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Background. In the present study, we aimed to find out whether luteolin (Lut) pretreatment could ameliorate myocardial ischemia/reperfusion (I/R) injury by regulating the lncRNA just proximal to XIST (JPX)/microRNA-146b (miR-146b) axis. Methods. We established the models in vitro (HL-1 cells) and in vivo (C57BL/6J mice) to certify the protection mechanism of Lut pretreatment on myocardial I/R injury. Dual luciferase reporter gene assay was utilized for validating that JPX could bind to miR-146b. JPX and miR-146b expression levels were determined by RT-qPCR. Western blot was utilized to examine apoptosis-related protein expression levels, including cleaved caspase-9, caspase-9, cleaved caspase-3, caspase-3, Bcl-2, Bax, and BAG-1. Apoptosis was analyzed by Annexin V-APC/7-AAD dualstaining, Hoechst 33342 staining, as well as flow cytometry. Animal echocardiography was used to measure cardiac function (ejection fraction (EF) and fractional shortening (FS) indicators). Results. miR-146b was demonstrated to bind and recognize the JPX sequence site by dual luciferase reporter gene assay. The expression level of miR-146b was corroborated to be enhanced by H/R using RT-qPCR (P < 0.001 vs. Con). Moreover, JPX could reduce the expression of miR-146b, whereas inhibiting JPX could reverse the alteration (P < 0.001 vs. H/R, respectively). Western blot analysis demonstrated that Lut pretreatment increased BAG-1 expression level and Bcl-2/Bax ratio, but diminished the ratio of cleaved caspase 9/caspase 9 and cleaved caspase 3/caspase 3 (P < 0.001 vs. H/R, respectively). Moreover, the cell apoptosis change trend, measured by Annexin V-APC/7-AAD dualstaining, Hoechst 33342 staining, along with flow cytometry, was consistent with that of apoptosis-related proteins. Furthermore, pretreatment with Lut improved cardiac function (EF and FS) (P < 0.001 vs. I/R, respectively), as indicated in animal echocardiography. Conclusion. Our results demonstrated that in vitro and in vivo, Lut pretreatment inhibited apoptosis via the JPX/miR-146b axis, ultimately improving myocardial I/R injury. [ABSTRACT FROM AUTHOR]

Schistosomiasis is a neglected tropical disease that affects humans and animals in tropical and subtropical regions worldwide. Schistosome eggs are responsible for the pathogenesis and transmission of schistosomiasis, thus reducing egg production is vital for prevention and control of schistosomiasis. However, the mechanisms underlying schistosome reproduction remain unclear. Annexin proteins (ANXs) are involved in the physiological and pathological functions of schistosomes, but the specific regulatory mechanisms and roles of ANX A13 in the development of Schistosoma japonicum and host–parasite interactions remain poorly understood. Therefore, in this study, the expression profiles of SjANX A13 at different life cycle stages of S. japonicum were assessed using quantitative PCR. In addition, the expression profiles of the homolog in S. mansoni were analyzed in reference to public datasets. The results of RNA interference showed that knockdown of SjANX A13 significantly affected the development and egg production of female worms in vivo. The results of an immune protection assay showed that recombinant SjANX A13 increased production of immunoglobulin G-specific antibodies. Finally, co-culture of S. japonicum exosomes with LX-2 cells using a transwell system demonstrated that SjANX A13 is involved in host–parasite interactions via exosomes. Collectively, these results will help to clarify the roles of SjANX A13 in the development of S. japonicum and host–parasite interactions as a potential vaccine candidate. [ABSTRACT FROM AUTHOR]

This study investigated the mechanism of action of ABT-263 in the treatment of neurogenic bladder fibrosis (NBF)and its protective effects against upper urinary tract damage (UUTD). Sixty 12-week-old Sprague-Dawley (SD) rats were randomly divided into sham, sham + ABT-263 (50 mg/kg), NBF, NBF + ABT-263 (25 mg/kg, oral gavage), and NBF + ABT-263 (50 mg/kg, oral gavage) groups. After cystometry, bladder and kidney tissue samples were collected for hematoxylin and eosin (HE), Masson, and Sirius red staining, and Western Blotting (WB) and qPCR detection. Primary rat bladder fibroblasts were isolated, extracted, and cultured. After co-stimulation with TGF-β1 (10 ng/mL) and ABT-263 (concentrations of 0, 0.1, 1, 10, and 100 µmol/L) for 24 h, cells were collected. Cell apoptosis was detected using CCK8, WB, immunofluorescence, and annexin/PI assays. Compared with the sham group, there was no significant difference in any physical parameters in the sham + ABT-263 (50 mg/kg) group. Compared with the NBF group, most of the markers involved in fibrosis were improved in the NBF + ABT-263 (25 mg/kg) and NBF + ABT-263 (50 mg/kg) groups, while the NBF + ABT-263 (50 mg/kg) group showed a significant improvement. When the concentration of ABT-263 was increased to 10 µmol/L, the apoptosis rate of primary bladder fibroblasts increased, and the expression of the anti-apoptotic protein BCL-xL began to decrease.ABT-263 plays an important role in relieving NBF and protecting against UUTD, which may be due to the promotion of myofibroblast apoptosis through the mitochondrial apoptosis pathway. [ABSTRACT FROM AUTHOR]

Background: This study aimed to explore the expression, function, and molecular mechanism of ANXA8, the gene for annexin 8, in cervical cancer. Methods: The gene expression of the ANX family members in cervical cancer tissues was classified via The Cancer Genome Atlas (TCGA) database. The expression of ANXA8 in paracancerous tissues, cervical cancer tissues, and cell lines was identified by fluorescence quantitative polymerase chain reaction (PCR) and immunohistochemistry. The effects of ANXA8 knockdown on the cellular growth and cell invasion of cervical cancer were examined by MTT, clone-formation assay, scratch test, and Transwell assay. The effect of ANXA8 knockdown on the proliferative potency of cervical cancer cells was assessed through an in vivo nude-mouse tumor-formation test. The gene expression levels of Ki-67 and ANXA8 in tumor-bearing tissues were measured through Immunohistochemical analysis. Results: TCGA data analysis and fluorescence quantitative PCR exhibited significantly increased expression of ANXA8 in cervical cancer tissues. Further examination exhibited that ANXA8 was expressed exceedingly in cervical cancer tissue, and it was associated with lymph node metastasis, FIGO stage, degree of differentiation, and infiltration depth of cancer patients. Cell-function assays revealed that knocking down ANXA8 may substantially suppress the propagation, colony formation, invasion, and migration of cervical cancer cells. In vivo experiments demonstrated that ANXA8 knockdown had a substantial inhibitory effect on the propagation of cervical cancer cells in nude mice and inhibited the expression of Ki-67. Conclusion: ANXA8 is specifically and significantly upregulated within cervical cancer tissues. The knockdown of this gene showed remarkable outcomes, as evidenced by the substantial inhibition of cervical cancer cell proliferation in in vitro and in vivo experimentations. Therefore, ANXA8 is a potential target or a marker that can serve as a therapeutic or diagnostic tool for cervical cancer. [ABSTRACT FROM AUTHOR]

Platelets are transfused to patients to prevent bleeding. Since both preparation and storage can impact the hemostatic functions of platelets, we studied platelet concentrates (PCs) with different initial composition in regard to platelet fragmentation and its impact on storage-induced changes in activation potential. Ten whole blood derived PCs were assessed over 7 storage days. Using flow cytometry, platelet (CD41+) subpopulations were characterized for activation potential using activation markers (PAC-1, P-selectin, and LAMP-1), phosphatidylserine (Annexin V), and mitochondrial integrity (DiIC1(5)). Aggregation response, coagulation, and soluble activation markers (cytokines and sGPVI) were also measured. Of the CD41+ events, the PCs contained a median of 82% normal-sized platelets, 10% small platelets, and 8% fragments. The small platelets exhibited procoagulant hallmarks (increased P-selectin and Annexin V and reduced DiIC1(5)). Normal-sized platelets responded to activation, whereas activation potential was decreased for small and abolished for fragments. Five PCs contained a high proportion of small platelets and fragments (median of 28% of CD41+ events), which was significantly higher than the other five PCs (median of 9%). A high proportion of small platelets and fragments was associated with procoagulant hallmarks and decreased activation potential, but, although diminished, they still retained some activation potential throughout 7 days storage. [ABSTRACT FROM AUTHOR]

Several fusion tags have been developed for non-chromatographic fusion protein purification. Previously, we identified that human annexin A1 as a novel N-terminal purification tag was used for purifying the fusion proteins produced in Escherichia coli through precipitation in 10 mM Ca2+ buffer, and redissolution of the precipitate in 15 mM EDTA buffer. In this work, we selected four metal-dependent enzymes including E. coli 5-aminolevulinate dehydratase, yeast 3-hydroxyanthranilate 3,4-dioxygenase, maize serine racemase and copper amine oxidase for investigating the annexin A1 tag applicability. Fusion of the His6-tag or the enzyme changed the behavior of precipitation-redissolution. The relatively high recovery yields of three tagged enzymes with the improved purities were obtained through two rounds of purification, whereas low recovery yield of the annexin A1 tagged maize amine oxidase was prepared. The added EDTA displayed different abilities to redissolve the fusion proteins precipitates in two precipitation-redissolution cycles. It inactivated three enzymes and obviously inhibited the activity of the fused maize serine racemase. Based on current findings, we believe that four enzymes could be applied for evaluating applicability of the proteins or peptides as affinity tags for chromatographic purification in a calcium dependent manner. [ABSTRACT FROM AUTHOR]

Due to the poor prognosis of T-cell acute lymphoblastic leukaemia (T-ALL), there is an urgent need to identify safer and more cost-effective drugs. This study evaluated the antitumour activity of Shuanghuanglian (SHL) on T-ALL cells and elucidated the mechanism. Jurkat and Molt4 cells were treated with SHL (0.1, 0.2 and 0.4 mg/mL) for 24 and 48 h. The controls were treated with RPMI 1640 containing 10% foetal bovine serum. Cell viability was evaluated through Cell Counting Kit-8 assay. Patterns of death and signalling pathway alterations caused by SHL were identified by network pharmacology combined with GO enrichment analysis and then were verified by Hoechst 33342 staining, Annexin V-FITC/PI staining and Western blotting. Interactions of the active ingredients with targets were analysed by molecular docking. The IC50 values of SHL in Jurkat and Molt4 cells were 0.30 ± 0.10 and 0.48 ± 0.07 mg/mL, respectively, at 24 h and 0.27 ± 0.05 and 0.30 ± 0.03 mg/mL at 48 h. In T-ALL, 117 target genes of SHL were mainly enriched in the apoptosis and NOTCH signalling pathways. SHL induced apoptosis was confirmed by Hoechst 33342 staining and flow cytometry. The protein levels of cleaved caspase-7 and cleaved PARP were significantly increased but those of cleaved NOTCH1 and MYC were reduced. The active ingredients of SHL can interact with γ-secretase. Discussion and conclusions: SHL induces apoptosis in T-ALL cells via the NOTCH1-MYC pathway and may be a potential drug for the treatment of T-ALL. [ABSTRACT FROM AUTHOR]

Introduction: Endometriosis is an inflammatory-related reproductive age disease characterized by the presence of endometrial cells outside the uterine cavity. Current laboratory practice does not provide specific markers for detecting and assessing the advancement of endometriosis in either plasma or peritoneal fluid. The severity of disease is assessed in stages from I to IV based on the results of laparoscopic inspection. The protein annexin A2 (ANXA2) has been reported to be associated with inflammatory processes.Aim of the Study: The study aimed to investigate and compare ANXA2 protein concentration using the ELISA method in plasma and peritoneal fluid in a group of women with endometriosis compared to controls.Materials and Methods: Biological material was collected during a multicenter, cross-sectional study, which was conducted at eight departments during elective laparoscopy from 53 women with and 40 women without endometriosis. Patients were divided by endometriosis stage and infertility status, and then compared with subgroups. Analysis included the Chi-square test for categorical variables, Mann–Whitney U-test and two-sided Wilcoxon rank-sum test for continuous variables.Results: Women with endometriosis had significantly elevated plasma ANXA2 levels compared to women without endometriosis (mean concentrations 28.69 vs 19.61 ng/L, p=0.01). Differences in peritoneal fluid ANXA2 levels were statistically insignificant (mean concentrations of 23.7 vs 22.97 ng/L, p=0.06). Plasma concentrations in patients with stage III and IV endometriosis were significantly higher compared to controls (mean concentrations of 24.19 vs 19.71 ng/L, p=0.03). No such differences were observed in plasma when comparing stages I–II vs III–IV, and stages I–II vs controls (mean concentrations of 33.82 vs 24.19 ng/L, p=0.72 and 33.82 vs 19.71 ng/L, p=0.12, respectively). Comparison of samples from patients with or without infertility, primary or secondary infertility, endometriosis with or without infertility, and non-endometriosis with or without infertility showed no significant differences in the plasma nor in the peritoneal fluid concentrations.Conclusion: ANXA2 is possibly involved in the pathogenesis of endometriosis, especially in advanced stages. Due to the limited group of tested samples, further studies are needed to confirm its role. [ABSTRACT FROM AUTHOR]

Women acquire HIV through sexual transmission, with increasing incidence in women >50 years old. Identifying protective mechanisms in the female genital tract (FGT) is important to prevent HIV-acquisition in women as they age. Human genital and blood neutrophils inactivate HIV by releasing neutrophil extracellular traps (NETs), an innate protective mechanism against HIV-infection. However, how NET formation is triggered by HIV in different tissues and whether this mechanism is affected by aging remain unknown. We demonstrate that the mechanisms that trigger NET release in response to HIV are different in blood and genital tissues, and that NET release decreases with aging. In blood neutrophils, HIV stimulation independently activated calcium pathways and endosomal TLR8, but aging reduced calcium responses, resulting in delayed NET release. In contrast, calcium responses were absent in genital neutrophils and NET release was triggered preferentially through TLR8 activation, but aging impaired this pathway. HIV induced NET formation through non-lytic pathways in blood and FGT neutrophils, except for a small subset of NETs that incorporated annexin V and lactoferrin predominantly in blood, suggesting proinflammatory and lytic NET release. Our findings demonstrate that blood neutrophils cannot model genital neutrophil responses which has important implications to understanding protection against HIV acquisition. [ABSTRACT FROM AUTHOR]

Mycoplasma bovis is responsible for various inflammatory diseases in cattle. The prevention and control of M. bovis are complicated by the absence of effective vaccines and the emergence of multidrug-resistant strains, resulting in substantial economic losses worldwide in the cattle industry. Lipoproteins, vital components of the Mycoplasmas cell membrane, are deemed potent antigens for eliciting immune responses in the host upon infection. However, the functions of lipoproteins in M. bovis remain underexplored due to their low sequence similarity with those of other bacteria and the scarcity of genetic manipulation tools for M. bovis. In this study, the lipoprotein LppA was identified in all examined M. bovis strains. Utilizing immunoelectron microscopy and Western blotting, it was observed that LppA localizes to the surface membrane. Recombinant LppA demonstrated dose-dependent adherence to the membrane of embryonic bovine lung (EBL) cells, and this adhesion was inhibited by anti-LppA serum. In vitro binding assays confirmed LppA's ability to associate with fibronectin, collagen IV, laminin, vitronectin, plasminogen, and tPA, thereby facilitating the conversion of plasminogen to plasmin. Moreover, LppA was found to bind and enhance the accumulation of Annexin A2 (ANXA2) on the cell membrane. Disrupting LppA in M. bovis significantly diminished the bacterium's capacity to adhere to EBL cells, underscoring LppA's function as a bacterial adhesin. In conclusion, LppA emerges as a novel adhesion protein that interacts with multiple host extracellular matrix proteins and ANXA2, playing a crucial role in M. bovis's adherence to host cells and dissemination. These insights substantially deepen our comprehension of the molecular pathogenesis of M. bovis. [ABSTRACT FROM AUTHOR]

(1) Annexins are proteins that bind phospholipids and calcium ions in cell membranes and mediate signal transduction between Ca2+ and cell membranes. They play key roles in plant immunity. (2) In this study, virus mediated gene silencing and the heterologous overexpression of TaAnn12 in Arabidopsis thaliana Col-0 trials were used to determine whether the wheat annexin TaAnn12 plays a positive role in plant disease resistance. (3) During the incompatible interaction between wheat cv. Suwon 11 and the Puccinia striiformis f. sp. tritici (Pst) race CYR23, the expression of TaAnn12 was significantly upregulated at 24 h post inoculation (hpi). Silencing TaAnn12 in wheat enhanced the susceptibility to Pst. The salicylic acid hormone contents in the TaAnn12-silenced plants were significantly reduced. The overexpression of TaAnn12 in A. thaliana significantly increased resistance to Pseudomonas syringae pv. tomato DC3000, and the symptoms of the wild-type plants were more serious than those of the transgenic plants; the amounts of bacteria were significantly lower than those in the control group, the accumulation of Reactive Oxygen Species (ROS)and callose deposition increased, and the expression of resistance-related genes (AtPR1, AtPR2, and AtPR5) significantly increased. (4) Our results suggest that wheat TaAnn12 resisted the invasion of pathogens by inducing the production and accumulation of ROS and callose. [ABSTRACT FROM AUTHOR]

Endothelial cells are the crucial inner lining of blood vessels, which are pivotal in vascular homeostasis and integrity. However, these cells are perpetually subjected to a myriad of mechanical, chemical, and biological stresses that can compromise their plasma membranes. A sophisticated repair system involving key molecules, such as calcium, annexins, dysferlin, and MG53, is essential for maintaining endothelial viability. These components orchestrate complex mechanisms, including exocytosis and endocytosis, to repair membrane disruptions. Dysfunctions in this repair machinery, often exacerbated by aging, are linked to endothelial cell death, subsequently contributing to the onset of atherosclerosis and the progression of cardiovascular diseases (CVD) and stroke, major causes of mortality in the United States. Thus, identifying the core machinery for endothelial cell membrane repair is critically important for understanding the pathogenesis of CVD and stroke and developing novel therapeutic strategies for combating CVD and stroke. This review summarizes the recent advances in understanding the mechanisms of endothelial cell membrane repair. The future directions of this research area are also highlighted. [ABSTRACT FROM AUTHOR]

Matrix vesicles (MVs) have shown strong effects in diseases such as vascular ectopic calcification and pathological calcified osteoarthritis and in wound repair of the skeletal system due to their membranous vesicle characteristics and abundant calcium and phosphorus content. However, the role of MVs in the progression of osteoporosis is poorly understood. Here, we report that annexin A5, an important component of the matrix vesicle membrane, plays a vital role in bone matrix homeostasis in the deterioration of osteoporosis. We first identified annexin A5 from adherent MVs but not dissociative MVs of osteoblasts and found that it could be sharply decreased in the bone matrix during the occurrence of osteoporosis based on ovariectomized mice. We then confirmed its potential in mediating the mineralization of the precursor osteoblast lineage via its initial binding with collagen type I to achieve MV adhesion and the subsequent activation of cellular autophagy. Finally, we proved its protective role in resisting bone loss by applying it to osteoporotic mice. Taken together, these data revealed the importance of annexin A5, originating from adherent MVs of osteoblasts, in bone matrix remodeling of osteoporosis and provided a new strategy for the treatment and intervention of bone loss. [ABSTRACT FROM AUTHOR]

Introduction: The extracellular matrix (ECM) has been heavily implicated in the development and progression of cancer. We have previously shown that Annexin A2 is integral in the migration and invasion of breast cancer cells and in the clinical progression of ER-negative breast cancer, processes which are highly influenced by the surrounding tumor microenvironment and ECM. Methods: We investigated how modulations of the ECM may affect the role of Annexin A2 in MDA-MB-231 breast cancer cells using western blotting, immunofluorescent confocal microscopy and immuno-precipitation mass spectrometry techniques. Results: We have shown that the presence of collagen-I, the main constituent of the ECM, increases the post-translational phosphorylation of Annexin A2 and subsequently causes the translocation of Annexin A2 to the extracellular surface. In the presence of collagen-I, we identified fibronectin as a novel interactor of Annexin A2, using mass spectrometry analysis. We then demonstrated that reducing Annexin A2 expression decreases the degradation of fibronectin by cancer cells and this effect on fibronectin turnover is increased according to collagen-I abundance. Discussion: Our results suggest that Annexin A2's role in promoting cancer progression is mediated by collagen-I and Annexin A2 maybe a therapeutic target in the bi-directional cross-talk between cancer cells and ECM remodeling that supports metastatic cancer progression. [ABSTRACT FROM AUTHOR]

Introduction: The extracellular matrix (ECM) has been heavily implicated in the development and progression of cancer. We have previously shown that Annexin A2 is integral in the migration and invasion of breast cancer cells and in the clinical progression of ER-negative breast cancer, processes which are highly influenced by the surrounding tumor microenvironment and ECM. Methods: We investigated how modulations of the ECM may affect the role of Annexin A2 in MDA-MB-231 breast cancer cells using western blotting, immunofluorescent confocal microscopy and immuno-precipitation mass spectrometry techniques. Results: We have shown that the presence of collagen-I, the main constituent of the ECM, increases the post-translational phosphorylation of Annexin A2 and subsequently causes the translocation of Annexin A2 to the extracellular surface. In the presence of collagen-I, we identified fibronectin as a novel interactor of Annexin A2, using mass spectrometry analysis. We then demonstrated that reducing Annexin A2 expression decreases the degradation of fibronectin by cancer cells and this effect on fibronectin turnover is increased according to collagen-I abundance. Discussion: Our results suggest that Annexin A2's role in promoting cancer progression is mediated by collagen-I and Annexin A2 maybe a therapeutic target in the bi-directional cross-talk between cancer cells and ECM remodeling that supports metastatic cancer progression. [ABSTRACT FROM AUTHOR]

Matrix vesicles are a special class of extracellular vesicles thought to actively contribute to both physiologic and pathologic mineralization. Proteomic studies have shown that matrix vesicles possess high amounts of annexin A5, suggesting that the protein might have multiple roles at the sites of calcification. Currently, Annexin A5 is thought to promote the nucleation of apatitic minerals close to the inner leaflet of the matrix vesicles' membrane enriched in phosphatidylserine and Ca2+. Herein, we aimed at unravelling a possible additional role of annexin A5 by investigating the ability of annexin A5 to adsorb on matrix-vesicle biomimetic liposomes and Langmuir monolayers made of dipalmitoylphosphatidylserine (DPPS) and dipalmitoylphosphatidylcholine (DPPC) in the absence and in the presence of Ca2+. Differential scanning calorimetry and dynamic light scattering measurements showed that Ca2+ at concentrations in the 0.5–2.0 mM range induced the aggregation of liposomes probably due to the formation of DPPS-enriched domains. However, annexin A5 avoided the aggregation of liposomes at Ca2+ concentrations lower than 1.0 mM. Surface pressure versus surface area isotherms showed that the adsorption of annexin A5 on the monolayers made of a mixture of DPPC and DPPS led to a reduction in the area of excess compared to the theoretical values, which confirmed that the protein favored attractive interactions among the membrane lipids. The stabilization of the lipid membranes by annexin A5 was also validated by recording the changes with time of the surface pressure. Finally, fluorescence microscopy images of lipid monolayers revealed the formation of spherical lipid-condensed domains that became unshaped and larger in the presence of annexin A5. Our data support the model that annexin A5 in matrix vesicles is recruited at the membrane sites enriched in phosphatidylserine and Ca2+ not only to contribute to the intraluminal mineral formation but also to stabilize the vesicles' membrane and prevent its premature rupture. [ABSTRACT FROM AUTHOR]

• Annexins are multigene of calcium dependent phospholipid binding proteins. • Annexins are characterized by unique annexin core domain,made up of four similar motifs approximately 70 amino acids long, each consisting of five α-helices. • Diverse functions including signal transduction, calcium homeostasis, regulation of redox poise, membrane organization and cell cycle control. • Implicated in stress tolerance. Plant annexins are an evolutionary conserved calcium (Ca2+)-dependent and phospholipid-binding protein family that are found in nearly all plant species. They are characterized by presence of a repeated annexin domain and are functionally specialized according to their Ca2+ channel-regulating, peroxidase, ATPase/GTPase, and other activities. They play a significant role in controlling a variety of biochemical and morphological processes, plant growth and development, and response to biotic and abiotic environmental stimuli. Despite the fact that numerous studies have studied the role of annexins, contemporary research on plant annexins has achieved significant strides. In this review, the fundamental characteristics of plant annexins and recent developments in understanding the various functions of annexins in plants are discussed. The role of annexins in biotic and abiotic stress response is also highlighted. [ABSTRACT FROM AUTHOR]

Objective: This study was to investigate whether annexin A7 (AnnexinA7, ANXA7) and its co-related protein tumor cell death domain silencer [suppressor of death domains (SODD)] regulates the migratory phenotype of liver cancer cells. Materials and Methods: In this experimental study, expression of ANXA7 in Hca-P cells, PANXA7 downregulated cells and PANXA7 unrelated sequence cells was detected by real-time quantitative polymerase chain reaction (PCR) at mRNA level and western blotting at protein level. Transwell migration and invasion assays were performed to determine the migratory phenotype. Results: After inhibition of ANXA7 expression, expression of SODD protein was also significantly decreased (P<0.05). Transwell cell transfer experiments showed that number of tumor cells that penetrated into the cell membrane was significantly reduced after ANXA7 silencing (P<0.05). Transwell cell invasion assay showed that number of tumor cells penetrating into Matrigel was significantly reduced after ANXA7 down-regulation (P<0.05). The CCK8 assay was measured at 0, 24 and 48 hours, and proliferation rate of PANXA7 lower weir cells was slower than that of Hca-P cells and PANXA7 non-related sequence cells (P<0.05). Conclusion: SODD expression was decreased with the down-regulation of ANXA7. Down-regulating ANXA7 in Hca-P cells decreased proliferation, migration and invasion of tumor cells. [ABSTRACT FROM AUTHOR]

The primary objective of this study was to elucidate the chemical composition, antioxidant properties, and antiproliferative activities of Eclipta prostrata extracts. Two flavonoids, 3′-O-methylorobol and apigenin 7-sulfate, were isolated from the ethyl acetate (EtOAc) extract of E. prostrata. The total phenolic and flavonoid contents of the E. prostrata extracts, as well as their overall antioxidant activities as measured using the 2,2-diphenyl-1-picrylhydrazyl and reducing power assays, were investigated. The E. prostrata EtOAc extract exhibited significantly greater antioxidant activities in both assays and higher phenol and flavonoid contents than the other extracts. The potential antiproliferative properties of the E. prostrata extracts and isolated compounds were investigated in vitro against the AGS, A549, and HT-29 cancer cell lines and the normal human HEK-293 cell line using the MTT assay. Annexin V-FITC/PI staining analysis and quantitative real-time PCR were used to assess AGS cell apoptosis. At a concentration of 100 µg/mL, the EtOAc extract of E. prostrata reduced AGS cell viability and proliferation by inducing apoptosis through the alteration of gene expression in the apoptotic cascade. These results highlight E. prostrata as a promising source of anticancer compounds. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) are known to have a therapeutic effect on nephrotoxicity. As animal models require significant time and resources to evaluate drug effects, there is a need for a new experimental technique that can accurately predict drug effects in humans. We evaluated the therapeutic effect of MSC-derived EVs in cisplatin nephrotoxicity using a three-dimensional, gravity-driven, two-layer tubule-on-a-chip (3D-MOTIVE chip). In the 3D-MOTIVE chip, 10 μM cisplatin decreased the number of attached cells compared to the vehicle. Conversely, annexin V and reactive oxygen species (ROS) were increased. Cell viability was increased 2.8-fold and 2.5-fold after treatment with EVs at 4 and 8 µg/mL, respectively, compared to the cisplatin-induced nephrotoxicity group. Cell attachment was increased 2.25-fold by treatment with 4 µg/mL EVs and 2.02-fold by 8 µg/mL EVs. Annexin V and ROS levels were decreased compared to those in the cisplatin-induced nephrotoxicity group. There were no significant differences in annexin V and ROS levels according to EV concentration. In sum, we created a cisplatin-induced nephrotoxicity model on a 3D-MOTIVE chip and found that MSC-derived EVs could restore cell viability. Thus, MSC-derived EVs may have the potential to ameliorate cisplatin-induced nephrotoxicity. [ABSTRACT FROM AUTHOR]

Cancer is one of the leading causes of death worldwide. With the help of current scientific developments and traditional knowledge about medicinal plants, great progress could be made in the fight against cancer. The present study attempts to scientifically validate the ethnomedicinal and anticancer effects of Mesua ferrea Linn from the Western Ghats of Karnataka, India. Using preparative HPLC, two main fractions (C1 and C2) were isolated from the methanolic extract of Mesua ferrea and their bioactivity was investigated. Gas chromatography/Mass spectrometry analysis of Mesua ferrea methanol extract revealed the presence of 7 phytocomponents. Fraction C2 shows maximum potency against liver HepG2 cancer cell lines. Fraction C2 was further purified and subjected to spectroscopic analysis by 13C NMR, 1H NMR, FTIR and liquid chromatography-mass spectrometry techniques. The structure is predicted and identified as 6-ethyl-3- hydroxy-4-methyl-8aH-xanthen-9(10aH)-one. This compound was found to have antiproliferative activity with an IC50 value of 352.007 µg/ml compared to the standard drug (camptothecin) with an IC50 value of 52.253 µg/ml and a crude drug of 177.006 µg/ml. Anticancer efficacy of the purified compound assessed by Annexin V apoptosis Study Using HepG2 cell lines. The compound exhibits significant early and late apoptosis of 13.6 % and 15.5 %, respectively, compared to the standard drug camptothecin with 28.7 % and 18.7 % early and late apoptosis and 7.02 % and 11.8 %. Caspase-3 expression analysis using HepG2 cell lines revealed that the purified compound inhibited early apoptosis in HepG2 cells at a concentration of 352 µg/ml, activating the caspase-3 cascade by 15.3 % compared with 17 % of the standard drug CTP. The result of the DNA fragmentation test supported the results of Annexin V and caspase 3 expression and showed significant protection. Our results indicate that 6-ethyl-3-hydroxy-4-methyl-8aH-xanthen-9(10aH)-one isolated from Mesua ferrea may be a new lead molecule for the development of an antiproliferative, DNA-protective and effective anticancer drugs. [ABSTRACT FROM AUTHOR]

Annexins exist widely in plants as multigene families and play critical roles in stress responses and a range of cellular processes. This study provides a comprehensive account of the cloning and functional characterization of the rice annexin gene OsAnn5. The findings reveal that a cold stress treatment at the seedling stage of rice induced OsAnn5 expression. GUS staining assay indicated that the expression of OsAnn5 was non tissue‐specific and was detected in almost all rice tissues. Subcellular localization indicated that OsAnn5‐GFP (green fluorescent protein) signals were found in the endoplasmic reticulum apparatus. Compared with wild type rice, knocking out OsAnn5 using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins) mediated genome editing resulted in sensitivity to cold treatments. These results indicate that OsAnn5 is involved in cold stress tolerance at the seedling stage. [ABSTRACT FROM AUTHOR]

Annexin A11 (ANXA11) is a calcium-dependent phospholipid-binding protein belonging to the annexin protein family and implicated in the neurodegenerative amyotrophic lateral sclerosis. Structurally, ANXA11 contains a conserved calcium-binding C-terminal domain common to all annexins and a putative intrinsically unfolded N-terminus specific for ANXA11. Little is known about the structure and functions of this region of the protein. By analogy with annexin A1, it was suggested that residues 38 to 59 within the ANXA11 N-terminus could form a helical region that would be involved in interactions. Interestingly, this region contains residues that, when mutated, may lead to clinical manifestations. In the present study, we have studied the structural features of the full-length protein with special attention to the N-terminal region using a combination of biophysical techniques which include nuclear magnetic resonance and small angle X-ray scattering. We show that the N-terminus is intrinsically disordered and that the overall features of the protein are not markedly affected by the presence of calcium. We also analyzed the 38–59 helix hypothesis using synthetic peptides spanning both the wild-type sequence and clinically relevant mutations. We show that the peptides have a remarkable character typical of a native helix and that mutations do not alter the behaviour suggesting that they are required for interactions rather than being structurally important. Our work paves the way to a more thorough understanding of the ANXA11 functions.

Abstract Background Cystic echinococcosis (CE) is a life-threatening zoonotic disease caused by the larval stage of Echinococcus granulosus sensu lato, which employs various strategies to evade the host immune system for survival. Recent advances have revealed the role of annexins as excretory/secretory products, providing new insights into the immune regulation by these proteins in the pathogenesis of CE. Methods Echinococcus granulosus annexin B proteins EgANXB2, EgANXB18, EgANXB20, and EgANXB23 were cloned, expressed, and analyzed using bioinformatic tools. Membrane binding analysis was used to assess their bioactivity, while their immunoreactivity and tissue distribution characteristics were determined experimentally using western blotting and immunofluorescence staining, respectively. Furthermore, quantitative real-time reverse transcription PCR (qRT-PCR) was used to analyze the mRNA expression profiles of EgANXBs in different developmental stages of E. granulosus. Finally, immunofluorescence staining, cell counting kit 8 assays, flow cytometry, transwell migration assays, and qRT-PCR were used to evaluate the functional effects of rEgANXB18 and rEgANXB20 on mouse peripheral blood mononuclear cells (PBMCs). Results In this study, we identified four EgANXBs with conserved protein structures and calcium-dependent phospholipid binding activities. rEgANXBs were recognized by serum from sheep infected with E. granulosus and distributed in the germinal layer of fertile cysts. Interestingly, transcription levels of the four EgANXBs were significantly higher in protoscoleces than in 28-day strobilated worms. Moreover, we demonstrated that rEgANXB18 and rEgANXB20 were secretory proteins that could bind to PBMCs and regulate their function. Specifically, rEgANXB18 inhibited cell proliferation and migration while promoting cell apoptosis, NO production, and cytokine profile shifting. In contrast, rEgANXB20 showed limited effects on apoptosis but inhibited NO production. Conclusions Our findings suggested that among the four identified EgANXBs, EgANXB2 and EgANXB23 might play a pivotal role for the development of protoscoleces, while EgANXB18 and EgANXB20, as secretory proteins, appeared to participate in the host-parasite interaction by regulating the function of immune cells. Graphical Abstract

Abstract 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.

The limitations of current cancer therapies, including the increasing prevalence of multidrug resistance, underscore the urgency for more effective treatments. One promising avenue lies in the repurposing of existing drugs. This review explores the impact of phenothiazines, primarily used as antipsychotic agents, on key mechanisms driving tumor growth and metastasis. The cationic and amphiphilic nature of phenothiazines allows interaction with the lipid bilayer of cellular membranes, resulting in alterations in lipid composition, modulation of calcium channels, fluidity, thinning, and integrity of the plasma membrane. This is especially significant in the setting of increased metabolic activity, a higher proliferative rate, and the invasiveness of cancer cells, which often rely on plasma membrane repair. Therefore, properties of phenothiazines such as compromising plasma membrane integrity and repair, disturbing calcium regulation, inducing cytosolic K-RAS accumulation, and sphingomyelin accumulation in the plasma membrane might counteract multidrug resistance by sensitizing cancer cells to membrane damage and chemotherapy. This review outlines a comprehensive overview of the mechanisms driving the anticancer activities of phenothiazines derivates such as trifluoperazine, prochlorperazine, chlorpromazine, promethazine, thioridazine, and fluphenazine. The repurposing potential of phenothiazines paves the way for novel approaches to improve future cancer treatment.

In various epithelial tissues, the epithelial monolayer acts as a barrier. To fulfill its function, the structural integrity of the epithelium is tightly controlled. When normal epithelial cells detach from the basal substratum and delaminate into the apical lumen, the apically extruded cells undergo apoptosis, which is termed anoikis. In contrast, transformed cells often become resistant to anoikis and able to survive and grow in the apical luminal space, leading to the formation of multilayered structures, which can be observed at the early stage of carcinogenesis. However, the underlying molecular mechanisms still remain elusive. In this study, we first demonstrate that S100A10 and ANXA2 (Annexin A2) accumulate in apically extruded, transformed cells in both various cell culture systems and murine epithelial tissues in vivo. ANXA2 acts upstream of S100A10 accumulation. Knockdown of ANXA2 promotes apoptosis of apically extruded RasV12-transformed cells and suppresses the formation of multilayered epithelia. In addition, the intracellular reactive oxygen species (ROS) are elevated in apically extruded RasV12 cells. Treatment with ROS scavenger Trolox reduces the occurrence of apoptosis of apically extruded ANXA2-knockdown RasV12 cells and restores the formation of multilayered epithelia. Furthermore, ROS-mediated p38MAPK activation is observed in apically delaminated RasV12 cells, and ANXA2 knockdown further enhances the p38MAPK activity. Moreover, the p38MAPK inhibitor promotes the formation of multilayered epithelia of ANXA2-knockdown RasV12 cells. These results indicate that accumulated ANXA2 diminishes the ROS-mediated p38MAPK activation in apically extruded transformed cells, thereby blocking the induction of apoptosis. Hence, ANXA2 can be a potential therapeutic target to prevent multilayered, precancerous lesions. [ABSTRACT FROM AUTHOR]

Multicomponent reactions have emerged as an important approach for the synthesis of diverse and complicated chemical compounds. They have various advantages over two-component reactions, including the convenience of one-pot procedures and the ability to modify the structure of agents. Here, we employed in vitro and in silico studies to explore the anticancer potential of novel aminobenzylnaphthols derived from the Betti reaction (MMZ compounds). MTT assay was used to explore the cytotoxic activity of the compounds in pancreatic (BxPC-3 cells) and colorectal (HT-29) cancer cell lines or normal human lung fibroblasts (WI-38 cells). Proapoptotic properties of two derivatives MMZ-45AA and MMZ-140C were explored using AO/EB and annexin V-FITC/PI staining. In silico studies including ADMET profiling, molecular target prediction, docking, and dynamics were employed. The compounds exhibited cytotoxic properties and showed proapoptotic properties in respective IC50 concentrations. As indicated by in silico investigations, anticancer activity of MMZs can be attributed to the inhibition of ADORA1, CDK2, and TRIM24. Furthermore, compounds exhibited favorable ADMET properties. MMZs constitute an interesting scaffold for the potential development of new anticancer agents. [ABSTRACT FROM AUTHOR]

Fungicides are widely used in agriculture for crop protection. Succinate dehydrogenase inhibitors (SDHIs) and strobilurins inhibit mitochondria electron transport chain (ETC) in fungi, by blocking complex II and complex III, respectively. Questions regarding their selectivity of action for fungi have been raised in the literature, and we previously showed that boscalid and bixafen (SDHIs) alter the mitochondrial function of human hepatocytes. Here, we analyzed the impact of the exposure of human hepatocytes to pyraclostrobin, a fungicide belonging to the class of strobilurins. Using electron paramagnetic resonance (EPR), we observed a decrease in oxygen consumption rate (OCR) and an increase in mitochondrial superoxide levels after 24 h exposure to 0.5 µM concentration. As a consequence, the content in ATP amount in the cells was reduced, the ratio reduced/oxidized glutathione was decreased, and a decrease in cell viability was observed using three different assays (PrestoBlue, crystal violet, and annexin V assays). In addition, as SDHIs and strobilurins are commonly associated in commercial preparations, we evaluated a potential "cocktail" toxic effect. We selected low concentrations of boscalid (0.5 µM) and pyraclostrobin (0.25 µM) that did not induce a mitochondrial dysfunction in liver cells when used separately. In sharp contrast, when both compounds were used in combination at the same concentration, we observed a decrease in OCR, an increase in mitochondrial superoxide production, a decrease in the ratio reduced/oxidized glutathione, and a decrease in cell viability in three different assays. [ABSTRACT FROM AUTHOR]

In this study, it was aimed to investigate the effects of melamine exposure since the weaning period on ovarian tissue and ovarian reserve. Melamine is illegally added to milk and formula to provide high false protein positivity. Female rats (the weaning period = 21 days old, n = 18) were divided into 3 groups. 0.1 mL saline was applied to the control group by gavage for 21 days. Fifty mg/kg and seventy-five mg/kg melamine was dissolved in 0.1 mL of saline and applied by gavage for 21 days, respectively. At the end of the experiment, plasma anti-Mullerian hormone (AMH) was measured, follicle count and ovarian diameter measurement were performed in the right ovaries, and flow cytometric analysis for apoptosis was performed in the left ovaries. While a statistically significant decrease was not observed in the number of the follicle and ovarian diameter between the control and melamine-treated groups (p > 0.05), a significant decrease in the corpus luteum and a significant increase in the number of atretic follicles were observed (p < 0.05). Apoptosis (Annexin V) increased in both melamine groups and AMH plasma level decreased significantly in the 75 mg/kg group (p < 0.05). Melamine exposure from the weaning (early postnatal) period may cause a decrease in ovarian reserve in parallel with a dose increase. [ABSTRACT FROM AUTHOR]

Background: Cystic echinococcosis (CE) is a life-threatening zoonotic disease caused by the larval stage of Echinococcus granulosus sensu lato, which employs various strategies to evade the host immune system for survival. Recent advances have revealed the role of annexins as excretory/secretory products, providing new insights into the immune regulation by these proteins in the pathogenesis of CE. Methods: Echinococcus granulosus annexin B proteins EgANXB2, EgANXB18, EgANXB20, and EgANXB23 were cloned, expressed, and analyzed using bioinformatic tools. Membrane binding analysis was used to assess their bioactivity, while their immunoreactivity and tissue distribution characteristics were determined experimentally using western blotting and immunofluorescence staining, respectively. Furthermore, quantitative real-time reverse transcription PCR (qRT-PCR) was used to analyze the mRNA expression profiles of EgANXBs in different developmental stages of E. granulosus. Finally, immunofluorescence staining, cell counting kit 8 assays, flow cytometry, transwell migration assays, and qRT-PCR were used to evaluate the functional effects of rEgANXB18 and rEgANXB20 on mouse peripheral blood mononuclear cells (PBMCs). Results: In this study, we identified four EgANXBs with conserved protein structures and calcium-dependent phospholipid binding activities. rEgANXBs were recognized by serum from sheep infected with E. granulosus and distributed in the germinal layer of fertile cysts. Interestingly, transcription levels of the four EgANXBs were significantly higher in protoscoleces than in 28-day strobilated worms. Moreover, we demonstrated that rEgANXB18 and rEgANXB20 were secretory proteins that could bind to PBMCs and regulate their function. Specifically, rEgANXB18 inhibited cell proliferation and migration while promoting cell apoptosis, NO production, and cytokine profile shifting. In contrast, rEgANXB20 showed limited effects on apoptosis but inhibited NO production. Conclusions: Our findings suggested that among the four identified EgANXBs, EgANXB2 and EgANXB23 might play a pivotal role for the development of protoscoleces, while EgANXB18 and EgANXB20, as secretory proteins, appeared to participate in the host-parasite interaction by regulating the function of immune cells. [ABSTRACT FROM AUTHOR]

Background and objectives: The relationship between the tumor microenvironment and the network of key signaling pathways in cancer plays a key role in the occurrence and development of tumors. Tumor-associated macrophages (TAMs) are important inflammatory cells in the tumor microenvironment and play an important role in tumorigenesis and progression. Macrophages in malignant tumors, mainly the M2 subtype, promote tumor progression by producing cytokines and down-regulating antiinflammatory immune responses. Several articles have investigated the effect of macrophages on the sensitivity of cancer chemotherapeutic agents, but few such articles have been reported in cholangiocarcinoma, so we investigated the effect of M2 macrophage on the sensitivity of cholangiocarcinoma cells to Lenvatinib compared to M1. Methods: THP-1 monocytes were polarized to M0 macrophage by phorbol 12-myristate 13-acetate (PMA) and then induced to differentiate into M1 and M2 macrophages by LPS, IFN-γ and IL-4 and IL-13, respectively. Macrophages and cholangiocarcinoma cells were co-cultured prior to 24 hours of Lenvatinib administration, cancer cell apoptosis was detected by western-blot, FACS analysis of Annexin V and PI staining. Furthermore, we use xCELLigence RTCA SP Instrument (ACEA Bio-sciences) to monitor cell viability of Lenvatinib administration in co-culture of cholangiocarcinoma cells and macrophages. After tumorigenesis in immunodeficient mice, Lenvatinib was administered, and the effects of M2 on biological characteristics of cholangiocarcinoma cells were investigated by immuno-histochemistry. Results: mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1 derived macrophages, which provided a successful and efficient model of monocyte polarization to TAMs. Lenvatinib-induced apoptosis of cholangiocarcinoma cells was significantly reduced when co-cultured with M2 macrophage, whereas apoptosis of cholangiocarcinoma cells co-cultured with M1 macrophage was increased. In the CDX model, Lenvatinib-induced cancer cell apoptosis was markedly reduced, and proliferative cells increased in the presence of M2 macrophages. Angiogenesis related factors was significantly increased in cholangiocarcinoma cells co-cultured with M2. Conclusion: Compared with M1, M2 macrophages can inhibit the anti-tumor effect of Lenvatinib on cholangiocarcinoma through immune regulation, which may be related to the tumor angiogenesis factor effect of M2 macrophage. [ABSTRACT FROM AUTHOR]

Background: Annexin A2 contributes to several key cellular functions and processes, including membrane repair. Effective repair prevents cell death and degeneration, especially in skeletal or cardiac muscle, epithelia, and endothelial cells. To maintain cell integrity after damage, mammalian cells activate multiple membrane repair mechanisms. One protein family that facilitates membrane repair processes are the Ca2+-regulated phospholipid-binding annexins. Body: Annexin A2 facilitates repair in association with S100A10 and related S100 proteins by forming a plug and linking repair to other physiologic functions. Deficiency of annexin A2 enhances cellular degeneration, exacerbating muscular dystrophy and degeneration. Downstream of repair, annexin A2 links the membrane with the cytoskeleton, calcium-dependent endocytosis, exocytosis, cell proliferation, transcription, and apoptosis to extracellular roles, including vascular fibrinolysis, and angiogenesis. These roles regulate cardiovascular disease progression. Finally, annexin A2 protects cancer cells from membrane damage due to immune cells or chemotherapy. Since these functions are regulated by post-translational modifications, they represent a therapeutic target for reducing the negative consequences of annexin A2 expression. Conclusion: Thus, connecting the roles of annexin A2 in repair to its other physiologic functions represents a new translational approach to treating muscular dystrophy and cardiovascular diseases without enhancing its pro-tumorigenic activities. [ABSTRACT FROM AUTHOR]

A series of thirty novel tetracarboxylatoplatinum(IV) complexes in trans-configuration featuring combinations of mixed ammine, methylamine, dimethylamine, and cyclopentylamine ligands as well as acetato/propanoato and trifluoropropanoato ligands was synthesised. The platinum(IV) complexes were characterised by one- and two-dimensional multinuclear NMR spectroscopy (1H, 13C, 15N, 19F, 195Pt), ESI-MS, elemental analysis, and X-ray diffraction. Additional parameters such as reduction behaviour and lipophilicity were measured via NMR spectroscopy and RP-HPLC, revealing slow reduction and a broad spectrum of log kw values in line with the respective ligand combination. In order to determine structure–activity relationships, cytotoxic activity was evaluated via the MTT assay in three human cancer cell lines (CH1/PA-1, ovarian teratocarcinoma, SW480, colon adenocarcinoma, A549, non-small-cell lung carcinoma). The induction of apoptosis and necrosis was determined in SW480 cells via the flow-cytometric annexin V/PI assay. In general, a tendency of higher lipophilicity leading to higher cytotoxicity was noticed. In contrast, lipophilicity alone plays a subordinate role for the induction of apoptosis, which strongly depends on the combination of am(m)ine and trifluoropropanoato ligands. [ABSTRACT FROM AUTHOR]

Imperatorin (IMP) is a secondary metabolite of plants and is the most abundant in Angelica dahurica. Previous studies showed that IMP exhibited anti-inflammatory activity in RAW264.7 cell line. Here, we aim to investigate the roles and mechanisms of IMP in bone marrow-derived macrophages (BMDMs), in view of the difference between primary macrophages and cell lines. BMDMs were stimulated with LPS for the inflammation model. Flow cytometry was performed with BMDMs treated with different doses of IMP (0–20mg/L) within staining Annexin V-APC for 5 min. The cytokines and inflammatory mediators were detected by RT-PCR or ELISA. RNA-seq was performed in IMP-treated BMDMs or control, stimulated with LPS for 6h. Western blotting is carried out to determine the phosphorylation of p65, ERK1/2, JNK1, p38, and Akt. Our results showed that IMP inhibited IL-12p40, IL-6, TNF-α and IL-1β in LPS-stimulated BMDMs. RNA-seq analysis suggested that IMP inhibits Toll-like receptor signaling pathway (KEGG), TNF signaling pathway (KEGG), NF-κB signaling pathway (KEGG), Inflammatory Response (GO). In addition, IMP inhibited myd88, tpl2, cxcl1, ptgs2(COX-2) expression in mRNA level. Finally, we found decreased phosphorylation of NF-κB p65 in IMP-treated BMDMs, after stimulated with LPS. IMP inhibits IL-12p40, IL-6, TNF-α, and IL-1β expression in LPS-stimulated BMDMs. IMP inhibits macrophage activation, which maybe resulted in decreased phosphorylation of NF-κB p65. Furthermore, IMP may protect against the progress of inflammatory-related diseases. [ABSTRACT FROM AUTHOR]

Bothrops venoms are rich in enzymes acting on platelets and coagulation. This action is dependent on two major co-factors, i.e., calcium and phospholipids, while antivenoms variably neutralize venom-related coagulopathy effects. Our aims were (i) to describe the composition of B. atrox and B. lanceolatus venoms; (ii) to study their activity on the whole blood using rotational thromboelastometry (ROTEM); (iii) to evaluate the contribution of calcium and phospholipids in their activity; and (iv) to compare the effectiveness of four antivenoms (Bothrofav™, Inoserp™ South America, Antivipmyn™ TRI, and PoliVal-ICP™) on the procoagulant activity of these two venoms. Venom composition was comparable. Both venoms exhibited hypercoagulant effects. B. lanceolatus venom was completely dependent on calcium but less dependent on phospholipids than B. atrox venom to induce in vitro coagulation. The four antivenoms neutralized the procoagulant activity of the two venoms; however, with quantitative differences. Bothrofav™ was more effective against both venoms than the three other antivenoms. The relatively similar venom-induced effects in vitro were unexpected considering the opposite clinical manifestations resulting from envenomation (i.e., systemic bleeding with B. atrox and thrombosis with B. lanceolatus). In vivo studies are warranted to better understand the pathophysiology of systemic bleeding and thrombosis associated with Bothrops bites. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most lethal brain cancer, causing inevitable deaths of patients owing to frequent relapses of cancer stem cells (CSCs). The significance of the NOTCH signaling pathway in CSCs has been well recognized; however, there is no NOTCH-selective treatment applicable to patients with GBM. We recently reported that Jagged1 (JAG1), a NOTCH ligand, drives a NOTCH receptor-independent signaling pathway via JAG1 intracellular domain (JICD1) as a crucial signal that renders CSC properties. Therefore, mechanisms regulating the JICD1 signaling pathway should be elucidated to further develop a selective therapeutic regimen. Here, we identified annexin A2 (ANXA2) as an essential modulator to stabilize intrinsically disordered JICD1. The binding of ANXA2 to JICD1 prevents the proteasomal degradation of JICD1 by heat shock protein-70/90 and carboxy-terminus of Hsc70 interacting protein E3 ligase. Furthermore, JICD1-driven propagation and tumor aggressiveness were inhibited by ANXA2 knockdown. Taken together, our findings show that ANXA2 maintains the function of the NOTCH receptor-independent JICD1 signaling pathway by stabilizing JICD1, and the targeted suppression of JICD1-driven CSC properties can be achieved by blocking its interaction with ANXA2. [ABSTRACT FROM AUTHOR]

Differentiated thyroid cancer is the most common malignancy of the endocrine system. Although most thyroid nodules are benign, given the high incidence of thyroid nodules in the population, it is important to understand the differences between benign and malignant thyroid cancer and the molecular alterations associated with malignancy to improve detection and signal potential diagnostic, prognostic, and therapeutic targets. Proteomics analysis of benign and malignant human thyroid tissue largely revealed changes indicating modifications in RNA regulation, a common cancer characteristic. In addition, changes in the immune system and cell membrane/endocytic processes were also suggested to be involved. Annexin A1 was considered a potential malignancy biomarker and, similarly to other annexins, it was found to increase in the malignant group. Furthermore, a bioinformatics approach points to the transcription factor Sp1 as being potentially involved in most of the alterations seen in the malignant thyroid nodules. [ABSTRACT FROM AUTHOR]

The purpose of this study was to confirm the antiproliferative and apoptotic induction potential of a saccharin and caffeine combination in ovarian cancer cells. The cell line used was Ovcar-3, and the cell viability was measured through a WST-8 assay, while a Chou–Talalay assay was used to confirm the synergistic effect of saccharin and caffeine on the ovarian cancer cells. A clonogenic assay, annexin V-FITC/PI-PE double-staining, and RT-PCR were performed to confirm the expression of genes that induce colony formation, cell viability, and apoptosis in ovarian cancer cells treated with the saccharin–caffeine combination. It was demonstrated that both saccharin and caffeine decreased the viability of Ovcar-3 cells, and the cell viability decreased even more significantly when the cells were treated with the combination of saccharin and caffeine. The clonogenic assay results showed that the number of colonies decreased the most when saccharin and caffeine were combined, and the number of colonies also significantly decreased compared to the single-treatment groups. Based on flow cytometry analysis using annexin V-FITC/PI-PE double-staining, it was confirmed that the decrease in cell viability caused by the combination of saccharin and caffeine was correlated with the induction of apoptosis. The results of the RT-PCR confirmed that the combined treatment of saccharin and caffeine promoted cell apoptosis by regulating the expression of apoptosis-inducing genes. These results demonstrate that the combination of saccharin and caffeine more efficiently inhibits the proliferation of Ovcar-3 cells and induces apoptosis in vitro. [ABSTRACT FROM AUTHOR]

Background: We aimed to assess the in vitro effects of the green synthesized silver nanoparticles (Ag NPs) via Thymus vulgaris (thyme) against Leishmania major infection. Methods: We have prepared T. vulgalis silver nanoparticles (TSNPs) by adding thyme extract to the silver nitrate aqueous solution (0.2 mM), and evaluated their antileishmanial activity. The viability of L. major promastigotes was assessed in the presence of various concentrations of TSNPs by direct counting after 24 h. The MTT assay was used to identify the viability of promastigotes. The same procedures were assessed in uninfected macrophage cells. The apoptotic effects of nanoparticles on L. major promastigotes were determined by flow cytometry assay using annexin staining. To evaluate anti-amastigotes activity of TSNPs, light microscopic observation was used to determine the number of parasites within the macrophages in each well. Results: The effect of TSNPs on promastigotes and amastigotes of L. major was effective and had a reverse relationship with its concentration. TSNPs, inhibited the growth rate of L. major amastigotes and, the IC50 value of these nanoparticles was estimated 3.02 µg/mL (28 µM) after 72h. The results of flow cytometry showed that the toxic effects of TSNPs on promastigotes after 24 hours were statistically significant (P<0.05) and showed 69.51% of apoptosis. Conclusion: TSNPs had an inhibitor effect on promastigote and amastigote forms of L. major in vitro. It might be considered as a candidate for the treatment of this infection. [ABSTRACT FROM AUTHOR]

Purpose: Although gemcitabine-based chemotherapy is most recommended for pancreatic ductal adenocarcinoma (PDAC), its effectiveness is limited because of drug resistance. Given thalidomide's anti-tumor effects in solid tumors, we investigated the effect of avadomide, a novel thalidomide analog, on PDAC and explored its anti-tumor mechanisms. Methods: PDAC cell lines, including gemcitabine-resistant (GR) clones derived from MiaPaCa2 cells, were used to evaluate the effects of avadomide. An annexin V assay, a cell cycle assay, and western blot analysis were performed to explain the mechanism of avadomide as an anti-tumor reagent. Moreover, we investigated the anti-tumor effect on tumor growth using a subcutaneous xenograft murine model. Results: Avadomide showed anti-tumor effects in human PDAC cell lines. The proportion of apoptotic cells and G0/G1 phase cells after avadomide treatment increased, especially in the GR PDAC clones. Western blot analysis also showed the induction of the apoptotic pathway by inhibiting the NF-κB process and G1 phase cell cycle arrest. The xenograft murine model revealed that the proportion of viable cells in the avadomide-treated group was lower than that in the untreated group. Conclusion: Our findings suggest that avadomide could be a novel therapeutic option to overcome gemcitabine resistance in patients with PDAC. [ABSTRACT FROM AUTHOR]

Simple Summary: Seminal plasma is a complex secretion that is produced in various organs of the male reproductive system. It is rich in proteins that play an essential role in the proper functioning of spermatozoa. Proteins found in seminal plasma are involved in processes such as sperm capacitation, the acrosome reaction, and providing antioxidant and immunological protection to spermatozoa. Differences in seminal plasma protein abundance may be linked to changes in the reproductive parameters of bulls. To investigate this possible association, we analyzed the proteomic profile of the seminal plasma of young Nellore bulls and its relationship with the results of the Breeding Soundness Evaluation (BSE). This study aimed to establish a link between the differential protein profile and the phenotypic characteristics of the semen of young bulls approved and not approved for reproduction. This study aimed to evaluate the proteomic profile of seminal plasma from young Nellore bulls. We used 20 bulls aged between 19.8 and 22.7 months, divided into two groups according to the results of the Breeding Soundness Evaluation (BSE): approved (FIT n = 10) and not approved (UNFIT n = 10). The scrotal perimeter was measured and a semen collection was performed through electroejaculation. The percentage of sperm motility, mass motility, and sperm vigor were calculated using conventional microscopy, and the percentage of sperm abnormalities was calculated using phase-contrast microscopy of all ejaculates. Seminal plasma was separated from spermatozoa using centrifugation and processed for proteomic analysis by LC-MS/MS. Seminal plasma proteins were identified using MASCOT Daemon software v.2.4.0 and label-free quantification analysis was carried out by SCAFFOLD Q+ software v.4.0 using the Exponentially Modified Protein Abundance Index (emPAI) method. Functional classification of proteins was performed based on their genetic ontology terms using KOG. Functional cluster analysis was performed on DAVID. There were no differences in scrotal perimeter and physical semen characteristics between FIT and UNFIT groups of bulls. The percentage of sperm abnormalities was higher (p < 0.05) in the UNFIT group of bulls. A total of 297 proteins were identified for the two groups. There were a total of 11 differentially abundant proteins (p < 0.05), two of them more abundant in FIT bulls (Spermadhesin-1 and Ig gamma-1 chain C region) and nine in UNFIT bulls (Vasoactive intestinal peptide, Metalloproteinase inhibitor 2, Ig lambda-1 chain C regions, Protein FAM3C, Hemoglobin beta, Seminal ribonuclease, Spermadhesin 2, Seminal plasma protein BSP-30kDa, and Spermadhesin Z13). Spermadhesin-1 was the protein with the highest relative abundance (36.7%) in the seminal plasma among all bulls, corresponding to 47.7% for the FIT bulls and 25,7% for the UNFIT bulls. Posttranslational modification, protein turnover, and chaperones were the functional categories with the highest number of classified proteins. Protein functional annotation clusters were related to Phospholipid efflux, ATP binding, and chaperonin-containing T-complex. The differentially abundant proteins in the group of FIT bulls were related to sperm capacitation and protection against reactive species of oxygen. In contrast, differentially expressed proteins in the group of UNFIT bulls were related to motility inhibition, intramembrane cholesterol removal and oxidative stress. In conclusion, the proteomic profile of the seminal plasma of FIT bulls presents proteins with participation in several biological processes favorable to fertilization, while the proteins of the seminal plasma of UNFIT bulls indicate a series of alterations that can compromise the fertilizing capacity of the spermatozoa. In addition, the relative abundance of spermadhesin-1 found in the seminal plasma of young Nellore bulls could be studied as a reproductive parameter for selection. [ABSTRACT FROM AUTHOR]

Polysaccharide extracts exhibit promise as potential anticancer agents. Among the fungi rich in polysaccharide content, G. applanatum stands out; however, its anticancer activity necessitates further investigation. This study aims to explore the impact of G. applanatum crude polysaccharide (GACP) extract by assessing its effects on cell viability, levels of proinflammatory cytokines such as TNF-α, IFN-γ, IL-2, and IL-12, and levels of proapoptotic markers including caspase-3 and caspase-9, as well as the percentages of necrosis and apoptosis in the HeLa cell line. Employing the HeLa cell line as a research model, four groups were studied: KN (media and DMSO), K+ (doxorubicin 10 μg/mL), P1 (G. applanatum extract 200 μg/mL), and P2 (G. applanatum extract 400 μg/mL). The G. applanatum extract was obtained via boiling distilled water. Anticancer activity was evaluated through the MTT test (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) conducted over three treatment durations (24, 48, and 72 hours). Cytokine levels and caspase-3 and caspase-9 levels were assessed using the ELISA test. Cell apoptosis was determined using the Annexin V-PI biomarker and analyzed through flow cytometry. The MTT test exhibited optimal results at the 48-hour treatment mark. Cytokine level analysis revealed significant reductions in TNF-α, IFN-γ, IL-2, and IL-12 levels (p < 0.005). Concurrently, caspase-3 and caspase-9 levels exhibited substantial increases (p < 0.005). Flow cytometry highlighted the highest percentage of apoptosis in HeLa cells. In conclusion, G. applanatum's polysaccharide extract demonstrates potential as an anticancer and therapeutic agent for cancer treatment. [ABSTRACT FROM AUTHOR]

Parasitic nematodes and their products are promising candidates for therapeutics against inflammatory bowel diseases (IBD). Two species of nematodes, the hookworm Necator americanus and the whipworm Trichuis suis, are being used in clinical treatment trials of IBD referred to as "helminth therapy". Heligmosomoides polygyrus is a well-known model for human hookworm infections. Excretory–secretory (ES) products of H. polygyrus L4 stage that developed during colitis show a different immunomodulatory effect compared to the ES of H. polgyrus from healthy mice. The aim of the study was to evaluate excretory–secretory proteins produced by H. polygyrus L4 stage males and females that developed in the colitic milieu. Mass spectrometry was used to identify proteins. Blast2GO was used to investigate the functions of the discovered proteins. A total of 387 proteins were identified in the ES of H. polygyrus L4 males (HpC males), and 330 proteins were identified in the ES of L4 females that developed in the colitic milieu (HpC females). In contrast, only 200 proteins were identified in the ES of L4 males (Hp males) and 218 in the ES of L4 females (Hp females) that developed in control conditions. Most of the proteins (123) were detected in all groups. Unique proteins identified in the ES of HpC females included annexin, lysozyme-2, apyrase, and galectin. Venom allergen/Ancylostoma-secreted protein-like, transthyretin-like family proteins, and galectins were found in the secretome of HpC males but not in the secretome of control males. These molecules may be responsible for the therapeutic effects of nematodes in DSS-induced colitis. [ABSTRACT FROM AUTHOR]

Background: To investigate the effects of osimertinib on hepatocellular carcinoma (HCC) and angiogenesis, and its combinatory effects with venetoclax in HCC. Methods: Viability was assessed by flow cytometry of Annexin V in multiple HCC cell lines after drug treatment. In vitro angiogenesis assay was performed using primary human liver tumor associated endothelial cell (HLTEC). HCC-bearing model was generated by subcutaneous implantation of Hep3B cells to investigate the efficacy of osimertinib alone and its combination with venetoclax. Results: Osimertinib significantly induced apoptosis in a panel of HCC cell lines regardless of EGFR expression level. It inhibited capillary network formation and induced apoptosis in HLTEC. Using HCC xenograft mouse model, we further showed that osimertinib at non-toxic dose inhibited tumor growth by ~ 50% and remarkably decreased blood vessel in tumor. Mechanism studies demonstrated that osimertinib acted on HCC cells in an EGFR-independent manner. It decreased level of VEGF and Mcl-1 in HCC cells via suppressed phosphorylation of eIF4E, thus leading to inhibition of eIF4E-mediated translation. Mcl-1 overexpression reversed pro-apoptotic effect of osimertinib, suggesting an important role of Mcl-1 in osimertinib's action in HCC cells. Of note, the combination of osimertinib and venetoclax achieved approximately complete HCC cell death and tumor growth in mice. Conclusions: We provide pre-clinical evidence that osimertinib is a promising candidate for the treatment of HCC via targeting tumor cells and angiogenesis. The combination of osimertinib and venetoclax is synergistic in inhibiting HCC. [ABSTRACT FROM AUTHOR]

We report the synthesis of new set of organometallic dinuclear ruthenium(II) arene complexes along with studies on their in vitro antiproliferative activity and cancer cell death mechanism. Specifically, dinuclear ruthenium complexes of the type [(η6-p-cymene)2Ru2(L)Cl2] (1–3) (L = benzil bis(furoylhydrazone derivatives)) were synthesized. The formation of the complexes was well examined by elemental analysis and spectroscopic studies, including FT-IR, UV-vis, NMR, and HR-MS. The existence of a piano-stool structure was evinced by single-crystal X-ray crystallographic technique and confirmed the coordination of furoylhydrazone to each ruthenium metal centre via a hydrazone nitrogen and imidolate oxygen. Further, the cancerous cell growth resistance property of all the complexes against human cancer cell lines, such as A549 (lung carcinoma) and HeLa (cervical cancer), was assessed by MTT assay along with the standard drug cisplatin. It was noted that complex 2 exhibited a two-fold higher activity in inhibition of all the cancer cells than cisplatin. This may be attributed to the existence of an electron-releasing methyl substituent of the ligand, which subsequently increased hydrophobicity. Further, fluorescent staining methods, including dual AO-EB and DAPI, confirmed apoptosis occurred through morphological changes. In addition, quantitative discrimination of late apoptosis by the externalization of phosphatidylserine was confirmed by flow cytometry using annexin V-FITC/propidium iodide (PI) double-staining. [ABSTRACT FROM AUTHOR]

Oncogenic activation of receptor tyrosine kinases (RTKs) such as MET is associated with cancer initiation and progression. We designed and synthesized a new series of quinazoline derivatives bearing 1,2,3-triazole moiety as targeted anticancer agents. The MET inhibitory effect of synthesized compounds was assessed by homogeneous time-resolved fluorescence (HTRF) assay and western blot analysis. Sulforhodamine B assay was conducted to examine the antiproliferative effects of synthetic compounds against 6 cancer cell lines from different origins including MET-dependent AsPC-1, EBC-1 and MKN-45 cells and also Mia-Paca-2, HT-29 and K562 cells. The growth inhibitory effect of compounds in a three-dimensional spheroid culture was examined by acid phosphatase (APH) assay, while apoptosis induction was evaluated by Annexin V/propidium iodide method. Compound 8c bearing p-methyl benzyl moiety on the triazole ring exhibited the highest MET inhibitory capacity among tested agents that was further confirmed by western blot findings. Derivatives 8c and 8h exhibited considerable antiproliferative effects against all tested cell lines, with more inhibitory effects against MET-positive cells with IC50 values as low as 6.1 μM. These two agents also significantly suppressed cell growth in spheroid cultures and induced apoptosis in MET overexpressing AsPC-1 cells. Moreover, among a panel of 24 major oncogenic kinases, the PDGFRA kinase was identified as a target of 8c and 8h compounds. The docking study results of compounds 8c and 8h were in agreement with experimental findings. The results of the present study suggest that quinazoline derivatives bearing 1,2,3-triazole moiety may represent promising targeted anticancer agents. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the mechanism and effect of Procyanidin A1 (PCA1) in gestational diabetes mellitus (GDM). Methods: Human trophoblast cell line HTR-8/Svneo was treated with 25 mM glucose for 24 h, and the effect of PCA1 on HTR-8/SVneo cell viability and proliferation was determined by CCK-8 assay and EdU proliferation kit. The expression of BAX, Bcl-2, cleaved Caspase-3 and cleaved caspase-9 was determined by western blotting. Cell apoptosis was assessed by Annexin V-FITC/PI staining. Cellular generation of reactive oxygen species (ROS) was determined by ROS assay kit while superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) were determined by the corresponding assay kits. The effect of PCA1 on Nrf2/HO-1 pathway was evaluated by determining the expression of Keap1, Nfr2, and HO-1. Results: Procyanidin A1 (PCA1) increased the viability of high glucose (HG) -induced HTR-8/SVneo cells and promoted cell proliferation. Furthermore, PCA1 significantly inhibited HG-induced BAX, cleaved caspase-3, and cleaved caspase-9 expression, resulting in further reduction in HG-induced cell apoptosis. High glucose (HG) induced a significant increase in intracellular ROS levels, and this HGinduced oxidative stress was inhibited by PCA1. Furthermore, PCA1 activated Nrf2/HO-1 pathway and this was responsible for its proliferative, anti-apoptosis and anti-oxidative effects. Conclusion: Procyanidin A1 promotes proliferation, and inhibits apoptosis and oxidative stress induced by high glucose in trophoblast cells by activating Nrf2/HO-1 signaling pathway. Therefore, it is a potential drug for the treatment of GDM. [ABSTRACT FROM AUTHOR]