Earle's Balanced Salt Solution Research Articles

Cezanne promoted autophagy through PIK3C3 stabilization and PIK3C2A transcription in lung adenocarcinoma

Osimertinib is a promising approved third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) for treating patients with lung adenocarcinoma (LUAD) harboring EGFR-activating mutations, however, almost all patients develop resistance to Osimertinib eventually limiting the long-term efficacy. Autophagy is a vital cellular recycling process promoting Osimertinib resistance. Identifying accurate and efficient autophagy-regulatory factors is of great significance in reducing Osimertinib resistance. This study identified Cezanne, a member of the ovarian tumor protease (OTU)-deubiquitinating family, as an autophagy regulator. Cezanne was highly expressed in Osimertinib-resistant cells, and Cezanne overexpression promoted Osimertinib resistance, while chloroquine (CQ), an autophagy inhibitor, reverted this process. In the Cezanne-overexpressing cells, autophagy was activated even in the absence of autophagy inducers rapamycin and Earle’s Balanced Salt Solution (EBSS). Further study showed that Cezanne stabilized PIK3C3 by deubiquitinating K48-linked ubiquitination at Lysine 322. Surprisingly, as a compensatory mechanism of PI3P generation, PIK3C2A was shown to be upregulated by Cezanne by promoting its transcription in a POLR2A-dependent way. Based on these results, Cezanne also accelerates EGFR recycling which may explain the mechanism mediating Cezanne expression and Osimertinib resistance. In conclusion, this study establishes a new model connecting Cezanne, autophagy, and Osimertinib resistance, opening new avenues to explore the effect of Cezanne and autophagy in LUAD.

Shielding AZ91D-1%Ca from corrosion through ultrasound melt treatment: A study for stent design

Magnesium-based materials show great potential for producing biodegradable stents, but their high corrosion rates are a roadblock.This study investigates whether ultrasound melt treatment can change the corrosion response of an extruded AZ91D-1.0%Ca (wt.%) in Earle's Balanced Salt Solution by tailoring the intermetallics' morphology in the as-extruded state.The results showed that the wires from ultrasound-treated ingots corroded faster than non-treated ones in immersion for up to 6 hours. This trend shifted for longer periods, and ultrasound-treated material showed lower corrosion rates and uniform corrosion, while the non-treated material displayed localized corrosion signs. Tensile testing of the wires demonstrated that immersion in EBSS lowered the tensile strength and elongation at fracture due to material degradation, regardless of the processing route. Nonetheless, this decline was sharper in the non-treated material.These findings suggest that ultrasound melt processing can be a promising method for improving the corrosion resistance of magnesium-based materials, paving the way for their use in manufacturing biodegradable stents.

Autophagy benefits the in vitro and in vivo clearance of Talaromyces marneffei

Talaromycosis, namely Talaromyces marneffei infection, is increasing gradually and has a high mortality rate even under antifungal therapy. Although autophagy acts differently on different pathogens, it is a promising therapeutic strategy. However, information on autophagy in macrophages and animals upon infection by T. marneffei is still limited. Therefore, several models were employed here to investigate the role of autophagy in host defense against T. marneffei, including RAW264.7 macrophages as in vitro models, different types of Caenorhabditis elegans and BALB/c mice as in vivo models. We applied the clinical T. marneffei isolate SUMS0152 in this study. T. marneffei-infected macrophages exhibit increased formation of autophagosomes. Further, macrophage autophagy promoted by rapamycin or Earle's balanced salt solution (EBSS) inhibited the viability of intracellular T. marneffei. In vivo, compared with uninfected Caenorhabditis elegans, the wild-type nematodes upregulated the expression of the autophagy-related gene lgg-1 and atg-18, and nematodes carrying GFP reporter were induced to form autophagosomes (GFP::LGG-1) after T. marneffei infection. Furthermore, the knockdown of lgg-1 significantly reduced the survival rate of T. marneffei-infected nematodes. Likewise, the autophagy activator rapamycin reduced the fungal burden and suppressed lung inflammation in a mouse model of infection. In conclusion, autophagy is essential for host defense against T. marneffei in vitro and in vivo. Therefore, autophagy may be an attractive target for developing new therapeutics to treat talaromycosis.

Silencing MYOT Expression May Inhibit Autophagy in Human Skeletal Muscle Cells.

Muscle diseases are closely related to autophagy disorders. Studies of autophagy inhibition indicated the importance of autophagy in muscle regeneration, while activation of autophagy can restore muscle function in some myopathies. Previous studies have revealed that mutations in the MYOT gene may lead to several kinds of hereditary myopathies. However, whether the autophagy played a crucial role in hereditary myopathy caused by MYOT mutations was still not clear. In this study, we established the MYOT knockdown human skeletal muscle cell models (HSkMCs) by small interfering RNA. Real-time PCR and Western blot studies found that the expression of p62 and LC3B-II was decreased dramatically, which suggested that silencing MYOT expression may regulate the autophagy in HSkMCs. Further immunofluorescence study on Ad-mCherry-GFP-LC3B adenovirus transfection and monodansylcadaverine (MDC) staining revealed that knocking down the expression of MYOT may inhibit the autophagy. Next, we used the autophagy inducer Earle's balanced salt solution (EBSS) and late-autophagy inhibitor bafilomycin A1 (BAF A1) to treat the HSkMCs, respectively, and found that silencing MYOT expression can inhibit the activation of autophagy by EBSS and aggravate the inhibition of autophagy by BAF A1. Finally, we also found that silencing MYOT expression can downregulate the expression of ATG7 and ATG5, two important autophagy regulatory molecules. Hence, our study may first reveal that knocking down the expression of MYOT may inhibit the autophagy. Hereditary myopathies caused by MYOT mutations may partly result from the inhibition of autophagy in HSkMCs.

Regulation of reactive oxygen species on the mitophagy of human periodontal ligament cells through the PINK1/Parkin pathway under starvation.

This study aimed to explore the specific mechanism, mediated by the reactive oxygen species (ROS) and PINK1/Parkin pathway, of the mitochondrial autophagy of human periodontal ligament cells (hPDLCs) under starvation conditions. hPDLCs were isolated and cultured from normal periodontal tissues. Earle's balanced salt solution (EBSS) was used to simulated a starvation environment and thus stimulate hPDLCs mitochondrial autophagy. N-Acetyl-L-cysteine (NAC) was used to inhibit ROS production to explore the role of ROS in hPDLC mitochondrial autophagy. Cyclosporin A was used to inhibit the PINK1/Parkin pathway to study the role of ROS and the PINK1/Parkin pathway in hPDLCs activation under starvation. The mitochondrial membrane potential was detected by flow cytometry with a JC-1 mitochondrial membrane potential detection kit. The morphological structure of mitochondria and the formation of mitochondrial autophagosome were observed by transmission electron microscopy. Mito tracker red cmxros and lyso tracker green staining were used to observe the localization of mitochondria and lysosomes. The formation intensity of ROS was detected with a DCFH-DA ROS fluorescent probe. The expression levels of mitochondrial autophagy genes (Tomm20 and Timm23) and the PINK1/Parkin pathway were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The expression levels of mitochondrial autophagy proteins (Tomm20 and Timm23) and PINK1/Parkin protein were detected by Western blot. EBSS starvation for 30 min induced the strongest activation of hPDLCs mitochondrial autophagy, increased the expression of ROS, downregulated the expression of mitochondrial autophagy-related genes (Tomm20 and Timm23) (P<0.001), and upregulated the PINK1/Parkin pathway (P<0.001). After NACinhibited ROS production, mitochondrial autophagy was also inhibited. Meanwhile, the expression of Tomm20 and Timm23 was upregulated (P<0.001 and P<0.05), and the expression of the PINK1/parkin pathway (P<0.001 and P<0.05) was down regulated. When cyclosporin A inhibited the expression of the PINK1/Parkin pathway (P<0.05 and P<0.05), it reversed the mitochondrial autophagy of hPDLCs (P<0.001 and P<0.01) and also upregulated the expression of Tomm20 and Timm23 (P<0.001 and P<0.01). ROS enhanced the mitochondrial autophagy of hPDLCs primarily through the PINK1/Parkin pathway under starvation conditions.

Neuroprotective Effects of a Cardioplegic Combination (Adenosine, Lidocaine, and Magnesium) in an Ischemic Stroke Model.

Adenosine, lidocaine, and magnesium (ALM) are clinically available cardioplegic solutions. We examined the effects of low-dose ALM on ischemic stroke in cell and animal models. Cobalt chloride (CoCl2)-treated SH-SY5Y cells were used as a surrogate model to mimic oxygen-glucose deprivation conditions. The cells were incubated with different dilutions of ALM authentic solution (1.0mM adenosine, 2.0mM lidocaine, and5 mM MgSO4 in Earle's balanced salt solution). At a concentration of 2.5%, ALM significantly reduced CoCl2-induced cell loss. This protective effect persisted even when ALM was administered 1h after the insult. We used transient middle cerebral artery occlusion to investigate the therapeutic effects of ALM in vivo. Rats were randomly assigned to two groups-the experimental (ALM) and control (saline) groups-and infusion was administered during the ischemia for 1h. The infarction area was significantly reduced in the ALM group compared with the control group (5.0% ± 2.0% vs. 23.5% ± 5.5%, p = 0.013). Neurological deficits were reduced in the ALM group compared with the control group (modified Longa score: 0 [0-1] vs. 2 [1-2], p = 0.047). This neuroprotective effect was substantiated by a reduction in the levels of various neuronal injury markers in plasma. These results demonstrate the neuroprotective effects of ALM and may provide a new therapeutic strategy for ischemic stroke.

Chaperone-mediated autophagy attenuates H2 O2 -induced cardiomyocyte apoptosis by targeting poly (ADP-ribose) polymerase 1 (PARP1) for lysosomal degradation.

Poly (ADP-ribose) polymerase 1 (PARP1)is a typical representative of the PARP enzyme family and is mainly related to DNA repair, gene transcription regulation, inflammation, and oxidative stress. Studies have found that PARP1 is involved in the pathophysiological processes of a variety of cardiovascular diseases. Chaperone-mediated autophagy (CMA)is involved in the molecular regulation of various diseases, including cardiovascular diseases, and plays a critical role in maintaining intracellular metabolism balance. However, the link between PARP1 and CMA in cardiomyocytes remains unclear. Therefore, the aims of this study were to investigate whether CMA is involved in PARP1 regulation and to further clarify the specific molecular mechanisms. Earle's balanced salt solution(EBSS)-induced activation of autophagy reduced PARP1 expression, whereas the autophagy lysosomal inhibitor CQ had the opposite effect. Correspondingly, treatment with the autophagy inhibitor 3-methyladeninedid not abolish the autophagy-inducing effects of EBSS. Additionally, PARP1 binds to heat shock cognate protein 70and lysosome-associated membrane protein 2A(LAMP2A). Moreover, adenovirus-mediated LAMP2A overexpression to activate the CMA signaling pathway in cardiomyocytes reduces PARP1 (cleaved) expression and further decreases cardiomyocyte apoptosis caused by oxidative stress. In contrast, downregulation of LAMP2A increased PARP1 (cleaved) expression and the degree of apoptosis. More importantly, we report that appropriate concentrations of H2 O2 triggered the nuclear translocation of PARP1, which subsequently promoted the degradation of PARP1 through the CMA pathway. In summary, our data are the first to reveal that CMA targeted PARP1 for lysosomal degradation in cardiomyocytes, which ultimately inhibited apoptosis by promoting the degradation of the PARP1 protein.

CRHBP is degraded via autophagy and exerts anti-hepatocellular carcinoma effects by reducing cyclin B2 expression and dissociating cyclin B2-CDK1 complex.

Autophagy is the predominant self-eating catabolic pathway activated in response to nutrient starvation and hypoxia within the microenvironment of varied malignancies, including hepatocellular carcinoma (HCC). SQSTM1/p62 links its cargos to autophagosomes for degradation, and reportedly acts as a contributor for hepatocarcinogenesis. Five GEO gene microarrays identified corticotropin releasing hormone (CRH) binding protein (CRHBP) as a significantly downregulated gene in HCC (log2 Fold change < -3 and p < 0.001), and an earlier human interactome study indicated that CRHBP may interact with p62. This study aimed to explore (1) the role of CRHBP in HCC development, and (2) whether p62-mediated autophagy was responsible for low CRHBP expression within HCC tissue. Following functional experiments first revealed an anti-proliferative, anti-metastatic, and anti-angiogenic role of CRHBP in HCC cells (Huh-7, Li-7 and HCCLM3) and xenografts. CRHBP negatively regulated cyclin B2 expression, and dissociated cyclin B2-CDK1 complex in HCC cells, thereby leading to cell cycle arrest at G2 phase. To simulate HCC microenvironment in vitro, Huh-7 cells were incubated in Earle's Balanced Salt Solution (nutrient starvation) or exposed to 1% O2 (hypoxic exposure). In addition to activating autophagy, nutrient starvation and hypoxic exposure also induced CRHBP degradation. Interestingly, CRHBP was demonstrated as a novel cargo targeted by p62 for degradation in autophagosomes. Blocking autophagy with 3-MA, chloroquine or siSQSTM1 prevented CRHBP degradation in HCC cells. Collectively, our study uncovers a role for CRHBP in retarding HCC development, reducing cyclin B2 expression and impairing cyclin B2-CDK1 interaction. CRHBP downregulation in HCC may attribute to p62-mediated autophagy.

PCV2 targets cGAS to inhibit type I interferon induction to promote other DNA virus infection.

Viruses use diverse strategies to impair the antiviral immunity of host in order to promote infection and pathogenesis. Herein, we found that PCV2 infection promotes the infection of DNA viruses through inhibiting IFN-β induction in vivo and in vitro. In the early phase of infection, PCV2 promotes the phosphorylation of cGAS at S278 via activation of PI3K/Akt signaling, which directly silences the catalytic activity of cGAS. Subsequently, phosphorylation of cGAS at S278 can facilitate the K48-linked poly-ubiquitination of cGAS at K389, which can been served as a signal for recognizing by the ubiquitin-binding domain of histone deacetylase 6 (HDAC6), to promote the translocation of K48-ubiquitinated-cGAS from cytosol to autolysosome depending on the deacetylase activity of HDAC6, thereby eventually resulting in a markedly increased cGAS degradation in PCV2 infection-induced autophagic cells relative to Earle's Balanced Salt Solution (EBSS)-induced autophagic cells (a typical starving autophagy). Importantly, we found that PCV2 Cap and its binding protein gC1qR act as predominant regulators to promote porcine cGAS phosphorylation and HDAC6 activation through mediating PI3K/AKT signaling and PKCδ signaling activation. Based on this finding, gC1qR-binding activity deficient PCV2 mutant (PCV2RmA) indeed shows a weakened inhibitory effect on IFN-β induction and a weaker boost effect for other DNA viruses infection compared to wild-type PCV2. Collectively, our findings illuminate a systematic regulation mechanism by which porcine circovirus counteracts the cGAS-STING signaling pathway to inhibit the type I interferon induction and promote DNA virus infection, and identify gC1qR as an important regulator for the immunosuppression induced by PCV2.

Intermittent Starvation Promotes Maturation of Human Embryonic Stem Cell-Derived Cardiomyocytes.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) represent an infinite cell source for cardiovascular disease modeling, drug screening and cell therapy. Despite extensive efforts, current approaches have failed to generate hPSC-CMs with fully adult-like phenotypes in vitro, and the immature properties of hPSC-CMs in structure, metabolism and electrophysiology have long been impeding their basic and clinical applications. The prenatal-to-postnatal transition, accompanied by severe nutrient starvation and autophagosome formation in the heart, is believed to be a critical window for cardiomyocyte maturation. In this study, we developed a new strategy, mimicking the in vivo starvation event by Earle’s balanced salt solution (EBSS) treatment, to promote hPSC-CM maturation in vitro. We found that EBSS-induced starvation obviously activated autophagy and mitophagy in human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Intermittent starvation, via 2-h EBSS treatment per day for 10 days, significantly promoted the structural, metabolic and electrophysiological maturation of hESC-CMs. Structurally, the EBSS-treated hESC-CMs showed a larger cell size, more organized contractile cytoskeleton, higher ratio of multinucleation, and significantly increased expression of structure makers of cardiomyocytes. Metabolically, EBSS-induced starvation increased the mitochondrial content in hESC-CMs and promoted their capability of oxidative phosphorylation. Functionally, EBSS-induced starvation strengthened electrophysiological maturation, as indicated by the increased action potential duration at 90% and 50% repolarization and the calcium handling capacity. In conclusion, our data indicate that EBSS intermittent starvation is a simple and efficient approach to promote hESC-CM maturation in structure, metabolism and electrophysiology at an affordable time and cost.

Autophagy promotes invadopodia formation in human ovarian cancer cells via the p62-extracellular signal-regulated kinase 1/2 pathway.

Invasiveness and metastatic potential are among the most essential characteristics of malignant tumors. Furthermore, it has been reported that autophagy and invasion are enhanced when tumor cells are grown in adverse conditions, such as nutritional deficiency and starvation. However, the association between autophagy and invasion remains largely unclear. In the present study, Earle's balanced salt solution (EBSS) was used to induce autophagy and an autophagy inhibitor was used to block autophagy. The results of Transwell assays revealed that autophagy inhibition limited the invasiveness of human ovarian cancer cells. Furthermore, the results of invadopodia formation assay indicated that autophagy stimulated invadopodia formation, and the selective autophagy receptor and signaling adaptor, sequestosome-1 (SQSTM1/p62 or simply p62), was closely associated with invadopodia formation in human ovarian cancer SKOV3 cells. The results of western blot analysis indicated that autophagy induced changes in p62 protein levels and p62 then functioned as a negative regulator of extracellular signal-regulated kinase 1/2 (ERK1/2) activity and invadopodia formation. The interaction between autophagy and invasion may thus be a self-protective mechanism for tumor cells in an unfavorable environment of nutritional deficiency, that maintains their survival and leads to increased invasiveness. An exploration of the intrinsic link between autophagy and invasion may provide a novel theoretical basis to reverse the resistance of tumor cells to a nutritional deficient environment.

β-Arrestin inhibition induces autophagy, apoptosis, G0/G1 cell cycle arrest in agonist-activated V2R receptor in breast cancer cells

Non-visual arrestins (β-arrestins) are endocytic proteins that mediate agonist-activated GPCRs internalization and signaling pathways in an independent manner. The involvement of β-arrestins in cancer invasion and metastasis is increasingly reported. So, it is hypothesized that inhibition of β-arrestins may diminish the survival chances of cancer cells. This study aimed to evaluate the in vitro impact of inhibiting β-arrestins on the autophagic and/or apoptotic responsiveness of breast cancer cells. We used Barbadin to selectively inhibit β-Arr/AP2 interaction in AVP-stimulated V2R receptor of triple-negative breast cancer cells (MDA MB-231). Autophagy was assessed by the microtubule-associated protein 1 light chain 3-II (LC3II), apoptosis was measured by Annexin-V/PI staining and cell cycle distribution was investigated based upon the DNA content using flow cytometry. Barbadin reduced cell viability to 69.1% and increased the autophagy marker LC3II and its autophagic effect disappeared in cells transiently starved in Earle's balanced salt solution (EBSS). Also, Barbadin mildly enhanced the expression of P62 mRNA and arrested 63.7% of cells in G0/G1 phase. In parallel, the drug-induced apoptosis in 29.9% of cells (by AV/PI) and 27.8% of cells were trapped in sub-G1 phase. The apoptotic effect of Barbadin was enhanced when autophagy was inhibited by the PI3K inhibitor (Wortmannin). Conclusively, the data demonstrate the dual autophagic and apoptotic effects of β-βArr/AP2 inhibition in triple-negative breast cancer cells. These observations nominate β-Arrs as selective targets in breast cancer treatment.

Regulation of FN1 degradation by the p62/SQSTM1-dependent autophagy\u2013lysosome pathway in HNSCC

Epithelial–mesenchymal transition (EMT) is involved in both physiological and pathological processes. EMT plays an essential role in the invasion, migration and metastasis of tumours. Autophagy has been shown to regulate EMT in a variety of cancers but not in head and neck squamous cell carcinoma (HNSCC). Herein, we investigated whether autophagy also regulates EMT in HNSCC. Analyses of clinical data from three public databases revealed that higher expression of fibronectin-1 (FN1) correlated with poorer prognosis and higher tumour pathological grade in HNSCC. Data from SCC-25 cells demonstrated that rapamycin and Earle’s balanced salt solution (EBSS) promoted autophagy, leading to increased FN1 degradation, while 3-methyladenine (3-MA), bafilomycin A1 (Baf A1) and chloroquine (CQ) inhibited autophagy, leading to decreased FN1 degradation. On the other hand, autophagic flux was blocked in BECN1 mutant HNSCC Cal-27 cells, and rapamycin did not promote autophagy in Cal-27 cells; also in addition, FN1 degradation was inhibited. Further, we identified FN1 degradation through the lysosome-dependent degradation pathway using the proteasome inhibitor MG132. Data from immunoprecipitation assays also showed that p62/SQSTM1 participated as an autophagy adapter in the autophagy–lysosome pathway of FN1 degradation. Finally, data from immunoprecipitation assays demonstrated that the interaction between p62 and FN1 was abolished in p62 mutant MCF-7 and A2780 cell lines. These results indicate that autophagy significantly promotes the degradation of FN1. Collectively, our findings clearly suggest that FN1, as a marker of EMT, has adverse effects on HNSCC and elucidate the autophagy–lysosome degradation mechanism of FN1.

Autophagy modulates mesenchymal-to-endothelial transition via p53.

Mesenchymal-to-endothelial transition (MEndT) is one of the mechanisms that influences cardiac fibrosis, which is a key process in cardiac remodeling. It has been reported that autophagy inhibits endothelial cell transition. However, whether autophagy could modulate MEndT in cardiac fibrosis has not yet been investigated. Here, we discussed the association between autophagy and MEndT and its possible mechanism. In this study, we induced endothelial-to-mesenchymal transition using transforming growth factor-β to generate mesenchymal cells and fibroblasts in wild-type human umbilical vein endothelial cells and cells with p53 knockout or overexpression. Then, autophagy was induced by Earle's balanced salt solution (EBSS) and was inhibited by bafilomycin A1 or lentivirus-ATG5-shRNA. The expression levels of MEndT and the autophagy markers CD31, VE-Cadherin, Vimentin, α-SMA, LC3, p62 and p53 were examined. We found that activation of autophagy could promote MEndT and increase cytoplasmic and total expression of p53, that but nuclear p53 expression was decreased, and that inhibition of autophagy activation could reverse the effect of EBSS. Moreover, after knockout of nuclear p53, autophagy promoted MEndT, while autophagy inhibited MEndT in p53 overexpressing cells. Our results demonstrate that autophagy modulate MEndT by nuclear p53 provide a new strategy for the treatment of fibrosis diseases.

WW and C2 domain-containing protein-3 promoted EBSS-induced apoptosis through inhibiting autophagy in non-small cell lung cancer cells.

BackgroundWW and C2 domain-containing protein-3 (WWC3) was identified in our previous studies as a tumor suppressor gene, which inhibits the proliferation and invasiveness of lung cancer cells. However, the relationship between WWC3 and autophagy and apoptosis in lung cancer cells is unclear. In this study, we aimed to investigate the potential role of WWC3 in starvation-induced autophagy and apoptosis in non-small cell lung carcinoma (NSCLC) cells.MethodsThe immunoblotting assay and quantitative real-time polymerase chain reaction (RT-qPCR) were used for observing the change of WWC3 protein and mRNA level under starvation condition. The immunoblotting assay and immunofluorescence assay were performed to detect the impact of WWC3 expression on autophagy process induced by Earle’s balanced salt solution (EBSS) in lung cancer cells; APC/propidium iodide (PI) apoptosis assay, caspase-3/7 activity assay and MTT assay were used for the apoptosis and proliferation detection of lung cancer cells.ResultsAfter starvation had been induced with EBSS, WWC3 expression was significantly decreased in the NSCLC cells. Ectopic WWC3 expression weakened the autophagy process in a Beclin1-independent manner and promoted non-small cell lung cancer cell apoptosis via EBSS starvation. Moreover, the inhibition of WWC3 gene knockout was weakened by 3-methyladenine (3-MA), an autophagy inhibitor.ConclusionsThese results indicate that WWC3 promotes apoptosis and death of starved lung cancer cells, at least partly through autophagy.

The molecular basis for p53 inhibition of autophagy in porcine fibroblast cells

Background: Autophagy regulation involves an intricate network that can degrade and recycle cytosolic components in autophagosomes when cells are subject to various stress signals. p53 plays a dual role of induction or inhibition in the regulation of autophagy. Recently, pigs have been considered an excellent large animal model for their many anatomical and physiological similarities to humans. Here, we investigated the relationship between p53 and autophagy, as well as the underling molecular basis, in porcine fibroblast cells (PFCs). Methods: Autophagy was induced by Earle’s balanced salt solution (EBSS) in p53 -/- and p53 wt PFCs. The autophagy response was assessed by immunoblotting, transmission electron microscopy (TEM) and fluorescent staining. The molecular basis for p53 regulation of autophagy was analyzed by qPCR. Results: We found that the increased expression of LC3-II and the decreased expression of P62 occurred earlier in p53 -/- PFCs than in p53 wt PFCs, the relative autophagic flux of p53 -/- PFCs was stronger than that of p53 wt PFCs in a time-dependent manner. Meanwhile, we observed a visualized increase of autophagosomes in p53 -/- PFCs. Moreover, we found greater accumulation of LC3 punctate and acidic vesicular organelle (AVOs) in the cytoplasm of p53 -/- PFCs than in that of p53 wt PFCs, and these effects were further augmented by Baf A1 treatment. Furthermore, we detected the expression of 6 autophagy signaling pathway-related genes and 14 autophagy-related ( ATG ) genes by qPCR. We found that the expression patterns of the 6 genes had significant differences in both groups of treated PFCs. These results demonstrated that p53 negatively regulated autophagy and involving the downregulation of LMNA gene by p53 via an unknown pathway, which causes the upregulation of the LC3, ULK1, ATG4B, ATG16L1 and ATG9A genes and the downregulation of the P62 gene. Conclusions: p53 -/- PFCs responded to autophagy earlier than p53 wt PFCs, which implied that p53 might inhibit autophagy. The expression patterns of autophagy signaling pathway-related genes and ATG genes were most different between p53 -/- and p53 wt PFCs. Our study will provide a new biological model and contribute to further study of the molecular basis for p53 and autophagy.

The molecular basis for p53 inhibition of autophagy in porcine fibroblast cells.

BackgroundAutophagy regulation involves an intricate network that can degrade and recycle cytosolic components in autophagosomes when cells are subject to various stress signals. p53 plays a dual role of induction or inhibition in the regulation of autophagy. Recently, pigs have been considered an excellent large animal model for their many anatomical and physiological similarities to humans. Here, we investigated the relationship between p53 and autophagy, as well as the underling molecular basis, in porcine fibroblast cells (PFCs).MethodsAutophagy was induced by Earle’s balanced salt solution (EBSS) in p53-/- and p53wt PFCs. The autophagy response was assessed by immunoblotting, transmission electron microscopy (TEM) and fluorescent staining. The molecular basis for p53 regulation of autophagy was analyzed by qPCR.ResultsWe found that the increased expression of LC3-II and the decreased expression of P62 occurred earlier in p53-/- PFCs than in p53wt PFCs, the relative autophagic flux of p53-/- PFCs was stronger than that of p53wt PFCs in a time-dependent manner. Meanwhile, we observed a visualized increase of autophagosomes in p53-/- PFCs. Moreover, we found greater accumulation of LC3 punctate and acidic vesicular organelle (AVOs) in the cytoplasm of p53-/- PFCs than in that of p53wt PFCs, and these effects were further augmented by Baf A1 treatment. Furthermore, we detected the expression of 6 autophagy signaling pathway-related genes and 14 autophagy-related (ATG) genes by qPCR. We found that the expression patterns of the 6 genes had significant differences in both groups of treated PFCs. These results demonstrated that p53 negatively regulated autophagy and involving the downregulation of LMNA gene by p53 via an unknown pathway, which causes the upregulation of the LC3, ULK1, ATG4B, ATG16L1 and ATG9A genes and the downregulation of the P62 gene.Conclusionsp53-/- PFCs responded to autophagy earlier than p53wt PFCs, which implied that p53 might inhibit autophagy. The expression patterns of autophagy signaling pathway-related genes and ATG genes were most different between p53-/- and p53wt PFCs. Our study will provide a new biological model and contribute to further study of the molecular basis for p53 and autophagy.

Starvation-induced autophagy is up-regulated via ROS-mediated ClC-3 chloride channel activation in the nasopharyngeal carcinoma cell line CNE-2Z.

Nutrient deficiency develops frequently in nasopharyngeal carcinoma cell (CNE-2Z) due to the characteristics of aggregation and uncontrolled proliferation. Therefore, starvation can induce autophagy in these cells. Chloride channel 3 (ClC-3), a member of the chloride channel family, is involved in various biological processes. However, whether ClC-3 plays an important role in starvation-induced autophagy is unclear. In this study, Earle's balanced salt solution (EBSS) was used to induce autophagy in CNE-2Z cells. We found that autophagy and the chloride current induced by EBSS were inhibited by chloride channel blockers. ClC-3 knockdown inhibited the degradation of LC3-II and P62. Furthermore, when reactive oxygen species (ROS) generation was suppressed by antioxidant N-acetyl-l-cysteine (L-NAC) pretreatment, EBSS-induced autophagy was inhibited, and the chloride current was unable to be activated. Nevertheless, ClC-3 knockdown had little effect on ROS levels, indicating that ROS acted upstream of ClC-3 and that both ROS and ClC-3 participated in EBSS-induced autophagy regulation in CNE-2Z.

SAT-LB064 Mitotane Induces Autophagy: A Mechanism to Promote Chemoresistance in Adrenocortical Carcinoma

Adrenocortical carcinoma (ACC) is an orphan malignancy with dismal survival rates. Although recent large genomic studies have shed insight into underlying molecular mechanisms, there are currently no targeted therapies available. Autophagy is an important cellular process to promote survival in a low nutrient tumor microenvironment or in the setting of physiologic stimuli such as endoplasmic reticulum stress (ER stress). In some settings, autophagy can result in drug resistance to cytotoxic chemotherapies and tyrosine kinase inhibitors. The role of autophagy in ACC has not been well characterized. Previous research in H295R ACC cells has shown that mitotane, the only FDA approved drug for ACC, promotes cell death in part through ER stress. Since autophagy has been shown to promote resistance to ER stress-mediated cell death, we hypothesized that autophagy may be activated in ACC cells as a compensatory mechanism, promoting cell survival in the face of mitotane treatment. CU-ACC2 cells demonstrated functional autophagy in response to Earle’s Balanced Salt Solution (EBSS), a potent stimulant of autophagy, indicated by a significant increase in the autophagy mediator LC3-II, as detected by immunoblot and enhanced autophagic flux detected by flow cytometry in cells expressing a tandem LC3-mCherry-GFP construct. To test if mitotane induces ER stress in our novel cell lines, qPCR was performed examining the expression of several ER stress pathway components including CHOP and XBP1 cleavage. In agreement with previous data in H295R ACC cells, there was significant cleavage of XBP1 and increased expression of CHOP in response to mitotane treatment in CU-ACC2, confirming ER stress induction. Mitotane also induced autophagy, as measured by LC3 detection by immunoblot as well as flow cytometry. ER stress induction was detected as early as 6 hours, while autophagy induction occurred later and peaked at 24 hours suggesting that ER stress precedes induction of autophagy in ACC cells. A functional effect of autophagy induction after mitotane treatment was demonstrated by treatment with mitotane with or without the autophagy inhibitor, chloroquine. Significantly more cleaved PARP, suggesting greater apoptosis, was detected after 24 and 48 hours of treatment with combination therapy than with than with either treatment alone. Together, these data suggest that mitotane activates ER stress to trigger apoptosis, but this treatment also activates autophagy, which abrogates the toxic effects. Thus, inhibition of the autophagy pathway may be a potential therapeutic adjunct to mitotane therapy in patients with ACC. Funding: VA Merit, Cancer League of Colorado, NIH K08, NIH R01 to AT. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

AMPK Activation Improves Survivability after Toxic Gas Exposure

IntroductionInhalation injury caused by irritant gases such as smoke, ozone, chlorine and bromine (Br2) results in an environmental and occupational hazards that leads to reactive airway disease syndrome (RADS), acute lung injury (ALI), and even death from respiratory failure. Development of interventional therapeutic agents, which when given post exposure, prevent acute inhalation injury and decrease mortality are urgently needed. AMP‐activated protein kinase (AMPK) is a metabolic sensor that maintains cellular ATP levels during metabolic stress. AMPK also acts independently of AMP and enhances vascular integrity by preventing actin stress fiber formation and limiting changes in cell morphology necessary for increased endothelial permeability. Here in the current study we tested the hypothesis that a single injection of an AMPK activator, such as 5‐aminoimidazole‐4‐carboxamide‐1‐beta‐D‐ribofuranoside (AICAR), administered intraperitoneally to C57BL/6 mice, post exposure to a lethal dose of inhaled Br2, decreases lung injury and mortality.MethodsC57BL/6 mice were exposed to Br2 (600 ppm, 45min) in environmental glass chambers and then returned to room air. Control mice breathed ambient air throughout this protocol. Six hours after exposure to Br2, mice were treated with either AICAR (10 mg/kg BW, intraperitoneally) or vehicle (Earle's balanced salt solution. Next seven days mice were monitored at interval of six hours. In another set of experiments, mice were sacrificed 24 hours post Br2 inhalation and were either lavaged with one ml of normal saline for protein and inflammatory cell counts or their lungs were fixed by instillation of formalin at 25 cm H20; and processed for histology.ResultsThe analysis of the Kaplan Meier survival curve showed that Br2 exposed mice developed progressive respiratory distress. Median survival after Br2 exposure was one day. However, mice receiving AICAR had a median survival of six days. Exposure to Br2 caused significant increase in total protein and number of inflammatory cells in BALF at 24 hours post exposure, consistent with severe injury to the blood gas barrier. AICAR returned these values to their corresponding air controls. Br2 exposure significantly reduced the level of AMPK phosphorylation whereas AICAR treatment brought its level to normal in animal lungs. Lung H&amp;E staining showed that AICAR attenuated Br2‐induced lung edema, alveolar congestion, septal thickening, neutrophil infiltration, hyaline membrane and perivascular cuffing. Results also showed that AICAR enhanced autophagy, which was significantly reduced after Br2 exposure suggesting a possible mechanism of AICAR‐mediated cyto‐protection.ConclusionsIn conclusion data suggest that activation of lung AMPK might be a useful target for a therapeutic intervention to reverse Br2‐induced acute lung injury.Support or Funding InformationThis study was supported by 1U01ES027697‐01 and 5U01ES026458‐02 to S. Matalon.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.