Nongenomic Signaling Pathways Research Articles

Testosterone acts through the membrane protein GPRC6A to cause cardiac edema in zebrafish embryos.

Androgens are classically thought to act through intracellular androgen receptors (AR/NR3C4), but they can also trigger non-genomic effects via membrane proteins. Although several membrane androgen receptors have been characterized in vitro, their functions in vivo remain unclear. Using a chemical-genetic screen in zebrafish, we found that GPRC6A, a G-protein-coupled receptor, mediates non-genomic androgen actions during embryonic development. Exposure to androgens (androstanedione, DHT and testosterone) caused cardiac edema or tail curvature in wild-type embryos, as well as in ar mutants, suggesting AR-independent pathways. We then mutated putative membrane androgen receptors [gprc6a, hcar1-4 and zip9 (slc39a9)] and found that only gprc6a mutants exhibited a significant reduction in cardiac edema after testosterone exposure. Additionally, co-treatment of wild-type embryos with testosterone and GPRC6A antagonists significantly suppressed the cardiac edema phenotype. Using RNA-seq and RNA rescue approaches, we found that testosterone and GPRC6A cause cardiac phenotypes by reducing Pak1 signaling. Our results indicate that testosterone induces cardiac edema in zebrafish embryos through GPRC6A, independent of nuclear androgen receptors, highlighting a previously unappreciated non-genomic androgen signaling pathway in embryonic development.

Triclocarban and triclosan promote breast cancer progression in vitro and in vivo via activating G protein-coupled estrogen receptor signaling pathways

Triclocarban (TCC) and triclosan (TCS) have been detected ubiquitously in human body and evoked increasing concerns. This study aimed to reveal the induction risks of TCC and TCS on triple negative breast cancer through non-genomic GPER-mediated signaling pathways. Molecular simulation indicated that TCC exhibited higher GPER binding affinity than TCS theoretically. Calcium mobilization assay displayed that TCC/TCS activated GPER signaling pathway with the lowest observed effective concentrations (LOEC) of 10 nM/100 nM. TCC and TCS also upregulated MMP-2/9, EGFR, MAPK3 but downregulated MAPK8 via GPER-mediated signaling pathway. Proliferation assay showed that TCC/TCS induced 4 T1 breast cancer cells proliferation with the LOEC of 100 nM/1000 nM. Wound-healing and transwell assays showed that TCC/TCS promoted 4 T1 cells migration in a concentration-dependent manner with the LOEC of 10 nM. The effects of TCC on breast cancer cells proliferation and migration were stronger than TCS and both were regulated by GPER. TCC/TCS induced migratory effects were more significantly than proliferative effect. Mechanism study showed that TCC/TCS downregulated the expression of epithelial marker (E-cadherin) but upregulated mesenchymal markers (snail and N-cadherin), which was reversed by GPER inhibitor G15. These biomarkers results indicated that TCC/TCS-induced 4 T1 cells migration was a classic epithelial to mesenchymal transition mechanism regulated by GPER signaling pathway. Orthotopic tumor model verified that TCC promoted breast cancer in-situ tumor growth and distal tissue metastasis via GPER-mediated signaling pathway at human-exposure level of 10 mg/kg/d. TCC-induced tissue metastasis of breast cancer was more significantly than in-situ tumor growth. Overall, we demonstrated for the first time that TCC/TCS could activate the GPER signaling pathways to induce breast cancer progression.

Abstract 3171: The effect of estrogen and phytoestrogens in Inflammatory breast cancer

Abstract Inflammatory breast cancer is an aggressive and lethal type of breast cancer. Frequently, this cancer is diagnosed in an advanced stage with metastasis. IBC diagnosis is associated with a worse survival rate than other types of breast cancer. The presence of dermal and stromal tumor emboli blocking the lymphatic vessels under the skin is considered a hallmark of IBC and assumed to be responsible for the high metastatic behavior and aggressiveness of IBC disease. To this date, there are no effective targeted therapeutics, especially for those patients that account for approximately 20-40% of IBC cases with triple-negative breast cancer (TNBC) classification. Several studies have shown that estrogen can exert non-genomic effects in IBC and non-IBC TNBC, mediated by the expression of alternate estrogen receptors including estrogen receptor alpha-36, and GPR30. In this context, estrogen can activate non-genomic signaling pathways involved in the acquisition of oncogenic phenotypes such as increased motility and invasion in IBC cells. Phytoestrogens, naturally derived structural analogs to 17β-estradiol (E2), were evaluated as an alternative treatment option using in vitro IBC cell models (SUM149 and SUM190). Given the estrogenic properties of phytoestrogens, the drugs were hypothesized to interact with targets in pathways similar to E2 in non-genomic signaling. Several phytoestrogens including coumestrol (Cou) can induce a cytotoxic effect in TNBC. We hypothesize (1) that estrogen non-genomic signaling has an active role in the aggressive metastatic phenotype of IBC, and (2) that Cou has anticancer activity by inhibiting estrogen non-genomic signaling in IBC. To test these hypotheses, IBC cells were treated with E2 or Cou to determine their effects on the activation of downstream kinases by western blot and phospho-kinase array assays. In addition, transcriptional effects upon treatment with Cou were identified using RNA-seq. A dose-response curve in 2D and 3D models was generated to determine the half-maximal inhibitory concentration (IC50) of Cou. Finally, treatment using the IC50 of Cou caused a decrease in cell viability in the TNBC cell line. Also, in comparison to E2, Cou decreases migration, proliferation, and tumor emboli formation in IBC cell lines. In addition, IBC cells were treated using monotherapy and combination therapy with phytoestrogens including Cou, genistein (GN), and/or 8-prenylnaringenin (8-PN). Observed reductions of cell viability in IBC cell lines treated with the different phytoestrogens and combinations make them worthy of further characterization as anti-cancer therapies in in vitro and in vivo models. In summary, IBC cells are responsive to E2 making this signaling pathway attractive for the development of innovative therapeutic strategies with phytoestrogens as potential candidates for treatment. Citation Format: Esther A. Peterson-Peguero, Keishla Rodriguez-Martir, Madeline Ganshert, Dalissa Negron-Figueroa, Ayeisha N. Colon-Ortiz. The effect of estrogen and phytoestrogens in Inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3171.

Estrogen and testosterone secretion from the mouse brain

Estrogen and testosterone are typically thought of as gonadal or adrenal derived steroids that cross the blood brain barrier to signal via both rapid nongenomic and slower genomic signalling pathways. Estrogen and testosterone signalling has been shown to drive interlinked behaviours such as social behaviours and cognition by binding to their cognate receptors in hypothalamic and forebrain nuclei. So far, acute brain slices have been used to study short-term actions of 17β-estradiol, typically using electrophysiological measures. For example, these techniques have been used to investigate, nongenomic signalling by estrogen such as the estrogen modulation of long-term potentiation (LTP) in the hippocampus. Using a modified method that preserves the slice architecture, we show, for the first time, that acute coronal slices from the prefrontal cortex and from the hypothalamus maintained in aCSF over longer periods i.e. 24 h can be steroidogenic, increasing their secretion of testosterone and estrogen. We also show that the hypothalamic nuclei produce more estrogen and testosterone than the prefrontal cortex. Therefore, this extended acute slice system can be used to study the regulation of steroid production and secretion by discrete nuclei in the brain.

Modeling Breast Cancer in Men

Background: In 2023, 2,800 men are expected to be diagnosed with breast cancer in the United States, and while it affects a smaller patient population, breast cancer in men has a 19% higher mortality rate. Even though 87% of male breast cancers are estrogen receptor positive (ER+), breast cancer in men is resistant to anti-estrogen therapies. In this study we set out to characterize the difference between male and female breast epithelial cells to elucidate potential treatment pathways.
 Project Methods: We compared male and female epithelial cell lines from ultrasound guided breast biopsies of healthy men and women from the Susan G. Komen Tissue Bank that were processed using a culturing method developed in the lab. RNA-Seq of breast epithelial cells was done to identify unique signaling networks and genes in men, and RT-qPCR was used to determine the expression of ER signaling associated genes including ESR1, TBX3, FOXA1, GATA3 and APOBEC3B. Immunofluorescence for ERα was done to characterize differential expression and localization between men and women.
 Result: RNA-Seq identified an upregulation of ESR1 which correlates with increased ER+ male breast cancer, along with upregulation of non-genomic ER signaling pathway, including SRC activation, which may affect treatment response. Male cells also showed high levels KDM5D which is implicated in immune system evasion in esophageal cancer and colon adenocarcinoma. RT-qPCR displayed a two-fold increase in ESR1 and increases in TBX3 and APOBEC3B. Immunofluorescence identified ERα and localization to the cell membrane in men.
 Conclusion: This first characterization and modeling of men’s breast epithelial cells provides the basis for determining the mechanism behind the lack of efficacy of anti-estrogen therapeutics. With further investigation of the increased non-genomic ER signaling in men, specifically the SRC pathway, we hope this will provide a pathway to treat male breast cancer through potential combination therapies.

Sex Differences in Colon Cancer: Genomic and Nongenomic Signalling of Oestrogen.

Colon cancer (CRC) is a prevalent malignancy that exhibits distinct differences in incidence, prognosis, and treatment responses between males and females. These disparities have long been attributed to hormonal differences, particularly the influence of oestrogen signalling. This review aims to provide a comprehensive analysis of recent advances in our understanding of the molecular mechanisms underlying sex differences in colon cancer and the protective role of membrane and nuclear oestrogen signalling in CRC development, progression, and therapeutic interventions. We discuss the epidemiological and molecular evidence supporting sex differences in colon cancer, followed by an exploration of the impact of oestrogen in CRC through various genomic and nongenomic signalling pathways involving membrane and nuclear oestrogen receptors. Furthermore, we examine the interplay between oestrogen receptors and other signalling pathways, in particular the Wnt/β-catenin proliferative pathway and hypoxia in shaping biological sex differences and oestrogen protective actions in colon cancer. Lastly, we highlight the potential therapeutic implications of targeting oestrogen signalling in the management of colon cancer and propose future research directions to address the current gaps in our understanding of this complex phenomenon.

Characteristic Binding Landscape of Estrogen Receptor-α36 Protein Enhances Promising Cancer Drug Design.

Breast cancer (BC) remains the most common cancer among women worldwide, and estrogen receptor-α expression is a critical diagnostic factor for BC. Estrogen receptor (ER-α36) is a dominant-negative effector of ER-α66-mediated estrogen-responsive gene pathways. ER-α36 is a novel target that mediates the non-genomic estrogen signaling pathway. However, the crystallized structure of ER-α36 remains unavailable for molecular studies. ER-positive and triple-negative BC tumors aggressively resist the FDA-approved drugs; therefore, highly potent structure-based inhibitors with preeminent benefits over toxicity will preferably replace the current BC treatment. Broussoflanol B (BFB), a B. papyrifera bark compound, exhibits potent growth inhibitory activity in ER-negative BC cells by inducing cell cycle arrest. For the first time, we unravel the comparative dynamic events of the enzymes' structures and the binding mechanisms of BFB when bound to the ER-α36 and ER-α66 ligand-binding domain using an all-atom molecular dynamics simulations approach and MM/PBSA-binding-free energy calculations. The dynamic findings have revealed that ER-α36 and ER-α66 LBD undergo timescale "coiling", opening and closing conformations favoring the high-affinity BFB-bound ER-α36 (ΔG = -52.57 kcal/mol) compared to the BFB-bound ER-α66 (ΔG = -42.41 kcal/mol). Moreover, the unbound (1.260 Å) and bound ER-α36 (1.182 Å) exhibit the highest flexibilities and atomistic motions relative to the ER-α66 systems. The RMSF (Å) of the unbound ER-α36 and ER-α66 exhibit lesser stabilities than the BFB-bound systems, resulting in higher structural flexibilities and atomistic motions than the bound variants. These findings present a model that describes the mechanisms by which the BFB compound induces downregulation-accompanied cell cycle arrest at the Gap0 and Gap1 phases.

Glucocorticoids modulate neural activity via a rapid non-genomic effect on Kv2.2 channels in the central nervous system

Glucocorticoids are primary stress hormones that exert neuronal effects via both genomic and non-genomic signaling pathways. However, their rapid non-genomic effects and underlying mechanisms on neural activities remain elusive. In the present study, we investigated the rapid non-genomic effect of glucocorticoids on Kv2.2 channels in cultured HEK293 cells and acute brain slices including cortical pyramidal neurons and calyx-type synapses in the brain stem. We found that cortisol, the endogenous glucocorticoids, rapidly increased Kv2.2 currents by increasing the single-channel open probability in Kv2.2-expressing HEK293 cells through activation of the membrane-associated glucocorticoid receptor. Bovine serum albumin-conjugated dexamethasone, a membrane-impermeable agonist of the glucocorticoid receptor, could mimic the effect of cortisol on Kv2.2 channels. The cortisol-increased Kv2.2 currents were induced by activation of the extracellular signal-regulated protein kinase (ERK) 1/2 kinase, which could be inhibited by U0126, an antagonist of the ERK signaling pathway. In layer 2 cortical pyramidal neurons and the calyx of Held synapses, cortisol suppressed the action potential firing frequency during depolarization and reduced the successful rate upon high-frequency stimulation by activating Kv2.2 channels. We further examined the postsynaptic responses and found that cortisol did not affect the mEPSC and evoked EPSC, but increased the activity-dependent synaptic depression induced by a high-frequency stimulus train. In conclusion, glucocorticoids can rapidly activate Kv2.2 channels through membrane-associated glucocorticoid receptors via the ERK1/2 signaling pathway, suppress presynaptic action potential firing, and inhibit synaptic transmission and plasticity. This may be a universal mechanism of the glucocorticoid-induced non-genomic effects in the central nervous system.

Chemical exploration of TGR5 functional hot-spots: Synthesis and structure-activity relationships of C7- and C23-Substituted cholic acid derivatives

The activation of TGR5 bestows on bile acids the ability to modulate nongenomic signaling pathways, which are responsible of physiological actions including immunosuppressive and anti-inflammatory properties as well as the regulation of glucose metabolism and energy homeostasis. TGR5 agonists have therefore emerged in drug discovery and preclinical appraisals as promising compounds for the treatment of liver diseases and metabolic syndrome. In this study, we have been devising site-selected chemical modifications of the bile acid scaffold to provide novel chemical tools able to modulate the functions of TGR5 in different tissues. Biological results of the tested collection of semisynthetic cholic acid derivatives were used to extend the structure-activity relationships of TGR5 agonists and to clarify the molecular basis and functional role of TGR5 hot-spots in the receptor activation and selectivity. Some unexpected properties deriving from the molecular structure of bile acids have been unveiled as relevant to the receptor activation and may hence be used to design novel, selective and potent TGR5 agonists.

Histopathological and transcriptomic analyses reveal the reproductive endocrine-disrupting effects of decabromodiphenyl ethane (DBDPE) in mussel Mytilus galloprovincialis

The novel brominated flame retardant DBDPE has become a widespread environmental contaminant and could affect reproductive endocrine system in vertebrates. However, information about reproductive endocrine-disrupting effects of DBDPE on invertebrates is totally unknown. In this study, mussels Mytilus galloprovincialis were exposed to 1, 10, 50, 200 and 500 μg/L DBDPE for 30 days. Histopathological and transcriptomic analyses were performed to assess the reproductive endocrine-disrupting effects of DBDPE in mussels and the potential mechanisms. DBDPE promoted the gametogenesis in mussels of both sexes according to histological observation, gender-specific gene expression (VERL and VCL) and histological morphometric parameter analysis. Transcriptomic analysis demonstrated that DBDPE suppressed the genes related to cholesterol homeostasis and transport in both sexes via different LRPs- and ABCs-mediated pathways. DBDPE also disturbed nongenomic signaling pathway including signaling cascades (GPR157-IP3-Ca2+) in males and secondary messengers (cGMP) in females, and subsequently altered the expression levels of reproductive genes (VMO1, ZAN, Banf1 and Hook1). Additionally, dysregulation of energy metabolism in male mussels induced by DBDPE might interfere with the reproductive endocrine system. Overall, this is the first report that DBDPE evoked reproductive endocrine-disruptions in marine mussels. These findings will provide important references for ecological risk assessment of DBDPE pollution in marine environment.

Icariin Exerts Estrogen-Like Actions on Proliferation of Osteoblasts in Vitro via Membrane Estrogen Receptors-Mediated Non-nuclear Effects.

According to reports, icariin (ICA) is a bone anabolic agent able to prevent osteoporosis in both ovariectomized rats and postmenopausal women. However, its effect on osteoblast proliferation remains to be determined, and the underlying mechanism remains to be elucidated. Icariin-bovine serum albumin (BSA) conjugates were purified by Sephadex G-25 gel chromatography technology. Primary osteoblasts from neonatal rats were used to evaluate the effects of ICA, ICA-BSA, ICA-BSA + ICI182780, and ICA-BSA + PD98059. 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and propidium iodide (PI)-staining assays were used to detect the proliferation of osteoblasts after drug exposure. The intracellular calcium ions were detected using a confocal microscope with Fluo-3/AM as the fluorescent indicator. Western blot was capitalized on to measure the relative content of phospho-extracellular signal-regulated kinase (p-ERK). Primary osteoblasts in culture were detected by histochemical staining of alkaline phosphatase, and calcified nodules were obtained by sequential digestion. Icariin and bovine serum albumin could form conjugate, which could be purified by Sephadex G-25 gel chromatography technology. MTT and flow cytometry results show that ICA-BSA conjugate significantly facilitated the proliferation of osteoblasts (P < 0.05). The intracellular calcium ions also ascended vastly in the cells treated with ICA-BSA conjugate (P < 0.01). Icariin-bovine serum albumin exposure rapidly activated the extracellular signal-regulated kinase (ERK) signaling. Furthermore, ICA- and ICA-BSA-mediated actions on osteoblasts were signally alleviated after dealing with ERK inhibitor PD98059 or estrogen receptor (ER) antagonist ICI182780, which might have a relation to the repression of ERK phosphorylation. Icariin could serve as estrogen in osteoblast cells by the rapid nongenomic ER signaling pathway independent of ligand and estrogen response element (ERE) and mediated by mitogen-activated protein kinase (MAPK).

Vitamin D supplementation worsens Alzheimer's progression: Animal model and human cohort studies.

Vitamin D deficiency has been epidemiologically linked to Alzheimer's disease (AD) and other dementias, but no interventional studies have proved causality. Our previous work revealed that the genomic vitamin D receptor (VDR) is already converted into a non‐genomic signaling pathway by forming a complex with p53 in the AD brain. Here, we extend our previous work to assess whether it is beneficial to supplement AD mice and humans with vitamin D. Intriguingly, we first observed that APP/PS1 mice fed a vitamin D‐sufficient diet showed significantly lower levels of serum vitamin D, suggesting its deficiency may be a consequence not a cause of AD. Moreover, supplementation of vitamin D led to increased Aβ deposition and exacerbated AD. Mechanistically, vitamin D supplementation did not rescue the genomic VDR/RXR complex but instead enhanced the non‐genomic VDR/p53 complex in AD brains. Consistently, our population‐based longitudinal study also showed that dementia‐free older adults (n = 14,648) taking vitamin D3 supplements for over 146 days/year were 1.8 times more likely to develop dementia than those not taking the supplements. Among those with pre‐existing dementia (n = 980), those taking vitamin D3 supplements for over 146 days/year had 2.17 times the risk of mortality than those not taking the supplements. Collectively, these animal model and human cohort studies caution against prolonged use of vitamin D by AD patients.

Abstract 329: Understanding the role of GPR30 in the estrogen non-genomic signaling in inflammatory breast cancer

Abstract Inflammatory Breast Cancer (IBC) is rare and the most aggressive subtype of all breast cancers, reporting rapid progression, poor prognosis, and a unique clinical diagnosis (blockage of lymph vessels by tumor emboli). Approximately, 40% of IBC cases are classified as triple-negative breast cancer (TNBC), meaning they test negative for hormone receptors (estrogen and progesterone) and the amplification of the epidermal growth factor receptor 2 (HER2), which leads to no effective targeted therapies availability. However, 17β-estrogen (E2) has been found to enhance the migration and invasion phenotypes of TN-IBC cells and it has been associated with the presence of a membrane G-protein coupled estrogen receptor (GPR30) and a novel cytoplasmic estrogen receptor (ERα36 isoform) that can undergo an estrogen non-genomic (rapid) cell’s response. Recent studies have also reported a crosstalk between these estrogen non-genomic receptors and the epidermal growth factor receptor (EGFR) signaling pathway in the presence of E2, inducing phosphorylation of the extracellular signal-regulated kinase (ERK1/2) and promoting oncogenic phenotypes (migration &amp; invasion) in TN-IBC cells. However, the exact role of GPR30 and ERα36 in the non-genomic signaling pathway remains elusive in TN-IBC. We hypothesis that GPR30 and ERα36 are necessary to undergo de estrogen non-genomic signaling and promote oncogenic phenotypes in TN-IBC SUM149 cells after E2 treatment. Methods: We have measured migratory activity and tumor emboli formation of SUM149 cells (by wound-healing and 3D-cell proliferation assay), and phosphorylation levels of ERK1/2 and AKT kinases (by western-blot) after treatments in time-points with E2 alone and in combination with G15 (GPR30 antagonist drug). We are also working to identify protein partners of GPR30 (by Ip and Mass-spec) after E2 treatment, including physical interaction with ERα36, in TN-IBC and other types of breast cancers. Results: G15 treatment in Sum149 cells significantly reduced E2-induced ERK1/2 phosphorylation, indicating that GPR30 is necessary for the estrogen non-genomic signaling pathway and has direct involvement with the EGFR signaling cascade. Data acquired from these experiments will contribute to a better understanding of TN-IBC’s molecular structure and create new opportunities to develop novel targeted therapies. Citation Format: Xavier S. Bittman Soto, Adrian J. Rivera Lopez, Esther A. Peterson. Understanding the role of GPR30 in the estrogen non-genomic signaling in inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 329.

Glucocorticoid regulation of clinical and functional manifestations of asthma in patients with different airway response to hyposmolar stimulus during anti-inflammatory therapy

Introduction. The role of an alternative adenylate cyclase pathway of hormonal signal transmission under the action of synthetic glucocorticosteroids with the participation of endogenous stress-limiting activity of the adrenal cortex in conjunction with the adaptive capabilities of airway homeostasis in patients with asthma under conditions of osmotic stress has not been studied at present. Aim. To assess the dynamics of cortisol and cyclic adenosin monophosphate (cAMP) in asthma patients with different airway responses to hypoosmolar stimuli when using anti-inflammatory combination therapy with inhaled corticosteroids/long-acting β2 -agonists (ICS/LABA). Materials and methods. 96 patients diagnosed with asthma received combined anti-inflammatory therapy with ICS/LABA for 24 weeks. Group 1 included patients (n=18) with airway hyperresponsiveness to hypoosmolar stimulus, group 2 (n=78) – with no reaction of the bronchi to a 3-minute ultrasonic inhalation of distilled water. At baseline and at the end of treatment, the lung function was studied; to assess the regulatory function of glucocorticoids using non-genomic signaling pathways, the levels of cortisol in blood serum and cAMP in blood leukocytes were determined. Results. Patients of group 1 in comparison with the second one initially had a lower FEV1 ‒ 88.2±5.3 and 98.5±1.7%, respectively (p&lt;0.05), after treatment in both groups there was a slight tendency to an increase in FEV1 (98.5±5.7 and 101.4±2.5%, respectively, p&gt;0.05). The concentration of cortisol and cAMP at baseline and after 24 weeks of therapy in patients of group 1 was 588.7±32.0 and 495.0±48.7 nmol/L, 61.7±5.1 and 76.5±5, 2 pmol/106 cells (p&lt;0.01); in group 2 − 610.5±20.1 and 522.2±15.60 nmol/L (p&lt;0.001), 76.2±2.2 and 90.6±2.5 pmol/106 cells (p&lt;0.001). Conclusion. In asthma patients with airway osmotic hyperresponsiveness, persistent adaptation to osmotic stress is traced, which is combined with a more significant impairment of the lung function and indicates insufficient therapeutic control over glucocorticoid regulation of osmotic stress by the selected volume of ICS/LABA therapy.

Cocoa flavonoids: a promising ingredient that potentiates vitamin d action in skeletal muscle cells

Rationale: Vitamin D (VitD) effects take place by binding to its receptor (VDR) which is a classical nuclear receptor responsible for genomic effects, but there is also a fraction of the VDR located in the muscle plasma membrane that is responsible for its non-genomic effects. In addition to the established effect of VitD in bone and mineral homeostasis, VitD insufficiency/deficiency is linked to a range of muscle disorders including frailty and muscle loss/dysfunction. Flavonoids decrease insulin resistance and have antioxidant and anti-inflammatory properties by activating Akt signaling pathways. Our study has aimed to evaluate, in VitD deficient conditions, if cocoa flavonoids (CF) are able to mediate the interacction between VitD and skeletal muscle glucose uptake by triggerring the modulation of its genomic and non-genomic signaling pathways. This could be useful in pathological deficient VitD situations, such as diabetes or sarcopenia where muscle function is compromised due to the onset of insulin resistance.

The Role of Androgen Receptor in Cross Talk Between Stromal Cells and Prostate Cancer Epithelial Cells.

Prostate cancer (PCa) lists as the second most lethal cancer for men in western countries, and androgen receptor (AR) plays a central role in its initiation and progression, which prompts the development of androgen deprivation therapy (ADT) as the standard treatment. Prostate tumor microenvironment, consisting of stromal cells and extracellular matrix (ECM), has dynamic interactions with PCa epithelial cells and affects their growth and invasiveness. Studies have shown that both genomic and non-genomic AR signaling pathways are involved in the biological regulation of PCa epithelial cells. In addition, AR signaling in prostate stroma is also involved in PCa carcinogenesis and progression. Loss of AR in PCa stroma is clinically observed as PCa progresses to advanced stage. Especially, downregulation of AR in stromal fibroblasts dysregulates the expression levels of ECM proteins, thus creating a suitable environment for PCa cells to metastasize. Importantly, ADT treatment enhances this reciprocal interaction and predisposes stromal cells to promote cell invasion of PCa cells. During this process, AR in PCa epithelium actively responds to various stimuli derived from the surrounding stromal cells and undergoes enhanced degradation while elevating the expression of certain genes such as MMP9 responsible for cell invasion. AR reduction in epithelial cells also accelerates these cells to differentiate into cancer stem-like cells and neuroendocrine cells, which are AR-negative PCa cells and inherently resistant to ADT treatments. Overall, understanding of the cross talk between tumor microenvironment and PCa at the molecular level may assist the development of novel therapeutic strategies against this disease. This review will provide a snapshot of AR’s action when the interaction of stromal cells and PCa cells occurs.

Evolution of non-genomic nuclear receptor function

Nuclear receptors (NRs) are responsible for the regulation of diverse developmental and physiological systems in metazoans. NR actions can be the result of genomic and non-genomic mechanisms depending on whether they act inside or outside of the nucleus respectively. While the actions of both mechanisms have been shown to be crucial to NR functions, non-genomic actions are considered less frequently than genomic actions. Furthermore, hypotheses on the origin and evolution of non-genomic NR signaling pathways are rarely discussed in the literature. Here we summarize non-genomic NR signaling mechanisms in the context of NR protein family evolution and animal phyla. We find that NRs across groups and phyla act via calcium flux as well as protein phosphorylation cascades (MAPK/PI3K/PKC). We hypothesize and discuss a possible synapomorphy of NRs in the NR1 and NR3 families, including the thyroid hormone receptor, vitamin D receptor, ecdysone receptor, retinoic acid receptor, steroid receptors, and others. In conclusion, we propose that the advent of non-genomic NR signaling may have been a driving force behind the expansion of NR diversity in Cnidarians, Placozoans, and Bilaterians.

Effects of thyroxine on apoptosis and proliferation of mammary tumors

Hypothyroidism is a protective factor against breast cancer but long-term exposure or overdoses of thyroid replacement therapy with thyroxine (T4) may increase breast cancer risk. Objective: to study, in vivo and in vitro, the effects of T4 on the proliferation and apoptosis of mammary tumors of hypo- and euthyroid rats, and the possible mechanisms involved in these effects. Material and Methods: Female Sprague-Dawley rats were treated with a single dose of dimethylbenzathracene (15 mg/rat) at 55 days of age and were divided into three groups: hypothyroidism (HypoT; 0.01% 6-N-propyl-2-thiouracil -PTU- in drinking water, n = 20), hypothyroidism treated with T4 (HypoT + T4; 0.01% PTU in drinking water and 0.25 mg/kg/day T4 via sc; n = 20) and EUT (untreated control, n = 20). At sacrifice, tumor explants from HypoT and EUT rats were obtained and treated either with 10−10 M T4 in DMEM/F12 without phenol red with 1% Charcoalized Fetal Bovine Serum or DMEM/F12 only for 15 min to evaluate intracellular signaling pathways associated with T4, and 24 h to evaluate changes in the expression of hormone receptors and proteins related to apoptosis and proliferation by immunohistochemistry and Western Blot. Results: In vivo, hypothyroidism retards mammary carcinogenesis but its treatment with T4 reverted the protective effects. In vitro, the proliferative and anti-apoptosis mechanisms of T4 were different regarding the thyroid status. In EUT tumors, the main signaling pathway involved was the cross-talk with other receptors, such as ERα, PgR, and HER2. In HypoT tumors, the non-genomic signaling pathway of T4 was the chief mechanism involved since αvβ3 integrin, HER2, β-catenin and, downstream, PI3K/AKT and ERK signaling pathways were activated. Conclusion: T4 can regulate mammary carcinogenesis by mainly activating its non-genomic signaling pathway and by interacting with other hormone or growth factor pathways endorsing that overdoses of thyroid replacement therapy with T4 can increase the risk of breast cancer.

Perfluorooctane sulfonate interferes with non-genomic estrogen receptor signaling pathway, inhibits ERK1/2 activation and induces apoptosis in mouse spermatocyte-derived cells

Perfluorooctane sulfonate (PFOS) is a widespread persistent organic pollutant. Both epidemiological survey and our previous in vivo study have revealed the associations between PFOS exposure and spermatogenesis disorder, while the underlying mechanisms are far from clear. In the present study, GC-2 cells, a mouse spermatocyte-derived cell line, was used to investigate the toxic effects of PFOS and its hypothetical mechanism of action. GC-2 cells were treated with PFOS (0, 50, 100 and 150 μM) for 24 h or 48 h. Results demonstrated that PFOS dose-dependently inhibited cell viability, induced G0/G1 cell cycle arrest and triggered apoptosis, which might be partly explained by the decrease in cyclin D1, PCNA and Bcl-2 protein expression; increase in Bax protein expression; and activation of caspase-9, -3. In addition, PFOS did not directly transactivate or repress estrogen receptors (ERs) in gene reporter assays, whereas the protein levels of both ERα and ERβ were significantly altered and the downstream ERK1/2 phosphorylation was inhibited by PFOS. Furthermore, pretreatment with specific ERα agonist PPT (1 μM) significantly attenuated the above PFOS-induced effects while specific ERβ agonist DPN (1 μM) accelerated them. These results suggest that PFOS may induce growth inhibition and apoptosis via non-genomic estrogen receptor/ERK1/2 signaling pathway in GC-2 cells, which provides a novel insight regarding the potential role of ERs in mediating PFOS-triggered spermatocyte toxicity.

Inhibitory Effects of Coumestrol in Triple‐Negative Inflammatory Breast Cancer

Breast cancer is the second leading cause of death in females in the United States, being Inflammatory Breast Cancer (IBC) the most aggressive subtype. Usually, this cancer is diagnosed in an advanced stage with metastasis. Experts in IBC pathogenesis think that IBC rapid metastasis is induces by tumor emboli formation, a hallmark of IBC. The poor prognosis for patients with IBC emphasizes the need to better understand the molecular signature of this disease to develop effective targeted therapeutics. To this date, there is no effective targeted therapeutics, especially for those patients that account for approximately 20-40% of IBC cases with triple-negative breast cancer (TNBC) classification. Importantly, several studies have shown that estrogen can exert non-genomic effects in IBC and non-IBC TNBC, mediated by the expression of an alternate isoform of estrogen receptor alpha-66, estrogen receptor alpha-36, and GPR30. In this context, estrogen can activate non-genomic signaling pathways involved in the acquisition of oncogenic phenotypes such as increased motility and invasion in IBC cells. Phytoestrogens, like coumestrol (Cou), are natural compounds found in plants, specifically soybeans or clover sprouts. Also, it is a polyphenolic compound that is structurally similar to estradiol (E2) and several studies have shown that coumestrol induces a cytotoxic effect in TNBC. We hypothesize (1) that estrogen non-genomic signaling has an active role in the aggressive metastatic phenotype of IBC, and (2) that Cou has anticancer activity by inhibiting estrogen non-genomic signaling in IBC. To test this hypothesis, IBC cells were treated with E2 or Cou, and activation of downstream kinases was analyzed by Western blot and using a Human Phospho-Kinase Array. A dose-response curve in 2D and 3D models were generated to determine the half-maximal inhibitory concentration (IC50) of Cou. Finally, treatment using the IC50 of Cou caused a decrease in cell viability in the TNBC cell line. Also, in comparison to E2, Cou decrease migration, proliferation, and tumor emboli formation in IBC cell line. In summary, IBC cells are responsive to E2 treatment making this signaling pathway attractive for the development of innovative therapeutic strategies. Importantly, these Cou preliminary studies show a potential anticancer activity in IBC cell lines.