Integration of network pharmacology and experimental verification to reveal the active components and molecular mechanism of modified Danzhi Xiaoyao San in the treatment of depression.

ETHNOPHARMACOLOGICAL RELEVANCE: Modified Danzhi Xiaoyao San (MDXS) is an effective clinical prescription for depression in China, which was deprived of Danzhi Xiaoyao San in the Ming Dynasty. MDSX has significant implications for the development of new antidepressants, but its pharmacological mechanism has been rarely studied. AIM OF THE STUDY: To reveal the active components and molecular mechanism of MDXS in treating depression through network pharmacology and experimental verification in vivo and in vitro. MATERIALS AND METHODS: UPLC-Q-TOF-MS/MS was used to identify the chemical components in the MDXS freeze-dried powder, drug-containing serum, and cerebrospinal fluid (CSF). Based on the analysis of prototype components in the CSF, the major constituents, potential therapeutic targets and possible pharmacological mechanisms of MDXS in treating depression were investigated using network pharmacological and molecular docking. Then corticosterone (CORT)-induced mice model of depression was established to investigate the antidepressant effects of MDXS. HT22 cells were cultured to verify the neuroprotective effects and core targets of the active components. RESULTS: There were 81 compounds in MDXS freeze-dried powder, 36 prototype components in serum, and 13 prototype components in CSF were identified, respectively. Network pharmacology analysis showed that these 13 prototype components in the CSF shared 190 common targets with depression, which were mainly enriched in MAPK and PI3K/AKT signaling pathways. PPI analysis suggested that AKT1 and MAPK1 (ERK1/2) were the core targets. Molecular docking revealed that azelaic acid (AA), senkyunolide A (SA), atractylenolide III (ATIII), and tokinolide B (TB) had the highest binding energy with AKT1 and MAPK1. Animal experiments verified that MDXS could reverse CORT-induced depression-like behaviors, improve synaptic plasticity, alleviate neuronal injury in hippocampal CA3 regions, and up-regulate the protein expression of p-ERK1/2 and p-AKT. In HT22 cells, azelaic acid, senkyunolide A, and atractylenolide III significantly protected the cell injury caused by CORT, and up-regulated the protein levels of p-ERK1/2 and p-AKT. CONCLUSIONS: These results suggested that MDXS may exert antidepressant effects partially through azelaic acid, senkyunolide A, and atractylenolide III targeting ERK1/2 and AKT.

Analysis of Calcium-Sensing Receptor Signaling Using Dual Luciferase Assays.

Luciferases catalyze a reaction that involves the emission of light, a phenomenon referred to as "bioluminescence". The calcium-sensing receptor (CaSR), a G protein-coupled receptor (GPCR), induces characteristic signaling pathways that stimulate extracellular signal-regulated kinase 1/2 (ERK1/2) and Ca2+ mobilization from the endoplasmic reticulum. ERK1/2 causes an activation of the serum response element (SRE), whereas Ca2+ causes an activation of the nuclear factor of activated T-cells response element (NFAT-RE). Transfection of cells with a vector containing a firefly luciferase reporter gene under the control of the SRE or NFAT-RE allows the monitoring of ERK1/2 activation and Ca2+ mobilization, respectively, by measuring luminescence. In a dual luciferase assay, firefly luminescence is normalized by co-transfecting an internal control vector, which contains a constitutively active promoter driving the expression of a second luciferase, namely, Renilla luciferase, whose activity can be quantified within the same sample. Here, a protocol for the analysis of CaSR signaling using dual luciferase assays in HEK293 cells is provided. The assays can, for example, be used to investigate functional consequences of mutations in the CaSR gene.

Nonylphenol displays immunotoxicity by triggering hemocyte extracellular traps in Manila clam via ROS burst, ERK pathway and glycolysis.

Nonylphenol (NP), an endocrine disruptor, has been demonstrated to be a harmful environmental contaminant and toxic to organisms. In this study, to address concerns regarding the immunotoxicity of NP, we treated clam Ruditapes philippinarum hemocytes with NP in vitro and explored the underlying mechanisms of NP-induced extracellular traps (ETs). NP could induce the formation of hemocytes ETs in a dose-dependent manner. Transcriptomics analysis revealed changes of signaling pathway involved in immunity and energy metabolism in hemocytes after NP stimulation. In this process, both reactive oxygen species (ROS) and myeloperoxidase (MPO) were up-regulated. Moreover, mitogen-activated protein kinase (MAPK) signaling pathway was proved to be activated in the formation of NP-induced ETs, manifested as enhanced phosphorylation of extracellular signal-regulated kinase (ERK) but not p38 or c-Jun N-terminal kinase (JNK). In the presence of U0126, an ERK phosphorylation inhibitor, the NP-induced expression of NADPH oxidase enzyme (NOX) was significantly decreased, which further alleviated the ROS production and ultimately limited the release of ETs. NP exposure increased glucose uptake, along with enhanced activities of glycolysis-related enzymes such as hexokinase (HK) and pyruvate kinase (PK). After inhibiting glycolysis by the inhibitor 2-DG, the formation of NP-induced ETs was significantly suppressed. ERK could regulate mTOR signaling and the PI3K/AKT pathway, potentially directing ETs formation by orchestrating the glycolysis through the activation of key transcription factors c-Myc and HIF-1α. Collectively, the results preliminary confirm that the ERK-NOX-ROS axis and glycolysis are involved in NP-induced ETs formation, contributing to the cellular immunotoxicity in clam.

Nuclear envelope budding inhibition slows down progerin-induced aging process.

Progerin causes Hutchinson-Gilford progeria syndrome (HGPS), but how progerin accelerates aging is still an interesting question. Here, we provide evidence linking nuclear envelope (NE) budding and accelerated aging. Mechanistically, progerin disrupts nuclear lamina to induce NE budding in concert with lamin A/C, resulting in transport of chromatin into the cytoplasm where it is removed via autophagy, whereas emerin antagonizes this process. Primary cells from both HGPS patients and mouse models express progerin and display NE budding and chromatin loss, and ectopically expressing progerin in cells can mimic this process. More excitingly, we screen a NE budding inhibitor chaetocin by high-throughput screening, which can dramatically sequester progerin from the NE and prevent this NE budding through sustaining ERK1/2 activation. Chaetocin alleviates NE budding-induced chromatin loss and ameliorates HGPS defects in cells and mice and significantly extends lifespan of HGPS mice. Collectively, we propose that progerin-induced NE budding participates in the induction of progeria, highlight the roles of chaetocin and sustained ERK1/2 activation in anti-aging, and provide a distinct avenue for treating HGPS.

FERMT1 promotes epithelial-mesenchymal transition of hepatocellular carcinoma by activating EGFR/AKT/ß-catenin and EGFR/ERK pathways.

OBJECTIVE: This study aimed to investigate the effects of fermitin family member 1 (FERMT1) on epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) via the EGFR/AKT/ß-catenin and EGFR/ERK pathways. METHODS: The expression of FERMT1 encoding protein kindlin-1 in HCC tissues was determined by immunohistochemistry, and FERMT1 mRNA expression in HCC tissues and cell lines was analyzed by qRT-PCR. After the FERMT1 expression of SNU182 and SNU387 interfered with siRNA, the cell viability, invasion, migration, and EMT were tested by CCK-8, transwell invasion, scratching, immunofluorescence/WB, respectively. Similarly, the effects of FERMT1 on the viability and metastasis of HCC were investigated in transplanted tumor and lung metastasis mouse models. The protein expressions of EGFR/AKT/ß-catenin and EGFR/ERK pathways were analyzed by WB. In addition, the relationship between FERMT1 and EGFR was further determined by immunofluorescence double staining and Co-IP. RESULTS: FERMT1 was significantly upregulated in HCC, and silencing FERMT1 inhibited the viability, invasion, migration, and EMT of HCC. Silencing FERMT1 also inhibited the activation of EGFR/AKT/ß-catenin and EGFR/ERK pathways. In addition, inhibition of EGFR, AKT, or ERK confirmed that EGFR/AKT/ß-catenin and EGFR/ERK pathways were involved in the promoting effects of FERMT1 on HCC. Co-IP and immunofluorescence experiments confirmed the targeting relationship between FERMT1 and EGFR. CONCLUSION: FERMT1 was highly expressed in HCC and promoted viability, invasion, migration, and EMT of HCC by targeting EGFR to activate the EGFR/AKT/ß-catenin and EGFR/ERK pathways. Our study revealed the role of FERMT1 in HCC and suggested that FERMT1 exerts biological effects through activating the EGFR/AKT/ß-catenin and EGFR/ERK pathways.

Investigation and Characterization of the RAS/RAF/MEK/ERK Pathway and Other Signaling Pathways in Chronic Sinusitis with Nasal Polyps.

BACKGROUND: The clinical outcomes of drug treatments and surgical interventions for chronic sinusitis with nasal polyps (CRSwNPs) are suboptimal, and the high recurrence rate remains a significant challenge in clinical practice. Targeted therapies such as biologics provide new perspectives and directions for treating CRSwNP. SUMMARY: With the continuous investigation of signaling pathways, RAS/RAF/MEK/ERK signaling pathway and other signaling pathways including Hippo, JAK-STAT, Wnt, TGF-ß, PI3K, Notch, and NF-κB were confirmed to play an important role in the progression of CRSwNP. Among them, the abnormality of RAS/RAF/MEK/ERK signaling pathway is accompanied by the abnormality of this apoptotic component, which may provide new research directions for targeting the components of signaling pathways to mediate apoptosis. KEY MESSAGES: Abnormalities in signaling pathways are particularly important in studying the pathogenesis and treatment of CRSwNP. Therefore, this review summarizes the ongoing investigation and characterization of RAS/RAF/MEK/ERK signaling pathway and other signaling pathways in CRSwNP, which provides constructive ideas and directions for improving the treatment of CRSwNP.

Clinicopathological characteristics of extranodal Rosai-Dorfman disease: A retrospective case series of 25 patients.

Rosai-Dorfman disease (RDD) is a rare non-Langerhans cell histiocytosis, classically affecting the lymph nodes. Even rarer extranodal disease is diagnostically challenging due to overlapping histologic features with other entities and lack of a universally agreed set of biomarkers. Cyclin D1 immunohistochemistry (IHC) may serve as a useful adjunct in diagnosing extranodal RDD. We present a retrospective case series of patients diagnosed with extranodal RDD between January 2013 and December 2023. IHC staining for cyclin D1 was performed on archived tissue samples. Baseline IHC results for biomarkers supporting the RDD diagnosis were recorded along with patient demographic characteristics, clinical features, and disease outcomes. A total of 25 patients with extranodal RDD were included: 21 women (84 %) and 4 men (16 %). The mean age at diagnosis was 42.6 years. Cutaneous and deep tissue involvement was seen in 5 (20 %) and 20 (80 %) patients, respectively. 11 patients (44 %) had disease localized to the trunk and extremities, and 13 had disease in the head and neck region (52 %), of which 5 occurred in the nose and paranasal tissues. Available follow-up data showed most patients fully recovered (n = 11; 78.6 %). However, 1 patient had disease recurrence, 1 developed blindness, and 1 developed deafness. Cyclin D1 IHC was positive in all samples (100 %), consistent with previous studies. The clinicopathologic findings in this study highlight the spectrum of potential disease sites, possible morbid outcomes related to disease site, and the diagnostic utility of cyclin D1 IHC.

Mechanism of DT-13 regulating macrophages in diabetic wound healing.

BACKGROUND: Diabetes is a complex metabolic system disease, and one of the main reasons why it is difficult to heal is that macrophages cannot realize the transition from pro-inflammatory M1 phenotype to anti-inflammatory M2 phenotype. Liriope spicata Lour. is a traditional Chinese medicine. According to the theory of traditional Chinese medicine, Liriope spicata Lour. has nourishing Yin Sheng Jin, moistening lung clear heart, used for lung dryness dry cough, Yin deficiency cough, throat arthralgia throat pain, thirst, internal heat thirst, upset insomnia, intestinal dryness constipation, is the classification of Yin tonifying drugs. Liriope muscari baily saponins C (DT-13) is one of the main active ingredients of Liriope spicata Lour., has significant anti-inflammatory, anti-tumor, and immunomodulatory effects. This thesis aims to explore the role of DT-13 in angiogenesis by regulating the polarization of macrophages, and ultimately play an anti-inflammatory role in regeneration by regulating immune cells. METHODS: We conducted in vivo experiments using a diabetic mouse ulcer model to verify the effect of DT-13 in promoting wound healing. Spatial transcriptome sequencing technology was utilized to perform RNA transcript analysis on wound tissues from type II diabetic mice and non-diabetic mice. Subsequently, we used the CCK-8 assay to evaluate the impact of DT-13 on the viability of THP-1 cells (human monocytes). ELISA, immunofluorescence, and Western blot techniques were employed to study the mechanisms by which DT-13 inhibits the sustained inflammation and polarization process of M1 macrophages induced by LPS. The Transwell assay was used to assess the influence of DT-13 treatment on the co-culture of M1 macrophages induced by LPS under high glucose conditions with HUVEC cells. Finally, the chick embryo chorioallantoic membrane (CAM) assay was applied to explore the effects of DT-13 on angiogenesis stimulated by M1 macrophages under high glucose conditions with LPS stimulation. RESULTS: We found that DT-13 can promote wound healing in a diabetic ulcer model in mice. Through spatial transcriptome sequencing results, we discovered that type II diabetic mice had higher levels of inflammation at the wound site and abnormal expression of macrophage characteristic proteins. The CCK-8 assay detected that DT-13 at 20 µmol/L had an effect on THP-1 cells. Through Q-PCR, ELISA, immunofluorescence, and Western blot results, we found that the mechanism by which DT-13 exerts anti-inflammatory effects on M1 macrophages with sustained inflammation induced by LPS under high glucose conditions may be through the TLR4-NFKB signaling pathway, and the mechanism for inducing the polarization of M1 macrophages to M2 type may be through the ERK-STAT3 signaling pathway. Interestingly, through the Transwell assay, we found that M1 macrophages induced by LPS under high glucose conditions, after treatment with DT-13 and co-cultured with HUVECs, could increase the migratory ability of HUVEC cells. This indicates that M1 macrophages induced by LPS under high glucose conditions, after treatment with DT-13, can promote angiogenesis. CONCLUSION: DT-13 can significantly promote healing wounds in diabetic mice, and its mechanism may be to participate in promoting the formation of blood vessels by changing the polarization of macrophages.

Flurbiprofen axetil is involved in basal-like breast cancer metastasis via suppressing the MEK/ERK signaling pathway.

Flurbiprofen axetil is commonly utilized in clinical practice as one of the nonsteroidal anti-inflammatory drugs (NSAIDs) and is included in multimodal analgesia regimens postbreast cancer surgery. Numerous NSAIDs have been studied for their potential to both promote and inhibit cancer. Given the variability in their effects on tumors, further investigation into the specific role of flurbiprofen axetil is warranted. Therefore, the primary objective of this study was to assess the impact of flurbiprofen axetil on basal-like breast cancer (BLBC) metastasis and elucidate the underlying molecular mechanisms involved. The BLBC metastasis mouse model was established by caudal vein injection of tumor cells. The lung metastasis of breast cancer in mice and the effect of flurbiprofen axetil were assessed by in vivo bioluminescence imaging, hematoxylin and eosin staining and immunohistochemistry. In vitro, the results of flurbiprofen axetil on the proliferation, migration, and invasion of MDA-MB-231 human breast cancer cells and BT-549 human breast cancer cells were assessed by colony formation assay and transwell assay. The effects of flurbiprofen axetil on several tumor metastasis-related signaling pathway proteins were examined by western blot, and the reversal extent of the flurbiprofen axetil effect by Ro 67-7476 (ERK phosphorylation agonist) was detected by transwell assay. The results showed that flurbiprofen axetil significantly inhibited BLBC lung metastasis in mice. Flurbiprofen axetil similarly inhibited breast cancer cell migration and invasion in vitro but did not affect their proliferation. Mechanistic investigations have revealed that flurbiprofen axetil exerts a noteworthy inhibitory influence on the MEK/ERK pathway while exhibiting no significant alteration in the expression of other pathway proteins intricately associated with epithelial-mesenchymal transition. In conclusion, the inhibitory effect of flurbiprofen axetil on BLBC metastasis is characterized by its selectivity in targeting the MEK/ERK signaling pathway rather than exerting a broad impact on the global signaling pathway.

Deletion of Mgat2 in spermatogonia blocks spermatogenesis.

Identifying factors required for spermatogenesis is important for understanding mechanisms of male fertility. Inactivation of either the Mgat1 or Man2a2 gene leads to a block in spermatogenesis causing infertility in male mice. MGAT1 GlcNAc-transferase initiates complex N-glycan synthesis and MAN2A2 mannosidase generates the substrate for MGAT2 GlcNAc-transferase to form a biantennary complex N-glycan. In this paper, we show that conditional deletion of Mgat2 in spermatogonia via Stra8-iCre caused a novel block in spermatogenesis, largely prior to the formation of round spermatids. Mgat2[-/-] germ cells did not bind the lectins Phaseolus vulgaris leucoagglutinin (L-PHA) or Griffonia simplicifolia II (GSA-II), similar to germ cells lacking MGAT1 and complex N-glycans. However, overall spermatogenic defects were distinct in germ cells with deleted Mgat2 versus Mgat1. In addition, RNA-seq analysis at 15 days after birth revealed a unique transcriptomic landscape in Mgat2[-/-] germ cells with genes required for sperm formation and functions being most downregulated. Bioinformatic analyses using the ingenuity pathway analysis (IPA) algorithm identified ERK and AKT as central activities. Western blot analyses of 15-day germ cell lysates confirmed that both AKT and ERK1/2 signaling were increased by loss of MGAT2 in germ cells. By contrast, Mgat1[-/-] germ cells were previously shown to have reduced ERK signaling and unchanged AKT activity. Therefore, since the loss of all complex N-glycans is common to each mutant model, the different immature N-glycans that accumulate in Mgat2[-/-] versus Mgat1[-/-] germ cells are proposed to be the basis of their unique spermatogenic phenotypes.

The major vault protein integrates adhesion-driven signals to regulate collagen remodeling.

DDR1 interacts with fibrillar collagen and can affect ß1 integrin-dependent signaling, but the mechanism that mediates functional interactions between these two different receptors is not defined. We searched for molecules that link DDR1 and ß1 integrin-dependent signaling in response to collagen binding. The activation of DDR1 by binding to fibrillar collagen reduced by 5-fold, ß1 integrin-dependent ERK phosphorylation that leads to MMP1 expression. In contrast, pharmacological inhibition of DDR1 or culturing cells on fibronectin restored ERK phosphorylation and MMP1 expression mediated by the ß1 integrin. A phospho-site screen indicated that collagen-induced DDR1 activation inhibited ß1 integrin-dependent ERK signaling by regulating autophosphorylation of focal adhesion kinase (FAK). Immunoprecipitation, mass spectrometry, and protein-protein interaction mapping showed that while DDR1 and FAK do not interact directly, the major vault protein (MVP) binds DDR1 and FAK depending on the substrate. MVP associated with DDR1 in cells expressing ß1 integrin that were cultured on collagen. Knockdown of MVP restored ERK activation and MMP1 expression in DDR1-expressing cells cultured on collagen. Immunostaining of invasive cancers in human colon showed colocalization of DDR1 with MVP. These data indicate that MVP interactions with DDR1 and FAK contribute to the regulation of ß1 integrin-dependent signaling pathways that drive collagen degradation.

Corynoxine exerts the anti-tumor effect on esophageal squamous cell carcinoma principally via the EZH2-DUSP5-ERK1/2-mediated cell growth inhibition.

BACKGROUND: Esophageal cancer is one of the most prevalent malignant tumors and the sixth largest cause of tumor-associated death worldwide. Squamous cell carcinoma (ESCC) accounts for 85 % of all esophageal cancer cases. ESCC treatment remains to be significantly difficult. Corynoxine (Cory) is a tetracyclic hydroxyindole alkaloid isolated from Uncaria macrophylla. It is unclear whether Cory has an anti-tumor effect on ESCC. PURPOSE: To determine the anti-tumor activity of Cory and the associated mechanisms in ESCC. STUDY DESIGN: Cory's effects on proliferation, apoptosis, migration, and invasion, as well as the underlying molecular causes were assessed using two ESCC cell lines, KYSE150 and TE-1. A xenograft mouse model was then applied to evaluate the anti-tumor activity of Cory in vivo. METHODS: Western blot, assays including CCK-8, colony formation, EdU staining, TUNEL staining, cell scratch and Transwell, and a xenograft mouse model were used in this study. RESULTS: Cory suppressed cell growth, provoked cell apoptosis, and hindered cell migration and invasion of ESCC cells. DUSP5 knockdown reduced the Cory-induced cell death and restored cell migration and invasion through ERK1/2 activation. Further analyses showed that Cory promoted DUSP5 expression via inhibiting EZH2 expression, leading to inactivation of ERK1/2 signaling and the subsequent cell growth inhibition of ESCC. In vivo experiments disclosed that Cory suppressed tumor growth of ESCC through upregulating DUSP5 expression. CONCLUSIONS: Cory plays an anti-tumor role in ESCC by regulating EZH2-DUSP5-ERK1/2 signaling pathway. Cory may be promising to be a novel therapy for treating ESCC.

Inflammatory priming of human mesenchymal stem cells induces osteogenic differentiation via the early response gene IER3.

Mesenchymal stem cells (MSCs) have gained tremendous interest due to their overall potent pro-regenerative and immunomodulatory properties. In recent years, various in vitro and preclinical studies have investigated different priming ("licensing") approaches to enhance MSC functions for specific therapeutic purposes. In this study, we primed bone marrow-derived human MSCs (hMSCs) with an inflammation cocktail designed to mimic the elevated levels of inflammatory mediators found in serum of patients with severe injuries, such as bone fractures. We observed a significantly enhanced osteogenic differentiation potential of primed hMSCs compared to untreated controls. By RNA-sequencing analysis, we identified the immediate early response 3 (IER3) gene as one of the top-regulated genes upon inflammatory priming. Small interfering RNA knockdown experiments established IER3 as a novel positive regulator of osteogenic differentiation. Mechanistic analysis further revealed that IER3 deletion significantly downregulated bone marrow stromal cell antigen 2 (BST2) expression and extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation in hMSCs, suggesting that IER3 regulates osteogenic differentiation through BST2 and ERK1/2 signaling pathway activation. On the basis of these findings, we propose IER3 as a novel therapeutic target to promote hMSC osteoblastogenesis, which might be of high clinical relevance, for example, in patients with osteoporosis or compromised fracture healing.

Analysis of the relationship between resistin with prognosis, cell migration, and p38 and ERK1/2 activation in breast cancer.

Obesity increases the risk and mortality of breast cancer through dysregulated secretion of proinflammatory cytokines and tumor adipokines that induce an inflammatory breast microenvironment. Resistin is an adipokine secreted by adipocytes, immune cells, and predominantly macrophages, which contributes to cancer progression, but its molecular mechanism in cancer is not completely described. In this study, we analyzed the relationship of resistin on breast cancer prognosis and tumor progression and the effect in vitro of resistin on p38 and ERK1/2 activation in breast cancer cell lines. By bioinformatic analysis, we found that resistin is overexpressed in the basal subtype triple-negative breast cancer and is related to poor prognosis. In addition, we demonstrated a positive correlation between RETN and MAPK3 expression in basal triple-negative breast cancer. Importantly, we found amplifications of the RETN gene in at least 20 % of metastatic samples from patients with breast cancer. Most samples with RETN amplifications metastasized to bone and showed high expression of IL-8 (CXCL8) and IL-6 (IL6). Finally, resistin could be considered a prognostic marker for basal triple-negative breast cancer, and we also proposed the possibility that resistin-induced cell migration involves the activation of MAPK in breast cancer cells.

Downregulation of microRNA­221­3p promotes angiogenesis of lipoprotein(a)­injured endothelial progenitor cells by targeting silent information regulator 1 to activate the RAF/MEK/ERK signaling pathway.

The present study aimed to investigate the role of microRNA (miR)­221­3p in endothelial progenitor cells (EPCs) treated with lipoprotein(a) [LP(a)]. EPCs were identified using immunofluorescence assays and miR­221­3p levels were measured using reverse transcription­quantitative PCR. EPC migration was detected using Transwell assays, proliferation was measured by staining with 5­ethynyl­2'­deoxyuridine and adhesion was assessed by microscopy. Flow cytometry was used to measure apoptosis and protein expression was detected using western blotting. A dual­luciferase reporter assay was used to confirm the target interactions. The proliferation, migration, adhesion and angiogenesis of EPCs were decreased, and apoptosis was increased after treatment with LP(a). These effects were weakened by transfection with miR­221­3p inhibitor. The negative effects of LP(a) on EPCs were also weakened by overexpression of silent information regulator 1 (SIRT1). Inhibition of the RAF/MEK/ERK signaling pathway blocked the effects of SIRT1 overexpression. In conclusion, miR­221­3p inhibitor transfection activated the RAF/MEK/ERK signaling pathway through SIRT1, promoted the proliferation, migration, adhesion and angiogenesis of EPCs, and reduced apoptosis.

Aldo-keto reductase family 1 member C3 mediates radioresistance of esophageal cancer cells through suppressing MAPK and AKT signaling.

BACKGROUND: Aldo-keto reductase family 1 member C3 (AKR1C3) is a radioresistance gene in esophageal cancer. This study aimed to investigate the signaling pathways that mediate the regulatory role of AKR1C3 in the radioresistance of esophageal cancer cells. METHODS: The protein levels of AKR1C3 in cancer tissue samples were compared between patients with radiosensitive and radioresistant esophageal cancer using immunohistochemical staining. AKR1C3-silenced stable KYSE170R esophageal cancer cells (KY170R-shAKR1C3) were established. Colony formation assay was employed to evaluate the radiosensitivity of cancer cells, while flow cytometry analysis was utilized to quantify reactive oxygen species (ROS) production in these cells. Additionally, Western blotting was conducted to determine protein expression levels. RESULTS: AKR1C3 protein exhibited significantly higher expression in radioresistant cancer tissue samples compared to radiosensitive samples. AKR1C3 silencing promoted radiosensitivity and ROS production of KYSE170R cells. At 32 h after X-ray radiation, the levels of total and phosphorylated ERK1/2, JNK, and AKT proteins were significantly elevated in KYSE170R-shAKR1C3 cells compared to untransfected KYSE170R cells. The inhibitor of AKR1C3 remarkably enhanced the radiosensitivity of KYSE170R cells. Conversely, treatment with either a MEK inhibitor or an AKT inhibitor significantly increased the radioresistance of KYSE170R-shAKR1C3 cells. CONCLUSIONS: Our results suggest that AKR1C3 mediates radioresistance of KYSE170R cells possibly through MAPK and AKT signaling.

ERK1/2 interaction with DHPS regulates eIF5A deoxyhypusination independently of ERK kinase activity.

This study explores a non-kinase effect of extracellular regulated kinases 1/2 (ERK1/2) on the interaction between deoxyhypusine synthase (DHPS) and its substrate, eukaryotic translation initiation factor 5A (eIF5A). We report that Raf/MEK/ERK activation decreases the DHPS-ERK1/2 interaction while increasing DHPS-eIF5A association in cells. We determined the cryoelectron microscopy (cryo-EM) structure of the DHPS-ERK2 complex at 3.5 Å to show that ERK2 hinders substrate entrance to the DHPS active site, subsequently inhibiting deoxyhypusination in vitro. In cells, impairing the ERK2 activation loop, but not the catalytic site, prolongs the DHPS-ERK2 interaction irrespective of Raf/MEK signaling. The ERK2 Ser-Pro-Ser motif, but not the common docking or F-site recognition sites, also regulates this complex. These data suggest that ERK1/2 dynamically regulate the DHPS-eIF5A interaction in response to Raf/MEK activity, regardless of its kinase function. In contrast, ERK1/2 kinase activity is necessary to regulate the expression of DHPS and eIF5A. These findings highlight an ERK1/2-mediated dual kinase-dependent and -independent regulation of deoxyhypusination.

Discovery of a potential bladder cancer inhibitor CHNQD-01281 by regulating EGFR and promoting infiltration of cytotoxic T cells.

As one of the common malignancies that threaten human life, bladder cancer occurs frequently with a high mortality rate in the world, due to its invasion, recurrence and drug resistance. Natural products from marine microorganisms are becoming the hotspots in discovery of new candidate drug entities, especially in the area of cancer. Brefeldin A (BFA) is a natural Arf-GEFs inhibitor, but due to the low aqueous solubility, strong toxicity, and poor bioavailability, it is urgent to conduct structural optimization research. Herein, a new BFA pyridine acrylate derivative CHNQD-01281 with improved solubility was prepared and found to exert moderate to strong antiproliferative activity on a variety of human cancer cell lines. It was noteworthy that CHNQD-01281 was most sensitive to two bladder cancer cell lines T24 and J82 (IC50 = 0.079 and 0.081 µmol/L) with high selectivity index (SI = 14.68 and 14.32), suggesting a superior safety to BFA. In vivo studies revealed that CHNQD-01281 remarkably suppressed tumor growth in a T24 nude mice xenograft model (TGI = 52.63%) and prolonged the survival time (ILS = 68.16%) in an MB49 allogeneic mouse model via inducing infiltration of cytotoxic T cells. Further mechanism exploration indicated that CHNQD-01281 regulated both EGFR/PI3K/AKT and EGFR/ERK pathways and mediated the chemotactic effect of chemokines on immune effector cells. Overall, CHNQD-01281 may serve as a potential therapeutic agent for bladder cancer through multiple mechanisms. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00246-w.

FBXO45 Knockdown Restrains the Progression of Bladder Cancer via the ERK/Cyclin D1/CDK4 Pathway.

BACKGROUNDS: F-box protein 45 (FBXO45) has been implicated in the progression of several diseases. Whether FBXO45 is involved in the development of bladder cancer remains unclear. Thus, this study focused on the effect of FBXO45 on the malignant progression of bladder cancer cells. METHODS: FBXO45 small-interference fragment was transfected into RT4 and 5637 cells by liposome-mediated transfection, and the knockdown efficiency of FBXO45 was verified by Western blot assay. The growth rate between FBXO45 knockdown cell lines and control cell lines was compared by counting kit 8 and plate cloning experiments. The motility of bladder cancer cells was observed via the Transwell test and Wound healing test. The effects of FBXO45 silencing on apoptosis and cell division were confirmed by flow cytometry. Western blot assay was performed to determine the function of FBXO45 knockdown on key proteins of cell apoptosis and the ERK/Cyclin D1/CDK4 pathway. RESULTS: After FBXO45 knockdown, the proliferation of bladder cancer cells was blocked (p < 0.01), and the migration and invasion abilities were reduced (p < 0.01). FBXO45 knockdown reduced the number of S-phase cells (RT4, p < 0.01; 5637, p < 0.05) and enhanced the apoptotic rate (p < 0.01). FBXO45 knockdown decreased the levels of p-ERK1/2, CDK4 and Cyclin D1 (p < 0.01). CONCLUSIONS: This study revealed that FBXO45 plays a carcinogenic role in bladder cancer via the ERK/Cyclin D1/CDK4 pathway, which provides a reference for the clinical treatment of patients with bladder cancer.

AOPPs induces EMT and fibrosis by activating oxidative stress through ERK/p38 MAPK signaling pathway in endometriosis.

Epithelial-mesenchymal transition (EMT) is known to play a crucial role in the development of endometriosis (EMs). However, the exact mechanisms involved in EMT regulation in EMs are not well understood. In this study, we performed comprehensive research using clinical samples, single-cell sequencing, and in vivo/in vitro models to investigate the effects of advanced oxidation protein products (AOPPs) on EMT and the underlying mechanisms in EMs. Combining bioinformatics analysis with experimental validation, our results show that AOPPs accumulate in EMs tissues, and their levels positively correlate with the expression of EMT markers in fibrotic lesions of EMs patients. Stimulation with AOPPs leads to a concentration- and time-dependent alteration of EMT markers expression in both in vitro and in vivo models. These effects are mainly mediated by the generation of reactive oxygen species and nitrite, along with the activation of the ERK and P38 signaling pathways. In chronic administration studies using normal rats, AOPPs induce EMT and enhance collagen deposition. These findings significantly contribute to our understanding of the molecular mechanisms of EMs and provide a foundation for future research and therapeutic development in this field.