Over-expression of gastrin inhibits apoptosis of gastric cancer cells via reactive oxygen species and annexin A2-mediated mitochondrial dysfunction / La sobreexpresión de gastrina inhibe la apoptosis de las células de cáncer gástrico a través de especies reactivas de oxígeno y disfunción mitocondrial mediada por anexina A2

SUMMARY: Gastrin plays a vital role in the development and progression of gastric cancer (GC). Its expression is up-regulated in GC tissues and several GC cell lines. Yet, the underlying mechanism remains to be investigated. Here, we aim to investigate the role and mechanism of gastrin in GC proliferation. Gastrin-overexpressing GC cell model was constructed using SGC7901 cells. Then the differentially expressed proteins were identified by iTRAQ analysis. Next, we use flow cytometry and immunofluorescence to study the effect of gastrin on the mitochondrial potential and mitochondria-derived ROS production. Finally, we studied the underlying mechanism of gastrin regulating mitochondrial function using Co-IP, mass spectrometry and immunofluorescence. Overexpression of gastrin promoted GC cell proliferation in vitro and in vivo. A total of 173 proteins were expressed differently between the controls and gastrin- overexpression cells and most of these proteins were involved in tumorigenesis and cell proliferation. Among them, Cox17, Cox5B and ATP5J that were all localized to the mitochondrial respiratory chain were down-regulated in gastrin-overexpression cells. Furthermore, gastrin overexpression led to mitochondrial potential decrease and mitochondria-derived ROS increase. Additionally, gastrin-induced ROS generation resulted in the inhibition of cell apoptosis via activating NF-kB, inhibiting Bax expression and promoting Bcl-2 expression. Finally, we found gastrin interacted with mitochondrial membrane protein Annexin A2 using Co-IP and mass spectrometry. Overexpr ession of gastrin inhibits GC cell apoptosis by inducing mitochondrial dysfunction through interacting with mitochondrial protein Annexin A2, then up-regulating ROS production to activate NF-kB and further leading to Bax/Bcl-2 ratio decrease.

La gastrina juega un papel vital en el desarrollo y progresión del cáncer gástrico (CG). Su expresión está regulada al alza en tejidos de CG y en varias líneas celulares de CG. Sin embargo, el mecanismo subyacente aun no se ha investigado. El objetivo de este estudio fue investigar el papel y el mecanismo de la gastrina en la proliferación de CG. El modelo de células CG que sobre expresan gastrina se construyó usando células SGC7901. Luego, las proteínas expresadas diferencialmente se identificaron mediante análisis iTRAQ. A continuación, utilizamos la citometría de flujo y la inmunofluorescencia para estudiar el efecto de la gastrina en el potencial mitocondrial y la producción de ROS derivada de las mitocondrias. Finalmente, estudiamos el mecanismo subyacente de la gastrina que regula la función mitocondrial utilizando Co-IP, espectrometría de masas e inmunofluorescencia. La sobreexpresión de gastrina promovió la proliferación de células CG in vitro e in vivo. Un total de 173 proteínas se expresaron de manera diferente entre los controles y las células con sobreexpresión de gastrina y la mayoría de estas proteínas estaban implicadas en la tumorigenesis y la proliferación celular. Entre estas, Cox17, Cox5B y ATP5J, todas localizadas en la cadena respiratoria mitocondrial, estaban reguladas a la baja en las células con sobreexpresión de gastrina. Además, la sobreexpresión de gastrina provocó una disminución del potencial mitocondrial y un aumento de las ROS derivadas de las mitocondrias. Por otra parte, la generación de ROS inducida por gastrina resultó en la inhibición de la apoptosis celular mediante la activación de NF-kB, inhibiendo la expresión de Bax y promoviendo la expresión de Bcl-2. Finalmente, encontramos que la gastrina interactuaba con la proteína de membrana mitocondrial Anexina A2 usando Co-IP y espectrometría de masas. La sobreexpresión de gastrina inhibe la apoptosis de las células CG al inducir la disfunción mitocondrial a través de la interacción con la proteína mitocondrial Anexina A2, luego regula el aumento de la producción de ROS para activar NF-kB y conduce aún más a la disminución de la relación Bax/Bcl-2.

Identification of natural compounds targeting Annexin A2 with an anti-cancer effect

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.

Papel da anexina A2 na progressão do câncer colorretal / [Role of annexin A2 in the progression of colorectal cancer]

Anexinas são proteínas ligantes de fosfolipídeos dependente de cálcio que exercem funções celulares como organização do citoesqueleto, transporte de íons e sinalização celular. Por estarem relacionadas com a organização de actina e com a proliferação celular;; alterações na sua expressão podem ser implicadas na tumorigênese de diferentes tipos de câncer. A anexina A2 (ANXA2), promove a proliferação, migração e invasão celular quando superexpressa. Em estudo recente, sugeriu-­se a utilização da anexina A2 como biomarcador do desenvolvimento do câncer colorretal (CCR). Contudo, existem poucas evidências sobre como a ANXA2 é regulada e seu papel na modulação de vias de sinalização é pouco conhecido. O objetivo desse estudo foi a elucidação do papel da ANXA2 em eventos relacionados com a progressão do CCR. Análise da expressão de ANXA2 em amostras de pacientes de CCR revelou aumento da expressão no tecido tumoral, corroborados pela análise do banco de dados do TCGA e por nossos resultados de IHC indicando relação da ANXA2 com estadios mais avançados e marcação diferencial na metástase. Foram realizados ensaios in vitro para avaliar os níveis de expressão e fosforilação (resíduo Y23) da proteína anexina A2, em linhagens celulares de adenocarcinoma de cólon (Caco-­2, HT-­29 e HCT-­116), através de imunoblotting. Para a avaliação do papel da ANXA2 na progressão tumoral, submetemos células HT-­29, silenciada ou não para a ANXA2, ao tratamento com TGF-­ß , avaliando o potencial proliferativo, migratório e invasivo destas células. Os resultados mostram que as células Caco-­2 apresentam um nível de fosforilação da ANXA2 menor do que os níveis observados nas células HT-­29 e HCT-­116, as quais apresentaram níveis similares de fosforilação desta proteína. Para avaliação de eventos relacionados com a EMT, foram analisados marcadores epiteliais e mesenquimas por meio de imunoblotting e imunofluorescência. Mediante tratamento com TGF-­ß , a linhagem HT-­29 exibiu alterações morfológicas características com o desenvolvimento da EMT, assim como redução de E-­caderina e aumento na expressão de marcadores mesenquimais como a vimentina. Houve aumento de expressão total de ANXA2, da sua forma fosforilada e relocalização da superfície celular para o citoplasma na linhagem HT-­29. Concomitantemente ao tratamento com TGF-­ß observou-­se a perda dos contatos intercelulares mediada pela internalização da E-­caderina e ANXA2. A análise de microscopia de iluminação estruturada confirmou a colocalização de ambas proteinas intracelularmente, assim como a transfecção de mutantes sítios específicos para a ANXA2 provou a sua interação com E-­caderina. O ensaio de proliferação mostrou que o TGF-­ß e o silenciamento de ANXA2 são anti-­proliferativos. O silenciamento causou redução na migração (Wound Healing), independente do aumento migratório causado pelo TGF-­ß . A indução da EMT por TGF-­ß levou ao aumento na capacidade invasiva destas células, aumento que não ocorreu nas células silenciadas para a ANXA2 ou tratadas com STA21 (inibidor de STAT3). O uso deste inibidor, STA21 e de PP2 (inibidor de Src) impediram a EMT nas células tratadas com TGF-­ß. Concluimos que a ANXA2 atua na manutenção das junções celulares mediadas pela E-­caderina;; do aumento da capacidade invasiva, características do processo de EMT, via Src/ANXA2/STAT3 e também na proliferação e migração celular.

Effect of annexin A2 on EGFR/NF-κB signal transduction and mucin expression in human airway epithelial cells treated with Mycoplasma pneumoniae / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To 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).</p><p><b>METHODS</b>H292 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).</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSIONS</b>AnxA2 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.</p>

Down-regulation of AnnexinⅡExpression in NB4 Cells Induced by all-trans Retinoic Acid and Its Mechanisms / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>NB4 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>All-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.</p>

Effects of Annexin A2 gene transcription down-regulation on human hepatoma cell biological behaviour / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects of Annexin A2 (ANXA2) deficiency on the malignant biological behaviour of hepatoma cells.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>Down-regulation of Annexin A2 gene transcription effectively changes the biological behaviours of hepatoma cells, and may represent a potential target of HCC molecular therapies.</p>

Effects of CD147 gene silencing on protein expression of ANXA2, MMP-2 and TIMP-2 by thyroid medullary carcinoma TT cells and biologic characteristics / 中华病理学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Protein 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>Through 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.</p>

Expression and function of Annexin II in lung cancer tissue

OBJECTIVE@#To explore the expression of Annexin II and its relationship with the cell differentiation, proliferation in lung cancer.@*METHODS@#RT-PCR and Western blot assays were used to detect the expression of Annexin II in lung cancer tissues and cell lines.@*RESULTS@#Annexin II was significantly up-regulated in lung cancer tissues, and in lung cancer cell lines, Annexin II had higher mRNA and protein expressions.@*CONCLUSIONS@#Annexin II is up-regulated in lung cancer, suggesting that the Annexin II has a potential value in the human lung cancer.

Apoptosis-inducing effect of annexin A2 on multiple myeloma cells and its related mechanisms / 中国实验血液学杂志

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 expression in intestinal mucosa of patients with inflammatory bowel disease and its clinical implications / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of annexin A2 (ANXA2) expression in the intestinal mucosa in the pathogenesis of inflammatory bowel disease (IBD).</p><p><b>METHODS</b>Intestinal 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.</p><p><b>RESULTS</b>Immunohistochemistry 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).</p><p><b>CONCLUSION</b>ANXA2 can serve as a marker for differential diagnosis of IBD, and its up-regulated expression is closely related to the pathogenesis of UC.</p>

Challenges and Pitfalls of Stroke Therapeutics Research / 대한뇌졸중학회지

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.

Silencing of the annexin II gene down- regulates the levels of S100A10, c-Myc, and plasmin and inhibits breast cancer cell proliferation and invasion

To explore the roles of annexin II in breast cancer progression, and to study the effect of annexin II on breast cancer cell proliferation and invasion. This study was conducted in the Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Chongqing Medical University, Chongqing, China from December 2006 to January 2009. First, we employed Western blot and reverse transcriptase polymerase chain reaction to detect the expression of annexin II and S100A10 in a panel of well-characterized human breast cancer cell lines, and investigated the localization of annexin II and S100A10 by use of immunofluorescence. We then silenced the expression of annexin II in MDA-MB-435s, which was found to over express annexin II, using the chemically-synthetic annexin II small interfering RNA [siRNA] duplexes [including 3 groups: blank MDA-MB-435s cells, cells transfected with negative control siRNA, and cells transfected with annexin II-siRNA]. Finally, the cell proliferation, invasion, and plasmin generation were assayed, and the cellular levels of S100A10 and c-Myc were also detected. All the tests were repeated 3 times. Annexin II and S100A10 were over expressed in invasive human breast cancer cell lines. The siRNA targeting annexin II of MDS-MB 435s cells did not only decrease annexin II messenger RNA and protein levels, but also down-regulated the levels of S100A10, and c-Myc. The treated cells were remarkably blocked in the G0/G1 phase, and cells in the S/G2+M phase decreased. Additionally, the treatment with siRNA resulted in reduction of plasmin generation as well as a loss of the invasive capacity of breast cancer cells. Annexin II might be a key contributor to breast cancer proliferation and invasion

The effect of granulocyte-macrophage-stimulating factor on the gene expression in human trophoblast of early pregnancy / 대한산부인과학회지

OBJECTIVE: Granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitates mammalian embryonic development and implantation. However, its biological function after implantation is not elucidated. The aim of this study is to assess the changes of gene expression by GM-CSF in human trophoblast obtained in early pregnancy. METHODS: Human trophoblast obtained in early pregnancy was cultured with or without GM-CSF. The difference of gene expression was evaluated with microarray and selected genes were reevaluated with real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Microarray analysis revealed that the expressions of 468 genes were increased while those of 40 genes were decreased by GM-CSF. These genes were evaluated according to the known biologic pathways. The regulation of actin cytoskeleton and focal adhesion pathways were mostly influenced by GM-CSF. Annexin A2, thymosin-like 3, vimentin, myogenin, ACK1, and tensin1 genes were selected for real-time RT-PCR. The increased expressions of of vimentin and ACK1, and decreased expressions of tensin1 were confirmed by real-time RT-PCR. CONCLUSION: GM-CSF activates focal adhesion pathway in human trophoblast by increasing the expression of vimentin and ACK1, and decreasing the expression of tensin1.

The effect of bortezomib on migration of endothelial cells and angiogenesis / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of bortezomib on the migration of endothelial cells and the expression of angiogenesis-related molecules, and explore the mechanism of its antiproliferation of tumor cells.</p><p><b>METHODS</b>Cell count kit CCK-8 was used to detect the relative proliferation activity of cells after treated by bortezomib at different concentrations for 12 h and 24 h, respectively. Transwell model was uesd to detect the migration rate of cells. Expression levels of VEGF and Annexin A2 genes were determined by real-time quantitative PCR. Annexin A2 protein was validated by Western blot.</p><p><b>RESULTS</b>After treated with bortezomib at concentrations of 2.5, 5.0 and 10 nmol/L for 12h, respectively, the HMEC-1 cell proliferation activity was 1.004 ± 0.002, 0.793 ± 0.021 and 0.874 ± 0.062, respectively, being no statistical difference from that of control group (1.000) P < 0.05); while the migration rates of them were 0.697 ± 0.060, 0.597 ± 0.090 and 0.874 ± 0.062, respectively, being significantly lower than that of control group (1.000) (P < 0.05) and so did for the expression of VEGF and Annexin A2 genes. After treated with 5 nmol/L bortezomib for 12 h, the Annexin A2 and VEGF gene relative expression level of HMEC-1 cells was 0.540 ± 0.001 and 0.793 ± 0.153, respectively, being of statistical difference from that of control group (1.000) P < 0.05). The conspicuous downregulation of Annexin A2 protein was also confirmed by Western Blot.</p><p><b>CONCLUSIONS</b>Bortezomib can inhibit migration of endothelial cell HMEC-1 by downregulating the expression of VEGF and Annexin A2, displaying a new mechanism of bortezomib for inhibition of tumor proliferation.</p>

Influence of arsenic trioxide and daunorubicin on the expression of annexin II and fibrinolytic activity in NB4 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To 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).</p><p><b>METHODS</b>Leukemia 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.</p><p><b>RESULTS</b>Compared 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.</p><p><b>CONCLUSION</b>NB4 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.</p>

Expression of annexin A1 (ANXA1) and A2 (ANXA2) and its significance in benign and malignant lesions of gallbladder / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the expression levels of ANXA1 and ANXA2 and elucidate their clinicopathological significance in adenocarcinoma, peritumoral tissues, adenomatous polyp and chronic cholecystitis of gallbladder.</p><p><b>METHODS</b>EnVision(TM) immunohistochemical staining was used to detect the expression of ANXA1 and ANXA2 in paraffin-embedded tissue sections from resected specimens of adenocarcinoma (n = 108), peritumoral tissue (n = 46), adenomatous polyp (n = 15) and chronic cholecystitis (n = 35).</p><p><b>RESULTS</b>The positive rates and scores of ANXA1 and ANXA2 were significantly higher in adenocarcinoma (59.3%, 56.5%; 3.2 ± 0.9, 3.4 ± 0.8) than those in peritumoral tissues (34.8%, 1.1 ± 0.8, P < 0.01; 30.4%, 1.0 ± 0.8, P < 0.01), adenomatous polyp (26.7%, 0.9 ± 0.7, P < 0.05 or P < 0.01; 26.7%, 0.9 ± 0.8, P < 0.05 or P < 0.01) and chronic cholecystitis (17.1%, 0.7 ± 0.9, P < 0.01; 20.0%, 0.8 ± 0.8, P < 0.01). The benign lesions with positive ANXA1 and/or ANXA2 expression showed mild to severe atypical hyperplasia of the gallbladder epithelium. The positive rates of ANXA1 and/or ANXA2 were significantly lower in the well-differentiated adenocarcinoma, in a maximal diameter of < 2 cm, with no metastasis to lymph nodes and no invasion to surrounding tissues than those in the moderately or poorly-differentiated adenocarcinoma, in a maximal diameter of ≥ 2 cm, with metastasis to lymph nodes and invasion in surrounding tissues (P < 0.05 or P < 0.01). A high consistence was found between the expression levels of ANXA1 and ANXA2 (χ(2) = 67.84, P < 0.01), and a close positive correlation between the scores of ANXA1 and ANXA2 (r = 0.78, P < 0.01) in gallbladder adenocarcinoma. Kaplan-Meier analysis and multivariate Cox regression analysis showed that ANXA1 or ANXA2 was not an independent prognostic predictor in gallbladder adenocarcinoma.</p><p><b>CONCLUSION</b>The expression levels of ANXA1 and/or ANXA2 may be important biological markers in the carcinogenesis, progression and biological behaviors of gallbladder adenocarcinoma.</p>

Annexin A2 promotes choroidal neovascularization by increasing vascular endothelial growth factor expression in a rat model of argon laser coagulation-induced choroidal neovascularization / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Choroidal neovascularization (CNV) is a common cause of visual loss in the elderly patients with age-related macular degeneration and represents the growth of subretinal new vessels in the macular region. This study aimed to investigate the relationship between annexin A2 (ANXA2) and vascular endothelial growth factor (VEGF) in CNV.</p><p><b>METHODS</b>In a rat model of argon laser coagulation-induced CNV, the mRNA expressions of the annexins and VEGF protein expression in the retina were detected using fluorescent real-time polymerase chain reaction (PCR) and immunohistochemistry, respectively. The interactions between ANXA2 and VEGF in both a retinal pigment epithelial cell line RPE-J and the rat model of CNV were examined by means of RNA interference, real-time PCR, Western blotting, enzyme-linked immunosorbent assay (ELISA) and histopathological examinations.</p><p><b>RESULTS</b>Fundus fluorescein angiography (FFA) showed that argon laser coagulation of the retina induced stable CNV models in the rats. Two to three weeks after the coagulation, ANXA2 and VEGF expressions in the coagulated area in the retina and choroid increased to the peak level, while the other annexin members (ANXA4, ANXA5, ANXA7 and ANXA11) showed no obvious changes. In RPE-J cells and the CNV model, RNA interference of ANXA2 gene significantly lowered the VEGF protein and mRNA expressions, and application of an adenoviral vector containing ANXA2 gene markedly increased VEGF expressions in the rat model of CNV, but produced no significant effects on the expressions of the kinase insert domain-containing receptor (KDR) or the fms-like tyrosine kinase (Flt-1). The expression of KDR inhibited the increment in ANXA2 expression, but VEGF and Flt-1 did not directly affect ANXA2 expression.</p><p><b>CONCLUSION</b>Besides the role as a plasminogen and the receptor of tissue plasminogen activator, ANXA2, which is under regulation of KDR via a negative feedback mechanism, also participates in neovascularization by regulating VEGF expression through a positive feedback mechanism.</p>

Effect of bortezomib on VEGF gene expression of endothelial cell line HMEC-1 and its possible mechanisms / 中国实验血液学杂志

This study was aimed to investigate the effects of bortezomib on VEGF gene expression of endothelial cell line HMEC-1, and to determine the changes of the transcriptional regulation activity of hypoxia-inducible factor 1 (HIF-1alpha) and expression intensity of Annexin A2, so as to analyze the possible mechanisms of the above expression of VEGF gene. Expression intensity of VEGF gene was determined by real-time quantitative PCR; the relative proliferation activity of cells was assayed by cell count kit CCK-8; the expression intensity of carbonic anhydrase IX (CA IX) gene was detected by RT-PCR; expression of Annexin A2 at gene and protein levels were determined by real-time quantitative PCR and Western blot respectively. The results showed that after being treated by bortezomib with 2.5, 5.0, 10 nmol/L for 12 hours, the expression intensity of VEGF gene of endothelial cell line HMEC-1 was as follows: 0.730 +/- 0.106, 0.673 +/- 0.153, 0.767 +/- 0.090 (as 1.0 was made in 0 nmol/L) (p < 0.05); the proliferation activity of cells was not significantly suppressed by bortezomib in 2.5, 5.0 nmol/L (p > 0.05), while that was significantly suppressed by bortezomib of 10 nmol/L (p = 0.024), The results from RT-PCR showed that expression intensity of CA IX gene was conspicuously down-regulated by bortezomib in different concentrations, which suggested that the transcriptional regulation activity of HIF-1alpha was inhibited by bortezomib. And down-regulated expression of Annexin A2 protein by bortezomib in different concentrations was confirmed by real-time quantitative PCR and Western blot. It is concluded that low doses of bortezomib has no significant inhibition effect on the activity of proteasome. Bortezomib may down-regulate the expression of VEGF gene of endothelial cell through regulating the activity of HIF-1alpha and the expression of Annexin A2.

Clinical study on the fibrinolytic activity in patients with acute promyelocytic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the fibrinolytic activity in patients with acute promyelocytic leukemia (APL) and its alteration in all-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO) treatment.</p><p><b>METHODS</b>Plasma fibrinogen concentration was determined with the conventional method, and the levels of fibrin degradation products (FDP) and D-dimer were quantified with ELISA. Plasminogen was measured by chromogenic assay. Cell surface expression of Annexin II and u-PAR and their mRNA levels were measured by flow cytometry and real time-PCR, respectively.</p><p><b>RESULTS</b>The levels of FDP and D-dimer in APL were remarkably higher in APL patients than that in normal controls, while fibrinogen and plasminogen were lower. Both Annexin II and u-PAR were highly expressed on APL cells, which declined after treatment with ATRA and/or ATO, but remained higher than those on normal bone marrow mononuclear cells.</p><p><b>CONCLUSION</b>Abnormally high levels of Annexin II and u-PAR expression on APL cells may contribute to the increased production of plasmin, leading to primary hyperfibrinolysis in APL. ATRA and ATO therapy induces down-regulation of Annexin II and u-PAR expression, which may be contribute, at least in part, to the relief of the hemorrhagic complications in APL.</p>

Effects of Annexin II gene silencing by siRNA on proliferation and invasive potential of Jurkat lymphoma cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Annexin II (AnxA2) gene silencing by siRNA on proliferation and invasive potential of lymphoma cell line Jurkat cells.</p><p><b>METHODS</b>A synthesized siRNA duplex targeting to AnxA2 was transfected into Jurkat cells. Transfection efficiency was analyzed by real-time PCR and flow cytometry. MTT assay for cell proliferation and transwell plates for invasive potential were performed.</p><p><b>RESULTS</b>Compared with the negative controls, the cell proliferation inhibitory rate of the AnxA2 siRNA transfected Jurkat cells was significantly increased at 24 h, 48 h and 72 h [(17.4 +/- 2.3)%, (22.4 +/- 3.8)%, (37.6 +/- 1.5)% vs (-1.3 +/- 5.1)%, (-5.5 +/- 4.4)%, (-10.8 +/- 5.5)%, respectively, P<0.05]. The cell invasive potential of the transfected Jurkat cells was inhibited remarkably at 48 h (11.3 +/- 4.2 vs 54.3 +/- 8.7, P<0.01).</p><p><b>CONCLUSION</b>AnxA2 gene silenced by siRNA can inhibit the proliferation and the invasive potential of Jurkat cells remarkably.</p>