Autophagy-related Genes Research Articles

Contribution of impaired autophagy, mitochondrial dysfunction and abnormal lipolysis to epididymal aging in mice

With the increase of the aged population in modern society, research on aging and aging-related diseases has attracted increasing attention. Unlike women, men experience changes gradually in the reproductive system during aging. The epididymis is an important organ for sperm maturation and storage, but less study has been conducted to investigate cellular senescence in aging epididymis and the corresponding influences on sperm. This study aims to explore cellular and molecular mechanisms underlying aging changes in epididymal tissues. Cellular senescence in the epididymis of 18-month-old C57BL/6 J mice was evaluated with SA (senescence-associated)-β-galactosidase staining and molecular markers such as P21 and Lamin B, compared to the 2-month-old young group. Western blot analysis and immunofluorescence staining were performed to examine the proteins expressions involved in AMPKα/SIRT1 pathway, autophagy/mitophagy, mitochondrial dynamics and lipolysis. The results showed that in old mice AMPKα/ SIRT1 pathway was downregulated with increased acetylation in the epididymal tissues. Reduced expressions of autophagy related genes and PINK1/PARK2 were detected as well as increased P62 protein level and decreased colocalization of LC3 and LAMP2, which indicated deficient autophagy and mitophagy occurred in aging epididymal tissues. Significant decreased expressions of MFN1, MFN2, p-DRP1(Ser637) and FIS1 showed an imbalance in mitochondrial dynamics in aging epididymal tissues. Additionally, intracellular lipid droplets accumulation occurred in epididymal epithelial cells in old mice, with reduced expressions of the lipolysis enzymes ATGL, HSL and Ascl4. Lipophagy impairment was further detected by minimal colocalization of lipid droplets with either LC3 or LAMP2 in the epididymal ductal epithelial cells of old mice. Our study provides new insights into the molecular mechanisms of impaired autophagy, imbalanced mitochondrial dynamics and disrupted lipolysis, which together contribute to senescent changes and may be detrimental to the epididymal function during aging.

Ceramide Ehux-C22 Targets the miR-199a-3p/mTOR Signaling Pathway to Regulate Melanosomal Autophagy in Mouse B16 Cells.

Melanosomes are specialized membrane-bound organelles where melanin is synthesized and stored. The levels of melanin can be effectively reduced by inhibiting melanin synthesis or promoting melanosome degradation via autophagy. Ceramide, a key component in the metabolism of sphingolipids, is crucial for preserving the skin barrier, keeping it hydrated, and warding off the signs of aging. Our preliminary study indicated that a long-chain C22-ceramide compound (Ehux-C22) isolated from the marine microalga Emiliania huxleyi, reduced melanin levels via melanosomal autophagy in B16 cells. Recently, microRNAs (miRNAs) were shown to act as melanogenesis-regulating molecules in melanocytes. However, whether the ceramide Ehux-C22 can induce melanosome autophagy at the post-transcriptional level, and which potential autophagy-dependent mechanisms are involved, remains unknown. Here, miR-199a-3p was screened and identified as a novel upregulated miRNA in Ehux-C22-treated B16 cells. An in vitro high melanin expression model in cultured mouse melanoma cells (B16 cells) was established by using 0.2 μM alpha-melanocyte-stimulating hormone(α-MSH) and used for subsequent analyses. miR-199a-3p overexpression significantly enhanced melanin degradation, as indicated by a reduction in the melanin level and an increase in melanosome autophagy. Further investigation demonstrated that in B16 cells, Ehux-C22 activated miR-199a-3p and inhibited mammalian target of rapamycin(mTOR) level, thus activating the mTOR-ULK1 signaling pathway by promoting the expression of unc-51-like autophagy activating kinase 1 (ULK1), B-cell lymphoma-2 (Bcl-2), Beclin-1, autophagy-related gene 5 (ATG5), and microtubule-associated protein light chain 3 (LC3-II) and degrading p62. Therefore, the roles of Ehux-C22-regulated miR-199a-3p and the mTOR pathway in melanosomal autophagy were elucidated. This research may provide novel perspectives on the post-translational regulation of melanin metabolism, which involves the coordinated control of melanosomes.

Autophagy-related biomarkers in hepatocellular carcinoma and their relationship with immune infiltration

BackgroundAutophagy regulation plays vital roles in many cancers. We aimed to investigate the expression, prognostic value, and immune infiltration of autophagy-related genes in hepatocellular carcinoma (HCC) by bioinformatics analysis.MethodHuman autophagy-related differentially expressed genes (DEGs) between adjacent and HCC tissues were identified. We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. We also evaluated immune infiltration and the response to tumor-sensitive drugs. Finally, we verified the expression of these proteins in clinical samples by immunohistochemistry (IHC), RNA isolation and real-time reverse transcription polymerase chain reaction (RT‒PCR).ResultsA total of 57 autophagy-related DEGs were identified. The HUB genes (BIRC5, CDKN2A, SPP1, and IGF1) were related to the diagnosis and prognosis of HCC. The HUB genes were significantly enriched in immune-related pathways. Furthermore, correlation analysis revealed that HUB gene expression was associated with immune infiltration. We identified 35 tumor-sensitive drugs targeting the HUB genes. Finally, by IHC, we discovered that the protein of CDKN2A, BIRC5, and SPP1 were upregulated in HCC tissues, while IGF1 was downregulated in HCC tissues compared with the levels in paracarcinoma tissues; by RT‒PCR, we discovered that the mRNA of CDKN2A, BIRC5, and SPP1 were upregulated in HCC tissues, while the mRNA of IGF1 was downregulated in HCC tissues compared with the levels in paracarcinoma tissues.ConclusionWe screened and validated four autophagy-related genes associated with immune infiltration and prognosis in patients with HCC.

Dual Guardians of Immunity: FoRab10 and FoRab29 in Frankliniella occidentalis Confer Resistance to Tomato Spotted Wilt Orthotospovirus.

Rab GTPase is critical for autophagy processes and is implicated in insect immunity against viruses. In this study, we aimed to investigate the role of FoRabs in the autophagic regulation of antiviral defense against tomato spotted wilt orthotospovirus (TSWV) in Frankliniella occidentalis. Transcriptome analysis revealed the downregulation of FoRabs in viruliferous nymph and adults of F. occidentalis in response to TSWV infection. Manipulation of autophagy levels with 3-MA and Rapa treatments resulted in a 5- to 15-fold increase and a 38-64% decrease in viral titers, respectively. Additionally, interference with FoRab10 in nymphs and FoRab29 in adults led to a 20-90% downregulation of autophagy-related genes, a decrease in ATG8-II (an autophagy marker protein), and an increase in the TSWV titers by 1.5- to 2.5-fold and 1.3- to 2.0-fold, respectively. In addition, the leaf disk and the living plant methods revealed increased transmission rates of 20.8-41.6 and 68.3-88.3%, respectively. In conclusion, FoRab10 and FoRab29 play a role in the autophagic regulation of the antiviral defense in F. occidentalis nymphs and adults against TSWV, respectively. These findings offer insights into the intricate immune mechanisms functional in F. occidentalis against TSWV, suggesting potential targeted strategies for F. occidentalis and TSWV management.

β-Hydroxybutyrate and melatonin suppress maladaptive UPR, excessive autophagy and pyroptosis in Aβ 1-42 and LPS-Induced SH-SY5Y cells.

Alzheimer's disease is a neurological disease characterized by the build-up of amyloid beta peptide (Aβ) and lipopolysaccharide (LPS), which causes synapse dysfunction, cell death, and neuro-inflammation. A maladaptive unfolded protein response (UPR), excessive autophagy, and pyroptosis aggravate the disease. Melatonin (MEL) and hydroxybutyrate (BHB) have both shown promise in terms of decreasing Aβ pathology. The goal of this study was to see how BHB and MEL affected the UPR, autophagy, and pyroptosis pathways in Aβ1-42 and LPS-induced SH-SY5Y cells. Human neuroblastoma SH-SY5Y cells were treated with BHB, MEL, or a combination of the two after being exposed to A β1-42 and LPS. Cell viability was determined using the MTT test, and gene expression levels of UPR (ATF6, PERK, and CHOP), autophagy (Beclin-1, LC3II, P62, and Atg5), and pyroptosis-related markers (NLRP3, TXNIP, IL-1β, and NFκB1) were determined using quantitative Real-Time PCR (qRT-PCR). For statistical analysis, one-way ANOVA was employed, followed by Tukey's post hoc test. BHB and MEL significantly increased SH-SY5Y cell viability in the presence of A β1-42 and LPS. Both compounds inhibited the expression of maladaptive UPR and autophagy-related genes, as well as inflammatory and pyroptotic markers caused by Aβ1-42 and LPS-induced SH-SY5Y cells. BHB and MEL rescue neurons in A β1-42 and LPS-induced SH-SY5Y cells by reducing maladaptive UPR, excessive autophagy, and pyroptosis. More research is needed to fully comprehend the processes behind their beneficial effects and to discover their practical applications in the treatment of neurodegenerative disorders.

Triclosan impairs spermatocyte cell proliferation and induces autophagy by regulating microRNA-20a-5 P by pargeting PTEN

BackgroundTriclosan (TCS), as an endocrine disrupter, has been found to affect male fertility. However, the potential molecular mechanism is still unknown. We aimed to investigate whether the toxic effects of TCS on spermatocyte cells was mediated by the regulation of microRNA-20a-5 P on PTEN. MethodsGC-2 and TM4 cells were treated with TCS (0.5–80 μM) for 24 or 48 hours. Effect of TCS on proliferation of GC-2 and TM4 cells was detected using a cell counting kit-8 (CCK8) assay. Expression of miR-17 family and autophagy genes were detected. The interaction between miR-20a-5 P and PTEN was determined by a dual-luciferase reporter assay. ResultsTCS decreased cell proliferation of GC-2 and TM4 cells. Expression of autophagy-related genes and miR-17 family was altered by TCS. PTEN expression was significantly increased, whereas the expression of miR-20a-5 P was significantly decreased in GC-2 and TM4 cells. As predicted in relevant databases, there is a binding site of miR-20a-5 P in PTEN. The expression of PTEN was significantly down-regulated by the miR-20a-5 P mimic. ConclusionAs a downstream target of miR-20a-5 P, PTEN functioned in the autophagy process of which TCS inhibited the proliferation of spermatocyte cells. Our results provided new ideas for revealing the molecular mechanism and protective strategy on male infertility.

Investigating the molecular mechanisms of Pseudalteromonas sp. LD-B1's algicidal effects on the harmful alga Heterosigma akashiwo

Heterosigma akashiwo is a harmful algal bloom species that causes significant detrimental effects on marine ecosystems worldwide. The algicidal bacterium Pseudalteromonas sp. LD-B1 has demonstrated potential effectiveness in mitigating these blooms. However, the molecular mechanisms underlying LD-B1’s inhibitory effects on H. akashiwo remain poorly understood. In this study, we employed the comprehensive methodology, including morphological observation, assessment of photosynthetic efficiency (Fv/Fm), and transcriptomic analysis, to investigate the response of H. akashiwo to LD-B1. Exposure to LD-B1 resulted in a rapid decline of H. akashiwo's Fv/Fm ratio, with cells transitioning to a rounded shape within 2 hours, subsequently undergoing structural collapse and cytoplasmic leakage. Transcriptomic data revealed sustained downregulation of photosynthetic genes, indicating impaired functionality of the photosynthetic system. Additionally, genes related to the respiratory electron transfer chain and antioxidant defenses were consistently downregulated, suggesting prolonged oxidative stress beyond the cellular antioxidative capacity. Notably, upregulation of autophagy-related genes was observed, indicating autophagic responses in the algal cells. This study elucidates the molecular basis of LD-B1's algicidal effects on H. akashiwo, advancing our understanding of algicidal mechanisms and contributing to the development of effective strategies for controlling harmful algal blooms.

Assessing DNA methylation of ATG 5 and MAP1LC3Av1 gene in oral squamous cell carcinoma and oral leukoplakia- a cross sectional study

BackgroundThe progression and pathogenesis of oral cancer is greatly impacted by epigenetic modifications, such as DNA methylation. Autophagy, is an adaptive mechanism used to maintain the survival and integrity of cells. Oral squamous cell carcinoma is linked to a number of autophagy indicators, although it is yet unknown if DNA methylation of autophagy-related genes promotes the development of oral leukoplakia (OL), oral squamous cell carcinoma (OSCC). AimOur study was aimed to assess, compare and evaluate the DNA methylation of ATG5 and MAP1LC3Av1 genes in oral leukoplakia, oral squamous cell carcinoma. Materials and methodsThis cross-sectional study was designed with sample size of 48 tissues which was clinically and histopathologically diagnosed as OL, OSCC and normal tissue. The samples were divided into three groups (Group A, Group B, and Group C; (n = 16 each). Following histopathological confirmation, the tissue was stored in the RNA reagent, then subjected to DNA extraction, methylation-sensitive polymerase chain reaction (MS-PCR). DNA methylation of the ATG5 and MAP1LC3Av1 genes were assessed. ResultsShapiro-Wilk and Kolmogorov-Smirnov tests showed that the values were normally distributed. Both the ATG5 and MAP1LC3Av1 genes were methylated in OSCC, OL tissues compared to normal tissues. A statistically significant results was seen among the three study groups. ConclusionA significant difference was noted in the hypermethylation status of the promoter regions of the ATG5 and MAP1LC3Av1 genes. This provides some insight into their crucial role in the development of tumors. Future research with larger sample is needed to assess its potential clinical implications in oral carcinoma.

Transcriptomic analysis reveals novel hub genes associated with astrocyte autophagy in intracerebral hemorrhage.

Neuroinflammation serves as a critical local defense mechanism against secondary brain injury following intracerebral hemorrhage (ICH), and astrocytes play a prominent role in this process. In this study, we investigated astrocytic changes during the inflammatory state after ICH to identify new targets for improving the inflammatory response. We stimulated mouse astrocytes with lipopolysaccharide (LPS) in vitro and analyzed their transcriptomes via ribonucleic acid sequencing. We created an ICH model in living organisms by injecting autologous blood. RNA sequencing revealed that 2,717 genes were differentially expressed in the LPS group compared to those in the saline group, with notable enrichment of the autophagic pathway. By intersecting the 2,717 differentially expressed genes (DEGs) with autophagy-related genes, we identified 36 autophagy-related DEGs and seven hub genes. Previous studies and quantitative reverse transcription-polymerase chain reaction results confirmed the increased expression of phosphatidylinositol 3-kinase catalytic subunit type 3 (Pik3c3), AKT serine/threonine kinase 1 (Akt1), and unc-51 like autophagy activating kinase 2 (Ulk2) in astrocytes after ICH. Transcription factors and target miRNAs were identified for the final three DEGs, and 3-methyladenine and leupeptin were identified as potential therapeutic agents for ICH. Our findings suggest that astrocyte autophagy plays a critical role in ICH complexity, and that Pik3c3, Akt1, and Ulk2 may be potential therapeutic targets.

Spermidine supplementation in honey bees: Autophagy and epigenetic modifications.

Polyamines (PAs), including putrescine (Put), spermidine (Spd), and spermine (Spm), are essential polycations with wide-ranging roles in cellular functions. PA levels decline with age, making exogenous PA supplementation, particularly Spd, an intriguing prospect. Previous research in honey bees demonstrated that millimolar Spd added to their diet increased lifespan and reinforced oxidative resilience. The present study is aimed to assess the anti-aging effects of spermidine supplementation at concentrations of 0.1 and 1 mM in honey bees, focusing on autophagy and associated epigenetic changes. Results showed a more pronounced effect at the lower Spd concentration, primarily in the abdomen. Spd induced site-specific histone 3 hypoacetylation at sites K18 and 27, hyperacetylation at K9, with no change at K14 in the entire body. Additionally, autophagy-related genes (ATG3, 5, 9, 13) and genes associated with epigenetic changes (HDAC1, HDAC3, SIRT1, KAT2A, KAT6B, P300, DNMT1A, DNMT1B) were upregulated in the abdomens of honey bees. In conclusion, our findings highlight profound epigenetic changes and autophagy promotion due to spermidine supplementation, contributing to increased honey bee longevity. Further research is needed to fully understand the precise mechanisms and the interplay between epigenetic alterations and autophagy in honey bees, underscoring the significance of autophagy as a geroprotective mechanism.

Association Between the Expression of Autophagy-Related Genes in Circulating Leukocytes and Aging: An Exploratory Modulating Effect of the Mediterranean Diet

Association Between the Expression of Autophagy-Related Genes in Circulating Leukocytes and Aging: An Exploratory Modulating Effect of the Mediterranean Diet

Bilirubin, once a toxin but now an antioxidant alleviating non-alcoholic fatty liver disease in an autophagy-dependent manner in high-fat diet-induced rats: a molecular and histopathological analysis.

As an endogenous antioxidant, bilirubin has surprisingly been inversely correlated with the risk of non-alcoholic fatty liver disease (NAFLD). Thereupon, the current evaluation was designed to assess the positive effects of bilirubin on the autophagy flux, as well as the other pathogenic processes and parameters involved in the expansion of NAFLD. Thirty adult male rats weighing 150-200 g with free access to sucrose solution (18%) were randomly subdivided into 5 groups (n = 6). Subsequently, the animals were euthanized, and their blood specimens and liver tissue samples were collected to measure serum biochemical indices, liver histopathological changes, intrahepatic triglycerides content, and tissue stereological alterations. Furthermore, the expression levels of autophagy-related genes (Atgs) were measured to assess the state of the autophagy flux. Fasting blood glucose, body weight, as well as liver weight, liver-specific enzyme activity, and serum lipid profile indices markedly decreased in rats that underwent a six-week bilirubin treatment compared to the control group. In addition, histopathological studies showed that hepatic steatosis, fibrosis, inflammation, and necrosis significantly decreased in the groups that received bilirubin compared to the control animals. Bilirubin also caused significant alterations in the expression levels of the Atgs, as well as the Beclin- 1 protein. Bilirubin may have potential ameliorative effects on NAFLD-associated liver damage. Moreover, the beneficial effects of bilirubin on intrahepatic lipid accumulation and steatosis were comparable with the group that did not ever receive bilirubin.

Autophagy, apoptosis, and inflammation response of Sebastiscus marmoratus to different concentrations of hydroxychloroquine

Hydroxychloroquine (HCQ) is an important public health therapeutic agent widely used in the prevention and treatment of malaria and autoimmune diseases, with some antiviral effects, as well as a common autophagy inhibitor. Its autophagy-inhibiting effect attracts great research interest in mammals but is still little studied in fish. We even have no idea about the effects of HCQ on different tissues of fish and what concentrations should be used for safety studies. This study investigated the effect of different concentration of HCQ treatments on the survival, tissue structure, and expressions of genes related to autophagy, apoptosis, and inflammation in the gill, spleen, testis, and ovary of Sebastiscus marmoratus. The results showed that the higher HCQ concentration (77.40 and 154.80 mg/mL) led to mass mortality within an hour. The half-lethal concentration (LC50, 24 h) of HCQ for S. marmoratus was approximately 48.95 mg/mL. When exposed to 30.96 mg/mL HCQ for 24 hours, autophagy was blocked as revealed by electron microscopy from gill, spleen and testis of fish. The expressions of autophagy-related genes (LC3/Cx43), apoptosis-related genes (Cas3/p53), and inflammation-related genes (TNF-α/IL8) exhibited tissue-specific and dose-dependent responses. The gonads showed preferential expression of all these genes and were found to be sensitive and regular after HCQ treatment. For example, at a concentration of 30.96 mg/mL, the testis demonstrated a regularity that suggests it is an ideal candidate tissue for studying the role of HCQ or autophagy. This study systematically revealed the response of S. marmoratus to different concentrations of HCQ and provided optional assay concentrations for key tissues, serving as an important reference for the future studies on HCQ and autophagy in S. marmoratus. Furthermore, the potential crosstalk between autophagy, apoptosis, and inflammatory pathways initially identified in this study could be helpful for the future research on autophagy regulation in marine fish.

Integrative Analysis of Transcriptional Regulation of the ATG4D, an Autophagy-Related Gene, Using JASPAR and STRING databases.

Autophagy is a catabolic cellular process that is conserved and plays an important role in sustaining cellular stability, adjusting to malnourishment, and responding to cellular stress. ATG4D is a gene that involves the formation of the autophagosome and is considered a key player in autophagy. Inclusive knowledge of how the ATG4D gene is regulated may provide insights into the gene's role in different cellular aspects. In this research, bioinformatics tools were utilized, specifically the JASPAR database, to predict the transcription factor binding sites (TFBS) and transcription factors (TFs) binding to the ATG4D promoter region. Multiple TFs were identified, and among these identified TFs, we focused on eight that were well-known for their established roles in regulating autophagy, responding to cellular stress, and promoting transcriptional regulation of ATG4D. These TFs include FOXO3, TFEB, TP53, SP1, STAT3, NF-κB, E2F1, and CREB1. To discover the functional interactions of these TFs, the STRING database was utilized to conduct protein-protein interaction analysis and functional enrichment. This analysis helped us to better comprehend the complicated regulatory network linked to autophagy and the precise role of ATG4D. Functional enrichment analysis revealed significant gene ontology terms and pathways linked with autophagy, cellular stress response, apoptosis, and transcriptional regulation. These findings indicate that the regulation of ATG4D gene expression is shaped by a complicated network of transcription factors that play significant roles in autophagy and sustaining cellular balance. Using a combination of bioinformatics tools such as JASPAR and STRING gave a thorough insight into the regulatory atmosphere of ATG4D, providing opportunities for treating diseases associated with autophagy dysregulation.

Exploring the prognostic analysis of autophagy and tumor microenvironment based on monocyte cells in lung cancer.

A deep understanding of the biological mechanisms of lung cancer offers more precise treatment options for patients. In our study, we integrated data from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) to investigate lung adenocarcinoma. Analyzing 538 lung cancer samples and 31 normal samples, we focused on 3076 autophagy-related genes. Using Seurat, dplyr, tidyverse, and ggplot2, we conducted single-cell data analysis, assessing the quality and performing Principal Component Analysis (PCA) and t-SNE analyses. Differential analysis of TCGA data using the "Limma" package, followed by immune infiltration analysis using the CIBERSORT algorithm, led us to identify seven key genes. These genes underwent further scrutiny through consensus clustering and gene set variation analysis (GSVA). We developed a prognostic model using Lasso Cox regression and multivariable Cox analysis, which was then validated with a nomogram, predicting survival rates for lung adenocarcinoma. The model's accuracy and universality were corroborated by ROC curves. Additionally, we explored the relationship between immune checkpoint genes and immune cell infiltration and identified two key genes, HLA-DQB1 and OLR1. This highlighted their potential as therapeutic targets. Our comprehensive approach sheds light on the molecular landscape of lung adenocarcinoma and offers insights into potential treatment strategies, emphasizing the importance of integrating single-cell and genomic data in cancer research.

ROS-Induced Autophagy of Skeletal Muscle Confers Resistance of Rice Flower Carp (Cyprinus carpio) to Short-Term Fasting.

Starvation is one of the main stresses for fish due to food shortage, the evasion of predators, and intraspecific competition. This research evaluated the impact of brief fasting periods on reactive oxygen species (ROS) levels, antioxidant response, mRNA expression of antioxidants, autophagy-related signaling genes, and autophagosome development in the muscle tissue of rice flower carp. Following a three-day fasting period, the levels of ROS and MDA rose. Additionally, after 3 d of fasting, there was a notable upregulation of NRF2 and significant increases in the levels of GSH and the activities of enzymes such as SOD, CAT, GST, GR, and GPX, while the expression of the autophagy marker gene LC3B did not change (p < 0.05). After 7 d of fasting, the content of the ROS, the activity of SOD and GR, and the GSH content reached the maximum (p < 0.05). Concurrently, there was a significant rise in the quantity of autophagosomes. An RT-qPCR analysis revealed that seven d of starvation significantly elevated the mRNA expression of genes associated with the initiation and expansion of autophagosome membranes, vesicle recycling, and cargo recruitment, including ULK1, BECLIN1, LC3B, ATG3, ATG4B, ATG4C, ATG5, ATG9, and P62. After feeding resumed for 3 d, the mRNA level of BECLIN1, ATG3, ATG4B, ATG4C, ATG5, LC3B, and P62 still remained at a high level. The LC3II protein reached its highest level. All autophagy-related gene expression decreased in the 7-day resumed feeding group. Our data implied that short-term fasting can cause oxidative stress and disrupt the antioxidant system first and then induce autophagy in the muscles of rice flower carp. These findings shed light on how fasting affects muscle homeostasis in fish. ROS-induced autophagy of the skeletal muscle may confer the resistance of rice flower carp to short-term fasting.

Neddylation activated TRIM25 desensitizes triple-negative breast cancer to paclitaxel via TFEB-mediated autophagy

BackgroundPaclitaxel (PTX) treatment resistance is an important factor leading to poor prognosis in triple-negative breast cancer (TNBC), therefore there is an urgent need to identify new target for combination therapy. Neddylation is a post-translational process that introduces a ubiquitin-like protein called neural precursor cell expressed developmentally downregulated protein 8 (NEDD8). Previous studies have found that neddylation is activated in multiple tumors, but its relationship with PTX chemotherapy sensitivity has not been reported.MethodsDifferences in UBC12 and NEDD8 expression levels between PTX-sensitive and PTX-insensitive TNBC tissues were validated using public databases and immunohistochemistry. The in vitro and in vivo functional experiments were used to observe the effect of neddylation inhibition combined with PTX therapy on tumor progression. Co-IP, western blot and PCR assays were used to investigate the molecular mechanisms. Molecular docking was used to simulate the protein binding of UBC12 and TRIM25. Molecular dynamics simulation was used to observe the changes in TRIM25 protein conformation.ResultsWe found that in TNBC that is insensitive to PTX, NEDD8 and NEDD8 conjugating enzyme UBC12 are highly expressed. Treatment with the NEDD8-activating enzyme (NAE) inhibitor mln4924 or knockdown of UBC12 significantly increased the sensitivity of the tumor to PTX, and this increase in sensitivity is related to UBC12-mediated autophagy activation. Mechanistically, UBC12 can transfer NEDD8 to E3 ubiquitin ligase tripartite motif containing 25 (TRIM25) at K117. Molecular dynamics simulations indicate that the neddylation modification of TRIM25 reduces steric hindrance in its RING domain, facilitating the binding of TRIM25 and ubiquitylated substrates. Subsequently, TRIM25 promotes the nuclear translocation of transcription factor EB (TFEB) and transcription of autophagy related genes by increasing K63-polyubiquitination of TFEB, thereby reducing tumor sensitivity to PTX.ConclusionsNeddylation is activated in PTX-insensitive TNBC. Specifically, autophagy gene transcriptional activation mediated by the UBC12/TRIM25/TFEB axis reduces TNBC sensitivity to PTX. Neddylation suppression combination with PTX treatment shows a synergistic anti-tumor effect.

Dramatic Suppression of Lipogenesis and No Increase in Beta-Oxidation Gene Expression Are among the Key Effects of Bergamot Flavonoids in Fatty Liver Disease.

Bergamot flavonoids have been shown to prevent metabolic syndrome, non-alcoholic fatty liver disease (NAFLD) and stimulate autophagy in animal models and patients. To investigate further the mechanism of polyphenol-dependent effects, we performed a RT2-PCR array analysis on 168 metabolism, transport and autophagy-related genes expressed in rat livers exposed for 14 weeks to different diets: standard, cafeteria (CAF) and CAF diet supplemented with 50 mg/kg of bergamot polyphenol fraction (BPF). CAF diet caused a strong upregulation of gluconeogenesis pathway (Gck, Pck2) and a moderate (>1.7 fold) induction of genes regulating lipogenesis (Srebf1, Pparg, Xbp1), lipid and cholesterol transport or lipolysis (Fabp3, Apoa1, Lpl) and inflammation (Il6, Il10, Tnf). However, only one β-oxidation gene (Cpt1a) and a few autophagy genes were differentially expressed in CAF rats compared to controls. While most of these transcripts were significantly modulated by BPF, we observed a particularly potent effect on lipogenesis genes, like Acly, Acaca and Fasn, which were suppressed far below the mRNA levels of control livers as confirmed by alternative primers-based RT2-PCR analysis and western blotting. These effects were accompanied by downregulation of pro-inflammatory cytokines (Il6, Tnfa, and Il10) and diabetes-related genes. Few autophagy (Map1Lc3a, Dapk) and no β-oxidation gene expression changes were observed compared to CAF group. In conclusion, chronic BPF supplementation efficiently prevents NAFLD by modulating hepatic energy metabolism and inflammation gene expression programs, with no effect on β-oxidation, but profound suppression of de novo lipogenesis.

Exploring the therapeutic potential of Nelumbo nucifera leaf extract against amyloid-beta-induced toxicity in the Caenorhabditis elegans model of Alzheimer's disease.

Introduction: Alzheimer's disease (AD) represents a critical global health challenge with limited therapeutic options, prompting the exploration of alternative strategies. A key pathology in AD involves amyloid beta (Aβ) aggregation, and targeting both Aβ aggregation and oxidative stress is crucial for effective intervention. Natural compounds from medicinal and food sources have emerged as potential preventive and therapeutic agents, with Nelumbo nucifera leaf extract (NLE) showing promising properties. Methods: In this study, we utilized transgenic Caenorhabditis elegans (C. elegans) models to investigate the potential of NLE in countering AD and to elucidate the underlying mechanisms. Various assays were employed to assess paralysis rates, food-searching capabilities, Aβ aggregate accumulation, oxidative stress, lifespan under stress conditions, and the expression of stress-resistance-related proteins. Additionally, autophagy induction was evaluated by measuring P62 levels and the formation of LGG-1+ structures, with RNAi-mediated inhibition of autophagy-related genes to confirm the mechanisms involved. Results: The results demonstrated that NLE significantly reduced paralysis rates in CL4176 and CL2006 worms while enhancing food-searching capabilities in CL2355 worms. NLE also attenuated Aβ aggregate accumulation and mitigated Aβ-induced oxidative stress in C. elegans. Furthermore, NLE extended the lifespan of worms under oxidative and thermal stress conditions, while concurrently increasing the expression of stress-resistance-related proteins, including SOD-3, GST-4, HSP-4, and HSP-6. Moreover, NLE induced autophagy in C. elegans, as evidenced by reduced P62 levels in BC12921 worms and the formation of LGG-1+ structures in DA2123 worms. The RNAi-mediated inhibition of autophagy-related genes, such as bec-1 and vps-34, negated the protective effects of NLE against Aβ-induced paralysis and aggregate accumulation. Discussion: These findings suggest that NLE ameliorates Aβ-induced toxicity by activating autophagy in C. elegans. The study underscores the potential of NLE as a promising candidate for further investigation in AD management, offering multifaceted approaches to mitigate AD-related pathology and stress-related challenges.

Silybin protected from avermectin-induced carp (Cyprinus carpio) nephrotoxicity by regulating PPAR-γ-involved inflammation, oxidative stress, ferroptosis and autophagy

Avermectin, a widely used deworming drug, poses a significant threat to fisheries. Silybin is recognized for its antioxidant and anti-inflammatory properties. The kidney, being crucial for fish survival, plays a vital role in maintaining ion balance, nitrogen metabolism, and hormone regulation. While residual avermectin in water could pose a risk to carp (Cyprinus carpio), it remains unclear whether silybin can alleviate the renal tissue toxicity induced by avermectin in this species. In current study, we developed a model of long-term exposure of carp to avermectin to investigate the potential protective effect of silybin against avermectin-induced nephrotoxicity. The results indicated that avermectin induced renal inflammation, oxidative stress, ferroptosis, and autophagy in carp. Silybin suppressed the mRNA transcript levels of pro-inflammatory factors, increased catalase (CAT) activity, reduced glutathione (GSH) activity, diminished reactive oxygen species (ROS) accumulation in renal tissues, and promoted the activation of the Nrf2-Keap1 signaling pathway. Furthermore, the transcript levels of ferroptosis-associated proteins, including gpx4 and slc7a11, were significantly reduced, while those of cox2, ftl, and ncoa4 were elevated. The transcript levels of autophagy-related genes, including p62 and atg5, were also regulated. Network pharmacological analysis revealed that silybin inhibited ROS accumulation and mitigated avermectin-induced renal inflammation, oxidative stress, ferroptosis, and autophagy in carp through the involvement of PPAR-γ. Silybin exerted its anti-inflammatory effect through the NF-κB pathway and antioxidant effect through the Nrf2-Keap1 pathway, induced renal cell iron efflux through the SLC7A11/GSH/GPX4, and suppressed autophagy initiation via the PI3K/AKT pathway. This study provides evidence of the protective effect of silybin against avermectin-induced nephrotoxicity in carp, highlighting its potential as a therapeutic agent to alleviate the adverse effects of avermectin exposure in fish.