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1.
Pharmaceuticals (Basel) ; 15(11)2022 Oct 31.
Article in English | MEDLINE | ID: mdl-36355519

ABSTRACT

Atopic dermatitis (AD) is a chronic inflammatory skin disease that results from eczema, itching, disrupted barrier function and aberrant cutaneous immune responses. The aim of the present study was to assess the efficacy of kushenol F as an effective treatment for AD via the suppression of thymic stromal lymphopoietin (TSLP) production. The results of the present study demonstrated that the clinical symptoms of AD were less severe and there was reduced ear thickening and scratching behavior in kushenol F-treated Dermatophagoides farinae extract (DFE)/1-chloro-2,4-dinitrochlorobenzene (DNCB)-induced AD mice. Histopathological analysis demonstrated that kushenol F decreased the DFE/DNCB-induced infiltration of eosinophil and mast cells and TSLP protein expression levels. Furthermore, kushenol F-treated mice exhibited significantly lower concentrations of serum histamine, IgE and IgG2a compared with the DFE/DNCB-induced control mice. Kushenol F also significantly decreased phosphorylated NF-κB and IKK levels and the mRNA expression levels of IL-1ß and IL-6 in cytokine combination-induced human keratinocytes. The results of the present study suggested that kushenol F may be a potential therapeutic candidate for the treatment of AD via reducing TSLP levels.

2.
Cell Biosci ; 12(1): 110, 2022 Jul 22.
Article in English | MEDLINE | ID: mdl-35869491

ABSTRACT

BACKGROUND: Spastin significantly influences microtubule regulation in neurons and is implicated in the pathogenesis of hereditary spastic paraplegia (HSP). However, post-translational regulation of the spastin protein remains nebulous. The association between E3 ubiquitin ligase and spastin provides a potential therapeutic strategy. RESULTS: As evidenced by protein chip analysis, FBXL17 inversely correlated with SPAST-M1 at the protein level in vitro and, also in vivo during embryonic developmental stage. SPAST-M1 protein interacted with FBXL17 specifically via the BTB domain at the N-terminus of SPAST-M1. The SCFFBXL17 E3 ubiquitin ligase complex degraded SPAST-M1 protein in the nuclear fraction in a proteasome-dependent manner. SPAST phosphorylation occurred only in the cytoplasmic fraction by CK2 and was involved in poly-ubiquitination. Inhibition of SCFFBXL17 E3 ubiquitin ligase by small chemical and FBXL17 shRNA decreased proteasome-dependent degradation of SPAST-M1 and induced axonal extension. The SPAST Y52C mutant, harboring abnormality in BTB domain could not interact with FBXL17, thereby escaping protein regulation by the SCFFBXL17 E3 ubiquitin ligase complex, resulting in loss of functionality with aberrant quantity. Although this mutant showed shortening of axonal outgrowth, low rate proliferation, and poor differentiation capacity in a 3D model, this phenotype was rescued by inhibiting SCFFBXL17 E3 ubiquitin ligase. CONCLUSIONS: We discovered that a novel pathway, FBXL17-SPAST was involved in pathogenicity of HSP by the loss of function and the quantitative regulation. This result suggested that targeting FBXL17 could provide new insight into HSP therapeutics.

3.
Cell Prolif ; 55(9): e13284, 2022 Sep.
Article in English | MEDLINE | ID: mdl-35723171

ABSTRACT

OBJECTIVES: The skin exhibits tremendous regenerative potential, as different types of progenitor and stem cells regulate skin homeostasis and damage. However, in vitro primary keratinocytes present with several drawbacks, such as high donor variability, short lifespan, and limited donor tissue availability. Therefore, more stable primary keratinocytes are needed to generate multiple uniform in vitro and in vivo skin models. RESULTS: We identified epidermal progenitor cells from primary keratinocytes using Integrin beta 1 (ITGB1) an epidermal stem cell marker markedly decreased after senescence in vitro. Epidermal progenitor cells exhibited unlimited proliferation and the potential for multipotent differentiation capacity. Moreover, they could completely differentiate to form an organotypic skin model including conversed mesenchymal cells in the dermis and could mimic the morphologic and biochemical processes of human epidermis. We also discovered that proliferation and the multipotent differentiation capacity of these cells relied on ITGB1 expression. Eventually, we examined the in vitro and in vivo wound healing capacity of these epidermal progenitor cells. CONCLUSIONS: Overall, the findings suggest that these stable and reproducible cells can differentiate into multiple lineages, including human skin models. They are a potentially powerful tool for studying skin regeneration, skin diseases, and are an alternative for in vivo experiments.


Subject(s)
Epidermal Cells , Skin , Cell Differentiation , Epidermal Cells/metabolism , Epidermis/metabolism , Humans , Integrin beta1/metabolism , Keratinocytes/metabolism , Skin/metabolism , Stem Cells
4.
J Back Musculoskelet Rehabil ; 35(2): 413-419, 2022.
Article in English | MEDLINE | ID: mdl-34250932

ABSTRACT

BACKGROUND: The medial hamstring (MH) and lateral hamstring (LH) can be selectively trained through tibial internal and external rotation during prone knee flexion. However, no study has identified how a combined tibial rotation and lumbo-pelvic stability strategy influences MH and LH muscle activities. OBJECTIVE: To investigate the combined effects of tibial rotation and the abdominal drawing-in maneuver (ADIM) on MH and LH muscle activities as well as pelvic rotation during prone knee flexion. METHODS: Fifteen female volunteers performed prone knee flexion with tibial internal and external rotation, with and without the ADIM. Under each condition, MH and LH muscle activities were measured by surface electromyography (EMG), and the pelvic rotation angle by a smartphone inclinometer application. RESULTS: The results showed increased MH (without the ADIM: p< 0.001, effect size (d) = 2.05; with the ADIM: p< 0.001, d= 1.71) and LH (without the ADIM: p< 0.001, d= 1.64; with the ADIM: p= 0.001, d= 1.58) muscle activities under internal and external tibial rotation, respectively. However, addition of the ADIM led to increased MH (internal tibial rotation: p= 0.001, d= 0.67; external tibial rotation: p= 0.019, d= 0.45) and LH (internal tibial rotation: p= 0.003, d= 0.79; external tibial rotation: p< 0.001, d= 1.05) muscle activities combined with reduced pelvic rotation (internal tibial rotation: p< 0.001, d= 3.45; external tibial rotation: p< 0.001, d= 3.01) during prone knee flexion. CONCLUSIONS: These findings suggest that the ADIM could be useful for reducing compensatory pelvic rotation and enhancing selective muscle activation in the MH and LH, according to the direction of tibial rotation, during prone knee flexion.


Subject(s)
Abdominal Muscles , Muscle, Skeletal , Abdominal Muscles/physiology , Biomechanical Phenomena , Electromyography , Female , Humans , Muscle, Skeletal/physiology , Pelvis , Tibia/physiology
5.
Int J Mol Sci ; 22(16)2021 Aug 17.
Article in English | MEDLINE | ID: mdl-34445562

ABSTRACT

Synaptonemal complex protein 3 (SCP3), a member of the Cor1 family, has been implicated in cancer progression, and therapeutic resistance, as well as cancer stem cell (CSC)-like properties. Previously, we demonstrated that SCP3 promotes these aggressive phenotypes via hyperactivation of the AKT signaling pathway; however, the underlying mechanisms responsible for SCP3-induced AKT activation remain to be elucidated. In this study, we demonstrated that the EGF-EGFR axis is the primary route through which SCP3 acts to activate AKT signaling. SCP3 triggers the EGFR-AKT pathway through transcriptional activation of EGF. Notably, neutralization of secreted EGF by its specific monoclonal antibody reversed SCP3-mediated aggressive phenotypes with a concomitant reversal of EGFR-AKT activation. In an effort to elucidate the molecular mechanisms underlying SCP3-induced transcriptional activation of EGF, we identified Jun activation domain-binding protein 1 (JAB1) as a binding partner of SCP3 using a yeast two-hybrid (Y2H) assay system, and we demonstrated that SCP3 induces EGF transcription through physical interaction with JAB1. Thus, our findings establish a firm molecular link among SCP3, EGFR, and AKT by identifying the novel roles of SCP3 in transcriptional regulation. We believe that these findings hold important implications for controlling SCP3high therapeutic-refractory cancer.


Subject(s)
COP9 Signalosome Complex/metabolism , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Drug Resistance, Neoplasm , Epidermal Growth Factor/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Neoplastic Stem Cells/drug effects , Peptide Hydrolases/metabolism , Uterine Cervical Neoplasms/drug therapy , COP9 Signalosome Complex/genetics , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Epidermal Growth Factor/genetics , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mutation , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Peptide Hydrolases/genetics , Phosphorylation , Protein Interaction Domains and Motifs , Signal Transduction , Tumor Cells, Cultured , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/pathology
6.
Exp Mol Med ; 53(3): 432-445, 2021 03.
Article in English | MEDLINE | ID: mdl-33731895

ABSTRACT

Cancer cachexia is a highly debilitating condition characterized by weight loss and muscle wasting that contributes significantly to the morbidity and mortality of pancreatic cancer. The factors that induce cachexia in pancreatic cancer are largely unknown. We previously showed that pancreatic adenocarcinoma upregulated factor (PAUF) secreted by pancreatic cancer cells is responsible for tumor growth and metastasis. Here, we analyzed the relation between pancreatic cancer-derived PAUF and cancer cachexia in mice and its clinical significance. Body weight loss and muscle weight loss were significantly higher in mice with Panc-1/PAUF tumors than in those with Panc-1/Mock tumors. Direct administration of rPAUF to muscle recapitulated tumor-induced atrophy, and a PAUF-neutralizing antibody abrogated tumor-induced muscle wasting in Panc-1/PAUF tumor-bearing mice. C2C12 myotubes treated with rPAUF exhibited rapid inactivation of Akt-Foxo3a signaling, resulting in Atrogin1/MAFbx upregulation, myosin heavy chain loss, and muscle atrophy. The neutrophil-to-lymphocyte ratio and body weight loss were significantly higher in pancreatic cancer patients with high PAUF expression than in those with low PAUF expression. Analysis of different pancreatic cancer datasets showed that PAUF expression was significantly higher in the pancreatic cancer group than in the nontumor group. Analysis of The Cancer Genome Atlas data found associations between high PAUF expression or a high DNA copy number and poor overall survival. Our data identified tumor-secreted circulating PAUF as a key factor of cachexia, causing muscle wasting in mice. Neutralizing PAUF may be a useful therapeutic strategy for the treatment of pancreatic cancer-induced cachexia.


Subject(s)
Adenocarcinoma/complications , Biomarkers, Tumor/metabolism , Cachexia/pathology , Gene Expression Regulation, Neoplastic , Intercellular Signaling Peptides and Proteins/metabolism , Muscular Atrophy/pathology , Pancreatic Neoplasms/complications , Animals , Apoptosis , Biomarkers, Tumor/genetics , Cachexia/etiology , Cachexia/metabolism , Cell Proliferation , Female , Humans , Male , Mice , Middle Aged , Muscular Atrophy/etiology , Muscular Atrophy/metabolism , Prognosis , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
7.
Biofabrication ; 12(4): 045029, 2020 09 25.
Article in English | MEDLINE | ID: mdl-32975216

ABSTRACT

Accurate prediction of pharmacokinetic (PK) and pharmacodynamic (PD) characteristics is critical for drug development. Oral drugs are particularly difficult because they are absorbed by the intestine and metabolized in the liver before systemic metabolism in vivo; this is called the first-pass effect and is a critical factor for predicting oral bioavailability (BA). Here, we fabricated a new networking and circulating cell culture system (NCCS), mimicking the circulatory system and interaction of organs for studying the pharmacokinetic and pharmacodynamics of oral drugs in vitro. NCCS consisted of a micro-pump for circulating fluids, two types of multi-insert culture dishes for culturing different cell types, and an orbital shaker for mixing; flow rate and shaking-speed were controlled by weight-sensors and drivers. A first-pass effect test was performed using functionally differentiated HepaRG and Caco-2 cell lines, using a new modified spheroid forming unit (SFU) protocol. To verify the similarity of PK (first-pass effect) data of NCCS with the data from the human body, 15 reference drugs were chosen and their associated data were obtained by liquid chromatography-mass spectrometry analysis. NCCS generated absorption and metabolism data showed >70% similarity to human data respectively. NCCS can also be used to demonstrate species differences. Animal models are the primary basis for drug discovery, development, and testing. However, the weak correlation between humans and animals, particularly regarding absorption and metabolism, is a substantial limitation for the use of animal models. Here we compare human and mouse acetaminophen (APAP) metabolism using NCCS, and its application can be extended to assess cellular responses, such as efficacy and toxicity, simultaneously.


Subject(s)
Models, Theoretical , Animals , Biological Availability , Caco-2 Cells , Humans , Intestines , Mice , Pharmaceutical Preparations
8.
Metabolism ; 110: 154302, 2020 09.
Article in English | MEDLINE | ID: mdl-32589900

ABSTRACT

BACKGROUND: Intracellular lipid accumulation is associated with various diseases, particularly cancer. Mitochondrial dysfunction is considered as a cause of lipid accumulation; however, the related underlying mechanism remains unclear. FINDINGS: We found that Von Hippel-Lindau (VHL)-deficiency led to lipid accumulation and mitochondrial dysfunction in renal cell carcinoma cells. Moreover, VHL downregulated ATP-citrate lyase (ACLY), a key enzyme in de novo lipid synthesis, at the transcriptional level, which inhibited intracellular lipid accumulation in human renal carcinoma tissues. We identified PPARγ as the transcription factor regulating ACLY expression by binding to the cis-regulatory site PPRE on its promoter. VHL directly interacted with and promoted ubiquitination of PPARγ, leading to its degradation both in vitro and in vivo, resulting in the downregulation of ACLY. Furthermore, adenovirus-mediated VHL overexpression substantially ameliorated hepatic steatosis induced by a high-fat diet in db/db mice. Importantly, low VHL expression was associated with high ACLY expression and poor prognosis in human liver carcinoma in a dataset in The Cancer Genome Atlas. CONCLUSIONS: VHL plays role in cellular lipid metabolism via regulating mitochondria and targeting PPARγ, a transcription factor for ACLY independent of hypoxia-inducible factor 1α. A novel VHL-PPARγ-ACLY axis and its implication in fatty liver disease and cancer were uncovered.


Subject(s)
ATP Citrate (pro-S)-Lyase/genetics , Lipid Metabolism/drug effects , Neoplasms/metabolism , PPAR gamma/metabolism , Ubiquitination , Von Hippel-Lindau Tumor Suppressor Protein/physiology , Animals , Cell Line, Tumor , Disease Progression , Fatty Liver/metabolism , Humans , Mice , Proteasome Endopeptidase Complex/physiology , Von Hippel-Lindau Tumor Suppressor Protein/genetics
9.
Sci Rep ; 9(1): 17610, 2019 11 26.
Article in English | MEDLINE | ID: mdl-31772214

ABSTRACT

Stable and reproducible kidney cellular models could accelerate our understanding of diseases, help therapeutics development, and improve nephrotoxicity screenings. Generation of a reproducible in vitro kidney models has been challenging owing to the cellular heterogeneity and structural complexity of the kidney. We generated mixed immortalized cell lines that stably maintained their characteristic expression of renal epithelial progenitor markers for the different lineages of kidney cellular compartments via the BMP7 signaling pathway from a mouse and a human whole kidney. These cells were used to generate functional and matured kidney spheroids containing multiple renal lineages, such as the proximal tubule, loop of Henle, distal tubules, and podocytes, using extracellular matrix and physiological force, named spheroid-forming unit (SFU). They expressed all apical and basolateral transporters that are important for drug metabolism and displayed key functional aspects of the proximal tubule, including protein endocytosis and increased gamma-glutamyltransferase activity, and cyclic AMP responded to external cues, such as parathyroid hormone. Following exposure, cells fluxed and took up drugs via proximal tubule-specific apical or basolateral transporters, and displayed increased cell death and expression of renal injury marker. Here, we developed a new differentiation method to generate kidney spheroids that structurally recapitulate important features of the kidney effectively and reproducibly using mixed immortalized renal cells, and showed their application for renal toxicity studies.


Subject(s)
Kidney/cytology , Spheroids, Cellular , Toxicity Tests/methods , Acyclovir/toxicity , Animals , Biological Transport/drug effects , Biomarkers , Bone Morphogenetic Protein 7/physiology , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/metabolism , Cell Culture Techniques , Cell Line, Transformed , Cell Lineage , Cimetidine/pharmacology , Cisplatin/toxicity , Cyclic AMP/metabolism , Cyclosporine/toxicity , Digoxin/pharmacology , Doxorubicin/toxicity , Drug Evaluation, Preclinical/methods , Endocytosis , Extracellular Matrix , Humans , Kidney/drug effects , Kidney/metabolism , Mice , Spheroids, Cellular/drug effects , Verapamil/pharmacology , gamma-Glutamyltransferase/metabolism
10.
Cell Death Dis ; 10(10): 758, 2019 10 07.
Article in English | MEDLINE | ID: mdl-31591383

ABSTRACT

Progranulin (PGRN) is a cysteine-rich secreted protein expressed in endothelial cells, immune cells, neurons, and adipocytes. It was first identified for its growth factor-like properties, being implicated in tissue remodeling, development, inflammation, and protein homeostasis. However, these findings are controversial, and the role of PGRN in liver disease remains unknown. In the current study, we examined the effect of PGRN in two different models of chronic liver disease, methionine-choline-deficient diet (MCD)-induced non-alcoholic steatohepatitis (NASH) and carbon tetrachloride (CCl4)-induced liver fibrosis. To induce long-term expression of PGRN, PGRN-expressing adenovirus was delivered via injection into the tibialis anterior. In the CCl4-induced fibrosis model, PGRN showed protective effects against hepatic injury, inflammation, and fibrosis via inhibition of nuclear transcription factor kappa B (NF-κB) phosphorylation. PGRN also decreased lipid accumulation and inhibited pro-inflammatory cytokine production and fibrosis in the MCD-induced NASH model. In vitro treatment of primary macrophages and Raw 264.7 cells with conditioned media from hepatocytes pre-treated with PGRN prior to stimulation with tumor necrosis factor (TNF)-α or palmitate decreased their expression of pro-inflammatory genes. Furthermore, PGRN suppressed inflammatory and fibrotic gene expression in a cell culture model of hepatocyte injury and primary stellate cell activation. These observations increase our understanding of the role of PGRN in liver injury and suggest PGRN delivery as a potential therapeutic strategy in chronic inflammatory liver disease.


Subject(s)
Inflammation/genetics , Liver Cirrhosis/genetics , Non-alcoholic Fatty Liver Disease/genetics , Progranulins/genetics , Animals , Endothelial Cells/immunology , Endothelial Cells/metabolism , Gene Expression Regulation/genetics , Hepatic Stellate Cells/immunology , Hepatic Stellate Cells/pathology , Hepatocytes/immunology , Hepatocytes/pathology , Humans , Inflammation/immunology , Inflammation/pathology , Liver/immunology , Liver/pathology , Liver Cirrhosis/pathology , Mice , NF-kappa B/genetics , Non-alcoholic Fatty Liver Disease/immunology , Non-alcoholic Fatty Liver Disease/pathology , Progranulins/immunology , RAW 264.7 Cells
11.
Cancers (Basel) ; 11(10)2019 Sep 30.
Article in English | MEDLINE | ID: mdl-31575084

ABSTRACT

Yes-associated protein (YAP)-1 is highly upregulated in pancreatic cancer and associated with tumor progression. However, little is known about the role of YAP1 and related genes in pancreatic cancer. Here, we identified target genes regulated by YAP1 and explored their role in pancreatic cancer progression and the related clinical implications. Analysis of different pancreatic cancer databases showed that Neuromedin U (NMU) expression was positively correlated with YAP1 expression in the tumor group. The Cancer Genome Atlas data indicated that high YAP1 and NMU expression levels were associated with poor mean and overall survival. YAP1 overexpression induced NMU expression and transcription and promoted cell motility in vitro and tumor metastasis in vivo via upregulation of epithelial-mesenchymal transition (EMT), whereas specific inhibition of NMU in cells stably expressing YAP1 had the opposite effect in vitro and in vivo. To define this functional association, we identified a transcriptional enhanced associate domain (TEAD) binding site in the NMU promoter and demonstrated that YAP1-TEAD binding upstream of the NMU gene regulated its transcription. These results indicate that the identified positive correlation between YAP1 and NMU is a potential novel drug target and biomarker in metastatic pancreatic cancer.

12.
Cell Death Dis ; 10(4): 279, 2019 03 22.
Article in English | MEDLINE | ID: mdl-30902965

ABSTRACT

Von Hippel Lindau (VHL) expression is significantly decreased in high-grade RCC, and autophagy, which is involved in tumor growth, invasion, differentiation, and metastasis, is activated in various human cancers. However, the relationship of autophagy and VHL in tumor progression remains controversial. Here, we showed that the expression levels of VHL and microtubule-associated protein 1 light chain 3B (MAP1LC3B, LC3B) were inversely correlated with various tumor grades of RCC tissues. pVHL was found to possess the LIR motif within a beta domain that interacted with MAP1LC3B and ubiquitinated it. The L101A VHL mutant failed to interact with MAP1LC3B, thereby failing to induce ubiquitination. MAP1LC3B-mediated autophagy was inhibited by functional pVHL and the ubiquitination of MAPLC3B was implicated in autophagy-induced cell death. We screened various autophagy inducers to determine the physiological function of the inhibition of LC3B-mediated autophagy by pVHL using VHL-deficient and VHL-expressing cell lines. MLN9708, a proteasome inhibitor, potently induced autophagy via the induction of MAP1LC3B and sensitized the cell to autophagy-mediated cell death in VHL-deficient and VHL-mutant (L101A) cells. In conclusion, our results showed that pVHL interacts with MAPL1LC3B and inhibits LC3B-mediated autophagy via MAP1LC3B ubiquitination. Furthermore, the activation of autophagy by the proteasome inhibitor MLN9708 induced cell death, indicating that MLN9708 can be used for VHL-deficient RCC therapy.


Subject(s)
Autophagy/drug effects , Boron Compounds/pharmacology , Carcinoma, Renal Cell/metabolism , Cell Death/drug effects , Glycine/analogs & derivatives , Kidney Neoplasms/metabolism , Microtubule-Associated Proteins/metabolism , Ubiquitination , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Animals , Boron Compounds/therapeutic use , Carcinoma, Renal Cell/drug therapy , Female , Glycine/pharmacology , Glycine/therapeutic use , HeLa Cells , Humans , Kidney Neoplasms/drug therapy , Mice , Mice, Inbred BALB C , Mice, Nude , Transfection , Tumor Burden/drug effects , Tumor Burden/genetics , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Xenograft Model Antitumor Assays
13.
Cell Mol Life Sci ; 76(13): 2647-2662, 2019 Jul.
Article in English | MEDLINE | ID: mdl-30903204

ABSTRACT

Hepatitis B virus (HBV) X protein (HBx) is associated with hepatocarcinogenesis. E2-EPF ubiquitin carrier protein (UCP) catalyzes ubiquitination of itself and von Hippel-Lindau protein (pVHL) for degradation and associates with tumor growth and metastasis. However, it remains unknown whether HBx modulates the enzyme activity of UCP and thereby influences hepatocarcinogenesis. Here, we show that UCP is highly expressed in liver tissues of HBx-transgenic mice, but not non-transgenic mice. UCP was more frequently expressed in HBV-positive liver cancers than in HBV-negative liver cancers. HBx binds to UCP specifically and serotype independently, and forms a ternary complex with UCP and pVHL. HBx inhibits self-ubiquitination of UCP, but enhances UCP-mediated pVHL ubiquitination, resulting in stabilization of hypoxia-inducible factor-1α and -2α. HBx and UCP stabilize each other by mutually inhibiting their ubiquitination. HBx promotes cellular proliferation and metastasis via UCP. Our findings suggest that UCP plays a key role in HBV-related hepatocarcinogenesis.


Subject(s)
Carcinoma, Hepatocellular/secondary , Hepatitis B/complications , Liver Neoplasms/pathology , Trans-Activators/metabolism , Ubiquitin-Conjugating Enzymes/chemistry , Animals , Apoptosis , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/virology , Cell Proliferation , Disease Progression , Female , Hepatitis B/virology , Hepatitis B virus/isolation & purification , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/virology , Mice , Mice, Inbred BALB C , Mice, Nude , Mice, Transgenic , Protein Stability , Signal Transduction , Trans-Activators/genetics , Tumor Cells, Cultured , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitination , Viral Regulatory and Accessory Proteins , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Xenograft Model Antitumor Assays
14.
Biochim Biophys Acta Mol Basis Dis ; 1864(10): 3221-3233, 2018 10.
Article in English | MEDLINE | ID: mdl-30006150

ABSTRACT

The spastin protein (SPAST) contains an ATPase with diverse cellular activities (AAA) domain and regulates microtubule dynamics. Missense mutations of the SPAST gene are frequently detected in patients with hereditary spastic paraplegias (HSPs) and represent the main reason of loss of SPAST function; however, the pathogenicity of mutant SPAST is heterogeneous. Here, SPAST variant with an I344K mutation (I344K-SPAST) was identified in a Korean family with autosomal dominant-type HSP. We investigated the role of the I344K-SPAST in HSP to provide a therapeutic mechanism. The I344K-SPAST mutation prolonged the half-life of the protein compared to wild-type SPAST (WT-SPAST) in cells by modulating post-translational modifications for proteasomal degradation. I344K-SPAST was localized in microtubule but defective in microtubule severing and ATPase activity compared to WT-SPAST in vitro and in cells. Mutant M87 isoform harboring the same mutation with I344K-M1 SPAST also increased protein stability and loss of MT severing activity, but the pathogenicity was not stronger than I344K-M1 SPAST in neurite outgrowth. Overexpression of I344K-SPAST resulted in microtubule accumulation following inhibited neurite growth in neuroblastoma, neural progenitor cells and mouse primary cortical neurons. Conversely, these pathogenic effects of I344K-SPAST were reduced by overexpression of WT-M1 SPAST in a dose dependent manner since WT-SPAST could interact with I344K-SPAST. Our data therefore provide proof-of-concept that gene transfer of WT-M1 SPAST may serve as a valid therapeutic option for HSPs.


Subject(s)
Mutation, Missense , Spastic Paraplegia, Hereditary/genetics , Spastin/genetics , Spastin/metabolism , Animals , Cell Line, Tumor , Female , HEK293 Cells , Half-Life , HeLa Cells , Humans , Male , Mice , Models, Molecular , Pedigree , Spastic Paraplegia, Hereditary/metabolism , Spastin/chemistry , Exome Sequencing
15.
Cancer Res ; 78(10): 2638-2653, 2018 05 15.
Article in English | MEDLINE | ID: mdl-29437706

ABSTRACT

Immunoediting caused by antitumor immunity drives tumor cells to acquire refractory phenotypes. We demonstrated previously that tumor antigen-specific T cells edit these cells such that they become resistant to CTL killing and enrich NANOGhigh cancer stem cell-like cells. In this study, we show that synaptonemal complex protein 3 (SCP3), a member of the Cor1 family, is overexpressed in immunoedited cells and upregulates NANOG by hyperactivating the cyclin D1-CDK4/6 axis. The SCP3-cyclin D1-CDK4/6 axis was preserved across various types of human cancer and correlated negatively with progression-free survival of cervical cancer patients. Targeting CDK4/6 with the inhibitor palbociclib reversed multiaggressive phenotypes of SCP3high immunoedited tumor cells and led to long-term control of the disease. Collectively, our findings establish a firm molecular link of multiaggressiveness among SCP3, NANOG, cyclin D1, and CDK4/6 and identify CDK4/6 inhibitors as actionable drugs for controlling SCP3high immune-refractory cancer.Significance: These findings reveal cyclin D1-CDK4/6 inhibition as an effective strategy for controlling SCP3high immune-refractroy cancer. Cancer Res; 78(10); 2638-53. ©2018 AACR.


Subject(s)
Cyclin D1/antagonists & inhibitors , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Nanog Homeobox Protein/metabolism , Neoplasms/drug therapy , Nuclear Proteins/metabolism , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Animals , Cell Cycle Proteins , Cell Line, Tumor , Cyclin D1/metabolism , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/metabolism , DNA-Binding Proteins , Female , HEK293 Cells , Humans , Immunotherapy/methods , Male , Mice , Mice, Inbred NOD , Mice, SCID , Nanog Homeobox Protein/genetics , Neoplasms/pathology , Nuclear Proteins/genetics , RNA Interference , RNA, Small Interfering/genetics , Zebrafish
16.
Sci Rep ; 7(1): 10499, 2017 09 05.
Article in English | MEDLINE | ID: mdl-28874716

ABSTRACT

We fabricated a spheroid-forming unit (SFU) for efficient and economic production of cell spheroids. We optimized the protocol for generating large and homogenous liver cancer cell spheroids using Huh7 hepatocellular carcinoma (HCC) cells. The large Huh7 spheroids showed apoptotic and proliferative signals in the centre and at the surface, respectively. In particular, hypoxia-induced factor-1 alpha (HIF-1α) and ERK signal activation were detected in the cell spheroids. To diminish core necrosis and increase the oncogenic character, we co-cultured spheroids with 2% human umbilical vein endothelial cells (HUVECs). HUVECs promoted proliferation and gene expression of HCC-related genes and cancer stem cell markers in the Huh7 spheroidsby activating cytokine signalling, mimicking gene expression in liver cancer. HUVECs induced angiogenesis and vessel maturation in Huh7 spheroids in vivo by activating epithelial-mesenchymal transition and angiogenic pathways. The large Huh7 cell spheroids containing HUVECs survived at higher concentrations of anti-cancer drugs (doxorubicin and sorafenib) than did monolayer cells. Our large cell spheroid provides a useful in vitro HCC model to enable intuitive observation for anti-cancer drug testing.


Subject(s)
Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Spheroids, Cellular , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Culture Techniques , Cell Line, Tumor , Coculture Techniques , Disease Models, Animal , Gene Expression Profiling , Human Umbilical Vein Endothelial Cells , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Mice , Transcriptome , Tumor Cells, Cultured , Tumor Microenvironment , Xenograft Model Antitumor Assays
17.
Cancer Res ; 77(18): 5039-5053, 2017 09 15.
Article in English | MEDLINE | ID: mdl-28716899

ABSTRACT

Cancer immunoediting drives the adaptation of tumor cells to host immune surveillance. Immunoediting driven by antigen (Ag)-specific T cells enriches NANOG expression in tumor cells, resulting in a stem-like phenotype and immune resistance. Here, we identify HDAC1 as a key mediator of the NANOG-associated phenotype. NANOG upregulated HDAC1 through promoter occupancy, thereby decreasing histone H3 acetylation on K14 and K27. NANOG-dependent, HDAC1-driven epigenetic silencing of cell-cycle inhibitors CDKN2D and CDKN1B induced stem-like features. Silencing of TRIM17 and NOXA induced immune and drug resistance in tumor cells by increasing antiapoptotic MCL1. Importantly, HDAC inhibition synergized with Ag-specific adoptive T-cell therapy to control immune refractory cancers. Our results reveal that NANOG influences the epigenetic state of tumor cells via HDAC1, and they encourage a rational application of epigenetic modulators and immunotherapy in treatment of NANOG+ refractory cancer types. Cancer Res; 77(18); 5039-53. ©2017 AACR.


Subject(s)
Breast Neoplasms/pathology , Drug Resistance, Multiple/immunology , Epigenesis, Genetic , Histone Deacetylase 1/metabolism , Nanog Homeobox Protein/metabolism , Neoplastic Stem Cells/pathology , Uterine Cervical Neoplasms/pathology , Acetylation , Animals , Apoptosis , Biomarkers, Tumor , Breast Neoplasms/drug therapy , Breast Neoplasms/immunology , Breast Neoplasms/metabolism , Cell Proliferation , Combined Modality Therapy , Drug Resistance, Multiple/drug effects , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/immunology , Female , Histone Deacetylase 1/genetics , Histones/metabolism , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Nanog Homeobox Protein/genetics , Neoplasm Staging , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/immunology , Neoplastic Stem Cells/metabolism , Prognosis , Transcriptional Activation , Tumor Cells, Cultured , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/immunology , Uterine Cervical Neoplasms/metabolism , Xenograft Model Antitumor Assays
18.
J Transl Med ; 15(1): 138, 2017 06 17.
Article in English | MEDLINE | ID: mdl-28623914

ABSTRACT

BACKGROUND: The interaction of vascular endothelial growth factor-C (VEGF-C)/VEGF-D/VEGF receptor-3 is considered to be a major driver of lymphangiogenesis, however the mechanism of this process remains unclear. We aimed to investigate the possible lymphangiogenic significance of synaptonemal complex protein 3 (SCP3) in non-small cell lung cancer (NSCLC). METHODS: The expression of SCP3, VEGF-C, and VEGF-D were measured and examined a correlation between SCP3 and VEGF-C or VEGF-D in various human lung cancer cell lines. Subsequently, we assessed SCP3, VEGF-A, VEGF-B, VEGF-C, and VEGF-D expression in archival tumor tissues from 89 NSCLC patients with lymph node (LN) metastasis by combined immunohistochemistry with quantitative digital image analysis. RESULTS: Positive correlations between SCP3 and VEGF-C expression (R 2  = 0.743) and VEGF-D expression (R 2  = 0.932) were detected in various human lung cancer cell lines. The high expression of SCP3, VEGF-A, VEGF-B, VEGF-C, and VEGF-D were detected in 24 (27.0%), 22 (24.7%), 27 (30.3%), 27 (30.3%), and 24 cases (27.0%), respectively. Notably, SCP3 positively correlated with VEGF-C and VEGF-D expression (for both, P < 0.001) and negatively correlated with VEGF-A and VEGF-B expression (P = 0.029 and P = 0.026, respectively). In multivariate analysis of patients with LN metastasis, SCP3 expression predicted worse overall survival (hazard ratio = 1.86, P = 0.008). CONCLUSIONS: SCP3 is associated with lymphangiogenesis and provides insight into the SCP3-VEGF-C/VEGF-D axis based cancer therapy strategy.


Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lymphangiogenesis , Lymphatic Metastasis/pathology , Nuclear Proteins/metabolism , Aged , Cell Cycle Proteins , Cell Line, Tumor , DNA-Binding Proteins , Female , Humans , Male , Middle Aged , Multivariate Analysis , Prognosis , Survival Analysis , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor B/metabolism , Vascular Endothelial Growth Factor C/metabolism , Vascular Endothelial Growth Factor D/metabolism
19.
J Phys Ther Sci ; 28(7): 2094-6, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27512272

ABSTRACT

[Purpose] Differences in scores on the Functional Movement Screen between patients with chronic lower back pain and healthy control subjects were investigated. [Subjects and Methods] In all, 20 chronic lower back pain patients and 20 healthy control subjects were recruited. Chronic lower back pain patients and healthy controls performed the Functional Movement Screen (deep squat, hurdle step, inline lunge, shoulder mobility, active straight leg raise, trunk stability pushup, and rotary stability). The Mann-Whitney test was used to analyze differences in Functional Movement Screen scores between the two groups. [Results] Chronic lower back pain patients scored lower on the Functional Movement Screen total composite compared with healthy control subjects. Chronic lower back pain patients scored lower on Functional Movement Screen subtests including the deep squat, hurdle step, active straight leg raise, and rotary stability tests. [Conclusion] The deep squat, hurdle step, active straight leg raise, and rotary stability tasks of the Functional Movement Screen can be recommended as a functional assessment tools to identify functional deficits in chronic lower back pain patients.

20.
Oncotarget ; 6(37): 40255-67, 2015 Nov 24.
Article in English | MEDLINE | ID: mdl-26517679

ABSTRACT

The fusion between anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) is a causative factor in a unique subset of patients with non-small cell lung carcinoma (NSCLC). Although the inhibitor crizotinib, as it blocks the kinase activity of the resulting EML4-ALK fusion protein, displays remarkable initial responses, a fraction of NSCLC cases eventually become resistant to crizotinib by acquiring mutations in the ALK domain or activating bypass pathways via EGFR, KIT, or KRAS. Cancer stem cell (CSC) theory provides a plausible explanation for acquisition of tumorigenesis and resistance. However, the question as to whether EML4-ALK-driven tumorigenesis is linked with the stem-like property and whether the stemness is an effective target in controlling EML4-ALK+ NSCLC including crizotinib-resistant NSCLC cells has not been addressed. Here, we report that stem-like properties stem from ALK activity in EML4-ALK+ NSCLC cells. Notably, treatment with rapamycin, a CSC targeting agent, attenuates stem-like phenotypes of the EML4-ALK+ cells, which increased capability of tumor formation and higher expression of stemness-associated molecules such as ALDH, NANOG, and OCT4. Importantly, combinational treatment with rapamycin and crizotinib leads to synergistic anti-tumor effects on EML4-ALK+ NSCLC cells as well as on those resistant to crizotinib. Thus, we provide a proof of principle that targeting stemness would be a novel strategy to control intractable EML4-ALK+ NSCLC.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/genetics , Neoplastic Stem Cells/metabolism , Oncogene Proteins, Fusion/genetics , RNA Interference , Animals , Antibiotics, Antineoplastic/pharmacology , Blotting, Western , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Crizotinib , Drug Synergism , Female , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Neoplastic Stem Cells/drug effects , Oncogene Proteins, Fusion/antagonists & inhibitors , Oncogene Proteins, Fusion/metabolism , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyridines/pharmacology , RNAi Therapeutics/methods , Sirolimus/pharmacology , Xenograft Model Antitumor Assays/methods
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