Western Blot Analysis Research Articles

Electroacupuncture Regulates Cannabinoid Receptor 1 Expression in a Mouse Fibromyalgia Model: Pharmacological and Chemogenetic Modulation

Fibromyalgia is a chronic illness usually accompanied by long-lasting, general pain throughout the body, often accompanied by anxiety, depression, fatigue, and sleep disruption. Meanwhile, doctors and scientists have not entirely discovered detailed mechanisms; patients always have an exaggerated sensation to pervasive pain without satisfied medical service. Given the lack of knowledge on its underlying mechanism, current treatments aim to provide pain and/or symptom relief. The present study aimed to clarify the role of cannabinoid receptor 1 (CB1) signaling in a mouse fibromyalgia pain model. To develop the mouse fibromyalgia model, mice were subjected to intermittent cold stress (ICS). Our results indicated that mechanical (2.09 ± 0.09 g) and thermal hyperalgesia (4.77 ± 0.29 s), which were evaluated by von Frey and Hargraves’ tests, were induced by ICS, suggesting successful modeling. The hurting replies were then provoked by electroacupuncture (EA) but not for sham EA mice. Further, in a Western blot analysis, we found significantly decreased CB1 protein levels in the thalamus, somatosensory cortex, and anterior cingulate cortex. In addition, the levels of pain-related protein kinases and transcription factor were increased. Treatment with EA reliably increased CB1 expression in various brain regions sequentially alleviated by nociceptive mediators. Furthermore, the administration of a CB1 agonist significantly attenuated fibromyalgia pain, reversed EA analgesia by the CB1 antagonist, and further reversed the chemogenetic inhibition of SSC. Our innovative findings evidence the role of CB1 signaling in the interaction of EA and fibromyalgia, suggesting its potential for clinical trials and as a treatment target.

Abstract B046: Pyk2 and MEK/ERK signaling regulate the extracellular vesicle release and tumor-associated myeloid Cells in glioblastoma

Abstract Glioblastoma (GBM) is often fatal within a year despite treatment, with immune checkpoint blockade therapies benefiting only about 10% of patients. Non-responsive GBMs exhibit an immunosuppressive profile and higher levels of tumor-associated myeloid cells (TAMs). Addressing this immunosuppressive microenvironment is a major challenge in GBM immunotherapy. Our recent studies have identified a positive correlation between Proline-Rich Tyrosine Kinase 2 (Pyk2) activation in GBM tumor cells and the cytokine expression profile of TAMs. This identified Pyk2 as a potential prognostic marker for the immune state in GBM tumor microenvironment. We hypothesize that Pyk2 and MEK/Erk signaling in GBM cells regulate the release of chemokines and cytokines through extracellular vesicles (EVs) by modulating the actin cytoskeleton, thereby impacting TAM activation. The study aimed to investigate the role of Pyk2/MEK/Erk signaling in EV release in GBM cells, potentially revealing new therapeutic targets. Two humans primary GBM cell lines, with and without Pyk2 CRISPR/Cas9 knockout (Pyk2KO), were used. Flow cytometric analysis of EVs, enriched from cell-conditioned medium, identified a shift towards larger in diameter EVs populations in Pyk2 KO cells, compared to wild-type (WT) cells. Analysis using Integrin as a plasma membrane marker showed that 84.70% of Integrin+ and 15.30% of Integrin- EVs were derived from WT cells, whereas Pyk2KO cells produced 89.07% Integrin+ and 10.93% Integrin- EVs. Treatment of WT cells with the Erk/MEK inhibitor Avutometinib (1µM) altered the EV population’s ratio to 79.11% Integrin+ and 20.89% Integrin-. Similarly, Avutometinib treatment of Pyk2KO cells resulted in 78.83% Integrin+ and 21.17% Integrin- EVs, similar to the ratio observed in Avutometinib-treated WT cells. Western blot and PCR analysis of EVs content revealed a significant reduction in CCL2, CCL5, tumor necrosis factor (TNF), and vascular endothelial growth factors (VEGF) in EVs from Pyk2KO GBM cells, compared to WT. In vivo studies using GL261/C57Bl/6 mouse glioma implantation model and flow cytometric analysis of TAMs from tumors generated with WT and Pyk2KO GL261 cells demonstrated increased infiltration of TNF+/IFNg+ CD8+ lymphocytes and Ly6C+/CD206- myeloid cells, alongside a reduction in CD45+/CD11b+/CD33+/MHCII- myeloid-derived suppressor cells. PCR analysis of TAM isolated from Pyk2KO tumors revealed lower gene expression of TNF, CCL5, CCL2, VEGFa, and epidermal growth factor (EGF) compared to WT tumors. The study highlights that Pyk2/MEK/Erk signaling regulates the immune microenvironment in GBM by influencing EV release, which impacts TAM cell activation. Pyk2 primarily regulates the release of Integrin- EVs, while MEK/Erk predominantly regulates EVs shed from the plasma membrane. The findings underscore the interplay between Pyk2 and MEK/Erk signaling in regulating EV biogenesis and composition. Citation Format: Neisha M. Ramirez. Pyk2 and MEK/ERK signaling regulate the extracellular vesicle release and tumor-associated myeloid Cells in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B046.

Bioinformatics combined with network pharmacology and experimental validation to identify key biomarkers of hepatocellular carcinoma and corresponding compounds in Radix Astragali and Pueraria Mirifica.

The occurrence and death rates of primary hepatocellular carcinoma (HCC) are increasing, and there remains a shortage of effective oral medications with minimal side effects. We aim to identify potential biomarkers and compounds from Radix Astragali (RA) and Pueraria Mirifica (PM) to treat liver cancer and improve prognosis. Differentially expressed genes (DEGs) associated with HCC were identified by bioinformatics analysis of three datasets, GSE112791, GSE101685, and GSE45114. Using public databases to predict the bioactive components and possible targets of RA and PM. Target crossover from Gene Expression Omnibus (GEO) and public databases were used to identify potential biomarkers for HCC. Subsequently, validation and prognostic value analyses were performed using the Gene Expression Profile Interaction Analysis (GEPIA) platform. The Cytoscape software created a network of "compound targets" to pinpoint compounds linked to the biomarkers. Molecular docking techniques were utilized to validate the connection between these compounds and the identified biomarkers. Ultimately, the HepG2 liver cancer cell line was chosen to assess the inhibitory effect of Hederagenin (HDG) and to confirm the expression of ADH1B through Western blot analysis. In this study, four key biomarkers (NR1I2, ADH1B, NQO1, GHR) were identified. Molecular docking showed that these four core targets could form stable conformations with the corresponding compounds. As the drug concentration decreases, the inhibitory effect on HepG2 diminishes, and the survival rate of HepG2 cells significantly declines following the administration of 100 µmol/L HDG. Compared to the control, the expression of ADH1B protein is significantly increased in HepG2 cells treated with 100 µmol/L HDG. The study identified four key biomarkers (ADH1B, GHR, NQO1, NR1I2) that have prognostic ability for HCC. This study provides biomarkers and potential targeted monomeric medicines for treating HCC.

Study on the mechanism of hyperoside in affecting the biological progression and radiosensitivity of esophageal carcinoma by modulating the STAT3/AKT/ERK pathway

Hyperoside (HYP) exhibits diverse pharmacological effects and holds potential for enhancing chemotherapy sensitivity. However, few studies have reported the impact of HYP on the malignant progression of esophageal carcinoma (EC) and its sensitivity to radiotherapy. The impact of HYP on the viability of esophageal carcinoma cells (TE-1 and KYSE-150) was assessed using Cell Counting Kit-8 (CCK-8) assays. The biological characteristics and radiosensitivity of esophageal carcinoma cells following HYP treatment were evaluated through colony formation experiments, flow cytometry, scratch wound-healing assays, and transwell migration and invasion assays. Western blot analysis was performed to determine the levels of proteins associated with cell death and epithelial-mesenchymal transition (EMT), as well as to explore whether HYP interferes with the radiosensitivity of esophageal carcinoma cells via the STAT3/AKT/ERK pathways. Finally, a subcutaneous graft tumor model was constructed to investigate the effects of HYP and X-ray treatments on in vivo tumor growth. The findings indicated a dose-dependent decrease in the survival rate of KYSE-150 and TE-1 cells following HYP treatment. HYP treatment also inhibited cell proliferation, invasion, migration, and EMT, while increasing the apoptotic rate and radiosensitivity of the cells. Notably, HYP suppressed the malignant progression of EC and enhanced radiosensitivity via the STAT3/AKT/ERK pathway. Moreover, HYP impaired the growth of esophageal carcinoma tumors in mice, with the combined HYP and X-ray treatment exerting a stronger inhibitory effect. In conclusion, HYP increases the radiosensitivity of esophageal carcinoma cells, offering considerable promise for application in the clinical treatment of EC.

Nuclear receptor subfamily 4 group a member 1 eases angiotensin II-arose oxidative stress in vascular smooth muscle cell by boosting nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 3 transcription

Objective: Hypertension significantly contributes to morbidity and mortality. Nuclear receptor subfamily 4 group a member 1 (Nur77) participates in regulating oxidative stress, but the mechanism in hypertension remains unclear. This study aimed to explore the function of Nur77 in oxidative stress induced by Angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs) in hypertension. Material and Methods: First, models of VSMC with Nur77, nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 3 (NLRC3) and tumor necrosis factor receptor-associated factor 6 (TRAF6) knockdown or overexpression were constructed using Short Hairpin RNA (Nur77) or pcDNA3.1 vector, respectively. Next, the putative-binding motifs between Nur77 and NLRC3 promoters were detected by dual luciferase assay. We conducted reverse transcription quantitative polymerase chain reaction (qPCR) and Western blot (WB) analysis to detect Nur77, NLRC3, and TRAF6 levels in VSMCs. Then, cell counting kit-8 assay, 5-ethynyl-2’-deoxyuridine assay, wound-healing assay, enzyme-linked immunosorbent assay, and 2’,7’-dichlorofluorescin diacetate were employed to examine the impact of the knockdown or overexpression of Nur77, NLRC3, and TRAF6 on VSMCs treated with Ang II. The assays measured cell viability and proliferation, cell migration, malondialdehyde levels, and reactive oxygen species levels. Results: The overexpression of Nur77 repressed cell growth (P < 0.001), migration (P < 0.01), and oxidative stress (P < 0.01) induced by Ang II in VSMCs. Nur77 transcriptionally promoted the expression of NLRC3 (P < 0.001), and the upregulation of NLRC3 suppressed cell proliferation (P < 0.05) and oxidative stress (P < 0.001) mediated by Ang II. Furthermore, NLRC3 negatively regulated the TRAF6/nuclear factor-kappa B (NF-κB) axis activated by Ang II, which resulted in the repression of hyperproliferation of VSMCs (P < 0.01) and oxidative stress (P < 0.001). Conclusion: Nur77 suppressed growth and oxidative stress induced by Ang II in VSMCs by promoting NLRC3 transcription, which, further, repressed the TRAF6/NF-κB axis. This understanding provides novel insights into the pathogenesis of hypertension.

MDM2 drives resistance to Osimertinib by contextually disrupting FBW7-mediated destruction of MCL-1 protein in EGFR mutant NSCLC.

Overcoming resistance to Osimertinib in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) is clinically challenging because the underlying mechanisms are not fully understood. The murine double minute 2 (MDM2) has been extensively described as a tumor promotor in various malignancies, mainly through a negative regulatory machinery on the p53 tumor suppressor. However, the significance of MDM2 on the sensitivity to Osimertinib has not been described. Osimertinib resistant cells were generated by standard dose escalation strategy and individual resistant clones were isolated for MDM2 testing. The MDM2 and its mutant constructs (ΔPBD, ΔRING, C464A) were introduced into PC-9, HCC827 and H1975 cells and evaluated for the sensitivity to Osimertinib by MTT assay, colony formation, EdU assay and TUNEL assay. MDM2 expression in resistant cells was manipulated by pharmacological and molecular approaches, respectively. Proteins that were implicated in PI3K/Akt, MAPK/Erk and apoptosis signaling were measured by Western blot analysis. Candidate proteins that interacted with MDM2 were captured by immunoprecipitation and probed with indicated antibodies. In comparison with parental PC-9 cells, the PC-9 OR resistant cells expressed high level of MDM2. Ectopic expression of MDM2 in PC-9, HCC827 and H1975 sensitive cells generated an Osimertinib resistant phenotype, regardless of p53 status. MDM2 promoted resistance to Osimertinib through a PI3K/Akt and MAPK/Erk-independent machinery, in contrast, MDM2 selectively stabilized MCL-1 protein to arrest Osimertinib-induced cancer cell apoptosis. Mechanistically, MDM2 acted as a E3 ligase to ubiquitinate FBW7, a well-established E3 ligase for MCL-1, at Lys412 residue, which resulted in FBW7 destruction and MCL-1 stabilization. Targeting MDM2 to augment MCL-1 protein breakdown overcame resistance to Osimertinib in vitro and in vivo. Finally, the clinical relevance of MDM2-FBW7-MCL-1 regulatory axis was validated in mouse xenograft tumor model and in NSCLC specimen. Overexpression of MDM2 is a novel resistant mechanism to Osimertinib in EGFR mutant NSCLC. MDM2 utilizes its E3 ligase activity to provoke FBW7 destruction and sequentially leads to MCL-1 stabilization. Cancer cells with aberrant MDM2 state are refractory to apoptosis induction and elicit a resistant phenotype to Osimertinib. Therefore, targeting MDM2 would be a feasible approach to overcome resistance to Osimertinib in EGFR mutant NSCLC.

Regulatory Effects of Copper on Ghrelin Secretion in Rat Fundic Glands.

Copper (Cu) is an effective additive in feed for promoting growth. Growth dan axis comprising growth hormone (GH), somatostatin (SS) and GH-releasing hormone (GHRH), with ghrelin regulating their release. The growth-promoting effects of Cu are closely related to ghrelin, but the specific mechanism behind the relationship remains unknown. We investigated the adjustment of ghrelin synthesis and secretion by Cu. Sprague-Dawley rats were fed basal diets with an addition of 0, 120 or 240 mg/kg Cu sulfate for 28 day to establish a growth-promoting model. Signalling molecules relevant to ghrelin synthesis and secretion were detected and mechanistically explored using enzyme-linked immunosorbent assay, quantitative reverse-transcription polymerase chain reaction and Western blot analysis. The 120 mg/kg supplement improved growth performance; significantly increased the serum levels of ghrelin, ghrelin O-acyltransferase (GOAT), acylated ghrelin (AG), GH, and reactive oxygen species (ROS) and decreased those of SS; significantly increased the mRNA and protein expression of ghrelin, GOAT, ghrelin receptor (GHS-R1α), and activator protein 1 (AP-1); increased the phosphorylation ratio of JNK and p38 MAPK; and inhibited the mRNA and protein expression of SS and SS receptor subtype 2 (SSTR2) in gastric fundic gland tissues. Thus, Cu may affect gastric ghrelin synthesis at the transcriptional level by activating the JNK/p38 MAPK pathway through increased ROS levels and regulating the activation of the downstream redox-sensitive transcription factor AP-1. SS plays a crucial determinant role in ghrelin regulation via intragastric Cu. Cu promotes GOAT activity and ghrelin secretion by inhibiting SS secretion, affecting AG levels, and promoting ghrelin acylation through ghrelin/GOAT/GHS-R1α system, modulating ghrelin secretion.

Ro 90-7501 suppresses colon cancer progression by inducing immunogenic cell death and promoting RIG-1-mediated autophagy.

Colon cancer is one of the leading causes of cancer-related mortality worldwide. Current treatments, including surgery, chemotherapy, and radiation therapy, often have limited efficacy due to tumor heterogeneity and resistance mechanisms. Therefore, there is a critical need for novel therapeutic strategies that can enhance the immune response against colon cancer cells while promoting their efficient clearance. In this study, we investigated the anti-tumor effects of Ro 90-7501, a specific small molecule, in colon cancer cell lines (HCT8 and SW480) and in vivo models. MTS, EdU, Scratch Wound and Transwell assays were performed to detect the cell proliferation and metastasis. Additionally, flow cytometry and immunofluorescence staining were used to analyze changes in apoptosis and necrosis. Furthermore, we examined its ability to induce immunogenic cell death (ICD) and promote retinoic acid-inducible gene I (RIG-I)-mediated autophagy. The expression levels of key molecules involved in ICD and autophagy were assessed using Western blot analysis, immunofluorescence staining, and enzyme-linked immunosorbent assay (ELISA). Our findings demonstrated that Ro 90-7501 significantly suppressed colon cancer cell proliferation and metastasis, inducing apoptosis and necrosis. Mechanistically, Ro 90-7501 triggered ICD by upregulating the exposure of calreticulin (CRT) on the cell surface and increasing the release of high mobility group box 1 (HMGB1) and extracellular ATP levels. Concurrently, it promoted RIG-I-mediated autophagy via the MAVS-TRAF6 signaling axis, evidenced by increased expression of autophagy-related genes, such as microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1 proteins, coupled with a reduction in P62 protein. Additionally, Ro 90-7501 treatment activated the RIG-I-MAVS-TRAF6 signaling axis in colon cancer cells. Furthermore, Ro 90-7501 enhanced the secretion of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFα), thereby activating the immune response. In conclusion, Ro 90-7501 exhibits potent anti-tumor activity against colon cancer by inducing ICD and promoting RIG-I-mediated autophagy. These results suggest that Ro 90-7501 may serve as a promising therapeutic candidate for colon cancer treatment by enhancing both intrinsic cellular processes and adaptive immune responses. Further studies are warranted to elucidate the detailed mechanisms underlying these effects and to evaluate its therapeutic potential in clinical settings.

Chondroitin Sulfate-Coated Heteroduplex-Molecular Spherical Nucleic Acids.

Molecular Spherical Nucleic Acids (MSNAs) are atomically uniform dendritic nanostructures and potential delivery vehicles for oligonucleotides. The radial formulation combined with covalent conjugation may hide the oligonucleotide content and simultaneously enhance the role of appropriate conjugate groups on the outer sphere. The conjugate halo may be modulated to affect the delivery properties of the MSNAs. In the present study, [60]fullerene-based molecular spherical nucleic acids, consisting of a 2'-deoxyribonucleotide and a ribonucleotide sequence, were used as hybridization-mediated carriers (''DNA and RNA-carriers'') for an antisense oligonucleotide, suppressing Tau protein, (i.e. Tau-ASO) and its conjugates with chondroitin sulfate tetrasaccharides (CS) with different sulfation patterns. The impact of the MSNA carriers, CS-moieties on the conjugates and the CS-decorations on the MSNAs on cellular uptake and - activity (Tau-suppression) of the Tau-ASO was studied with hippocampal neurons in vitro. The formation and stability of these heteroduplex ASO-MSNAs were evaluated by UV melting profile analysis, polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS) and size exclusion chromatography equipped with a multi angle light scattering detector (SEC-MALS). The cellular uptake and - activity were studied by confocal microscopy and Western blot analysis, respectively.

Helicobacter pylori promotes gastric cancer progression by activating the TGF-β/Smad2/EMT pathway through HKDC1.

Helicobacter pylori (H. pylori) infection is widely acknowledged as the primary risk factor for gastric cancer, facilitating its progression via the Correa cascade. Concurrently, Hexokinase Domain Containing 1 (HKDC1) has been implicated in the mediation of aerobic glycolysis, contributing to tumorigenesis across various cancers. However, the precise role of HKDC1 in the inflammatory transformation associated with H. pylori-induced gastric cancer remains elusive. In this study, transcriptome sequencing revealed a significant correlation between HKDC1 and H. pylori-induced gastric cancer. Subsequent validation using qRT-PCR, immunohistochemistry, and Western blot analysis confirmed elevated HKDC1 expression in both human and murine gastritis and gastric tumors. Moreover, in vitro and in vivo experiments demonstrated that H. pylori infection up-regulates TGF-β1 and p-Smad2, thereby activating the epithelial-mesenchymal transition (EMT) pathway, with HKDC1 playing a pivotal role. Suppression of HKDC1 expression or pharmacological inhibition of TGF-β1 reversed EMT activation, consequently reducing gastric cancer cell proliferation and metastasis. These results underscore HKDC1's essential contribution to H. pylori-induced gastric cancer progression via EMT activation.

Fibroblast growth factor 10 alleviates LPS-induced acute lung injury by promoting recruited macrophage M2 polarization.

Acute lung injury (ALI) is characterized by damage to the alveoli and an overabundance of inflammation. Representing a serious inflammatory condition, ALI lacks a precise treatment approach. Despite the recognized benefit impacts of Fibroblast growth factor-10 (FGF10) on ALI, the underlying mechanisms remain unelucidated. To study the role of FGF10 in ALI, C57BL/6J mice were intratracheally injected with 5mg/kg Lipopolysaccharide (LPS) with FGF10 (5mg/kg) or an equal volume of PBS. Inflammatory factors were quantified in bronchoalveolar lavage fluid (BALF) and plasma using ELISA. RNA sequencing of F4/80+Ly6G- macrophages in BALF explored changes in macrophage phenotype and potential mechanisms. Macrophage polarization in BALF was assessed using qRT-PCR, flow cytometry, and Western blot analysis. In vitro, a Transwell co-culture of mouse lung epithelial cells (MLE12) and bone marrow macrophages (BMDM) validated the role of FGF10 in modulating LPS-induced macrophage phenotypic changes. FGF10 ameliorated LPS-induced ALI by diminishing pro-inflammatory factors (IL-1β, TNF-α, and IL-6) and the neutrophil accumulation in BALF. FGF10 also increased the levels of anti-inflammatory factor IL-10. The FGF10 intervention group exhibited enhanced gene expression of macrophage arginine biosynthesis marker (ARG1), and expression of M2-type marker CD206 in monocytes and macrophages. In addition, phosphorylated STAT3 expression increased in isolated monocyte-derived macrophages. Experiments in vitro confirmed that FGF10 could elevate macrophage M2 marker ARG1 expression through the JAK2/STAT3 pathway. FGF10 ameliorates acute LPS-induced lung injury by modulating the polarization of monocyte-derived macrophages recruited in the alveolar space to the M2 type.

Exosomal PSM-E inhibits macrophage M2 polarization to suppress prostate cancer metastasis through the RACK1 signaling axis.

It is well-established that understanding the mechanism of prostate cancer (PCa)-associated metastasis is paramount for improving its prognosis. Metastasis is known to involve the communication between tumor-associated macrophages (TAMs) and tumor cells. Exosomes are crucial in mediating this intercellular communication within the tumor microenvironment. Nonetheless, the role of exosomal proteins in PCa metastasis is not yet fully understood. Here, we investigated the mechanisms of prostate cancer-derived exosomal PSM-E on regulating macrophage M2 polarization to suppress tumor invasion and metastasis. PSM-E levels in exosomes were detected by transmission electron microscopy and Western blotting analysis. The diagnostic value of urine-derived exosomal PSM-E in PCa were evaluated by LC-MS/MS, correlation analysis, and ROC curves analysis. The mechanisms underlying the inhibitory effect of exosomal PSM-E on the M2 polarization of macrophages was investigated by co-IP, IHC staining, and PCa tumorigenesis model, etc. RESULTS: We revealed that exosomal PSM-E is upregulated in exosomes derived from the serum and urine of PCa patients. Clinically, an elevated exosomal PSM-E expression in urine is significantly correlated with an advanced pathological tumor stage and a high Gleason score. Our research also revealed that exosomal PSM-E inhibits prostate cancer cell proliferation, invasion, and metastasis by suppressing macrophage polarization in vitro and in vivo. Furthermore, we provided compelling evidence that exosomal PSM-E inhibits M2 polarization of macrophages by recruiting RACK1 and suppressing the FAK and ERK signaling pathways, consequently suppressing PCa invasion and metastasis. Furthermore, we found that the protease-associated domain of PSM-E and the fourth tryptophan-aspartate repeat of RACK1 are crucial for the interaction between PSM-E and RACK1. Notably, exosomes carrying PSM-E from PCa urine could potentially serve as a biomarker for PCa, and targeting exosomal PSM-E may represent a strategy for preventing tumor progression in this patient population.

The LINC01094/miR-545-3p/SLC7A11 Signaling Axis Promotes the Development of Gastric Cancer by Regulating Cell Growth and Ferroptosis.

This study aimed to investigate the role and mechanism of action of LINC01094 in the development of gastric cancer (GC). The expression levels of LINC01094 in GC patients and healthy individuals were analyzed online using the Cancer Genome Atlas database. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to determine the expression of LINC01094/miR-545-3p/SLC7A11 in GC tissues and cells. Functional experiments (MTT assay, colony formation assay, and flow cytometry) were conducted to assess the effect of LINC01094 and miR-545-3p on cell proliferation, viability, apoptosis, cell cycle, and reactive oxygen species. Correlations between LINC01094 and miR-545-3p, as well as SLC7A11, were analyzed and validated using the dual-luciferase reporter assay and RNA immunoprecipitation. The levels of Fe2+, malondialdehyde, and glutathione in the cells were measured biochemically, and the protein expression levels of Bcl-2, cleaved caspase3, Cyclin D1, and p21 were detected by Western blotting. LINC01094 was significantly upregulated in the GC tissues and cells with a targeting relationship with miR-545-3p; the expression levels of LINC01094 and miR-545-3p were negatively correlated. Knockdown of LINC01094 notably inhibited the proliferation and viability of GC cells and promoted cell ferroptosis, which, however, was abrogated by the silencing of miR-545-3p. These findings indicate that miR-545-3p could target and positively correlate with SLC7A11 expression. Additionally, LINC01094 could promote GC cell progression and affect cellular ferroptosis by regulating the miR-545-3p/SLC7A11 signaling axis.

Tasquinimod promotes the sensitivity of ovarian cancer cells to cisplatin by down-regulating the HDAC4/p21 pathway.

To investigate whether Tasquinimod can influence cisplatin resistance in drug-resistant ovarian cancer (OC) cell lines by regulating histone deacetylase 4 (HDAC4) or p21, we explored its effects on the cell cycle, and associated mechanisms. RT-PCR and Western blot analyses, flow cytometry, CCK8 assay, and immunofluorescence were utilized to investigate the effects of Tasquinimod on gene expression, cell cycle, apoptosis, viability, and protein levels in OC cells. The results showed that Tasquinimod inhibited cell viability and promoted apoptosis in SKOV3/DDP (cisplatin) and A2780/DDP cells more effectively than DDP alone. In combination with cisplatin, Tasquinimod further enhanced cell apoptosis and reduced cell viability in these cell lines, an effect that could be reversed following HDAC4 overexpression. Tasquinimod treatment down-regulated HDAC4, Bcl-2, and cyclin D1, and CDK4 expression and up-regulated the cleaved-Caspase-3, and p21 expression in SKOV3/DDP and A2780/ DDP cells. Additionally, Tasquinimod inhibited DDP resistance in OC/DDP cells. These effects were similarly observed in OC mouse models treated with Tasquinimod. In conclusion, Tasquinimod can improve OC cells' sensitivity to DDP by down-regulating the HDAC4/p21 axis, offering insights into potential strategies for overcoming cisplatin resistance in OC.

The role of the KEAP1-NRF2 signaling pathway in form deprivation myopia guinea pigs.

In recent years, the global prevalence of myopia has reached an unprecedented level, especially‌ in East Asia. Multitude of studies has shown that the etiology of myopia is complex. Some researchers have suggested that oxidative stress (OS) may contribute to myopia, although there are limited reports on the alterations of related signaling pathways. Notably, the Kelch-like ECH-associated protein 1 (KEAP1) -nuclear factor erythroid 2-related factor 2 (NRF2), which plays a significant role in regulating OS and the mechanism, has not been explored in myopia. To investigate the modulation of KEAP1-NRF2 signaling pathway and its downstream superoxide dismutase (SOD) during the development of form-deprivation myopia, three-week-old guinea pigs were randomly assigned to four groups: negative control (NC), self-control (SC), form-deprivation myopia (FDM), and FDM group treated with tert-butylhydroquinone (TBHQ). Spherical equivalent (SE) and axial length (AL) were measured by retinoscopy and A-scan ultrasound, respectively. The results revealed that TBHQ treatment decelerated the progression in SE and AL changes. Immunohistochemistry (IHC) assessed the distribution and expression of KEAP1, NRF2, and SOD. The results shown that they located in the retinal ganglion cells (RGC). Subsequently, retinal mRNA and protein expression levels of KEAP1, NRF2, and SOD were quantified using real-time polymerase chain reaction (RT-PCR) and Western blot (WB) analysis. The RT-PCR and WB results demonstrated that TBHQ could activate NRF2, induce KEAP1 degradation, and enhance the expression of the antioxidant SOD. In summary, the modulation of KEAP1-NRF2 and it downstream SOD expression could alter the retinal antioxidant capacity and influence the development of myopia.

The Glucagon-Like Peptide-1 (GLP-1) Receptor Agonist Liraglutide Regulates Sirtuin-1-Mediated Neutrophil Extracellular Traps to Improve Diabetes-Induced Bone Metabolism Imbalance

Background: Diabetes mellitus (DM) is a chronic metabolic disorder that disrupts normal bone remodeling. Objectives: This study aimed to investigate how the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide (LIR) addresses bone metabolism imbalances induced by type-II diabetes. Methods: Type-II diabetic rat models were established through a single intraperitoneal injection of streptozotocin (STZ). Blood glucose levels were measured using a blood glucose meter, and insulin levels were assessed using an assay kit. Bone formation markers [alkaline phosphatase (ALP), osteocalcin (OCN), and procollagen I N-terminal propeptide (PINP)] and bone resorption markers [tartrate-resistant acid phosphatase (TRACP) and CTX-1] were monitored using assay kits. Bone marrow mesenchymal stem cells (BMSCs) were cultured in vitro under high-fat and high-glucose (HFHS) conditions to mimic diabetic bone metabolism dysregulation. Neutrophil extracellular traps (NETs) formation was examined through immunofluorescent staining and Western blot analysis. Results: Liraglutide was found to reduce STZ-induced NETs formation, as indicated by decreased expression of cit-H3 by 36.90% - 53.57%, myeloperoxidase (MPO) by 55.81% - 65.12%, NE by 53.95% - 65.17%, and PAD4 by 46.81% - 63.83%, alongside increased Sirtuin-1 (SIRT1) expression in femur tissue (70.71% - 91.19%). In vitro, LIR enhanced osteogenesis and inhibited apoptosis, effects that were partially reversed by SIRT1 knockdown. Additionally, SIRT1 knockdown partially restored LIR-induced reductions in oxidative stress, inflammation, and NETs formation. Conclusions: LIR mitigates diabetes-induced bone metabolism imbalance by inhibiting NETs formation through SIRT1 mediation.

Low-intensity pulsed ultrasound promotes cell viability of hUSCs in volumetric bioprinting scaffolds via PI3K/Akt and ERK1/2 pathways.


The study aimed to investigate the impact of low-intensity pulsed ultrasound (LIPUS) on human urinary-derived stem cells (hUSCs) viability within 3D cell-laden gelatin methacryloyl (GelMA) scaffolds.
Methods:
hUSCs were integrated into GelMA bio-inks at concentrations ranging from 2.5% to 10% (w/v) and then bioprinted using a volumetic-based method. Subsequent exposure of these scaffolds to LIPUS under varying parameters or sham irradiation aimed at optimizing the LIPUS treatment. Assessment of hUSCs viability employed Cell Counting Kit-8 (CCK8), cell cycle analysis, and live&dead cell double staining assays. Additionally, Western blot analysis was conducted to determine protein expression levels.
Result:
With 3D bio-printed cell-laden GelMA scaffolds successfully constructed, LIPUS promoted the proliferation of hUSCs. Optimal LIPUS conditions, as determined through CCK8 and live&dead cell double staining assays, was achieved at a frequency of 1.5 MHz, a spatial-average temporal-average intensity (ISATA) of 150 mW/cm2, with an exposure duration of 10 minutes per session administered consecutively for two sessions. LIPUS facilitated the transition from G0/G1 phase to S and G2/M phases and enhanced the phosphorylation of ERK1/2 and PI3K-Akt. Inhibition of ERK1/2 (U0126) and PI3K (LY294002) significantly attenuated LIPUS-induced phosphorylation of ERK1/2 and PI3K-Akt respectively, both of which decreased the hUSC viability within 3D bio-printed GelMA scaffolds.
Conclusion:
Applying a LIPUS treatment at an ISATA of 150 mW/cm2 promotes the growth of hUSCs within 3D bio-printed GelMA scaffolds through modulating ERK1/2 and PI3K-Akt signaling pathways.&#xD.

Mitigating ibrutinib-induced ventricular arrhythmia and cardiac dysfunction with metformin.

Ibrutinib is a first-line drug that targets Bruton's tyrosine kinase for the treatment of B cell cancer. However, cardiotoxicity induced by ibrutinib is a major side effect that limits its clinical use. This study aimed to investigate the mechanism of ibrutinib-induced cardiotoxicity and evaluate the protective role of metformin. The study utilized male C57BL/6 J mice, which were administered ibrutinib at a dosage of 30 mg/kg/day via oral gavage for 4 weeks to induce cardiotoxicity. Metformin was administered orally at 200 mg/kg/day for 5 weeks, starting 1 week before ibrutinib treatment. Cardiac function was assessed using echocardiography and electrophysiological studies, including surface electrocardiography and epicardial electrical mapping. Blood pressure was measured using a tail-cuff system. Western blot analysis was conducted to evaluate the activity of the PI3K-AKT and AMPK pathways, along with apoptosis markers. C57BL/6 J mice were treated with ibrutinib for 4 weeks to assess its effect on cardiac function. We observed that ibrutinib induced ventricular arrhythmia and abnormal conduction while reducing the left ventricular ejection fraction. Furthermore, pretreatment with metformin reversed ibrutinib-induced cardiotoxicity. Mechanistically, ibrutinib decreased PI3K-AKT activity, resulting in apoptosis of cardiomyocytes. Administration of metformin upregulated AMPK and PI3K-AKT activity, which contributed to the improvement of cardiac function. The study concludes that metformin effectively mitigates ibrutinib-induced cardiotoxicity, including ventricular arrhythmia and cardiac dysfunction, by enhancing AMPK and PI3K-AKT pathway activity. These findings suggest that metformin holds potential as a therapeutic strategy to protect against the adverse cardiac effects associated with ibrutinib treatment, offering a promising approach for improving the cardiovascular safety of patients undergoing therapy for B cell cancers.

Melittin suppresses aerobic glycolysis by regulating HSF1/PDK3 to increase chemosensitivity of NSCLC.

Non-small cell lung cancer (NSCLC), although considered non-immunogenic, is often resistant to chemotherapy agents during the course of treatment in clinical patients. Melittin (C131H229N39O31, CAS: 20449-79-0), the major component of honey bee venom, is a promising anticancer drug. However, the mechanism employed by melittin to reverse chemotherapy resistance of NSCLC cells remains unknown. In this study, the Cell Counting Kit 8, ethynyl deoxyuridine assay, and other assays were utilized to elucidate the melittin effects upon cell proliferation. Proteomics, lung cancer (LC) tissue chip, and western blot analysis were used to identify potential targets of melittin. A549/DDP cells were employed to investigate the melittin effects against cisplatin resistance. Also, an in vivo animal experiment was conducted to further clarify the regulatory function of melittin towards cisplatin resistance of A549/DDP cells. Results showed that melittin inhibited malignant progression of A549/DDP cells by down-regulation of pyruvate dehydrogenase kinase 3 (PDK3)-mediated aerobic glycolysis and inhibition of heat shock factor 1 (HSF1) expression. The therapeutic effect of melittin was increased by combination with KNK437 and impaired chemotherapy resistance regarding A549/DDP cells via reversing aerobic glycolysis. The in vivo experiments confirmed that melittin incremented A549/DDP cell cisplatin sensitivities. Collectively, the data suggested that melittin suppressed aerobic glycolysis by regulating HSF1/PDK3, which incremented cisplatin sensitivity of A549/DDP cells. It may provide a new treatment method for chemotherapy resistance in clinical NSCLC patients.

Solasodine Downregulates ABCB1 Overexpression in Multidrug Resistant Cancer Cells Via Inhibiting Nrf2/Keap1 Signaling Pathway.

Multidrug-resistant (MDR) cancer cells maintain redox homeostasis to eliminate oxidative stress-mediated cell death. This study explores the effects of solasodine on regulating P-glycoprotein (P-gp) expression through the Nrf2/Keap1 signaling pathway and oxidative stress-induced sensitization of drug-resistant cancer cells to chemotherapeutics. Initially, the oxidative stress indicators such as intracellular ROS generation, the levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) and gamma-H2AX (γ-H2AX) in the KBChR-8-5 drug-resistant cells were measured. Additionally, the protein expression levels of Nuclear factor erythroid 2-related factor 2 (Nrf-2), Kelch-like ECH-associated protein 1 (Keap1), and ATP Binding Cassette Subfamily B Member 1 (ABCB1)/P-gp were measured at various concentrations of solasodine (1, 5, & 10 µM) through immunofluorescence and western blot analysis. The antioxidant activities in the KBChR-8-5 cells were assessed using established protocols. In this investigation, the treatment with solasodine and doxorubicin combination showed a notable increase in intracellular ROS generation in KBChR-8-5 cells. Furthermore, this combination treatment led to enhanced nuclear condensation, elevated levels of 8-OHdG, and increased γ-H2AX foci formation in the KBChR-8-5 cells. Solasodine treatment effectively inhibited the nuclear translocation of Nrf2 and activation of the ABCB1 gene, consequently preventing overexpression of P-gp in KBChR-8-5 cells. Additionally, the combination therapy increased the lipid peroxidation levels while simultaneously reducing the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and the levels of glutathione (GSH). These results demonstrated that solasodine disrupts redox balance, and overcomes drug resistance by downregulating P-gp via regulating Nrf2/Keap1 signaling pathway in MDR cancer cells.