Identification of ANXA3 as a biomarker associated with pyroptosis in ischemic stroke.

BACKGROUND: Pyroptosis plays an important role in the pathological process of ischemic stroke (IS). However, the exact mechanism of pyroptosis remains unclear. This paper aims to reveal the key molecular markers associated with pyroptosis in IS. METHODS: We used random forest learning, gene set variation analysis, and Pearson correlation analysis to screen for biomarkers associated with pyroptosis in IS. Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen and glucose deprivation/reoxygenation (OGD/R) models were constructed in vitro and in vivo. Cells were transfected with an Annexin A3 silencing (si-ANXA3) plasmid to observe the effects of ANXA3 on OGD/R + lipopolysaccharides (LPS)-induced pyroptosis. qRT‒PCR and western blotting were used to detect the expression of potential biomarkers and pyroptotic pathways. RESULTS: Samples from a total of 170 IS patients and 109 healthy individuals were obtained from 5 gene expression omnibus databases. Thirty important genes were analyzed by random forest learning from the differentially expressed genes. Then, we investigated the relationship between the above genes and the pyroptosis score, obtaining three potential biomarkers (ANXA3, ANKRD22, ADM). ANXA3 and ADM were upregulated in the MCAO/R model, and the fold difference in ANXA3 expression was greater. Pyroptosis-related factors (NLRP3, NLRC4, AIM2, GSDMD-N, caspase-8, pro-caspase-1, cleaved caspase-1, IL-1ß, and IL-18) were upregulated in the MCAO/R model. Silencing ANXA3 alleviated the expression of pyroptosis-related factors (NLRC4, AIM2, GSDMD-N, caspase-8, pro-caspase-1, cleaved caspase-1, and IL-18) induced by OGD/R + LPS or MCAO/R. CONCLUSION: This study identified ANXA3 as a possible pyroptosis-related gene marker in IS through bioinformatics and experiments. ANXA3 could inhibit pyroptosis through the NLRC4/AIM2 axis.

ANXA3, associated with YAP1 regulation, participates in the proliferation and chemoresistance of cervical cancer cells.

BACKGROUND: Cervical cancer, as one of the most common cancers in women, remains a major health threat worldwide. Annexin A3 (ANXA3), a component of the annexin family, is upregulated in numerous cancers, with no explicit role in cervical cancer. OBJECTIVE: This study aims to investigate the function of ANXA3 in cervical cancer. METHODS: Differential expression genes between the cervical cancer tissues of patients and the controls were analyzed in The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA) database. Using transfection approaches to either upregulate or downregulate ANXA3, its role in cell proliferation and chemosensitivity of human cervical cancer cell lines (HeLa and C33A) was evaluated. Furthermore, the binding activity between YAP1 and ANXA3 was also explored. RESULTS: Genomics analysis indicated that differential genes were mostly associated with cell cycle progression and DNA replication. ANXA3 was highly expressed in the cervical cancer tissues and closely linked to malignancy degree. Knockdown of ANXA3 in cervical cancer cells inhibited cell cycle progression. A similar result was observed in the reduction of cyclin D, CDK4, cyclin E, and CDK2 in cervical cancer cells with ANXA3 silencing. Cervical cancer cells obtained high sensitivity to cisplatin (DDP) when ANXA3 was downregulated. Conversely, these capabilities were the opposite in cervical cancer cells overexpressing ANXA3. Furthermore, the expression levels of ANXA3 and YAP1 were positively correlated. YAP1 upregulation was positively connected with malignant behaviors, which were reversed by ANXA3 downregulation. CONCLUSION: In light of our findings, targeting ANXA3 expressed in cervical cancer might contribute to more potential therapeutic strategies.

Downregulation of annexin A3 promotes ionizing radiation-induced EGFR activation and nuclear translocation and confers radioresistance in nasopharyngeal carcinoma.

Radioresistance currently poses a significant challenge to successful disease control of nasopharyngeal carcinoma (NPC). We previously uncovered that annexin A3 (ANXA3), a calcium-dependent phospholipid binding protein, is underexpressed in radioresistant NPC cells and mouse xenografts. This study aims to further unravel the mechanistic basis underlying ANXA3-mediated radioresistance in NPC. We show that either innate ANXA3 downregulation or short hairpin RNA(shRNA)-based knockdown of ANXA3 confers resistance to ionizing radiation (IR) in NPC both in vitro and in mouse xenograft models in vivo, whereas radiosensitization was observed when ANXA3 was ectopically expressed. Mechanistically, ANXA3 knockdown dramatically enhances IR-induced epidermal growth factor receptor (EGFR) phosphorylation and nuclear translocation, leading to increased post-IR phosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) concomitant with markedly accelerated DNA DSB repair. In addition, pretreatment with cetuximab efficiently abrogated the radioresistant phenotype of ANXA3-low cells as well as the ANXA3 knockdown-induced post-IR EGFR nuclear accumulation, suggesting that EGFR is an essential mediator for ANXA3 depletion-mediated radioprotection in NPC. Collectively, this work reveals for the first time a critical role of ANXA3 in radiation survival and DNA repair mechanism of NPC and provides mechanistic evidence to support ANXA3 as a potential therapeutic target to improve radiocurability for NPC.

Annexin A3 as a Marker Protein for Microglia in the Central Nervous System of Rats.

The parenchymal microglia possess different morphological characteristics in cerebral physiological and pathological conditions; thus, visualizing these cells is useful as a means of further investigating parenchymal microglial function. Annexin A3 (ANXA3) is expressed in microglia, but it is unknown whether it can be used as a marker protein for microglia and its physiological function. Here, we compared the distribution and morphology of parenchymal microglia labeled by ANXA3, cluster of differentiation 11b (CD11b), and ionized calcium-binding adaptor molecule 1 (Iba1) and measured the expression of ANXA3 in nonparenchymal macrophages (meningeal and perivascular macrophages). We also investigated the spatiotemporal expression of ANXA3, CD11b, and Iba1 in vivo and in vitro and the cellular function of ANXA3 in microglia. We demonstrated that ANXA3-positive cells were abundant and evenly distributed throughout the whole brain tissue and spinal cord of adult rats. The morphology and distribution of ANXA3-labeled microglia were quite similar to those labeled by the microglial-specific markers CD11b and Iba1 in the central nervous system (CNS). ANXA3 was expressed in the cytoplasm of microglia, and its expression was significantly increased in activated microglia. ANXA3 was almost undetectable in the nonparenchymal macrophages. Meanwhile, the protein and mRNA expression levels of ANXA3 in different regions of the CNS were different from those of CD11b and Iba1. Moreover, knockdown of ANXA3 inhibited the proliferation and migration of microglia, while overexpression of ANXA3 enhanced these activities. This study confirms that ANXA3 may be a novel marker for parenchymal microglia in the CNS of adult rats and enriches our understanding of ANXA3 from expression patterns to physiological function.

Identification of Key Candidate Genes Involved in the Progression of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal, agnogenic interstitial lung disease with limited therapeutic options. To investigate vital genes involved in the development of IPF, we integrated and compared four expression profiles (GSE110147, GSE53845, GSE24206, and GSE10667), including 87 IPF samples and 40 normal samples. By reanalyzing these datasets, we managed to identify 62 upregulated genes and 20 downregulated genes in IPF samples compared with normal samples. Differentially expressed genes (DEGs) were analyzed by gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to illustrate relevant pathways of IPF, biological processes, molecular function, and cell components. The DEGs were then subjected to protein-protein interaction (PPI) for network analysis, serving to find 11 key candidate genes (ANXA3, STX11, THBS2, MMP1, MMP9, MMP7, MMP10, SPP1, COL1A1, ITGB8, IGF1). The result of RT-qPCR and immunohistochemical staining verified our finding as well. In summary, we identified 11 key candidate genes related to the process of IPF, which may contribute to novel treatments of IPF.

Microglial Annexin A3 promoted the development of melanoma via activation of hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway.

BACKGROUND: Melanoma, a relatively common malignancy, has become one of the tumors with the fastest rising incidence in recent years. The purpose of this study was to investigate the effect of Microglial Annexin A3 (ANXA3) on melanoma. METHODS: Serum samples were obtained from 20 patients with melanoma or 20 healthy controls. Kaplan-Meier survival analysis was performed. Transcriptome were used to analyze the correlation between ANXA3 expression and overall survival in patients with melanoma. Human melanoma cell lines WM-115 cells were transfected with ANXA3, si-ANXA3, ANXA3 + si-hypoxia inducible factor-1α (HIF-1α), si-ANXA3 + HIF-1α, and negative plasmids. Cell proliferation assay, cell invasion assay, and wound healing assay were performed on WM-115 cells. Lactate dehydrogenase (LDH) and caspase-3/9 activities were detected by commercial kits. Western blot and RT-PCR were used to detect the protein and mRNA expression of relation factors. RESULTS: ANXA3 expression was up-regulated in patients with melanoma in comparison with healthy controls. Over-expression of ANXA3 promoted cell growth and migration, and reduced cytotoxicity of WM-115 cells. Overall survival (OS) and disease-free survival (DFS) of patients with high ANXA3 expression were both lower than those of patients with low ANXA3 expression. Down-regulation of ANXA3 reduced cell growth and migration, and promoted cytotoxicity of WM-115 cells. ANXA3 induced vascular endothelial growth factor (VEGF) signaling pathway by activation of HIF-1α. CONCLUSION: In conclusion, our results indicated that ANXA3 promoted cell growth and migration of melanoma via activation of HIF-1α/VEGF signaling pathway.

Annexin A3 in sepsis: novel perspectives from an exploration of public transcriptome data.

According to publicly available transcriptome datasets, the abundance of Annexin A3 (ANXA3) is robustly increased during the course of sepsis; however, no studies have examined the biological significance or clinical relevance of ANXA3 in this pathology. Here we explored this interpretation gap and identified possible directions for future research. Based on reference transcriptome datasets, we found that ANXA3 expression is restricted to neutrophils, is upregulated in vitro after exposure to plasma obtained from septic patients, and is associated with adverse clinical outcomes. Secondly, an increase in ANXA3 transcript abundance was also observed in vivo, in the blood of septic patients in multiple independent studies. ANXA3 is known to mediate calcium-dependent granules-phagosome fusion in support of microbicidal activity in neutrophils. More recent work has also shown that ANXA3 enhances proliferation and survival of tumour cells via a Caspase-3-dependent mechanism. And this same molecule is also known to play a critical role in regulation of apoptotic events in neutrophils. Thus, we posit that during sepsis ANXA3 might either play a beneficial role, by facilitating microbial clearance and resolution of the infection; or a detrimental role, by prolonging neutrophil survival, which is known to contribute to sepsis-mediated organ damage.

Microglial annexin A3 downregulation alleviates bone cancer-induced pain through inhibiting the Hif-1α/vascular endothelial growth factor signaling pathway.

Bone cancer-induced pain (BCP) is a challenging clinical problem because traditional therapies are often only partially effective. Annexin A3 (ANXA3) is highly expressed in microglia in the spinal cord, and its expression is upregulated during BCP. However, the roles of microglial ANXA3 in the development and maintenance of BCP and the underlying molecular mechanisms remain unclear. This study was performed on male mice using a metastatic lung BCP model. Adeno-associated virus shANXA3 (AAV-shANXA3) was injected intrathecally 14 days before and 7 days after bone cancer induction, and relevant pain behaviors were assessed by measuring the paw withdrawal mechanical threshold, paw withdrawal thermal latency, and spontaneous hind limb lifting. ANXA3 protein expression was downregulated in microglial N9 cells by lentiviral transfection (LV-shANXA3). ANXA3, hypoxia-inducible factor-1α (Hif-1α), vascular endothelial growth factor (VEGF) expression levels, and Hif-1α transactivation activity regulated by ANXA3 were measured. As a result, ANXA3 was expressed in microglia, and its expression significantly increased during BCP. ANXA3 knockdown reversed pain behaviors but did not prevent pain development. Moreover, ANXA3 knockdown significantly reduced Hif-1α and VEGF expression levels in vitro and in vivo. And overexpression of Hif-1α or VEGF blocked the effects of AAV-shANXA3 on BCP. ANXA3 knockdown in N9 cells significantly decreased the p-PKC protein expression in the cocultured neurons. Finally, ANXA3 overexpression significantly increased Hif-1α transactivation activity in 293T cells. Therefore, microglial ANXA3 downregulation alleviates BCP by inhibiting the Hif-1α/VEGF signaling pathway, which indicates that ANXA3 may be a potential target for the treatment of BCP.

miR-18b attenuates cerebral ischemia/reperfusion injury through regulation of ANXA3 and PI3K/Akt signaling pathway.

Recent, research has displayed that the disorders of miR-18b are related to ischemic stroke. Here, we aimed to investigate the underlying neuroprotective mechanism of miR-18b in cerebral ischemia/reperfusion (I/R) injury. Oxygen-glucose deprivation/reperfusion (OGDR) model in vitro and middle cerebral artery occlusion (MCAO) model in vivo were established to simulate cerebral I/R injury. RT-PCR, western blotting, CCK-8, TUNEL, and TTC staining assays were applied in this study to explore the effect of miR-18b on cerebral I/R injury. Results displayed that miR-18b expression was reduced after cerebral I/R injury. Besides, miR-18b showed neuroprotective effects on cerebral I/R injury both in vitro and in vivo, These neuroprotective effects included promoting cell viability, decreasing cell apoptosis, reducing the production of inflammatory cytokines in SH-SY 5Y cells after OGDR and depressing MCAO-induced infarct size, neurological deficits and apoptotic cells in mice. Moreover, miR-18b negatively regulated ANXA3 expression, and its neuroprotection on cerebral I/R injury was overturned by ANXA3. Additionally, increasing miR-18b or decreasing ANXA3 promoted the activation of the PI3K/Akt signaling pathway in SH-SY 5Y cells after cerebral I/R injury. In conclusion, these data indicate that miR-18b protects against cerebral I/R injury by inhibiting ANXA3 and activating PI3K/Akt pathway, which provides a promising therapeutic target for ischemic stroke therapy.

microRNA-382 suppresses the progression of pancreatic cancer through the PI3K/Akt signaling pathway by inhibition of Anxa3.

Pancreatic cancer (PC) is a lethal cancer in the digestive system. microRNAs (miRNAs) have been demonstrated to participate in PC progression. In this context, we, thus, aimed to explore the mechanism of miR-382 in epithelial mesenchymal transition (EMT) and lymph node metastasis in PC in relation to Anxa3 and the PI3K/Akt signaling pathway. Gene expression data sets GSE16515, GSE71989, and GSE32676 were screened out, with the findings showing the significance of miR-382 and annexin A3 (Anxa3) in PC. A total of 115 PC patients were selected for determination of miR-382 and Anxa3 expression with lowly expressed miR-382 and highly expressed Anxa3 found via RT-quantitative PCR and Western blot analysis. Additionally, negative correlation was found between miR-382 and Anxa3 in PC. Dual-luciferase reporter gene assay and in situ hybridization results confirmed that miR-382 negatively regulated Anxa3. miR-382 targeted Anxa3 and suppressed PC progression by blocking the PI3K/Akt signaling pathway. After a series of gain- and loss-of function approaches, upregulation of miR-382 or silencing of Anxa3 inhibited the EMT and lymph node metastasis, as evidenced by increased level of E-cadherin and decreased level of N-cadherin, vimentin, vascular endothelial growth factor(VEGFR)-3, VEGF-C, and VEGF-D. Overexpression of miR-382 or downregulation of Anxa3 was shown to inhibit colony formation, migration, and invasion abilities of PC cells. Further, tumor xenograft in nude mice in vivo also confirmed the inhibitory role of miR-382 and silenced Anxa3 in lymph node metastasis in PC. Thus, this study provides promising therapeutic targets for PC treatment.NEW & NOTEWORTHY This study focused on the mechanism of miR-382 in epithelial mesenchymal transition and lymph node metastasis in PC in relation to Anxa3 and the PI3K/Akt signaling pathway. We found the inhibitory role of miR-382 in PC in vitro and in vivo.

ANXA3 deletion inhibits the resistance of lung cancer cells to oxaliplatin.

OBJECTIVE: The purpose of this study was to explore the role of ANXA3 in lung cancer cell resistance to oxaliplatin (OXA). MATERIALS AND METHODS: After adding different concentrations of Ox, A549, and A549/Ox cell viability were examined using cell counting kit-8 (CCK-8) assay, and the mRNA and protein expressions of ANXA3 were analyzed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. After treating cells with 5 µg/mL and 15 µg/mL Ox for 24 hours and knocking down ANXA3, qRT-PCR, CCK8, flow cytometry, transwell, and BrdU assays were performed to examine ANXA3 expression level, cell viability, apoptosis, migration, and proliferative capacities, respectively. In addition, Western blot was performed to detect the protein expression of c-caspase 3. RESULTS: The higher the concentration of Ox added, the worse the cell viability. Meanwhile, ANXA3 expression in A549/Ox cells was found remarkably higher than that in normal A549 cells. After treated with different concentrations of Ox for 24 hours, the cell viability, migration capacity and cell proliferation of A549 cells were found remarkably decreased, while the opposite results were observed in cell apoptosis and C-caspase 3 protein expression, and the Ox treatment group was evidently lower than control group. CONCLUSIONS: Knockdown of ANXA3 may be able to inhibit the resistance of LCa cells to OXA.

Identification of the downstream molecules of agrin/Dok-7 signaling in muscle.

The development of the neuromuscular junction depends on signaling processes that involve protein phosphorylation. Motor neuron releases agrin to activate muscle protein Dok-7, a key tyrosine kinase essential for the formation of a mature and functional neuromuscular junction. However, the signaling cascade downstream of Dok-7 remains poorly understood. In this study, we combined the clustered regularly interspaced short palindromic repeats/Cas9 technique and quantitative phosphoproteomics analysis to study the tyrosine phosphorylation events triggered by agrin/Dok-7. We found tyrosine phosphorylation level of 36 proteins increased specifically by agrin stimulation. In Dok-7 mutant myotubes, however, 13 of the 36 proteins failed to be enhanced by agrin stimulation, suggesting that these 13 proteins are Dok-7-dependent tyrosine-phosphorylated proteins, could work as downstream molecules of agrin/Dok-7 signaling. We validated one of the proteins, Anxa3, by in vitro and in vivo assays. Knocking down of Anxa3 in the cultured myotubes inhibited agrin-induced AChR clustering, whereas reduction of Anxa3 in mouse muscles induced abnormal postsynaptic development. Collectively, our phosphoproteomics analysis provides novel insights into the complicated signaling network downstream of agrin/Dok-7.

Annexin A3 is necessary for parallel artery-vein alignment in the mouse retina.

BACKGROUND: Annexin A3 (Anxa3) is a member of the calcium-regulated, cell membrane-binding family of annexin proteins. We previously confirmed that Anxa3 is expressed in the endothelial lineage in vertebrates and that loss of anxa3 in Xenopus laevis leads to embryonic blood vessel defects. However, the biological function of Anxa3 in mammals is completely unknown. In order to investigate Anxa3 vascular function in mammals, we generated an endothelial cell-specific Anxa3 conditional knockout mouse model (Anxa3f/f ;Tie2-Cre). RESULTS: Anxa3f/f ;Tie2-Cre mice are born at Mendelian ratios and display morphologically normal blood vessels during development. However, loss of Anxa3 leads to artery-vein (AV) misalignment characterized by atypical AV crossovers in the postnatal and adult retina. CONCLUSIONS: Anxa3 is not essential for embryonic blood vessel formation but is required for proper parallel AV alignment in the murine retina. AV crossovers associated with Anxa3f/f ;Tie2-Cre mice are similar to AV intersections observed in patients with branch retinal vein occlusion (BRVO), although we did not observe occluded vessels. This new Anxa3 mouse model may provide a basis for understanding AV crossover formation associated with BRVO.

Differential Expression of Inflammation-Related Genes in Down Syndrome Patients with or without Periodontal Disease.

AIM: Aware that Down Syndrome patients present among their clinical characteristics impaired immunity, the aim of this study is to identify the statistically significant differences in inflammation-related gene expression by comparing Down Syndrome patients with Periodontal Disease (DS+PD+) with Down Syndrome patients without Periodontal Disease (DS+PD-), and their relationship with periodontitis as a chronic oral inflammatory clinical feature. MATERIALS AND METHODS: Case study and controls on eleven Down Syndrome patients (DS+PD+ vs. DS+PD-). RNA was extracted from peripheral blood using a Qiagen PAXgene Blood miRNA Kit when performing an oral examination. A search for candidate genes (92 selected) was undertaken on the total genes obtained using a Scientific GeneChip® Scanner 3000 (Thermo Fisher Scientific) and Clariom S solutions for human, mouse, and rat chips, with more than 20,000 genes annotated for measuring expression levels. RESULTS: Of the 92 inflammation-related genes taken initially, four genes showed a differential expression across both groups with a p value of <0.05 from the data obtained using RNA processing of the patient sample. Said genes were TNFSF13B (p = 0.0448), ITGB2 (p = 0.0033), ANXA3 (p = 0.0479), and ANXA5 (p = 0.016). CONCLUSIONS: There are differences in inflammation-related gene expression in Down Syndrome patients when comparing patients who present a state of chronic oral inflammation with patients with negative rates of periodontal disease.

Expression and significance of Annexin A3 in the osteosarcoma cell lines HOS and U2OS.

Annexin A3 (ANXA3) is highly expressed in different types of cancers, but the impact of ANXA3 in bone tumors is still not clear. In the present study, the expression of ANXA3 in osteosarcoma cells was first confirmed by cellular immunofluorescence. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analysis were used to detect the expression of ANXA3 in osteoblasts in the osteosarcoma cell lines U2OS and HOS. Furthermore, small interfering (si)­RNA were transfected into U2OS and HOS cells via a liposome­mediated method. Then once ANXA3 had been successfully downregulated in U2OS and HOS cells, the cells were collected and total protein was extracted after 48 h of transfection. Western blot analysis was used to confirm successful ANXA3 transfection into osteosarcoma cells and the apoptotic rate of HOS and U2OS was detected by flow cytometry. The expression of ANXA3 in the osteosarcoma cell lines HOS and U2OS were first observed by confocal laser scanning microscopy, and was then detected by RT­qPCR and western blotting. The mRNA and protein levels of ANXA3 in the osteosarcoma cell lines HOS and U2OS were significantly increased compared with osteoblasts, particularly in HOS cells. When siRNA was transfected into HOS and U2OS cells, the protein expression level of ANXA3 was measured via western blotting. The results indicated that the expression of ANXA3 was significantly decreased. In addition, to determine whether ANXA3 knockdown induced cell apoptosis, the present study analyzed the apoptotic rate by flow cytometry. The results revealed that ANXA3 knockdown markedly increased HOS and U2OS cell apoptosis. To the best of our knowledge, the present study is the first to confirm that ANXA3 is highly expressed in the osteosarcoma cell lines HOS and U2OS. In addition, downregulation of ANXA3 expression in HOS and U2OS cells could increase apoptotic ability.

Annexin A3 may play an important role in ochratoxin-induced malignant transformation of human gastric epithelium cells.

Ochratoxin A (OTA), one of the most abundant food-contaminating mycotoxins, is a possible carcinogen to humans. We previously demonstrated that long-term (40 weeks) OTA exposure induces the malignant transformation of human gastric epithelium cells (GES-1) in vitro. However, the specific mechanism underlying OTA-induced gastric carcinogenesis is complex. In the present study, we used 2-DE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF MS) combined with bioinformatics and immunoblotting to investigate the differentially expressed proteins between GES-1 and OTA-malignant transformed GES-1 cells (OTA-GES-1T cells) in vitro. We found that four differentially expressed proteins were identified after malignant transformation, including actin, cytoplasmic 1 (ACTB), F-actin-capping protein subunit alpha-1 (CAPZA1), Annexin A3 (ANXA3), thioredoxin peroxidase B from red blood cells (TPx-B) and Fibrinogen beta B (Fibrinogen ß). Among the differentially expressed proteins, the effect of Annexin A3 was analyzed by MTT assay, western blot, cell cycle analysis, wound healing assay, Transwell assay, and colony formation assay in OTA-GES-1T cells. The results showed that inhibition of Annexin A3 by siRNA effectively prevented the proliferation, migration, and invasion abilities of OTA-GES-1T cells. Collectively, the results of this study will guide future research on OTA carcinogenicity.

Efficacy of neoadjuvant chemotherapy and Annexin A3 expression in breast cancer.

PURPOSE: To observe the efficacy of neoadjuvant chemotherapy (NAC) in patients with breast cancer (BC) and to investigate the effect of Annexin A3 (ANXA3) expression. METHODS: 158 patients with BC treated in Yantai Yuhuangding Hospital from September 2015 to December 2017 were retrospectively analyzed. Among them, 83 cases were treated with epirubicin + cyclophosphamide + 5-fluorouracil (CEF group), 75 cases with epirubicin + cyclophosphamide + docetaxel (TEC group), with 3 cycles of chemotherapy. The efficacy and adverse reactions of the two NAC regimens were compared and analyzed. Tissue specimens were collected before and 10 days after the administration of chemotherapy in order to detect the expression of ANXA3 by qRT-PCR in each group. RESULTS: There were significant differences in the rates of complete remission (CR), partial remission (PR), stable disease (SD), and progressive disease (PD) between the two groups (z=10.716, p=0.013). The clinical effectiveness rate in the TEC group was significantly higher than that in the CEF group (p<0.05). There was no difference in the pathology grade between the two groups (p>0.05); however, the pathological effective rate in the TEC group was significantly higher than that in the CEF group (p<0.05). There was no difference in the rate of bone marrow suppression between the two groups (p>0.05). While there was no difference in the relative expression of ANXA3 between the two groups before chemotherapy, the relative expression of ANXA3 in the TEC group was lower than that in the CEF group after NAC (p<0.05). The relative expression in both groups after chemotherapy was lower than that before NAC (P<0.05). CONCLUSION: Compared with CEF regimen, NAC with TEC regimen can improve the clinical and pathological effectiveness rate, inhibit the expression of ANXA3, and improve the prognosis of patients, thus having a certain application prospect in NAC.

Annexin A3 depletion overcomes resistance to oxaliplatin in colorectal cancer via the MAPK signaling pathway.

Colorectal cancer (CRC) is a common disease with high mortality and morbidity. Annexin A3 (ANXA3) belongs to the structurally homologous family of Ca2+ and phospholipid-binding proteins. This study aimed to investigate the effects and potential mechanisms of ANXA3 on oxaliplatin (Ox) resistance in CRC. We generated two human CRC cell lines (HCT116/Ox and SW480/Ox) with acquired Ox resistance and determined their resistance properties. ANXA3 expression and cell apoptosis, migration and invasion also were evaluated. We found that cell viability of HCT116/Ox and SW480/Ox was higher than that in parental cells in the presence of Ox. ANXA3 was highly expressed in HCT116/Ox and SW480/Ox cells. ANXA3 downregulation diminished cell survival, migration and invasion, while increased the apoptosis of HCT116 and SW480 with or without Ox. Moreover, depletion of ANXA3 reduced cell viability and BrdU incorporation, increased cell apoptosis and c-caspase 3 expression in HCT116/Ox with or without Ox. A transwell assay determined that knockdown of ANXA3 impeded the migration and invasion of HCT116/Ox and SW480/Ox cells. Additionally, phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) decreased upon ANXA3 depletion in HCT116/Ox cells, and ANXA3 silencing suppressed Ox-induced activation of ERK and JNK signaling pathway. ANXA3 downregulation reduced Ox resistance in CRC, and treatment with the ERK inhibitor PD098059 or JNK inhibitor SP600125 contributed to this process. These results indicate that silencing ANXA3 could overcome Ox resistance in CRC via the mitogen-activated protein kinase signaling pathway.

Cancer-associated fibroblasts contribute to cisplatin resistance by modulating ANXA3 in lung cancer cells.

Cancer tissues consist of cancer cells, surrounding stromal cells and the extracellular matrix. Cancer-associated fibroblasts (CAF) are one of the key components of stromal cells. CAF have a great impact on the behavior of cancer cells, including proliferation, invasion, metastasis and chemoresistance in many ways. However, the underlying mechanism had not been fully elucidated. In this study, we investigated the role of CAF in cisplatin resistance of lung cancer cells. By using conditioned medium from CAF (CAF-CM), we found that CAF decreased the sensitivity of lung cancer cells to cisplatin. RNA sequencing results showed that CAF expressed a higher level of Annexin A3 (ANXA3) than normal fibroblasts (NF), and CAF-CM incubation increased the ANXA3 level in lung cancer cells. Overexpression of ANXA3 in lung cancer cells increased cisplatin resistance and activated c-jun N-terminal kinase (JNK), whereas knockdown of ANXA3 increased cisplatin sensitivity. Further study showed that CAF-CM enhanced cisplatin resistance by inhibiting cisplatin-induced apoptosis, determined by repression of caspase-3 and caspase-8, through activation of the ANXA3/JNK pathway. Conversely, suppression of JNK activation by specific inhibitor retarded the effect of CAF-CM and ANXA3 on cisplatin sensitivity. Taken together, our study demonstrated that CAF potentiated chemoresistance of lung cancer cells through a novel ANXA3/JNK pathway both in vitro and in vivo, suggesting ANXA3 could be a potential therapeutic target for the treatment of chemoresistant cancer.