Bispecific aptamer-decorated and light-triggered nanoparticles targeting tumor and stromal cells in breast cancer derived organoids: implications for precision phototherapies.

BACKGROUND: Based on the established role of cancer-stroma cross-talk in tumor growth, progression and chemoresistance, targeting interactions between tumor cells and their stroma provides new therapeutic approaches. Dual-targeted nanotherapeutics selectively acting on both tumor and stromal cells may overcome the limits of tumor cell-targeting single-ligand nanomedicine due to the complexity of the tumor microenvironment. METHODS: Gold-core/silica-shell nanoparticles embedding a water-soluble iridium(III) complex as photosensitizer and luminescent probe (Iren-AuSiO2_COOH) were efficiently decorated with amino-terminated EGFR (CL4) and PDGFRß (Gint4.T) aptamers (Iren-AuSiO2_Aptamer). The targeting specificity, and the synergistic photodynamic and photothermal effects of either single- and dual-aptamer-decorated nanoparticles have been assessed by confocal microscopy and cell viability assays, respectively, on different human cell types including mesenchymal subtype triple-negative breast cancer (MES-TNBC) MDA-MB-231 and BT-549 cell lines (both EGFR and PDGFRß positive), luminal/HER2-positive breast cancer BT-474 and epidermoid carcinoma A431 cells (only EGFR positive) and adipose-derived mesenchymal stromal/stem cells (MSCs) (only PDGFRß positive). Cells lacking expression of both receptors were used as negative controls. To take into account the tumor-stroma interplay, fluorescence imaging and cytotoxicity were evaluated in preclinical three-dimensional (3D) stroma-rich breast cancer models. RESULTS: We show efficient capability of Iren-AuSiO2_Aptamer nanoplatforms to selectively enter into target cells, and kill them, through EGFR and/or PDGFRß recognition. Importantly, by targeting EGFR+ tumor/PDGFRß+ stromal cells in the entire tumor bulk, the dual-aptamer-engineered nanoparticles resulted more effective than unconjugated or single-aptamer-conjugated nanoparticles in either 3D spheroids cocultures of tumor cells and MSCs, and in breast cancer organoids derived from pathologically and molecularly well-characterized tumors. CONCLUSIONS: Our study proposes smart, novel and safe multifunctional nanoplatforms simultaneously addressing cancer-stroma within the tumor microenvironment, which are: (i) actively delivered to the targeted cells through highly specific aptamers; (ii) localized by means of their luminescence, and (iii) activated via minimally invasive light, launching efficient tumor death, thus providing innovative precision therapeutics. Given the unique features, the proposed dual targeted nanoformulations may open a new door to precision cancer treatment.

Juan-tong-yin potentially impacts endometriosis pathophysiology by enhancing autophagy of endometrial stromal cells via unfolded protein reaction-triggered endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Endometriosis (EMs) is characterized by inflammatory lesions, dysmenorrhea, infertility, and chronic pelvic pain. Single-target medications often fail to provide systemic therapeutic results owing to the complex mechanism underlying endometriosis. Although traditional Chinese medicines-such as Juan-Tong-Yin (JTY)-have shown promising results, their mechanisms of action remain largely unknown. AIM OF THE STUDY: To elucidate the therapeutic mechanism of JTY in EMs, focusing on endoplasmic reticulum (ER) stress-induced autophagy. MATERIALS AND METHODS: The major components of JTY were detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS). The potential mechanism of JTY in EMs treatment was predicted using network pharmacological analysis. Finally, the pathogenesis of EMs was validated in a clinical case-control study and the molecular mechanism of JTY was validated in vitro using endometrial stromal cells (ESCs). RESULTS: In total, 241 compounds were analyzed and identified from JTY using UPLC-MS. Network pharmacology revealed 288 targets between the JTY components and EMs. Results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses indicated that regulating autophagy, migration, apoptosis, and inflammation were the key mechanisms of JTY in treating EMs. Meanwhile, we found that protein kinase R-like endoplasmic reticulum kinase (PERK), Beclin-1, and microtubule-associated protein light chain 3 B (LC3B) expressions were lower in endometria of patients with EMs than in those with normal eutopic endometria (p < 0.05). Additionally, during in vitro experiments, treatment with 20% JTY-containing serum significantly suppressed ESC proliferation, achieving optimal effects after 48 h. Electron microscopy revealed significantly increased autophagy flux in the JTY group compared with the control group. Moreover, JTY treatment significantly reduced the migratory and invasive abilities of ESCs and upregulated protein expression of PERK, eukaryotic initiation factor 2α (eIF2α)/phospho-eukaryotic initiation factor 2α (p-eIF2α), activating Transcription Factor-4 (ATF4), Beclin-1, and LC3BII/I, while subsequently downregulating NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and interleukin 18 (IL-18) expression. However, administration of GSK2656157-a highly selective PERK inhibitor-reversed these changes. CONCLUSION: JTY ameliorates EMs by activating PERK associated with unfolded protein reaction, enhancing cell ER stress and autophagy, improving the inflammatory microenvironment, and decreasing the migration and invasion of ESCs.

ITRAQ-based proteomics analysis of human ectopic endometrial stromal cells treated by Maqian essential oil.

BACKGROUND: Endometriosis is a common and complex syndrome characterized by the presence of endometrial-like tissue outside the uterus. Chinese medicine has been recently found to show good efficacy in treating endometriosis. Our previous results revealed that Maqian fruit essential oil (MQEO) could inhibit the proliferation and induce apoptosis of ectopic endometrial stromal cells (EESCs), but the mechanisms remain unclear. In this study, we aim to explore the molecular mechanism of MQEO's specific effects in EESCs. METHODS: We conducted a quantitative proteomics analysis by iTRAQ on EESCs treated with MQEO or DMSO. Then deep analysis was performed based on differentially expressed proteins, including Gene Ontology enrichment analysis, pathway enrichment analysis and protein interaction analysis. Candidate protein targets were subsequently verified by western blotting. RESULTS: Among 6575 identified proteins, 435 proteins exhibited altered expression levels in MQEO-treated EESCs. Of these proteins, most were distributed in signal transduction as well as immune system and the most significantly altered pathway was complement and coagulation cascades. Moreover, two differentially expressed proteins (Heme oxygenase 1 and Acyl-CoA 6-desaturase) were verified and they can be potential biomarkers for endometriosis treatment. CONCLUSIONS: Our proteomic analysis revealed distinct protein expression patterns induced by MQEO treatment in EESCs, highlighting the potential of MQEO for endometriosis treatment and biomarker discovery.

Exosomes and their miRNA/protein profile in keratoconus-derived corneal stromal cells.

Keratoconus (KC) is a corneal thinning disorder and a leading cause of corneal transplantation worldwide. Exosomes are small, secreted extracellular vesicles (30-150 nm) that mediate cellular communication via their protein, lipid, and nucleic acid content. We aimed to characterize the exosomes secreted by primary corneal fibroblasts from subjects with or without KC. Using human keratoconus stromal fibroblast cells (HKC, n = 4) and healthy stromal fibroblasts (HCF, n = 4), we collected and isolated exosomes using serial ultracentrifugation. Using nanoparticle tracking analysis (NTA) with ZetaView®, we compared the size and concentration of isolated exosomes. Different exosomal markers were identified and quantified using a transmission electron microscope (TEM) (CD81) and Western blot (CD9 and CD63). Exosomal miRNA profiles were determined by qRT-PCR using Exiqon Human panel I miRNA assays of 368 pre-selected miRNAs. Proteomic profiles were determined using a label-free spectral counting method with mass spectrometry. Differential expression analysis for miRNAs and proteins was done using student's t-test with a significance cutoff of p-value ≤0.05. We successfully characterized exosomes isolated from HCFs using several complementary techniques. We found no significant differences in the size, quantity, or morphology between exosomes secreted by HCFs with or without KC. Expression of CD81 was confirmed by immuno-EM, and expression of CD63 and CD9 with western blots in all exosome samples. We detected the expression of 72-144 miRNAs (threshold cycle Ct < 36) in all exosome samples. In HKC-derived exosome samples, miR-328-3p, miR-532-5p, miR-345-5p, and miR-424-5p showed unique expression, while let-7c-5p and miR-665 have increased expression. Protein profiling identified 157 proteins in at least half of the exosome samples, with 38 known exosomal proteins. We identified 12 up- and 2 down-regulated proteins in HKC-derived exosomes. The proteins are involved in membrane-bounded vesicles, cytoskeletal, calcium binding, and nucleotide binding. These proteins are predicted to be regulated by NRF2, miR-205, and TGF-ß1, which are involved in KC pathogenesis. We successfully characterized the HKC-derived exosomes and profiled their miRNA and protein contents, suggesting their potential role in KC development. Further studies are necessary to determine if and how these exosomes with differential protein/miRNA profiles contribute to the pathogenesis of KC.

Melatonin modulates endometrial decidualization via NOTCH1-NRF2-FOXO1-GSH pathway†.

Melatonin is important for oocyte maturation, fertilization, early embryonic development, and embryo implantation, but less knowledge is available regarding its role in decidualization. The present study found that melatonin did not alter the proliferation of human endometrial stromal cells (ESCs), as well as cell cycle progress, but suppressed stromal differentiation after binding to the melatonin receptor 1B (MTNR1B), which was visualized in decidualizing ESCs. Further analysis evidenced that application of melatonin resulted in the diminishment for NOTCH1 and RBPJ expression. Supplementation of recombinant NOTCH1 protein (rNOTCH1) counteracted the impairment of stromal differentiation conferred by melatonin, while the addition of the NOTCH signaling pathway inhibitor DAPT aggravated the differentiation progress. Meanwhile, melatonin might restrain the expression and transcriptional activity of nuclear factor erythroid 2-related factor 2 (NRF2), whose blockage accelerated the fault of stromal differentiation under the context of melatonin, but this restraint was subsequently ameliorated by rNOTCH1. Forkhead box O 1 (FOXO1) was identified as a downstream target of melatonin in decidualization. Repression of NRF2 antagonized the retrieval of rNOTCH1 due to aberrant FOXO1 expression elicited by melatonin. Moreover, melatonin brought about the occurrence of oxidative stress accompanied by an obvious accumulation of intracellular reactive oxygen species and a significant reduction in glutathione (GSH) content, as well as enzymatic activities of glutathione peroxidase and glutathione reductase, whereas supplementation of rNOTCH1 improved the above-mentioned effects. Nevertheless, this improvement was disrupted by the blockage of NRF2 and FOXO1. Furthermore, addition of GSH rescued the defect of stromal differentiation by melatonin. Collectively, melatonin might impair endometrial decidualization by restraining the differentiation of ESCs dependent on NOTCH1-NRF2-FOXO1-GSH pathway after binding to the MTNR1B receptor.

Antioxidant Supplementation Alleviates Mercury-Induced Cytotoxicity and Restores the Implantation-Related Functions of Primary Human Endometrial Cells.

Mercury (Hg) cytotoxicity, which is largely mediated through oxidative stress (OS), can be relieved with antioxidants. Thus, we aimed to study the effects of Hg alone or in combination with 5 nM N-Acetyl-L-cysteine (NAC) on the primary endometrial cells' viability and function. Primary human endometrial epithelial cells (hEnEC) and stromal cells (hEnSC) were isolated from 44 endometrial biopsies obtained from healthy donors. The viability of treated endometrial and JEG-3 trophoblast cells was evaluated via tetrazolium salt metabolism. Cell death and DNA integrity were quantified following annexin V and TUNEL staining, while the reactive oxygen species (ROS) levels were quantified following DCFDA staining. Decidualization was assessed through secreted prolactin and the insulin-like growth factor-binding protein 1 (IGFBP1) in cultured media. JEG-3 spheroids were co-cultured with the hEnEC and decidual hEnSC to assess trophoblast adhesion and outgrowth on the decidual stroma, respectively. Hg compromised cell viability and amplified ROS production in trophoblast and endometrial cells and exacerbated cell death and DNA damage in trophoblast cells, impairing trophoblast adhesion and outgrowth. NAC supplementation significantly restored cell viability, trophoblast adhesion, and outgrowth. As these effects were accompanied by the significant decline in ROS production, our findings originally describe how implantation-related endometrial cell functions are restored in Hg-treated primary human endometrial co-cultures by antioxidant supplementation.

Decidual Stromal Cell Ferroptosis Associated with Abnormal Iron Metabolism Is Implicated in the Pathogenesis of Recurrent Pregnancy Loss.

Iron is necessary for various critical biological processes, but iron overload is also dangerous since labile iron is redox-active and toxic. We found that low serum iron and decidual local iron deposition existed simultaneously in recurrent pregnancy loss (RPL) patients. Mice fed with a low-iron diet (LID) also showed iron deposition in the decidua and adverse pregnancy outcomes. Decreased ferroportin (cellular iron exporter) expression that inhibited the iron export from decidual stromal cells (DSCs) might be the reason for local iron deposition in DSCs from low-serum-iron RPL patients and LID-fed mice. Iron supplementation reduced iron deposition in the decidua of spontaneous abortion models and improved pregnancy outcomes. Local iron overload caused ferroptosis of DSCs by downregulating glutathione (GSH) and glutathione peroxidase 4 levels. Both GSH and cystine (for the synthesis of GSH) supplementation reduced iron-induced lipid reactive oxygen species (ROS) and cell death in DSCs. Ferroptosis inhibitor, cysteine, and GSH supplementation all effectively attenuated DSC ferroptosis and reversed embryo loss in the spontaneous abortion model and LPS-induced abortion model, making ferroptosis mitigation a potential therapeutic target for RPL patients. Further study that improves our understanding of low-serum-iron-induced DSC ferroptosis is needed to inform further clinical evaluations of the safety and efficacy of iron supplementation in women during pregnancy.

Synergistic effects of electroacupuncture and bone marrow stromal cells transplantation therapy in ischemic stroke.

OBJECTIVE: Animal studies and clinical trials demonstrated the effectiveness of a combination of transplanted bone marrow stromal cells (BMSC) and electroacupuncture (EA) treatment in improving neurological deficits. However, the ability of the BMSC-EA treatment to enhance brain repair processes or the neuronal plasticity of BMSC in ischemic stroke model is unclear. The purpose of this study was to investigate the neuroprotective effects and neuronal plasticity of BMSC transplantation combined with EA in ischemic stroke. MATERIALS AND METHODS: A male Sprague-Dawley (SD) rat middle cerebral artery occlusion (MCAO) model was used. Intracerebral transplantation of BMSC, transfected with lentiviral vectors expressing green fluorescent protein (GFP), was performed using a stereotactic apparatus after modeling. MCAO rats were treated with BMSC injection alone or in combination with EA. After the treatment, proliferation and migration of BMSC were observed in different groups by fluorescence microscopy. Quantitative real-time PCR (qRT-PCR), Western blotting, and immunohistochemistry were performed to examine changes in the levels of neuron-specific enolase (NSE) and nestin in the injured striatum. RESULTS: Epifluorescence microscopy revealed that most BMSC in the cerebrum were lysed; few transplanted BMSC survived, and some living cells migrated to areas around the lesion site. NSE was overexpressed in the striatum of MCAO rats, illustrating the neurological deficits caused by cerebral ischemia-reperfusion. The combination of BMSC transplantation and EA attenuated the expression of NSE, indicating nerve injury repair. Although the qRT-PCR results showed that BMSC-EA treatment elevated nestin RNA expression, less robust responses were observed in other tests. CONCLUSIONS: Our results show that the combination treatment significantly improved restoration of neurological deficits in the animal stroke model. However, further studies are required to see if EA could promote the rapid differentiation of BMSC into neural stem cells in the short term.

Effects of EZH2 on Invasion and Migration of Endometrial Stromal Cells in Endometriosis Patients by Regulating PCDH10 Gene H3K27 Methylation.

Context: Endometriosis refers to the appearance of ectopic endometrioid tissue outside the uterus. Low PCDH10 expression has been associated with enhancer of zeste homolog 2 (EZH2), which catalyzes histone 3 (H3K27me3). H3K27me3 is an epigenetic marker associated with endometriosis. Objective: The study intended to explore the influence of protocadherin 10 (PCDH10) on the invasion and migration of endometrial stromal cells in endometriosis as well as its mechanism. Design: The research team designed a laboratory study using endometrial tissue. Setting: The study took place in Department of Obstetrics and Gynecology at South University of Science and Technology Hospital in Shenzhen, Guangdong Province, China. Participants: Participants were 10 patients with ovarian endometriosis (ovarian chocolate cysts) who were undergoing surgical treatment at the hospital between January and December 2019. The endometrial tissue of those participants became the endometriosis group. Other participants with normal endometrial tissue became the controls (n=10). Outcome Measures: The research team collected tissues from participants and used immunofluorescence, real-time quantitative polymerase chain reaction (qPCR), and Western blot assay to determine the expression levels of PCDH10, enhancer of zeste homolog 2 (EZH2), and histone H3 (H3K27me3). The team cultured endometrial stromal cells from participants primarily to detect the effects of silencing EZH2 on PCDH10 and H3K27me3 expression. The team used a Transwell assay and scratch test to examine the influence of silencing EZH2 on invasion and migration of endometrial stromal cells and applied chromatin immunoprecipitation to determine H3K27me3 enrichment in the PCDH10 gene promoter region. Results: PCDH10 in heterotopic endometrial tissues of endometriosis patients had low expression, while EZH2 and H3K27me3 were highly expressed. Silencing EZH2 inhibited EZH2 protein expression, increased PCDH10 expression, and inhibited invasion and migration of endometrial stromal cells by increasing PCDH10 expression. Silencing EZH2 also reduced H3K27me3 enrichment in PCDH10 promoter region. Conclusions: Low PCDH10 expression may be associated with high EZH2 expression and H3K27me3 enrichment in endometriosis patients, which promotes the migration and invasion of endometrial stromal cells. This connection provides a theoretical basis for the treatment of endometriosis.

Aloe polysaccharide promotes osteogenesis potential of adipose-derived stromal cells via BMP-2/Smads and prevents ovariectomized-induced osteoporosis.

BACKGROUND: Aloe polysaccharide (AP) is a type of an active macromolecule of Aloe vera, which contributes to its function. However, whether AP possesses anti-osteoporosis properties is unknown. METHODS: Adipose-derived stromal cells were treated with different concentrations of AP. Early and late osteogenesis were, respectively, evaluated by ALP and Alizarin Red S staining. The effect of AP on the processes of adipogenesis inhibition in ADSCs was analyzed by oil red O staining. Western blot was used to assess the expression of osteogenic and adipogenic related factors. Then, Noggin was administered to further confirm the mechanism by which AP promotes the osteogenesis of ADSCs. Finally, 40 female SD rats were classified into a bilateral laparotomy group (Sham group) and three bilateral ovariectomy groups: OVX group, OVX + AP group, and OVX + AP + Noggin group. The bilateral rat femurs were collected to perform micro-CT scanning, HE, Masson trichrome, and Oil red O staining. RESULTS: The results indicated that AP could increase ALP expression and calcium deposition. Through molecular mechanisms, AP promotes the protein expression of COL1A1, OPN, and ALP in ADSCs, but downregulates the expression of PPARγ. Also, AP directs ADSCs' fate by stimulating the BMP2/Smads signaling pathway. In vivo, the rat AP-treated had more trabecular bone than the OVX rat, indicating partial protection from cancellous bone loss after treatment with AP. CONCLUSION: Our results show that AP may promote osteogenesis of ADSCs through BMP-2/Smads signaling pathway and inhibits lipogenic differentiation. Thus, AP might be a promising alternative medicine to treat postmenopausal osteoporosis.

Permixon®, hexane-extracted Serenoa repens, inhibits human prostate and bladder smooth muscle contraction and exerts growth-related functions in human prostate stromal cells.

AIMS: Recently, the European Association of Urology recommended hexane-extracted fruit of Serenoa repens (HESr) in their guidelines on management of non-neurogenic male lower urinary tracts symptoms (LUTS). Despite previously lacking recommendations, Permixon® is the most investigated HESr in clinical trials, where it proved effective for male LUTS. In contrast, underlying mechanisms were rarely addressed and are only marginally understood. We therefore investigated effects of Permixon® on human prostate and detrusor smooth muscle contraction and on growth-related functions in prostate stromal cells. MAIN METHODS: Permixon® capsules were dissolved using n-hexane. Contractions of human prostate and detrusor tissues were induced in organ bath. Proliferation (EdU assay), growth (colony formation), apoptosis and cell death (flow cytometry), viability (CCK-8) and actin organization (phalloidin staining) were studied in cultured human prostate stromal cells (WPMY-1). KEY FINDINGS: Permixon® inhibited α1-adrenergic and thromboxane-induced contractions in prostate tissues, and methacholine-and thromboxane-induced contractions in detrusor tissues. Endothelin-1-induced contractions were not inhibited. Neurogenic contractions were inhibited in both tissues in a concentration-dependent manner. In WPMY-1 cells, Permixon® caused concentration-dependent breakdown of actin polymerization, inhibited colony formation, reduced cell viability, and proliferation, without showing cytotoxic or pro-apoptotic effects. SIGNIFICANCE: Our results provide a novel basis that allows, for the first time, to fully explain the ubiquitous beneficial effects of HESr in clinical trials. HESr may inhibit at least neurogenic, α1-adrenergic and thromboxane-induced smooth muscle contraction in the prostate and detrusor, and in parallel, prostate stromal cell growth. Together, this may explain symptom improvements by Permixon® in previous clinical trials.

Ginsenoside Rb1 Modulates the Migration of Bone-Derived Mesenchymal Stem Cells through the SDF-1/CXCR4 Axis and PI3K/Akt Pathway.

Methods: Wound-healing assay and Transwell assay were utilized to evaluate the effect of ginsenoside Rb1 on the migration of BMSCs. RT-PCR and Western blotting were performed to evaluate the expression of stromal-derived factor 1 (SDF-1), C-X-C chemokine receptor type 4 (CXCR4), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (PKB; AKT). Results: Ginsenoside Rb1 significantly enhanced the migration of BMSCs through the activation of SDF-1, CXCR4, p-PI3K/PI3K, and p-Akt/Akt relative expression. Furthermore, this stimulus was blocked by the pretreatment with AMD3100 and LY294002. Conclusions: Ginsenoside Rb1 facilitated the migration of BMSCs through the activation of the SDF-1/CXCR4 axis and PI3K/Akt pathway.

Modulation of the tumor microenvironment by natural agents: implications for cancer prevention and therapy.

The development of cancer is not just the growth and proliferation of a single transformed cell, but its surrounding environment also coevolves with it. Indeed, successful cancer progression depends on the ability of the tumor cells to develop a supportive tumor microenvironment consisting of various types of stromal cells. The interactions between the tumor and stromal cells are bidirectional and mediated through a variety of growth factors, cytokines, metabolites, and other biomolecules secreted by these cells. Tumor-stromal crosstalk creates optimal conditions for the tumor growth, metastasis, evasion of immune surveillance, and therapy resistance, and its targeting is being explored for clinical management of cancer. Natural agents from plants and marine life have been at the forefront of traditional medicine. Numerous epidemiological studies have reported the health benefits imparted on the consumption of certain fruits, vegetables, and their derived products. Indeed, a significant majority of anti-cancer drugs in clinical use are either naturally occurring compounds or their derivatives. In this review, we describe fundamental cellular and non-cellular components of the tumor microenvironment and discuss the significance of natural compounds in their targeting. Existing literature provides hope that novel prevention and therapeutic approaches will emerge from ongoing scientific efforts leading to the reduced tumor burden and improve clinical outcomes in cancer patients.

Gut microbiota-derived short-chain fatty acids protect against the progression of endometriosis.

Worldwide, ∼196 million are afflicted with endometriosis, a painful disease in which endometrial tissue implants and proliferates on abdominal peritoneal surfaces. Theories on the origin of endometriosis remained inconclusive. Whereas up to 90% of women experience retrograde menstruation, only 10% develop endometriosis, suggesting that factors that alter peritoneal environment might contribute to endometriosis. Herein, we report that whereas some gut bacteria promote endometriosis, others protect against endometriosis by fermenting fiber to produce short-chain fatty acids. Specifically, we found that altered gut microbiota drives endometriotic lesion growth and feces from mice with endometriosis contained less of short-chain fatty acid and n-butyrate than feces from mice without endometriosis. Treatment with n-butyrate reduced growth of both mouse endometriotic lesions and human endometriotic lesions in a pre-clinical mouse model. Mechanistic studies revealed that n-butyrate inhibited human endometriotic cell survival and lesion growth through G-protein-coupled receptors, histone deacetylases, and a GTPase activating protein, RAP1GAP. Our findings will enable future studies aimed at developing diagnostic tests, gut bacteria metabolites and treatment strategies, dietary supplements, n-butyrate analogs, or probiotics for endometriosis.

Porcine endometrial 3D co-culture: Morphological changes in 3D endometrium tissues according to hormonal changes.

Cells cultured as monolayers proliferate well, but do not sustain their differentiation characteristics. Previous studies have investigated the interactions between cells and growth factors or cytokines by establishing either in vivo or in vitro three-dimensional (3D) cultures. Using porcine uterine epithelial cells and endometrial cells, the current study was designed to develop a 3D uterine culture system and investigate the response to hormone treatment. Formation of the 3D uterine model was similar to that of uterus from the group supplemented with calcium and magnesium, and the addition of these ions altered the spectrum of basement membrane degrading enzyme expression and activity. In particular, the epithelial cell junctions in the 3D model most closely resembled those of an actual uterus when the medium was supplemented with calcium and magnesium; the intercellular basement membrane structure was also tall under these conditions. The study confirmed that Casp-3 expression was lowest in the P4 (progesterone) treatment group, and this hormone was the most potent stimulus for formation of the endometrial cell layer. Therefore, the addition of calcium and magnesium plays an important role in the formation of a 3D uterine model, and the addition of P4 hormone mimics uterine thickening by stimulating growth of the epithelial cell layer.

[Molecular mechanism study of Shouhui Tongbian Capsules on promoting energy metabolism of gastrointestinal stromal cells].

Mechanism study was performed to explore how Shouhui Tongbian Capsules promotes energy metabolism of gastrointestinal stromal cells. In this study, gastrointestinal stromal cells line GIST-882 was used as the model to explore energy metabolism regulation effects of Shouhui Tongbian Capsules extract(10, 20, 50 and 100 µg·mL~(-1)) by measuring the cell proliferation, ATP level, mitochondrial membrane potential, and mitochondrial isocitrate dehydrogenase activity. Meanwhile, Western blot was used to detect the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Our results showed that Shouhui Tongbian Capsules promoted cell proliferation and increased ATP level of gastrointestinal stromal cells. In addition, Shouhui Tongbian Capsules obviously improved mitochondrial structural integrity, and increased mitochondrial membrane potential in GIST-882 cells. Mechanism study revealed that Shouhui Tongbian Capsules increased mitochondrial isocitrate dehydrogenase activity and up-regulated the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Collectively, our study indicated that Shouhui Tongbian Capsules promoted the energy metabolism for gastrointestinal stromal cells proliferation by activating mitochondrial isocitrate dehydrogenase to induce ATP production, as well as activating SCF/c-Kit and CDK2/cyclin A signaling pathways.

Regional Hyperthermia Enhances Mesenchymal Stem Cell Recruitment to Tumor Stroma: Implications for Mesenchymal Stem Cell-Based Tumor Therapy.

The tropism of mesenchymal stem cells (MSCs) for tumors forms the basis for their use as delivery vehicles for the tumor-specific transport of therapeutic genes, such as the theranostic sodium iodide symporter (NIS). Hyperthermia is used as an adjuvant for various tumor therapies and has been proposed to enhance leukocyte recruitment. Here, we describe the enhanced recruitment of adoptively applied NIS-expressing MSCs to tumors in response to regional hyperthermia. Hyperthermia (41°C, 1 h) of human hepatocellular carcinoma cells (HuH7) led to transiently increased production of immunomodulatory factors. MSCs showed enhanced chemotaxis to supernatants derived from heat-treated cells in a 3D live-cell tracking assay and was validated in vivo in subcutaneous HuH7 mouse xenografts. Cytomegalovirus (CMV)-NIS-MSCs were applied 6-48 h after or 24-48 h before hyperthermia treatment. Using 123I-scintigraphy, thermo-stimulation (41°C, 1 h) 24 h after CMV-NIS-MSC injection resulted in a significantly increased uptake of 123I in heat-treated tumors compared with controls. Immunohistochemical staining and real-time PCR confirmed tumor-selective, temperature-dependent MSC migration. Therapeutic efficacy was significantly enhanced by combining CMV-NIS-MSC-mediated 131I therapy with regional hyperthermia. We demonstrate here for the first time that hyperthermia can significantly boost tumoral MSC recruitment, thereby significantly enhancing therapeutic efficacy of MSC-mediated NIS gene therapy.

Inhibiting NTRK2 signaling causes endometriotic lesion regression.

Endometriosis is a common gynecological disease in reproductive-age women. Although the hormone-dependent therapy is the first line treatment for endometriosis, it is not a curative regimen and associated with severe side-effects, which significantly decrease the life quality of affected individuals. To seek a target for treatment of endometriosis, we focused on plasma membrane proteins that are elevated in ectopic cells and exert beneficial effects in cell growth and survival. We performed bioinformatics analysis and identified the neurotrophic receptor tyrosine kinase 2 (NTRK2) as a potential candidate for treatment. The expression levels of NTRK2 were markedly upregulated in the lesions of clinical specimen as well as in the mouse endometriotic-like lesion. Mechanistic investigation demonstrated that upregulation of NTRK2 is induced by hypoxia in a hypoxia-inducible factor 1 alpha-dependent manner. Knockdown of NTRK2 or administration of ANA-12, a selective antagonist of NTRK2, significantly induced endometriotic stromal cells death, suggesting it may be a potential therapeutic agent. In vivo study using surgery-induced endometriosis mice model showed ANA-12 (1.5 mg/kg body weight) treatment induced apoptosis of endometriotic cells and caused the regression of ectopic lesions. Taken together, our findings suggest a possible mechanism responsible for the aberrant expression of NTRK2 in endometriotic lesions and this may be involved in the pathogenesis of endometriosis.

The Effect of Rutin and Extracts of Uncaria guianensis (Aubl.) J. F. Gmeland on Primary Endometriotic Cells: A 2D and 3D Study.

There is increasing interest in the potential of natural compounds to treat diseases, such as endometriosis, a gynecological disorder that affects 10-15% of women of reproductive age, and it is related to severe pelvic pain and infertility. We have evaluated the in vitro effects of rutin and the aqueous bark, roots, and leaf extracts (ABE, ARE, and ALE, respectively) and isolated components of Uncaria guianensis on stromal cells from eutopic endometrium and lesions of patients with endometriosis. Two- and three-dimensional cultures were used to assess the cell death and production of reactive oxygen species (ROS), cytokines and growth factors of cells following exposure to these natural products. The applied treatments did not reduce cellular viability, but ROS production did increase. In addition, significant increases in the levels of interleukin (IL)-15, IL-17A, IL-4, IL-6, tumor necrosis factor-α, and vascular endothelium growth factor were observed when 2D-cells from endometrium of patients with endometriosis were treated with ABE, while exposure to ALE induced significant increases in epidermal growth factor in lesion cells.

Evidence from systematic reviews on photobiomodulation of human bone and stromal cells: Where do we stand?

This study summarizes the available evidence from systematic reviews on the in vitro effects of photobiomodulation on the proliferation and differentiation of human bone and stromal cells by appraising their methodological quality. Improvements for future studies are also highlighted, with particular emphasis on in vitro protocols and cell-related characteristics. Six reviews using explicit eligibility criteria and methods selected in order to minimize bias were included. There was no compelling evidence on the cellular mechanisms of action or treatment parameters of photobiomodulation; compliance with quality assessment was poor. A rigorous description of laser parameters (wavelength, power, beam spot size, power density, energy density, repetition rate, pulse duration or duty cycle, exposure duration, frequency of treatments, and total radiant energy), exposure conditions (methods to ensure a uniform irradiation and to avoid cross-irradiation, laser-cell culture surface distance, lid presence during irradiation) and cell-related characteristics (cell type or line, isolation and culture conditions, donor-related factors where applicable, tissue source, cell phenotype, cell density, number of cell passages in culture) should be included among eligibility criteria for study inclusion. These methodological improvements will maximize the contribution of in vitro studies on the effects of photobiomodulation on human bone and stromal cells to evidence-based translational research.