Expression Levels Of Autophagy-related Proteins Research Articles

β-Elemene Reverses Gefitinib Resistance in NSCLC Cells by Inhibiting lncRNA H19-Mediated Autophagy.

Lung cancer is a leading cause of mortality worldwide, especially among Asian patients with non-small cell lung cancer (NSCLC) who have epidermal growth factor receptor (EGFR) mutations. Initially, first-generation EGFR tyrosine kinase inhibitors (TKIs) are commonly administered as the primary treatment option; however, encountering resistance to these medications poses a significant obstacle. Hence, it has become crucial to address initial resistance and ensure continued effectiveness. Recent research has focused on the role of long noncoding RNAs (lncRNAs) in tumor drug resistance, especially lncRNA H19. β-elemene, derived from Curcuma aromatic Salisb., has shown strong anti-tumor effects. However, the relationship between β-elemene, lncRNA H19, and gefitinib resistance in NSCLC is unclear. This study aims to investigate whether β-elemene can enhance the sensitivity of gefitinib-resistant NSCLC cells to gefitinib and to elucidate its mechanism of action. The impact of gefitinib and β-elemene on cell viability was evaluated using the cell counting kit-8 (CCK8) assay. Furthermore, western blotting and qRT-PCR analysis were employed to determine the expression levels of autophagy-related proteins and genes, respectively. The influence on cellular proliferation was gauged through a colony-formation assay, and apoptosis induction was quantified via flow cytometry. Additionally, the tumorigenic potential in vivo was assessed using a xenograft model in nude mice. The expression levels of LC3B, EGFR, and Rab7 proteins were examined through immunofluorescence. Our findings elucidate that the resistance to gefitinib is intricately linked with the dysregulation of autophagy and the overexpression of lncRNA H19. The synergistic administration of β-elemene and gefitinib markedly attenuated the proliferative capacity of resistant cells, expedited apoptotic processes, and inhibited the in vivo proliferation of lung cancer. Notably, β-elemene profoundly diminished the expression of lncRNA H19 and curtailed autophagic activity in resistant cells, thereby bolstering their responsiveness to gefitinib. Moreover, β-elemene disrupted the Rab7-facilitated degradation pathway of EGFR, facilitating its repositioning to the plasma membrane. β-elemene emerges as a promising auxiliary therapeutic for circumventing gefitinib resistance in NSCLC, potentially through the regulation of lncRNA H19-mediated autophagy. The participation of Rab7 in this dynamic unveils novel insights into the resistance mechanisms operative in lung cancer, paving the way for future therapeutic innovations.

Abstract 5866: TESC mediates resistance to lenvatinib in hepatocellular carcinoma by regulating cell autophagy

Abstract Objective: To investigate the mechanism by which TESC (Tescalcin) induces lenvatinib resistance in hepatocellular carcinoma (HCC) through autophagy mediation. Methods:1) Utilized established liver cancer organoids from over 70 patients for lenvatinib drug sensitivity screening and transcriptome sequencing to identify lenvatinib-resistant genes. 2) Constructed stable TESC overexpression and downregulation liver cancer cell lines. Employed CCK8 assays to plot IC50 drug sensitivity curves, elucidating changes in lenvatinib sensitivity in liver cancer cells after TESC overexpression or downregulation. 3) Used TESC-stable cells to establish subcutaneous tumor models and intrahepatic tumor models in nude mice. After tumor formation, administered lenvatinib orally to observe whether TESC affects liver cancer sensitivity to lenvatinib in vivo. 4) Conducted Western blot (WB) analysis to assess the expression of autophagy-related molecules (LC3B II/I, P62, and Beclin1 proteins) in TESC downregulated or overexpressed stable cell lines and their control cell lines. Utilized the LC3-GFP-mRFP fluorescence dual-labeling lentivirus system and laser confocal microscopy to examine autophagic flux. 5)Treated TESC downregulated cells with the autophagy inhibitor 3-MA. Conducted WB to assess the expression levels of autophagy-related proteins and plotted drug sensitivity curves using IC50 experiments to evaluate the impact of 3-MA on reversing liver cancer cell sensitivity to lenvatinib. Results: Compared to the control group, HCC cells overexpressing TESC showed an increased IC50 value and reduced sensitivity to lenvatinib, while TESC downregulation resulted in a decreased IC50 value and decreased sensitivity to lenvatinib. Subcutaneous tumor experiments in nude mice indicated that, compared to the control group, tumors with reduced TESC expression were more sensitive to lenvatinib. Additionally, in HCC cells overexpressing TESC, protein expression levels of LC3B II/I and Beclin1 significantly decreased, while P62 expression increased. Conversely, TESC downregulation significantly increased protein expression levels of LC3B II/I and Beclin1, while P62 expression decreased. Furthermore, results from the LC3-GFP-mRFP autophagy dual-labeling lentivirus system showed a significant reduction in fluorescence spots in the TESC overexpression group compared to the control group, indicating weakened autophagic flux. Treatment of TESC downregulated liver cancer cells with 3-MA resulted in an increased IC50 value compared to the control group. Conclusion: TESC expression levels are associated with the sensitivity of liver cancer cells to lenvatinib, possibly by inhibiting autophagy and affecting the sensitivity of liver cancer cells to lenvatinib. Citation Format: Hao Zhuang, Tingting Zhang, Jiaqi Chen. TESC mediates resistance to lenvatinib in hepatocellular carcinoma by regulating cell autophagy [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 5866.

GW9508 ameliorates cognitive dysfunction via autophagy pathway in streptozotocin-induced mouse model of Alzheimer's disease.

G protein-coupled receptor 40 (GPR40) is a potential drug target for Alzheimer's disease (AD), and its agonist GW9508 ameliorates cognitive impairment by intravenous administration. The present study was conducted to investigate the efficacy of GW9508 administered peripherally on cognitive dysfunction in streptozotocin (STZ)-induced AD mice. Seventy male ICR mice were randomly divided into seven groups: vehicle sham group, model, Donepezil, GW9508-L, GW9508-M, GW9508-H, and GW1100 + GW9508-H groups, and administered either vehicle (artificial cerebrospinal fluid [aCSF]) or STZ (3mg/kg in the vehicle) once a day (9:00 a.m.) by intracerebroventricular injection bilaterally on day 1 and day 3, respectively. After 2weeks of recovery, all mice were given drug treatment. Behavioral experiments were applied to test the recognition and spatial memory of mice, while molecular biology experiments such as Western blot, ELISA, and Nissl staining were used to detect the corresponding changes of signaling pathways. Intraperitoneal administration of GW9508 prevented STZ-induced cognitive impairment as well as decreased the level of p-tau and Aβ1-42 in plasma and brain. GW9508 upregulated the expression of gut-brain peptides like PYY, CCK, IGF-1, and GLP-1 both in blood circulation and brain and downregulated the expression level of autophagy-related proteins through activating Akt/mTOR signaling pathway. Meanwhile, the treatment effect of GW9508 was reversed by GPR40 antagonist GW1100 significantly. Peripheral administration of GW9508 exhibits neuroprotective effects, and it could be a promising therapy for AD. The neuroprotective mechanism of GW9508 was based on promoting gut-brain peptide secretion, activating Akt/mTOR signal pathway, and regulating neuronal autophagy.

Homogeneous Polyporus polysaccharide exerts anti-bladder cancer effects via autophagy induction

Context Polyporus polysaccharide (PPS), the leading bioactive ingredient extracted from Polyporus umbellatus (Pers.) Fr. (Polyporaceae), has been demonstrated to exert anti-bladder cancer and immunomodulatory functions in macrophages. Objective To explore the effects of homogeneous Polyporus polysaccharide (HPP) on the proliferation and autophagy of bladder cancer cells co‐cultured with macrophages. Materials and methods MB49 bladder cancer cells and RAW264.7 macrophages were co-cultured with or without HPP intervention (50, 100, or 200 μg/mL) for 24 h. The cell counting kit-8 (CCK-8) assay and 5-ethynyl-2″-deoxyuridine (EdU) staining evaluated MB49 cell proliferation. Monodansylcadaverine (MDC) staining and transmission electron microscopy (TEM) observed autophagosomes. Western blotting detected the expression levels of autophagy-related proteins and PI3K/Akt/mTOR pathway proteins. Results HPP inhibited the proliferation of MB49 cells co-cultured with RAW264.7 cells but not MB49 cells alone. HPP altered the expression of autophagy-related proteins and promoted the formation of autophagosomes in MB49 cells in the co-culture system. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) not only antagonized HPP-induced autophagy but also attenuated the inhibitory effects of HPP on MB49 cell proliferation in the co-culture system. HPP or RAW264.7 alone was not sufficient to induce autophagy in MB49 cells. In addition, HPP suppressed the protein expression of the PI3K/Akt/mTOR pathway in MB49 cells in the co-culture system. Discussion and conclusions HPP induced bladder cancer cell autophagy by regulating macrophages in the co-culture system, resulting in the inhibition of cancer cell proliferation. The PI3K/Akt/mTOR pathway was involved in HPP-induced autophagy in the co-culture system.

MiRNA-363-3p Hinders Proliferation, Migration, Invasion and Autophagy of Thyroid Cancer Cells by Controlling SYT1 Transcription to affect NF-κB.

Thyroid cancer (TC) is a frequent endocrine malignant tumor with various pathologic types. miRNA-363-3p plays a pivotal part in the occurrence, development, prognosis, and treatment of cancer. To explore the mechanism of miRNA-363-3p in TC and provide a new idea for targeted therapy of TC. Differential miRNAs and downstream target mRNAs in TC tissues were predicted with bioinformatics analysis. Expression levels of miRNA-363-3p and Synaptotagmin I (SYT1) in TC cells were ascertained by qRT-PCR. Cell migration, invasion, and proliferation were detected by wound healing assay, transwell assay, colony formation assay, CCK-8, and BrdU fluorescence experiment, respectively. Flow cytometry was utilized to detect the levels of apoptosis and necrosis. Immunofluorescence assay was used for detecting autophagosome formation in cells, and the expression levels of autophagy-related proteins, as well as NF-κB related proteins, were measured by western blot. Dual-luciferase reporter gene assay was applied for detecting the interaction between miRNA-363-3p and SYT1. miRNA-363-3p was prominently down-regulated in TC cells. miRNA-363-3p overexpression suppressed migration, invasion, and proliferation, promoting apoptosis and necrosis of TC cells. As the downstream target of miRNA-363-3p, SYT1 was up-regulated in TC cells. SYT1 overexpression reversed the inhibition of TC cell proliferation, invasion, migration, and autophagy mediated by miRNA-363-3p overexpression. In addition, miRNA-363-3p overexpression inhibited the activation of the NF-κB pathway in cells, while further overexpression of SYT1 weakened the inhibition of miRNA-363-3p overexpression on the NF-κB pathway. miRNA-363-3p affected the NF-κB signaling pathway by down-regulating SYT1 expression to inhibit the malignant progression of TC cells, providing theoretical support for the treatment of TC.

MiR-125b reverses cisplatin resistance by regulating autophagy via targeting RORA/BNIP3L axis in lung adenocarcinoma.

The platinum-based chemotherapy is one of the most frequently used treatment protocols for lung adenocarcinoma (LUAD), and chemoresistance, however, usually results in treatment failure and limits its application in the clinic. It has been shown that microRNAs (miRNAs) play a significant role in tumor chemoresistance. In this study, miR-125b was identified as a specific cisplatin (DDP)-resistant gene in LUAD, as indicated by the bioinformatics analysis and the real-time quantitative PCR assay. The decreased serum level of miR-125b in LUAD patients was correlated with the poor treatment response rate and short survival time. MiR-125b decreased the A549/DDP proliferation, and the multiple drug resistance- and autophagy-related protein expression levels, which were all reversed by the inhibition of miR-125b. In addition, xenografts of human tumors in nude mice were suppressed by miR-125b, demonstrating that through autophagy regulation, miR-125b could reverse the DDP resistance in LUAD cells, both in vitro and in vivo. Further mechanistic studies indicated that miR-125b directly repressed the expression levels of RORA and its downstream BNIP3L, which in turn inhibited autophagy and reversed chemoresistance. Based on these findings, miR-125b in combination with DDP might be an effective treatment option to overcome DDP resistance in LUAD.

Liuwei Dihuang pills attenuate ovariectomy-induced bone loss by alleviating bone marrow mesenchymal stem cell (BMSC) senescence via the Yes-associated protein (YAP)-autophagy axis

Context Liuwei Dihuang pill (LWDH) has been used to treat postmenopausal osteoporosis (PMOP). Objective To explore the effects and mechanisms of action of LWDH in PMOP. Materials and methods Forty-eight female Sprague-Dawley rats were divided into four groups: sham-operated (SHAM), ovariectomized (OVX), LWDH high dose (LWDH-H, 1.6 g/kg/d) and LWDH low dose (LWDH-L, 0.8 g/kg/d); the doses were administered after ovariectomy via gavage for eight weeks. After eight weeks, the bone microarchitecture was evaluated. The effect of LWDH on the differentiation of bone marrow mesenchymal stem cells (BMSCs) was assessed via osteogenesis- and lipogenesis-induced BMSC differentiation. The senescence-related biological indices were also detected using senescence staining, cell cycle analysis, quantitative real-time polymerase chain reaction and western blotting. Finally, the expression levels of autophagy-related proteins and Yes-associated protein (YAP) were evaluated. Results LWDH-L and LWDH-H significantly modified OVX-induced bone loss. LWDH promoted osteogenesis and inhibited adipogenesis in OVX-BMSCs. Additionally, LWDH decreased the positive ratio of senescence OVX-BMSCs and improved cell viability, cell cycle, and the mRNA and protein levels of p53 and p21. LWDH upregulated the expression of autophagy-related proteins, LC3, Beclin1 and YAP, in OVX-BMSCs and downregulated the expression of p62. Discussion and conclusions LWDH improves osteoporosis by delaying the BMSC senescence through the YAP-autophagy axis.

The environmental enrichment ameliorates chronic cerebral hypoperfusion-induced cognitive impairment by activating autophagy signaling pathway and improving synaptic function in hippocampus

BackgroundChronic cerebral hypoperfusion (CCH) is a frequently observed underlying pathology of both Alzheimer's disease (AD) and vascular dementia (VD), which is a common consequence of cerebral blood flow (CBF) dysregulation. Synaptic damage has been proven as a crucial causative factor for CCH-related cognitive impairment. This study aimed to investigate the neuroprotective impact of environmental enrichment (EE) intervention on CCH-induced synaptic destruction and the consequent cognitive impairment. Furthermore, the underlying mechanism of this neuroprotective effect was explored to provide new insights into therapeutic interventions for individuals suffering from AD or VD. MethodsIn this experiment, all rats were initially acclimatized to a standard environment (SE) for a period of one week. On the seventh day, rats underwent either bilateral common carotid artery occlusion (2VO) surgery or sham surgery (Sham) before being subjected to a four-week procedure of exposure to an EE, except for the control group. During the EE or SE procedure, intraperitoneal injection of chloroquine (CQ) into rats was performed once daily for four weeks. Following this, cognitive function was assessed using the Morris water maze (MWM) test. The synapse ultrastructure was subsequently observed using transmission electron microscopy. Expression levels of autophagy-related proteins (LC3, LAMP1, and P62) and synapse-related proteins (Synapsin I and PSD-95) were detected through Western blotting. Finally, immunofluorescence was used to examine the expression levels of Synapsin I and PSD-95 and the colocalization of LAMP-1 and LC3 in the hippocampus. ResultsAfter undergoing 2VO, rats exposed to SE exhibited cognitive impairment, autophagic dysfunction, and synapse damage. The synapse damage was evidenced by ultrastructural damage and degradation of synapse-related proteins. However, these effects were significantly mitigated by exposure to an EE intervention. Moreover, the intervention led to an improvement in autophagic dysfunction. ConclusionThe study found that EE had a positive impact on CCH-induced synaptic damage. Specifically, EE was found to increase synaptic plasticity-associated proteins and postsynaptic density thickness, while decreasing synaptic space. This multifaceted effect resulted in an amelioration of CCH-induced cognitive impairment. It was shown that this beneficial outcome was mediated via the activation of the autophagy-lysosomal pathway. Overall, the findings suggest that EE may have a therapeutic potential for cognitive impairments associated with CCH through autophagy-mediated synaptic improvement.

Exosomal Linc00969 induces trastuzumab resistance in breast cancer by increasing HER-2 protein expression and mRNA stability by binding to HUR

BackgroundBreast cancer (BC) is the most common malignant disease in female patients worldwide. In HER-2+ BC patients, trastuzumab therapy is associated with a better prognosis. However, many HER-2+ BC patients experience recurrence or metastasis because of trastuzumab resistance. The mechanisms underlying trastuzumab resistance remain unclear. Recently, substantial evidence has suggested that exosomes are associated with drug resistance, and lncRNAs have attracted increasing attention due to their potential role in the regulation of trastuzumab resistance.MethodsWe collected the exosomes from the plasma of BC patients with and without trastuzumab resistance, sequenced the whole transcriptomes, identified differentially expressed lncRNAs, and identified lncRNA Linc00969, which was overexpressed in trastuzumab-resistant patients. Then, we established trastuzumab-resistant BC cell lines and explored the role of exosomal Linc00969 in trastuzumab resistance in vitro and in vivo by silencing or overexpressing Linc00969 and performing a series of functional analyses. Furthermore, to explore the mechanism by which exosomal Linc00969 contributes to trastuzumab resistance, we measured changes in HER-2, HUR and autophagy-related protein expression levels after regulating Linc00969 expression. In addition, we investigated the interaction between Linc00969 and HUR via pull-down and RIP assays and the effect of HUR on HER-2 expression and trastuzumab resistance after blocking HUR.ResultsWe first found that exosomal lncRNA Linc00969 was overexpressed in trastuzumab-resistant BC patients and that exosome-mediated Linc00969 transfer could disseminate trastuzumab resistance in BC. Then, we found that silencing Linc00969 could reduce trastuzumab resistance and that overexpressing Linc00969 could enhance trastuzumab resistance. Furthermore, our results showed that Linc00969 could upregulate HER-2 expression at the protein level and maintain the stability of HER-2 mRNA by binding to HUR. Additionally, we found that exosomal Linc00969 could regulate trastuzumab resistance by inducing autophagy.ConclusionsIn this study, we first identified that exosomal lncRNA Linc00969 could induce trastuzumab resistance by increasing HER-2 protein expression and mRNA stability by binding to HUR, and Linc00969 might also be involved in trastuzumab resistance by inducing autophagy. Our results elucidate a novel mechanism underlying trastuzumab resistance, and Linc00969 might be a new target for improving the treatment of HER-2+ BC patients.

Function and autophagy of monocyte-derived dendritic cells is affected by hepatitis B virus infection

BackgroundThe role of dendritic cells and the autophagy state of dendritic cells in the immune response of hepatitis B virus (HBV) infection was still controversial. In this study, we carefully examined the phenotype, function and autophagy pathway of dendritic cells in HBV infection.MethodsMonocyte-derived dendritic cells from healthy blood donors and patients with chronic HBV infection were stimulated by lipopolysaccharide, supernatant of HepG2.2.15 cells or supernatant of HepG2 cells respectively. Phenotype of dendritic cells was examined by flow cytometry and cytokines secretion was detected by enzyme-linked immunosorbent assay. Autophagy related proteins were detected by western blot and immunofluorescence analysis.ResultsOur results showed that the expression of both major histocompatibility complex II molecules and co-stimulated molecules including cluster of differentiation antigen 80, cluster of differentiation antigen 86 in the monocyte-derived dendritic cells from patients with chronic HBV infection was significantly higher than that from healthy donors when cultured with supernatant of HepG2.2.15 cells. The amount of cytokines, including tumour necrosis factor-α, interleukin-10 and interleukin-12, secreted by monocyte-derived dendritic cells from patients with chronic HBV infection was also significantly higher than that from healthy donors when stimulate by HBV. Interestingly, the expression level of autophagy-related proteins including autophagy-related protein5 and associated protein 1 light chain in dendritic cells from patients with chronic HBV infection was significantly increased when compared with that from healthy donors when re-exposed to HBV.ConclusionsOur results indicated that dendritic cells from patients with chronic HBV infection could intensively present antigen and express co-stimulatory molecules. The increased activation of dendritic cells might be related to the enhanced autophagy of dendritic cells in HBV infection.

A new insight of cadmium-induced cellular evidence of autophagic-associated spermiophagy during spermatogenesis.

Autophagy plays a dynamic role in spermatozoa development during spermatogenesis. However, the disruption of autophagic flux induces cell death under metal toxicity and severe oxidative stress. Therefore, we hypothesized that cadmium-induced autophagy might be involved in this mechanism. To verify this hypothesis, we studied cadmium-induced cellular evidence of autophagic-associated spermiophagy within the testis. In the present study, treatment with cadmium caused nuclear depressive disorders and vacuolated mitochondrial damage of Sertoli cells. In addition, spermiophagy through the cellular evidence of spermatozoa phagocytosis, the high lysosomal activity (lysosome engulfment and phagolysosome), and autophagy activity (autolysosome and autophagosome) were observed in the Sertoli cells. The immunohistochemistry of lysosomal membrane protein (LAMP2) to target the phagocytosis of spermatozoa revealed that the immunoreactivity of LAMP2 was overstimulated in the luminal compartment of testis's seminiferous tubules. In addition, the immunohistochemistry and immunofluorescence of autophagy-related protein and microtubule-associated light chain (LC3) results showed the strong immunoreactivity and immunosignaling of LC3 in the Sertoli cells of the testis. Moreover, cadmium caused the overactivation of the expression level of autophagy-related proteins, autophagy-related gene (ATG7), (ATG5), beclin1, LC3, sequestosome 1 (P62), and LAMP2 which were confirmed by western blotting. In summary, this study demonstrated that hazards related to cadmium-induced autophagic-associated spermiophagy with the disruption of autophagic flux, providing new insights into the toxicity of cadmium in mammals and representing a high risk to male fertility.

Protective Effect and Mechanism of Autophagy in Endothelial Cell Injury Induced by Hyperoxia.

Bronchopulmonary dysplasia is a chronic lung disease in premature infants with alveolar simplification and pulmonary vascular development disorder as the main pathological feature and hyperoxia as the main etiology. Autophagy is a highly conserved cytological behavior of self-degrading cellular components and is accompanied by oxidative stress. Studies have reported that autophagy is regulated by FOXO1 posttranslational modification. However, whether autophagy can be involved in the regulation of endothelial cell injury induced by hyperoxia and its mechanism are still unclear. We have activated and inhibited autophagy in human umbilical vein endothelial cells under hyperoxia and verified the role of autophagy in endothelial cell-related functions from both positive and negative aspects. Our research showed that the expression level of autophagy-related proteins decreased, accompanied by decreased cell migration ability and tube formation ability and increased cell reactive oxygen species level and cell permeability under hyperoxia conditions. Using an autophagy agonist alleviated hyperoxia-induced changes and played a protective role. However, inhibition of autophagy aggravated the cell damage induced by hyperoxia. Moreover, the decrease in autophagy proteins was accompanied by the upregulation of FOXO1 phosphorylation and acetylation. We concluded that autophagy was a protective mechanism against endothelial cell injury caused by hyperoxia. Autophagy might participate in this process by coregulating posttranslational modifications of FOXO1. · Hyperoxia induces vascular endothelial cell injury.. · Autophagy may has a protective role under hyperoxia conditions.. · FOXO1 posttranslational modification may be involved in the regulation of autophagy..

COTE-1 promotes the proliferation and invasion of small cell lung cancer by regulating autophagy activity via the AMPK/mTOR signaling pathway

BackgroundCOTE-1 has been found to promote the proliferation and invasion of non-small cell lung cancer. However, the mechanism of COTE-1 in SCLC is still unclear. Exploring the role of COTE-1 in SCLC is expected to provide a potential target for the prognosis and treatment of SCLC. MethodsThe expression of COTE-1 and ki-67 was detected by immunohistochemical staining. PCR detected COTE-1 expression level. Cell proliferation activity was detected by CCK8 assay. A wound healing test detected cell migrative ability. Transwell invasion assay detected cell invasive ability. The numbers of autophagosomes were observed by transmission electron microscopy. WB detected the expression levels of autophagy-related proteins and AMPK/mTOR pathway-related proteins. The effect of COTE-1 expression level on the proliferation of SCLC tumor tissues was investigated by establishing a mouse SCLC xenograft tumor model. ResultsThe expression of COTE-1 in SCLC tissues and cells was higher than that in normal tissues and cells. In SCLC cells with high COTE-1 expression, the expression level of autophagy proteins was notably increased, the number of intracellular autophagosomes increased, and the proliferative activity, migration and invasion abilities were enhanced. COTE-1 promotes autophagy, proliferation, and invasion of SCLC cells under nutrient deprivation by activating the AMPK/mTOR signaling pathway. Activation of autophagy by COTE-1 promotes the proliferation and development of xenograft tumors in a mouse model of SCLC. ConclusionCOTE-1 promotes the proliferation, migration and invasion of small cell lung cancer by mediating autophagy based on the AMPK/mTOR pathway.

Dinotefuran exposure induces autophagy and apoptosis through oxidative stress in Bombyx mori

As a third-generation neonicotinoid insecticide, dinotefuran is extensively used in agriculture, and its residue in the environment has potential effects on nontarget organisms. However, the toxic effects of dinotefuran exposure on nontarget organism remain largely unknown. This study explored the toxic effects of sublethal dose of dinotefuran on Bombyx mori. Dinotefuran upregulated reactive oxygen species (ROS) and malondialdehyde (MDA) levels in the midgut and fat body of B. mori. Transcriptional analysis revealed that the expression levels of many autophagy and apoptosis-associated genes were significantly altered after dinotefuran exposure, consistent with ultrastructural changes. Moreover, the expression levels of autophagy-related proteins (ATG8-PE and ATG6) and apoptosis-related proteins (BmDredd and BmICE) were increased, whereas the expression level of an autophagic key protein (sequestosome 1) was decreased in the dinotefuran-exposed group. These results indicate that dinotefuran exposure leads to oxidative stress, autophagy, and apoptosis in B. mori. In addition, its effect on the fat body was apparently greater than that on the midgut. In contrast, pretreatment with an autophagy inhibitor effectively downregulated the expression levels of ATG6 and BmDredd, but induced the expression of sequestosome 1, suggesting that dinotefuran-induced autophagy may promote apoptosis. This study reveals that ROS generation regulates the impact of dinotefuran on the crosstalk between autophagy and apoptosis, laying the foundation for studying cell death processes such as autophagy and apoptosis induced by pesticides. Furthermore, this study provides a comprehensive insight into the toxicity of dinotefuran on silkworm and contributes to the ecological risk assessment of dinotefuran in nontarget organisms.

α-amanitin induces autophagy through AMPK-mTOR-ULK1 signaling pathway in hepatocytes

Amanitin poisoning is one of the most life-threatening mushroom poisonings. α-Amanitin plays a key role in Amanita phalloides intoxication. α-Amanitin shows toxic effects on the liver. However, the mechanism by which α-amanitin induces liver injury has not been elucidated. Autophagy plays a crucial role in maintaining cellular homeostasis and is closely related to the occurrence of a variety of diseases. Studies have shown that autophagy may play an important role in the process of α-amanitin-induced liver injury. However, the mechanism of α-amanitin-induced autophagy remains unclear. Thus, this study aimed to explore the mechanisms of α-amanitin in inducing hepatotoxicity in Sprague Dawley (SD) rats and the normal human liver cell line L02 cells. The SD rats and L02 cells exposed to α-amanitin were observed to determine whether α-amanitin could induce the autophagy of rat liver and L02 cells. The regulatory relationship between autophagy and the AMPK-mTOR-ULK pathway by exposing the autophagy agonist (rapamycin (RAPA)), autophagy inhibitor (3-methylademine (3-MA)), and AMPK inhibitor (compound C) was also explored. Autophagy-related proteins and AMPK-mTOR-ULK pathway-related proteins were detected using Western blot. The results of the study indicated that exposure to different concentrations of α-amanitin led to morphological changes in liver cells and significantly elevated levels of ALT and AST in the serum of SD rats. Additionally, the expression levels of LC3-II, Beclin-1, ATG5, ATG7, AMPK, p-AMPK, mTOR, p-mTOR, and ULK1 were significantly increased in the rat liver. And we found that L02 cells exposed to 0.5 μM α-amanitin for 6 h significantly induced autophagy and activated the AMPK-mTOR-ULK1 pathway. Pretreated with RAPA, 3-MA, and compound C for 1 h, the expression levels of autophagy-related proteins and AMPK-mTOR-ULK pathway-related proteins significantly changed. Our results indicates that autophagy and the AMPK-mTOR-ULK pathway are involved in the process of α-amanitin-induced liver injury. This study may foster the identification of actionable therapeutic targets for A. phalloides intoxication.

Hippo-YAP signaling pathway regulates autophagy of human periodontal ligament cells under cyclic tensile stress.

This work aimed to investigate the molecular mechanism of cyclic tensile stress (CTS) stimulating autophagy in human periodontal ligament cells (hPDLCs). hPDLCs were isolated and cultured from normal periodontal tissues. hPDLCs were loaded with tensile stress by force four-point bending extender to simulate the autophagy of hPDLCs induced by orthodontic force du-ring orthodontic tooth movement. XMU-MP-1 was used to inhibit the Hippo signaling pathway to explore the role of the Hippo-YAP signaling pathway in activating hPDLC autophagy by tensile stress. The expression levels of autophagy-related genes (Beclin-1, LC3, and p62) in hPDLCs were detected by real-time quantitative polymerase chain reaction. Western blot was used to detect the expression levels of autophagy-related proteins (Beclin-1, LC3-Ⅱ/LC3-Ⅰ, and p62) and Hippo-YAP pathway proteins (active-YAP and p-YAP) in hPDLCs. Immunofluorescence was used to locate autophagy-related proteins (LC3-Ⅱand p62) and Hippo-YAP pathway proteins (active-YAP) of hPDLCs. CTS-activated autophagy in hPDLCs and expression of autophagy-related proteins initially increased and then decreased; it began to increase at 30 min, peaked at 3 h, and decreased (P<0.05). CTS increased the expression of active-YAP protein and decreased the expression of p-YAP protein (P<0.05). When XMU-MP-1 inhibited the Hippo-YAP signaling pathway (P<0.05), active-YAP protein was promoted to enter the nucleus and autophagy expression was enhanced (P<0.05). The Hippo-YAP signaling pathway is involved in the regulation of autophagy activation in hPDLCs under CTS.

Blueberry Stem Extract Prevents Lacrimal Hyposecretion in Non-obese Diabetic Mice via Activation of AMPK.

Tears secreted from the lacrimal gland are essential for preserving the ocular surface. Thus, dysfunction of the lacrimal gland in Sjögren's syndrome (SS) can lead to dry eye, resulting in a reduced quality of life. We previously reported that blueberry 'leaf' water extract prevents lacrimal hyposecretion in male non-obese diabetic (NOD) mice in a SS-like model. In this study, we investigated the effect of blueberry 'stem' water extract (BStEx) on lacrimal hyposecretion in NOD mice. Male NOD mice were fed 1% BStEx or control (AIN-93G) for 2, 4, or 6 weeks from 4 weeks of age. Pilocarpine-induced tear secretion was measured using a phenol red-impregnated thread. The lacrimal glands were histologically evaluated by HE staining. Inflammatory cytokine levels in the lacrimal glands were measured using ELISA. Immunostaining was performed to examine aquaporin 5 (AQP5) localization. The expression levels of autophagy-related proteins, AQP5, and phosphorylated AMPK were measured using western blotting. After feeding BStEx to mice for 4 or 6 weeks, tear volume was observed to have increased in the BStEx group compared with that in the control group. There were no significant differences in inflammatory cell infiltration, autophagy-related protein expression, or the localization and expression of AQP5 in the lacrimal glands between the two groups. In contrast, AMPK phosphorylation increased in the BStEx group. BStEx prevented lacrimal hyposecretion in the SS-like model of male NOD mice, probably by opening tight junctions via the activation of AMPK in lacrimal acinar cells.

More light components and less light damage on rats' eyes: evidence for the photobiomodulation and spectral opponency.

The blue-light hazard (BLH) has raised concerns with the increasing applications of white light-emitting diodes (LEDs). Many researchers believed that the shorter wavelength or more light components generally resulted in more severe retinal damage. In this study, based on the conventional phosphor-coated white LED, we added azure (484nm), cyan (511nm), and red (664nm) light to fabricate the low-hazard light source. The low-hazard light sources and conventional white LED illuminated 68 Sprague-Dawley (SD) rats for 7 days. Before and after light exposure, we measured the retinal function, thickness of retinal layers, and fundus photographs. The expression levels of autophagy-related proteins and the activities of oxidation-related biochemical indicators were also measured to investigate the mechanisms of damaging or protecting the retina. With the same correlated color temperature (CCT), the low-hazard light source results in significantly less damage on the retinal function and photoreceptors, even if it has two times illuminance and blue-light hazard-weighted irradiance ([Formula: see text]) than conventional white LED. The results illustrated that [Formula: see text] proposed by IEC 62471 could not exactly evaluate the light damage on rats' retinas. We also figured out that more light components could result in less light damage, which provided evidence for the photobiomodulation (PBM) and spectral opponency on light damage.

Nano-selenium Alleviates Cadmium-Induced Mouse Leydig Cell Injury, via the Inhibition of Reactive Oxygen Species and the Restoration of Autophagic Flux.

Cadmium (Cd) is a well-known environmental pollutant that can contribute to male reproductive toxicity through oxidative stress. Nano-selenium (Nano-se) is an active single body of selenium with strong antioxidant properties and low toxicity. Some studies have addressed the potential ameliorative effect of Nano-se against Cd-induced testicular toxicity; however, the underlying mechanisms remain to be investigated. This study aimed to explore the protective effect of Nano-se on Cd-induced mouse testicular TM3 cell toxicity by regulating autophagy process. We showed that cadmium exposure to TM3 cells inhibited cell viability and elevated the level of reactive oxygen species (ROS) generation. Morphology observation by transmission electron microscope and the presence of mRFP-GFP-LC3 fluorescence puncta demonstrated that cadmium increased autophagosome formation and accumulation in TM3 cells, resulting in blocking the autophagic flux of TM3 cells. Meanwhile, cadmium remarkably increased the ratio of LC3-II to LC3-I protein expression (2.07 ± 0.31) and the Beclin-1 protein expression (1.97 ± 0.40) in TM3 cells (P < 0.01). Pretreatment with Nano-se significantly reduced Cd-induced TM3 cell toxicity (P < 0.01). Furthermore, Nano-se treatment reversed Cd-induced ROS production and autophagosome accumulation, and autophagy as evidenced by the ratio of LC3-II to LC3-I and Beclin-1 expression. In addition, ROS scavenger, N-acetyl-L-cysteine (NAC) or autophagy inhibitor, 3-methyladenine (3-MA) reversed cadmium-induced ROS generation, autophagosome accumulation, and autophagy-related protein expression levels, which confirmed that cadmium induced TM3 cell injury via ROS signal pathway and blockage of autophagic flux. Collectively, our results reveal that Nano-se attenuates Cd-induced TM3 cell toxicity through the inhibition of ROS production and the amelioration of autophagy disruption.

RTFPI Protects Cardiomyocytes from Hypoxia/Reoxygenation Injury through Inhibiting Autophagy and the Class III PI3K/Beclin-1 Pathway.

Autophagy plays various roles at different stages of ischemia reperfusion (I/R) injury in cardiomyocytes. It has been reported that tissue factor pathway inhibitor (TFPI) has a protective effect on I/R injury. This study aimed to determine the roles of TFPI in autophagy during the I/R injury process in cardiomyocytes and the possible mechanisms. An isolated hypoxia/reoxygenation (H/R) pattern of cardiomyocytes was established by the MIC101 system. The cell viability and oxidative stress of cardiomyocytes were detected by an MTT assay and ROS assay, respectively. The autophagy level was measured by Ad-mCherry-GFP-LC3B and MDC. We detected the expression levels of autophagy-related proteins by western blotting. After 2 h of hypoxia and 12 h of reoxygenation, the cardiomyocyte viability in the H/R group was significantly lower than that in the control group (p < 0.05) than in the H/R group. According to intracellular ROS production, the fluorescence intensity in the H/R group was enhanced compared with that in the negative control group, and it was weaker in the H/R + rTFPI group compared with the H/R group. The level of autophagy and the expression levels of autophagy-related proteins (LC3-II/LC3-I, Beclin-1 and PI3K) were markedly increased in the H/R group compared to the control group (p < 0.05) whereas the levels were markedly decreased in the H/R + rTFPI group compared to the H/R group (p < 0.05). TFPI could relieve cardiomyocyte injury by inhibiting the Class III PI3K/Beclin-1 pathway and oxidative stress; thus, TFPI decreased autophagy and protected cardiomyocytes induced by H/R injury. In conclusion, TFPI may be a new direction for the prevention of myocardial I/R injury.