YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) such as sunitinib are multitarget antiangiogenic agents in clear cell renal cell carcinoma (ccRCC). They are widely used in the treatment of advanced/metastatic renal cancer. However, resistance to TKIs is common in the clinic, particularly after long-term treatment. YTHDC1 is the main nuclear reader protein that binds with m6A to regulate the splicing, export and stability of mRNA. However, the specific role and corresponding mechanism of YTHDC1 in renal cancer cells are still unclear. METHODS: The Cancer Genome Atlas (TCGA) dataset was used to study the expression of YTHDC1 in ccRCC. Cell counting kit-8 (CCK-8), wound healing, Transwell and xenograft assays were applied to explore the biological function of YTHDC1 in ccRCC. Western blot, quantitative real time PCR (RT‒qPCR), RNA immunoprecipitation PCR (RIP-qPCR), methylated RIP-qPCR (MeRIP-qPCR) and RNA sequencing (RNA-seq) analyses were applied to study the YY1/HDAC2/YTHDC1/ANXA1 axis in renal cancer cells. The CCK-8 assay and xenograft assay were used to study the role of YTHDC1 in determining the sensitivity of ccRCC to sunitinib. RESULTS: Our results demonstrated that YTHDC1 is downregulated in ccRCC tissues compared with normal tissues. Low expression of YTHDC1 is associated with a poor prognosis in patients with ccRCC. Subsequently, we showed that YTHDC1 inhibits the progression of renal cancer cells via downregulation of the ANXA1/MAPK pathways. Moreover, we also showed that the YTHDC1/ANXA1 axis modulates the sensitivity of tyrosine kinase inhibitors. We then revealed that HDAC2 inhibitors resensitize ccRCC to tyrosine kinase inhibitors through the YY1/HDAC2 complex. We have identified a novel YY1/HDAC2/YTHDC1/ANXA1 axis modulating the progression and chemosensitivity of ccRCC. CONCLUSION: We identified a novel YY1/HDAC2/YTHDC1/ANXA1 axis modulating the progression and chemosensitivity of ccRCC.

Electroacupuncture promotes microglial M2 polarization in ischemic stroke via annexin A1.

BACKGROUND: Neuroinflammation is the leading cause of neurological sequelae in ischemic stroke. Recently, we reported that the anti-inflammatory mediator annexin A1 (ANXA1) favored microglial M2 polarization in brain injury. The purpose of this study was to investigate electroacupuncture (EA) treatment and its potentially ANXA1-mediated anti-inflammatory effects in the middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model of stroke. METHODS: Treatment with EA consisted of dense-sparse frequencies (alternating 4 Hz sparse waves for 1.5 s and 16 Hz dense waves for 1.5 s) at CV24 and GV26. Intracerebroventricular (ICV) injection of Boc-2 (5 µM) or short hairpin RNA (sh)ANXA1 (2 µL) 3 days before EA was performed to block the effects of ANXA1. RESULTS: EA pretreatment enhanced expression of ANXA1 and its receptor, formyl peptide receptor (FPR), when compared to MCAO/R alone. EA treatment also rescued MCAO/R-induced deficits in neurological performance, and learning and memory, and reduced infarct volume. Double immunofluorescent labeling showed that EA prevented MCAO/R-induced changes in microglial activation and morphology. EA also reduced the release of pro-inflammatory cytokines, such as interleukin (IL)-1ß, inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α, while increasing the release of anti-inflammatory cytokines, such as arginase-1 (Arg-1) and brain-derived neurotrophic factor (BDNF). All EA-induced effects were either partially or completely prevented by prior administration of FPR antagonist Boc-2 or shANXA1. CONCLUSION: The current study provides strong evidence that EA treatment has protective effects against ischemic stroke in the MCAO/R mouse model and that the mechanism likely involves the promotion of M2 polarization in microglia via ANXA1.

Methanolic extract of Cariniana rubra Gardner ex Miers stem bark negatively regulate the leukocyte migration and TNF-α and up-regulate the annexin-A1 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Cariniana rubra Gardner ex Miers (Lecythidaceae), is a native and endemic tree in Brazil, whose inner stem bark decoction preparation is used in folk medicine to treat various inflammatory disorders. Previous scientific reports confirmed its popular use as an anti-inflammatory, without, however, evaluating its action mechanisms. AIM: The objective of this study was to determine the cytotoxicity and anti-inflammatory mechanism of action of the methanolic extract of Cariniana rubra (MECr), using experimental models in vivo and in vitro, as well as to identify secondary metabolites present in the extract. MATERIAL AND METHODS: The MECr was prepared by maceration of inner stem bark powder in methanol (1:10 w/v). The in vitro cytotoxicity effect was evaluated in CHO-k1 cells. The Hippocratic screening test was conducted to evaluate the acute toxicity of MECr in mice. The actions of MECr on leukocyte migration, cytokine levels (IL-1ß and TNF-α) and annexin-A1 (AnxA1) expression, were carried out on lambda-type carrageenan air pouch inflammation model in Swiss mice. Additionally, the phytochemical analysis of MECr was carried out by thin-layer chromatography (TLC) and spectrometric mass analysis with electrospray ionization ESI(-)/MS and gas chromatography-mass spectrometry (GC-MS). RESULTS: Treatment of CHO-k1 cells for 24 h with MECr did not cause cytotoxicity (IC50 > 200 µg/mL), however, the MECr was shown to be cytotoxic after 72 h of cell exposure (IC50 = 19.90 ± 3.51 µg/mL). In the Hippocratic test, oral treatment of mice with 750, 1500, or 3000 mg/kg of MECr did not show any histopathological changes and mortality during the 14 days of observation. In the carrageenan air pouch inflammation model, MECr reduced (p < 0.001) polymorphonuclear migration (57.7% and 57.8%), leukocyte monocyte migration (74.5% and 61.8%) in the air pouch cavity and in the skin tissue, respectively. MECr also inhibited TNF-α concentration in the air cavity wash (3.2%, p < 0.01) and increased expression of the AnxA1 protein (26.9%, p < 0.01) in the skin tissue, particularly in neutrophils. ß-sitosterol (1.95%), gallic acid (1.24%), ß-amyrin (0.87%) and stigmasterol (0.66%) were identified as the major constituents in methanolic extract. CONCLUSION: MECr exhibits significant anti-inflammatory action at least by increasing AnxA1 expression and by inhibiting the release of TNF-α pro-inflammatory cytokine and leukocyte migration, which is probably linked to the presence of identified biologically active compounds, especially gallic acid and terpenes. We believe that the results of this study provide a pharmacological basis for the MECr to be considered as a potential therapeutic agent for the treatment of inflammatory diseases.

Evaluation of phytoestrogens in inducing cell death mediated by decreasing Annexin A1 in Annexin A1-knockdown leukemia cells.

BACKGROUND: Phytoestrogens are plant compounds that are structurally similar to estrogen and that possess anti-cancer properties. Previous studies have reported that coumestrol, daidzein and genistein could induce cell death by reducing Annexin A1 protein in leukemic cell lines. Annexin A1 (ANXA1) is involved in cell progression, metastasis, and apoptosis in several types of cancer cells. The present study sought to investigate if the effects of phytoestrogens on apoptosis, cell cycle arrest and phagocytosis in ANXA1-knockdown leukemic cells are mediated through ANXA1 or occurred independently. METHODS: Transfection of ANXA1 siRNA was conducted to downregulate ANXA1 expression in Jurkat, K562 and U937 cells. Apoptosis and cell cycle assays were conducted using flow cytometry. Western blot was performed to evaluate ANXA1, caspases and Bcl-2 proteins expression. Phagocytosis was determined using hematoxylin and eosin staining. RESULTS: The expression of ANXA1 after the knockdown was significantly downregulated in all cell lines. Genistein significantly induced apoptosis associated with an upregulation of procaspase-3, -9, and - 1 in Jurkat cells. The Bcl-2 expression showed no significant difference in Jurkat, K562 and U937 cells. Treatment with phytoestrogens increased procaspase-1 expression in Jurkat and U937 cells while no changes were detected in K562 cells. Flow cytometry analysis demonstrated that after ANXA1 knockdown, coumestrol and genistein caused cell cycle arrest at G2/M phase in selected type of cells. The percentage of phagocytosis and phagocytosis index increased after the treatment with phytoestrogens in all cell lines. CONCLUSION: Phytoestrogens induced cell death in ANXA1-knockdown leukemia cells, mediated by Annexin A1 proteins. Graphical abstract.

Hydroalcoholic crude extract of Casearia sylvestris Sw. reduces chronic post-ischemic pain by activation of pro-resolving pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Casearia sylvestris Sw. is widely used in popular medicine to treat conditions associated with pain. AIM OF THE STUDY: The present study investigated the influence of hydroalcoholic crude extract of Casearia sylvestris (HCE-CS) and contribution of pro-resolving mediators on mechanical hyperalgesia in a mouse model of chronic post-ischemia pain (CPIP). METHODS AND RESULTS: Male Swiss mice were subjected to ischemia of the right hind paw (3h), then reperfusion was allowed. At 10min, 24h or 48h post-ischemia/reperfusion (I/R), different groups of animals were treated with HCE-CS (30mg/Kg, orally [p.o]), selected agonists at the pro-resolving receptor ALX/FPR2 (natural molecules like resolvin D1 and lipoxin A4 or the synthetic compound BML-111; 0.1-1µg/animal) or vehicle (saline, 10mL/Kg, s.c.), in the absence or presence of the antagonist WRW4 (10µg, s.c.). Mechanical hyperalgesia (paw withdrawal to von Frey filament) was asseseed together with histological and immunostainning analyses. In these settings, pro-resolving mediators reduced mechanical hyperalgesia and HCE-CS or BML-111 displayed anti-hyperalgesic effects which was markedly attenuated in animals treated with WRW4. ALX/FPR2 expression was raised in skeletal muscle or neutrophils after treatment with HCE-CS or BML-111. CONCLUSION: These results reveal significant antihyperalgesic effect of HCE-CS on CPIP, mediated at least in part, by the pathway of resolution of inflammation centred on the axis modulated by ALX/FPR2.

Proteomic analysis for anti-atherosclerotic effect of tetrahydroxystilbene glucoside in rats.

2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (TSG) extracted from Polygonum multiflorum (a traditional Chinese medicinal herb) has been proved to exhibit significant anti-atherosclerotic activity. In this study, we firstly used proteomic analyses to investigate the molecular events occurring in the atherosclerotic rats after TSG treatment. Aortic samples were collected from the atherosclerotic rat group and the TSG-treated group, and its proteome was analyzed by two-dimensional gel electrophoresis (2-DE). Proteins showing significant changes in expression were identified and analyzed by matrix-assisted desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). As a result, 21 protein spots were found with significant differential expression after the treatment with TSG. A total of 18 spots were identified by database searching, and 17 spots matched with known proteins. Among these proteins (11 proteins up-regulated and six proteins down-regulated), five proteins were mainly involved in inflammation, cholesterol transport, cell apoptosis and adhesion. TSG treatment enhanced the expression of HSP 70, lipocortin 1 and Apo A-I, and inhibited the expression of calreticulin, vimentin. Furthermore, we randomly selected four proteins and confirmed the results of proteomic analysis by RT-PCR and western blotting. In conclusion, TSG treatment suppresses atherosclerosis by altering the expression of different proteins. Calreticulin, vimentin, HSP 70, lipocortin 1, and Apo A-I, are key proteins that may be novel molecular targets responsible for atherogenesis suppression induced by TSG treatment.

GADD45α and annexin A1 are involved in the apoptosis of HL-60 induced by resveratrol.

Resveratrol (3,4',5-trihydroxy-trans-stilbene), one of secondary metabolites of low molecular weight present in plant, has various important biological effects. It can induce apoptosis in human leukemia cell types in vitro, although the mechanism is not fully understood. In the present study, we demonstrated reduced viability and DNA synthesis, as well as increased proportion of the subdiploid cell population, in HL-60 cells as determined by cell cycle analysis with resveratrol. Resveratrol treatment resulted in a gradual time-dependent decrease in the expression of anti-apoptotic Bcl-2 and increase in that of Bax, annexin A1, growth arrest- and DNA damage-induced gene 45α (GADD45α), and cleaved caspase-3. In addition, resveratrol markedly increased caspase-3 activity in cells. Our results suggest that resveratrol could inhibit the proliferation and induce apoptosis of HL-60 cells through a GADD45α and annexin A1/caspase-3 pathway.

Comprehensive interaction of dicalcin with annexins in frog olfactory and respiratory cilia.

Dicalcin (renamed from p26olf) is a dimer form of S100 proteins found in frog olfactory epithelium. S100 proteins form a group of EF-hand Ca(2+)-binding proteins, and are known to interact with many kinds of target protein to modify their activities. To determine the role of dicalcin in the olfactory epithelium, we identified its binding proteins. Several proteins in frog olfactory epithelium were found to bind to dicalcin in a Ca(2+)-dependent manner. Among them, 38 kDa and 35 kDa proteins were most abundant. Our analysis showed that these were a mixture of annexin A1, annexin A2 and annexin A5. Immunohistochemical analysis showed that dicalcin and all of these three subtypes of annexin colocalize in the olfactory cilia. Dicalcin was found to be present in a quantity almost sufficient to bind all of these annexins. Colocalization of dicalcin and the three subtypes of annexin was also observed in the frog respiratory cilia. Dicalcin facilitated Ca(2+)-dependent liposome aggregation caused by annexin A1 or annexin A2, and this facilitation was additive when both annexin A1 and annexin A2 were present. In this facilitation effect, the effective Ca(2+) concentrations were different between annexin A1 and annexin A2, and therefore the dicalcin-annexin system in frog olfactory and respiratory cilia can cover a wide range of Ca(2+) concentrations. These results suggested that this system is associated with abnormal increases in the Ca(2+) concentration in the olfactory and other motile cilia.

Green tea induces annexin-I expression in human lung adenocarcinoma A549 cells: involvement of annexin-I in actin remodeling.

Green tea polyphenols exhibit multiple antitumor activities in various in vitro and in vivo tumor models, and the mechanisms of action are not clear. Previously, we found that green tea extract (GTE) regulates actin remodeling in different cell culture systems. Actin remodeling plays an important role in cancer cell morphology, cell adhesion, motility, and invasion. Using proteomic approaches, we found GTE-induced expression of annexin-I, a multifunctional actin binding protein, in these cell lines. In this study, we aimed to further define the functional role of GTE-induced annexin-I expression in actin remodeling, cell adhesion, and motility in lung adenocarcinoma A549 cells. We found that GTE stimulates the expression of annexin-I in a dose-dependent fashion. The GTE-induced annexin-I expression appears to be at the transcription level, and the increased annexin-I expression mediates actin polymerization, resulting in enhanced cell adhesion and decreased motility. Annexin-I specific interference resulted in loss of GTE-induced actin polymerization and cell adhesion, but not motility. In fact, annexin-I specific interference itself inhibited motility even without GTE. Together, annexin-I plays an important role in GTE-induced actin remodeling, and it may serve as a potential molecular target associated with the anticancer activities of green tea.

Annexin-I as a potential target for green tea extract induced actin remodeling.

Using a multistep human urothelial model, we previously showed that green tea extract (GTE) selectively modulates actin remodeling in transformed cells (MC-T11), which resulted in increased cell adhesion and reduced cell motility (Lu et al., Clin Cancer Res 2005;11:1675-83). This study further analyzed which actin binding proteins (ABPs) might be involved in this process. Proteomic profiles of GTE treated and untreated MC-T11 cells using two-dimensional gel electrophoresis coupled with liquid chromatography tandem mass spectrometry (LC/MS/MS) and matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) identified 20 GTE-induced proteins. Among them, 3 were ABPs (tropomodulin, cofilin and annexin-I), and only annexin-I showed a dose- and time-dependent expression. The increased annexin-I correlated with actin remodeling, and was the result of transcription level up-regulation, as determined by RT-PCR, pull-down immunoblot and siRNA analyses. 5-Azacytidine, a DNA methylation inhibitor, exhibited no effect on annexin-I expression when used alone, but had an additive effect for GTE-induced annexin-I expression. Immunohistochemistry of bladder cancer tissue array showed a decrease of annexin-I expression in carcinoma in situ and low grade papillary carcinoma (n = 32, 0% positive) compared to nontumor urothelium (n = 18, 89% positive) (p < 0.001 by Fisher exact test), but increased in some (6 of 15, 40%) high-grade tumors. Together, GTE induced annexin-I expression plays a role in regulating actin remodeling and decreased annexin-I expression is a common event in early stage of bladder cancer development.

Annexin-I expression modulates drug resistance in tumor cells.

The use of anti-cancer chemotherapy often leads to the rise of multidrug-resistant (MDR) tumors. We have previously reported the overexpression of a 40kDa protein (P-40) in several MDR tumor cell lines. In this report we describe the cloning of a 1.4kb cDNA with an open reading frame of 344 amino acids that encodes the P-40 protein. Analysis of the P-40 amino acid sequence showed it is identical to the human annexin I (Anx-I) protein. The identity of the isolated P-40 cDNA as Anx-I was confirmed by the specific binding of IPM96 mAb to a 40kDa protein following the in vitro expression of P-40 full-length cDNA. Northern blot analysis of total RNA from drug-sensitive and -resistant cells revealed an increase in P-40 (or Anx-I) mRNA in drug-resistant cells relative to drug-sensitive cells. Transfection of Anx-I cDNA into drug-sensitive MCF-7 cells was carried out without further drug selection and showed 2- to 5-fold increase in resistance of transfected cells to adriamycin, melphalan, and etoposide. Conversely, transfection of reverse Anx-I cDNA into SKOV-3 cells decreased the expression of Anx-I without affecting the expression of other members of the annexin family and showed a 3- to 8-fold increase in sensitivity to these drugs. Of interest was the correlation between the presence of Anx-I and MDR in MDA-MB-231 cells when compared to MCF-7 cells. MDA-MB-231 cells show 3- to 20-fold increase in resistance to adriamycin, melphalan, and etoposide in the absence of detectable levels of P-glycoprotein (P-gp1), the multidrug resistance protein (MRP1) or the breast cancer resistance protein (BCRP). Taken together, these results provide the first direct evidence for the role of Anx-I in MDR of tumor cells.

Arachidonic acid metabolism in cells transfected with sense and anti-sense cDNA to annexin I.

Thymic epithelial cell line TEA3A1 produce prostaglandin E2 (PGE2) and express high levels of annexin I. In order to study the relationship between the levels of annexin I expression and the arachidonic acid metabolism, TEA3A1 cells transfected with expression vector containing full length annexin I cDNA in either sense or anti-sense orientation was studied. In the anti-sense cells where the level of annexin I was reduced, the PGE2 production was significantly lower. On the other hand, PGE2 production was significantly higher in sense cells where the production of annexin I was also higher than in control cells. Our results show that increased PGE2 production in sense cells was accompanied by higher levels of PLA2 enzymatic activities. These results suggest that the production of annexin I is positively associated with the phospholipase A2 enzymatic activity and the prostaglandin production in thymic epithelial cells.