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Cell Cycle (Georgetown, Tex.) Apr 2023This study aimed to explore the role of lipopolysaccharide-binding protein (LBP) in adipose browning. Mouse embryonic fibroblasts (MEFs) were treated with...
Deficiency of lipopolysaccharide binding protein facilitates adipose browning, glucose uptake and oxygen consumption in mouse embryonic fibroblasts via activating PI3K/Akt/mTOR pathway and inhibiting autophagy.
This study aimed to explore the role of lipopolysaccharide-binding protein (LBP) in adipose browning. Mouse embryonic fibroblasts (MEFs) were treated with differentiation induction reagents and Perifosine (Akt inhibitor), with the transfection of Atg5, short hairpin RNA targeting LBP (shLBP), and Atg5 (shAtg5). The expression levels of LBP, inflammatory markers , brown fat markers, lipid metabolism marker, autophagy markers, insulin signaling-related molecules , p-mTOR, mTOR, p-Akt, Akt, p-PI3K, and PI3K were quantified or determined by Western blot, qRT-PCR, and immunofluorescence assay. The formation of lipid was examined through Oil red O staining assay. The consumption of oxygen was assessed using a Seahorse XF96 analyzer, and the uptake of glucose was evaluated by [H]-2-deoxy-D-glucose uptake assay. Deficiency of LBP promoted adipose browning, oxygen consumption, glucose uptake, and insulin sensitivity in differentiated MEFs, where it inhibited inflammation and autophagy. All of the effects above were reversed by Atg5 overexpression. Meanwhile, the knockdown of Atg5 strengthened the activation of PI3K/Akt/mTOR pathway induced by the depletion of LBP, while Perifosine partly reversed the activation of differentiated MEFs. The knockdown of LBP facilitated adipose browning, glucose uptake, and oxygen consumption in MEFs via the activation of PI3K/Akt/mTOR pathway and the inhibition of autophagy.
Topics: Animals; Mice; Autophagy; Fibroblasts; Glucose; Lipopolysaccharides; Obesity; Oxygen Consumption; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases
PubMed: 36710409
DOI: 10.1080/15384101.2023.2169521 -
Molecular Immunology Mar 2023Previously, we revealed a crucial role of 5-HT degradation system (5DS), consisting of 5-HT2A receptor (5-HT2AR), 5-HT synthases and monoamine oxidase A (MAO-A), in...
Myocardial ischemia-reperfusion injury is probably due to the excessive production of mitochondrial ROS caused by the activation of 5-HT degradation system mediated by PAF receptor.
AIM
Previously, we revealed a crucial role of 5-HT degradation system (5DS), consisting of 5-HT2A receptor (5-HT2AR), 5-HT synthases and monoamine oxidase A (MAO-A), in ischemia-reperfusion (IR)-caused organ injury. Whereas, platelet activating factor receptor (PAFR) also mediates myocardial ischemia-reperfusion injury (MIRI). Here, we try to clarify the relationship between 5DS and PAFR in mediating MIRI.
METHODS
H9c2 cell injury and rat MIRI were caused by hypoxia/reoxygenation (H/R) or PAF, and by ligating the left anterior descending coronary artery then untying, respectively. 5-HTR and PAFR antagonists [sarpogrelate hydrochloride (SH) and BN52021], MAO-A, AKT, mTOR and 5-HT synthase inhibitors (clorgyline, perifosine, rapamycin and carbidopa), and gene-silencing PKCε were used in experiments RESULTS: The mitochondrial ROS production, respiratory chain damage, inflammation, apoptosis and myocardial infarction were significantly prevented by BN52021, SH and clorgyline in H/R and PAF-treated cells and in IR myocardium. BN52021 also significantly suppressed the upregulation of PAFR, 5-HTR, 5-HT synthases and MAO-A expression (mRNA and protein), and Gα and PKCε (in plasmalemma) expression induced by H/R, PAF or IR; the effects of SH were similar to that of BN52021 except for no affecting the expression of PAFR and 5-HTR. Gene-silencing PKCε suppressed H/R and PAF-induced upregulation of 5-HT synthases and MAO-A expression in cells; perifosine and rapamycin had not such effects; however, clorgyline suppressed H/R and PAF-induced phosphorylation of AKT and mTOR.
CONCLUSION
MIRI is probably due to PAFR-mediated 5-HTR activation, which further activates PKCε-mediated 5-HT synthesis and degradation, leading to mitochondrial ROS production.
Topics: Animals; Rats; Apoptosis; Clorgyline; Monoamine Oxidase; Myocardial Reperfusion Injury; Platelet Membrane Glycoproteins; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Receptors, G-Protein-Coupled; Serotonin; TOR Serine-Threonine Kinases
PubMed: 36682136
DOI: 10.1016/j.molimm.2023.01.004 -
International Journal of Molecular... Jan 2023Numerous hematologic neoplasms, including acute B-lymphoblastic leukemia (B-ALL), are characterized by overexpression of anti-apoptotic BCL-2 family proteins. Despite...
Numerous hematologic neoplasms, including acute B-lymphoblastic leukemia (B-ALL), are characterized by overexpression of anti-apoptotic BCL-2 family proteins. Despite the high clinical efficacy of the specific BCL-2 inhibitor venetoclax in acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL), dose limitation and resistance argue for the early exploration of rational combination strategies. Recent data indicated that BCL-2 inhibition in B-ALL with rearrangements is a promising intervention option; however, combinatorial approaches have not been in focus so far. The PI3K/AKT pathway has emerged as a possible target structure due to multiple interactions with the apoptosis cascade as well as relevant dysregulation in B-ALL. Herein, we demonstrate for the first time that combined BCL-2 and PI3K/AKT inhibition has synergistic anti-proliferative effects on B-ALL cell lines. Of note, all tested combinations (venetoclax + PI3K inhibitors idelalisib or BKM-120, as well as AKT inhibitors MK-2206 or perifosine) achieved comparable anti-leukemic effects. In a detailed analysis of apoptotic processes, among the PI3K/AKT inhibitors only perifosine resulted in an increased rate of apoptotic cells. Furthermore, the combination of venetoclax and perifosine synergistically enhanced the activity of the intrinsic apoptosis pathway. Subsequent gene expression studies identified the pro-apoptotic gene as a possible player in synergistic action. All combinatorial approaches additionally modulated extrinsic apoptosis pathway genes. The present study provides rational combination strategies involving selective BCL-2 and PI3K/AKT inhibition in B-ALL cell lines. Furthermore, we identified a potential mechanistic background of the synergistic activity of combined venetoclax and perifosine application.
Topics: Humans; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-bcl-2; Bridged Bicyclo Compounds, Heterocyclic; Apoptosis Regulatory Proteins; Apoptosis; Leukemia, Myeloid, Acute; Cell Line, Tumor; Precursor Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 36674872
DOI: 10.3390/ijms24021359 -
Journal of Cellular and Molecular... Jan 2023An acidic environment and hypoxia within the tumour are hallmarks of cancer that contribute to cell resistance to therapy. Deregulation of the PI3K/Akt pathway is common...
An acidic environment and hypoxia within the tumour are hallmarks of cancer that contribute to cell resistance to therapy. Deregulation of the PI3K/Akt pathway is common in colon cancer. Numerous Akt-targeted therapies are being developed, the activity of Akt-inhibitors is, however, strongly pH-dependent. Combination therapy thus represents an opportunity to increase their efficacy. In this study, the cytotoxicity of the Akt inhibitor perifosine and the Bcl-2/Bcl-xL inhibitor ABT-737 was tested in colon cancer HT-29 and HCT-116 cells cultured in monolayer or in the form of spheroids. The efficacy of single drugs and their combination was analysed in different tumour-specific environments including acidosis and hypoxia using a series of viability assays. Changes in protein content and distribution were determined by immunoblotting and a "peeling analysis" of immunohistochemical signals. While the cytotoxicity of single agents was influenced by the tumour-specific microenvironment, perifosine and ABT-737 in combination synergistically induced apoptosis in cells cultured in both 2D and 3D independently on pH and oxygen level. Thus, the combined therapy of perifosine and ABT-737 could be considered as a potential treatment strategy for colon cancer.
Topics: Humans; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Colonic Neoplasms; Drug Synergism; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Tumor Microenvironment; Phosphorylcholine
PubMed: 36523175
DOI: 10.1111/jcmm.17636 -
Frontiers in Oncology 2022[This corrects the article DOI: 10.3389/fonc.2021.686898.].
Corrigendum: Downregulation of ATXN3 enhances the sensitivity to AKT inhibitors (Perifosine or MK-2206), but decreases the sensitivity to chemotherapeutic drugs (etoposide or cisplatin) in neuroblastoma cells.
[This corrects the article DOI: 10.3389/fonc.2021.686898.].
PubMed: 35992874
DOI: 10.3389/fonc.2022.984514 -
Pharmacological Research Sep 2022The serine/threonine kinase Akt is a major player in the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, and its modulation... (Review)
Review
The serine/threonine kinase Akt is a major player in the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, and its modulation impacts multiple cellular processes such as growth, proliferation, and survival. Several abnormalities in this pathway have been documented over the years, and these alterations were shown to have great implications in tumorigenesis and resistance to chemotherapy. Thus, multiple Akt inhibitors have been developed and tested in adult tumors, and some of them are currently undergoing phase I, II, and III clinical trials for distinct cancers that arise during adulthood. Despite that, the impact of these inhibitors is still not fully understood in pediatric tumors, and Akt-specific targeting seems to be a promising approach to treat children affected by cancers. This review summarizes recent available evidence of Akt inhibitors in pediatric cancers, from both preclinical and clinical studies. In short, we demonstrate the impact that Akt inhibition provides in tumorigenesis, and we suggest targeting the PI3K/Akt/mTOR signaling pathway, alone or in combination with other inhibitors, is a feasible tool to achieve better outcomes in pediatric tumors.
Topics: Adult; Carcinogenesis; Child; Humans; Neoplasms; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases
PubMed: 35987481
DOI: 10.1016/j.phrs.2022.106403 -
Journal of Immunology Research 2022Ischemia/reperfusion (I/R) is a primary cause of morbidity and mortality in acute myocardial infarction (AMI). L-Borneol...
L-Borneol 7-O-[-D-Apiofuranosyl-(1→6)]--D-Glucopyranoside Alleviates Myocardial Ischemia-Reperfusion Injury in Rats and Hypoxic/Reoxygenated Injured Myocardial Cells via Regulating the PI3K/AKT/mTOR Signaling Pathway.
Ischemia/reperfusion (I/R) is a primary cause of morbidity and mortality in acute myocardial infarction (AMI). L-Borneol 7-O-[-D-apiofuranosyl-(1→6)]--D-glucopyranoside (LBAG), extracted from the Radix Ophiopogonis, is the main bioactive component that may be exerting cardiovascular protection in AMI. The purpose was to examine the effects of LBAG on myocardial I/R injury (MIRI) in rats and H9c2 cells treated with hypoxia/reoxygenation (H/R). MIRI was induced through the combination of ischemia with reperfusion for 30 min and 24 h, respectively. LBAG was administered 7 days before vascular ligation. Myocardial function was detected by an electrocardiograph, histological, TTC, and TUNEL staining analyses. The influences of LBAG on the content concentration of cardiac enzymes in the serum were measured by ELISA. Moreover, H9c2 cells were exposed to LBAG or combined with AKT inhibitor (perifosine) and then exposed to H/R for simulating the cardiac injury process. Afterward, cell viability, LDH, CD-KM release, apoptosis, and autophagy were evaluated by CCK-8 and ELISA assays, flow cytometry, TUNEL, and immunofluorescence staining, respectively. Additionally, the proteins of apoptosis, autophagy, and PI3K/mTOR pathway were determined by western blotting. In I/R rats, LBAG pretreatment significantly ameliorated cardiac function, as illustrated by reducing the infarct size, myocardial autophagy, and apoptosis levels. In H/R-induced H9c2 cells, LBAG pretreatment significantly decreased cell apoptosis, LC3 II/I, and Beclin 1 levels, elevated the Bcl-2 levels, attenuated LDH, and CD-KM production. Moreover, LBAG pretreatment markedly increased the PI3K/mTOR pathway activation, and the protective influences of LBAG were partly abolished with the AKT inhibitor perifosine treatment. These findings demonstrated the protective functions of LBAG on I/R by regulating apoptosis and autophagy in vitro and in vivo by activating the PI3K/mTOR pathway.
Topics: Animals; Apoptosis; Camphanes; Hypoxia; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; TOR Serine-Threonine Kinases
PubMed: 35600046
DOI: 10.1155/2022/5758303 -
Zhonghua Yu Fang Yi Xue Za Zhi [Chinese... Feb 2022To explore the biofilm inhibitory efficacy of perifosine against () and its mechanisms. Twenty-fourwell plate was used to form biofilms at the bottom and crystal violet...
To explore the biofilm inhibitory efficacy of perifosine against () and its mechanisms. Twenty-fourwell plate was used to form biofilms at the bottom and crystal violet staining was used to determine the biofilm inhibitory effects of perifosine against , the wells without perifosine was set as control group. Glass tubes combined with crystal violet staining was used to detect the gas-liqud interface related bioiflm inhibitory effects of perifosine, the wells without perifosine was set as control group. Time-growth curved was used to detect the effects of perifosine on the bacteial planktonic cells growth of , the wells without perifosine was set as control group. The interaction model between perifosine and PqsE was assessed by molecular docking assay. The inhibitory effects of perifosine on the catalytic activity of PqsE was determined by detection the production of thiols, the wells without perifosine was set as control group. Binding affinity between perifosine and PqsE was detected by plasma surface resonance. The biofims at the bottom of the microplates and air-liquid interface were effectively inhibited by perifosine at the concentration of 4-8 μg/ml. There was no influence of perifosine on the cells growth of . The resuts of molecular docking assay indicates that perifosine could interacted with PqsE with the docking score of -10.67 kcal/mol. Perifosine could inhibit the catalytic activity of PqsE in a dose-dependent manner. The binding affinity between perifosine and PqsE was comfirmed by plasma surface resonance with KD of 6.65×10mol/L. Perifosine could inhibited the biofilm formation of by interacting with PqsE.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Molecular Docking Simulation; Phosphorylcholine; Pseudomonas aeruginosa; Quorum Sensing
PubMed: 35184449
DOI: 10.3760/cma.j.cn112150-20211020-00970 -
Molecular Biology Reports Apr 2022Melatonin can regulate apoptosis and autophagy of mouse Leydig cells, but its specific mechanism is still unclear.
BACKGROUND
Melatonin can regulate apoptosis and autophagy of mouse Leydig cells, but its specific mechanism is still unclear.
METHODS
In this study, we used the TM3 cell line as the research object, and used H2O2 to induce autophagy. After adding 10 ng/ml melatonin, we used qRT-PCR and western-blot to detect autophagy-related gene and protein expression, and flow cytometry to detect cellular ROS level.
RESULTS
The results showed that melatonin can significantly inhibit the occurrence of autophagy, accompanied by a significant decrease in the expression of Becn1, LC3, and FOXO1 (P < 0.05), a significant increase in the expression of p62 and pAKT (P < 0.05), and a significant decrease in ROS level (P < 0.05). After added the inhibitor of AKT perifosine, the effect of melatonin on inhibiting autophagy was reversed. On this basis, we used small RNA interference technology to knock down the expression of FOXO1, and found that there was no significant change of the expression of genes and proteins related to autophagy and ROS level.
CONCLUSIONS
In summary, melatonin can inhibit H2O2-induced autophagy in TM3 cells through the AKT/FOXO1 pathway.
Topics: Animals; Apoptosis; Autophagy; Forkhead Box Protein O1; Hydrogen Peroxide; Male; Melatonin; Mice; Proto-Oncogene Proteins c-akt
PubMed: 34997871
DOI: 10.1007/s11033-021-07107-0 -
Human Reproduction Update Feb 2022Autophagy is an intracellular catabolic process of degrading and recycling proteins and organelles to modulate various physiological and pathological events, including... (Review)
Review
BACKGROUND
Autophagy is an intracellular catabolic process of degrading and recycling proteins and organelles to modulate various physiological and pathological events, including cell differentiation and development. Emerging data indicate that autophagy is closely associated with male reproduction, especially the biosynthetic and catabolic processes of sperm. Throughout the fate of sperm, a series of highly specialized cellular events occur, involving pre-testicular, testicular and post-testicular events. Nonetheless, the most fundamental question of whether autophagy plays a protective or harmful role in male reproduction, especially in sperm, remains unclear.
OBJECTIVE AND RATIONALE
We summarize the functional roles of autophagy in the pre-testicular (hypothalamic-pituitary-testis (HPG) axis), testicular (spermatocytogenesis, spermatidogenesis, spermiogenesis, spermiation) and post-testicular (sperm maturation and fertilization) processes according to the timeline of sperm fate. Additionally, critical mechanisms of the action and clinical impacts of autophagy on sperm are identified, laying the foundation for the treatment of male infertility.
SEARCH METHODS
In this narrative review, the PubMed database was used to search peer-reviewed publications for summarizing the functional roles of autophagy in the fate of sperm using the following terms: 'autophagy', 'sperm', 'hypothalamic-pituitary-testis axis', 'spermatogenesis', 'spermatocytogenesis', 'spermatidogenesis', 'spermiogenesis', 'spermiation', 'sperm maturation', 'fertilization', 'capacitation' and 'acrosome' in combination with autophagy-related proteins. We also performed a bibliographic search for the clinical impact of the autophagy process using the keywords of autophagy inhibitors such as 'bafilomycin A1', 'chloroquine', 'hydroxychloroquine', '3-Methyl Adenine (3-MA)', 'lucanthone', 'wortmannin' and autophagy activators such as 'rapamycin', 'perifosine', 'metformin' in combination with 'disease', 'treatment', 'therapy', 'male infertility' and equivalent terms. In addition, reference lists of primary and review articles were reviewed for additional relevant publications. All relevant publications until August 2021 were critically evaluated and discussed on the basis of relevance, quality and timelines.
OUTCOMES
(i) In pre-testicular processes, autophagy-related genes are involved in the regulation of the HPG axis; and (ii) in testicular processes, mTORC1, the main gate to autophagy, is crucial for spermatogonia stem cell (SCCs) proliferation, differentiation, meiotic progression, inactivation of sex chromosomes and spermiogenesis. During spermatidogenesis, autophagy maintains haploid round spermatid chromatoid body homeostasis for differentiation. During spermiogenesis, autophagy participates in acrosome biogenesis, flagella assembly, head shaping and the removal of cytoplasm from elongating spermatid. After spermatogenesis, through PDLIM1, autophagy orchestrates apical ectoplasmic specialization and basal ectoplasmic specialization to handle cytoskeleton assembly, governing spermatid movement and release during spermiation. In post-testicular processes, there is no direct evidence that autophagy participates in the process of capacitation. However, autophagy modulates the acrosome reaction, paternal mitochondria elimination and clearance of membranous organelles during fertilization.
WIDER IMPLICATIONS
Deciphering the roles of autophagy in the entire fate of sperm will provide valuable insights into therapies for diseases, especially male infertility.
Topics: Autophagy; Humans; Infertility, Male; Male; Spermatids; Spermatogenesis; Spermatozoa
PubMed: 34967891
DOI: 10.1093/humupd/dmab043