Remodeling of sorafenib as an orally bioavailable ferroptosis inducer for Lung Cancer by chemical modification of adenine-binding motif.

Sorafenib (BAY 43-9006) was developed as a multi-kinase inhibitor to treat advanced renal cell, hepatocellular, and thyroid cancers. The cytotoxic effect of sorafenib on cancer cells results from not only inhibiting the MEK/ERK signaling pathway (the on-target effect) but also inducing oxidative damage (the off-target effect). The inhibitory effect of sorafenib on system Xc- (xCT), a cystine/glutamate antiporter, promotes ferroptosis induction and accounts for oxidative damage. While emerging studies on ferroptosis in cancers have garnered increasing attention, the lack of consideration for ferroptosis inducers (FINs) with favorable pharmacokinetics could be problematic. Herein, we remodeled the chemical structure of sorafenib, of which pharmacokinetics and safety are already assured, to customize the off-target effect (i.e., ferroptosis induction) to on-target by disrupting the adenine-binding motif. JB3, a sorafenib derivative (i.e., JB compounds), with a tenfold higher IC50 toward RAF1 because of chemical remodeling, induced strong cytotoxicity in the elastin-sensitive lung cancer cells, while it was markedly reduced by ferrostatin-1. The 24% oral bioavailability of JB3 in rats accounted for a significant anti-tumor effect of orally administrated JB3 in xenograft models. These results indicate that JB3 could be further developed as an orally bioavailable FIN in novel anti-cancer therapeutics.

Optimizing Embryo Collection for Application of CRISPR/Cas9 System and Generation of Fukutin Knockout Rat Using This Method.

Rat animal models are widely used owing to their relatively superior cognitive abilities and higher similarity compared with mouse models to human physiological characteristics. However, their use is limited because of difficulties in establishing embryonic stem cells and performing genetic modifications, and insufficient embryological research. In this study, we established optimal superovulation and fertilized-egg transfer conditions, including optimal hormone injection concentration (≥150 IU/kg of PMSG and hCG) and culture medium (mR1ECM), to obtain high-quality zygotes and establish in vitro fertilization conditions for rats. Next, sgRNA with optimal targeting activity was selected by performing PCR analysis and the T7E1 assay, and the CRISPR/Cas9 system was used to construct a rat model for muscular dystrophy by inducing a deficiency in the fukutin gene without any off-target effect detected. The production of fukutin knockout rats was phenotypically confirmed by observing a drop-in body weight to one-third of that of the control group. In summary, we succeeded in constructing the first muscular dystrophy disease rat model using the CRISPR/CAS9 system for increasing future prospects of producing various animal disease models and encouraging disease research using rats.

Aptamers and Nanobodies as New Bioprobes for SARS-CoV-2 Diagnostic and Therapeutic System Applications.

The global challenges posed by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic have underscored the critical importance of innovative and efficient control systems for addressing future pandemics. The most effective way to control the pandemic is to rapidly suppress the spread of the virus through early detection using a rapid, accurate, and easy-to-use diagnostic platform. In biosensors that use bioprobes, the binding affinity of molecular recognition elements (MREs) is the primary factor determining the dynamic range of the sensing platform. Furthermore, the sensitivity relies mainly on bioprobe quality with sufficient functionality. This comprehensive review investigates aptamers and nanobodies recently developed as advanced MREs for SARS-CoV-2 diagnostic and therapeutic applications. These bioprobes might be integrated into organic bioelectronic materials and devices, with promising enhanced sensitivity and specificity. This review offers valuable insights into advancing biosensing technologies for infectious disease diagnosis and treatment using aptamers and nanobodies as new bioprobes.

Discovery and Feasibility Study of Medical Fluorophore 33 as a Novel Theranostic Agent.

Fluorescent dyes have garnered significant attention as theranostic platforms owing to their inherent characteristics. In this study, we present the discovery of Medical Fluorophore 33 (MF33), a novel and potent theranostic agent with a phenaleno-isoquinolinium salt structure that can serve as a cancer therapeutic strategy. The synthesis of MF33 is readily achievable through a simple Rh(III)-catalyzed reaction. Moreover, MF33 displayed strong fluorescence signals, excellent microsomal stability, and high biocompatibility in vivo. It induces significant apoptosis in cancer cells via the p53/p21/caspase-3 signaling pathway, leading to selective cytotoxicity in various cancer cells. In vivo fluorescence imaging with MF33 enabled the visualization of sentinel lymph nodes in living mice. Notably, repeated intraperitoneal administration of MF33 resulted in antitumor activity in mice with colorectal cancer. Collectively, our findings suggest that phenaleno-isoquinolinium salt-based MF33 is a viable theranostic agent for biomedical imaging and cancer treatment.

Visualizing mast cell migration to tumor sites using sodium iodide symporter of nuclear medicine reporter gene.

PURPOSE: Owing to the close relationship between mast cells and cancer progression, an imaging technique that can be applied in a clinical setting to explore the biological behavior of mast cells in the tumor microenvironment is needed. In this study, we visualized mast cell migration to lung tumor lesions in live mice using sodium iodide symporter (NIS) as a nuclear medicine reporter gene. EXPERIMENTAL DESIGN: The murine mast cell line MC-9 was infected with retrovirus including NIS, luciferase (as a surrogate marker for NIS), and Thy1.1 to generate MC-9/NFT cells. Radioiodine uptake was measured in MC-9/NFT cells, and an inhibition assay of radioiodine uptake using KCLO4 was also performed. Cell proliferation and FcεRI expression was examined in MC-9 and MC-9/NFT cells. The effect of mast cell-conditioned media (CM) on the proliferation of Lewis lung cancer (LLC) cells was examined. The migration level of MC-9/NFT cells was confirmed in the presence of serum-free media (SFM) and CM of cancer cells. After intravenous injection of MC-9/NFT cells into mice with an LLC tumor, I-124 PET/CT and biodistribution analysis was performed. RESULTS: MC-9/NFT cells exhibited higher radioiodine avidity compared to parental MC-9 cells; this increased radioiodine avidity in MC-9/NFT cells was reduced to basal level by KCLO4. Levels of FcεRI expression and cell proliferation were not different in parental MC-9 cell and MC-9/ NFT cells. The CM of MC-9/NFT cells increased cancer cell proliferation relative to that of the SFM. The migration level of MC-9/NFT cells was higher in the CM than the SFM of LLC cells. PET/CT imaging with I-124 clearly showed infiltration of reporter mast cells in lung tumor at 24 h after transfer, which was consistent with the findings of the biodistribution examination. CONCLUSION: These findings suggest that the sodium iodide symporter can serve as a reliable nuclear medicine reporter gene for non-invasively imaging the biological activity of mast cells in mice with lung tumors. Visualizing mast cells in the tumor microenvironment via a nuclear medicine reporter gene would provide valuable insights into their biological functions.

An Inverse Agonist of Estrogen-Related Receptor Gamma, GSK5182, Enhances Na+/I- Symporter Function in Radioiodine-Refractory Papillary Thyroid Cancer Cells.

Previously, we reported that an inverse agonist of estrogen-related receptor gamma (ERRγ), GSK5182, enhances sodium iodide (Na+/I-) symporter (NIS) function through mitogen-activated protein (MAP) kinase signaling in anaplastic thyroid cancer cells. This finding helped us to further investigate the effects of GSK5182 on NIS function in papillary thyroid cancer (PTC) refractory to radioactive iodine (RAI) therapy. Herein, we report the effects of ERRγ on the regulation of NIS function in RAI-resistant PTC cells using GSK5182. RAI-refractory BCPAP cells were treated with GK5182 for 24 h at various concentrations, and radioiodine avidity was determined with or without potassium perchlorate (KClO4) as an NIS inhibitor. We explored the effects of GSK5182 on ERRγ, the mitogen-activated protein (MAP) kinase pathway, and iodide metabolism-related genes. We examined whether the MAP pathway affected GSK5182-mediated NIS function using U0126, a selective MEK inhibitor. A clonogenic assay was performed to evaluate the cytotoxic effects of I-131. GSK5182 induced an increase in radioiodine avidity in a dose-dependent manner, and the enhanced uptake was completely inhibited by KClO4 in BCPAP cells. We found that ERRγ was downregulated and phosphorylated extracellular signal-regulated kinase (ERK)1/2 was upregulated in BCPAP cells, with an increase in total and membranous NIS and iodide metabolism-related genes. MEK inhibitors reversed the increase in radioiodine avidity induced by GSK5182. Clonogenic examination revealed the lowest survival in cells treated with a combination of GSK5182 and I-131 compared to those treated with either GSK518 or I-131 alone. We demonstrate that an inverse agonist of ERRγ, GSK5182, enhances the function of NIS protein via the modulation of ERRγ and MAP kinase signaling, thereby leading to increased responsiveness to radioiodine in RAI-refractory papillary thyroid cancer cells.

Repositioning Trimebutine Maleate as a Cancer Treatment Targeting Ovarian Cancer Stem Cells.

The overall five-year survival rate for late-stage patients of ovarian cancer is below 29% due to disease recurrence and drug resistance. Cancer stem cells (CSCs) are known as a major contributor to drug resistance and recurrence. Accordingly, therapies targeting ovarian CSCs are needed to overcome the limitations of present treatments. This study evaluated the effect of trimebutine maleate (TM) targeting ovarian CSCs, using A2780-SP cells acquired by a sphere culture of A2780 epithelial ovarian cancer cells. TM is indicated as a gastrointestinal motility modulator and is known to as a peripheral opioid receptor agonist and a blocker for various channels. The GI50 of TM was approximately 0.4 µM in A2780-SP cells but over 100 µM in A2780 cells, demonstrating CSCs specific growth inhibition. TM induced G0/G1 arrest and increased the AV+/PI+ dead cell population in the A2780-SP samples. Furthermore, TM treatment significantly reduced tumor growth in A2780-SP xenograft mice. Voltage gated calcium channels (VGCC) and calcium-activated potassium channels (BKCa) were overexpressed on ovarian CSCs and targeted by TM; inhibition of both channels reduced A2780-SP cells viability. TM reduced stemness-related protein expression; this tendency was reproduced by the simultaneous inhibition of VGCC and BKCa compared to single channel inhibition. In addition, TM suppressed the Wnt/ß-catenin, Notch, and Hedgehog pathways which contribute to many CSCs characteristics. Specifically, further suppression of the Wnt/ß-catenin pathway by simultaneous inhibition of BKCa and VGCC is necessary for the effective and selective action of TM. Taken together, TM is a potential therapeutic drug for preventing ovarian cancer recurrence and drug resistance.

Tunicamycin as a Novel Redifferentiation Agent in Radioiodine Therapy for Anaplastic Thyroid Cancer.

The silencing of thyroid-related genes presents difficulties in radioiodine therapy for anaplastic thyroid cancers (ATCs). Tunicamycin (TM), an N-linked glycosylation inhibitor, is an anticancer drug. Herein, we investigated TM-induced restoration of responsiveness to radioiodine therapy in radioiodine refractory ATCs. 125I uptake increased in TM-treated ATC cell lines, including BHT101 and CAL62, which was inhibited by KClO4, a sodium-iodide symporter (NIS) inhibitor. TM upregulated the mRNA expression of iodide-handling genes and the protein expression of NIS. TM blocked pERK1/2 phosphorylation in both cell lines, but AKT (protein kinase B) phosphorylation was only observed in CAL62 cells. The downregulation of glucose transporter 1 protein was confirmed in TM-treated cells, with a significant reduction in 18F-fluorodeoxyglucose (FDG) uptake. A significant reduction in colony-forming ability and marked tumor growth inhibition were observed in the combination group. TM was revealed to possess a novel function as a redifferentiation inducer in ATC as it induces the restoration of iodide-handling gene expression and radioiodine avidity, thereby facilitating effective radioiodine therapy.

In Vivo Optical Reporter-Gene-Based Imaging of Macrophage Infiltration of DNCB-Induced Atopic Dermatitis.

This study was conducted to monitor the macrophage infiltration of atopic dermatitis (AD)-like skin lesions and to evaluate the effects of anti-AD therapeutic agents in immunocompetent mice via optical reporter-gene-based molecular imaging. The enhanced firefly luciferase (effluc)-expressing macrophage cell line (Raw264.7/effluc) was intravenously introduced into mice with 2,4-dinitrochlorobenzene (DNCB)-induced AD, followed by bioluminescent imaging (BLI). After in vivo imaging, AD-like skin lesions were excised, and ex vivo imaging and Western blotting were conducted to determine the presence of infused macrophages. Finally, the therapeutic effect of dexamethasone (DEX), an AD-modulating agent, was evaluated via macrophage tracking. In vivo imaging with BLI revealed the migration of the reporter macrophages to DNCB-induced AD-like skin lesions on day 1 post-transfer. The greatest recruitment was observed on day 3, and a decline in BLI signal was observed on day 14. Notably, in vivo BLI clearly showed the inhibition of the reporter macrophage infiltration of DNCB-induced AD-like skin lesions by DEX, which was consistent with the reduced AD symptoms observed in DEX-treated mice. We successfully visualized the macrophage migration to DNCB-induced AD-like skin lesions, proving the feasibility of macrophage imaging for evaluating AD-regulating drugs in living organisms.

An orally available inverse agonist of estrogen-related receptor gamma showed expanded efficacy for the radioiodine therapy of poorly differentiated thyroid cancer.

Estrogen-related receptor gamma (ERRγ) is the NR3B subgroup of associated transcription factors. In this report, a new generation of a potent and selective ERRγ inverse agonist (25) with good biocompatibility was proposed. We also explored the potential of the newly developed compound 25 in the PDTC model to expand the original indications from ATC. In addition, an X-ray crystallographic study of the ligand and ERRγ co-complex showed that 25 completely binds to the target protein (PDB 6KNR). Its medicinal chemistry, including a distinctive structural study to in vivo results, denotes that 25 may be directed towards the development of a pivotal treatment for ERRγ-related cancers.

The Expression and Contribution of SRCs with Preeclampsia Placenta.

The steroid hormones act by binding to their receptors and subsequently interacting with coactivators. Several classes of coactivators have been identified and shown to be essential in estradiol (E2) responsiveness. The major coregulators are the p160 steroid receptor coactivator (SRC) family. Although the function of SRCs in other organs has been well studied, it has not been thoroughly studied in the placenta. In addition, the correlation between preeclampsia (PE) and SRCs has not been examined previously. Therefore, we compared the expression patterns of SRCs in normal and PE placentas. In human PE placental tissues, SRC-1 mRNA, and protein levels were downregulated in the PE group. In addition, to assess the expression of SRCs in a PE environment, we used Reduced Uterine Perfusion Pressure (RUPP) model and placental cells were cultured in hypoxia condition. SRC-1 proteins were reduced in the placenta of PE-like rat RUPP model. Furthermore, SRCs proteins were significantly downregulated in hypoxia-grown placental cells. To examine the interaction between estrogen receptors (ERs) and SRC-1 protein, we performed co-immunoprecipitation. The interaction of SRC-1 with ERα was significantly stronger than that with ERß. In PE placenta, the interaction of both ERα and ERß with SRC-1 was stronger than that in normal placenta. In summary, our results demonstrate that expression levels of SRC-1, not SRC-2 and SRC-3, were decreased in hypoxia-induced PE placenta, which may further reduce the signaling of sex steroid hormones such as E2. The dysregulated signaling of E2 by SRC-1 expression could be associated with the PE placental symptoms of patients.

Medical fluorophore 1 (MF1), a benzoquinolizinium-based fluorescent dye, as an inflammation imaging agent.

Structure-based targeting of fluorescent dyes is essential for their use as imaging agents for disease diagnosis. Here, we describe the development of the benzoquinolizinium compound Medical fluorophore 1 (MF1) as a novel biomedical imaging agent that allows the visualization of inflammation by virtue of its unique chemical structure. Lipopolysaccharide treatment stimulated the uptake of MF1 by bone marrow-derived macrophages, with no adverse effects on cell proliferation. In vivo fluorescence lifetime imaging revealed the accumulation of MF1 in carrageenan-induced acute inflammatory lesions in mice, which peaked at 6 h. MF1-based imaging also allowed monitoring of the response to the anti-inflammatory drugs dexamethasone and sulfasalazine. Thus, MF1 can be used to diagnose diseases characterized by inflammation as well as treatment efficacy.

A Novel Orally Active Inverse Agonist of Estrogen-related Receptor Gamma (ERRγ), DN200434, A Booster of NIS in Anaplastic Thyroid Cancer.

PURPOSE: New strategies to restore sodium iodide symporter (NIS) expression and function in radioiodine therapy-refractive anaplastic thyroid cancers (ATCs) are urgently required. Recently, we reported the regulatory role of estrogen-related receptor gamma (ERRγ) in ATC cell NIS function. Herein, we identified DN200434 as a highly potent (functional IC50 = 0.006 µmol/L), selective, and orally available ERRγ inverse agonist for NIS enhancement in ATC. EXPERIMENTAL DESIGN: We sought to identify better ERRγ-targeting ligands and explored the crystal structure of ERRγ in complex with DN200434. After treating ATC cells with DN200434, the change in iodide-handling gene expression, as well as radioiodine avidity was examined. ATC tumor-bearing mice were orally administered with DN200434, followed by 124I-positron emission tomography/CT (PET/CT). For radioiodine therapy, ATC tumor-bearing mice treated with DN200434 were administered 131I (beta ray-emitting therapeutic radioiodine) and then bioluminescent imaging was performed to monitor the therapeutic effects. Histologic analysis was performed to evaluate ERRγ expression status in normal tissue and ATC tissue, respectively. RESULTS: DN200434-ERRγ complex crystallographic studies revealed that DN200434 binds to key ERRγ binding pocket residues through four-way interactions. DN200434 effectively upregulated iodide-handling genes and restored radioiodine avidity in ATC tumor lesions, as confirmed by 124I-PET/CT. DN200434 enhanced ATC tumor radioiodine therapy susceptibility, markedly inhibiting tumor growth. Histologic findings of patients with ATC showed higher ERRγ expression in tumors than in normal tissue, supporting ERRγ as a therapeutic target for ATC. CONCLUSIONS: DN200434 shows potential clinical applicability for diagnosis and treatment of ATC or other poorly differentiated thyroid cancers.

Discovery of Potent, Selective, and Orally Bioavailable Estrogen-Related Receptor-γ Inverse Agonists To Restore the Sodium Iodide Symporter Function in Anaplastic Thyroid Cancer.

An inverse agonist of estrogen-related receptor-γ (ERRγ), an orphan nuclear receptor encoded by E srrg, enhances sodium iodide symporter-mediated radioiodine uptake in anaplastic thyroid cancer (ATC) cells, thereby facilitating responsiveness to radioiodine therapy in vitro. We synthesized potent, selective, and orally bioavailable ERRγ-inverse agonists and evaluated their activity by analyzing in vitro pharmacology and absorption, distribution, metabolism, excretion, and toxicity profiles. X-ray crystallographic analysis of the ligand and ERRγ complex showed that 35 completely binds to the target protein (PDB 6A6K ). Our results showed improved radioiodine avidity in ATC cells through compound 35-mediated upregulation of iodide-handling genes, leading to enhanced responsiveness to radioiodine therapy in vitro. Importantly, in vivo 124I-positron emission tomography/computed tomography imaging revealed that 35 increases radioiodine avidity in CAL62 tumors. Collectively, these results demonstrated that 35 can be developed as a promising treatment for ERRγ-related cancer in the future.

Crushed Gold Shell Nanoparticles Labeled with Radioactive Iodine as a Theranostic Nanoplatform for Macrophage-Mediated Photothermal Therapy.

Plasmonic nanostructure-mediated photothermal therapy (PTT) has proven to be a promising approach for cancer treatment, and new approaches for its effective delivery to tumor lesions are currently being developed. This study aimed to assess macrophage-mediated delivery of PTT using radioiodine-124-labeled gold nanoparticles with crushed gold shells (124I-Au@AuCBs) as a theranostic nanoplatform. 124I-Au@AuCBs exhibited effective photothermal conversion effects both in vitro and in vivo and were efficiently taken up by macrophages without cytotoxicity. After the administration of 124I-Au@AuCB-labeled macrophages to colon tumors, intensive signals were observed at tumor lesions, and subsequent in vivo PTT with laser irradiation yielded potent antitumor effects. The results indicate the considerable potential of 124I-Au@AuCBs as novel theranostic nanomaterials and the prominent advantages of macrophage-mediated cellular therapies in treating cancer and other diseases.

The expression and activation of sex steroid receptors in the preeclamptic placenta.

Estrogen and progesterone are the main pregnancy hormones produced by the placenta. It is well understood that estrogen stimulates angiogenesis in the uterus during the reproductive cycle. Although the estrogen and progesterone signaling pathways are assumed to be associated with placental vascularization and preeclampsia, expression of estrogen receptors (ESRs) and progesterone receptor (PGR) in the placenta have not been well studied. The present study examined the expression patterns of steroid hormone receptors in placentas. Human placenta samples were collected and divided into normal and preeclampsia groups. Results revealed that expression levels of ESR1 were reduced, whereas ESR2 and PGR were elevated in preeclamptic placentas. To generate an in vitro preeclampsia environment, human placenta­derived BeWo cells were incubated under hypoxic conditions, or treated with catechol­O­methyl transferase inhibitor (COMT­in) or L­NG­nitroarginine methyl ester (L­NAME). Expression levels of ESR1, ESR2 and PGR in hypoxic cells demonstrated similar regulation as those in placentas from women with preeclampsia. Although COMT­in and L­NAME did not significantly regulate the expression levels of the receptors, COMT­in translocated ESR2 and PGR from the nucleus to the cytoplasm, indicating that these receptors were inactivated. These results suggested that ESRs and PGR are associated with symptoms of preeclampsia in the placenta. The expression of ESR1 was reduced in preeclamptic placenta and hypoxic BeWo cells. In addition, the activation of ESR2 and PGR was blocked in placenta cells subjected to COMT­in treatment. The reduced ESR1 expression and inactivation of ESR2 and PGR proteins may affect the physiological complications of preeclampsia in the placenta.

Regulation of steroid hormones in the placenta and serum of women with preeclampsia.

Preeclampsia (PE) is a pregnancy­specific hypertensive syndrome that results in substantial maternal and fetal morbidity and mortality. The exact cause of PE has not been completely elucidate, although abnormal formation of the placenta has been considered. The placenta connects the developing fetus to the uterine wall, producing a large quantity of steroid hormones to maintain pregnancy. Although steroid hormones, particularly progesterone (P4) and estrogen (E2), in the serum of women with PE have been studied, steroidogenesis in the placenta has not well been established. The present study compared the concentrations of steroid hormones, including pregnenolone (PG), P4, dehydroepiandrosterone (DHEA), testosterone (T) and E2, in the serum and placenta of women with PE. PG, P4, DHEA and E2 concentrations tended to be decreased in PE serum and placentas, and the results were statistically significant for P4 and E2 in the serum. Quantification of genes associated with steroidogenesis in the placenta was performed, and the expression of the P4­ and E2­synthesizing enzymes testosterone 17­ß­dehydrogenase 3 and 3 ß­hydroxysteroid dehydrogenase/δ5 4­isomerase type 1 was reduced. Notably, aromatase, an enzyme required for the production of E2, was upregulated in the PE placenta, suggesting that steroidogenic enzymes may be dynamically regulated and may affect the symptoms of PE. In conclusion, the results of the present study suggested that the levels of steroid hormones, including P4 and E2, in the serum and placenta of women with PE are downregulated, which may be mediated by the regulation of steroidogenic enzyme expression in the PE placenta.

The regulation of oxytocin and oxytocin receptor in human placenta according to gestational age.

Oxytocin (OXT) is a peptide hormone that plays a central role in the regulation of parturition and lactation. OXT signaling is mediated by OXT receptor (OXTR), which shows species- and tissue-specific expressions and gene regulation. In the present study, we examined the synthesis of OXT and OXTR in human placenta tissue according to gestational age. A total of 48 placentas were divided into early preterm, late preterm and term groups depending on gestational age, and expression of OXT and OXTR was evaluated. First, OXT and OXTR mRNA and protein were detected in normal placenta tissue via Q-PCR, Dot-blot and Western blot assay. Both OXT and OXTR levels in normal placenta increased gradually in the late stage of pregnancy, suggesting that local OXT may play a critical role in the function of the placenta. To determine the regulatory mechanism of OXT, placental BeWo cells were administrated estrogen (E2) or progesterone (P4), and expression of OXT and OXTR was tested. The mRNA and protein levels of OXT and OXTR were upregulated by E2 but blocked by co-treatment with P4 In order to confirm the estrogen receptor (ESR)-mediated signaling, we administrated ESR antagonists together with E2 to BeWo cells. As a result, both OXT and OXTR were significantly altered by ESR1 antagonist (MPP) while moderately regulated by ESR2 antagonist (PHTPP). These results suggest that OXT and OXTR are controlled mainly by E2 in the placenta via ESR1 and thus may play physiological functions in the human placenta during the late stage of pregnancy.

Expression of reproductive hormone receptors and contraction­associated genes in porcine uterus during the estrous cycle.

Contraction of uterus tissue frequently occurs throughout the estrous cycle and is regulated by several endogenous factors, including estradiol, progesterone, luteinizing hormone, follicle­stimulating hormone, oxytocin (OXT) and contraction­associated proteins (CAPs). Contraction activity of uterus tissue according to the estrous cycle is important, due to the fact that it is directly associated with balanced implantation and stable pregnancy. However, few studies have examined the mechanism of uterus contraction activity in a porcine model. In the current study, porcine uterus tissue was separated into the follicular and luteal phases by histological analysis. To investigate regulation of contraction­associated factors according to the estrous cycle, mRNA and protein expression levels of reproductive hormonal receptors, including estrogen receptors, progesterone receptor and luteinizing hormone/choriogonadotropin receptor in addition to CAPs including OXT, OXT receptor (OXTR), hydroxyprostaglandin dehydrogenase 15­(NAD) and gap junction α­1 protein, were examined in the porcine uterus according to the follicular and luteal phases. For the results, hormonal receptors and CAPs were dynamically regulated depending on the estrous cycle. In conclusion, genes associated with uterine contraction and its regulatory hormonal receptors in the porcine uterus were differently regulated in the follicular and luteal phases, suggesting that these genes are critically involved in the remodeling and contraction of uterine tissue and may be required to modulate the physiological status of the uterus.