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Hepatobiliary Surgery and Nutrition Jun 2024Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated death. Emerging evidence suggests that autophagy plays a critical role in HCC...
BACKGROUND
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated death. Emerging evidence suggests that autophagy plays a critical role in HCC tumorigenesis, metastasis, and prognosis. Choline is an essential nutrient related to prolonged survival and reduced risk of HCC. However, it remains unclear whether this phenomenon is mediated by autophagy.
METHODS
Two HCC cell lines (HUH-7 and Hep3B) were used in the present study. Cell growth was evaluated by cell counting kit 8 (CCK-8), colony formation, and mouse xenografts assays. Cell motility was calculated by wound healing and transwell assays. Autophagosomes were measured by transmission electron microscope (TEM), and autophagy flux was detected by mRFP-GFP-labeled LC3 protein. The mRNA level of genes was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels were detected by Western blotting (WB).
RESULTS
We found that choline inhibited the proliferation, migration, and invasion of HCC cells by downregulating autophagy and . Upregulated expression of the solute carrier family 5 member 7 (SLC5A7), a specific choline transporter, correlated with better HCC prognosis. We further discovered that choline could promote SLC5A7 expression, upregulate cytoplasm p53 expression to impair the AMPK/mTOR pathway, and attenuate autophagy. Finally, we found that choline acted synergistically with sorafenib to attenuate HCC development and .
CONCLUSIONS
Our findings provide novel insights into choline-mediated autophagy in HCC, providing the foothold for its future application in HCC treatment.
PubMed: 38911213
DOI: 10.21037/hbsn-22-476 -
Ferroptosis inhibitor improves outcome after early and delayed treatment in mild spinal cord injury.Acta Neuropathologica Jun 2024We show that redox active iron can induce a regulated form of non-apoptotic cell death and tissue damage called ferroptosis that can contribute to secondary damage and...
We show that redox active iron can induce a regulated form of non-apoptotic cell death and tissue damage called ferroptosis that can contribute to secondary damage and functional loss in the acute and chronic periods after spinal cord injury (SCI) in young, adult, female mice. Phagocytosis of red blood cells at sites of hemorrhage is the main source of iron derived from hemoglobin after SCI. Expression of hemeoxygenase-1 that induces release of iron from heme, is increased in spinal cord macrophages 7 days after injury. While iron is stored safely in ferritin in the injured spinal cord, it can, however, be released by NCOA4-mediated shuttling of ferritin to autophagosomes for degradation (ferritinophagy). This leads to the release of redox active iron that can cause free radical damage. Expression of NCOA4 is increased after SCI, mainly in macrophages. Increase in the ratio of redox active ferrous (Fe) to ferric iron (Fe) is also detected after SCI by capillary electrophoresis inductively coupled mass spectrometry. These changes are accompanied by other hallmarks of ferroptosis, i.e., deficiency in various elements of the antioxidant glutathione (GSH) pathway. We also detect increases in enzymes that repair membrane lipids (ACSL4 and LPCAT3) and thus promote on-going ferroptosis. These changes are associated with increased levels of 4-hydroxynonenal (4-HNE), a toxic lipid peroxidation product. Mice with mild SCI (30 kdyne force) treated with the ferroptosis inhibitor (UAMC-3203-HCL) either early or delayed times after injury showed improvement in locomotor recovery and secondary damage. Cerebrospinal fluid and serum samples from human SCI cases show evidence of increased iron storage (ferritin), and other iron related molecules, and reduction in GSH. Collectively, these data suggest that ferroptosis contributes to secondary damage after SCI and highlights the possible use of ferroptosis inhibitors to treat SCI.
Topics: Ferroptosis; Animals; Spinal Cord Injuries; Mice; Female; Mice, Inbred C57BL; Iron; Treatment Delay
PubMed: 38907771
DOI: 10.1007/s00401-024-02758-2 -
Life Science Alliance Sep 2024Apolipoprotein E4, the most important genetic risk factor for Alzheimer's disease, is shown to internalize into neurons and intersect with amyloid-β in...
Apolipoprotein E4, the most important genetic risk factor for Alzheimer's disease, is shown to internalize into neurons and intersect with amyloid-β in endosomes-autophagosomes of neurites and modulate intraneuronal amyloid-β-42.
PubMed: 38906679
DOI: 10.26508/lsa.202402875 -
International Journal of Biological... 2024Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the critical pathological mechanisms of pulmonary hypertension (PH), and therefore is...
Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the critical pathological mechanisms of pulmonary hypertension (PH), and therefore is gradually being adopted as an important direction for the treatment of PH. Metallothioneins (MTs) have been reported to be associated with PH, but the underlying mechanisms are not fully understood. Here, we demonstrated that the expression level of metallothionein 3 (MT3) was significantly increased in pulmonary arterioles from PH patients and chronic hypoxia-induced rat and mouse PH models, as well as in hypoxia-treated human PASMCs. Knockdown of MT3 significantly inhibited the proliferation of human PASMCs by arresting the cell cycle in the G1 phase, while overexpression of MT3 had the opposite effect. Mechanistically, we found that MT3 increased the intracellular zinc (Zn) concentration to enhance the transcriptional activity of metal-regulated transcription factor 1 (MTF1), which promoted the expression of autophagy-related gene 5 (ATG5), facilitating autophagosome formation. More importantly, MT3-induced autophagy and proliferation of human PASMCs were largely prevented by knockdown of MTF1 and ATG5. Therefore, in this study, we identified MT3-Zinc-MTF1-ATG5 as a novel pathway that affects PASMC proliferation by regulating autophagosome formation, suggesting that MT3 may be a novel target for the treatment of PH.
Topics: Pulmonary Artery; Animals; Cell Proliferation; Humans; Zinc; Mice; Rats; Myocytes, Smooth Muscle; Metallothionein 3; Male; Autophagosomes; Autophagy-Related Protein 5; Rats, Sprague-Dawley; Transcription Factors; Autophagy; Hypertension, Pulmonary; Mice, Inbred C57BL; DNA-Binding Proteins; Transcription Factor MTF-1; Metallothionein
PubMed: 38904023
DOI: 10.7150/ijbs.92992 -
ELife Jun 2024The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including a-synuclein (aSyn)...
The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including a-synuclein (aSyn) associated with the pathogenesis of Parkinson's disease (PD). However, the activation of this pathway is mainly by targeting lysosomal enzymic activity. Here, we focused on the autophagosome-lysosome fusion process around the microtubule-organizing center (MTOC) regulated by lysosomal positioning. Through high-throughput chemical screening, we identified 6 out of 1,200 clinically approved drugs enabling the lysosomes to accumulate around the MTOC with autophagy flux enhancement. We further demonstrated that these compounds induce the lysosomal clustering through a JIP4-TRPML1-dependent mechanism. Among them, the lysosomal-clustering compound albendazole promoted the autophagy-dependent degradation of Triton-X-insoluble, proteasome inhibitor-induced aggregates. In a cellular PD model, albendazole boosted insoluble aSyn degradation. Our results revealed that lysosomal clustering can facilitate the breakdown of protein aggregates, suggesting that lysosome-clustering compounds may offer a promising therapeutic strategy against neurodegenerative diseases characterized by the presence of aggregate-prone proteins.
PubMed: 38899618
DOI: 10.7554/eLife.98649 -
Nature Communications Jun 2024Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require...
Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca-dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes.
Topics: Arabidopsis; Autophagosomes; Endosomal Sorting Complexes Required for Transport; Arabidopsis Proteins; Calcium-Binding Proteins; Autophagy; Phosphatidylinositol Phosphates; Autophagy-Related Protein 8 Family; Vacuoles; Phase Separation
PubMed: 38898014
DOI: 10.1038/s41467-024-49485-6 -
International Journal of Molecular... Jun 2024Sprouty-related enabled/vasodilator-stimulated phosphoprotein homology 1 domain containing 2 (SPRED2) is an inhibitor of the mitogen-activated protein kinase...
Sprouty-related enabled/vasodilator-stimulated phosphoprotein homology 1 domain containing 2 (SPRED2) is an inhibitor of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway and has been shown to promote autophagy in several cancers. Here, we aimed to determine whether SPRED2 plays a role in autophagy in hepatocellular carcinoma (HCC) cells. The Cancer Genome Atlas (TCGA) Liver Cancer Database showed a negative association between the level of SPRED2 and p62, a ubiquitin-binding scaffold protein that accumulates when autophagy is inhibited. Immunohistochemically, accumulation of p62 was detected in human HCC tissues with low SPRED2 expression. Overexpression of SPRED2 in HCC cells increased the number of autophagosomes and autophagic vacuoles containing damaged mitochondria, decreased p62 levels, and increased levels of light-chain-3 (LC3)-II, an autophagy marker. In contrast, SPRED2 deficiency increased p62 levels and decreased LC3-II levels. SPRED2 expression levels were negatively correlated with translocase of outer mitochondrial membrane 20 (TOM20) expression levels, suggesting its role in mitophagy. Mechanistically, SPRED2 overexpression reduced ERK activation followed by the mechanistic or mammalian target of rapamycin complex 1 (mTORC1)-mediated signaling pathway, and SPRED2 deficiency showed the opposite pattern. Finally, hepatic autophagy was impaired in the liver of SPRED2-deficient mice with hepatic lipid droplet accumulation in response to starvation. These results indicate that SPRED2 is a critical regulator of autophagy not only in HCC cells, but also in hepatocytes, and thus the manipulation of this process may provide new insights into liver pathology.
Topics: Humans; Carcinoma, Hepatocellular; Liver Neoplasms; Autophagy; Hepatocytes; Animals; Mice; Cell Line, Tumor; Mechanistic Target of Rapamycin Complex 1; MAP Kinase Signaling System; Mitophagy; Repressor Proteins
PubMed: 38892460
DOI: 10.3390/ijms25116269 -
International Journal of Molecular... Jun 2024This study demonstrated the anticancer efficacy of chalcones with indole moiety (MIPP, MOMIPP) in fibrosarcoma cells for the first time. The results showed that MIPP and...
This study demonstrated the anticancer efficacy of chalcones with indole moiety (MIPP, MOMIPP) in fibrosarcoma cells for the first time. The results showed that MIPP and MOMIPP reduced the viability of HT-1080 cells in a concentration-dependent manner. MOMIPP was more active than MIPP in HT-1080 cells, showing lower IC values (3.67 vs. 29.90 μM). Both compounds at a concentration of 1 μM induced apoptosis in HT-1080 cells, causing death strictly related to caspase activation, as cell viability was restored when the caspase inhibitor Z-VAD was added. Reactive oxygen species production was approximately 3-fold higher than in control cells, and cotreatment with the inhibitor of mitochondrial ATPase oligomycin diminished this effect. Such effects were also reflected in mitochondrial dysfunction, including decreased membrane potential. Interestingly, the compounds that were studied caused massive vacuolization in HT-1080 cells. Immunocytochemical staining and TEM analysis showed that HT-1080 cells exhibited increased expression of the LC3-II protein and the presence of autophagosomes with a double membrane, respectively. Both compounds induced apoptosis, highlighting a promising link between autophagy and apoptosis. This connection could be a new target for therapeutic strategies to overcome chemoresistance, which is a significant cause of treatment failure and tumour recurrence in fibrosarcoma following traditional chemotherapy.
Topics: Humans; Apoptosis; Fibrosarcoma; Autophagy; Indoles; Cell Line, Tumor; Reactive Oxygen Species; Chalcones; Membrane Potential, Mitochondrial; Cell Survival; Antineoplastic Agents; Mitochondria
PubMed: 38892288
DOI: 10.3390/ijms25116100 -
International Journal of Molecular... May 2024Periodontitis development arises from the intricate interplay between bacterial biofilms and the host's immune response, where macrophages serve pivotal roles in defense...
Periodontitis development arises from the intricate interplay between bacterial biofilms and the host's immune response, where macrophages serve pivotal roles in defense and tissue homeostasis. Here, we uncover the mitigative effect of copper chelator Tetrathiomolybdate (TTM) on periodontitis through inhibiting cuproptosis, a newly identified form of cell death which is dependent on copper. Our study reveals concurrent cuproptosis and a macrophage marker within murine models. In response to lipopolysaccharide (LPS) stimulation, macrophages exhibit elevated cuproptosis-associated markers, which are mitigated by the administration of TTM. TTM treatment enhances autophagosome expression and mitophagy-related gene expression, countering the LPS-induced inhibition of autophagy flux. TTM also attenuates the LPS-induced fusion of autophagosomes and lysosomes, the degradation of lysosomal acidic environments, lysosomal membrane permeability increase, and cathepsin B secretion. In mice with periodontitis, TTM reduces cuproptosis, enhances autophagy flux, and decreases levels. Our findings underscore the crucial role of copper-chelating agent TTM in regulating the cuproptosis/mitophagy/lysosome pathway during periodontitis inflammation, suggesting TTM as a promising approach to alleviate macrophage dysfunction. Modulating cuproptosis through TTM treatment holds potential for periodontitis intervention.
Topics: Animals; Lysosomes; Mice; Periodontitis; Autophagy; Molybdenum; Copper; Chelating Agents; Lipopolysaccharides; Macrophages; Chelation Therapy; Inflammation; Mice, Inbred C57BL; Male
PubMed: 38892077
DOI: 10.3390/ijms25115890 -
Molecular Neurodegeneration Jun 2024Age-related macular degeneration (AMD) is the leading cause of blindness in elderly people in the developed world, and the number of people affected is expected to...
BACKGROUND
Age-related macular degeneration (AMD) is the leading cause of blindness in elderly people in the developed world, and the number of people affected is expected to almost double by 2040. The retina presents one of the highest metabolic demands in our bodies that is partially or fully fulfilled by mitochondria in the neuroretina and retinal pigment epithelium (RPE), respectively. Together with its post-mitotic status and constant photooxidative damage from incoming light, the retina requires a tightly-regulated housekeeping system that involves autophagy. The natural polyphenol Urolithin A (UA) has shown neuroprotective benefits in several models of aging and age-associated disorders, mostly attributed to its ability to induce mitophagy and mitochondrial biogenesis. Sodium iodate (SI) administration recapitulates the late stages of AMD, including geographic atrophy and photoreceptor cell death.
METHODS
A combination of in vitro, ex vivo and in vivo models were used to test the neuroprotective potential of UA in the SI model. Functional assays (OCT, ERGs), cellular analysis (flow cytometry, qPCR) and fine confocal microscopy (immunohistochemistry, tandem selective autophagy reporters) helped address this question.
RESULTS
UA alleviated neurodegeneration and preserved visual function in SI-treated mice. Simultaneously, we observed severe proteostasis defects upon SI damage induction, including autophagosome accumulation, that were resolved in animals that received UA. Treatment with UA restored autophagic flux and triggered PINK1/Parkin-dependent mitophagy, as previously reported in the literature. Autophagy blockage caused by SI was caused by severe lysosomal membrane permeabilization. While UA did not induce lysosomal biogenesis, it did restore upcycling of permeabilized lysosomes through lysophagy. Knockdown of the lysophagy adaptor SQSTM1/p62 abrogated viability rescue by UA in SI-treated cells, exacerbated lysosomal defects and inhibited lysophagy.
CONCLUSIONS
Collectively, these data highlight a novel putative application of UA in the treatment of AMD whereby it bypasses lysosomal defects by promoting p62-dependent lysophagy to sustain proteostasis.
Topics: Animals; Mice; Coumarins; Autophagy; Macular Degeneration; Retina; Mitophagy; Sequestosome-1 Protein; Lysosomes; Humans; Disease Models, Animal; Neuroprotective Agents; Mice, Inbred C57BL; Iodates
PubMed: 38890703
DOI: 10.1186/s13024-024-00739-3