenia is progressive loss of muscle mass and strength, occurring during normal aging with significant consequences on the quality of life for elderly. Neurotrophin...
Sarcopenia is progressive loss of muscle mass and strength, occurring during normal aging with significant consequences on the quality of life for elderly. Neurotrophin 3 (NT-3) is an important autocrine factor supporting Schwann cell survival and differentiation and stimulating axon regeneration and myelination. NT-3 is involved in the maintenance of neuromuscular junction (NMJ) integrity, restoration of impaired radial growth of muscle fibers through activation of the Akt/mTOR pathway. We tested the efficacy of NT-3 gene transfer therapy in wild type (WT)-aged C57BL/6 mice, a model for natural aging and sarcopenia, via intramuscular injection 1 × 1011 vg AAV1.tMCK.NT-3, at 18 months of age. The treatment efficacy was assessed at 6 months post-injection using run to exhaustion and rotarod tests, muscle contractility assay, and histopathological studies of the peripheral nervous system, including NMJ connectivity and muscle. AAV1.NT-3 gene therapy in WT-aged C57BL/6 mice resulted in functional and muscle physiology improvements, supported by quantitative histology from muscle, peripheral nerves and NMJ. Hindlimb and forelimb muscles in the untreated cohort showed the presence of a muscle- and sex-dependent remodeling and fiber size decrease with aging, which was normalized toward values obtained from 10 months old WT mice with treatment. The molecular studies assessing the NT-3 effect on the oxidative state of distal hindlimb muscles, accompanied by western blot analyses for mTORC1 activation were in accordance with the histological findings. Considering the cost and quality of life to the individual, we believe our study has important implications for management of age-related sarcopenia.
Topics: Mice; Animals; Sarcopenia; Muscle, Skeletal; Axons; Quality of Life; Mice, Inbred C57BL; Nerve Regeneration; Aging; Genetic Therapy
PubMed: 36897179
DOI: 10.18632/aging.204577