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1.
J Clin Invest ; 130(8): 4301-4319, 2020 08 03.
Article in English | MEDLINE | ID: mdl-32396532

ABSTRACT

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers and is highly resistant to current treatments. ESCC harbors a subpopulation of cells exhibiting cancer stem-like cell (CSC) properties that contribute to therapeutic resistance including radioresistance, but the molecular mechanisms in ESCC CSCs are currently unknown. Here, we report that ribosomal S6 protein kinase 4 (RSK4) plays a pivotal role in promoting CSC properties and radioresistance in ESCC. RSK4 was highly expressed in ESCC CSCs and associated with radioresistance and poor survival in patients with ESCC. RSK4 was found to be a direct downstream transcriptional target of ΔNp63α, the main p63 isoform, which is frequently amplified in ESCC. RSK4 activated the ß-catenin signaling pathway through direct phosphorylation of GSK-3ß at Ser9. Pharmacologic inhibition of RSK4 effectively reduced CSC properties and improved radiosensitivity in both nude mouse and patient-derived xenograft models. Collectively, our results strongly suggest that the ΔNp63α/RSK4/GSK-3ß axis plays a key role in driving CSC properties and radioresistance in ESCC, indicating that RSK4 is a promising therapeutic target for ESCC treatment.


Subject(s)
Esophageal Neoplasms/enzymology , Esophageal Squamous Cell Carcinoma/enzymology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/biosynthesis , Radiation Tolerance , Ribosomal Protein S6 Kinases, 90-kDa/biosynthesis , Signal Transduction , Animals , Esophageal Neoplasms/genetics , Esophageal Neoplasms/pathology , Esophageal Neoplasms/therapy , Esophageal Squamous Cell Carcinoma/genetics , Esophageal Squamous Cell Carcinoma/pathology , Esophageal Squamous Cell Carcinoma/therapy , HEK293 Cells , Humans , Mice , Neoplasm Proteins/genetics , Ribosomal Protein S6 Kinases, 90-kDa/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Xenograft Model Antitumor Assays
2.
J Huazhong Univ Sci Technolog Med Sci ; 33(6): 810-816, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24337840

ABSTRACT

Autophagy is a conserved and programmed catabolic process that degrades damaged proteins and organelles. But the underlying mechanism and functions of autophagy in the ischemia-reperfusion (IR)-induced injury are unknown. In this study, we employed simulated IR of N2a cells as an in vitro model of IR injury to the neurons and monitored autophagic processes. It was found that the levels of Beclin-1 (a key molecule of autophay complex, Beclin-1/class III PI3K) and LC-3II (an autophagy marker) were remarkably increased with time during the process of ischemia and the process of reperfusion after 90 min of ischemia, while the protein kinases p70S6K and mTOR which are involved in autophagy regulation showed delayed inactivation after reperfusion. Administration of 3-methyladenine (3MA), an inhibitor of class III PI3K, abolished autophagy during reperfusion, while employment of rapamycin, an inhibitor of mTORC1 (normally inducing autophagy), surprisingly weakened the induction of autophagy during reperfusion. Analyses of mitochondria function by relative cell viability demonstrated that autophagy inhibition by 3-MA attenuated the decline of mitochondria function during reperfusion. Our data demonstrated that there were two distinct dynamic patterns of autophagy during IR-induced N2a injury, Beclin-1/class III PI3K complex-dependent and mTORC1-dependent. Inhibition of over-autophagy improved cell survival. These suggest that targeting autophagy therapy will be a novel strategy to control IR-induced neuronal damage.


Subject(s)
Autophagy , Neurons/metabolism , Reperfusion Injury/metabolism , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Beclin-1 , Cell Line, Tumor , Cell Survival , Mechanistic Target of Rapamycin Complex 1 , Mice , Mitochondria/metabolism , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/metabolism , Neurons/drug effects , Neuroprotective Agents/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism
3.
Acta Pharmacol Sin ; 33(2): 230-6, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22212389

ABSTRACT

AIM: Aquaporin-2 (AQP2) is a vasopressin-regulated water channel located in the collecting tubule and collecting duct cells of mammalian kidney. The aim of this study is to investigate whether PKCα plays a role in vasopressin-induced AQP2 trafficking in mouse inner medullary collecting duct 3 (mIMCD3) cells. METHODS: AQP2-mIMCD3 stable cell line was constructed by transfection of mouse inner medullary collecting duct 3 (mIMCD3) cells with AQP2-GFP construct. Then the cells were transfected with PKCα shRNA, PKCα A/25E, or PKCα scrambled shRNA. The expression levels of PKCα, AQP2, and phospho-S256-AQP2 were analyzed using Western blot. The interaction between AQP2 and PKCα was examined using immunoprecipitation. The distribution of AQP2 and microtubules was studied using immunocytochemistry. The AQP2 trafficking was examined using the biotinylation of surface membranes. RESULTS: Treatment of AQP2-mIMCD3 cells with 100 µmol/L of 1-desamino-8-D-arginine vasopressin (DdAVP) for 30 min stimulated the translocation of AQP2 from the cytoplasm to plasma membrane through influencing the microtubule assembly. Upregulation of active PKCα by transfection with PKCα A/25E plasmids resulted in de-polymerization of α-tubulin and redistributed AQP2 in the cytoplasm. Down-regulation of PKCα by PKCα shRNA partially inhibited DdAVP-stimulated AQP2 trafficking without altering α-tubulin distribution. Although 100 µmol/L of DdAVP increased AQP2 phosphorylation at serine 256, down-regulation of PKCα by PKCα shRNA did not influence DdAVP-induced AQP2 phosphorylation, suggesting that AQP2 phosphorylation at serine 256 was independent of PKCα. Moreover, PKCα did not physically interact with AQP2 in the presence or absence of DdAVP. CONCLUSION: Our results suggested that PKCα regulates AQP2 trafficking induced by DdAVP via microtubule assembly.


Subject(s)
Antidiuretic Agents/pharmacology , Aquaporin 2/metabolism , Deamino Arginine Vasopressin/pharmacology , Kidney/cytology , Protein Kinase C-alpha/metabolism , Tubulin/metabolism , Animals , Aquaporin 2/genetics , Cell Line , Mice , Phosphorylation/drug effects , Protein Kinase C-alpha/genetics , Protein Transport/drug effects , RNA, Small Interfering/genetics , Up-Regulation
4.
Article in Chinese | MEDLINE | ID: mdl-19288912

ABSTRACT

OBJECTIVE: To construct DNA vaccine (pIRES-Sj97-Sj14-Sj26) and study its immunogenicity and protective immunity against Schistosoma japonicum. METHODS: The plasmid pIRES-Sj97-Sj14-Sj26 containing fatty binding protein (Sj14), GST (Sj26) and paramyosin (Sj97) was constructed and expressed on the membrane. RT-PCR was used to detect the expression of Sj14 mRNA, Sj26 mRNA and Sj97 mRNA in the Hela cells, and IFA for detecting the expression of trans-membrane Sj14, Sj26 and Sj97. Sixty BALB/c mice were randomly divided into 3 groups. Mice in each group respectively received normal saline, pIRES blank vector and pIRES-Sj97-Sj14-Sj26 by intramuscular injection. Two weeks after the 3rd immunization, 10 mice from each group were sacrificed and total IgG in serum and the level of IFN-y were detected by ELISA and lymphocyte stimulating index (SI) by MTt. FCM was used to analyze the subgroups of splenocytes. The level of NO secreted by peritoneal macrophages was determined by nitrate reductase approaches. The left 10 mice in each group were challenged with (40 +/- 1) cercariae of S. japonicum by abdominal skin penetration. Forty-five days after challenge, mice were sacrificed, and numbers of recovered worms and hepatic eggs were counted. RESULTS: RT-PCR showed the expression of Sj14 mRNA, Sj26 mRNA and Sj97 mRNA. IFA proved the expression of Sj26, Sj14 and Sj97 protein. Level of total IgG in the vaccination group, saline group and pIRES blank vector group was (5.62 +/- 0.64), (1.22 +/- 0.20) and (1.48 +/- 0.36) mg/ml respectively, showing a statistical significance (P < 0.01, P < 0.05). The NO level in macrophages was (321.19 +/- 18.03), (184.12 +/- 11.05) and (213.51 +/- 15.93) nmol/ml in the 3 groups respectively (P < 0.05), and the lymphocyte stimulating index in the 3 groups was (2.25 +/- 0.29), (1.18 +/- 0.07) and(1.22 +/- 0.09) respectively (P < 0.01). The INF-gamma level was higher in the vaccination group than others (P < 0.01). The percentage of CD4+ and CD8+ T cells increased considerably (P < 0.01). The vaccination group showed a worm reduction rate of 39.9% (P < 0.01) and an egg reduction rate of 43.9% (P < 0.01). CONCLUSION: The vaccine candidate pIRES-Sj97-Sj14-Sj26 induces an immune protection in BALB/c mice against Schistosoma japonicum.


Subject(s)
Antigens, Helminth/immunology , Membrane Proteins/immunology , Schistosoma japonicum/immunology , Vaccines, DNA/immunology , Animals , Antibodies, Helminth/blood , Antibodies, Helminth/immunology , Antigens, Helminth/genetics , HeLa Cells , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Interferon-gamma/immunology , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Male , Membrane Proteins/genetics , Mice , Mice, Inbred BALB C , Nitric Oxide/metabolism , Plasmids , Schistosoma japonicum/genetics , Transfection
5.
Am J Pathol ; 160(4): 1269-78, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11943712

ABSTRACT

We have developed an ultrasensitive bienzyme-substrate-recycle enzyme-linked immunosorbent assay for the measurement of Alzheimer's disease (AD) abnormally hyperphosphorylated tau in cerebrospinal fluid (CSF). The assay, which recognizes attomolar amounts of tau, is approximately 400 and approximately 1300 times more sensitive than conventional enzyme-linked immunosorbent assay in determining the hyperphosphorylated tau and total tau, respectively. With this method, we measured both total tau and tau phosphorylated at Ser-396/Ser-404 in lumbar CSFs from AD and control patients. We found that the total tau was 215 +/- 77 pg/ml in cognitively normal control (n = 56), 234 +/- 92 pg/ml in non-AD neurological (n = 37), 304 +/- 126 pg/ml in vascular dementia (n = 46), and 486 +/- 168 pg/ml (n = 52) in AD patients, respectively. However, a remarkably elevated level in phosphorylated tau was only found in AD (187 +/- 84 pg/ml), as compared with normal controls (54 +/- 33 pg/ml), non-AD (63 +/- 34 pg/ml), and vascular dementia (72 +/- 33 pg/ml) groups. If we used the ratio of hyperphosphorylated tau to total tau of > or =0.33 as cutoff for AD diagnosis, we could confirm the diagnosis in 96% of the clinically diagnosed patients with a specificity of 95%, 86%, 100%, and 94% against nonneurological, non-AD neurological, vascular dementia, and all of the three control groups combined, respectively. It is suggested that the CSF level of tau phosphorylated at Ser-396/Ser-404 is a promising diagnostic marker of AD.


Subject(s)
Alzheimer Disease/cerebrospinal fluid , Enzyme-Linked Immunosorbent Assay/methods , tau Proteins/cerebrospinal fluid , Aged , Dementia, Vascular/cerebrospinal fluid , Enzyme-Linked Immunosorbent Assay/standards , Female , Humans , Male , Middle Aged , Phosphorylation , Reference Values , Sensitivity and Specificity , tau Proteins/metabolism
6.
Neurosci Lett ; 320(3): 156-60, 2002 Mar 08.
Article in English | MEDLINE | ID: mdl-11852185

ABSTRACT

Neurofilament (NF) subunits NF-H, NF-M and NF-L are hyperphosphorylated and elevated in Alzheimer disease (AD) brain. We investigated the level and phosphorylation states of NF subunits in lumbar cerebrospinal fluid (CSF) from living patients by bienzyme substrate-recycle enzyme-linked immunosorbent assay. We found: (i), that the levels of phosphorylated NF-H/M (pNF-H/M), non-phosphorylated NF-H/M (npNF-H/M) and NF-L were significantly higher (pNF-H/M, approximately 12-24-fold; npNF-H/M, approximately 3-4-fold) in neurologically healthy aged people than young control individuals; (ii), that in AD, the levels of npNF-H/M, and NF-L were similar to vascular dementia (VaD), and higher than in age-matched controls; and (iii), that the levels of pNF-H/M were significantly higher than in aged controls, non-AD neurological disorders and VaD. Based on these findings, it is suggested that the increased level of total NF proteins in CSF could be used as a marker for brain aging and neurodegenerative disorders in general, and the levels of pNF-H/M as a marker to discriminate AD from normal brain aging and as well as neurological conditions including VaD.


Subject(s)
Alzheimer Disease/cerebrospinal fluid , Brain/metabolism , Neurofilament Proteins/cerebrospinal fluid , Neurons/metabolism , Up-Regulation/physiology , Adolescent , Age Factors , Aged , Brain/pathology , Brain/physiopathology , Child , Child, Preschool , Female , Humans , Immunoassay , Male , Middle Aged , Neurons/pathology , Phosphorylation , tau Proteins/cerebrospinal fluid
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