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![Effect of compound C and AMPK DN on autophagic proteolysis in HeLa cells. A, HeLa cells were co-transfected with the cDNAs encoding GFP-LKB1 and FLAG-STRAD (T) or with control vectors (C). LKB1 and STRAD were detected by immunoblotting with an anti-GFP and an anti-FLAG, respectively, in co-transfected cells. HeLa LKB1/STRAD cells were transfected with the cDNA encoding Myc-AMPK DN (T1) or with control vector (C1). The transfected protein was detected by immunoblotting with the anti-c-Myc. B, the AMPK-dependent phosphorylation of ACC was detected by the anti-ACC/phospho-Ser 79 in complete medium (CM) and nutrientfree medium (HBSS). The quantification of ACC phosphorylation was expressed by the ratio of ACC-P per total actin in arbitrary units. When required AICAR and CC were added at 250 M and 40 M, respectively. C, [ 14 C]valine-labeled protein degradation in HeLa cells, HeLa LKB1/STRAD, and HeLa LKB1/STRAD/AMPK DN cells. After labeling overnight with radioactive valine, long-lived protein degradation was measured as described under "Experimental Procedures" in complete medium (CM) and nutrient-free medium (HBSS). Cells were used 48-h post-transfection. When required, amino acids (AA, 4x), 3-MA (10 mM), and CC (40 M) were added to the chase medium. Values are the mean S.E. of three independent experiments. *, p 0.05 versus control cells and HeLa LKB1/STRAD cells incubated in nutrient-free medium.](profile/Alfred-Meijer/publication/6804749/figure/fig5/AS:669272615419924@1536578434896/Effect-of-compound-C-and-AMPK-DN-on-autophagic-proteolysis-in-HeLa-cells-A-HeLa-cells.png)
Effect of compound C and AMPK DN on autophagic proteolysis in HeLa cells. A, HeLa cells were co-transfected with the cDNAs encoding GFP-LKB1 and FLAG-STRAD (T) or with control vectors (C). LKB1 and STRAD were detected by immunoblotting with an anti-GFP and an anti-FLAG, respectively, in co-transfected cells. HeLa LKB1/STRAD cells were transfected with the cDNA encoding Myc-AMPK DN (T1) or with control vector (C1). The transfected protein was detected by immunoblotting with the anti-c-Myc. B, the AMPK-dependent phosphorylation of ACC was detected by the anti-ACC/phospho-Ser 79 in complete medium (CM) and nutrientfree medium (HBSS). The quantification of ACC phosphorylation was expressed by the ratio of ACC-P per total actin in arbitrary units. When required AICAR and CC were added at 250 M and 40 M, respectively. C, [ 14 C]valine-labeled protein degradation in HeLa cells, HeLa LKB1/STRAD, and HeLa LKB1/STRAD/AMPK DN cells. After labeling overnight with radioactive valine, long-lived protein degradation was measured as described under "Experimental Procedures" in complete medium (CM) and nutrient-free medium (HBSS). Cells were used 48-h post-transfection. When required, amino acids (AA, 4x), 3-MA (10 mM), and CC (40 M) were added to the chase medium. Values are the mean S.E. of three independent experiments. *, p 0.05 versus control cells and HeLa LKB1/STRAD cells incubated in nutrient-free medium.
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Interruption of mTOR-dependent signaling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast.
Because activation of AMPK also inhibits mTOR-dependent signaling one would expect stimulation of autophagy by AMPK activation.
According to the literature, this is true for yeast but, unexpectedly, not for mammalian cells on...
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... In this cell line, which does not express the AMPK kinase LKB1 (28), AICAR is not able to stimulate AMPK unless LKB1 is expressed (Fig. 8B and Ref. 29). LKB1 is present in a complex with the regula- tory proteins STRAD and MO25 (29). As the amount of STRAD is low in HeLa cells (29), we co-transfected HeLa cells with cDNAs encoding LKB1 and STRAD (Fig. 8A). In transfected cells we observed an increase in AMPK activity, which was further stimulated in the presence of AICAR (Fig. 8B and Ref. 29). Next we have analyzed the rate of degrada- tion of long-lived [ 14 C]valine-labeled proteins in cells trans- fected with cDNAs encoding LKB1 and STRAD and in con- trol HeLa cells (cells ...
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... observed an increase in AMPK activity, which was further stimulated in the presence of AICAR (Fig. 8B and Ref. 29). Next we have analyzed the rate of degrada- tion of long-lived [ 14 C]valine-labeled proteins in cells trans- fected with cDNAs encoding LKB1 and STRAD and in con- trol HeLa cells (cells transfected with empty vectors). As shown in Fig. 8C, in both cell lines amino acid-and 3-MA- sensitive autophagic proteolysis was stimulated in HBSS. However, no significative difference in the rate of starvation- stimulated autophagic proteolysis was observed depending on the activity of AMPK. Although the activity of AMPK was not stimulated by AICAR, an inhibitory effect of AICAR on ...
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... 3-MA- sensitive autophagic proteolysis was stimulated in HBSS. However, no significative difference in the rate of starvation- stimulated autophagic proteolysis was observed depending on the activity of AMPK. Although the activity of AMPK was not stimulated by AICAR, an inhibitory effect of AICAR on autophagic proteolysis was still observed (Fig. 8C). Next we analyzed the effect of inhibition of AMPK activity on the rate of proteolysis. In a first series of experiments, HeLa cells expressing LKB1 and STRAD were transfected with the cDNA encoding AMPK DN (Fig. 8A). Under these conditions, we observed a decrease in AMPK activity and no stimulatory effect of AICAR (Fig. 8B). We ...
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... the activity of AMPK was not stimulated by AICAR, an inhibitory effect of AICAR on autophagic proteolysis was still observed (Fig. 8C). Next we analyzed the effect of inhibition of AMPK activity on the rate of proteolysis. In a first series of experiments, HeLa cells expressing LKB1 and STRAD were transfected with the cDNA encoding AMPK DN (Fig. 8A). Under these conditions, we observed a decrease in AMPK activity and no stimulatory effect of AICAR (Fig. 8B). We observed an almost complete inhibition of starvation-induced autophagic degradation of long-lived [ 14 C]valine-labeled proteins in these cells (Fig. 8C). We then investigated the effect of compound C in HeLa cells and in ...
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... was still observed (Fig. 8C). Next we analyzed the effect of inhibition of AMPK activity on the rate of proteolysis. In a first series of experiments, HeLa cells expressing LKB1 and STRAD were transfected with the cDNA encoding AMPK DN (Fig. 8A). Under these conditions, we observed a decrease in AMPK activity and no stimulatory effect of AICAR (Fig. 8B). We observed an almost complete inhibition of starvation-induced autophagic degradation of long-lived [ 14 C]valine-labeled proteins in these cells (Fig. 8C). We then investigated the effect of compound C in HeLa cells and in cells expressing LKB1 and STRAD. Compound C inhibited AMPK activity in HeLa cells expressing LKB1 and STRAD and ...
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... cells expressing LKB1 and STRAD were transfected with the cDNA encoding AMPK DN (Fig. 8A). Under these conditions, we observed a decrease in AMPK activity and no stimulatory effect of AICAR (Fig. 8B). We observed an almost complete inhibition of starvation-induced autophagic degradation of long-lived [ 14 C]valine-labeled proteins in these cells (Fig. 8C). We then investigated the effect of compound C in HeLa cells and in cells expressing LKB1 and STRAD. Compound C inhibited AMPK activity in HeLa cells expressing LKB1 and STRAD and also in control HeLa cells, which have a low but detectable AMPK activity (Fig. 8B). In agreement with the results obtained in hepatocytes and HT-29 cells, ...
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... autophagic degradation of long-lived [ 14 C]valine-labeled proteins in these cells (Fig. 8C). We then investigated the effect of compound C in HeLa cells and in cells expressing LKB1 and STRAD. Compound C inhibited AMPK activity in HeLa cells expressing LKB1 and STRAD and also in control HeLa cells, which have a low but detectable AMPK activity (Fig. 8B). In agreement with the results obtained in hepatocytes and HT-29 cells, compound C was a very potent inhibitor of starvation-induced autoph- agic proteolysis in HeLa cells, independent of the amount of LKB1 and STRAD (Fig. 8C). As autophagy is a vacuolar mechanism that sequesters cytoplasmic material to deliver it to the lysosomal ...
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... in HeLa cells expressing LKB1 and STRAD and also in control HeLa cells, which have a low but detectable AMPK activity (Fig. 8B). In agreement with the results obtained in hepatocytes and HT-29 cells, compound C was a very potent inhibitor of starvation-induced autoph- agic proteolysis in HeLa cells, independent of the amount of LKB1 and STRAD (Fig. 8C). As autophagy is a vacuolar mechanism that sequesters cytoplasmic material to deliver it to the lysosomal compartment, we have investigated the presence of autophagic vacuoles by staining with MDC, a dye A, left, effect of AICAR and metformin on AMPK activity. When required, metformin (2 mM) or AICAR (250 M) was added for 15 min to ...
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![Fig. 2. Effect of compound C on basal [control (C)] and AICAR-induced...](profile/Rolando-Ceddia/publication/7465158/figure/fig2/AS:280248320053250@1443827814497/Effect-of-compound-C-on-basal-control-C-and-AICAR-induced-A-AMPK-A-and-ACC-B_Q320.jpg)
The purpose of this study was to investigate the effects of long-chain fatty acids (LCFAs) on AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation and beta-oxidation in skeletal muscle. L6 rat skeletal muscle cells were exposed to various concentrations of palmitate (1-800 microM). Subsequently, ACC and AMPK p...
We tested the hypothesis that 5'AMP-activated protein kinase (AMPK) plays an important role in regulating the acute, exercise-induced activation of metabolic genes in skeletal muscle, which were dissected from whole-body alpha2- and alpha1-AMPK knockout (KO) and wild-type (WT) mice at rest, after treadmill running (90 min), and in recovery. Running...
AMPK (AMP-activated protein kinase) responds to intracellular ATP depletion, while PPARalpha (peroxisome proliferator-activated receptor alpha) induces the expression of genes coding for enzymes and proteins involved in increasing cellular ATP yields. PPARalpha-mediated transcription is shown here to be co-activated by the alpha subunit of AMPK, as...
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Citations
... 42 AMPK also promotes catabolism of amino acids by inhibiting translation, either through inhibiting the target of rapamycin complex 1 (TORC1), 43,44 or through promoting the inhibition of the eukaryotic elongation factor 2 (eEF2) by eEF2 kinase (eEF2K). 45 In addition, AMPK helps release free amino acids from cellular proteins either by promoting autophagy, [46][47][48] or through increasing proteasomal degradation of labile proteins. 49 However, the mechanisms underlying the prioritization of alternative carbon source utilization remain unclear. ...
The shift of carbon utilization from primarily glucose to other nutrients is a fundamental metabolic adaptation to cope with decreased blood glucose levels and the consequent decline in glucose oxidation. AMP-activated protein kinase (AMPK) plays crucial roles in this metabolic adaptation. However, the underlying mechanism is not fully understood. Here, we show that PDZ domain containing 8 (PDZD8), which we identify as a new substrate of AMPK activated in low glucose, is required for the low glucose-promoted glutaminolysis. AMPK phosphorylates PDZD8 at threonine 527 (T527) and promotes the interaction of PDZD8 with and activation of glutaminase 1 (GLS1), a rate-limiting enzyme of glutaminolysis. In vivo, the AMPK-PDZD8-GLS1 axis is required for the enhancement of glutaminolysis as tested in the skeletal muscle tissues, which occurs earlier than the increase in fatty acid utilization during fasting. The enhanced glutaminolysis is also observed in macrophages in low glucose or under acute lipopolysaccharide (LPS) treatment. Consistent with a requirement of heightened glutaminolysis, the PDZD8-T527A mutation dampens the secretion of pro-inflammatory cytokines in macrophages in mice treated with LPS. Together, we have revealed an AMPK-PDZD8-GLS1 axis that promotes glutaminolysis ahead of increased fatty acid utilization under glucose shortage.
... The regulation of glucose deprivation-induced protective autophagy involves multiple pathways [45,46,78,79,81,82], with the mTORC1 complex being a well-known negative regulator of autophagy [53,83]. In addition to the classical AMPK-mTORC1 pathway [45,84,85], other mechanisms have been identified. For instance, HK-II binding to TORC1 suppresses mTORC1 activity and promotes protective autophagy in response to glucose deprivation [8]. ...
Low glucose is a common microenvironment for rapidly growing solid tumors, which has developed multiple approaches to survive under glucose deprivation. However, the specific regulatory mechanism remains largely elusive. In this study, we demonstrate that glucose deprivation, while not amino acid or serum starvation, transactivates the expression of DCAF1. This enhances the K48-linked polyubiquitination and proteasome-dependent degradation of Rheb, inhibits mTORC1 activity, induces autophagy, and facilitates cancer cell survival under glucose deprivation conditions. This study identified DCAF1 as a new cellular glucose sensor and uncovered new insights into mechanism of DCAF1-mediated inactivation of Rheb-mTORC1 pathway for promoting cancer cell survival in response to glucose deprivation.
... Activated ULK complex leads to the phosphorylation of BECN1 (a constituent of class III phosphatidylinositol 3-kinase complexes) and subsequently autophagy [25e27]. Following this, the AMPK protein and the TSC1-TSC2 complex direct the growth factors and signaling cascades [28,29]. On the other hand, mTORC1 also inhibits autophagy through cytosolic retention of the transcriptional factor, TFEB, thus stressing the need for mTORC1 inhibition [30]. ...
The overexpression of glutaminase is reported to influence cancer growth and metastasis through glutaminolysis. Upregulation of glutamine catabolism is recently recognized as a critical feature of cancer, and cancer cells are observed to reprogram glutamine metabolism to maintain its survival and proliferation. Special focus is given on the glutaminase isoform, GLS1 (kidney type glutaminase), as the other isoform GLS2 (Liver type glutaminase) acts as a tumour suppressor in some conditions. Glutaminolysis linked with autophagy, which is mediated via mTORC1, also serves as a promising target for cancer therapy. Glutamine also plays a vital role in maintaining redox homeostasis. Inhibition of glutaminase aggravates oxidative stress by reducing glutathione level, thus leading to apoptotic-mediated cell death in cancer cells Therefore, inhibiting the glutaminase activity using glutaminase inhibitors such as BPTES, DON, JHU-083, CB-839, compound 968, etc. may answer many intriguing questions behind the uncontrolled proliferation of cancer cells and serve as a prophylactic treatment for cancer. Earlier reports neither discuss nor provide perspectives on exact signaling gene or pathway. Hence, the present review highlights the plausible role of glutaminase in cancer and the current therapeutic approaches and clinical trials to target and inhibit glutaminase enzymes for better cancer treatment.
... Ulk1 kinase complex (Ulk1/Atg1, Atg13, FIP200, and Atg101) is tightly regulated positively and negatively [18][19][20][21][22]. The initiation of autophagy requires the ULK1 kinase complex, which is tightly regulated by AMPK and mTOR which acts as an activator and inhibitor, respectively [1, [5][6][7][23][24][25]. AMPK activates ULK1 through a series of phosphorylation events, while active mTOR inhibits ULK1 through phosphorylation of a different set of serine/threonine residues [1, 3,23,26,27]. ...
... These researchers suggested that transportation of mir-83 across tissue i.e., from intestine to BWM is possibly through extracellular vesicles (EVs). 6 Modulation of signaling molecules within one tissue can lead to systemic upregulation/downregulation of autophagy [4,87,88]. This has been elegantly demonstrated recently in D. melanogaster. ...
Macro-autophagy (autophagy hereafter) is an evolutionarily conserved cellular process that has long been recognized as an intracellular mechanism for maintaining cellular homeostasis. It involves the formation of a membraned structure called the autophagosome, which carries cargo that includes toxic protein aggregates and dysfunctional organelles to the lysosome for degradation and recycling. Autophagy is primarily considered and studied as a cell-autonomous mechanism. However, recent studies have illuminated an underappreciated facet of autophagy, i.e., non-autonomously regulated autophagy. Non- autonomously regulated autophagy involves the degradation of autophagic components, including organelles, cargo, and signaling molecules, and is induced in neighboring cells by signals from primary adjacent or distant cells/tissues/organs. This review provides insight into the complex molecular mechanisms governing non-autonomously regulated autophagy, highlighting the dynamic interplay between cells within tissue/organ or distinct cell types in different tissues/organs. Emphasis is placed on modes of intercellular communication that include secreted molecules, including microRNAs, and their regulatory roles in orchestrating this phenomenon. Furthermore, we explore the multidimensional roles of non-autonomously regulated autophagy in various physiological contexts, spanning tissue development and aging, as well as its importance in diverse pathological conditions, including cancer and neurodegeneration. By studying the complexities of non-autonomously regulated autophagy, we hope to gain insights into the sophisticated intercellular dynamics within multicellular organisms, including mammals. These studies will uncover novel avenues for therapeutic intervention to modulate intercellular autophagic pathways in altered human physiology.
... Autophagy is a cell-autonomous mechanism, i.e., occurs within the cell's cytoplasm. Autophagy is regulated by several growth signaling pathways like mTOR, AMPK, Insulin signaling, etc. [1][2][3][4][5][6][7]. There are three main types of autophagy classified based on the mode of cargo delivery for degradation: microautophagy, chaperon-mediated autophagy (CMA), and macroautophagy [8][9][10][11]. ...
... Ulk1 kinase complex (Ulk1/Atg1, Atg13, FIP200, and Atg101) is tightly regulated positively and negatively [17][18][19][20][21]. The initiation of autophagy requires the ULK1 kinase complex, which is tightly regulated by AMPK and mTOR which acts as an activator and inhibitor, respectively [1, [5][6][7][22][23][24]. AMPK activates ULK1 through a series of phosphorylation events, while active mTOR inhibits ULK1 through phosphorylation of a different set of serine/threonine residues [1, 3,22,25,26]. ...
... Modulation of signaling molecules within one tissue can lead to systemic upregulation/downregulation of autophagy [4,86,87]. This has been elegantly demonstrated recently 6 in D. melanogaster. D. melanogaster 3 rd instar larvae exhibit reduced dTOR activity and a strong autophagy response in their fat body (the mammalian equivalent of the liver) in response to starvation [88]. ...
Macro-autophagy (autophagy hereafter) is an evolutionarily conserved cellular process that has long been recognized as an intracellular mechanism for maintaining cellular homeostasis. It involves the formation of a membraned structure called the autophagosome, which carries cargo that includes toxic protein aggregates and dysfunctional organelles to the lysosome for degradation and recycling. Autophagy is primarily considered and studied as a cell-autonomous mechanism. However, recent studies have illuminated an underappreciated facet of autophagy, i.e., non-autonomously regulated autophagy. Non- autonomously regulated autophagy involves the degradation of autophagic components, including organelles, cargo, and signaling molecules, and is induced in neighboring cells by signals from primary adjacent or distant cells/tissues/organs. This review provides insight into the complex molecular mechanisms governing non-autonomously regulated autophagy, highlighting the dynamic interplay between cells within tissue/organ or distinct cell types in different tissues/organs. Emphasis is placed on modes of intercellular communication that include secreted molecules, including microRNAs, and their regulatory roles in orchestrating this phenomenon. Furthermore, we explore the multidimensional roles of non-autonomously regulated autophagy in various physiological contexts, spanning tissue development and aging, as well as its importance in diverse pathological conditions, including cancer and neurodegeneration. By studying the complexities of non-autonomously regulated autophagy, we hope to gain insights into the sophisticated intercellular dynamics within multicellular organisms, including mammals. These studies will uncover novel avenues for therapeutic intervention to modulate intercellular autophagic pathways in altered human physiology.
... Additionally, AMPK activation significantly reduces in vivo myocardial I/R injury. AMPK activation thought to be a key regulator of autophagy in reaction to energy deficiency (Meley et al., 2006). SIRT1 and AMPK can activate each other, but there is an inhibition relationship between SIRT1 and mTOR (Giovannini, Bianchi, 2017). ...
... activity and autophagy induction. Our findings offer a mechanistic explanation for the previously reported inhibitory effects of AMPK on autophagy [1][2][3][10][11][12][13][14][15]17 . The key aspect of our elucidated mechanism is that AMPK-mediated phosphorylations of ULK1 at Ser556 and Thr660 suppress ULK1 activity and autophagy induction. ...
Autophagy maintains cellular homeostasis during low energy states. According to the current understanding, glucose-depleted cells induce autophagy through AMPK, the primary energy-sensing kinase, to acquire energy for survival. However, contrary to the prevailing concept, our study demonstrates that AMPK inhibits ULK1, the kinase responsible for autophagy initiation, thereby suppressing autophagy. We found that glucose starvation suppresses amino acid starvation-induced stimulation of ULK1-Atg14-Vps34 signaling via AMPK activation. During an energy crisis caused by mitochondrial dysfunction, the LKB1-AMPK axis inhibits ULK1 activation and autophagy induction, even under amino acid starvation. Despite its inhibitory effect, AMPK protects the ULK1-associated autophagy machinery from caspase-mediated degradation during energy deficiency, preserving the cellular ability to initiate autophagy and restore homeostasis once the stress subsides. Our findings reveal that dual functions of AMPK, restraining abrupt induction of autophagy upon energy shortage while preserving essential autophagy components, are crucial to maintain cellular homeostasis and survival during energy stress.
... Autophagy is a specific cellular mechanism for recycling cellular components such as proteins, macromolecules, organelles and pathogens, which mediates the engulfment of material in autophagic vesicles by membrane structures, fusion with lysosomes and subsequent degradation of material in autophagic vesicles (Bento et al., 2016). AMPK has been proved to both regulate autophagy in yeast (Wang et al., 2001) and mammalian cells (Meley et al., 2006;Hoyer-Hansen et al., 2007). First, AMPK directly phosphorylates Thr1227 and Ser1345 of the mTOR upstream regulator TSC2 (Inoki et al., 2003) and Ser722 and Ser792 of the mTORC1 subunit RAPTOR (Gwinn et al., 2008). ...
Background: Delta-like ligand 3 (DLL3) is one of the NOTCH family of ligands, which plays a pro- or anti-carcinogenic role in some cancers. But the role of DLL3 in colon adenocarcinoma (COAD) has not been studied in depth.
Materials and methods: First, we used Kaplan-Meier (K-M) curve to evaluate the effect of DLL3 on the prognosis of COAD in The Cancer Genome Atlas (TCGA), which was further validated in clinical samples for immunohistochemistry. Then we screened for differentially expressed genes (DEGs) of DLL3 by analyzing datasets of COAD samples from Gene Expression Omnibus (GEO) and TCGA. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, and Gene Set Enrichment Analysis (GSEA) were conducted to explore the underlying mechanisms of DLL3-related in the development and prognosis of COAD. On the basis of DLL3-related signature genes, a prognostic model and a nomogram were constructed. Finally, CIBERSORT was applied to assess the proportion of immune cell types in COAD sample.
Results: Survival analysis showed a significant difference in overall survival between high- and low-expression group (p = 0.0092), with COAD patients in the high-group having poorer 5-year survival rate. Gene functional enrichment analysis revealed that DLL3-related DEGs were mainly enriched in tumor- and immunity-related signaling pathways, containing AMPK pathway and mitophagy-animal. The comparison of COAD tumor and normal, DLL3 high- and low-expression groups by GSEA found that AMPK signaling pathway and mitophagy-animal were inhibited. Nomogram constructed from DLL3-related signature genes had a good predictive effect on the prognosis of COAD. We found the highest correlation between DLL3 and interstitial dendritic cell (iDC), natural killer (NK) cell and Interstitial dendritic cell (Tem). DLL3 was also revealed to be diagnostic for COAD. In clinical sample, we identified higher DLL3 expression in colon cancer tissue than in adjacent control (p < 0.0001) and in metastasis than in primary lesion (p = 0.0056). DLL3 expression was associated with stage and high DLL3 expression was observed to predict poorer overall survival (p = 0.004).
Conclusion: It suggested that DLL3 may offer prognostic value and therapeutic potential for individualized treatment of COAD, and that it may has a diagnostic role in COAD.
... Autophagy is significantly involved in the systemic homeostasis of an organism [3]. This process is mainly modulated by AMP-activated protein kinase (AMPK) and mammalian/ mechanistic target of rapamycin (mTOR) [4][5][6]. The mTOR, as a master regulator of autophagy, activates the ATG1 complex [7], and AMPK (as a significant energy sensor) regulates autophagy via inhibition of mTOR signaling [8]. ...
Background:
Autophagy is an important part of pathogenesis of IBD. Thiopurines such as azathioprine (AZA) are approved drugs for clinical practices in IBD patients. Besides, as an escape strategy, Toxoplasma gondii can use the mTORC1 complex to inactivate autophagy.
Methods:
In this study, we investigated whether T. gondii tachyzoites may modulate autophagy and interfere the effects of azathioprine in IBD treatment. PMA-activated human monocyte cell line (THP-1) was infected with fresh T. gondii RH tachyzoites. After 5 h of infection, the cells were treated with AZA for 6 h. The expression of atg5, atg7, atg12, lc3b, and β-actin (BACT) genes was evaluated using quantitative real-time PCR. To analyze the phosphorylation of ribosomal protein S6 (rpS6), western blot using specific primary antibodies was performed.
Results:
The results of real-time PCR revealed that AZA, T. gondii tachyzoites, and a combination of AZA and T. gondii tachyzoites upregulated atg5 gene for 4.297-fold (P-value = 0.014), 2.49-fold (P-value = 0.006), and 4.76-fold (P-value = 0.001), respectively. The atg7 gene showed significant upregulation (2.272-fold; P-value = 0.014) and (1.51-fold; P-value = 0.020) in AZA and AZA / T. gondii, respectively. The expression of atg12 gene was significantly downregulated in AZA and T. gondii tachyzoites for (8.85-fold; P-value = 0.004) and (2.005-fold; P-value = 0.038), respectively, but upregulated in T. gondii/AZA (1.52-fold; P-value = 0.037). In addition, the lc3b gene was only significantly changed in AZA / T. gondii (3.028-fold; P-value = 0.001). Western blot analysis showed that T. gondii tachyzoites significantly phosphorylated rpS6, and tachyzoites did not interfere the effects of AZA to phosphorylate the rpS6.
Conclusion:
Taken together, although AZA and T. gondii similarly affects the expression levels of atg5, atg7, and atg12, but T. gondii does not seem to modulate the effects of AZA via mTORC functions.
... The AMPK-mTOR pathway is an important regulator of autophagy in response to ischemic injury [15,16]. ...
... FGF10 reduces MIRI via the AMPK/mTOR/TFEB pathway We next explored the signaling pathways involved in FGF10-mediated cardioprotection. The AMPK-mTOR pathway is an important regulator of autophagy in response to ischemic injury [15,16]. However, AMPK is no longer activated during the reperfusion phase, resulting in mTOR activation [17,18]. ...
... The AMPK-mTOR pathway is an important regulator of autophagy in response to ischemic injury [15,16]. AMPK is activated during ischemia [38] but is no longer activated during the reperfusion phase, resulting in mTOR activation [17,18]. ...
Myocardial ischemia/reperfusion injury (MIRI) is a common clinical complication that occurs upon reperfusion therapy for acute myocardial infarction. Recent studies indicated that impaired autophagic flux contributes to MIRI-induced apoptosis of cardiomyocytes (CMs). Fibroblast growth factor 10 (FGF10), a multifunctional FGF family member, was reported to protect against renal and hepatic ischemia/reperfusion injury. However, it has not been investigated whether FGF10 has a similar beneficial effect against MIRI, and if so, whether autophagy is involved in this effect. Herein, we found that FGF10 was upregulated in mice with MIRI and neonatal rat cardiomyocytes (NRCMs) with hypoxia/reoxygenation injury. FGF10 treatment decreased infarct size and improved cardiac function upon MIRI. FGF10 attenuated MIRI-induced apoptosis of CMs and impairment of autophagic flux mainly through the AMPK/mTOR/TFEB pathway. Therefore, FGF10 may have potential applications for the prevention and treatment of MIRI.
