P53 Levels Research Articles

In vitro and in vivo evaluation of the cyclophosphamide effect along with oncolytic Newcastle disease virus (NDV): An animal preclinical research

AbstractBackgroundBreast cancer is a major cause of mortality globally. Oncolytic virotherapy is a promising treatment modality that directly destroys cancer cells and induces an immune response against them. Among natural oncolytic viruses, Newcastle disease virus (NDV) has shown selective tumor cell infection.Materials and methodsIn this study, we investigated the efficacy of variable doses of NDV and cyclophosphamide on 4T1 cancer cell line and BALB/c mouse tumors for the first time.ResultsCompared with the control group, the combination treatment group with NDV and cyclophosphamide showed a significant increase in the expression levels of P21, P27, and P53 genes by 38%, 46%, and 81%, respectively (p < 0.05). In contrast, the expression levels of CD34, integrin α5, vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor (VEGFR) genes significantly decreased by 47%, 45%, 42%, and 23%, respectively (p < 0.05). The reactive oxygen species (ROS) generation assay evaluated with 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) staining showed a significant increase in ROS levels within 4T1 cells treated with NDV compared with the untreated group after 24 h (p < 0.01). Furthermore, Annexin V/propidium iodide (PI) double staining analysis showed that the proportion of apoptotic cells in the NDV‐treated group decreased by 0.61% and 1.63% after 6 h and 12 h, respectively (p < 0.05). After 12 days, tumor volume in the NDV‐treated groups decreased by 72%−87% compared with a 48% increase in the control group, reflecting a net reduction in tumor volume relative to the control group (p < 0.001).ConclusionThese findings demonstrate that NDV in combination with chemotherapy drugs may be a promising therapeutic option for cancer patients. However, several other factors need to be considered. These results indicate that NDV may have potential effects on cancer treatment.

Camptothecin Triggers Apoptosis in Human and Mouse Drug-resistant Glioblastoma Cells via ROS-mediated Activation of the p53-p21-CD1/CDK2-E2F1-Bcl-xL Signaling Axis.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor. Temozolomide (TMZ) is the first-line treatment for GBM. However, most patients with GBM develop drug resistance. Our previous study showed the effects of camptothecin (CPT) and CRLX101, a nanoparticle of CPT, in suppressing GBM growth by targeting drug-sensitive glioblastoma cells. This study evaluated the effects of CPT on drug-resistant glioblastoma cells and explored the underlying molecular mechanisms. Expression of type I topoisomerase (Topo-1) gene in GBM was analyzed using the UALCAN database. Human U87MG-R and mouse GL261-R TMZ-resistant glioblastoma cells were developed. After CPT treatment, apoptotic events were successively determined. The role of the p53-p21-cyclin D1 (CD1)/cyclin-dependent kinase 6 (CDK6)-E2F1-Bcl-xL signaling axis was subsequently investigated. The expression of Topo-1 gene was up-regulated in human GBM compared to normal human brains. Treatment of human U87MG-R cells with CPT decreased cell viability. Sequentially, exposure to CPT led to activation of caspase-3, fragmentation of chromosomal DNA, and cell apoptosis. Furthermore, intracellular reactive oxygen species (iROS) were augmented following CPT treatment. Suppression of iROS production concurrently alleviated CPT-triggered apoptotic insults. CPT enhanced the levels of p53, phosphorylated p53, and p21. In contrast, levels of CDK6, CD1, E2F1, and Bcl-xL were decreased by CPT. Attenuating p53 transactivation activity using pifithrin-α also mitigated the CPT-induced apoptosis. The effects of CPT on killing drug-resistant glioblastoma cells were further confirmed in mouse GL261-R cells. CPT could effectively induce apoptosis in drug-resistant glioblastoma cells via iROS-mediated activation of the p53-p21-CD1/CDK6-E2F1-Bcl-xL axis.

Effect of propolis supplementation on cadmium toxicity associated with renal and hepatic dysfunction in rats.

Cadmium (Cd), one of the toxic heavy metals, is of great importance for public health due to its use in many industrial areas. Propolis is a natural product with antioxidant and anti-inflammatory properties collected from plants by honey bees. The aim of this study was to investigate the protective role of propolis against the potential toxic effects of cadmium chloride in blood, liver and kidney tissues. For this purpose, rats were divided into four groups. Control group, propolis (150 mg/kg b.w.) treated group, cadmium chloride (1.76 mg/kg b.w. 1/50 LD50) treated group and cadmium chloride plus propolis (1.76 mg/kg b.w. and 150 mg/kg b.w. respectively) treated group. The substances were given to rats by gavage for 28 days. After 28 days of treatment, a statistically significant change was observed in serum biochemical parameters such as ALT, AST, BUN, LDH, urea, uric acid and creatinine, hematological parameters such as erythrocyte, hemoglobin and hematocrit, and IL-1β and IL-6 cytokine levels of cadmium chloride treated rats compared to the control group. In addition, significant changes were observed in mRNA expression levels of Casp-3, p53 and Tnf-α genes, HSP70, HSP90 and GRP78 protein levels and histopathological/immunohistochemical examinations. Improvement in biochemical and hematologic parameters, cytokines, mRNA expression, heat shock proteins and immunohistochemistry changes were observed in cadmium plus propolis treated group compared to cadmium treated group.

RES-CMCNPs Enhance Antioxidant, Proinflammatory, and Sensitivity of Tumor Solids to γ-irradiation in EAC-Bearing Mice.

Resveratrol (Res) is a bifunctional compound found in numerous plants, including grapes and mulberries. Nanotechnology has promising applications in medicine. The ability of various nanomaterials to serve as radiosensitizers against tumor cells were reported in several manuscripts. The present investigation aimed to assess the antitumor and radiosensitizing effects of Res-CMCNPs on EAC-bearing mice. Res-CMCNPs have been developed using the CMC emulsification cross-linking technique. Entrapment efficiency (%), particle size, Polydispersity index and ZETA potential, UV, FTIR spectra, and drug release were evaluated and described for RES-CMCNPs. The radiosensitizing properties of RES-CMCNPs were also evaluated in vitro and in vivo against EAC-carrying rodents. The LD50 of Res-CMCNPs was estimated and its 1/20 LD50 was prepared for treating EAC transplanted mice. The results revealed that the Res-CMCNPs exhibited a high entrapment efficiency (85.46%) and a size of approximately 184.60 ±17.36 nm with zeta potential value equals -51.866 mv. Also, the UV spectra of Res and Res-CMCNPs have strong absorption at 225 and 290 nm. The percentage of resveratrol release at pHs 5.8 and 7.4 was found to be 56.73% and 51.60%, respectively, after 24 h at 100 rpm. Also, the FTIR analysis confirmed the chemical stability of resveratrol in Res-CMCNPs cross-linking. The IC50 values of Res-CMCNPs against EAC cells viability were 32.99, 25.46, and 22.21 μg after 24-, 48- and 72 h incubation, respectively, whereas those of Res- CMCNPs in combination with γ-irradiation after 6-, 10 and 12-mins exposure were 24.07, 16.06 and 7.48 μg, respectively. Also, the LD50 of Res-CMCNPs was 2180 mg/kg.b.w. The treatment of EAC-bearing mice with Res-CMCNPs plus γ-irradiation improved plasma levels of NO, caspase-3, P53 and NF-kB levels as well as liver MDA, GSH, SOD, CAT, LT-B4, aromatase, Bax, Bcl2 and TGF-β levels and exhibited more significant anticancer activity than administration of Res- CMCNPs and/or exposure to γ-irradiation individually. On the other hand, administration of Res- CMCNPs in combination with γ-irradiation attenuated liver mRNAs (21, 29b, 181a, and 451) gene expression. Grafting resveratrol onto carboxymethyl chitosan appears to be a promising strategy for cancer therapy as a radiosensitizer, potentiating tumor cells' sensitivity to radiation by improving levels of proinflammatory features and antioxidant biomarkers.

Curcumin nanoparticles alleviate brain mitochondrial dysfunction and cellular senescence in γ-irradiated rats

Despite the diverse applications of γ radiation in radiotherapy, industrial processes, and sterilization, it causes hazardous effects on living organisms, such as cellular senescence, persistent cell cycle arrest, and mitochondrial dysfunction. This study evaluated the efficacy of curcumin nanoparticles (CNPs) in mitigating mitochondrial dysfunction and cellular senescence induced by γ radiation in rat brain tissues. Four groups of male Wistar albino rats (n = 8 per group) were included: (Gr1) the control group; (Gr2) the CNPs group (healthy rats receiving oral administration of curcumin nanoparticles at a dose of 10 mg/kg/day, three times per week for eight weeks); (Gr3) the irradiated group (rats exposed to a single dose of 10 Gy head γ irradiation); and (Gr4) the irradiated + CNPs group (irradiated rats treated with CNPs). The data obtained demonstrated that oral administration of CNPs for eight weeks attenuated oxidative stress in γ-irradiated rats by lowering the brain’s lipid peroxidation level [malondialdehyde (MDA)] and enhancing antioxidant markers [superoxide dismutase (SOD), reduced glutathione (GSH), and total antioxidant capacity (TAC)] (P < 0.05). In addition, CNPs significantly increased mitochondrial function by improving complex I, complex II, and ATP production levels compared to the irradiated group. In irradiated rats, CNPs also showed anti-neuroinflammatory effects by reducing brain interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and nuclear factor-kappa B (NF-ĸB) levels (P < 0.05). Moreover, CNPs administered to irradiated rats significantly reduced brain β-galactosidase activity and the expression levels of p53, p21, and p16 genes (P < 0.05) while concurrently inducing a significant increase in AMPK mRNA expression compared to the irradiated group. In conclusion, CNPs ameliorated the neurotoxicity of γ radiation and hold promise as a novel agent to delay cellular senescence via their combined antioxidant, anti-inflammatory, and mitochondrial-enhancing properties.

Quercetin Suppresses Glioma Stem Cells via Activating p16-INK4 Gene Expression through Epigenetic Regulation.

Our study aimed to explore the effects of quercetin on glioma stem cells in patients with brain tumors. Human glioblastoma cell line, U373MG, or glioma stem cell lines, were treated with quercetin. Cell viability was determined by using the cell counting kit 8 assays. Cell apoptosis was determined by using the Annexin-V reagent. Western blotting and qPCR were used to detect the protein and mRNA levels of cyclindependent kinase inhibitor 2A (p16INK4a). Chromatin immunoprecipitation analysis was used to determine the enrichment of H3K27me3 on the p16-INK4 locus with or without quercetin. Treatment with quercetin inhibited cell viability and induced cell apoptosis in U373MG cells. Moreover, treatment with quercetin inhibited the cell viability of four glioma stem cell lines (G3, G10, G15, and G17) from brain tumor samples at high concentrations while having no obvious effects for the other two glioma stem cell lines (G9 and G21). Treatment with quercetin increased the mRNA and protein levels of p16- INK4 in glioma stem cell lines. The study of the underlying mechanism revealed that treatment with quercetin reduced H3K27me3 (an epigenetic modification to the DNA packaging protein histone H3) levels at the p16-INK4 locus. In conclusion, quercetin inhibits glioma cell growth by activating p16-INK4 gene expression through epigenetic regulation.

Inhibition of p75NTR/p53 axis by ambrisentan suppresses apoptosis and oxidative stress-mediated renal damage in a cisplatin AKI model.

Cisplatin (CP) is a potent antineoplastic agent that triggers nephrotoxicity as a major adverse effect which can cause treatment interruptions and limitations to its clinical use. Nephrotoxicity associated with CP involves inflammation, oxidative stress and apoptosis in kidney tubules. The objective of this work was to assess the effect of the blockade of endothelin-1 (ET-1) receptor with ambrisentan (Amb) on altered renal function induced by CP. Swiss albino mice were assigned into control, CP, CP/Amb-5 and CP/Amb-10 groups. Ambrisentan improved kidney function (serum creatinine and BUN) and histopathological changes in comparison to CP-treated group. Ambrisentan significantly reduced protein expression of p75NTR and JNK influencing renal apoptosis as evidenced by reducing p53, caspase-3 and Bax level and elevating Bcl-2 level (p<0.05 vs CP group). Moreover, vasodilatory effect of ambrisentan was indicated by significant increase in level of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) (p<0.05 vs CP group). Ambrisentan also significantly restored oxidative balance in renal tissues as evidenced by reduced malondialdehyde and increased total antioxidant capacity and superoxide dismutase activity, in addition to decreasing nitric oxide levels (p<0.05 vs CP group). This protective effect of ambrisentan might be mediated through the downregulation of death receptor, P75NTR that in turn restores renal blood flow and oxidative balance and regulates p53, VEGF/eNOS, NF-κB, and bcl-2/Bax/caspase-3 signaling.

Synergistic Anticancer Activity of Quercetin Combined with Luteolin in MCF-7 Breast Cancer Cells via Induction of Apoptosis

Cancer is a major public health problem with its increasing incidence and mortality. The striking role of phytochemicals in the prevention and treatment of cancer is undeniable in studies. In this study, the effects of the combination of quercetin (QUR) and luteolin (LTN) on the MCF-7 human breast cancer cell line were investigated in vitro. WST-1 cell cytotoxicity assay was used to determine cytotoxic activity. The effects on colony survival and cell migration were determined by clonogenic assay and wound scratch assay, respectively. For the determination of apoptosis, AO/EB dual staining, and Caspase-3 ELISA analysis, and genomic analyses revealing p53, Bcl-2, and Bax expression levels were performed. As a result of the analyses, it was seen that especially QUR plus LTN treatment exhibited a great cytotoxic activity in cells. It was determined that the combination treatment suppressed colony survival and significantly inhibited cell migration. Quantitative analysis results show that QUR+LTN treatment triggers cellular apoptosis by upregulating Caspase-3, p53, and Bax, and downregulating Bcl-2. Supporting these findings with further in vitro and in vivo analyses may contribute significantly to revealing the promising efficacy of combined phytochemical treatment approaches on cancer.

Ribosomal protein L6 suppresses hepatocellular carcinoma by modulating FBXO22-mediated p53 degradation.

The ribosomal protein L6 (RPL6) is significant in the progression of different cancer types. However, its precise role in hepatocellular carcinoma (HCC) remains unclear. This research demonstrated that the expression levels of RPL6 are notably decreased in HCC tissues. The decreased expression of RPL6 is strongly linked to tumor size, the presence of vascular invasion, and a worse prognosis. Functional experiments revealed that the expression of RPL6 impedes the proliferation of HCC cells and the advancement of xenograft tumors. Mechanistically, we found that RPL6 binds to and is degraded by the E3 ubiquitin ligase FBXO22, thereby inhibiting the polyubiquitination and subsequent degradation of p53 by FBXO22. The enhanced activity of p53 further contributes to cell growth inhibition. In contrast, the levels of p53 decreased significantly following RPL6 depletion, indicating that RPL6 is essential for the stabilization of p53. In summary, RPL6 inhibits the proliferation of HCC cells via the FBXO22/p53 signaling pathway, suggesting its potential as a biomarker and a therapeutic target for HCC.

Synephrine Inhibits Oxidative Stress and H2O2-Induced Premature Senescence.

Synephrine, a protoalkaloid found in Citrus aurantium (CA) peels, exerts lipolytic, anti-inflammatory, and vasoconstrictive effects; however, its antioxidant activity remains unclear. In this study, electron spin resonance spectroscopy revealed that synephrine scavenged both hydroxyl and superoxide anion radicals. Several external stimuli, such as H2O2, X-rays, and ultraviolet (UV) radiation, cause stress-induced premature senescence (SIPS). As oxidative stress induces SIPS, we hypothesized that synephrine, an antioxidant, would suppress H2O2-induced premature senescence in WI-38 cells. Synephrine significantly decreased the reactive oxygen species levels induced by H2O2, thereby reducing lipid peroxidation, and oxidative DNA damage and preventing SIPS. Additionally, synephrine inhibited mitochondrial dysfunction in H2O2-treated WI-38 cells. The expression levels of p53, p21, and p16-INK4A, which are involved in the induction of cell cycle arrest in SIPS, were significantly lower in synephrine-treated cells than in untreated cells. Our results indicate that synephrine inhibits H2O2-induced oxidative stress and mitochondrial dysfunction, suppressing premature senescence by inhibiting activation of the p53-p21 and p16-INK4A-pRB pathways.

Lung endothelial cell senescence impairs barrier function and promotes neutrophil adhesion and migration.

Cellular senescence contributes to inflammation and organ dysfunction during aging. While this process is generally characterized by irreversible cell cycle arrest, its morphological features and functional impacts vary in different cells from various organs. In this study, we examined the expression of multiple senescent markers in the lungs of young and aged humans and mice, as well as in mouse lung endothelial cells cultured with a senescence inducer, suberoylanilide hydroxamic acid (SAHA), or doxorubicin (DOXO). We detected increased levels of p21, γH2AX, and SA-β-Gal and decreased Ki-67 and Lamin B1 in aged lungs and senescent lung endothelial cells. Importantly, the expression of senescent markers was associated with an inflammatory response in aged mouse lungs characterized by neutrophil infiltration, increased expression of intercellular adhesion molecule 1 (ICAM-1), and decreased protein levels of VE-cadherin and ZO-1. As the latter two are critical constituents of endothelial cell-cell junctions, we hypothesized that their decreased expression could lead to compromised junction barrier integrity. Indeed, senescent endothelial cells (ECs) exhibited impaired barrier properties, as measured by increased permeability to solutes of small size (3-kD) and albumin (70-kD). When co-cultured with neutrophils, senescent ECs and their supernatant promoted neutrophil chemotaxis and trans-endothelial migration. Taken together, our results suggest that lung EC senescence weakens cell-cell junctions, impairs barrier function, and promotes neutrophil adhesion and migration, which may contribute to the development of inflammation and related pathologies in the lungs during aging.

Klebsiella pneumoniae-derived extracellular vesicles impair endothelial function by inhibiting SIRT1

BackgroundThe potential role of Klebsiella pneumoniae (K.pn) in hypertension development has been emphasized, although the specific mechanisms have not been well understood. Bacterial extracellular vesicles (BEVs) released by Gram-negative bacteria modulate host cell functions by delivering bacterial components to host cells. Endothelial dysfunction is an important early event in the pathogenesis of hypertension, yet the impact of K.pn-secreted EVs (K.pn EVs) on endothelial function remains unclear. This study aimed to investigate the effects of K.pn EVs on endothelial function and to elucidate the underlying mechanisms.MethodsK.pn EVs were purified from the bacterial suspension using ultracentrifugation and characterized by transmission electron microscopy nanoparticle tracking analysis, and EV marker expression. Endothelium-dependent relaxation was measured using a wire myograph after in vivo or ex vivo treatment with K.pn EVs. Superoxide anion production was measured by confocal microscopy and HUVEC senescence was assessed by SA-β-gal activity. SIRT1 overexpression or activator was utilized to investigate the underlying mechanisms.ResultsOur data showed that K.pn significantly impaired acetylcholine-induced endothelium-dependent relaxation and increased superoxide anion production in endothelial cells in vivo. Similarly, in vivo and ex vivo studies showed that K.pn EVs caused significant endothelial dysfunction, endothelial provocation, and increased blood pressure. Further examination revealed that K.pn EVs reduced the levels of SIRT1 and p-eNOS and increased the levels of NOX2, COX-2, ET-1, and p53 in endothelial cells. Notably, overexpression or activation of SIRT1 attenuated the adverse effects and protein changes induced by K.pn EVs on endothelial cells.ConclusionThis study reveals a novel role of K.pn EVs in endothelial dysfunction and dissects the relevant mechanism involved in this process, which will help to establish a comprehensive understanding of K.pn EVs in endothelial dysfunction and hypertension from a new scope.

High glucose induces renal tubular epithelial cell senescence by inhibiting autophagic flux.

Autophagy, a cellular degradation process involving the formation and clearance of autophagosomes, is mediated by autophagic proteins, such as microtubule-associated protein 1 light chain 3 (LC3) and sequestosome 1 (p62), and modulated by 3-methyladenine (3-MA) as well as chloroquine (CQ). Senescence, characterised by permanent cell cycle arrest, is marked by proteins such as cyclin-dependent kinase inhibitor 1 (p21) and tumour protein 53 (p53). This study aims to investigate the relationship between cell senescence and renal function in diabetic kidney disease (DKD) and the effect of autophagy on high-glucose-induced cell senescence. We categorised 46 patients with DKD diagnosed by renal biopsy into classes I, IIa, IIb, III and IV and used four normal kidney specimens from patients with renal trauma as controls. We evaluated pathological changes, LC3 and p21. We used streptozotocin-induced DKD models in rats and 35mM glucose-cultured human proximal tubular epithelial cells (HK-2) with or without 3-MA and CQ. We assessed p53, p21, LC3 and p62. We observed autophagosomes and detected senescence-associated galactosidase (SA-β-gal) activity. In patients with DKD, p21 and LC3 expression levels increased over time and correlated positively with blood creatinine and proteinuria. In DKD rats and HK-2 cells, p21, p53, LC3 and p62 expression levels were higher than in the controls, as were SA-β-gal-positive cells, renal tubular autophagosomes and co-expression of p21 and LC3. The 3-MA reduced p16, p21 and p53 expression compared with the high glucose group, whereas CQ had the opposite effect. These results suggest that renal tubular cell senescence is associated with the progression of DKD. Additionally, autophagic flux may play a role in mediating high-glucose-induced senescence in renal tubular cells.

Ethyl Acetate Extract of Cichorium glandulosum Activates the P21/Nrf2/HO-1 Pathway to Alleviate Oxidative Stress in a Mouse Model of Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is a significant global health concern, primarily resulting from chronic alcohol consumption, with oxidative stress as a key driver. The ethyl acetate extract of Cichorium glandulosum (CGE) exhibits antioxidant and hepatoprotective properties, but its detailed mechanism of action against ALD remains unclear. This study investigates the effects and mechanisms of CGE in alleviating alcohol-induced oxidative stress and liver injury. Ultra-Performance Liquid Chromatography coupled with Quadrupole-Orbitrap Mass Spectrometry (UPLC-Q-Orbitrap-MS) was used to identify CGE components. A C57BL/6J mouse model of ALD was established via daily oral ethanol (56%) for six weeks, with CGE treatment at low (100 mg/kg) and high doses (200 mg/kg). Silibinin (100 mg/kg) served as a positive control. Liver function markers, oxidative stress indicators, and inflammatory markers were assessed. Transcriptomic and network pharmacology analyses identified key genes and pathways, validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. UPLC-Q-Orbitrap-MS identified 81 CGE compounds, mainly including terpenoids, flavonoids, and phenylpropanoids. CGE significantly ameliorated liver injury by reducing alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels and enhancing antioxidative markers such as total antioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD) while lowering hepatic malondialdehyde (MDA) levels. Inflammation was mitigated through reduced levels of Tumor Necrosis Factor Alpha (TNF-α), Interleukin-1 Beta (IL-1β), and C-X-C Motif Chemokine Ligand 10 (CXCL-10). Transcriptomic and network pharmacology analysis revealed seven key antioxidant-related genes, including HMOX1, RSAD2, BCL6, CDKN1A, THBD, SLC2A4, and TGFβ3, validated by RT-qPCR. CGE activated the P21/Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)/Heme Oxygenase-1 (HO-1) signaling axis, increasing P21, Nrf2, and HO-1 protein levels while suppressing Kelch-like ECH-associated Protein 1 (Keap1) expression. CGE mitigates oxidative stress and liver injury by activating the P21/Nrf2/HO-1 pathway and regulating antioxidant genes. Its hepatoprotective effects and multi-target mechanisms highlight CGE's potential as a promising therapeutic candidate for ALD treatment.

MiR-507 Acts as a Tumor Suppressor in Renal Cell Carcinoma Cells by Targeting STEAP3.

In recent years, there has been a rise in the incidence of renal cell carcinoma (RCC), with metastatic RCC being a prevalent and significant contributor to mortality. While a regulatory role for microRNAs (miRNAs) in the development and progression of RCC has been recognized, their precise functions, molecular mechanisms, and potential clinical implications remain inadequately elucidated. Hence, this study aimed to explore the role of miR-507 in RCC and identify STEAP3 as a downstream target of miR-507. Bioinformatics analysis was used to analyze the expression of miR-507 and STEAP3 in RCC specimens. CCK-8, Transwell, and flow cytometry assays were used to assess the function of miR-507 in RCC cells. The connection between miR-507 and STEAP3 was confirmed through a luciferase reporter assay. The expression level of STEAP3, p53, and xCT was analyzed by western blotting. Bioinformatics analysis showed that miR-507 was expressed at low levels in RCC tissues and was linked to poor overall survival. STEAP3 was found to be significantly upregulated in RCC. Further, STEAP3 was shown to be targeted by miR-507. High levels of miR-507 reduced the expression of STEAP3, leading to stagnant cell viability, apoptosis, and migrative capacity. Whereas miR-507 knockdown reverted such a tendency. The study also discovered that miR-507 exerted its inhibitory effect through the op53/xCT pathway. Within RCC, miR-507 modulates the expression of SETAP3/p53/xCT axis, exhibiting a tumor suppressive effect. These discoveries offer present prospective biomarkers for both surveillance and treatment of RCC.

USP38 exacerbates myocardial injury and malignant ventricular arrhythmias after ischemia/reperfusion by promoting ferroptosis through the P53/SLC7A11 pathway

IntroductionMyocardial ischemia–reperfusion (I/R) leads to myocardial injury and malignant ventricular arrhythmias (VAs). Ferroptosis is a novel form of cell death that plays a role in myocardial injury after I/R. Ubiquitin-specific protease 38 (USP38), a member of the deubiquitinating enzyme family, is involved in regulating the progression of inflammation and tumors, but its role in myocardial I/R and ferroptosis is unclear. ObjectivesHerein, we explored whether USP38 regulates myocardial I/R-induced ferroptosis and the development of malignant arrhythmias and underlying mechanisms. MethodsIn this study, cardiac I/R mice model was established by ligating/loosening the left anterior descending artery, and the effects of USP38 on I/R-induced ferroptosis and VAs susceptibility were investigated using USP38 cardiac conditional knockout (USP38-CKO) mice and USP38 cardiac specific overexpression (USP38-TG) mice. In addition, an in vitro I/R model was constructed by hypoxia/reoxygenation for further validation. ResultsOur study showed that USP38 expression was significantly increased after I/R. USP38-CKO significantly inhibited I/R-induced iron overload, ROS production, and lipid peroxidation. In addition, USP38-CKO ameliorates post-I/R electrophysiologic abnormalities and reduces susceptibility to VAs. USP38-TG showed the opposite effect, exacerbating ferroptosis and increasing susceptibility to VAs after I/R. In vivo experiments similarly demonstrated that USP38 significantly exacerbated H/R-induced ferroptosis. Mechanistically, USP38 directly interacts with P53 and regulates the ubiquitination level of P53 and downstream SLC7A11 expression. ConclusionWe found that ferroptosis was significantly associated with VAs after I/R. USP38 can modulate myocardial injury and VAs susceptibility by affecting ferroptosis, which may be related to the P53/SLC7A11 pathway.

Modeling senescence in ARPE19 cells to investigate the role of small extracellular vesicles in age‐related macular degeneration

Aims/Purpose: Age‐related macular degeneration (AMD) is a degenerative disease causing significant vision loss in the elderly. The molecular mechanisms of AMD onset and progression are not fully understood. Senescent cells are known to secrete increased amounts of small extracellular vesicles (sEV). However, the impact of sEVs on RPE dysfunction and AMD through senescence spreading remains unclear. Our study aimed to develop an in vitro model of RPE cell senescence.Methods: We used the retinal pigment epithelial cell line, ARPE‐19. Senescence induction in RPE cells was achieved through two different approaches: repeated passaging (replicative senescence) and ionizing radiation (DNA damage‐induced senescence). Characterization of sEVs released by RPE cells was performed and markers of senescence were assessed.Results: RPE cells subjected to replicative exhaustion or ionizing radiation showed higher H2AX foci accumulation compared to p9 cells. Ionizing radiation significantly increased nucleus area and deformation, with a similar trend at p21 and p31. Levels of p21, critical for establishing senescence, increased at p31 and showed a tendency to rise in ionizing radiation‐exposed cells. Levels of p16, maintaining the senescence phenotype, also increased in ionizing radiation‐exposed cells and tended to rise at p21 and p31. Activities of active MMP‐2 and ‐9 increased in the conditioned medium of senescent cells, with MMP‐2 activity correlating with higher protein levels. sEVs from senescent cell‐conditioned medium showed significant increases in concentration and mean size compared to p9 cells.Conclusions: We successfully induced senescence in ARPE19 cells, creating a valuable model to investigate the role of sEVs in spreading senescence to neighboring cells, especially in the context of AMD onset.Support: FCT, Portugal: 2020.04811.BD; PEst UIDB/04539/Base/2020 and UIDP/04539/Programatico/2020.European Union's Horizon 2020 Teaming project MIA‐Portugal (no 857524) and CHAngeing – Connected Hubs in Ageing: Healthy Living to Protect Cerebrovascular Function (no 101087071), funded by Excellence Hubs Call – Horizon‐ WIDERA‐2022‐ACCESS‐04‐01.

Modeling senescence in ARPE19 cells to investigate the role of small extracellular vesicles in age‐related macular degeneration

Aims/Purpose: Age‐related macular degeneration (AMD) is a degenerative disease causing significant vision loss in the elderly. The molecular mechanisms of AMD onset and progression are not fully understood. Senescent cells are known to secrete increased amounts of small extracellular vesicles (sEV). However, the impact of sEVs on RPE dysfunction and AMD through senescence spreading remains unclear. Our study aimed to develop an in vitro model of RPE cell senescence.Methods: We used the retinal pigment epithelial cell line, ARPE‐19. Senescence induction in RPE cells was achieved through two different approaches: repeated passaging (replicative senescence) and ionizing radiation (DNA damage‐induced senescence). Characterization of sEVs released by RPE cells was performed and markers of senescence were assessed.Results: RPE cells subjected to replicative exhaustion or ionizing radiation showed higher H2AX foci accumulation compared to p9 cells. Ionizing radiation significantly increased nucleus area and deformation, with a similar trend at p21 and p31. Levels of p21, critical for establishing senescence, increased at p31 and showed a tendency to rise in ionizing radiation‐exposed cells. Levels of p16, maintaining the senescence phenotype, also increased in ionizing radiation‐exposed cells and tended to rise at p21 and p31. Activities of active MMP‐2 and ‐9 increased in the conditioned medium of senescent cells, with MMP‐2 activity correlating with higher protein levels. sEVs from senescent cell‐conditioned medium showed significant increases in concentration and mean size compared to p9 cells.Conclusions: We successfully induced senescence in ARPE19 cells, creating a valuable model to investigate the role of sEVs in spreading senescence to neighboring cells, especially in the context of AMD onset.Support: FCT, Portugal: 2020.04811.BD; PEst UIDB/04539/Base/2020 and UIDP/04539/Programatico/2020.European Union's Horizon 2020 Teaming project MIA‐Portugal (no 857524) and CHAngeing – Connected Hubs in Ageing: Healthy Living to Protect Cerebrovascular Function (no 101087071), funded by Excellence Hubs Call – Horizon‐ WIDERA‐2022‐ACCESS‐04‐01.

JAG1/Notch Pathway Inhibition Induces Ferroptosis and Promotes Cataractogenesis.

Cataracts remain the leading cause of visual impairment worldwide, yet the underlying molecular mechanisms, particularly in age-related cataracts (ARCs), are not fully understood. The Notch signaling pathway, known for its critical role in various degenerative diseases, may also contribute to ARC pathogenesis, although its specific involvement is unclear. This study investigates the role of Notch signaling in regulating ferroptosis in lens epithelial cells (LECs) and its impact on ARC progression. RNA sequencing of anterior lens capsule samples from ARC patients revealed a significant downregulation of Notch signaling, coupled with an upregulation of ferroptosis-related genes. Notch1 expression decreased, while ferroptosis markers increased in an age-dependent manner. In vitro, upregulation of Notch signaling alleviated ferroptosis by decreasing ferritin heavy chain 1 (FTH1) and p53 levels while enhancing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione peroxidase 4 (GPX4), and solute carrier family 7 member 11 (SLC7A11). Conversely, inhibition of Notch signaling exacerbated ferroptosis, as evidenced by reduced Nrf2, GPX4, and SLC7A11 expression. These findings suggest that downregulation of Notch signaling promotes ferroptosis in LECs by impairing the Nrf2/GPX4 antioxidant pathway, thereby contributing to ARC development. This study offers new insights into ARC pathogenesis and highlights the Notch signaling pathway as a potential therapeutic target for preventing or mitigating ARC progression.

TOE1 deadenylase inhibits gastric cancer cell proliferation by regulating cell cycle progression.

TOE1, also known as hCaf1z, belongs to the DEDD superfamily of deadenylases and a newly identified isoenzyme of hCaf1 deadenylases. Previous research has demonstrated that TOE1 has deadenylase activity, which can catalyze the degradation of poly(A) substrates and interact with hCcr4d to form the unconventional human Ccr4-Caf1 deadenylase complex. Our recent research indicates that hCaf1a and hCaf1b isoenzymes, highly expressed in gastric cancer, promote gastric cancer cell proliferation and tumorigenicity via modulating cell cycle progression. However, no studies have yet explored the relationship between TOE1 deadenylase and tumor development. In our study, we systematically investigated the functions and mechanisms of TOE1 in gastric cancer progression. Our findings revealed that overexpression of TOE1 inhibited gastric cancer cell proliferation, invasion and migration, promoted cell apoptosis, and led to cell cycle arrest in G0/G1 phase, while TOE1 knockdown had the opposite biological effects on these processes in gastric cancer cells. Further results indicated that TOE1 suppressed gastric cancer progression by inhibiting EMT process and MMPs expression. Moreover, our study clarified that TOE1 blocked gastric cancer cell cycle progression by up-regulating the expression level of the key cell cycle factors p21 and p53 through different regulatory mechanisms. Specifically, TOE1 up-regulated p53 expression by enhancing p53 promoter activity, and up-regulated p21 expression by enhancing p21 mRNA stability. Collectively, our findings first contribute to further elucidating the molecular mechanisms by which TOE1 participates in the regulation of gastric cancer progression, and are expected to provide a theoretical basis for diagnosis and targeted treatment of gastric cancer.