BACKGROUND: Fluoranthene (FR) is a common environmental pollutant that exists in a complex mixture with other polycyclic aromatic hydrocarbons (PAHs). We identified biomarkers for monitoring FR exposure and investigated the rescue effect of FR-induced cellular toxicity via aryl hydrocarbon receptor (AHR) antagonist activity in bone marrow derived mesenchymal stem cells (BM-MSCs).METHODS: Morphological changes, viability, and rescue effects of an AHR antagonist (CH223191) were examined in BM-MSCs after exposure to FR. Cytotoxic effects were assayed using the tetrazolium-based colorimetric assay. Apoptosis was measured by annexin V and propidium iodide dye-based flowcytometry assay, mitochondrial membrane potential assay, and nuclear DNA fragmentation assay. Molecular signaling pathways of apoptosis and autophagy were investigated using immunoblotting. Proteomics were performed in order to reveal the spectra of cellular damage and identify biomarkers for FR exposure.RESULTS: Exposing BM-MSCs to FR (IC₅₀=50 µM) induced cell death and morphological changes, while the AHR antagonist showed rescue effects. Autophagy was activated and mitochondrial membrane potential was decreased. Proteomic analysis identified 48 deregulated proteins (26 upregulated and 22 downregulated). Among them, annexin A6, pyruvate kinase, UDP-glucose dehydrogenase, and phospholipase A2 could be potential biomarkers for FR exposure.CONCLUSION: The exposure of BM-MSCs to FR induced remarkable alterations in cellular biology and the proteome, allowing for identification of novel biomarkers for FR exposure. Furthermore, AHR antagonists might be able to prevent cellular damage due to FR exposure.
Annexin A5 ; Annexin A6 ; Apoptosis ; Autophagy ; Biomarkers ; Bone Marrow ; Cell Death ; DNA Fragmentation ; Immunoblotting ; Membrane Potential, Mitochondrial ; Mesenchymal Stromal Cells ; Oxidoreductases ; Phospholipases A2 ; Polycyclic Hydrocarbons, Aromatic ; Propidium ; Proteome ; Proteomics ; Pyruvate Kinase ; Receptors, Aryl Hydrocarbon

Annexin A2, a multifunctional tumor associated protein, promotes nuclear factor-kappa B (NF-κB) activation by interacting with NF-κB p50 subunit and facilitating its nuclear translocation. Here we demonstrated that two ginsenosides Rg5 (G-Rg5) and Rk1 (G-Rk1), with similar structure, directly bound to Annexin A2 by molecular docking and cellular thermal shift assay. Both Rg5 and Rk1 inhibited the interaction between Annexin A2 and NF-κB p50 subunit, their translocation to nuclear and NF-κB activation. Inhibition of NF-κB by these two ginsenosides decreased the expression of inhibitor of apoptosis proteins (IAPs), leading to caspase activation and apoptosis. Over expression of K302A Annexin A2, a mutant version of Annexin A2, which fails to interact with G-Rg5 and G-Rk1, effectively reduced the NF-κB inhibitory effect and apoptosis induced by G-Rg5 and G-Rk1. In addition, the knockdown of Annexin A2 largely enhanced NF-κB activation and apoptosis induced by the two molecules, indicating that the effects of G-Rg5 and G-Rk1 on NF-κB were mainly mediated by Annexin A2. Taken together, this study for the first time demonstrated that G-Rg5 and G-Rk1 inhibit tumor cell growth by targeting Annexin A2 and NF-κB pathway, and G-Rg5 and G-Rk1 might be promising natural compounds for targeted cancer therapy.
Active Transport, Cell Nucleus ; drug effects ; Annexin A2 ; chemistry ; deficiency ; genetics ; metabolism ; Antineoplastic Agents ; chemistry ; metabolism ; pharmacology ; Apoptosis ; drug effects ; Biological Products ; chemistry ; metabolism ; pharmacology ; Cell Nucleus ; drug effects ; metabolism ; Down-Regulation ; drug effects ; Drug Discovery ; Gene Knockdown Techniques ; Ginsenosides ; chemistry ; Hep G2 Cells ; Humans ; Molecular Docking Simulation ; Molecular Targeted Therapy ; NF-kappa B p50 Subunit ; metabolism ; Protein Conformation

OBJECTIVETo investigate the effect of annexin A2 (AnxA2) on epithelial growth factor receptor (EGFR)/nuclear factor-κB (NF-κB) signal transduction and mucin expression in human airway epithelial H292 cells treated with Mycoplasma pneumoniae (MP).

METHODSH292 cells were divided into control group, MP group, NC-siRNA+MP group, and AnxA2 siRNA+MP group. The cells in the MP group were incubated with 5 μg/mL MP antigen for 2 hours. The cells in the NC-siRNA+MP and AnxA2 siRNA+MP groups were transfected with NC-siRNA and AnxA2 siRNA for 24 hours, followed by MP antigen stimulation for 2 hours. The MTT method was used to measure cell viability; quantitative real-time PCR was used to measure the mRNA expression of AnxA2; Western blot was used to measure the protein expression of AnxA2, phosphorylated EGFR (p-EGFR), and phosphorylated p65 NF-κB (p-p65 NF-κB); ELISA was used to measure the secretion of mucin 5AC (MUC5AC) and mucin 5B (MUC5B).

RESULTSThe MP and NC-siRNA+MP groups had lower cell viability than the control group (P<0.05). The AnxA2 siRNA+MP group had higher cell viability than the MP and NC-siRNA+MP groups and lower cell viability than the control group (P<0.05). The MP and NC-siRNA+MP groups had significantly higher mRNA and protein expression of AnxA2 than the AnxA2 siRNA+MP group (P<0.05). Compared with the control group, the MP and NC-siRNA+MP groups had significant increases in the protein expression of p-EGFR, p-p65 NF-κB, MUC5AC, and MUC5B (P<0.05); the AnxA2 siRNA+MP group had lower protein expression than the MP and NC-siRNA+MP groups, but higher protein expression than the control group (P<0.05).

CONCLUSIONSAnxA2 is involved in the airway lesion induced by MP antigen via mediating EGFR/NF-κB signaling activation and mucin expression in human airway epithelial cells.

Annexin A2 ; physiology ; Bronchi ; physiology ; Cells, Cultured ; Epithelial Cells ; microbiology ; Humans ; Mucins ; analysis ; Mycoplasma pneumoniae ; pathogenicity ; NF-kappa B ; physiology ; Receptor, Epidermal Growth Factor ; physiology ; Signal Transduction ; physiology

OBJECTIVETo explore effect of all-trans retinoic acid(ATRA) on annexin Ⅱ expression in NB4 cells and to analyze the luciferase activity of annexinⅡ promoter in condition of ATRA-induced treatment.

METHODSNB4 cells were cultured in vitro, the transcriptional or translational expression levels of Annexin Ⅱ in NB4 cells treated with 1 µmol/L ATRA at different time points were detected by RT-PCR or Western blot respectively. Annexin Ⅱ-promoter was constructed, the recombinant plasmids pGL4.15 -Annexin Ⅱ -promoter were transfected into NB4 cells with electroporation, and after being treated with 1 µmol/L ATRA for 24 hours the luciferase acttivity of Annexin Ⅱ promoter was determined by luciferase activity assay.

RESULTSThe transcriptional expression of Annexin Ⅱ was down-regulated after 48 h. The translation expression of Annexin Ⅱ was slowly weakened after 24 h, and it was seriously reduced after 48 h. Further, Luciferase activity of AnnexinⅡ promoter in NB4 cells treated with 1 µmol/L ATRA was down-regulated, and showed a decreased tendency at indicated time points.

CONCLUSIONAll-trans retinoic acid can induce the down-regulation of AnnexinⅡ expression on the membrane of NB4 cells, and the activity of Annexin Ⅱpromoter is down-regulated too. This study provide a basis for further study of molecular mechanism.

Annexin A2 ; Cell Differentiation ; Cell Line, Tumor ; Down-Regulation ; Humans ; Tretinoin

OBJECTIVETo investigate the effects of Annexin A2 (ANXA2) deficiency on the malignant biological behaviour of hepatoma cells.

METHODSThe human hepatocellular carcinoma (HCC) cells lines MHCC97-H, HepG2, SMMC-7721, SMMC-7402 and L02 were evaluated. The expression and distribution of ANXA2 were analysed by western blotting, real-time PCR, immunofluorescence and immunohistochemistry.Cell cycle was assessed by flow cytometry and propidium iodide staining. Effects of ANXA2 silencing on invasion and migration potential were assessed by transwell assay and wound healing assay, respectively. Proliferative potential was assessed by CCK-8 kit in vitro and xenograft tumour-growth assay in vivo. The t-test, chi square test, rank sum test, q-test and F-test were used for statistical analyses.

RESULTSThe expression level of ANXA2 was markedly higher in the MHCC97-H cells with high metastasis potential than in the HepG2, SMMC-7721, SMMC-7402 and L02 cells. The efficiency of shRNA-mediated ANXA2 deficiency was more than 80%. Immunofluorescence analysis of the MHCC97-H cells indicated that ANXA2 expression was mainly localized to the cellular membrane and cytoplasm, with some nuclear localization. Down-regulation of ANXA2 led to S-phase arrest of HCC cells (q =8.001, P =0.002) and an inhibition of proliferation (q =17.140, P less than 0.01), migration (q =12.808, P less than 0.01) and invasion potential (q =9.069, P =0.002). Xenograft tumour-growth assay indicated that shRNA targeting of ANXA2 led to lower tumour weight (q =11.968, P < 0.001) and down-regulated ANXA2 expression (Z =2.530, P =0.011).

CONCLUSIONDown-regulation of Annexin A2 gene transcription effectively changes the biological behaviours of hepatoma cells, and may represent a potential target of HCC molecular therapies.

Animals ; Annexin A2 ; genetics ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Liver Neoplasms ; pathology ; Neoplasm Transplantation ; RNA, Small Interfering ; genetics ; Signal Transduction ; Transcription, Genetic

OBJECTIVETo study the influence of CD147 gene silencing on the expression of ANXA2, MMP-2 and TIMP-2 of thyroid medullary carcinoma TT cells and related biological characteristics.

METHODSProtein expression of CD147, ANXA2, MMP-2 and TIMP-2 was detected by immunocytochemistry.RNAi technology was used to identify the specific siRNA sequences and the optimal time point of effective inhibition of CD147 gene. The expression of ANXA2, MMP-2 and TIMP-2 at mRNA and protein levels was detected with RT-PCR and Western blot, respectively. MTT method was used to detect the proliferation of the TT cells, flow cytometry (FCM) to detect the cell cycle and apoptosis changes of TT cells and transwell chamber assays to document the influence of CD147 gene silencing on migration and invasion of the TT cells.

RESULTSThe protein expression of CD147, ANXA2, MMP-2 and TIMP-2 proteins was variable in the TT cells. Two siRNA sequences were identified to effectively silence CD147 gene in the TT cells, in which relative expression of MMP-2 was reduced at both mRNA and protein levels; although the expression of ANXA2 mRNA and protein did not change significantly. TIMP-2 protein expression markedly decreased in an absence of its mRNA expression. The proliferation of the TT cells was inhibited upon the CD147 gene silencing along with a significant increase of G(0)/G(1) phase cells and a decrease of G(2)/M phase cells.However, the proportion of the apoptotic cells in all experimental groups did not change. The number of the penetrating cells through the membrane filters did not show significant changes in all experimental groups in the Transwell chamber assays.

CONCLUSIONSThrough RNAi technology, two CD147 siRNA sequences are identified and shown to effectively inhibit CD147 gene expression of the TT cells. CD147 gene silencing leads to growth inhibition of the TT cells and alteration of the cell cycle. However, silencing CD147 does not significantly affect the apoptosis, migration and invasion of the TT cells.

Annexin A2 ; genetics ; metabolism ; Basigin ; genetics ; metabolism ; Carcinoma, Medullary ; metabolism ; pathology ; Cell Cycle ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Humans ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Thyroid Neoplasms ; metabolism ; pathology ; Tissue Inhibitor of Metalloproteinase-2 ; genetics ; metabolism ; Transfection ; Tumor Cells, Cultured

This study was purposed to investigate the apoptosis-inducing effect of Annexin A2 gene (AnxA2) on multiple myeloma (MM) cells and its mechanisms. The human MM cell lines U266 and RPMI8226 were transfected by using siRNA targeting at AnxA2; the expressions of AnxA2 mRNA and protein in the siRNA-transfected cells were detected by real-time PCR and Western blot, respectively; the cell apoptosis was assayed by flow cytometry. The results showed that silencing AnxA2 gene by siRNA resulted in decreased expressions of AnxA2 gene and protein, increased apoptosis of U266 and RPMI8226 cell lines (P < 0.05), at the same time resulted in down-regulation of apoptosis-related gene expressions including p65NF-κB, IL-2, IL-6 (P < 0.05), and up-regulation of P53 gene expression (P < 0.05). It is concluded that the AnxA2 silence plays a promoting role in apoptosis of MM cell lines U266 and RPMI8226.
Annexin A2 ; genetics ; Apoptosis ; genetics ; Cell Line, Tumor ; Humans ; Multiple Myeloma ; genetics ; pathology ; RNA, Small Interfering ; genetics

OBJECTIVETo explore the role of annexin A2 (ANXA2) expression in the intestinal mucosa in the pathogenesis of inflammatory bowel disease (IBD).

METHODSIntestinal or colonic mucosal biopsy samples were obtained from 54 patients with ulcerative colitis (UC), 37 with Crohn's disease (CD), and 15 healthy control subjects. Immunohistochemistry was employed to examine the expression of ANXA2 in the intestinal mucosa, and mRNA expression of ANXA2 was detected using real-time PCR.

RESULTSImmunohistochemistry showed a ANXA2 positivity rate of 83.3% (45/54) in patients with UC, 27.0% (10/37) in patients with CD, and 53.3% (8/15) in the control subjects. ANXA2 expression in the intestinal or colonic mucosa was significantly up-regulated in patients with UC compared with the patients with CD and healthy control subjects, but was significantly lower in patients with CD than in the healthy controls (P<0.05). The expression levels of ANXA2 were strongly associated with the severity of clinical manifestations and the histopathological grades of UC (P<0.05). Compared with the healthy controls and patients with CD, patients with UC showed a significantly increased ANXA2 mRNA expression level in the inflamed mucosa of UC (P<0.05).

CONCLUSIONANXA2 can serve as a marker for differential diagnosis of IBD, and its up-regulated expression is closely related to the pathogenesis of UC.

Annexin A2 ; metabolism ; Case-Control Studies ; Colitis, Ulcerative ; metabolism ; pathology ; Crohn Disease ; metabolism ; pathology ; Female ; Humans ; Inflammatory Bowel Diseases ; metabolism ; pathology ; Intestinal Mucosa ; metabolism ; pathology ; Male

Ischemia-reperfusion injury is complicated with multiple injury pathways. If a particular agent is used to restore blood flow and prevent cell death, many damaged neurons may come back to life. However, for stroke victims, there are no effective curative therapeutic approaches available other than thrombolytic treatment. The efficacy of neuroprotective agents are limited by low diffusion, narrow time window, strict dose titration, and lack of confidence at the preclinical level. NXY-059 reflects the fundamental limitation of neuroprotectant. There are recent attempts to overcome these limitations, with use of annexin A2, fingolimod, hydrogen, nitrite, and more. By covering two components, this report reviews what we have recently learned. In addition, it sheds light on some of the newer issues in clinical application.
Annexin A2 ; Benzenesulfonates ; Cell Death ; Diffusion ; Hydrogen ; Light ; Multiple Trauma ; Neurons ; Neuroprotective Agents ; Propylene Glycols ; Reperfusion Injury ; Sphingosine ; Stroke ; Fingolimod Hydrochloride

OBJECTIVETo study the expression of annexin II (AnnII) and the fibrinolytic activity in NB4 cells and their alterations in the presence of arsenic trioxide (ATO) and daunorubicin (DNR).

METHODSLeukemia cell line NB4 was treated with ATO or DNR for 24 ∼ 72 h. Cell surface expression of AnnII and its mRNA were analysed by flow cytometry and real time PCR, respectively, the fibrinolytic activity by chromogenic assay.

RESULTSCompared with other acute leukemia cell lines, the expression of AnnII on untreated NB4 cells was relatively higher. The AnnII positive cell rates on NB4, HL-60, K562, and A3 cells were (94.5 ± 1.6)%, (40.1 ± 2.1)%, (36.3 ± 1.5)% and (11.8 ± 2.5)%, respectively. The fibrinolytic activity of NB4 cells was the greatest with a A value of 0.68 ± 0.02. The fibrinolytic activity of NB4 cells was obviously decreased by ATO, DNR or monoclonal antibody against AnnII, being decreased by 60.4%, 35.8% and 26.0% of the pretreatment level, respectively. The expressions of AnnII and its mRNA in NB4 cells were decreased dramatically after ATO and DNR treated for 48 h. Annexin II positive cells rate were (55.46 ± 4.72)% and (27.00 ± 6.18)%, respectively.

CONCLUSIONNB4 cells have strong ability to enhance the catalytic efficiency of the t-PA-dependent plasminogen activation and AnnII on the cell membrane contributes to this activity. Its high fibrinolytic activity can be corrected by ATO and DNR through down-regulating AnnII.

Annexin A2 ; Apoptosis ; Daunorubicin ; HL-60 Cells ; Humans ; Leukemia ; metabolism