Effect of a disintegrin and metalloprotease 17 deletion on production of reactive oxygen species and mitochondrial function in nasopharyngeal carcinoma cells / 中国生物制品学杂志

@#Objective To investigate the effects of a disintegrin and metalloproteinase 17(ADAM17) deletion on the production of reactive oxygen species(ROS) and mitochondrial function in nasopharyngeal carcinoma(NPC) cells.Methods Three groups of ADAM1 7 interfering plasmid ADAM17 shRNA and empty plasmid ADAM17-shRNA-NC were transfected into NPC cell line(CNE1) and detected for the interference efficiency by RT-PCR and Western blot to select shRNA with the best interference effect for the follow-up experiments.The cell proliferation was detected by CCK-8 assay,while the cell growth by clone formation test,the apoptosis and changes in mitochondrial membrane potential(MMP) by flow cytometry,the level of mitochondrial oxidative damage product ROS by fluorescence microscope,the contents of oxidative stress markers MDA and SOD by malondialdehyde(MDA) kit and superoxide dismutase(SOD) kit and the expression of mitochondrial damage markers Bax/Bcl-2,cleaved-caspase 9/caspase 9,cleaved-caspase 3/caspase 3 and c-Myc by Western blot.Results ADAM17-shRNA2 group showed the best interference effect.Compared with shRNA-NC group,the proliferation rate of cell in ADAM17-shRNA 2 group decreased significantly(t=8.964,P=0.036);the number of colonies were significantly reduced(t=10.351,P=0.014);the number of apoptosis increased significantly(t=11.25,P=0.008);the fluorescence intensity representing ROS level in cells increased obviously;the mitochondrial membrane potential decreased significantly(t=9.233,P=0.013);the SOD content decreased(t=7.233,P=0.034) and MDA content increased(t=7.415,P=0.038) significantly;the levels of Bax/Bcl-2,cleaved-caspase 9/caspase 9 and cleaved-caspase 3/caspase 3 significantly increased(t=8.985,9.021 and 7.789,P=0.023,0.011 and 0.031,respectively),while the expression of c-Myc proteins significantly decreased(t=10.352,P=0.004).Conclusion Interfering with ADAM1 7 induced SOD decrease and MDA increase by promoting oxidation,thereby alleviating oxidative damage of cell membrane,which also promoted the expression level of ROS in mitochondrion,reduced MMP,inhibited cell proliferation in vitro,and promoted apoptosis.

Identification of new human mesenchymal stem cell biomarker by aptamers / Identificação de novos biomarcadores de células tronco mesenquimais de origem humana por meio de aptâmeros

Stem cells are undifferentiated cells that can be distinguished from others by their ability to self-renew and to differentiate into new specific cell types. Mesenchymal stem cells (MSC) are adult stem cells that can be obtained from different sources, such as adipose tissue, bone marrow, dental pulp, and umbilical cord. They can either replicate, originating new identical cells, or differentiate into cells of mesodermal origin and from other germ layers. MSC have been studied as new tools for regenerative therapy. Although encouraging results have been demonstrated, MSC-based therapies still face a great barrier: the difficulty of isolating these cells from heterogeneous environments. MSC are currently characterized by immunolabelling through a set of multiple surface membrane markers, including CD29, CD73, CD90 and CD105, which are also expressed by other cell types. Hence, the present work aimed to identify new specific biomarkers for the characterization of human MSC using DNA aptamers produced by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique. Our results showed that MSC from different origins bound to DNA candidate aptamers, that is, DNA or RNA oligonucleotides selected from random libraries that bind specifically to biological targets. Aptamer-bound MSC could be isolated by fluorescenceactivated cell sorting (FACS) procedures, enhancing the induction of differentiation into specific phenotypes (chondrocytes, osteocytes and adipocytes) when compared to the whole MSC population. Flow cytometry analyses revealed that candidate aptamers bound to 50% of the MSC population from dental pulp and did not present significant binding rates to human fibroblasts or lymphocytes, both used as negative control. Moreover, immunofluorescence images and confocal analyses revealed staining of MSC by aptamers localized in the surfacemembrane of these cells. The results also showed internal staining of human monocytes by our investigated aptamers. A non-specific control aptamer (CNTR APT) obtained from the random pool was then utilized to compare the specificity of the aptamers bound to the analyzed non-apoptotic cells, showing no staining for MSC. However, 40% of the monocytes bound to the CNTR APT. Normalized data based on the cells bound to candidate aptamers compared to those bound to the CNTR APT, revealed a 10 to 16-fold higher binding rate for MSC against 2-fold for monocytes. Despite its low specificity, monocyte-aptamer binding occurs probably due to the expression of shared markers with MSC, since monocytes are derived from hematopoietic stem cells and are important for the immune system ability to internalize/phagocyte external molecules. Given that, we performed a pull-down assay followed by mass spectrometry analysis to detect which MSC-specific protein or other target epitope not coexpressed by monocytes or the CNTR APT would bind to the candidate aptamer. Distinguishing between MSC and monocyte epitopes is important, as both cells are involved in immunomodulatory effects after MSC transplantations. ADAM17 was found to be a target of the APT10, emerging as a possible biomarker of MSC, since its involvement in the inhibition of the TGF signaling cascade, which is responsible for the differentiation of MSC. Thus, MSC with a higher stemness profile should overexpress the protein ADAM17, which presents a catalytic site with affinity to APT10. Another target of Apt 10 is VAMP3, belonging to a transmembrane protein complex that is involved in endocytosis and exocytosis processes during immune and inflammatory responses. Overall, proteins identified as targets of APT10 may be cell surface MSC biomarkers, with importance for MSC-based cell and immune therapies

Células tronco são células indiferenciadas que podem ser distinguidas de outros tipos celulares por meio da habilidade de se auto renovarem e de se diferenciarem em novos tipos celulares. Células tronco mesenquimais (MSC) são células tronco adultas encontradas em diferentes tecidos como tecido adiposo, polpa de dente e cordão umbilical. Estas células podem se autodividir em células idênticas ou se diferenciarem em células de origem mesodermal. Estas células têm sido estudadas em novas aplicações que envolvem terapia regenerativas. Embora resultados encorajadores tenham sido demonstrados, terapias que utilizam MSC ainda encontram uma grande barreira: a dificuldade no isolamento destas células a partir de um ambiente heterogêneo. MSC são caracterizadas por populações positivas em ensaios de imunomarcação para os epítopos membranares CD29, CD73, CD90 e CD105, presentes também em outros tipos celulares. Assim, o presente trabalho tem o objetivo de identificar novos biomarcadores de MSC de origem humana, utilizando aptâmeros de DNA produzidos pela técnica SELEX (Systematic Evolution of Ligands by EXponential Enrichment) como ferramenta. Nossos resultados mostraram que MSC de diferentes origens ligam-se a aptâmeros (oligonucleotídeos de DNA ou RNA que atuam como ligantes específicos de alvos moleculares) de DNA candidatos que atuam no isolamento de MSC por meio da técnica FACS de separação celular, promovendo uma maior indução de diferenciação em células específicas (condrócitos, osteócitos e adipócitos) comparada com a população total de MSC. Análises de citometria de fluxo mostraram que os aptâmeros candidatos se ligam a 50% das MSC de polpa de dente e não apresentam taxa de ligação significante para fibroblastos e linfócitos de origem humana - utilizados como controles negativo. Além domais, imagens de imunofluorescência e confocal mostraram ligação na superfície da membrana de MSC e a marcação interna de monócitos a estes aptâmeros. Portanto, um aptâmero controle (CNTR APT) foi utilizado para comparar a especificidade dos aptâmeros ligados a células viáveis, mostrando a não ligação deste aptâmero a MSC. Porém, 40% da população de monócitos ligou-se ao CNTR APT. Uma normalização baseada na comparação entre as taxas de ligação entre células ligadas com aptâmeros candidatos e o aptâmero controle gerou uma taxa de especificidade entre 10-16 vezes maior para MSC contra 2,5 vezes para os monócitos. Deste modo, embora os resultados tenham mostrado uma taxa de ligação entre monócitos e aptâmeros, as MSC ligadas aos aptâmeros candidatos possuem uma maior taxa de especificidade devido a uma maior presença de antígenos que são expressos em ambas as células. Um ensaio de Pull Down seguido de espectrometria de massas foi utilizado para a identificação de biomarcadores que se ligariam aos aptâmeros candidatos, e que não seriam co-expressos por monócitos e por antígenos ligados ao aptâmero controle. Deste modo, a proteína ADAM17 foi identificada nas amostras de APT10 ligadas às MSC. Tal proteína está relacionada à inibição de uma cascata de sinalização da família de proteínas TGF, responsável pela diferenciação de MSC. Assim, MSC com maior potencial tronco deveriam expressar ADAM17 em maior quantidade. Tal proteína apresenta um sítio catalítico que demonstra interagir com o APT10, de acordo com predição Docking entre proteína e DNA. Foi identificada também, a proteína VAMP3, que pertence a um complexo proteico transmembranar responsável pelos processos de endocitose e exocitose, e que podem ter um papel importante na liberação de citocinas e outras moléculas relacionadas às respostas imune e inflamatórias. Deste modo, o APT10 identificou proteínas importantes que devem estar relacionas com a melhora de imunoterapias que utilizam MSC

The generation of PD-L1 and PD-L2 in cancer cells: From nuclear chromatin reorganization to extracellular presentation

The immune checkpoint blockade (ICB) targeting on PD-1/PD-L1 has shown remarkable promise in treating cancers. However, the low response rate and frequently observed severe side effects limit its broad benefits. It is partially due to less understanding of the biological regulation of PD-L1. Here, we systematically and comprehensively summarized the regulation of PD-L1 from nuclear chromatin reorganization to extracellular presentation. In PD-L1 and PD-L2 highly expressed cancer cells, a new TAD (topologically associating domain) (chr9: 5,400,000-5,600,000) around CD274 and CD273 was discovered, which includes a reported super-enhancer to drive synchronous transcription of PD-L1 and PD-L2. The re-shaped TAD allows transcription factors such as STAT3 and IRF1 recruit to PD-L1 locus in order to guide the expression of PD-L1. After transcription, the PD-L1 is tightly regulated by miRNAs and RNA-binding proteins via the long 3'UTR. At translational level, PD-L1 protein and its membrane presentation are tightly regulated by post-translational modification such as glycosylation and ubiquitination. In addition, PD-L1 can be secreted via exosome to systematically inhibit immune response. Therefore, fully dissecting the regulation of PD-L1/PD-L2 and thoroughly detecting PD-L1/PD-L2 as well as their regulatory networks will bring more insights in ICB and ICB-based combinational therapy.

Expresión génica de ligandos mica, micb y ulbp (1-6) del receptor NKG2D de células natural killer y metaloproteinasas adam10, adam17 y mmp14 en lineas celulares de cáncer de cervical / Gene expression of mica, micb and ulbp (1-6) ligands of the NKG2D receptor of natural killer cells and metalloproteinases adam10, adam17 and mmp14 in cells lines of cervical cancer

RESUMEN El CCU es la segunda causa de muerte en mujeres de nuestro país. Dentro de los primeros mecanismos de defensa del hospedero se encuentra la respuesta inmune de las células NK y su función lítica a expensas de su receptor activador NKG2D, el cual posee como ligandos mica, micb y ulbp (1-6), los cuales se expresan en células transformadas y/o infectadas por virus. Uno de los mecanismos de evasión por parte de la célula tumoral es el clivaje de estas proteínas a través de metaloproteinasas como adam10, adam17 y mmp14. Se analizó la expresión de estos ligandos y metaloproteinasas mediante PCR tiempo real, en lineas celulares de referencia para cáncer cervical como HeLa (positiva para VPH-18) y C33A (negativa para VPH). Se obtuvieron valores representativos de expresion relativa genica con diferencias significativas asi: mmp14 en linea HeLa (p= 0.006); y mica y ulbp-3 en la linea C33A (p= 0.020 y p=0.003 respectivamente). Por lo tanto, se podría sugerir que la expresión de mmp14 se encuentra posiblemente involucrados con la presencia de VPH causante del cancer cervical y la respuesta inmunne innata desarrollada.

ABSTRACT Cervical cancer is the second leading cause of death in women in our country. Within the first host defense mechanisms is the immune response of NK cells and their lytic function at the expense of its NKG2D receptor activator which has as ligands mica, micb and ulbp (1-6), which are expressed in transformed cells and / or virally infected. One of the mechanisms of evasion by the tumor cell is the cleavage of these proteins through metalloproteinases as adam10, adam17 and mmp14. We analyzed the expression of these ligands and metalloproteinases by real time PCR, in reference to cell lines HeLa cervical cancer (positive for HPV-18) and C33A (negative for HPV). We obtained representing relative gene expression with significant differences from the other lines of study as follows: mmp14 in HeLa (p = 0.006); and mica and ulbp-3 in C33A (p = 0.020 and p = 0.003 respectively). Thus one might suggest that the expression of mmp14 is possible involved with HPV presence causing high risk of cervical cancer and innate inmunne response developed.

Effect of paraquat on the expression of a disintegrin and metalloproteinase-17 in A549 cells / 中华劳动卫生职业病杂志

Objective@#Construct a paraquat (PQ) cell fibrosis model in vitro, observe the effect of PQ on the expression of a disintegrin and metalloproteinase-17 (ADAM17) in A549 cells, and explore the role of ADAM17 in the pulmonary fibrosis induced by PQ poisoning.@*Methods@#A549 cells are divided into normal control group, different concentration of PQ groups, CCK-8 is used to detect cell viability, screening concentration and time of PQ, cell morphology is observed under microscope; Enzyme-linked immunosorbent assay (ELISA) detectes fibrosis markers of collagen type I (Col I) and fibronectin (FN) expression. Establishment of cell model of fibrosis; distribution by immunocytochemical detection of ADAM17 in A549 cells, Reverse transcription-polymerase chain reaction and Western blot are used to detect the expression of ADAM17 mRNA and protein.@*Results@#1. With the increase of PQ concentration and the prolongation of the action time, the activity of A549 cells decreased (P< 0.05) , which is dose-dependent and time dependent. 2.The normal A549 cells fusion is paving stone growth and arranged more closely. After PQ induction, the cell arrangement was loose, the intercellular connection became loose, and some cells dissolved and died. 3.ELISA showed that with the increase of PQ concentration, the expression of Col I and FN increased (P<0.05) , and Col I and FN expression gradually increased with the prolongation of PQ time (P<0.05) , and the fibroblast model is successfully established. 4. Immunocytochemistry showes that ADAM17 is expressed in the cytoplasm of A549 cells. 5. RT-PCR and Western blot showed that the expression of ADAM17 mRNA and protein increased significantly with the increase of PQ concentration (P<0.05) , which is most obvious at PQ 200 μmol/L. With the prolonged action of PQ, the expression level of ADAM17 mRNA and protein also increased significantly (P<0.05) , and reached the peak in 24 h.@*Conclusion@#PQ can induce morphological changes of alveolar epithelial cells, cause cell damage, and successfully establish a cell fibrosis model, which has a dose and time dependence on the toxicity of A549 cells. ADAM17 is overexpressed in the A549 cells induced by PQ and may be involved in the process of pulmonary fibrosis induced by paraquat.

Experimental study of regulatory effect of hydrogen sulfide on sFlt-1 production in adipocytes through ADAM17 / 中华内分泌代谢杂志

Objective To study the effect of hydrogen sulfide on the production of soluble fms-like tyrosine kinase 1 (sFlt-1) through a distintegrin and metalloproteinase 17 (ADAM17) in adipocytes. Methods 3T3-L1 cells were cultured and induced to differentiate into adipocytes, then treated with different doses of sodium hydrogen sulfide (NaHS), L-cysteine or transfected with cystathionine-γ-lyase ( CSE) siRNA, ADAM17 siRNA or treated with ADAM17 inhibitor, monoclonal antibody. 24 hours after treatment, the expression of ADAM17, CSE, and the production of sFlt-1 were determined. Results After the treatment of 10, 25, 50 nmol/L NaHS or 0. 5, 1. 0, 2. 0 μmol/L L-cysteine, the expression of ADAM17 and the production of sFlt-1 in adipocytes were significantly decreased, the higher dose of L-cysteine and sFlt-1, the lower expression of ADAM17 and the production of sFlt-1; the effect of 2.0 μmol/L L-cysteine decreasing the expression of ADAM17 and the production of sFlt-1 were reversed by transfection of CSE siRNA; after the transfection of ADAM17 siRNA and treatment of ADAM17 inhibitor or monoclonal antibody, the production of sFlt-1 in adipocytes were significantly decreased. Conclusion Hydrogen sulfide can reduce the production of sFlt-1 in adipocytes by downregulating the expression of ADAM17.

ADAM17 knockdown increases sensitivity of SW480 cells to cetuximad / 南方医科大学学报

OBJECTIVE@#To explore the association between expression of ADAM17 and cetuximad resistance in human colorectal cancer SW480 cells.@*METHODS@#The expression of ADAM17 was detected using Western blotting in different human colorectal cancer cell lines, and the cells highly expressing ADAM17 were selected as the target cells. SW480 cells were transfected with ADAM17-siRNA 1 and ADAM17-siRNA 2 and the changes in the expression of ADAM17 protein were detected using Western blotting. SW480 cells were exposed to cetuximad for 24 h and the cell apoptosis was analyzed using flow cytometry. Transwell assay was used to examine the migration ability of SW480 cells with different expression levels of ADAM17; Western blotting was used to analyze the changes in the expressions of AKT signaling pathway-related proteins in the treated cells.@*RESULTS@#The baseline expressions of ADAM17 were significantly higher in SW480 cells than in the other human colorectal cancer cell lines tested ( < 0.05). Both ADAM17-siRNA 1 and 2 effectively reduced the expression of ADAM17 protein in SW480 cells. Knockdown of ADAM17 with siRNA 1 significantly increased the sensitivity of SW480 cells to tocetuximad ( < 0.05), obviously inhibited the cell proliferation, migration and invasion, and significantly reduced the expressions of p-EGFR and p-AKT in the cells ( < 0.001).@*CONCLUSIONS@#ADAM17 knockdown obviously inhibits EGFR-AKT signaling pathway and increases the sensitivity of SW480 cells to tocetuximad.

Effects of ADAM17 on high-glucose mediated permeability, proliferation, migration and proteins expression in human retinal microvascular endothelial cells / 眼科新进展

Objective To investigate the effects and mechanisms of a disintegrin and metalloproteinase 17 (ADAM17) on high-glucose mediated permeability,proliferation and migration in human retinal microvascular endothelial cells (HRMECs).Methods HRMECs were divided into 4 groups:normal group (5 mmol · L-1 glucose),high glucose group (25 mmol · L-1 glucose),NC (Negative control for siRNA) + high glucose group and siADAM17 (ADAM17 siRNA) + high glucose group.The expression of ADAM17 was detected using real time PCR and Western blot.Horseradish Peroxidase (HRP) was used to detect the permeability of HRMECs.Cell Counting Kit-8 (CCK-8)and BrdU were used to evaluate cell proliferation.Cell migration was determined using Transwell assay.In addition,the expression of p-EGFR,p-ERK and MMP9 was assayed using Western blot.Results Compared with normal group,the mRNA and protein levels of ADAM17 were increased in high glucose group (P ＜ 0.01).ADAM17 expression of siADAM17 + high glucose group was markedly reduced compared with NC + high glucose group.High glucose increased the permeability of HRP comparison to normal group,whereas in siADAM17 + high glucose group the permeability of HRP was reduced compared with NC + high glucose group.The optical density of HRMECs was decreased in siADAM17 + high glucose group 1.53 ± 0.29 in comparison with NC + high glucose group 2.43 ± 0.25,as well as the content of BrdU-incorporation(P ＜ 0.05).The number of migrated cells in high glucose group,NC + high glucose group,siADAM17 + high glucose group and normal group were 157.00 ± 7.93,169.00 ± 10.12,121.00 ± 9.28,110.00 ±8.25,respectively.Moreover,the expression of p-EGFR,p-ERK and MMP9 in siADAM17 +high glucose group was decreased compared with NC + high glucose group (all P ＜0.01).Conclusion SiADAM17 can reduce the cell permeability,suppressed and migration induced by high glucose via EGFR/ERK/MMP9 signaling pathway.

Tetraspanin CD9 modulates ADAM17-mediated shedding of LR11 in leukocytes

LR11, also known as SorLA or SORL1, is a type-I membrane protein from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding on cleavage with a disintegrin and metalloproteinase 17 (ADAM17). A shedding mechanism is presumed to have a key role in the functions of LR11, but the evidence for this has not yet been demonstrated. Tetraspanin CD9 has been recently shown to regulate the ADAM17-mediated shedding of tumor necrosis factor-alpha and intercellular adhesion molecule-1 on the cell surface. Here, we investigated the role of CD9 on the shedding of LR11 in leukocytes. LR11 was not expressed in THP-1 monocytes, but it was expressed and released in phorbol 12-myristate 13-acetate (PMA)-induced THP-1 macrophages (PMA/THP-1). Confocal microscopy showed colocalization of LR11 and CD9 proteins on the cell surface of PMA/THP-1. Ectopic neo-expression of CD9 in CCRF-SB cells, which are LR11-positive and CD9-negative, reduced the amount of sLR11 released from the cells. In contrast, incubation of LR11-transfected THP-1 cells with neutralizing anti-CD9 monoclonal antibodies increased the amount of sLR11 released from the cells. Likewise, the PMA-stimulated release of sLR11 increased in THP-1 cells transfected with CD9-targeted shRNAs, which was negated by treatment with the metalloproteinase inhibitor GM6001. These results suggest that the tetraspanin CD9 modulates the ADAM17-mediated shedding of LR11 in various leukemia cell lines and that the association between LR11 and CD9 on the cell surface has an important role in the ADAM17-mediated shedding mechanism.

Contribution of environmental pollutants to male infertily: A working model of germ cell apoptosis induced by plasticizers

Bisphenol A [2,2-bis(4-hydroxyphenyl)propane] (BPA), 4-nonylphenol (NP) and di(2-ethylhexyl)phthalate (DEHP), and its metabolite mono-2-ethylhexyl phthalate (MEHP) are chemicals found in plastics, which act as endocrine disruptors (EDs) in animals, including human. EDs act like hormones in the endocrine system, and disrupt the physiologic function of endogenous hormones. Most people are exposed to different endocrine disruptors and concern has been raised about their true effect on reproductive organs. In the testis, they seem to preferentially attack developing testis during puberty rather than adult organs. However, the lack of information about the molecular mechanism, and the apparently controversial effect observed in different models has hampered the understanding of their effects on mammalian spermatogenesis. In this review, we critically discuss the available information regarding the effect of BPA, NP and DEHP/ MEHP upon mammalian spermatogenesis, a major target of EDs. Germ cell sloughing, disruption of the blood-testis-barrier and germ cell apoptosis are the most common effects reported in the available literature. We propose a model at the molecular level to explain the effects at the cellular level, mainly focused on germ cell apoptosis.

Expression and clinicopathological significance of ADAM17 mRNA in esophageal squamous cell carcinoma / 肿瘤研究与临床

ObjectiveTo investigate the clinicopathologic significance of a disintegrin and metalloproteinase 17(ADAM 17) mRNA in esophageal squamous cell carcinoma (ESCC),and to evaluate its relation to clinicopathological features. MethodsThe expression of ADAM17 mRNA in 50 ESCC and 50 normal esophageal tissues were detected by RT-PCR. ResultsThe mRNA expression of ADAM17 in 50 ESCC and 50 normal esophageal tissues were 0.937±0.241 and 0.225±0.077,respectively.The expression of ADAM17 mRNA in the ESCC was much higher than in normal esophageal tissues (t=-19.899,P＜0.01).The expression of ADAM 17 mRNA was positively correlated with lymph node metastasis(t=-4.703,P＜0.01 ) and TNM staging (t=-2.652,P＜0.05).There were no correlation between the expression of ADAM17 mRNA and sex,age and histological grade (t=0.299,-0.907,-3.163,all P＞0.05).ConclusionADAM17 mRNA was higherly express in ESCC than in normal esophageal tissues and may play an important role in the development,invasion and metastasis of ESCC.It may be used as a prognostic factor.