Hepatitis C Virus Disrupts Annexin 5-Mediated Occludin Integrity through Downregulation of Protein Kinase Cα (PKCα) and PKCη Expression, Thereby Promoting Viral Propagation.

Annexins (ANXs) comprise a family of calcium- and phospholipid-binding proteins and are implicated in the hepatitis C virus (HCV) life cycle. Here, we demonstrate a novel role of ANX5 in the HCV life cycle. Comparative analysis by quantitative PCR in human hepatoma cells revealed that ANX2, ANX4, and ANX5 were highly expressed among the ANX family proteins. Knockdown of ANX5 mRNA resulted in marked enhancement of HCV RNA replication but had no effect on either HCV translation or assembly. Using the HCV pseudoparticle (HCVpp) system, we observed enhancement of HCVpp infectivity in ANX5 knockdown Huh-7OK1 cells, suggesting that ANX5 is involved in suppression of HCV entry. Additionally, we observed that subcellular localizations of tight-junction proteins, such as claudin 1 (CLDN1) and occludin (OCLN), were disrupted in the ANX5 knockdown cells. It was reported that HCV infection was facilitated by disruption of OCLN distribution and that proper distribution of OCLN was regulated by its phosphorylation. Knockdown of ANX5 resulted in a decrease of OCLN phosphorylation, thereby disrupting OCLN distribution and HCV infection. Further analysis revealed that protein kinase C (PKC) isoforms, including PKCα and PKCη, play important roles in the regulation of ANX5-mediated phosphorylation and distribution of OCLN and in the restriction of HCV infection. HCV infection reduced OCLN phosphorylation through the downregulation of PKCα and PKCη expression. Taken together, these results suggest that ANX5, PKCα, and PKCη contribute to restriction of HCV infection by regulating OCLN integrity. We propose a model that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting HCV propagation. IMPORTANCE Host cells have evolved host defense machinery to restrict viral infection. However, viruses have evolved counteracting strategies to achieve their infection. In the present study, we obtained results suggesting that ANX5 and PKC isoforms, including PKCα and PKCη, contribute to suppression of HCV infection by regulating the integrity of OCLN. The disruption of OCLN integrity increased HCV infection. We also found that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting viral infection. We propose that HCV disrupts ANX5-mediated OCLN integrity to establish a persistent infection. The disruption of tight-junction assembly may play important roles in the progression of HCV-related liver diseases.

Annexin-A5 and annexin-A6 silencing prevents metastasis of breast cancer cells in zebrafish.

BACKGROUND INFORMATION: During tumor invasion and metastasis processes, cancer cells are exposed to major compressive and shearing forces, due to their migration through extracellular matrix, dense cell areas, and complex fluids, which may lead to numerous plasma membrane damages. Cancer cells may survive to these mechanical stresses thanks to an efficient membrane repair machinery. Consequently, this machinery may constitute a relevant target to inhibit cancer cell dissemination. RESULTS: We show here that annexin-A5 (ANXA5) and ANXA6 participate in membrane repair of MDA-MB-231 cells, a highly invasive triple-negative breast cancer cell line. These crucial components of the membrane repair machinery are substantially expressed in breast cancer cells in correlation with their invasive properties. In addition, high expression of ANXA5 and ANXA6 predict poor prognosis in high-grade lung, gastric, and breast cancers. In zebrafish, the genetic inhibition of ANXA5 and ANXA6 leads to drastic reduction of tumor cell dissemination. CONCLUSION: We conclude that the inhibition of ANXA5 and ANXA6 prevents membrane repair in cancer cells, which are thus unable to survive to membrane damage during metastasis. SIGNIFICANCE: This result opens a new therapeutic strategy based on targeting membrane repair machinery to inhibit tumor invasion and metastasis.

Annexin A5 suppression promotes the progression of cervical cancer.

BACKGROUND: Cervical cancer is a common malignant gynecological disease that threatens the health of women all over the world. The abnormal expression of Annexin A5 (ANXA5) is closely related to the biological behavior of various malignant tumors, however, the relationship between ANXA5 and cervical cancer is still unclear. Therefore, the effects of low expression of ANXA5 on the proliferation, apoptosis, migration and invasion of cervical cancer cells (HeLa) and its related mechanism were explored. METHODS: The cells were divided into three groups: ANXA5-si group, negative control group and blank group. RNA interference was used to suppress ANXA5 expression. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, flow cytometry and propidium iodide (PI) staining, wound healing assay and transwell assay were employed to detect cell proliferation, apoptosis, migration and invasion respectively. Meanwhile, gene expression was detected by qPCR and Western blotting. RESULTS: ANXA5 suppression lead to the increase of proliferation, migration, invasion and the decrease of apoptosis of cervical cancer HeLa cells. Furthermore, the expression of both pPI3K and pAkt increased. CONCLUSION: ANXA5 might inhibit Hela cells proliferation and metastasis by regulating PI3K/Akt signal pathway.

Effects of Curcumin and Lactoferrin to Inhibit the Growth and Migration of Prostatic Cancer Cells.

Prostate cancer remains one of the main causes of death for men worldwide. Despite recent advances in cancer treatment, patients develop resistance after an initial period of optimal efficacy. Nowadays, it is accepted that natural compounds can result in health benefits with a preventive or adjuvant effect. The purpose of this study was to evaluate the effects of curcumin (CU), a bioactive compound in the spice turmeric, and lactoferrin (LF), a natural glycoprotein with immunomodulatory properties, on DU145 and PC3. Prostate cancer cells were cultured with and without LF (175 µM) and CU (2.5 µg/mL and 5 µg/mL), alone and in combination. Cell viability, migration ability, death receptors (DRs), and integrins (α3, ß1) gene expression were evaluated, as well as human annexin V quantification and Akt phosphorylation. Differences among cells group, defined according to the treatment used, were assessed with ANOVA. The results showed that the effects of CU and LF are different between the two prostatic cell lines analyzed. In DU145, a reduction in cell proliferation and migration is reported both in the presence of single and combined treatments. In PC3 cells, there is a significant reduction in proliferation in the presence of CU alone, while the inhibition of migration is mainly related to the LF treatment and its combination with CU, compared to untreated cells. Moreover, the reduction in gene expression of integrins and Akt pathway activation were observed mostly in the presence of the CU and LF combination, including the upregulation of DR and annexin V levels, with greater significance for the DU145 cells. In conclusion, our results suggest that CU and LF may have a potentially beneficial effect, mainly when administered in combination, leading to a reduction in cancer cells' aggressiveness.

LncRNA MYMLR promotes pituitary adenoma development by upregulating carbonyl reductase 1 via sponging miR-197-3p.

Long noncoding RNAs (lncRNAs) have been demonstrated to participate in various biological processes and play key roles in tumorigenesis and metastasis. Pituitary adenoma (PA) is one of the most common malignancies in central nervous system. Recently, multiple lncRNAs have been identified to regulate PA initiation, progression and metastasis. we aimed to elucidate the expression pattern and function of lncRNA MYMLR in PA development. The expression of lncRNA MYMLR in PA tissues and cells was examined by real-time quantitative PCR. Knockdown of MYMLR expression was achieved by using shRNA. The function of MYMLR and regulatory network were analyzed using CCK-8 assay, wound-healing assay, migration assay and Annexin V/PI staining. Xenograft tumor model was used to explore the function of MYMLR in vivo . Bioinformatics analysis and luciferase reporter assay were conducted to investigate the interaction between MYMLR and its regulatory network. LncRNA MYMLR was highly expressed in PA tissues compared with that in normal tissues. Knockdown of MYMLR suppressed cell proliferation, migration and invasion, while promoting PA cell apoptosis. Mechanistically, MYMLR functioned as a competing endogenous RNA (ceRNA) sponging microRNA miR-197-3p. Furthermore, miR-197-3p exerted its tumor inhibitory role via negatively regulating carbonyl reductase 1 (CBR1). Overexpression of CBR1 antagonized the inhibitory effect of lncRNA MYMLR knockdown or miR-197-3p overexpression. In addition, xenograft tumor model revealed that knockdown of lncRNA MYMLR suppressed PA tumor development in vivo via regulating CBR1. Our findings suggest a regulatory network of lncRNA MYMLR/miR-197-3p/CBR1, which benefits the understanding of PA development and provides a promising lncRNA-direct therapeutic strategy against PA.

Exploiting DNA Ligase III addiction of multiple myeloma by flavonoid Rhamnetin.

BACKGROUND: DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM. METHODS: Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. RESULTS: Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo. CONCLUSION: Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.

Vegetative forms of Clostridium chauvoei trigger apoptotic and inflammatory responses on macrophages.

BACKGROUND: Clostridium chauvoei is a gram-positive, spore-forming, strictly anaerobic bacterium that causes blackleg, a disease that affects cattle by inducing fulminant myonecrosis, thereby leading to high and constant losses of cattle. Macrophages (Mɸs) are depleted in tissues infected with the vegetative form of C. chauvoei, but the mechanism remains partially known. Consequently, Mɸs may be a critical target in the pathogenicity of C. chauvoei. AIM: The objective of this work was to study the mechanism of death of mouse-primary Mɸs infected in vitro for 24 h with the vegetative form of C. chauvoei. METHODS: Mouse peritoneal Mɸs were infected in vitro with different multiplicities of infection (MOIs) of C. chauvoei (i.e., 5:1, 20:1, and 100:1). After 24 h post-infection, cell viability (MTT reduction assay), apoptosis (apoptotic bodies, DNA ladder, and Annexin V assays), and inflammatory cell response (iNOS and TNF-α expression) were assessed. RESULTS: All the MOIs investigated decreased cell viability. An MOI of 20:1 caused the highest production of apoptotic bodies and an electrophoretic DNA-ladder pattern typical of an apoptosis cell death process. These results were corroborated using the Annexin V-flow cytometry assay. Concurrently with apoptotic cell death, Mφs expressed iNOS and TNF-α. CONCLUSION: Inflammation-mediated apoptosis of Mφs can be a potential mechanism of evasion of the immune response used by C. chauvoei in tissues for depleting phagocytic cells at the site of infection.

Increased ANXA5 expression in stomach adenocarcinoma infers a poor prognosis and high level of immune infiltration.

BACKGROUND: The prognostic role of annexin A5 (ANXA5) in stomach adenocarcinoma (STAD) has not been studied, and its relationship with immune infiltration is still unclear. OBJECTIVE: This investigation aimed at exploring the role of ANXA5 in STAD using an integrated bioinformatics analysis. METHODS: The expression of ANXA5 in STAD and the correlations between the effect of ANXA5 and survival of STAD patients were investigated using database. The clusterProfiler package in R software was used to perform enrichment analysis on the top 100 co-expressed genes of ANXA5 from the COXPRESdb online database. Correlations between ANXA5 and immune cell infiltrates were analyzed using the TIMER database. RESULTS: In STAD, ANXA5 expression was significantly upregulated and increased ANXA5 expression was significantly correlated with poor overall survival (P< 0.05). In multivariate analysis, upregulated ANXA5 expression was an independent predictive factors of poor prognosis (P< 0.05). The co-expressed genes were involved in extracellular matrix (ECM)-related processes. In STAD, ANXA5 expression was significantly correlated with various infiltrating immune cells (P< 0.05). CONCLUSIONS: Together with our findings, ANXA5 could serve as a potential biomarker to assess prognosis and immune infiltration level in STAD.

Activin A specifically suppresses the expression of annexin A5 mRNA and augments gonadotropin-releasing hormone stimulation of A1 expression in LßT2 gonadotrope cells.

Recently, we reported that gonadotropin-releasing hormone (GnRH) stimulates annexin A1 (Anxa1) and A5 (Anxa5) mRNA expression through the GnRH-receptor-mitogen-activated protein kinase cascade in LßT2 cells. As LßT2 cells respond to activin, we examined the effect of activin A on Anxa1 and a5 expression in LßT2 cells. Activin A (0.4 and 4 ng/mL) treatment decreased Anxa5 mRNA levels in a dose-dependent manner, but did not affect Anxa1 mRNA levels at concentrations up to 40 ng/mL. After activin A treatment (4 ng/mL), Anxa5 mRNA levels significantly decreased at 6 h, gradually declined until 24 h, and remained low until 48 h, whereas Anxa1 mRNA levels did not significantly change following treatment. Pretreatment with activin A for 24 h increased GnRH agonist (GnRHa)-induced Anxa1 increase by approximately 7-fold compared with GnRHa stimulation alone, but Anxa5 was not affected. As previously reported, these activin A treatments increased gonadotropin ß subunit and GnRH receptor mRNA levels and slightly decreased common α-glycoprotein subunit mRNA levels. Furthermore, we examined the effect of activin A on Nr4a3, which is repressed by ANXA5 and which reduces Fshb expression, and found that activin A augmented Nr4a3 expression and slightly decreased the GnRHa-induced increase in Nr4a3. These results suggest that in gonadotrope cells, the mechanism regulating Anxa1 and a5 expression is differentially coupled with activin A signal transduction. Activin A suppresses Anxa5 expression under increased Nr4a3 expression, whereas activin A and GnRH synergistically stimulate Anxa1 expression. These GnRH-inducible annexins may have different specific functions in gonadotropes.

Cell Dissociation Enzymes Affect Annexin V/Flow-Cytometric Apoptotic Assay Outcomes After miRNA-based Transient Transfection.

BACKGROUND/AIM: miRNA functional analysis involves transfection with miRNA-based oligos to identify gain-of or loss-of function cellular phenotypes. Apoptosis is a common phenotypic endpoint for miRNA functional analysis. We report that four common cell dissociation enzymes, TrypLE, Accutase, Trypsin, and Accumax, can differentially impact cell viability and apoptosis in Annexin V flow cytometric analysis after miRNA-based transient transfection. MATERIALS AND METHODS: We transiently transfected a nonsense oligo into an epithelial cancer cell line (UM-SCC-12) for 24 h. Cells were harvested with either TrypLE, Accutase, Accumax, or Trypsin after 5 min. The Annexin V/7-AAD assay via flow cytometry was employed. Studies were performed in triplicate. Significant effects were detected by ANOVA, followed by Tukey's Multiple Comparison tests. RESULTS: Trypsin produced the lowest cell viability and lowest percentage of apoptotic cells, specifically when compared to TrypLE and Accutase, respectively (p<0.01). Importantly, transfected trypsinized cells had a significant difference in cell viability and necrosis (p<0.05) when compared with non-transfected trypsinized cells, highlighting the influence of miRNA-based transfection on Annexin V flow cytometric outcomes. Interassay variability was lowest with TrypLE (1.13 %). As such, TrypLE provided the greatest reproducibility and reliability in our cell line. CONCLUSION: Our study highlights the variable effects of cell dissociation enzymes on transfected cells. Overall, the variability may lead to errors in detection of apoptotic cells using the Annexin V assay after miRNA-based transfection. Before assay use, we recommend pretesting cell dissociation enzymes on transfected cells to ensure reliable and reproducible results.

Leber's hereditary optic neuropathy-associated ND6 14484T > C mutation caused pleiotropic effects on the complex I, RNA homeostasis, apoptosis and mitophagy.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mitochondrial DNA (mtDNA) mutations. LHON-linked ND6 14484T > C (p.M64V) mutation affected structural components of complex I but its pathophysiology is poorly understood. The structural analysis of complex I revealed that the M64 forms a nonpolar interaction Y59 in the ND6, Y59 in the ND6 interacts with E34 of ND4L, and L60 of ND6 interacts with the Y114 of ND1. These suggested that the m.14484T > C mutation may perturb the structure and function of complex I. Mutant cybrids constructed by transferring mitochondria from lymphoblastoid cell lines of one Chinese LHON family into mtDNA-less (ρo) cells revealed decreases in the levels of ND6, ND1 and ND4L. The m.14484T > C mutation may affect mitochondrial mRNA homeostasis, supported by reduced levels of SLIRP and SUPV3L1 involved in mRNA degradation and increasing expression of ND6, ND1 and ND4L genes. These alterations yielded decreased activity of complex I, respiratory deficiency, diminished mitochondrial ATP production and reduced membrane potential, and increased production of reactive oxygen species in the mutant cybrids. Furthermore, the m.14484T > C mutation promoted apoptosis, evidenced by elevating Annexin V-positive cells, release of cytochrome c into cytosol, levels in apoptotic proteins BAX, caspases 3, 7, 9 and decreasing levels in anti-apoptotic protein Bcl-xL in the mutant cybrids. Moreover, the cybrids bearing the m.14484T > C mutation exhibited the reduced levels of autophagy protein LC3, increased levels of substrate P62 and impaired PINK1/Parkin-dependent mitophagy. Our findings highlighted the critical role of m.14484T > C mutation in the pathogenesis of LHON.

Gonadotropin-releasing hormone stimulation of annexin A5 expression in the thymus of male rats.

As gonadotropin-releasing hormone (GnRH) is expressed in the thymus, its direct action on thymic cells, including thymic involution, has been suggested. Annexin A5 (ANXA5), a biomarker of GnRH, was used to determine whether GnRH affects the thymus of male rats. Immunohistochemistry showed positive reactions for ANXA5 in large medullary epithelial cells at 30 days of age, and the expression continued until 180 days of age. Organ culture of thymus pieces was performed to examine the direct action of a GnRH agonist (GnRHa) on the expression of Anxa5 and Gnrh mRNA. Thymus tissues obtained from male rats (40-60 days old) were cut into small pieces (2-3 mm3) and incubated for 3 hr with the GnRHa. The expression levels of Anxa5 and Gnrh mRNA were augmented by the GnRHa. Immunohistochemistry of these tissue fragments showed that ANXA5 expression was enhanced, especially in medullary epithelial cells. These results revealed that GnRH synthesis in the thymus could affect thymic epithelial cells after puberty.

Genetic analysis of ANXA5 haplotype and its effect on recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is often associated with dysregulated Annexin A5 (ANXA5) expression. Moreover, the variants of Anxa5, a protein that is enriched in the placenta to prevent coagulation, have been reported to affect RPL risks. The haplotypes M1 [including single nucleotide polymorphisms (SNPs) 1A/C and 27T/C] and M2 (including SNPs 19G/A, 1A/C, 27T/C and 76G/A) of ANXA5 were also reported to affect RPL risks. The present study aimed to investigate the association between the haplotype located in the promoter region of ANXA5 and the risk of RPL. Patients with RPL (n=235) or intrauterine fetus death (IUFD; n=154), as well as healthy control subjects (n=375) were enrolled in the current research. Their haplotypes of ANXA5 were determined using genotyping, and the association between ANXA5 haplotypes and the risk of RPL was accordingly analyzed. A luciferase assay was conducted to investigate the haplotype responsible for ANXA5 activity. Reverse transcription­quantitative PCR, western blot analysis, immunohistochemistry and ELISA were performed to assess the expression level and activity of ANXA5 in patients with RPL. Consequently, the majority (n=214) of patients with RPL had a history of early RPL, whereas 31 patients with RPL had a history of both early and late RPL episodes. A significant difference was found between cases and controls in terms of gravidity and parity, whereas no significant differences were found in terms of age. The percentage of patients with RPL carrying the M2 haplotype of ANXA5 was significantly higher compared with that in control subjects, indicating that the M2 haplotype of ANXA5 was an independent risk of RPL as it influenced the transcription efficiency of ANXA5 promoter. In patients with RPL, ANXA5 activity was suppressed and the mRNA and protein expression levels of Anxa5 were decreased. Thus, the ANXA5 M2 haplotype may be an independent risk factor of RPL by affecting Anxa5 activity.

Development of Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper(roy-/-,nacre-/-) Transparent Transgenic In Vivo Zebrafish Model to Study the Cardiomyocyte Function.

The zebrafish provided an excellent platform to study the genetic and molecular approach of cellular phenotype-based cardiac research. We designed a novel protocol to develop the transparent transgenic zebrafish model to study annexin-5 activity in the cardiovascular function by generating homozygous transparent skin Casper(roy-/-,nacre-/-); myl7:RFP; annexin-5:YFP transgenic zebrafish. The skin pigmentation background of any vertebrate model organism is a major obstruction for in vivo confocal imaging to study the transgenic cellular phenotype-based study. By developing Casper(roy-/-,nacre-/-); myl7; annexin-5 transparent transgenic zebrafish strain, we established time-lapse in vivo confocal microscopy to study cellular phenotype/pathologies of cardiomyocytes over time to quantify changes in cardiomyocyte morphology and function over time, comparing control and cardiac injury and cardio-oncology. Casper contributes to the study by integrating a transparent characteristic in adult zebrafish that allows for simpler transparent visualization and observation. The Casper(roy-/-,nacre-/-) transgenic progenies developed through cross-breeding with the transgenic strain of Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP). Confocal and fluorescent microscopy were being used to obtain accurate, precise imaging and to determine fluorescent protein being activated. This study protocol was conducted under two sections; 1.1: Generation of homozygous Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper(roy-/-,nacre-/-) zebrafish (generation F01-F06) and 1.2: Screening and sorting the transparent transgenic progeny and in vivo imaging to validate cardiac morphology through in vivo confocal imaging. We coined the newly developed strain as Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper(roy-/-,nacre-/-)gmc1. Thus, the newly developed strain maintains transparency of the skin throughout the entire life of zebrafish and is capable of application of a non-invasive in vivo imaging process. These novel results provide an in vivo whole organism-based platform to design high-throughput screening and establish a new horizon for drug discovery in cardiac cell death and cardio-oncology therapeutics and treatment.

Long-circulating XTEN864-annexin A5 fusion protein for phosphatidylserine-related therapeutic applications.

Annexin A5 (anxA5) is a marker for apoptosis, but has also therapeutic potential in cardiovascular diseases, cancer, and, due to apoptotic mimicry, against dangerous viruses, which is limited by the short blood circulation. An 864-amino-acid XTEN polypeptide was fused to anxA5. XTEN864-anxA5 was expressed in Escherichia coli and purified using XTEN as tag. XTEN864-anxA5 was coupled with DTPA and indium-111. After intravenous or subcutaneous injection of 111In-XTEN864-anxA5, mouse blood samples were collected for blood half-life determination and organ samples for biodistribution using a gamma counter. XTEN864-anxA5 was labeled with 6S-IDCC to confirm binding to apoptotic cells using flow cytometry. To demonstrate targeting of atherosclerotic plaques, XTEN864-anxA5 was labeled with MeCAT(Ho) and administered intravenously to atherosclerotic ApoE-/- mice. MeCAT(Ho)-XTEN864-anxA5 was detected together with MeCAT(Tm)-MAC-2 macrophage antibodies by imaging mass cytometry (CyTOF) of aortic root sections. The ability of anxA5 to bind apoptotic cells was not affected by XTEN864. The blood half-life of XTEN864-anxA5 was 13 h in mice after IV injection, markedly longer than the 7-min half-life of anxA5. 96 h after injection, highest amounts of XTEN864-anxA5 were found in liver, spleen, and kidney. XTEN864-anxA5 was found to target the adventitia adjacent to atherosclerotic plaques. XTEN864-anxA5 is a long-circulating fusion protein that can be efficiently produced in E. coli and potentially circulates in humans for several days, making it a promising therapeutic drug.

Sequential preovulatory expression of a gonadotropin-releasing hormone-inducible gene, Nr4a3, and its suppressor Anxa5 in the pituitary gland of female rats.

Functional relationship between nuclear receptor subfamily 4 group A member 3 (Nr4a3) and annexin A5 (Anxa5), which are two gonadotropin-releasing hormone (GnRH)-inducible genes, has been established while evaluating pituitary gonadotropes in relation to follicle-stimulating hormone beta (Fshb) expression. However, the physiological variations that arise due to the differential expression of these genes in the pituitary gland during rat estrous cycle remain unknown. This study aimed to evaluate the Nr4a3 and Anxa5 mRNA expression during the estrous cycle in rats in comparison with the expression of the gonadotropin subunit genes, luteinizing hormone beta (Lhb) and Fshb. Nr4a3 mRNA expression showed a single peak at 1400 h of proestrus during the 4-d estrous cycle. Anxa5 mRNA level was elevated along with increased Fshb mRNA expression after the decline of Nr4a3 mRNA until 2300 h. Lhb mRNA expression levels were not significantly changed during the estrous cycle. Notably, addition of a GnRH antagonist at 1100 h completely eradicated luteinizing hormone secretion at 1400 h and 1700 h of proestrus, and significantly reduced the Nr4a3 mRNA expression level at both the time points. These results suggest that GnRH is, at least partly, responsible for the increase in pituitary Nr4a3, and that the interaction between NR4A3 and ANXA5 is required to regulate Fshb expression during the preovulatory gonadotropin surge.

Intrinsic and extrinsic apoptosis responses in leukaemia cells following daunorubicin treatment.

BACKGROUND: Daunorubicin is used clinically in the treatment of myeloma, acute lymphatic and myelocytic leukaemia. The toxic lesions caused by daunorubicin induce various modes of cell death, including apoptosis. Apoptosis is highly regulated programmed cell death that can be initiated mainly via two pathways, through death receptors (extrinsic) or involvement of the mitochondria (intrinsic). Induction of apoptosis via these pathways has been alluded following treatment with daunorubicin, but never compared in acute lymphoblastic leukaemia over a time course. METHODS: This study investigated the mechanisms of daunorubicin induced apoptosis in the treatment of CCRF-CEM, MOLT-4 (acute T-lymphoblastic leukaemia) and SUP-B15 (acute B-lymphoblastic leukaemia) cells. Cells were treated with daunorubicin for 4 h, and then placed in recovery medium (without daunorubicin) for 4 h, 12 h and 24 h. Apoptotic response was analysing using annexin-V expression, caspase activity, mitochondrial membrane potential change and an array to detect 43 apoptotic proteins. RESULTS: Daunorubicin induced apoptosis in all leukemic cell lines, but with different levels and duration of response. Both apoptosis levels and caspase activity increased after four hours recovery then declined in CCRF-CEM and MOLT-4 cells. However, SUP-B15 cells displayed initially comparable levels but remained elevated over the 24 h assessment period. Changes in mitochondrial membrane potential occurred in both MOLT-4 and CCRF-CEM cells but not in SUP-B15 cells. Expression of apoptotic proteins, including Bcl-2, Bax, caspase 3 and FADD, indicated that daunorubicin potentially induced both extrinsic and intrinsic apoptosis in both CCRF-CEM and MOLT-4 cells, but only extrinsic apoptosis in SUP-B15 cells. CONCLUSIONS: This study describes variations in sensitivities and timing of apoptotic responses in different leukaemia cell lines. These differences could be attributed to the lack of functional p53 in coordinating the cells response following cytotoxic treatment with daunorubicin, which appears to delay apoptosis and utilises alternative signalling mechanisms that need to be further explored.

Synergistic effect of the herbal mixture C5E on gemcitabine treatment in PANC­1 cells.

The present study aimed to determine the anticancer effect of the herbal mixture extract C5E in the pancreatic cancer cell line, PANC­1, in the absence or presence of gemcitabine treatment, a chemotherapeutic drug used for the treatment of pancreatic cancer. The anticancer effects of C5E, gemcitabine and C5E plus gemcitabine in PANC­1 cells following 72 h of treatment were investigated. The effect of each treatment on cell cycle arrest, apoptosis and the proportion of side population (SP) cells was determined using flow cytometric analysis following propidium iodide (PI), Annexin V­FITC/PI double staining and Hoechst 33342 staining, respectively. SP cells share similar characteristics to cancer stem­like cells, and a reduction in the SP is considered to be indicative of an anticancer effect. The percentage of SP cells and the cell viability of general PANC­1 cells were significantly decreased in response to all treatments. The percentage of SP cells was reduced from 8.2% (control) to 3.9, 7.2 and 5.1% following the treatment with C5E, gemcitabine and the co­treatment, respectively. All three treatments were discovered to inhibit cell viability by arresting the cell cycle at the S phase and promoted cell death by inducing early apoptosis, with the levels of apoptosis being increased from 1.9% (control) to 7.3, 2.5 and 12.0% following the treatment with C5E, gemcitabine and the co­treatment, respectively. The mRNA expression levels of sonic hedgehog, which is implicated in the development of certain types of cancer, were downregulated to a greater extent following the co­treatment with C5E and gemcitabine compared with the treatment with either C5E or gemcitabine alone. As the co­treatment with gemcitabine and C5E was more effective than each individual treatment, the present study suggested that the combined treatment may exhibit synergistic effects in PANC­1 cells.

Secretion of Recombinant Human Annexin V in Fusion with the Super Folder GFP for Labelling Phosphatidylserine-Exposing Membranes.

Annexin V (ANXV), mostly characterized by its ability to interact with biological membranes in a calcium-dependent manner. ANXV interacts mainly with phosphatidylserine (PS), for that fluorescent ANXV widely produced and used as a sensitive and specific probe to mark apoptotic cells or any PS-containing bilayers membranes. Many reports described the prokaryotic expression of recombinant human ANXV. To overcome some of E. coli expression limitations, we aimed in this work to investigate unconventional alternative expression system in mammalian cells for producing secreted human ANXV in fusion with the super folder green fluorescent protein (sfGFP). HEK239T cells were transfected using polyethylenimine (PEI) and pcDNA-sfGFP-ANXV plasmid. Forty-eight hours post transfection, direct fluorescence measurement, immunoblotting and ELISA confirmed the presence of secreted sfGFP-ANXV in cells supernatant. The yield of secreted 6 × His-tagged sfGFP-ANXV after affinity purification was estimated to be around 2 µg per 1 ml of cells supernatant. The secretion system was proper to produce a fully functional sfGFP-ANXV fusion protein in quantities enough to recognize and bind PS-containing surfaces or liposomes. Besides, biological assays such as flow cytometry and fluorescent microscopy confirmed the capacity of the secreted sfGFP-ANXV to detect PS exposure on apoptotic cells. Taken together, we present mammalian expression as a quick, affordable and endotoxin-free system to produce sfGFP-ANXV fusion protein. The secreted sfGFP-ANXV in eukaryotic system is a promising biotechnological tool, it opens up new horizons for additional applications in the detection of PS bearing surfaces and apoptosis in vitro and in vivo assays.

Kevetrin induces apoptosis in TP53 wild­type and mutant acute myeloid leukemia cells.

Tumor protein p53 is a key regulator of several cellular pathways, including DNA repair, cell cycle and angiogenesis. Kevetrin exhibits p53­dependent as well as­independent activity in solid tumors, while its effects on leukemic cells remain unknown. The aim of the present study was to analyze the response of acute myeloid leukemia (AML) cell lines (TP53 wild­type: OCI­AML3 and MOLM­13; and TP53­mutant: KASUMI­1 and NOMO­1) to kevetrin at a concentration range of 85­340 µM. The cellular and molecular effects of the treatment were analyzed in terms of cell growth, viability [Annexin V­propidium iodide (PI) staining] and cell cycle alterations (PI staining). Gene expression profiling, western blotting and immunofluorescence were performed to elucidate the pathways underlying kevetrin activity. Pulsed exposure exerted no effect on the wild­type cells, but was effective on mutant cells. After continuous treatment, significant cell growth arrest and apoptosis were observed in all cell lines, with TP53­mutant models displaying a higher sensitivity and p53 induction. Kevetrin also displayed efficacy against TP53 wild­type and mutant primary AML, with a preferential cytotoxic activity against blast cells. Gene expression profiling revealed a common core transcriptional program altered by drug exposure and the downregulation of glycolysis, DNA repair and unfolded protein response signatures. These findings suggest that kevetrin may be a promising therapeutic option for patients with both wild­type and TP53­mutant AML.