Expression Of Autophagy-related Genes Research Articles

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.

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-5P 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-5P on PTEN. MethodsGC-2 and TM4 cells were treated with TCS (0.5-80μM) for 24 or 48hours. 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-5P 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-5P was significantly decreased in GC-2 and TM4 cells. As predicted in relevant databases, there is a binding site of miR-20a-5P in PTEN. The expression of PTEN was significantly down-regulated by the miR-20a-5P mimic. ConclusionAs a downstream target of miR-20a-5P, 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.

Autophagy-related molecular clusters identified as indicators for distinguishing active and latent TB infection in pediatric patients

BackgroundAutophagy is crucial for controlling the manifestation of tuberculosis. This study intends to discover autophagy-related molecular clusters as biomarkers for discriminating between latent tuberculosis (LTBI) and active tuberculosis (ATB) in children through gene expression profile analysis. MethodsThe expression of autophagy modulators was examined in pediatric patients with LTBI and ATB utilizing public datasets from the Gene Expression Omnibus (GEO) collection (GSE39939 and GSE39940).ResultsIn a training dataset (GSE39939), patients with LTBI and ATB exhibited the expression of autophagy-related genes connected with their active immune responses. Two molecular clusters associated with autophagy were identified. Compared to Cluster 1, Cluster 2 was distinguished through decreased adaptive cellular immune response and enhanced inflammatory activation, according to single-sample gene set enrichment analysis (ssGSEA). Per the study of gene set variation, Cluster 2’s differentially expressed genes (DEGs) played a role in synthesizing transfer RNA, DNA repair and recombination, and primary immunodeficiency. The peak variation efficiency, root mean square error, and area under the curve (AUC) (AUC = 0.950) were all lowered in random forest models. Finally, a seven-gene-dependent random forest profile was created utilizing the CD247, MAN1C1, FAM84B, HSZFP36, SLC16A10, DTX3, and SIRT4 genes, which performed well against the validation dataset GSE139940 (AUC = 0.888). The nomogram calibration and decision curves performed well in identifying ATB from LTBI.ConclusionsIn summary, according to the present investigation, autophagy and the immunopathology of TB might be correlated. Furthermore, this investigation established a compelling prediction expression profile for measuring autophagy subtype development risks, which might be employed as possible biomarkers in children to differentiate ATB from LTBI.

Dynamic changes in autophagy activity in different degrees of pulmonary fibrosis in mice.

The aim of this study is to observe the changes in autophagy activities in lung tissues of mice with different degrees of pulmonary fibrosis (PF), and explore the association between PF and autophagy activity. The PF model was established by bleomycin (BLM, 25 and 35 mg/kg) atomization inhalation in C57BL/6 mice, samples were collected on the 7, 14, and 28 days after BLM administration. Hematoxylin-eosin staining was used to observe the pathological changes in lung tissues. Masson staining was utilized to assess areas of blue collagen fiber deposition in lung tissues. Quantitative real time polymerase chain reaction was used to detect the mRNA expressions of autophagy-related genes, including Atg5, Atg7, and Atg10 in lung tissues. Western blot was used to detect the protein expressions of autophagy-related genes, including p62 and LC3II/LC3I in lung tissues. Compared with control group, BLM dose-dependently decreased PaO2, mRNA expressions of Atg5, Atg7, Atg10, and LC3II/LC3I, while increased lung wet weight, lung coefficient, PF score, the blue area of collagen fibers, and p62 protein on the 7th, 14th, and 28th days. In conclusion, the more severe the PF induced by BLM, the lower the autophagy activity.

Hydatid fluid from Echinococcus granulosus induces autophagy in dendritic cells and promotes polyfunctional T-cell responses.

Parasites possess remarkable abilities to evade and manipulate the immune response of their hosts. Echinococcus granulosus is a parasitic tapeworm that causes cystic echinococcosis in animals and humans. The hydatid fluid released by the parasite is known to contain various immunomodulatory components that manipulate host´s defense mechanism. In this study, we focused on understanding the effect of hydatid fluid on dendritic cells and its impact on autophagy induction and subsequent T cell responses. Initially, we observed a marked downregulation of two C-type lectin receptors in the cell membrane, CLEC9A and CD205 and an increase in lysosomal activity, suggesting an active cellular response to hydatid fluid. Subsequently, we visualized ultrastructural changes in stimulated dendritic cells, revealing the presence of macroautophagy, characterized by the formation of autophagosomes, phagophores, and phagolysosomes in the cell cytoplasm. To further elucidate the underlying molecular mechanisms involved in hydatid fluid-induced autophagy, we analyzed the expression of autophagy-related genes in stimulated dendritic cells. Our results demonstrated a significant upregulation of beclin-1, atg16l1 and atg12, indicating the induction of autophagy machinery in response to hydatid fluid exposure. Additionally, using confocal microscopy, we observed an accumulation of LC3 in dendritic cell autophagosomes, confirming the activation of this catabolic pathway associated with antigen presentation. Finally, to evaluate the functional consequences of hydatid fluid-induced autophagy in DCs, we evaluated cytokine transcription in the splenocytes. Remarkably, a robust polyfunctional T cell response, with inhibition of Th2 profile, is characterized by an increase in the expression of il-6, il-10, il-12, tnf-α, ifn-γ and tgf-β genes. These findings suggest that hydatid fluid-induced autophagy in dendritic cells plays a crucial role in shaping the subsequent T cell responses, which is important for a better understanding of host-parasite interactions in cystic echinococcosis.

Exploring the Therapeutic Potential of β-Hydroxybutyrate (BHB) in Clear Cell Renal Cell Carcinoma: A Journey into Fat Browning, Autophagy, and Tumor Slimming

This study delves into the therapeutic potential of β-hydroxybutyrate (BHB) in clear cell renal cell carcinoma (ccRCC), a cancer known for its complex pathogenesis and resistance to conventional treatments. The research specifically explores the impact of BHB on cell viability, autophagy induction, and lipid metabolism in Caki-1 cells. The findings reveal that BHB significantly reduces ccRCC cell viability, particularly under low-glucose conditions. The combination of glucose and BHB treatment activates autophagy pathways, as evidenced by increased expression of autophagy-related genes (Beclin-1, LC3IIβ, and ATG5) and decreased expression of P62 after 48 and 72 hours. Moreover, the combined therapy enhances lipid metabolism, as indicated by elevated expression of PGC-1α and UCP-1, along with upregulation of ACSL3 and CPT1A, which are associated with lipid droplet formation and facilitate lipid breakdown within cells. The study concludes that BHB holds promise as a therapeutic agent for ccRCC, targeting abnormal lipid metabolism, inducing autophagy-mediated cell death, and promoting fat browning. The results suggest potential avenues for precision-guided nutritional therapies in ccRCC treatment, highlighting the innovative role of BHB in addressing the challenges posed by this cancer.

Autophagy receptor ZmNBR1 promotes the autophagic degradation of ZmBRI1a and enhances drought tolerance in maize.

Drought stress is a crucial environmental factor that limits plant growth, development, and productivity. Autophagy of misfolded proteins can help alleviate the damage caused in plants experiencing drought. However, the mechanism of autophagy-mediated drought tolerance in plants remains largely unknown. Here, we cloned the gene for a maize (Zea mays) selective autophagy receptor, NEXT TO BRCA1 GENE 1 (ZmNBR1), and identified its role in the response to drought stress. We observed that drought stress increased the accumulation of autophagosomes. RNA sequencing and reverse transcription-quantitative polymerase chain reaction showed that ZmNBR1 is markedly induced by drought stress. ZmNBR1 overexpression enhanced drought tolerance, while its knockdown reduced drought tolerance in maize. Our results established that ZmNBR1 mediates the increase in autophagosomes and autophagic activity under drought stress. ZmNBR1 also affects the expression of genes related to autophagy under drought stress. Moreover, we determined that BRASSINOSTEROID INSENSITIVE 1A (ZmBRI1a), a brassinosteroid receptor of the BRI1-like family, interacts with ZmNBR1. Phenotype analysis showed that ZmBRI1a negatively regulates drought tolerance in maize, and genetic analysis indicated that ZmNBR1 acts upstream of ZmBRI1a in regulating drought tolerance. Furthermore, ZmNBR1 facilitates the autophagic degradation of ZmBRI1a under drought stress. Taken together, our results reveal that ZmNBR1 regulates the expression of autophagy-related genes, thereby increasing autophagic activity and promoting the autophagic degradation of ZmBRI1a under drought stress, thus enhancing drought tolerance in maize. These findings provide new insights into the autophagy degradation of brassinosteroid signaling components by the autophagy receptor NBR1 under drought stress.

Investigating the Effects of Perampanel on Autophagy-mediated Regulation of GluA2 and PSD95 in Epilepsy.

Epilepsy is a chronic neurological disorder characterized by recurrent seizures. Despite various treatment approaches, a significant number of patients continue to experience uncontrolled seizures, leading to refractory epilepsy. The emergence of novel anti-epileptic drugs, such as perampanel (PER), has provided promising options for effective epilepsy treatment. However, the specific mechanisms underlying the therapeutic effects of PER remain unclear. This study aimed to investigate the intrinsic molecular regulatory mechanisms involved in the downregulation of GluA2, a key subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, following epileptic seizures. Primary mouse hippocampal neurons were cultured and subjected to an epilepsy cell model. The expression levels of GluA2 and autophagy-related proteins were assessed using Western blotting and real-time fluorescent quantitative PCR. Immunofluorescence and immunohistochemistry techniques were employed to investigate the nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1). Additionally, status epilepticus animal models were established to further validate the findings. The epilepsy cell model exhibited a significant decrease in GluA2 expression, accompanied by elevated levels of autophagy-related proteins. Immunofluorescence analysis revealed the nuclear translocation of CRTC1, which correlated with the expression of autophagy-related genes. Treatment with an autophagy inhibitor reversed the decreased expression of GluA2 in the epilepsy cell model. Furthermore, the calcium/calmodulin-dependent protein phosphatase inhibitor FK506 and CaN overexpression affected the dephosphorylation and nuclear translocation of CRTC1, consequently influencing GluA2 expression. Animal model results further supported the involvement of these molecular mechanisms in epilepsy. Our findings suggest that the downregulation of GluA2 following epileptic seizures involves the activation of autophagy and the regulation of CRTC1 nuclear translocation. These intrinsic molecular regulatory mechanisms provide potential targets for developing novel therapeutic strategies to alleviate refractory epilepsy and preserve cognitive functions in patients.

Quercetin attenuates lipopolysaccharide-induced hepatic inflammation by modulating autophagy and necroptosis

Lipopolysaccharide (LPS) from Gram-negative bacteria initially induces liver inflammation with proinflammatory cytokines expressions. However, the underlying hepatoprotective mechanism of quercetin on LPS-induced hepatic inflammation remains unclear. Specific pathogen-free chicken embryos (n = 120) were allocated control vehicle, PBS with or without ethanol vehicle, LPS (125 ng/egg) with or without quercetin treatment (10, 20, or 40 nmol/egg, respectively), quercetin groups (10, 20, or 40 nmol/egg). Fifteen-day-old embryonated eggs were inoculated abovementioned solutions via the allantoic cavity. At embryonic d 19, the livers of the embryos were collected for histopathological examination, RNA extraction, real-time polymerase chain reaction, and immunohistochemistry investigation. We found that the liver presented inflammatory response (heterophils infiltration) after LPS induction. The LPS-induced mRNA expressions of inflammation-related factors (TLR4, TNFα, IL-1β, IL-10, IL-6, MYD88, NF-κB1, p38, and MMP3) were upregulated after LPS induction when compared with the PBS group, while quercetin could downregulate these expressions as compared with the LPS group. Quercetin significantly decreased the immunopositivity to TLR4 and MMP3 in the treatment group when compared with the LPS group. Quercetin could significantly downregulate the mRNA expressions of autophagy-related genes (ATG5, ATG7, Beclin-1, LC3A, and LC3B) and necroptosis-related genes (Fas, Bcl-2, Drp1, and RIPK1) after LPS induction. Quercetin significantly decreased the immunopositivity to LC3 in the treatment group when compared with the LPS group; meanwhile, quercetin significantly decreased the protein expressions of LC3-I, LC3-II, and the rate of LC3-II/LC3-I. In conclusions, quercetin can alleviate hepatic inflammation induced by LPS through modulating autophagy and necroptosis.

Research progress in the regulation of autophagy and mitochondrial homeostasis by AMPK signaling channels

AMP-activated protein kinase (AMPK) is a widely distributed and evolutionarily conserved serine/threonine protein kinase present in eukaryotic cells. In regulating cellular energy metabolism, AMPK plays an extremely important role as an energy metabolic kinase. When the body is in a low energy state, AMPK is activated in response to changes in intracellular adenine nucleotide levels and is bound to adenosine monophosphate (AMP) or adenosine diphosphate (ADP). Activated AMPK regulates various metabolic processes, including lipid and glucose metabolism and cellular autophagy. AMPK directly promotes autophagy by phosphorylating autophagy-related proteins in the mammalian target of rapamycin complex 1 (mTORC1), serine/threonine protein kinase-dysregulated 51-like kinase 1 (ULK1) and type III phosphatidylinositol 3-kinase-vacuolar protein-sorting 34 (PIK3C3-VPS34) complexes. AMPK also indirectly promotes autophagy by regulating the expression of downstream autophagy-related genes of transcription factors such as forkhead box O3 (FOXO3), lysosomal function transcription factor EB (TFEB) and bromodomain protein 4 (BRD4). AMPK also regulates mitochondrial autophagy, induces the division of damaged mitochondria and promotes the transfer of the autophagic response to damaged mitochondria. Another function of AMPK is to regulate mitochondrial health by stimulating mitochondrial biogenesis and participating in various aspects of mitochondrial homeostasis regulation. This review discusses the specific regulation of mitochondrial biology and internal environmental homeostasis by AMPK signaling channels as central to the cellular response to energy stress and regulation of mitochondria, highlighting the key role of AMPK in regulating cellular autophagy and mitochondrial autophagy, as well as advances in research on the regulation of mitochondrial homeostasis.

ALKBH5 suppresses autophagic flux via N6-methyladenosine demethylation of ZKSCAN3 mRNA in acute pancreatitis.

Increasing evidence has demonstrated that N6-methyladenosine (m6A) RNA modification plays an essential role in a wide range of pathological conditions. Impaired autophagy is a critical hallmark of acute pancreatitis (AP). To explore the role of the m6A modification of ZKSCAN3 in the regulation of autophagy in AP. The AP mouse cell model was established by cerulein-treated mouse pancreatic acinar cells (MPC-83), and the results were confirmed by the levels of amylase and inflammatory factors. Autophagy activity was evaluated by specific identification of the autophagy-related microstructure and the expression of autophagy-related genes. ZKSCAN3 and ALKBH5 were knocked down to study the function in AP. A m6A RNA binding protein immunoprecipitation assay was used to study how the m6A modification of ZKSCAN3 mRNA is regulated by ALKBH. The increased expression of amylase and inflammatory factors in the supernatant and the accumulation of autophagic vacuoles verified that the AP mouse cell model was established. The downregulation of LAMP2 and upregulation of LC3-II/I and SQSTM1 demonstrated that autophagy was impaired in AP. The expression of ZKSCAN3 was upregulated in AP. Inhibition of ZKSCAN3 increased the expression of LAMP2 and decreased the expression of the inflammatory factors, LC3-II/I and SQSTM1. Furthermore, ALKBH5 was upregulated in AP. Knockdown of ALKBH5 downregulated ZKSCAN3 expression and restored decreased autophagic flux in AP. Notably, the bioinformatic analysis revealed 23 potential m6A modification sites on ZKSCAN3 mRNA. The m6A modification of ZKSCAN3 mRNA was significantly decreased in AP. Knockdown of ALKBH5 increased the modification of ZKSCAN3 mRNA, which confirmed that ALKBH5 upregulated ZKSCAN3 expression in a m6A-dependent manner. ALKBH5 inhibits autophagic flux through m6A demethylation of ZKSCAN3 mRNA in AP, thereby aggravating the severity of the disease.

Phloretic acid requires the insulin/IGF-1 pathway and autophagy to enhance stress resistance and extend the lifespan of Caenorhabditis elegans.

Objective: In humans, aging is associated with increased susceptibility to most age-related diseases. Phloretic acid (PA), a naturally occurring compound found in Ginkgo biloba and Asparagus, exhibits has potential as an anti-aging agent and possesses antioxidant, anti-inflammatory, and immunomodulatory properties. This study aimed to investigate the effects of PA on longevity and stress resistance in Caenorhabditis elegans (C.elegans) and the mechanisms that underlie its effects. Methods: First, we examined the effects of PA on lifespan and healthspan assay, stress resistance and oxidative analysis, lipofuscin levels. Second, we examined the insulin/insulin-like pathway, mitochondria, autophagy-related proteins, and gene expression to explain the possible mechanism of PA prolonging lifespan. Results: Our findings demonstrated that PA dose-dependently extended the C.elegans lifespan, with 200μMPA showing the greatest effect and increased the C.elegans lifespan by approximately 16.7%. PA enhanced motility and the pharyngeal pumping rate in senescent C.elegans while reducing the accumulation of aging pigments. Further investigations revealed that daf-16, skn-1, and hsf-1 were required for mediating the lifespan extension effect of PA in C.elegans since its impact was suppressed in mutant strains lacking these genes. This suggests that PA activates these genes, leading to the upregulation of downstream genes involved in stress response and senescence regulation pathways. Furthermore, PA did not extend the lifespan of the RNAi atg-18 and RNAi bec-1 but it attenuated SQST-1 accumulation, augmented autophagosome expression, upregulated autophagy-related gene expression, and downregulated S6K protein levels. These findings suggest that the potential life-extending effect of PA also involves the modulation of the autophagy pathway. Conclusion: These findings results highlight the promising anti-aging effects of PA and warrant further investigation into its pharmacological mechanism and medicinal development prospects.

Lipopeptide C17 Fengycin B Exhibits a Novel Antifungal Mechanism by Triggering Metacaspase-Dependent Apoptosis in Fusarium oxysporum.

Fusarium wilt is a worldwide soil-borne fungal disease caused by Fusarium oxysporum that causes serious damage to agricultural products. Therefore, preventing and treating fusarium wilt is of great significance. In this study, we purified ten single lipopeptide fengycin components from Bacillus subtilis FAJT-4 and found that C17 fengycin B inhibited the growth of F. oxysporum FJAT-31362. We observed early apoptosis hallmarks, including reactive oxygen species accumulation, mitochondrial dysfunction, and phosphatidylserine externalization in C17 fengycin B-treated F. oxysporum cells. Further data showed that C17 fengycin B induces cell apoptosis in a metacaspase-dependent manner. Importantly, we found that the expression of autophagy-related genes in the TOR signaling pathway was significantly upregulated; simultaneously, the accumulation of acidic autophagy vacuoles in F. oxysporum cell indicated that the autophagy pathway was activated during apoptosis induced by C17 fengycin B. Therefore, this study provides new insights into the antifungal mechanism of fengycin.

Exploring the combined anti-cancer effects of sodium butyrate and celastrol in glioblastoma cell lines: a novel therapeutic approach

Glioblastoma, a highly aggressive and lethal brain cancer, lacks effective treatment options and has a poor prognosis. In our study, we explored the potential anti-cancer effects of sodium butyrate (SB) and celastrol (CEL) in two glioblastoma cell lines. SB, a histone deacetylase inhibitor, and CEL, derived from the tripterygium wilfordii plant, act as mTOR and proteasome inhibitors. Both can cross the blood–brain barrier, and they exhibit chemo- and radiosensitive properties in various cancer models. GB cell lines LN-405 and T98G were treated with SB and CEL. Cell viability was assessed by MTT assay and IC50 values were obtained. Gene expression of DNA repair, apoptosis, and autophagy-related genes was analyzed by RT-PCR. Cell cycle distribution was determined using flow cytometry. Viability assays using MTT assay revealed IC50 values of 26 mM and 22.7 mM for SB and 6.77 μM, and 9.11 μM for CEL in LN-405 and T98G cells, respectively. Furthermore, we examined the expression levels of DNA repair genes (MGMT, MLH-1, MSH-2, MSH-6), apoptosis genes (caspase-3, caspase-8, caspase-9), and an autophagy gene (ATG-6) using real-time polymerase chain reaction. Additionally, flow cytometry analysis revealed alterations in cell cycle distribution following treatment with SB, CEL and their combination. These findings indicate that SB and CEL may act through multiple mechanisms, including DNA repair inhibition, apoptosis induction, and autophagy modulation, to exert their anti-cancer effects in glioblastoma cells. This is the first study providing novel insights into the potential therapeutic effects of SB and CEL in glioblastoma.

Guasha improves knee osteoarthritis by inhibiting chondrocyte apoptosis and regulating expression of autophagy-related genes and proteins in rats.

To observe the effect of Guasha on inflammation factors, apoptosis and autophagy in the cartilage tissue of knee joint in rats with knee osteoarthritis (KOA), so as to explore its mechanisms underlying improvement of KOA. A total of 51 male SD rats were randomized into three groups：blank control, KOA model and Guasha (n= 17 in each group) . The rats in the blank control group received intra-articular injection of 0.9% NaCl solution in the right knee joint. The KOA model was established by intraarticular injection of glutamate sodium iodoacetic acid in the right knee joint. For rats of the Guasha group, Guasha (at a frequency of 1 time/s, and an applied pressure of 0.3-0.5 kgf) was applied to "Yanglingquan" (GB34) and "Xuehai"(SP10) areas of the right leg, once every other day, for 7 consecutive sessions. The circumference of the right knee was measured, The histopathological changes of right knee cartilage were observed after H.E. staining. The contents of inflammatory factors interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the right knee articular cartilage tissue were assayed using ELISA. The expression levels of autophagy-related key molecule Beclin-1 (homologous series of yeast Atg6), light chain protease complication 3 type II/I (LC3II/LC3 I), ubiquitin binding factor 62 (P62) and cysteine aspartate protease-3 (Caspase-3) mRNAs and proteins of the right knee articular cartilage tissue were measured using real-time fluorescent quantitative PCR and Western blot, separately. The apoptosis of chondrocytes was assayed using TUNEL staining, and the immunoactivity of LC3 determined using immunofluorescence staining. After modeling, the right knee circumfe-rence of the model and Guasha groups was significantly increased compared with the blank control group (P<0.01), and after the intervention, the knee circumference of the Guasha group was markedly decreased in comparison with that of the model group (P<0.05). Results of H.E. staining showed obvious degeneration and defects in the cartilage tissue, necrosis of a large number of chondrocytes, fibrous hyperplasia, accompanied by inflammatory cell infiltration, osteoclast increase, fibroplasia and bone trabecular destruction in the model group, which was relatively milder in the Guasha group. Compared with the blank control group, the expression of Beclin-1 and LC3 mRNAs and proteins, and LC immunofluorescence intensity in the right knee articular cartilage tissue were significantly down-regulated (P<0.01, P<0.001), whereas the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, contents of IL-1β and TNF-α in the right knee articular cartilage tissue considerably increased (P<0.01, P<0.001) in the model group. In contrast to the model group, the Guasha group had an apparent increase in the expression levels of Beclin-1 and LC3 mRNAs and proteins and LC immunofluorescence intensity in the right knee articular cartilage tissue (P<0.05), and a pronounced decrease in the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, and contents of IL-1β and TNF-α in the right knee articular cartilage tissue (P<0.05, P<0.01). Guasha stimulation of GB34 and SP10 can improve joint cartilage damage in KOA rats, which may be associated with its functions in inhibiting the excessive release of inflammatory factors and apoptosis, possibly by down-regulating the expression of P62 and Caspase-3 mRNAs and proteins and up-regulating the expression of Beclin-1 and LC3 mRNAs and proteins, and by promoting autophagy of chondrocytes.

Abstract 4775: Unveiling caveolin-1's crucial role in breast cancer progression and metastasis: A molecular perspective

Abstract Background: Breast cancer, particularly its metastatic form, constitutes a significant portion of oncological mortality. Caveolin-1 (Cav-1), a caveolae structural protein, has been implicated in cellular processes such as proliferation, apoptosis, autophagy, and cellular motility (aberrant in breast cancer). With its dual role as a tumor suppressor and promoter, Cav-1's function in breast cancer pathophysiology requires elucidation for potential therapeutic targeting. Methods: We utilized a syngeneic mouse model to engineer Cav-1 deficient 4T1 murine mammary carcinoma cells which mirror the characteristics of stage IV breast cancer to investigate Cav-1's role in tumor growth and metastasis. We assessed cellular proliferation and lung metastasis, compared with wild-type controls, and performed molecular analyses to evaluate epithelial and mesenchymal marker expression, stemness-related markers, and autophagy-related gene expression. Results: Cav-1 ablation corresponded with a marked decrease in tumor proliferation and lung metastasis, indicating a suppressive role in tumor progression. Molecular characterization revealed downregulated epithelial markers (E-cadherin, β-catenin), transcription factors (TWIST1/2), and stem cell markers (NANOG, OCT4, SOX2, c-MYC, SALL4), with concurrent upregulation of the mesenchymal marker vimentin. Enhanced expression of autophagy-related genes (Beclin-1, ATG5, ATG16) was also observed, suggesting a disruption in autophagic pathways. Conclusion: Our findings underscore the critical role of Cav-1 in breast cancer progression, specifically its function in suppressing metastasis and modulating cellular phenotypes. Targeting Cav-1 could represent a novel therapeutic strategy to mitigate breast cancer growth and dissemination. Further research is warranted to explore the therapeutic potential of modulating Cav-1 in breast cancer treatment. Citation Format: Rizwana Begum, Shilpa Thota, Dhirendra Pratap Singh, Naveen Chintala, Abhishek Pandit, Biplov Sapkota, Joseph Francis. Unveiling caveolin-1's crucial role in breast cancer progression and metastasis: A molecular perspective [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4775.

Overexpression of TRPV1 activates autophagy in human lens epithelial cells under hyperosmotic stress through Ca2+-dependent AMPK/mTOR pathway.

To explore whether autophagy functions as a cellular adaptation mechanism in lens epithelial cells (LECs) under hyperosmotic stress. LECs were treated with hyperosmotic stress at the concentration of 270, 300, 400, 500, or 600 mOsm for 6, 12, 18, 24h in vitro. Polymerase chain reaction (PCR) was employed for the mRNA expression of autophagy-related genes, while Western blotting detected the targeted protein expression. The transfection of stub-RFP-sens-GFP-LC3 autophagy-related double fluorescence lentivirus was conducted to detect the level of autophagy flux. Scanning electron microscopy was used to detect the existence of autolysosome. Short interfering RNA of autophagy-related gene (ATG) 7, transient receptor potential vanilloid (TRPV) 1 overexpression plasmid, related agonists and inhibitors were employed to their influence on autophagy related pathway. Flow cytometry was employed to test the apoptosis and intracellular Ca2+ level. Mitochondrial membrane potential was measured by JC-1 staining. The cell counting kit-8 assay was used to calculate the cellular viability. The wound healing assay was used to evaluate the wound closure rate. GraphPad 6.0 software was utilized to evaluate the data. The hyperosmotic stress activated autophagy in a pressure- and time-dependent manner in LECs. Beclin 1 protein expression and conversion of LC3B II to LC3B I increased, whereas sequestosome-1 (SQSTM1) protein expression decreased. Transient Ca2+ influx was stimulated caused by hyperosmotic stress, levels of mammalian target of rapamycin (mTOR) phosphorylation decreased, and the level of AMP-activated protein kinase (AMPK) phosphorylation increased in the early stage. Based on this evidence, autophagy activation through the Ca2+-dependent AMPK/mTOR pathway might represent an adaptation process in LECs under hyperosmotic stress. Hyperosmotic stress decreased cellular viability and accelerated apoptosis in LECs and cellular migration decreased. Inhibition of autophagy by ATG7 knockdown had similar results. TRPV1 overexpression increased autophagy and might be crucial in the occurrence of autophagy promoted by hyperosmotic stress. A combination of hyperosmotic stress and autophagy inhibition may be a promising approach to decrease the number of LECs in the capsular bag and pave the way for improving prevention of posterior capsular opacification and capsular fibrosis.

Inhibition of SIRT1 in the nucleus accumbens attenuates heroin addiction-related behavior by decreasing D1 neuronal autophagy.

This study aimed to investigate the effects of SIRT1 modulation on heroin addiction-like behavior and its possible biological mechanisms. Wild-type C57BL/6J and Sirt1loxp/loxp D1-Cre mice were used in this experiment, and Sirt1 loxp/loxp D1-Cre(-) mice were used as a control for conditional knockout mice. Mice were divided into saline control and heroin-dependent groups. Behavioral methods were used to record the withdrawal response, conditioned place preference (CPP) changes, and open field test results. Transmission electron microscopy (TEM) was used to observe the structure of autophagosomes in nucleus accumbens (NAc) neurons. The expression of SIRT1 and autophagy-related proteins and genes, such as LC3Ⅱ, ATG5 , and ATG7 , was detected in the NAc of each mouse group via western blot, real-time quantitative PCR (qPCR) analyzes, and immunofluorescence. The results of this experiment showed that compared with the saline group, mice in the wild-type heroin-dependent group showed marked withdrawal symptoms, with more autophagosomes observed in NAc via TEM. Compared with wild-type and Sirt1loxp/loxp D1-Cre(-) heroin-dependent groups, CPP formation was found to be reduced in the conditional knockout mouse group, with a significant decrease in spontaneous activity. Western blot, qPCR, and immunofluorescence results indicated that the expression of LC3Ⅱ, ATG-5, and ATG-7 was significantly reduced in the NAc of the Sirt1loxp/loxp D1-Cre(+) group. It was still, however, higher than that in the saline control group. These results suggest that inhibition of Sirt1 expression may prevent heroin-induced addiction-related behaviors via reducing D1 neuronal autophagy.

Melatonin reduces lung injury in type 1 diabetic mice by the modulation of autophagy

BackgroundIn recent years, the role of autophagy has been highlighted in the pathogenesis of diabetes and inflammatory lung diseases. In this study, using a diabetic model of mice, we investigated the expression of autophagy-related genes in the lung tissues following melatonin administration.ResultsData showed histopathological remodeling in lung tissues of the D group coincided with an elevated level of IL-6, Becline-1, LC3, and P62 compared to the control group (p < 0.05). After melatonin treatment, histopathological remodeling was improved D + Mel group. In addition, expression levels of IL-6, Becline-1, LC3, and P62 were decreased in D + Mel compared to D group (P < 0.05). Statistically significant differences were not obtained between Mel group and C group (p > 0.05).ConclusionOur results showed that melatonin injection can be effective in the amelioration of lung injury in diabetic mice presumably by modulating autophagy-related genes.