SQSTM1 Expression Research Articles

Measurement of SQSTM1 by flow cytometry

ABSTRACT Macroautophagy/autophagy is a regulated cellular degradation process essential as a pro-survival mechanism and integral to the regulation of diverse cellular processes in eukaryotes. During cellular stress and nutrient sensing, SQSTM1/p62 (sequestosome 1) functions as a key receptor for selective autophagy by shuttling ubiquitinated cargoes toward autophagic degradation making it a useful marker for monitoring autophagic flux. We present a straightforward and rapid flow cytometric assay for the quantitative measurement of intracellular SQSTM1 with improved sensitivity to conventional immunoblotting and with the benefit of higher throughput and reduced requirements for starting cellular materials for adequate analysis. We demonstrate that flow cytometry is able to detect similar trends in the measurement of intracellular SQSTM1 levels following serum starvation, genetic manipulations, and bafilomycin A1/chloroquine treatments. The assays utilizes readily available reagents and equipment without the need for transfection and utilizes standard flow cytometry equipment. In the present studies, expression of reporter proteins was applied to a range of SQSTM1 expression levels generated by genetic and chemical manipulation in both mouse as well as human cells. In combination with appropriate controls and attention to cautionary issues, this assay offers the ability to assess an important measure of autophagic capacity and flux. Abbreviations: ATG5: autophagy related 5 ATG7: autophagy related 7 BafA: bafilomycin A1 BMDM: bone marrow-derived macrophages CQ: chloroquine EBV: Epstein-Barr Virus EDTA: ethylenediaminetetraacetic acid FBS: fetal bovine serum gMFI: geometric mean fluorescent intensity HD: healthy donor MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3 MedianFI: median fluorescent intensity NTC: non-target control PBMC: peripheral blood mononuclear cells RPMI: Roswell Park Memorial Institution SQSTM1/p62: sequestosome 1 WT: wild type

Tl(I) and Tl(III) induce reticulum stress in MDCK cells

The effects of the exposure of proliferating MDCK cells to thallium [Tl(I) or Tl(III)] on cell viability and proliferation were investigated. Although Tl stopped cell proliferation, the viability was > 95%. After 3 h, two autophagy markers (SQSTM-1 expression and LC3β localization) were altered, and at 48 h increased expression of SQSTM-1 (60%) and beclin-1 (50–100%) were found. At 24 h, the expression of endoplasmic reticulum (ER) stress markers ATF-6 and IRE-1 were increased in 100% and 150%, respectively, accompanied by XBP-1 splicing and nuclear translocation. At 48 h, major ultrastructure abnormalities were found, including ER enlargement and cytoplasmic vacuolation which was not prevented by protein synthesis inhibition. Increased PHB (85% and 40% for Tl(I) and Tl(III), respectively) and decreased β-tubulin (45%) expression were found which may be related to the promotion of paraptosis. In summary, Tl(I) and Tl(III) promoted ER stress and probably paraptosis in MDCK cells, impairing their proliferation.

Calycosin alleviates cerebral ischemia/reperfusion injury by repressing autophagy via STAT3/FOXO3a signaling pathway

BackgroundAs a common cerebrovascular disease (CVD) of the elderly, ischemic stroke (IS) is characterized by high disability and mortality. Excessive autophagy induced by IS is implicated in neuronal death, therefore, the inhibition of immoderate autophagy is viewed as a potential therapeutic avenue to treat IS. Calysoin (CA) is a bioactive component of Radix Astragali, which has been widely used to treat CVDs. However, the mechanism of the treatment of IS by CA is still problematic. PurposeBased on the result of network pharmacology, whether CA inhibited autophagy by regulating the STAT3/FOXO3a pathway to alleviate cerebral ischemia-reperfusion injury (CIRI) was investigated in vivo and in vitro for the first time. Study designIntegrate computational prediction and experimental validation based on network pharmacology. MethodsIn current study, network pharmacology was applied to predict the mechanism of the treatment of IS by CA, and it was shown that CA alleviated CIRI by inhibiting autophagy via STAT3/FOXO3a signaling pathway. One hundred and twenty adult male specific pathogen-free Sprague-Dawley rats in vivo and PC12 cells in vitro were used to verify the above prediction results. The rat middle cerebral artery occlusion/reperfusion (MCAO/R) model was established by suture method, and oxygen glucose deprivation/re-oxygenation (OGD/R) model was used to simulate cerebral ischemia in vivo. The content of MDA, TNF-α, ROS and TGF-β1 in rat serum were detected by ELISA kits. The mRNA and protein expressions in brain tissue were detected by RT-PCR and Western Blotting. The expressions of LC3 in brain were detected immunofluorescent staining. ResultsThe experimental results demonstrated that administration of CA dosage-dependently improved rat CIRI as evidenced by the reduction in the cerebral infarct volume, amelioration of the neurological deficits. HE staining and transmission electron microscopy results revealed that CA ameliorated cerebral histopathological damage, abnormal mitochondrial morphology, and damaged mitochondrial cristae structure in MCAO/R rats. CA treatment exerted protective effects in CIRI by inhibiting inflammation response, oxidative stress injury, and cell apoptosis in rat and PC12 cells. CA relieved excessive autophagy induced by MCAO/R or OGD/R through downregulating the LC3Ⅱ/LC3Ⅰ ratio and upregulating the SQSTM1 expression. CA treatment also decreased p-STAT3/STAT3 and p-FOXO3a/FOXO3a ratio in the cytoplasm and modulated the autophagy-related gene expression both in vivo and in vitro. ConclusionTreatment with CA attenuated CIRI by reducing excessive autophagy via STAT3/FOXO3a signal pathway in rat and PC12 cells.

Umbelliferone protects against cerebral ischemic injury through selective autophagy of mitochondria

Effective therapeutic treatments for ischemic stroke are limited. Previous studies suggest selective activation of mitophagy alleviates cerebral ischemic injury while excessive autophagy is detrimental. However, few compounds are available to selectively activate mitophagy without affecting autophagy flux. Here, we found that acute administration of Umbelliferone (UMB) upon reperfusion exerted neuroprotective effects against ischemic injury in mice subjected to transient middle cerebral artery occlusion (tMCAO) and suppressed oxygen-glucose deprivation reperfusion (OGD-R)-induced apoptosis in SH-SY5Y cells. Interestingly, UMB promoted the translocation of mitophagy adaptor SQSTM1 to mitochondria and further reduced the mitochondrial content as well as the expression of SQSTM1 in SHSY5Y cells after OGD-R. Importantly, both the mitochondrial loss and reduction of SQSTM1 expression after UMB incubation can be reversed by autophagy inhibitor chloroquine and wortmannin, proving the mitophagy activation by UMB. Nevertheless, UMB failed to further affect neither LC3 lipidation nor the number of autophagosomes after cerebral ischemia in vivo and in vitro. Furthermore, UMB facilitated OGD-R-induced mitophagy in a Parkin-dependent manner. Inhibition of autophagy/mitophagy either pharmaceutically or genetically abolished the neuroprotective effects of UMB. Taken all, these results suggest that UMB protects against cerebral ischemic injury, both in vivo and in vitro, via promoting mitophagy without increasing the autophagic flux. UMB might serve as a potential leading compound for selectively activating mitophagy and the treatment of ischemic stroke.

New in vitro findings about halogenated boroxine cytotoxicity and deregulation of cell death-related genes in GR-M melanoma cells.

Anti-proliferative effects of halogenated boroxine - K2(B3O3F4OH) (HB) - have been confirmed in multiple cancer cell lines, including melanoma, but the exact mechanism of action is still unknown. This study aimed to determine its cytotoxic effects on human Caucasian melanoma (GR-M) cell growth in vitro as well as on the expression of cell death-related genes BCL-2, BECN1, DRAM1, and SQSTM1. GR-M and peripheral blood mononuclear (PBM) cells were treated with different HB concentrations and their growth inhibition and relative gene expression profiles were determined using the Alamar blue assay and real-time PCR. HB significantly inhibited cell growth of both GR-M and PBM cells but was even more effective in GR-M melanoma cells, as significant inhibition occurred at a lower HB concentration of 0.2 mg/mL. GR-M BCL-2 expression was significantly downregulated (P=0.001) at HB concentration of 0.4 mg/mL, which suggests that HB is a potent tumour growth inhibitor. At the same time, it upregulated BCL-2 expression in normal (PBM) cells, probably by activating protective mechanisms against induced cytotoxicity. In addition, all but the lowest HB concentrations significantly upregulated SQSTM1 (P=0.001) in GR-M cells. Upregulated BECN1 expression suggests early activation of autophagy at the lowest HB concentration in SQSTM1 cells and at all HB concentrations in PBM cells. Our findings clearly show HB-associated cell death and, along with previous cytotoxicity studies, reveal its promising anti-tumour potential.

Autophagic-Related Anticancer Effect of Grapes Extract and Tomatoes Extract: Ex-Vivo Study

Cells undergo autophagy to save themselves from injury, but progressive autophagy can cause cell death. This study characterized and compared the effect of grape (resveratrol) and tomato (lycopene) extracts and their combination on modulating autophagy-related miRNA and its target gene in squamous cell carcinoma cell line. Docking analysis for extracts and selected genes was performed. Methyl Thiazol Tetrazolium assays were used to assess the cytotoxicity of extracts and their combination toward HEp-2 cells. qRT-PCR was used to quantify changes in gene expression. Data were statistically analyzed. miRNA-20a was identified as a potential effector in laryngeal cancer, and sequestosome-1 (SQSTM1) was its target gene. Docking analysis showed that resveratrol interacted with miRNA-20a and showed less affinity toward SQSTM1. Hydrogen bonds and hydrophobic interactions were predicted. In contrast, lycopene showed less affinity toward miRNA-20a than resveratrol. Increasing doses of resveratrol, lycopene, and their combination induced a statistically significant reduction in mean percent viability and mean fold changes of miRNA-20a and SQSTM1 expression in treated HEp-2 cells. Pearson’s correlation showed a statistically significant positive correlation between miRNA-20a and SQSTM1 (R=0.812, p≤0.001). Grape and tomato extracts and their combination display promising cytotoxicity against HEp-2 cells in a dose- and time-dependent fashion. Both extracts reduce the expression of miRNA-20a and SQSTM1 with subsequent inhibition autophagy and promotion of apoptosis in HEp-2 cells.

SCAMP3 promotes breast cancer progression through the c-MYC-β-Catenin-SQSTM1 growth and stemness axis

The cellular trafficking protein secretory-carrier-membrane-protein 3 (SCAMP3) has been previously shown to promote hepatocellular carcinoma, melanoma, glioma and pancreatic adenocarcinoma. Moreover, previous work has shown that SCAMP3 regulates the epidermal growth factor receptor (EGFR) in triple negative breast cancer (TNBC). However, the oncogenic role of SCAMP3 in different molecular subtypes of breast cancer (BRCA) remains largely unknown. In this study, the role of SCAMP3 in different molecular subtypes of BRCA was investigated using in silico, in vitro and in vivo approaches. In silico analysis of BRCA patient samples showed that SCAMP3 is highly overexpressed in different BRCA molecular subtypes, advanced disease grades and lymph node metastatic stages. Depletion of SCAMP3 inhibited BRCA cell growth, stemness, clonogenic potential and migration and promoted autophagy and cellular senescence. The expression of stemness markers CD44 and OCT4A was reduced in SCAMP3-silenced MDA-MB-231 cells. SCAMP3 overexpression promoted cell proliferation, clonogenicity, tumor spheroid formation and migration in vitro and tumor growth in vivo. SCAMP3 promoted epithelial-mesenchymal-transition (EMT) by regulating E-cadherin expression. SCAMP3 enhanced in vivo tumor growth in MDA-MB-231 tumor xenograft mouse model. Mechanistically, SCAMP3 depletion inhibited β-Catenin, c-MYC and SQSTM1 expression, while its overexpression increased the expression of the same oncogenic proteins. Increased SCAMP3 expression associated with increased chemoresistance in BRCA cells while its depletion associated with increased sensitivity to chemotherapy. BRCA patients with high SCAMP3 expression showed poor prognosis, decreased overall survival and relapse free survival relative to counterparts with reduced SCAMP3 expression. These findings suggest that SCAMP3 exerts a wide range of oncogenic effects in different molecular subtypes of BRCA by modulating the c-MYC-β-Catenin-SQSTM1 axis that targets tumor growth, metastasis, stemness and chemoresistance.

The autophagic protein p62 is a target of reactive aldehydes in human and murine cholestatic liver disease.

Inflammatory cholestatic liver diseases, including Primary Sclerosing Cholangitis (PSC), are characterized by periportal inflammation with progression to cirrhosis. The objective of this study was to examine interactions between oxidative stress and autophagy in cholestasis. Using hepatic tissue from male acute cholestatic (bile duct ligated) as well as chronic cholestatic (Mdr2KO) mice, localization of oxidative stress, the antioxidant response and induction of autophagy were analyzed and compared to human PSC liver. Concurrently, the ability of reactive aldehydes to post-translationally modify the autophagosome marker p62 was assessed in PSC liver tissue and in cell culture. Expression of autophagy markers was upregulated in human and mouse cholestatic liver. Whereas mRNA expression of Atg12, Lamp1, Sqstm1 and Map1lc3 was increased in acute cholestasis in mice, it was either suppressed or not significantly changed in chronic cholestasis. In human and murine cholestasis, periportal hepatocytes showed increased IHC staining of ubiquitin, 4-HNE, p62, and selected antioxidant proteins. Increased p62 staining colocalized with accumulation of 4-HNE-modified proteins in periportal parenchymal cells as well as with periportal macrophages in both human and mouse liver. Mechanistically, p62 was identified as a direct target of lipid aldehyde adduction in PSC hepatic tissue and in vitro cell culture. In vitro LS-MS/MS analysis of 4-HNE treated recombinant p62 identified carbonylation of His123, Cys128, His174, His181, Lys238, Cys290, His340, Lys341 and His385. These data indicate that dysregulation of autophagy and oxidative stress/protein damage are present in the same periportal hepatocyte compartment of both human and murine cholestasis. Thus, our results suggest that both increased expression as well as ineffective autophagic degradation of oxidatively-modified proteins contributes to injury in periportal parenchymal cells and that direct modification of p62 by reactive aldehydes may contribute to autophagic dysfunction.

Abstract 15056: Cardiac P62/sqstm1 Deficiency Exacerbates Hypoxic Stress vi a Impaired Hypoxia Inducible Factor 1α Signaling

Ischemic heart disease (IHD), a major cause of heart failure, is characterized by metabolic dysfunction and myocardial cell death. Cellular hypoxia activates hypoxia inducible factor 1α (HIF1α) to initiate metabolic, angiogenic, and growth-factor related responses. Under normoxia, HIF1α is degraded by prolyl hydroxylase (PHD) domain-containing proteins via the proteasome. Due to the short half-life of HIF1α (<5mins), PHD inhibitors are utilized to stabilize HIF1α and improve cardiac function in pre-clinical models of ischemia/infarction. However, evidence exists that PHD inhibitors exert off-target effects. Based on prior studies in cancer cells demonstrating that the autophagy adaptor protein p62 prevents HIF1α degradation, we hypothesized that p62 stabilizes cardiac HIF1α and provides cardioprotection from hypoxia. We generated mice with tamoxifen-inducible cardiomyocyte-specific p62 deletion (p62icKO mice; 8 week male and female) and exposed them to 7% O 2 for 6h. Hypoxia increased cardiac mRNA expression of Sqstm1 , Bnip3 , and Hmox1 in wild-type (WT) but not p62icKO mice. Underscoring functional relevance, ejection fraction and fractional shortening were less, oxidative stress was elevated, and apoptosis was greater, in p62icKO vs. WT mice exposed to hypoxia. Mechanisms were evaluated using cultured H9c2 cardiomyoblasts exposed to 21% (normoxia) or 1% (hypoxia) O 2 for 24h. Hypoxia increased both HIF1α and p62 protein expression. To determine the role of p62, H9c2 cells were transfected with p62 or scrambled (ctrl) siRNA for 48h, followed by 24 h hypoxia. Hypoxia-induced HIF1α protein accrual and expression of HIF1α downstream targets Egln1 , Vegfa , Bnip3 and Hmox1 mRNA was attenuated in H9c2 cells transfected with p62 vs. control siRNA. These data indicate p62 stabilizes cardiac HIF1α and regulates its transcriptional activation. Thus, p62 could be a novel therapeutic target for treatment of IHD.

Excessive fluoride impairs autophagy flux in ameloblasts which is prevented by the autophagy activator rapamycin.

Excessive fluoride intake can cause dental fluorosis during teeth development and growth. However, the mechanisms underlying fluoride-induced enamel damage are still not fully elucidated. Previously, we observed fluoride-induced autophagy in ameloblasts, but the effects of fluoride on autophagy flux in ameloblasts remain unclear. Hence, this study aimed to clarify the effects of fluoride and rapamycin, an autophagy activator, on autophagy flux in ameloblasts. This in vitro study used the murine ameloblast-derived cell line LS8. Cells were treated with different concentrations of sodium fluoride (NaF) to evaluate NaF-induced cytotoxicity. Using transmission electron microscopy, we observed an increase in the number of autophagosomes with increasing fluoride concentrations. Western blot analyses showed increases in microtubule-associated protein 1 light chain 3 (LC3) and SQSTM1 (p62) expression after NaF treatment and an increase in LC3II expression after bafilomycin A1 administration. Together with changes in RFP-GFP-LC3 lentivirus expression, this demonstrated that fluoride impaired autophagy flux. Furthermore, we evaluated whether rapamycin can alleviate fluoride-induced cytotoxicity by restoring autophagy flux. Compared to the NaF-treated group, LS8 cells cotreated with NaF and rapamycin grew considerably better and had significantly decreased p62 expression. Taken together, these data suggest that fluoride-induced impaired autophagosome degradation may damage ameloblasts. This provides experimental in vitro evidence and an explanation for the observed NaF-induced toxicity of ameloblasts. Rapamycin probably alleviates this impairment by decreasing the expression of p62, thereby preventing autophagy defects.

IL-10 cytokine family secretion is associated with the activity of mitophagy components in visceral adipose tissue in obese with and without type 2 diabetes mellitus

Autophagy is required to maintain cellular homeostasis and organ function by selectively ridding cells of potentially toxic proteins, lipids, and organelles. Impaired homeostasis of autophagic processes is associated with metabolic disorders such as obesity and type 2 diabetes mellitus. In obesity, a violation of autophagy in adipose tissue and its inflammation contributes to the formation of type 2 diabetes mellitus.
 The aim of the study was to analyze the expression of autophagy genes in the adipose tissue of the greater omentum and to search for their relationship with the levels of cytokines of the IL-10 family in blood plasma in obese patients, depending on the presence or absence of type 2 diabetes mellitus.
 Blood plasma and visceral adipose tissue samples were studied from 347 obese patients with and without type 2 diabetes. A biochemical analysis of the patients' blood was carried out. The level of cytokines was detected by flow fluorometry. Gene expression was determined by real-time PCR, and tissue-specific protein production was determined by immunoblotting. Statistical processing of the results was carried out using GraphPad Prism 9.0.0 software.
 Plasma levels of IL-10, IL-20, IL-22, IL-28A, and IL-29 are increased in obese patients without type 2 diabetes compared with patients with type 2 diabetes. In patients with type 2 diabetes mellitus, the expression of the SQSTM1_p62 and MAP1LC3B genes in the greater omentum increased compared to patients without it.
 High plasma levels of IL-22 and IL-26 are associated with the presence of type 2 diabetes mellitus. In patients without type 2 diabetes mellitus, an increase in the level of IL-28A in blood plasma is associated with a decrease in the expression of autophagy genes SQSTM1_p62 and MAP1LC3B in the adipose tissue of the greater omentum.

Abstract 1284: The tumor scaffold protein SQSTM1 at the crossroads of DNA damage and immunotherapy responses in solid tumors

Abstract Introduction After the recent advent of Immune Checkpoint Inhibitors (ICIs), one of current challenge of clinical cancer trials is certainly to develop the optimal combinations of ICIs with DNA-Damaging Agents (chemotherapy and/or radiotherapy, named DDAs hereafter). Elucidating resistance mechanisms to these different treatments is pivotal to propose new predictive biomarkers and to develop therapeutic strategies to improve ICI efficiency. We hypothesized that resistance to DDA and ICIs is mediated in part by intrinsic tumor mechanisms, some of which may be shared. Methods To address this knowledge gap, we compared RNA expression signatures, antigen presentation, and PD-L1 expression from cohorts of cancer patients treated with radiotherapy, chemotherapy, and immunotherapy to identify shared molecular pathways that may mediate cross-resistance. Using a panel of lung cancer cell lines, we then confirmed that the tumor-cell-intrinsic expression of SQSTM1 is positively correlated with antigen presentation, and inversely with DNA damage repair and DDA/ICIs resistance gene signatures. This SQSTM1/HERV pathway was then supported in vitro using engineered silenced cell-lines (SQSTM1, MAVS, and STING ShRNA) treated with FDA-approved DDA (cisplatin, oxaliplatin, doxorubicin, and ionizing radiations) or synthetic double-stranded RNA (5’ppp dsRNAs and poly IC). Results Through three complementary approaches (in silico, ex vivo on patient cohorts, and in vitro), we identify the p62/SQSTM1 scaffold protein as a key molecular mediator able of predicting and controlling sensitivity for DDA and ICIs. Mechanistically, in response to DNA damage, we found that SQSTM1 is essential for the inhibition of DNA repair and the reactivation of human endogenous retroviruses (hERV). Analogous to a “viral alarm”, the hERV are sensed and activate IFN responses that drive the expression of MHC-I and PD-L1, leading to tumor immune evasion. Targeting the hERV pathways in SQSTM1-depleted tumor cells by poly(I:C)/Lyovec, or docetaxel treatment can rescue the hERV, IFN, and MHC pathway, providing a promising therapeutic avenue turning a “cold” into a “hot” tumor in non-responders cancer patients. Conclusion Depending on its levels, we thus propose SQSTM1 as a predictive biomarker for guiding treatment decisions from i) ICI alone, ii) ICI combined with cisplatin, or; iii) ICI combined with poly(I:C)/Lyovec or docetaxel, with the aim to increase immunotherapy efficacy. Citation Format: Iris Grosjean, Grégoire D’Andréa, Nathalie Yazbeck, Amine Belaid, Barnabé Roméo, Marie Angela Domdom, Nicolas Guillot, Eric Gilson, Emmanuel Chamorey, Gérard Milano, Véronique Hofman, Marius Ilié, Isabelle Benard-Thiery, Simon Heeke, Patrick Brest, François Ghiringhelli, Paul Hofman, Baharia Mograbi. The tumor scaffold protein SQSTM1 at the crossroads of DNA damage and immunotherapy responses in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1284.

DDIT3 antagonizes innate immune response to promote bovine alphaherpesvirus 1 replication via the DDIT3-SQSTM1-STING pathway

ABSTRACT DNA damage-inducible transcript 3 (DDIT3), a transcription factor, is typically involved in virus replication control. We are the first to report that DDIT3 promotes the replication of bovine viral diarrhea virus, an RNA virus, by inhibiting innate immunity. However, whether the DDIT3 gene participates in DNA virus replication by regulating innate immunity remains unclear. This study reported that DDIT3 suppressed the innate immune response caused by DNA viruses to promote bovine herpesvirus 1 (BoHV-1) replication. After BoHV-1 infection of Madin-Darby bovine kidney (MDBK) cells, upregulated expression of DDIT3 induced SQSTM1-mediated autophagy and promoted STING degradation. Overexpression of the SQSTM1 protein effectively reduced STING protein levels, whereas SQSTM1 knockdown increased STING protein levels. Coimmunoprecipitation experiments and confocal laser scanning microscopy revealed that the SQSTM1 protein interacts with and colocalizes with STING. Knockdown of SQSTM1 expression in DDIT3-overexpressing cell lines restored STING protein levels. Moreover, a dual-luciferase reporter assay revealed that DDIT3 directly binds to the bovine SQSTM1 promoter and induces SQSTM1 transcription. Overexpression of SQSTM1 promoted BoHV-1 replication by inhibiting IFN-β and IFN-stimulated genes (ISGs) production; silencing of SQSTM1 promoted the expression of IFN-β and ISGs to inhibit BoHV-1 replication. In conclusion, DDIT3 targets STING via SQSTM1-mediated autophagy to promote BoHV-1 replication. These results suggest a novel mechanism by which DDIT3 regulates DNA virus replication by targeting innate immunity. DDIT3 antagonizes the innate immune response to promote bovine alphaherpesvirus 1 replication via the DDIT3-SQSTM1-STING pathway.

Chicoric Acid Attenuated Renal Tubular Injury in HFD-Induced Chronic Kidney Disease Mice through the Promotion of Mitophagy via the Nrf2/PINK/Parkin Pathway.

As the main factor in the pathogenesis of chronic kidney disease (CKD), the excessive apoptosis of renal tubular epithelial cells (RTECs) and its underlying mechanism of action are worth further investigation. Chicoric acid (CA), a major active constituent of the Uyghur folk medicine chicory, was recorded to possess a renal protective effect. The precise effect of CA on renal tubular injury in obesity-related CKD remains unknown. In the current study, CA was proven to ameliorate metabolic disorders including overweight, hyperglycemia, hyperlipidemia, and hyperuricemia in high fat diet (HFD)-fed mice. Furthermore, the reverse effect of CA on renal histological changes and functional damage was confirmed. In vitro, the alleviation of lipid accumulation and cell apoptosis was observed in palmitic acid (PA)-exposed HK2 cells. Treatment with CA reduced mitochondrial damage and oxidative stress in the renal tubule of HFD-fed mice and PA-treated HK2 cells. Finally, CA was observed to activate the Nrf2 pathway; increase PINK and Parkin expression; and regulate LC3, SQSTM1, Mfn2, and FIS1 expression; therefore, it would improve mitochondrial dynamics and mitophagy to alleviate mitochondrial damage in RTECs of obesity-related CKD. These results may provide fresh insights into the promotion of mitophagy in the prevention and alleviation of obesity-related CKD.

Mechanism of Electroacupuncture at Baihui and Shenting Alleviating Cerebral Ischemia-reperfusion Injury via Regulating BNIP3L-Mediated Mitophagy

<sec><title>Objective</title> The purpose of this study is to explore the mechanism of electroacupuncture at Baihui and Shenting acupoints to alleviate cerebral ischemia-reperfusion injury by regulating BNIP3L-mediated mitophagy. </sec><sec><title>Methods</title> A total of 60 SD rats were randomly divided into sham operation group (<italic>n</italic>=15) and operation group (<italic>n</italic>=45) by using the random number table method. Only the blood vessels were separated and exposed in the sham operation group, without ligation and no nylon thread inserted. The rats in the operation group were treated with the middle cerebral artery occlusion (MCAO) method to prepare the focal cerebral ischemia-reperfusion (I/R) model, and the Zea Longa method was used to score the neurological function 2h after the operation. Finally, 30 rats were included in the operation group. The included operation group rats were further randomly divided into the model group, electroacupuncture group, and electroacupuncture+3-MA group, with 10 rats in each group. After grouping, each group was treated with different methods such as electroacupuncture for 7 days, and the Zea Longa method was used to evaluate the neurological function again on the 1, 3, 5, and 7 days. After 7 days of intervention, the left cerebral cortex tissue of each group was obtained, hematoxylin-eosin (HE) staining was used to observe the pathological changes of the brain tissue, the expression level of LC3-Ⅱ/LC3-Ⅰ protein was detected by Western blot, and the tissue immunofluorescence technology was applied to detect the expression and colocalization of mitophagy-related protein BNIP3L and SQSTM1 labeled. The TUNEL method detected the apoptosis rate of rat nerve cells in each group. </sec><sec><title>Results</title> 1) The neurological scores of the rats in the operation group were significantly increased 2 hours after the MCAO, and the rats in the sham-operation group did not show symptoms of neurological deficit. On the 7th day after the intervention, the neurological scores of the rats in the model group and the electroacupuncture+3-MA group still increased significantly, which were significantly different from those in the sham operation group (<italic>P</italic><0.05). However, the neurological scores of rats in the electroacupuncture group reduced after electroacupuncture intervention, which was significantly different from the model group and electroacupuncture+3-MA group (<italic>P</italic><0.05). 2) The results of HE staining showed that the cell body of neurons in the sham operation group was large, polygonal (multiple protrusions), the nucleus was lightly colored, and the characteristic structure of Nissl body could be seen in the cytoplasm. In the model group, neurons shrank, the cytoplasmic structure was unclear, cytoplasmic eosinophilic staining was enhanced, Nissl bodies gathered or disappeared, nuclear pyknosis, and some cell bodies deformed and shrunk to triangles and the spaces around the cells widened. The electroacupuncture group could reduce the pathological damage caused by ischemia-reperfusion to a certain extent. 3) Western blot results showed that the autophagy level (LC3-Ⅱ/LC3-Ⅰ) of the model group decreased on the 7th day after MCAO, there wass a significant difference compared with the sham operation group (<italic>P</italic><0.05), while the electroacupuncture group could activate the level of autophagy, which was significantly different from the model group (<italic>P</italic><0.05). 4) In the model group, the level of mitophagy in the cortical infarction area was absent, as evidenced by the weakened red fluorescence and the intensity of green fluorescence. However, the intervention of electroacupuncture could activate the mitophagy of nerve cells in the infarct area, which was manifested by increased expression of red fluorescence and green fluorescence and enhanced colocalization (orange). 5) TUNEL test results indicated the apoptotic rate of rats in the model group rising, and there was a significant difference compared with the sham operation group (<italic>P</italic><0.05). However, the apoptotic rate of the electroacupuncture group decreased, which was significantly different from that of the model group (<italic>P</italic><0.05). The results of the electroacupuncture+3-MA group showed that the autophagy inhibitor 3-MA could reverse the neuroprotective effect of electroacupuncture in MCAO model rats. </sec><sec><title>Conclusion</title> Electroacupuncture at Baihui and Shenting acupoints can alleviate neurological damage in rats with cerebral ischemia-reperfusion. The specific mechanism is related to regulating BNIP3L-mediated mitophagy to reduce cerebral ischemia-reperfusion injury and cell apoptosis. </sec>

β-Elemene induces apoptosis and autophagy in colorectal cancer cells through regulating the ROS/AMPK/mTOR pathway.

β-Elemene is an effective anti-cancer ingredient extracted from the genus Curcuma (Zingiberaceae familiy). In the present study, we demonstrated that β-elemene inhibited the proliferation of colorectal cancer cells and induced cell cycle arrest in the G2/M phase. In addition, β-elemene induced nuclear chromatin condensation and cell membrane phosphatidylserine eversion, decreased cell mitochondrial membrane potential, and promoted the cleavage of caspase-3, caspase-9 and PARP proteins, indicating apoptosis in colorectal cancer cells. At the same time, β-elemene induced autophagy response, and the treated cells showed autophagic vesicle bilayer membrane structure, which was accompanied by up-regulation of the expression of LC3B and SQSTM1. Furthermore, β-elemene increased ROS levels in colorectal cancer cells, promoted phosphorylation of AMPK protein, and inhibited mTOR protein phosphorylation. In the experiments in vivo, β-elemene inhibited the tumor size and induced apoptosis and autophagy in nude mice. In summary, β-elemene inhibited the occurrence and development of colon cancer xenografts in nude mice, and significantly induced apoptosis and autophagy in colorectal cancer cells in vitro. These effects were associated with regulation of the ROS/AMPK/mTOR signaling. We offered a molecular basis for the development of β-elemene as a promising anti-tumor drug candidate for colorectal cancer.

SQSTM1-mediated clearance of cytoplasmic mutant TARDBP/TDP-43 in the monkey brain

ABSTRACT The cytoplasmic accumulation and aggregates of TARDBP/TDP-43 (TAR DNA binding protein) are a pathological hallmark in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We previously reported that the primate specific cleavage of TARDBP accounts for its cytoplasmic mislocalization in the primate brains, prompting us to further investigate how the cytoplasmic TARDBP mediates neuropathology. Here we reported that cytoplasmic mutant TARDBP reduced SQSTM1 expression selectively in the monkey brain, when compared with the mouse brain, by inducing SQSTM1 mRNA instability via its binding to the unique 3ʹUTR sequence (GU/UG)n of the primate SQSTM1 transcript. Overexpression of SQSTM1 could diminish the cytoplasmic C-terminal TARDBP accumulation in the monkey brain by augmenting macroautophagy/autophagy activity. Our findings provide additional clues for the pathogenesis of cytoplasmic TARDBP and a potential therapy for mutant TARDBP-mediated neuropathology.

SQSTM1 Expression in Hepatocellular Carcinoma and Relation to Tumor Recurrence After Radiofrequency Ablation

Autophagy is a process that allows the degradation of detrimental components through the lysosome to maintain cellular homeostasis under variable stimuli. SQSTM1 is a key molecule involved in functional autophagy and is linked to different signaling pathways, oxidative responses, and inflammation. Dysregulation of autophagy is reported in a broad spectrum of diseases. Accumulation of SQSTM1 reflects impaired autophagy, which is related to carcinogenesis and progression of various tumors, including hepatocellular carcinoma (HCC). This study investigated SQSTM1 protein expression in HCC and its relation to the clinicopathological features and the likelihood of tumor recurrence after radiofrequency ablation (RFA). This study included 50 patients with cirrhotic HCC of Barcelona Clinic Liver Cancer stages 0/A-B eligible for RFA. Tumor and peritumor biopsies were obtained just prior to local ablation and assessed for tumor pathological grade and SQSTM1 expression by immunohistochemistry. Patients were followed for one year after achieving complete ablation to detect any tumor recurrence. Serum alpha-fetoprotein level (U=149.50, P=0.027∗) and pathological grade of the tumor (χ2=12.702, P=0.002∗) associated significantly with the tumor response to RFA. SQSTM1 expression level was significantly increased in HCC compared to the adjacent peritumor cirrhotic liver tissues (Z=5.927, P<0.001∗). Significant direct relation was found between SQSTM1 expression level in HCC and the pathological grade of the tumor (H=33.789, P<0.001∗). On follow-up, tumor and peritumor SQSTM1 expression levels performed significantly as a potential predictor of the overall survival, but not the disease recurrence. SQSTM1 expression could determine aggressive HCC, even with reasonable tumor size and number, and identify the subset of HCC patients with short overall survival and unfavorable prognosis. SQSTM1 expression could not predict post-RFA intrahepatic HCC recurrence. SQSTM1 may be a potential biomarker and target for the selection of HCC patients for future therapies.

CRISPR/Cas9 genome-wide screening identifies LUC7L2 that promotes radioresistance via autophagy in nasopharyngeal carcinoma cells

Radioresistance emerges as the major obstacle to nasopharyngeal carcinoma (NPC) treatment, further understanding of underlying mechanisms is necessary to overcome the radioresistance and improve the therapeutic effect. In this study, we first identified a candidate radioresistant-related gene LUC7L2 via CRISPR/Cas9 high-throughput screening and quantitative proteomic approach. Overexpression of LUC7L2 in NPC cells promoted cell viability following exposure to ionizing radiation (IR), while knockdown of LUC7L2 significantly slowed down the DNA replication and impaired cell survival, sensitized NPC-radioresistant cells to IR. Using immunoprecipitation assay, we found SQSTM1, an autophagy receptor, was a potential binding partner of LUC7L2. Down-regulation of LUC7L2 in NPC-radioresistant cells led to reduction of SQSTM1 expression and enhancement of autophagy level. Furthermore, LUC7L2 knockdown in combination with autophagy inhibitor, chloroquine (CQ), resulted in more NPC-radioresistant cell death. Besides, LUC7L2 was obviously distributed in NPC tissues, and high LUC7L2 expression correlated with shorter survival in NPC patients. Our data suggest that LUC7L2 plays a huge part in regulating radioresistance of NPC cells, and serves as a promising therapeutic target in re-sensitizing NPC to radiotherapy.

Effect of EGFR on SQSTM1 Expression in Malignancy and Tumor Progression of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most common types of malignant tumor. Sequestosome 1 (SQSTM1) serves as an adaptor of autophagy for degrading protein aggregates. The regulation of autophagy by EGFR and its clinical impacts are indicated in various types of cancer. However, the association of EGFR and SQSTM1 in OSCC is still unknown. Our results show that the expression levels of SQSTM1 and EGFR proteins are higher in tumor tissues than in the corresponding tumor-adjacent (CTAN) tissues of OSCC patients. The expression levels of SQSTM1 were positively associated with the EGFR expression level. High co-expression of SQSTM1 and EGFR is associated with poor prognosis in OSCC patients. Moreover, SQSTM1 expression is decreased in EGFR-knockdown cells. Cell growth and invasion/migration are also decreased in cells with single/combined knockdowns of EGFR and SQSTM1 or in SQSTM1-knockdown cells without EGFR kinase inhibitor Lapatinib treatment compared to that in scrambled cells. However, cell growth and invasion/metastasis were not significantly different between the scrambled cells and SQSTM1-knockdown cells in the presence of Lapatinib. This study is the first to indicate the biological roles and clinical significance of SQSTM1 regulation by EGFR in OSCC.