Non-canonical RNA-binding protein ANXA11 regulates microRNA resorting into small extracellular vesicles to mediate cisplatin resistance.

The sensitivity of laryngeal squamous cell carcinoma (LSCC) to chemotherapy shows large heterogeneity. The role of miRNA in small extracellular vesicles (sEV) in chemotherapy resistance is under investigation. However, the regulation and sorting mechanism of sEV miRNAs remains unclear. In this study, small RNA sequencing was used to explore miRNA expression profiles in sEV of LSCC after cisplatin stimulation; RNA pull-down, mass spectrometry, and EMSA were used to clarify the binding of candidate RNA-binding protein (RBP) and candidate miRNA. Immunostaining and microRNA fluorescence in situ hybridization were performed to identify how candidate RBP affects miRNA stability and nuclear/cytoplasmic distribution. In vivo experiments were performed to verify the biological functions and response to cisplatin of candidate RBP. We found that cisplatin stimulation induced increased expression of miR-148a-3p and sEV sorting. ANXA11 binds to miR-148a-3p in a sequence-specific manner. ANXA11 inhibits tumor cell proliferation and drug resistance by binding to and retaining miR-148a-3p. Cisplatin stimulation reduced ANXA11 expression and promoted miR-148a-3p efflux through sEV pathways. ANXA11 overexpression reduced in vivo tumor proliferation and cisplatin-resistance. Taken together, ANXA11 mediates cisplatin resistance through sEV miRNA resorting. Mechanically, ANXA11 binds to miR-148a-3p in a sequence-specific manner to regulate its resorting and thus influences tumor proliferation and chemoresistance.

Genome-wide association study indicates novel associations of annexin A13 to secretory and GAS2L2 with mucous otitis media.

To evaluate the genetics of chronic nonsuppurative otitis media (OM). We performed a genome-wide association study of 429,599 individuals included in the FinnGen study using three different case definitions: combined chronic nonsuppurative OM (7034 cases) (included serous and mucous chronic OM), mucous chronic OM (5953 cases), and secretory chronic OM (1689 cases). Individuals without otitis media were used as controls (417,745 controls). We used immunohistochemistry (IHC) of the murine middle ear to evaluate the expression of annexin A13. Four loci were significantly associated (p < 1.7 × 10-8) with nonsuppurative OM. Three out of the four association signals included missense variants in genes that may play a role in otitis media pathobiology. According to our subtype-specific analyses, one novel locus, located near ANXA13, was associated with secretory OM. Three loci (near TNFRSF13B, GAS2L2, and TBX1) were associated with mucous OM. Immunohistochemistry of murine middle ear samples revealed annexin A13 expression at the apical pole of the Eustachian tube epithelium as well as variable intensity of the secretory cells of the glandular structure in proximity to the Eustachian tube. We demonstrated that secretory and mucous OM have distinct and shared genetic associations. The association of GAS2L2 with ciliary epithelium function and the pathogenesis of dysfunctional mucosa in mucous OM is suggested. The abundant expression of annexin A13 in the Eustachian tube epithelium, along with its role in apical transport for the binding and transfer of phospholipids, indicates the role of annexin A13 and phospholipids in Eustachian tube dysfunction.

Interference with ANXA8 inhibits the malignant progression of ovarian cancer by suppressing the activation of the Wnt/ß-catenin signaling pathway via UCHL5.

Ovarian cancer (OC), which threatens women's lives, is a common tumor of the female reproductive system. Annexin A8 (ANXA8) is highly expressed in OC. However, the mechanism of ANXA8 in OC remains unclear. This study investigated the potential mechanisms of ANXA8 in OC. The expression of ANXA8 in OC cells was determined by qRT-PCR and western blotting. ANXA8 interference plasmid was constructed. Moreover, CCK-8, EDU staining, TUNEL staining, western blotting, wound healing, and transwell assays were used to detect cell proliferation, apoptosis, migration, and invasion, respectively. Next, the relationship between ANXA8 and ubiquitin C-terminal hydrolase L5 (UCHL5) was verified through Co-IP. Finally, western blotting was used to detect the expression of Wnt/ß-catenin signaling-related proteins. Additionally, we further interfered ANXA8 in nude mice with OC, and detected the expression of ANXA8, UCHL5 and the signaling pathway-related proteins by immunohistochemistry and western blotting. Our results suggested that ANXA8 expression was significantly increased in OC cells. ANXA8 interference significantly attenuated the proliferative, invasive, and migratory capabilities and promoted the apoptotic ability of OC cells. Moreover, the expression of UCHL5 in OC was significantly increased. ANXA8 bound to UCHL5 in OC cells. Knockdown of ANXA8 attenuated OC cell malignant progression by downregulating the expression of UCHL5. Furthermore, ANXA8 affected the expression of Wnt/ß-catenin signaling pathway-related proteins in OC cells via UCHL5. Collectively, ANXA8 interference suppressed the activation of Wnt/ß-catenin signaling pathway via UCHL5 to inhibit cell proliferation, invasion, migration and induce cell apoptosis in OC, thus presenting a potential therapeutic strategy for OC treatment.

Knockdown of sulfotransferase 2B1 suppresses cell migration, invasion and promotes apoptosis in ovarian carcinoma cells via targeting annexin A9.

BACKGROUND: Sulfotransferase family 2B member 1 (SULT2B1) has been reported to play oncogenic role in many types of cancers. Nevertheless, the role that SULT2B1 played in ovarian cancer (OC) and the hidden molecular mechanism is obscure. METHODS: Expression of SULT2B1 in OC was analyzed by GEPIA database. qRT-PCR and western blot (WB) was applied for the appraisement of SULT2B1 and Annexin A9 (ANXA9) in OC cell lines. The capabilities of cells to proliferate, migrate and invade were assessed with CCK-8 assay, wound healing assay, along with transwell assay. Cell apoptotic level was estimated utilizing flow cytometry. WB was employed for the evaluation of migration- and apoptosis-related proteins. Bioinformatic analysis and co-immunoprecipitation were used to predict and verify the combination of SULT2B1 and ANXA9. RESULTS: The data showed that SULT2B1 and ANXA9 were upregulated in OC cells. SULT2B1 depletion suppressed the proliferative, migrative, and invasive capabilities of SKOV3 cells but facilitated the cell apoptosis. SULT2B1-regulated ANXA9 expression and were proved to bind to ANXA9. Additionally, ANXA9 deficiency exhibited the same impacts on cell migrative, invasive capability and apoptotic level as SULT2B1 silencing. Moreover, ANXA9 overexpression reversed the inhibitory impacts of SULT2B1 silencing on the proliferative, migrative, invasive, and apoptotic capabilities of SKOV3 cells. CONCLUSION: In summary, SULT2B1 silencing repressed OC progression by targeting ANXA9.

The role of annexins in central nervous system development and disease.

Annexins, a group of Ca2+-dependent phospholipid-binding proteins, exert diverse roles in neuronal development, normal central nervous system (CNS) functioning, neurological disorders, and CNS tumors. This paper reviews the roles of individual annexins (A1-A13) in these contexts. Annexins possess unique structural and functional features, such as Ca2+-dependent binding to phospholipids, participating in membrane organization, and modulating cell signaling. They are implicated in various CNS processes, including endocytosis, exocytosis, and stabilization of plasma membranes. Annexins exhibit dynamic roles in neuronal development, influencing differentiation, proliferation, and synaptic formation in CNS tissues. Notably, annexins such as ANXA1 and ANXA2 play roles in apoptosis and blood-brain barrier (BBB) integrity. Neurological disorders, including Alzheimer's disease, multiple sclerosis, and depression, involve annexin dysregulation, influencing neuroinflammation, blood-brain barrier integrity, and stress responses. Moreover, annexins contribute to the pathogenesis of CNS tumors, either promoting or suppressing tumor growth, angiogenesis, and invasion. Annexin expression patterns vary across different CNS tumor types, providing potential prognostic markers and therapeutic targets. This review underscores the multifaceted roles of annexins in the CNS, highlighting their importance in normal functioning, disease progression, and potential therapeutic interventions.

Bending of a lipid membrane edge by annexin A5 trimers.

Plasma membrane damage occurs in healthy cells and more frequently in cancer cells where high growth rates and metastasis result in frequent membrane damage. The annexin family of proteins plays a key role in membrane repair. Annexins are recruited at the membrane injury site by Ca+2 and repair the damaged membrane in concert with several other proteins. Annexin A4 (ANXA4) and ANXA5 form trimers at the bilayer surface, and previous simulations show that the trimers induce high local negative membrane curvature on a flat bilayer. The membrane-curvature-inducing property of ANXA5 is presumed to be vital to the membrane repair mechanism. A previously proposed descriptive model hypothesizes that ANXA5-mediated curvature force is utilized at the free edge of the membrane at a wound site to pull the wound edges together, resulting in the formation of a "neck"-shaped structure, which, when combined with a constriction force exerted by ANXA6, leads to membrane repair. The molecular details and mechanisms of repair remain unknown, in part because the membrane edge is a transient structure that is difficult to investigate both experimentally and computationally. For the first time, we investigate the impact of ANXA5 near a membrane edge, which is modeled by a bicelle under periodic boundary conditions. ANXA5 trimers induce local curvature on the membrane leading to global bending of the bicelle. The global curvature depends on the density of annexins on the bicelle, and the curvature increases with the ANXA5 concentration until it reaches a plateau. The simulations suggest that not only do annexins induce local membrane curvature, but they can change the overall shape of a free-standing membrane. We also demonstrate that ANXA5 trimers reduce the rate of phosphatidylserine lipid diffusion from the cytoplasmic to the exoplasmic leaflet along the edge of the bicelle. In this way, membrane-bound annexins can potentially delay the apoptotic signal triggered by the presence of phosphatidylserine lipids in the outer leaflet, thus biding time for repair of the membrane hole. Our findings provide new insights into the role of ANXA5 at the edges of the membrane (the injury site) and support the curvature-constriction model of membrane repair.

The role of the annexin A protein family at the maternal-fetal interface.

Successful pregnancy requires the tolerance of the maternal immune system for the semi-allogeneic embryo, as well as a synchrony between the receptive endometrium and the competent embryo. The annexin family belongs to calcium-regulated phospholipid-binding protein, which functions as a membrane skeleton to stabilize the lipid bilayer and participate in various biological processes in humans. There is an abundance of the annexin family at the maternal-fetal interface, and it exerts a crucial role in embryo implantation and the subsequent development of the placenta. Altered expression of the annexin family and dysfunction of annexin proteins or polymorphisms of the ANXA gene are involved in a range of pregnancy complications. In this review, we summarize the current knowledge of the annexin A protein family at the maternal-fetal interface and its association with female reproductive disorders, suggesting the use of ANXA as the potential therapeutic target in the clinical diagnosis and treatment of pregnancy complications.

Effects of annexin B18 from Echinococcus granulosus sensu lato on mouse macrophages.

Cystic echinococcosis (CE) is a zoonotic disease, caused by Echinococcus granulosus sensu lato (E. granulosus s. l.), which posed significant public health concern globally. E. granulosus s. l. annexin B18 (EgANXB18) acts as a secretory protein, exerting a crucial influence in mediating host-parasite interactions. Recombinant annexin B18 (rEgANXB18) was expressed by Escherichia coli and the immunoreactivity was assessed by western blotting. The binding affinity between rEgANXB18 and total protein of RAW264.7 cells was assessed by ELISA. The impact of rEgANXB18 on the metabolic activity of RAW264.7 cells was assayed by Cell Counting Kit-8 assay. The mRNA levels of polarization markers (inducible nitrous oxide synthase (iNOS) and arginase 1 (Arg1)) and key cellular factors (IL-1ß，IL-6，IL-10 and TNFα) were evaluated by qRT-PCR. rEgANXB18 was successfully expressed and recognized by E. granulosus s.l. infected canine sera, as well as could bind to the total protein of RAW264.7 cells. Additionally, rEgANXB18 could promote metabolic activity at 5, 10, 20, and 40 µg/mL while no significant impact on metabolic activity was observed at 80 µg/mL. Co-culture RAW264.7 cells with rEgANXB18 resulted in significantly upregulation of the transcript levels of polarization markers iNOS and Arg1. Moreover, rEgANXB18 significantly upregulated the transcript levels of IL-1ß, IL-6, TNFα, and IL-10, while dose-effect relationship was observed in IL-1ß, IL-6, and IL-10. Our results indicated that EgANXB18 showed the potential to regulate immune response of macrophages by shifting the cell polarization and cytokine profile, thereby promoting the parasitism of CE.

Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC.

BACKGROUND: Rosmarinic acid (RA) possesses promising anticancer potential, but further development of chemotherapeutic agents is hindered by their toxicity to off-target tissue. In particular, chemotherapy-related anemia is a major obstacle in cancer therapy, which may be aggravated by hemolysis and eryptosis. This work presents a toxicity assessment of RA in human RBCs and explores associated molecular mechanisms. METHODS: RBCs isolated from healthy donors were treated with anticancer concentrations of RA (10-800 µM) for 24 h at 37 °C, and hemolysis and related markers were photometrically measured. Flow cytometry was used to detect canonical markers of eryptosis, including phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, cell size by FSC, and oxidative stress by H2DCFDA. Ions and pH were assessed by an ion-selective electrode, while B12 was detected by chemiluminescence. RESULTS: RA elicited concentration-dependent hemolysis with AST and LDH release but rescued the cells from hypotonic lysis at sub-hemolytic concentrations. RA also significantly increased annexin-V-positive cells, which was ameliorated by extracellular Ca2+ removal and isosmotic sucrose. Furthermore, a significant increase in Fluo4-positive cells and B12 content and a decrease in FSC and extracellular pH with KCl efflux were noted upon RA treatment. Hemolysis was augmented by blocking KCl efflux and was blunted by ATP, SB203580, staurosporin, D4476, isosmotic urea, and PEG 8000. CONCLUSIONS: RA stimulates Ca2+-dependent and sucrose-sensitive hemolysis and eryptosis characterized by PS exposure, Ca2+ accumulation, loss of ionic regulation, and cell shrinkage. These toxic effects were mediated through energy deprivation, p38 MAPK, protein kinase C, and casein kinase 1α.

Tamarix articulata extract offers protection against toxicity induced by beauty products in Hs27 human skin fibroblasts.

The current study evaluates the cytotoxicity, mode of cell death and chemical analysis of selected beauty products and evaluation of the protective effect of Tamarix articulata (TA) extract against toxicity induced by beauty products in skin fibroblasts (Hs27). MTT and Crystal violet (CV) assays were used to determine the dose-dependent cytotoxic effects of beauty products against Hs27 fibroblasts. DNA fragmentation assay and annexin-V staining were conducted to determine the mode of cell killing induced by evaluated beauty products. Quantification of reactive oxygen species (ROS) and antioxidant enzyme levels were used to evaluate the oxidative stress. Chemical analysis and heavy metals were evaluated to determine beauty products. Pre-treatment with TA extract for different time points followed by time-dependent exposure with beauty products to assess the protective effect of TA extract in Hs27 cells was analyzed by MTT and CV assays. Owing to the presence of various harmful heavy metals such as arsenic (As), chromium (Cr), cadmium (Cd), nickel (Ni), and lead (Pb) in beauty products, our results revealed that all beauty products induce significant cytotoxicity over time (1, 4 h) in a dose-dependent (125, 250, 500 µg/mL) manner. DNA fragmentation assay, quantification of apoptosis by annexin-V staining, determination of ROS and antioxidant enzymes (CAT, GSH-Px and SOD) revealed that the induced cytotoxicity was caused by oxidative stress-mediated apoptosis. However, pre-incubation with a safe dose (50 µg/mL) of TA for different times (24, 48 h) followed by exposure to various doses (62.5, 125, 250, 500 µg/mL) of beauty products for different times (1, 4 h) revealed significant (*p≤0.05, **p≤0.01) protection against beauty product-mediated cytotoxicity. The effect was more pronounced for 1 h exposure to beauty products compared to 4 h. Our study demonstrates that the due to the presence of heavy metals in synthetic beauty products exhibit marked toxicity to skin fibroblasts due to oxidative stress-mediated apoptosis. However, the presence of abundant bioactive polyphenols with promising antiscavenging activity in TA extracts significantly nullifies cytotoxicity promoted by examined beauty products in skin fibroblasts (Hs27).

Desipramine induces eryptosis in human erythrocytes, an effect blunted by nitric oxide donor sodium nitroprusside and N-acetyl-L-cysteine but enhanced by Calcium depletion.

Background: Desipramine a representative of tricyclic antidepressants (TCAs) promotes recovery of depressed patients by inhibition of reuptake of neurotransmitters serotonin (SER) and norepinephrine (NE) in the presynaptic membrane by directly blocking their respective transporters SERT and NET.Aims: To study the effect of desipramine on programmed erythrocyte death (eryptosis) and explore the underlying mechanisms.Methods: Phosphatidylserine (PS) exposure on the cell surface as marker of cell death was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry. Hemolysis was determined photometrically, and intracellular glutathione [GSH]i from high performance liquid chromatography.Results: Desipramine dose-dependently significantly enhanced the percentage of annexin-V-binding cells and didn´t impact glutathione (GSH) synthesis. Desipramine-induced eryptosis was significantly reversed by pre-treatment of erythrocytes with either nitric oxide (NO) donor sodium nitroprusside (SNP) or N-acetyl-L-cysteine (NAC). The highest inhibitory effect was obtained by using both inhibitors together. Calcium (Ca2+) depletion aggravated desipramine-induced eryptosis. Changing the order of treatment, i.e. desipramine first followed by inhibitors, could not influence the inhibitory effect of SNP or NAC.Conclusion: Antidepressants-caused intoxication can be treated by SNP and NAC, respectively. B) Patients with chronic hypocalcemia should not be treated with tricyclic anti-depressants or their dose should be noticeably reduced.

Potential prognostic and therapeutic value of ANXA8 in renal cell carcinoma: based on the comprehensive analysis of annexins family.

BACKGROUND: Annexins are a family of proteins involved in a wide variety of cellular processes such as inflammation, proliferation, differentiation, apoptosis, migration and membrane repair. However, the role of most Annexins in renal cell carcinoma (RCC) remained unclear. METHODS: The differentially expressed Annexins in RCC compared with normal controls were screened applying the TCGA database. The correlation of differentially expressed Annexins with clinical stages, grades and overall survival was analyzed to explore the clinical significance of Annexins in RCC. Then ANXA8 was selected and further stained in the discover and validation RCC cohort. The correlation of ANXA8 expression with clinical parameter was verified at the protein level. To explore the potential function of ANXA8, ANXA8 was knockdown in the RCC cell line and further analyzed using transcriptome and bioinformatic analysis. RESULTS: mRNA expression of ANXA1, ANXA2R, ANXA4, ANXA8, ANXA8L1 and ANXA13 were significantly upregulated in RCC compared with normal kidney tissues. In contrast, ANXA3 and ANXA9 mRNA expression was significantly downregulated. Higher expression of ANXA2R, ANXA8 and ANXA8L1 were correlated with worse overall survival, while lower expression of ANXA3, ANXA9 and ANXA13 were associated with worse clinical outcomes in RCC patients. We further demonstrated that ANXA8 expression was significantly increased in RCC compared with normal renal tissues at the protein level. And higher protein expression of ANXA8 was associated with higher clinical grades. Through the bioinformatics analysis and cell cycle analysis, we found knockdown of ANXA8 mainly influenced the cell cycle and DNA replication. The top ten hub genes consist of CDC6, CDK2, CHEK1, CCNB1, ORC1, CHEK2, MCM7, CDK1, PCNA and MCM3. CONCLUSIONS: Multiple members of Annexins were abnormally expressed and associated with the prognosis of RCC. The expression of ANXA8 was significantly increased in RCC and associated with poor prognosis. ANXA8 might influence the cell cycle and could be a potential biomarker and therapeutic target for RCC.

Exosome-derived ANXA9 functions as an oncogene in breast cancer.

Breast cancer (BCA) is one of the most prevalent cancers among women. Emerging evidence has revealed that Annexin A-9 (ANXA9) plays a crucial function in the development of some cancers. Notably, ANXA9 has been reported to be a new prognostic biomarker for gastric and colorectal cancers. However, its expression and biological function in BCA have not yet been investigated. Using online bioinformatics tools such as TIMER, GEPIA, HPA, and UALCAN, we predicted ANXA9 expression and its correlation with the clinicopathological characteristics of BCA patients. RT-qPCR and western blot were utilized to measure ANXA9 mRNA and ANXA9 protein expression in BCA patient tissues and cells. BCA-derived exosomes were identified by transmission electron microscopy. Functional assays were employed to evaluate the biological role of ANXA9 in BCA cell proliferation, migration, invasion, and apoptosis. A tumor xenograft in vivo model was utilized to assess the role of ANXA9 in tumor growth in mice. Bioinformatics and functional screening analysis revealed that ANXA9 was highly expressed in BCA patient tissues, with median ANXA9 expression 1.5- to 2-fold higher than in normal tissues (p < 0.05). RT-qPCR confirmed that ANXA9 expression in BCA tissues was around 1.5-fold higher than the adjacent normal tissues (p < 0.001). ANXA9 expression in different subtypes of BCA also showed a difference, and ANXA9 was found to be mostly significantly upregulated in luminal BCA relative to normal tissues or other histological subtypes (p < 0.001). Moreover, ANXA9 expression was elevated in different races, ages, clinical stages, node metastasis status, and menopause status groups relative to the normal group (p < 0.001). Furthermore, ANXA9 was found to be secreted by BCA tissue-derived exosomes and its expression was upregulated 1- to 7-fold in BCA cells treated with exosomes (p < 0.001), while its expression in MCF10A cells was not significantly altered by treatment with exosomes (p > 0.05). ANXA9 silencing induced a significant decrease of around 30% in the colony number of BCA cells (p < 0.01). The number of migrated and invaded BCA cells also decreased by around 65 and 68%, respectively, after silencing ANXA9 (p < 0.01). Tumor size was significantly reduced (nearly half) in the LV-sh-ANXA9 group relative to the LV-NC group in the xenograft model (p < 0.01), suggesting that ANXA9 silencing repressed tumor progression in BCA progression in vitro and in vivo. In conclusion, exosome-derived ANXA9 functions as an oncogene that facilitates the proliferation, migration, and invasiveness of BCA cells and enhances tumor growth in BCA development, which may provide a new prognostic and therapeutic biomarker for BCA patients.

Annexin A9 promotes cell proliferation by regulating the Wnt signaling pathway in colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. Expression of Annexin A9 (ANXA9), a member of the annexin A family, is upregulated in CRC. However, the molecular role of ANXA9 in CRC remains unknown. In the present study, we aimed to investigate the function of ANXA9 and to elucidate the mechanisms underlying its regulation in CRC. In this study, mRNA expression data and clinical information were downloaded from The Cancer Genome Atlas (TCGA) and GEPIA database, respectively. Kaplan-Meier analysis was used to analyze the survival rates. LinkedOmics and Metascape databases were used to explore the potential mechanisms of regulation of ANXA9 and to identify genes co-expressed with ANXA9. Finally, in vitro experiments were used to evaluate the function of ANXA9 and explore potential mechanisms. We found that ANXA9 expression was significantly elevated in CRC tissue and cells. High ANXA9 expression was associated with shorter overall survival, poorer disease specific survival, as well as with patient age, clinical stage, M stage, and OS events in CRC. Knockdown of ANXA9 inhibited cell proliferation, invasion, migratory potential, and cell cycle arrest. Mechanistically, functional analysis revealed that genes co-expressed with ANXA9 were mainly enriched in the Wnt signaling pathway. ANXA9 deletion suppressed cell proliferation via the Wnt signaling pathway, while Wnt activation reversed the effects of ANXA9. In conclusion, ANXA9 may promote CRC progression by regulating the Wnt signaling pathway and may be a potential diagnostic biomarker in the clinical management of CRC.

Engineering a membrane-binding protein to trimerize and induce high membrane curvature.

The annexins are a family of Ca2+-dependent peripheral membrane proteins. Several annexins are implicated in plasma membrane repair and are overexpressed in cancer cells. Annexin A4 (ANXA4) and annexin A5 (ANXA5) form trimers that induce high curvature on a membrane surface, a phenomenon deemed to accelerate membrane repair. Despite being highly homologous to ANXA4, annexin A3 (ANXA3) does not form trimers on the membrane surface. Using molecular dynamics simulations, we have reverse engineered an ANXA3-mutant to trimerize on the surface of the membrane and induce high curvature reminiscent of ANXA4. In addition, atomic force microscopy images show that, like ANXA4, the engineered protein forms crystalline arrays on a supported lipid membrane. Despite the trimer-forming and curvature-inducing properties of the engineered ANXA3, it does not accumulate near a membrane lesion in laser-punctured cells and is unable to repair the lesion. Our investigation provides insights into the factors that drive annexin-mediated membrane repair and shows that the membrane-repairing property of trimer-forming annexins also necessitates high membrane binding affinity, other than trimer formation and induction of negative membrane curvature.

Annexins A2 and A5 are potential early biomarkers of hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is a highly lethal liver cancer with late diagnosis; therefore, the identification of new early biomarkers could help reduce mortality. We determine the tissue and plasma status of five annexins during hepatocarcinogenesis by diethylnitrosamine-induced cirrhosis-HCC. We found that Anxa5 was the earliest upregulated gene at week 12 after HCC initiation, while Anxa1 and Anxa2 were upregulated in advanced HCC stages (weeks 18 and 22). Furthermore, the protein level of Annexin A1, A2, A5 and A10 was increased from the early stages. Immunofluorescence and subcellular fractionation revealed Annexin A1, A2, and A5 in the cytoplasm and nuclei of tumor cells. Notably, increased plasma levels of Annexin A5 significantly (r2 = 0.8203) correlated with Annexin A5 levels in liver tissue from week 12 and gradually increased until week 22. Using the TCGA database, we found that the expression of ANXA2 (HR = 1.7, p = 0.0046) and ANXA5 (HR = 1.8, p = 0.00077) was associated with poor survival in HCC patients. In conclusion, we have identified Annexin A1 and A5 as potentially useful early biomarkers for poor prognosis in HCC patients.

Myricetin-induced suicidal erythrocyte death.

BACKGROUND: Myricetin, a type of flavonol commonly found in fruits and herbs, has demonstrated anticancer properties by triggering the process of apoptosis or programmed cell death in tumor cells. Despite the absence of mitochondria and nuclei, erythrocytes can undergo programmed cell death, also known as eryptosis.This process is characterized by cell shrinkage, externalization of phosphatidylserine (PS) on the cell membrane, and the formation of membrane blebs. The signaling of eryptosis involves Ca2+ influx, the formation of reactive oxygen species (ROS), and the accumulation of cell surface ceramide. The present study explored the effects of myricetin on eryptosis. METHODS AND RESULTS: Human erythrocytes were exposed to various concentrations of myricetin (2-8 µM) for 24 h. Flow cytometry was used to assess the markers of eryptosis, including PS exposure, cellular volume, cytosolic Ca2+ concentration, and ceramide accumulation. In addition, the levels of intracellular ROS were measured using the 2',7'-dichlorofluorescin diacetate (DCFDA) assay. The myricetin-treated (8 µM) erythrocytes significantly increased Annexin-positive cells, Fluo-3 fluorescence intensity, DCF fluorescence intensity, and the accumulation of ceramide. The impact of myricetin on the binding of annexin-V was significantly reduced, but not completely eliminated, by the nominal removal of extracellular Ca2+. CONCLUSION: Myricetin triggers eryptosis, which is accompanied and, at least in part, caused by Ca2+ influx, oxidative stress and increase of ceramide abundance.

Fufang Shengdi mixture alleviates psoriasis-like skin inflammation via promoting Annexin-A proteins expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine believes that "blood fever" is an important cause of psoriasis. Fufang Shengdi mixture (FFSD), based on the Hongban Decoction, is composed of Rehmannia glutinosa (Gaertn.) DC., Raw gypsum (Chinese: Sheng Shi Gao), and Lonicera japonica Thunb (Caprifoliaceae). FFSD has effects on nourishing Yin, clearing heat, connecting collaterals, and cooling blood. In modern medical explanation, FFSD has the effects of anti-inflammatory and immunosuppression. Our study proved that FFSD can suppress immunity and ameliorate the symptoms of imiquimod-induced psoriasis in mice. AIM OF THE STUDY: This study evaluated the efficacy and possible mechanism of FFSD in psoriasis mice. METHODS AND MATERIALS: First, the main components of FFSD were analyzed using high-performance liquid chromatography-tandem high-resolution mass spectrometry (HPLC-HRMS). An imiquimod (IMQ)-induced psoriasis mouse model was used to evaluate the efficacy of FFSD orally. Psoriasis area and severity index (PASI) scores were recorded throughout the course of the mice to reflect the severity of psoriasis. Hematoxylin-eosin staining was used to observe the pathological changes in skin lesions. Enzyme-linked immunosorbent assay (ELISA) was performed to test the level of IFN-γ and TNF-α in plasma. To further investigate the immunopharmacological effect of FFSD, we used chicken ovalbumin (OVA) to induce immunoreaction in mice. ELISA was used to detect the levels of anti-OVA antibody, IFN-γ and TNF-α in mice. Flow cytometry was performed to quantify the ratio of cell types in peripheral blood mononuclear cells (PBMCs) to evaluate the effect of FFSD on immunosuppression. Proteomics and bioinformatics analyzes were performed to find the regulation pathway of the immunosuppressive effect of FFSD. Finally, quantitative PCR (qPCR) and immunohistochemistry were used to measure the upregulation of Annexin-A proteins (ANXAs) in the skin lesion tissue of IMQ-induced mouse. RESULTS: On the basis of knowing the composition of FFSD, we first proved the efficacy of FFSD in alleviating IMQ-induced psoriasis in mice. Second, we further clarified the pharmacological effect of FFSD on immunosuppression via OVA-induced mice. Subsequently, it was found that the significant up-regulation of ANXAs was caused by FFSD through proteomics analysis, and the finding was proved in the IMQ-induced psoriasis mouse model. CONCLUSIONS: This study elucidates the immunosuppressive pharmacological effect of FFSD on improving psoriasis through up-regulating ANXAs.

Strong Annexin A10 Expression Supports a Pancreatic Primary and Combined Annexin A10, Claudin 18, and SOX2 Expression Supports an Esophagogastric Origin in Carcinomas of Unknown Primary.

Primary tumor site determination for gastrointestinal (GI) tract and pancreaticobiliary (PB) tree carcinomas that present as metastasis of unknown primary can be problematic. Annexin A10 (ANXA10), claudin 18 (CLDN18), and trefoil factor 1 (TFF1) have been identified through expression profiling as markers of gastric lineage commitment; sex-determining region Y (SRY)-box transcription factor 2 (SOX2) expression has been reported in several tumor types, including gastric adenocarcinomas. We evaluated the diagnostic utility of immunohistochemistry for ANXA10, CLDN18, SOX2, and TFF1 for determining the site of origin for GI/PB adenocarcinomas. Immunohistochemistry for all 4 markers was performed on tissue microarrays including 559 GI/PB tumors and 421 other tumors. H-scores were calculated as the product of the intensity (0 to 3) and extent (percentage, 0% to 100%) of staining. Positive staining was defined as >5% staining. ANXA10 expression was most frequent in pancreatic adenocarcinomas when compared with all other GI/PB tumors (96.4% vs. 43.5%, P <0.001). Strong staining for ANXA10 (H-score ≥200) distinguished pancreatic ductal adenocarcinoma from intrahepatic cholangiocarcinoma and adenocarcinomas of the gallbladder and colorectum (69.6% vs. 0%, P <0.001). Triple positivity for ANXA10, CLDN18, and SOX2 was more frequent in esophagogastric tumors than in other GI/PB tumors (22.6% vs. 4.1%; P <0.001). TFF1 expression was observed in nearly all tumor types. Staining for ANXA10, CLDN18, and SOX2 as part of a panel may aid in distinguishing esophagogastric adenocarcinomas from lower GI/PB tumors. ANXA10 staining may be particularly useful in distinguishing pancreatic adenocarcinomas from intrahepatic cholangiocarcinoma and adenocarcinomas of the gallbladder and colorectum.

ANXA10 Expression Is Inversely Associated with Tumor Stage, Grade, and TP53 Expression in Upper and Lower Urothelial Carcinoma.

INTRODUCTION: Urothelial carcinoma (UC) is a common type of malignant disease, but little is known about the diagnostic and prognostic markers of upper urinary tract urothelial cancer (UTUC) because of its rarity. To clarify the significance of ANXA10 in UTUC, we studied ANXA10 expression with immunohistochemistry (IHC). METHODS: The expression of ANXA10 was analyzed in the upper and lower urinary tract of UC by IHC in combination with The Cancer Genome Atlas (TCGA) data analysis. The association between ANXA10 expression and representative cancer-related molecules was also evaluated. RESULTS: ANXA10 expression was weak in normal upper tract urothelium but was positive in 39/117 (33%) UTUCs. ANXA10 was more frequently positive in tumors with pure UC (36%, p < 0.05), papillary morphology (50%, p < 0.01), low grade (G1/2: 57%, p < 0.01), and pTa/is/1 stage (55%, p < 0.01) than in those with histological variants (0%), nodular morphology (9%), G3 (16%), and pT2/3/4 (13%), respectively. ANXA10-positive patients showed better cancer-specific survival and progression-free survival than ANXA10-negative patients (p < 0.05). IHC showed that ANXA10 positivity was detected more in cases with the low expression of TP53 (p < 0.01) and Ki-67 labeling index <20% (p < 0.01). In TCGA dataset of muscle-invasive bladder cancer, higher ANXA10 expression correlated with papillary morphology, lower grade/stage, luminal papillary subtype, wild-type TP53, and FGFR3 gene mutation. CONCLUSION: We revealed that ANXA10 expression was increased during carcinogenesis and was observed more frequently in papillary UC of lower grade and stage. However, its expression decreased as cancer progressed. Therefore, the ANXA10 expression in UTUC might be clinically useful for decision-making.