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Nan Fang Yi Ke Da Xue Xue Bao = Journal... Aug 2021To investigate neuregulin 2 (NRG2) expression in gliomas and its role in glioma development.
OBJECTIVE
To investigate neuregulin 2 (NRG2) expression in gliomas and its role in glioma development.
METHODS
We compared the expression levels of NRG2 and glial fibrillary acidic protein (GFAP) in low-grade glioma (LGG) and glioblastoma multiforme (GBM) with those in normal control samples using GEPIA database.The correlation between NRG2 and GFAP expression and their association with the overall survival of patients with LGG and GBM were analyzed.Immunohistochemical staining was used to detect NRG2 protein expression levels in a tissue microarray consisting of human gliomas of different grades, and potential co-localization of NRG2 and GFAP was analyzed using a double-labeling immunofluorescence assay.Western blotting was used to investigate the effect of perifosine (an AKT inhibitor) on the regulation of GFAP expression by NRG2 in human glioblastoma U-87 MG cells.
RESULTS
Both LGG and GBM tissues, especially the former, exhibited high expressions of NRG2 ( < 0.01).In GBM samples, patients with low NRG2 levels had slightly higher overall survival after 30 months than patients with high NRG2 levels.The expression level of NRG2 mRNA was negatively correlated with that of GFAP in LGG samples ( < 0.01) but positively correlated with GFAP expression in GBM samples ( < 0.01).Immunofluorescence assay showed that NRG2 and GFAP were co-expressed in the same tumor cells of LGG tissues but were separately expressed in different tumor cells in GBM tissues.In U-87 MG cells, treatment with recombinant human NRG2 obviously promoted the expression of GFAP, and this effect was significantly inhibited by perifosine ( < 0.01).
CONCLUSION
NRG2 is highly expressed in gliomas of different grades and regulates GFAP expression in glioma cells at least partly the Akt signaling pathway to affect the survival of glioma patients.
Topics: Biomarkers, Tumor; Brain Neoplasms; Glial Fibrillary Acidic Protein; Glioblastoma; Glioma; Humans; Nerve Growth Factors; Neuregulins; Proto-Oncogene Proteins c-akt; Signal Transduction
PubMed: 34549707
DOI: 10.12122/j.issn.1673-4254.2021.08.07 -
Cell Stress & Chaperones Nov 20205-Hydroxytryptamine receptor 2A (HTR2A) is a central regulator of fetal brain development and cognitive function in adults. However, the roles of HTR2A in the...
5-Hydroxytryptamine receptor 2A (HTR2A) is a central regulator of fetal brain development and cognitive function in adults. However, the roles of HTR2A in the cardiovascular system are not fully understood. Here in this study, we explored the function of HTR2A in cardiac hypertrophy. Significantly, the expression levels of HTR2A mRNA and protein levels were upregulated in hypertrophic hearts of human patients. Besides, the expression of HTR2A was also upregulated in isoproterenol (ISO)-induced cardiac hypertrophy in the mouse. Next, the expression of HTR2A was knocked down with shRNA or overexpressed with adenovirus in neonatal rat cardiomyocytes, and ISO was used to induce cardiomyocyte hypertrophy. We showed that HTR2A knockdown repressed ISO-induced cardiomyocyte hypertrophy, which was demonstrated by decreased cardiomyocyte size and repressed expression of hypertrophic fetal genes (e.g., myosin heavy chain beta (β-Mhc), atrial natriuretic peptide (Anp), and brain natriuretic peptide (Bnp)). By contrast, HTR2A overexpression promoted cardiomyocyte hypertrophy. Of note, we observed that HTR2A promoted the activation (phosphorylation) of AKT-mTOR (mammalian target of rapamycin) signaling in cardiomyocytes, and repression of AKT-mTOR with perifosine or rapamycin blocked the effects of HTR2A on cardiomyocyte hypertrophy. Finally, we showed that HTR2A regulated AKT-mTOR signaling through activating the PI3K-PDK1 pathway, and inhibition of either PI3K or PDK1 blocked the roles of HTR2A in regulating AKT-mTOR signaling and cardiomyocyte hypertrophy. Altogether, these findings demonstrated that HTR2A activated PI3K-PDK1-AKT-mTOR signaling and promoted cardiac hypertrophy.
Topics: 3-Phosphoinositide-Dependent Protein Kinases; Animals; Animals, Newborn; Cardiomegaly; Humans; Isoproterenol; Male; Mice, Inbred C57BL; Models, Biological; Myocytes, Cardiac; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Receptor, Serotonin, 5-HT2A; Signal Transduction; TOR Serine-Threonine Kinases
PubMed: 32519137
DOI: 10.1007/s12192-020-01124-x -
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 -
ACS Pharmacology & Translational Science Feb 2020-amplified neuroblastoma is one of the deadliest forms of childhood cancer and remains a significant clinical challenge. Direct pharmacological inhibition of MYCN is not...
-amplified neuroblastoma is one of the deadliest forms of childhood cancer and remains a significant clinical challenge. Direct pharmacological inhibition of MYCN is not currently achievable. One strategy could be to target the AKT/GSK3β pathway, which directly regulates the stability of the MYCN protein. Numerous potent and isoform-specific small-molecule AKT inhibitors have been developed. However, the selection of the right drug combinations in the relevant indication will have a significant impact on AKT inhibitor clinical success. To maximally exploit the potential of AKT inhibitors, a better understanding of AKT isoform functions in cancer is crucial. Here using RNAi to downregulate specific AKT isoforms, we demonstrated that loss of total AKT activity rather than isoform-specific expression was necessary to decrease MYCN expression and cause a significant decrease in neuroblastoma cell proliferation. Consistent with these observations, isoform-specific pharmacological inhibition of AKT was substantially less effective than pan-AKT inhibition in combination with cytotoxic drugs in -amplified neuroblastoma. The allosteric pan-AKT inhibitor perifosine had promising and activity in combination with conventional cytotoxic drugs in -amplified neuroblastoma cells. Our results demonstrated that perifosine drug combination was able to induce apoptosis and downregulate ABC transporter expression. Collectively, this study shows that selecting pan-AKT inhibitors rather than isoform-specific drugs to synergize with first-line chemotherapy treatment should be considered for clinical trials for aggressive neuroblastoma and, potentially, other MYCN -driven cancers.
PubMed: 32259094
DOI: 10.1021/acsptsci.9b00085 -
Molecular Medicine Reports Aug 2020Vascular complications are the primary reason for disability and mortality associated with diabetes mellitus (DM), and numerous microRNAs (miRNAs/miRs) are involved in...
Vascular complications are the primary reason for disability and mortality associated with diabetes mellitus (DM), and numerous microRNAs (miRNAs/miRs) are involved in the process, such as miR‑122, miR‑24 and miR‑423. It has been reported that miR‑328 regulates DM and cardiovascular disease; however, the role and mechanism of action underlying miR‑328 in HUVECs is not completely understood. The present study aimed to investigate the role and mechanism of action underlying the effects of miR‑328 on the functions of HUVECs. To simulate hyperglycemia combined with ischemia‑induced tissue starvation, HUVECs were cultured in endothelial cell medium with 25 mmol/l D‑glucose and 2% FBS for 24 h [high glucose (HG) + 2% FBS group]. HUVEC miR‑328 expression levels were detected by reverse transcription‑quantitative PCR. Cell migration, cytotoxicity and tube‑like structure formation were analyzed using wound healing, Cell Counting Kit‑8 and tube formation assays, respectively. Following transfection with miR‑328 inhibitor, miR‑328 expression was downregulated in HUVECs. Protein expression levels were determined by western blotting. Compared with the control group, the migration and tube‑like structure formation of HUVECs were decreased, and cell cytotoxicity was increased in the HG + 2% FBS group. The protein expression levels of vascular endothelial growth factor were also decreased, and the expression levels of miRNA‑328 in the HG + 2% FBS group were increased compared with the control group. However, miRNA‑328 downregulation reversed the aforementioned effects. Further experiments indicated that the AKT signaling pathway was inhibited in the HG + 2% FBS group; however, miR‑328 downregulation activated the AKT/mTOR signaling pathway, which was blocked by the AKT signaling pathway inhibitor, perifosine. Gene prediction and western blotting demonstrated that miR‑328 displayed a regulatory role via Pim‑1 proto‑oncogene, serine/threonine kinase (PIM1). In conclusion, miR‑328 expression was upregulated and angiogenesis was inhibited when HUVECs were subjected to high glucose and low serum conditions. miR‑328 downregulation enhanced angiogenesis by increasing PIM1 expression and activating the AKT/mTOR signaling pathway in HUVECs under high glucose and low serum conditions.
Topics: Binding Sites; Cell Movement; Cell Survival; Cells, Cultured; Culture Media; Databases, Genetic; Down-Regulation; Glucose; Human Umbilical Vein Endothelial Cells; Humans; MicroRNAs; Neovascularization, Physiologic; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-pim-1; Serum; Signal Transduction; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A
PubMed: 32626978
DOI: 10.3892/mmr.2020.11141 -
Molecules (Basel, Switzerland) Oct 2021Excessive host inflammation following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with severity and mortality in...
Standardized Extract of Stem Attenuates SARS-CoV-2 Spike Protein-Induced IL-6 and IL-1β Production by Suppressing p44/42 MAPK and Akt Phosphorylation in Murine Primary Macrophages.
Excessive host inflammation following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with severity and mortality in coronavirus disease 2019 (COVID-19). We recently reported that the SARS-CoV-2 spike protein S1 subunit (S1) induces pro-inflammatory responses by activating toll-like receptor 4 (TLR4) signaling in macrophages. A standardized extract of stem (EAS) is a unique functional food that elicits anti-photoaging effects by suppressing pro-inflammatory signaling in hydrogen peroxide and ultraviolet B-exposed skin fibroblasts. To elucidate its potential in preventing excessive inflammation in COVID-19, we examined the effects of EAS on pro-inflammatory responses in S1-stimulated macrophages. Murine peritoneal exudate macrophages were co-treated with EAS and S1. Concentrations and mRNA levels of pro-inflammatory cytokines were assessed using enzyme-linked immunosorbent assay and reverse transcription and real-time polymerase chain reaction, respectively. Expression and phosphorylation levels of signaling proteins were analyzed using western blotting and fluorescence immunomicroscopy. EAS significantly attenuated S1-induced secretion of interleukin (IL)-6 in a concentration-dependent manner without reducing cell viability. EAS also markedly suppressed the S1-induced transcription of IL-6 and IL-1β. However, among the TLR4 signaling proteins, EAS did not affect the degradation of inhibitor κBα, nuclear translocation of nuclear factor-κB p65 subunit, and phosphorylation of c-Jun N-terminal kinase p54 subunit after S1 exposure. In contrast, EAS significantly suppressed S1-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and Akt. Attenuation of S1-induced transcription of IL-6 and IL-1β by the MAPK kinase inhibitor U0126 was greater than that by the Akt inhibitor perifosine, and the effects were potentiated by simultaneous treatment with both inhibitors. These results suggest that EAS attenuates S1-induced IL-6 and IL-1β production by suppressing p44/42 MAPK and Akt signaling in macrophages. Therefore, EAS may be beneficial in regulating excessive inflammation in patients with COVID-19.
Topics: Animals; Asparagus Plant; Butadienes; Cell Survival; Interleukin-1beta; Interleukin-6; Macrophages; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Plant Extracts; Plant Stems; Proto-Oncogene Proteins c-akt; Signal Transduction; Spike Glycoprotein, Coronavirus; Toll-Like Receptor 4; Transcription, Genetic
PubMed: 34684771
DOI: 10.3390/molecules26206189 -
Analytical Chemistry Nov 2019In this paper, we present an easy-to-follow procedure for the analysis of tissue sections from 3D cell cultures (spheroids) by matrix-assisted laser...
In this paper, we present an easy-to-follow procedure for the analysis of tissue sections from 3D cell cultures (spheroids) by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and laser scanning confocal microscopy (LSCM). MALDI MSI was chosen to detect the distribution of the drug of interest, while fluorescence immunohistochemistry (IHC) followed by LSCM was used to localize the cells featuring specific markers of viability, proliferation, apoptosis and metastasis. The overlay of the mass spectrometry (MS) and IHC spheroid images, typically without any morphological features, required fiducial-based coregistration. The MALDI MSI protocol was optimized in terms of fiducial composition and antigen epitope preservation to allow MALDI MSI to be performed and directly followed by IHC analysis on exactly the same spheroid section. Once MS and IHC images were coregistered, the quantification of the MS and IHC signals was performed by an algorithm evaluating signal intensities along equidistant layers from the spheroid boundary to its center. This accurate colocalization of MS and IHC signals showed limited penetration of the clinically tested drug perifosine into spheroids during a 24 h period, revealing the fraction of proliferating and promigratory/proinvasive cells present in the perifosine-free areas, decrease of their abundance in the perifosine-positive regions, and distinguishing between apoptosis resulting from hypoxia/nutrient deprivation and drug exposure.
Topics: Animals; Cell Culture Techniques; Fiducial Markers; Fluorescent Antibody Technique; HT29 Cells; Humans; Imaging, Three-Dimensional; Microscopy, Confocal; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 31584797
DOI: 10.1021/acs.analchem.9b02462 -
Einstein (Sao Paulo, Brazil) 2023To determine the role of the AKT pathway in the regulating of natural Killer-induced apoptosis of acute myeloid leukemia cells and to characterize the associated...
OBJECTIVE
To determine the role of the AKT pathway in the regulating of natural Killer-induced apoptosis of acute myeloid leukemia cells and to characterize the associated molecular mechanisms.
METHODS
BALB/c nude mice were injected with HL60 cells to induce a xenogenic model of subcutaneous leukemic tumors. Mice were treated with perifosine, and their spleens were analyzed using biometry, histopathology, and immunohistochemistry. Gene expression analysis in leukemia cells was performed by real-time PCR. Protein analysis of leukemia and natural Killer cells was performed by flow cytometry. AKT inhibition in HL60 cells, followed by co-culture with natural Killer cells was performed to assess cytotoxicity. Apoptosis rate was quantified using flow cytometry.
RESULTS
Perifosine treatment caused a reduction in leukemic infiltration in the spleens of BALB/c nude mice. In vitro , AKT inhibition reduced HL60 resistance to natural Killer-induced apoptosis. AKT inhibition suppressed the immune checkpoint proteins PD-L1, galectin-9, and CD122 in HL60 cells, but did not change the expression of their co-receptors PD1, Tim3, and CD96 on the natural Killer cell surface. In addition, the death receptors DR4, TNFR1, and FAS were overexpressed by AKT inhibition, thus increasing the susceptibility of HL60 cells to the extrinsic pathway of apoptosis.
CONCLUSION
The AKT pathway is involved in resistance to natural Killer-induced apoptosis in HL60 cells by regulating the expression of immune suppressor receptors. These findings highlight the importance of AKT in contributing to immune evasion mechanisms in acute myeloid leukemia and suggests the potential of AKT inhibition as an adjunct to immunotherapy.
Topics: Animals; Mice; Humans; Immune Checkpoint Proteins; HL-60 Cells; Mice, Nude; Proto-Oncogene Proteins c-akt; Leukemia, Myeloid, Acute
PubMed: 37341216
DOI: 10.31744/einstein_journal/2023AO0171 -
Molecular Biology Reports Sep 2021Epithelial mesenchymal transition (EMT) is a critical process involved in the invasion and metastasis of cancer, including lung cancer (LC). Transforming growth factor...
BACKGROUNDS
Epithelial mesenchymal transition (EMT) is a critical process involved in the invasion and metastasis of cancer, including lung cancer (LC). Transforming growth factor (TGF)-β is one of factors capable of inducing EMT. Polyinosinic-polycytidylic acid (polyI:C), a synthetic agonist for toll-like receptor (TLR) 3, can enhance immune responses and has been used as an adjuvant for cancer vaccines; however, it remains unclear whether it influences other process, such as EMT. In the present study, we examined the effects of polyI:C on TGF-β-treated A549 human LC cells.
METHODS AND RESULTS
By in vitro cell proliferation assay, polyI:C showed no effect on the growth of A549 cells treated with TGF-β1 at the concentration range up to 10 μg/ml; however, it markedly suppressed the motility in a cell scratch and a cell invasion assay. By Western blotting, polyI:C dramatically decreased TGF-β1-induced Ak strain transforming (Akt) phosphorylation and increased phosphatase and tensin homologue (PTEN) expression without affecting the Son of mothers against decapentaplegic (Smad) 3 phosphorylation or the expression level of E-cadherin, N-cadherin or Snail, indicating that polyI:C suppressed cell motility independently of the 'cadherin switching'. The Akt inhibitor perifosine inhibited TGF-β1-induced cell invasion, and the PTEN-specific inhibitor VO-OHpic appeared to reverse the inhibitory effect of polyI:C.
CONCLUSION
PolyI:C has a novel function to suppress the motility of LC cells undergoing EMT by targeting the phosphatidylinositol 3-kinase/Akt pathway partly via PTEN and may prevent or reduce the metastasis of LC cells.
Topics: A549 Cells; Adenocarcinoma, Bronchiolo-Alveolar; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Organometallic Compounds; PTEN Phosphohydrolase; Phosphatidylinositol 3-Kinase; Phosphorylation; Phosphorylcholine; Poly I-C; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Signal Transduction; Toll-Like Receptor 3; Transforming Growth Factor beta1
PubMed: 34390443
DOI: 10.1007/s11033-021-06625-1 -
Bioengineered Dec 2021Chronic skin ulcers are a primary global health problem. Velvet antler polypeptide (VAP) regulates endothelial cell migration and angiogenic sprout. Adipose-derived stem...
Chronic skin ulcers are a primary global health problem. Velvet antler polypeptide (VAP) regulates endothelial cell migration and angiogenic sprout. Adipose-derived stem cells (ADSCs) are reported to make pivotal impacts upon wound healing. This study aimed to explore the role of VAP combined with ADSCs in wound healing of chronic skin ulcers. The effect of VAP on phenotypes of ADSCs, and VAP (PLGA microspheres) combining with ADSCs on wound healing of chronic skin ulcers was evaluated. VAP generally promoted the proliferation, migration and invasion of ADSCs, and ADSC-induced angiogenesis in human umbilical vein endothelial cells (HUVECs) through PI3K/Akt/HIF-1α pathway. VAP-PLGA (PLGA microspheres) enhanced the promoting effect of ADSCs on wound healing, pathological changes, and angiogenesis in chronic skin ulcers . VAP-PLGA intensified the effect of ADSCs on up-regulating the levels of p-PI3K/PI3K, p-Akt/Akt, HIF-1α, vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), C-X-C motif chemokine receptor 4 (CXCR4), angiopoietin-4 (Ang-4), VEGF receptor (VEGFR), and transforming growth factor-β1 (TGF-β1), and down-regulating the levels of interleukin-1 β (IL-1β), IL-18 and IL-6 in wound tissues in chronic skin ulcers . Collectively, VAP promoted the growth, migration, invasion, and angiogenesis of ADSCs through activating PI3K/Akt/HIF-1α pathway, and VAP-PLGA enhanced the function of ADSCs in promoting wound healing , which was associated with angiogenesis, inflammation inhibition, and dermal collagen synthesis.
Topics: Adipose Tissue; Animals; Antlers; Cell Movement; Cell Proliferation; Cell Shape; Chromones; Chronic Disease; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Microspheres; Morpholines; Neovascularization, Physiologic; Peptides; Phenotype; Phosphatidylinositol 3-Kinases; Phosphorylcholine; Polylactic Acid-Polyglycolic Acid Copolymer; Proto-Oncogene Proteins c-akt; Signal Transduction; Skin Ulcer; Stem Cells; Wound Healing
PubMed: 34720043
DOI: 10.1080/21655979.2021.1990193