Determination of the levels and possible associations of alpha2-macroglobulin with autoantibodies in the serum of patients with various forms of autoimmune thyroiditis.

Antibodies to thyroid peroxidase (AB-TPO), antibodies to thyroglobulin (AB-TG), and the content of α2-macroglobulin (α2-MG) have been studied in serum samples of patients with autoimmune thyroiditis (AIT). All the patients were divided into 3 groups depending on age: 25-35, 36-50, 51-65 years. We found a significant change in the thyroid panel parameters in AIT, but without significant changes in the average concentration of α2-MG in the age groups of patients. This may be due to the accumulation and retention of complexes of defective forms of α2-MG in the circulation associated with their decreased ability to bind to receptors.

Alpha-2-Macroglobulin Is a Novel Anticancer Agent.

INTRODUCTION: Melanoma is the most aggressive skin cancer, with an increasing occurrence. Despite the recent important improvements due to novel immunotherapy approaches, when late diagnosed, melanoma prognosis is poor due to the metastatic progression and drug-resistance onset. Therefore, there is an urgent need to identify additional therapeutic targets. Melanoma invasive behavior is related to the activity of metalloproteases, able to degrade extracellular matrix leading to tumor dissemination. A recent study suggested that the most potent proteases inhibitor alpha-2-macroglobulin (A2MG) from plasma of hibernating fishes exerts potent antiproliferative effects. Our previous studies showed a significant reduction of A2MG in sera from mice/human melanoma models. METHODS: Gene and protein expression studies have been performed by using platforms and databases available online containing expression data from thousands of patients and healthy controls. RESULTS: We carried out an extensive bioinformatics analysis to evaluate the A2MG gene/protein expression on a large cohort of patients affected by many different cancer types, compared to healthy control subjects, and we found a highly significant difference of A2MG expression in 20 out of 31 cancer types (including melanoma) compared to healthy controls. Similar results were also confirmed at the proteomic level using another platform available online. Further, we found that higher A2MG expression is significantly related to overall survival in different cancers including melanoma. CONCLUSION: Our results strongly suggest A2MG as a novel molecular target in melanoma therapy, as well as in other cancer types.

Effect of Short PSG Peptides on Inflammatory Markers in Allogeneic Bone Marrow Cell Transplantation in Wistar Rats.

Short linear peptide fragments of placental trophoblastic ß1-glycoprotein (PSG) (YECE, YQCE, YVCS, and YACS) were studied in the context of their immunomodulatory effects at the level of inflammatory markers. The original host-versus-graft model was used in male Wistar rats without prior conditioning of recipient bone marrow. A composition of PSG peptide fragments was injected to animals after allogeneic transplantation of bone marrow cells in a dynamic experiment, inflammatory markers α1-acid glycoprotein (AGP, orosomucoid), α2-macroglobulin (α2M) were assayed by ELISA, and biochemical parameters (total protein, glucose, creatinine, and urea) were measured. The levels of α2M and AGP increased in response to allotransplantation, whereas administration of PSG peptides normalized serum α2M levels by the end of the experiment. The decrease in α2M level coincided with the independent effect of PSG peptide administration. The levels of total protein, glucose, creatinine, and urea in rat serum after allotransplantation were reduced throughout the experiment. Administration of PSG peptides contributed to normalization of serum total protein, creatinine, and urea levels by the end of the experiment. Administration of PSG peptides after allogeneic transplantation of bone marrow suspension contributed to normalization of the levels of α2M, total protein, creatinine, and urea, which can be interpreted as an anti-inflammatory effect of these peptides.

Mesenchymal Stem Cell-Originated Exosomal Lnc A2M-AS1 Alleviates Hypoxia/Reperfusion-Induced Apoptosis and Oxidative Stress in Cardiomyocytes.

BACKGROUND: Mesenchymal stem cell (MSC)-derived exosomes play significant roles in ameliorating cardiac damage after myocardial ischemia-reperfusion (I/R) injury. Long non-coding RNA alpha-2-macroglobulin antisense RNA 1 (Lnc A2M-AS1) was found that might protect against myocardial I/R. However, whether Lnc A2M-AS1 delivery via MSC-derived exosomes could also regulate myocardial I/R injury remains unknown. METHODS: Exosomes were isolated by ultracentrifugation, and qualified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Hypoxia/reoxygenation (H/R) treatment in human cardiomyocytes was used to mimic the process of myocardial I/R in vitro. The viability and apoptosis of cardiomyocytes were detected using cell counting kit-8, flow cytometry, and Western blot assays. The contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were evaluated using corresponding commercial kits. The quantitative real-time polymerase chain reaction and Western blot were used to determine the expression levels of Lnc A2M-AS1, microRNA (miR)-556-5p, and X-linked inhibitor of apoptosis protein (XIAP). The binding interaction between miR-556-5p and Lnc A2M-AS1 or XIAP was confirmed by the dual-luciferase reporter, RIP and pull-down assays. RESULTS: Exosomes isolated from hMSCs (hMSCs-exo) attenuated H/R-induced apoptosis and oxidative stress in cardiomyocytes. Lnc A2M-AS1 was lowly expressed in AMI patients and H/R-induced cardiomyocytes. Besides, Lnc A2M-AS1 was detectable in hMSCs-exo, exosomes derived from Lnc A2M-AS1-transfected hMSCs weakened H/R-induced apoptosis and oxidative stress, and enhanced the protective action of hMSCs-exo on H/R-induced cardiomyocytes. Further mechanism analysis showed that Lnc A2M-AS1 acted as a sponge for miR-556-5p to increase XIAP expression level. Importantly, miR-556-5p overexpression or XIAP knockdown reversed the action of exosomal Lnc A2M-AS1 on H/R-induced cardiomyocytes. CONCLUSION: Lnc A2M-AS1 delivery via MSC-derived exosomes ameliorated H/R-induced cardiomyocyte apoptosis and oxidative stress via regulating miR-556-5p/XIAP, opening a new window into the pathogenesis of myocardial I/R injury.

Overexpression of lncRNA A2M-AS1 inhibits cell growth and aggressiveness via regulating the miR-587/bone morphogenetic protein 3 axis in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a malignant tumor that occurs in the lungs. Numerous reports have substantiated the participation of long non-coding RNAs (lncRNAs) in the tumorigenesis of LUAD. Previously, lncRNA alpha-2-macroglobulin antisense RNA 1 (A2M-AS1) was confirmed to be an important regulator in the biological processes of LUAD and dysregulation of A2M-AS1 was associated with non-small cell lung cancer (NSCLC) progression. However, the precise mechanism of A2M-AS1 in LUAD has not been elucidated. Therefore, our study was designed to investigate the detailed molecular mechanism of A2M-AS1 in LUAD. Herein, the expression of lncRNA A2M-AS1, microRNA (miRNA) miR-587, and bone morphogenetic protein 3 (BMP3) in LUAD cell lines and tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. The viability, proliferation, migration and invasion of LUAD cells were tested by cell counting kit-8 (CCK-8), colony formation and Transwell assays. In vivo tumor growth was investigated by xenograft animal experiment. Interactions among A2M-AS1, miR-587 and BMP3 were measured by RNA pulldown and luciferase reporter assays. In this study, A2M-AS1 was downregulated in LUAD tissues and cells and related to poor prognosis in LUAD patients. A2M-AS1 overexpression suppressed LUAD cell proliferation, migration and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, A2M-AS1 directly bound with miR-587 to promote BMP3 expression in LUAD cells. Low expression of BMP3 was found in LUAD tissues and cells and was closely correlated with poor prognosis in LUAD patients. BMP3 deficiency reserved the inhibitory influence of A2M-AS1 overexpression on LUAD cell behaviors. Overall, A2M-AS1 inhibits cell growth and aggressiveness via regulating the miR-587/BMP3 axis in LUAD.

LncRNA A2M-AS1 Promotes Ferroptosis in Pancreatic Cancer via Interacting With PCBP3.

Ferroptosis is a newly-discovered cell death mechanism involved in the progression of various tumors, the role of noncoding RNAs (ncRNAs) in it was relatively less explored. This study identified the low levels of a recently studied long noncoding RNA (lncRNA), A2M-AS1, in pancreatic cancer and suggested its positive correlation with the overall survival time of patients with pancreatic cancer. A2M-AS1 was mainly localized in the cytoplasm, inhibiting the cellular proliferation, migration, and invasion as well as the tumor growth of the pancreatic cancer cells. Moreover, the Erastin-induced ferroptosis increased the expression levels of A2M-AS1. The overexpression of A2M-AS1 promoted ferroptosis in the pancreatic cancer, which was inhibited by the silencing of A2M-AS1. Mechanically, A2M-AS1 could directly interact with the poly (rC) binding protein 3 (PCBP3), which plays an important role in the process of iron metabolism, thereby promoting the ferroptosis in pancreatic cancer. In addition, the A2M-AS1/PCBP3 axis could facilitate the p38 activation and inhibit the phosphorylation of the AKT-mTOR signaling pathway; all these participate in regulating ferroptosis. In conclusion, the regulation of ferroptosis by targeting the A2M-AS1/PCBP3 axis might provide a novel target for the treatment of pancreatic cancer in the future. IMPLICATIONS: A2M-AS1 might be a potential novel therapeutic target for patients with pancreatic cancer in the future.

Indomethacin Disrupts the Formation of ß-Amyloid Plaques via an α2-Macroglobulin-Activating lrp1-Dependent Mechanism.

Epidemiological studies have implied that the nonsteroidal anti-inflammatory drug (NSAID) indomethacin slows the development and progression of Alzheimer's disease (AD). However, the underlying mechanisms are notably understudied. Using a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9) (APP/PS1) expressing transgenic (Tg) mice and neuroblastoma (N) 2a cells as in vivo and in vitro models, we revealed the mechanisms of indomethacin in ameliorating the cognitive decline of AD. By screening AD-associated genes, we observed that a marked increase in the expression of α2-macroglobulin (A2M) was markedly induced after treatment with indomethacin. Mechanistically, upregulation of A2M was caused by the inhibition of cyclooxygenase-2 (COX-2) and lipocalin-type prostaglandin D synthase (L-PGDS), which are responsible for the synthesis of prostaglandin (PG)H2 and PGD2, respectively. The reduction in PGD2 levels induced by indomethacin alleviated the suppression of A2M expression through a PGD2 receptor 2 (CRTH2)-dependent mechanism. Highly activated A2M not only disrupted the production and aggregation of ß-amyloid protein (Aß) but also induced Aß efflux from the brain. More interestingly, indomethacin decreased the degradation of the A2M receptor, low-density lipoprotein receptor-related protein 1 (LRP1), which facilitated the brain efflux of Aß. Through the aforementioned mechanisms, indomethacin ameliorated cognitive decline in APP/PS1 Tg mice by decreasing Aß production and clearing Aß from the brains of AD mice.

Effects of ethanolic extracts of Quercus, Cirsium vulgare, and Falcaria vulgaris on gastric ulcer, antioxidant and inflammatory indices, and gene expression in rats.

INTRODUCTION: Gastric ulcer is a multifaceted process and is usually caused by mucosal damage. Herbal medicines have received much attention considering the side effects of chemical drugs. Nowadays, the use of herbal medicines has received much attention considering the side effects of chemical drugs. Quercus brantii Lindl, Cirsium vulgare (Savi) Ten, and Falcaria vulgaris Bernh are plants used as traditional phytomedicine for gastric ulcer diseases. AIM OF THE STUDY: This study was aimed to investigate the protective effects of hydroalcoholic extracts of these herbs on ethanol-induced gastric ulceration, in addition, to investigate the antioxidant, anti-inflammatory, and gene expression. MATERIALS AND METHODS: Thirty Sprague Dawley rats, (200-250 g), were divided into six groups: Control: intact animals; sham: gavaged with distilled water (14 days); negative control: gavaged with 20 mg/kg of omeprazole (14 days); experimental groups I, II, and III: gavaged with 500 mg/kg of the extract of Falcaria vulgaris, Quercus brantii, and Cirsium vulgare, respectively, (14 days). The number of ulcers and pathological parameters were assessed. The serum superoxide dismutase, catalase, glutathione peroxidase, malondialdehyde, total antioxidant capacity, albumin, total protein, haptoglobin, alpha-1-acid glycoprotein, total globulin, alpha-2-macroglobulin, C-fos, C-myc, and Caspase-9 were measured by ELISA and RT-PCR. RESULTS: The extracts significantly reduced gastric ulcer (52.33%). The results showed that the Quercus brantii extract was more effective. There were significant differences between the serum levels of alpha-1-acid glycoprotein and those of alpha-2-macroglobulin. Also, there was a significant difference in the serum level of antioxidant parameters. Changes in the expression of the genes also confirmed the results suggested by other parameters. The expression levels of C-fos, C-myc, and caspase-9 were decreased, but the Bcl-2 expression increased. CONCLUSION: The hydro-alcoholic extracts revealed various protection and noticeable change in the expression of caspase-9, C-myc, C-fos, and Bcl-2 genes in rats.

α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin-like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity-conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

GALNT6 promotes breast cancer metastasis by increasing mucin-type O-glycosylation of α2M.

Breast cancer is the most lethal malignancy in women. N-acetylgalactosaminyltransferase 6 (GALNT6) is an enzyme which mediates the initial step of mucin-type O-glycosylation, and has been reported to be involved in mammary carcinogenesis. However, the molecular mechanism of GALNT6 in breast cancer metastasis has not been fully explored. In this study, based on online database analyses and tissue microarrays, the overall survival (OS) of breast cancer patients with high expression of GALNT6 was found to be shorter than those with low expression of GALNT6. Also, high GALNT6 expression was positively correlated with advanced pN stage and pTNM stage. GALNT6 was shown to be able to promote the migration and invasion of breast cancer cells, and enhance the level of mucin-type O-glycosylation of substrates in the supernatants of breast cancer cells. Qualitative mucin-type glycosylomics analysis identified α2M as a novel substrate of GALNT6. Further investigation showed that GALNT6 increased O-glycosylation of α2M, and the following activation of the downstream PI3K/Akt signaling pathway was involved in the promotion of migration and invasion of breast cancer cells. This study identified a new substrate of GALNT6 and provides novel understanding of the role of GALNT6 in promoting metastasis and poor prognosis in breast cancer.

Activated α2-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion.

The villous cytotrophoblastic cells have the ability to fuse and differentiate, forming the syncytiotrophoblast (STB). The syncytialisation process is essential for placentation. Nevertheless, the mechanisms involved in cell fusion and differentiation are yet to be fully elucidated. It has been suggested that cell surface glucose-regulated protein 78 (GRP78) was involved in this process. In multiple cancer cells, cell membrane-located GRP78 has been reported to act as a receptor binding to the active form of α2-macroglobulin (α2M*), activating thus several cellular signalling pathways implicated in cell growth and survival. We hypothesised that GRP78 interaction with α2M* may also activate signalling pathways in trophoblastic cells, which, in turn, may promote cell fusion. Here, we observed that α2M mRNA is highly expressed in trophoblastic cells, whereas it is not expressed in the choriocarcinoma cell line BeWo. We thus took advantage of forskolin-induced syncytialisation of BeWo cells to study the effect of exogenous α2M* on syncytialisation. We first demonstrated that α2M* induced trophoblastic cell fusion. This effect is dependent on α2M*-GRP78 interaction, ERK1/2 and CREB phosphorylation, and unfolded protein response (UPR) activation. Overall, these data provide novel insights into the signalling molecules and mechanisms regulating trophoblastic cell fusion.

The Role of Alpha 2 Macroglobulin in IgG-Aggregation and Chronic Activation of the Complement System in Patients With Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the western world. One of the treatments offered for CLL is immunotherapy. These treatments activate various cellular and biochemical mechanisms, using the complement system. Recently it was shown that the complement system in CLL patients is persistently activated at a low level through the classical pathway (CP). The mechanism of chronic CP activation involves the formation of IgG-hexamers (IgG-aggregates). According to recent studies, formation of ordered IgG-hexamers occurs on cell surfaces via specific interactions between Fc regions of the IgG monomers, which occur after antigen binding. The present study investigated the formation of IgG-hexamers in CLL patients and normal (non-malignant) controls (NC), their ability to activate complement, their incidence as cell-free and cell-bound forms and the identity of the antigen causing their formation. Sera from 30 patients and 12 NC were used for separation of IgG- aggregates. The obtained IgG- aggregates were measured and used for assessment of CP activation. For evaluation of the presence of IgG- aggregates on blood cells, whole blood samples were stained and assessed by flow cytometry. Serum levels of IgG- aggregates were higher in CLL and they activated the complement system to a higher extent than in NC. Alpha 2 macroglobulin (A2M) was identified as the antigen causing the hexamerization/aggregation of IgG, and was found to be part of the hexamer structure by mass spectrometry, Western blot and flow cytometry analysis. The presence of A2M-IgG-hexamers on B-cells suggests that it may be formed on B cells surface and then be detached to become cell-free. Alternatively, it may form in the plasma and then attach to the cell surface. The exact time course of A2M-IgG-hexamers formation in CLL should be further studied. The results in this study may be useful for improvement of current immunotherapy regimens.

Relationship of α2-Macroglobulin with Steroid-Induced Femoral Head Necrosis: A Chinese Population-Based Association Study in Southeast China.

OBJECTIVE: The present study aimed to identify the relationship of α-2-macroglobulin and microvascular vessel pathology with steroid-induced femoral head necrosis in the Southeast Chinese population. METHODS: This study enrolled 40 patients diagnosed with steroid-induced necrosis of the femoral head. Patients had various stages of femoral head necrosis. The differential expression of serum proteins and mRNA from patients with steroid-induced necrosis of the femoral head (SINFH) and healthy volunteers was analyzed by western blot and quantitative polymerase chain reaction (QT-PCR). The pathological change in osteocyte necrosis was indicated by hematoxylin and eosin stain and immunohistochemistry. RESULTS: Hematoxylin and eosin stain showed histopathology changes in the necrotic area of patients with steroid-induced INFH: bone trabeculae were fewer and thinner, became broken, fragmented and structurally disordered; intraosseous adipose cells became enlarged; the arrangement of the osteoblasts became irregular; and vacant bone lacunae increased. QT-PCR showed significantly lower levels of α-2-macroglobulin in the serum of patients with SINFH than in controls (P < 0.05). Immunohistochemical staining and western blotting demonstrated that the expression of α-2-macroglobulin was significantly decreased in the necrotic area of SINFH patients (P < 0.05). CONCLUSION: The α-2-macroglobulin may be associated with the pathology of SINFH. The multiple pathological reactions occur in SINFH and α-2-macroglobulin may serve as a potential biomarker for the diagnosis of SINFH or a promising therapeutic target.

Recombinant production of human α2-macroglobulin variants and interaction studies with recombinant G-related α2-macroglobulin binding protein and latent transforming growth factor-ß2.

α2-Macroglobulins (α2Ms) regulate peptidases, hormones and cytokines. Mediated by peptidase cleavage, they transit between native, intact forms and activated, induced forms. α2Ms have been studied over decades using authentic material from primary sources, which was limited by sample heterogeneity and contaminants. Here, we developed high-yield expression systems based on transient transfection in Drosophila Schneider 2 and human Expi293F cells, which produced pure human α2M (hα2M) at ~1.0 and ~0.4 mg per liter of cell culture, respectively. In both cases, hα2M was mainly found in the induced form. Shorter hα2M variants encompassing N-/C-terminal parts were also expressed and yielded pure material at ~1.6/~1.3 and ~3.2/~4.6 mg per liter of insect or mammalian cell culture, respectively. We then analyzed the binding of recombinant and authentic hα2M to recombinant latent human transforming growth factor-ß2 (pro-TGF-ß2) and bacterial G-related α2M binding protein (GRAB) by surface plasmon resonance, multiple-angle laser light scattering, size-exclusion chromatography, fluorogenic labelling, gel electrophoresis and Western-blot analysis. Two GRAB molecules formed stable complexes of high affinity with native and induced authentic hα2M tetramers. The shorter recombinant hα2M variants interacted after preincubation only. In contrast, pro-TGF-ß2 did not interact, probably owing to hindrance by the N-terminal latency-associated protein of the cytokine.

Changes Due to Ageing in the Glycan Structure of Alpha-2-Macroglobulin and Its Reactivity with Ligands.

Alpha-2-macroglobulin (α2M) is a molecule generally associated with inflammation, and chronic inflammation is associated with ageing and cancer. The degree of inflammation was recently proposed to be considered as a biomarker of biological ageing. In this study, glycans attached to α2M were analysed in a human population of different ages by lectin-based protein microarray. Higher reactivity of α2M with several lectins was detected in older individuals indicating an increased content of specific monosaccharides: α2,6 sialic acid, mannose and N-acetylglucosamine, and multiantennary complex type N-glycans. The increased glycosylation of α2M was accompanied by reduced binding of Zn ions and insulin-like growth factor-binding protein 2 (IGFBP-2). Glycosylation of α2M and its reactivity with IGFBP-2 is similarly affected by ageing and incidence of colon cancer, but the reactivity of α2M with Zn ions is differently affected, as the binding of Zn ions remains unaltered in patients with colon cancer compared to healthy middle-aged individuals. Thus, the binding of IGFBP-2 to α2M seems to be related to structural changes in the glycan moieties of α2M, whereas binding of Zn ions, most likely, is not.

Membrane-bound Glucose regulated protein 78 interacts with alpha-2-macroglobulin to promote actin reorganization in sperm during epididymal maturation.

Glucose regulated protein 78 (GRP78) is expressed on cell surface in exceptional conditions as seen in cancer cells and macrophages. We have reported its membrane localization in sperm. The functional significance of its surface localization in sperm is an enigma. Alpha-2-macroglobulin (α2M) reportedly binds surface GRP78, regulating macrophage motility. Additionally, seminal plasma α2M levels are low in cases of asthenozoospermia. We investigated the functional relevance of sperm surface GRP78 and α2M crosstalk using testicular sperm (Tsp; immature) and caudal sperm (Cdsp; mature) from adult Holtzman rats. α2M colocalized and interacted with GRP78 and was significantly higher in Cdsp. Cofilin pathway proteins were detected in Tsp and Cdsp, however the pathway was highly active in Cdsp. Tsp surface GRP78 tyrosine phosphorylation and [Ca2+]i levels increased significantly on exposure to activated α2M (α2M*). This binding activated Rac/Cdc42, and consequently PAK, leading to LIMK and cofilin phosphorylation and thus promoting actin reorganization. Cofilin translocation from the sperm tail to the head in the presence of α2M* possibly prevented F-actin depolymerization in the tail. Thus, profiles observed with Cdsp could be re-created upon exposure of Tsp with α2M*. We conclude that α2M secreted into seminiferous tubule fluid by Sertoli cells, may be activated by proteinases in the epididymis and may bind to sperm surface GRP78 during epididymal transit, thereby facilitating sperm motility via actin reorganization. As F-actin is required for maintaining structural integrity and hyperactivated motility in sperm, our finding has significant implications in light of our previous reports of reduced GRP78 phosphorylation and the actin-based motility pathway being significantly altered in asthenozoospermia.

Validation of Serum Test for Advanced Liver Fibrosis in Patients With Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: We analyzed markers of fibrosis in serum samples from patients with nonalcoholic fatty liver disease (NAFLD), assessed by liver biopsy. We used serum levels of markers to develop an algorithm to discriminate patients with advanced fibrosis from those with mild or moderate fibrosis and validated its performance in 2 independent cohorts of patients with NAFLD. METHODS: We performed a retrospective analysis of serum samples from 396 patients with NAFLD and different stages of fibrosis (F0-F4), collected from 2007 through 2017 on the day of liver biopsy (training cohort 1). We measured serum concentrations of alpha-2 macroglobulin (A2M), hyaluronic acid (HA), and TIMP metallopeptidase inhibitor 1 (TIMP1), and used measurements to develop an algorithm that could discriminate patients with NAFLD with advanced fibrosis (F3-F4; 24.1% of cohort) from those with mild or moderate fibrosis (F0-F2; 79.5% of cohort). We validated the algorithm using serum samples collected from a separate 396 patients from the same time period and location (validation cohort 1), as well as 244 patients with NAFLD evaluated at a separate location, from 2011 through 2017, within a median of 11 days of liver biopsy (cohort 2). RESULTS: The algorithm identified patients with advanced fibrosis vs mild or moderate fibrosis in training cohort 1 with an area under the receiver operating characteristic (AUROC) curve of 0.867 (95% CI, 0.827-0.907), 84.8% sensitivity (95% CI, 75.5%-91.0%), and 72.3% specificity (95% CI, 66.9%-77.3%), at a cutoff score of 17. The AUROC for the combined validation cohorts 1 and 2 (n=640) was 0.856 (95% CI, 0.820-0.892), identifying patients with 79.7% sensitivity (95% CI, 71.9%-86.2%) and 75.7% specificity (95% CI, 71.8%-79.4%) at the predetermined cutoff score of 17. The algorithm had negative predictive values that ranged from 92.5% to 94.7% in the validation cohorts; it correctly classified 90.0% of F0 samples, 75.0% of F1 samples, 77.4% of F3 samples, and 94.4% of F4 samples. CONCLUSION: We developed an algorithm that identifies patients with advanced fibrosis from those with mild to moderate fibrosis in patients with NAFLD with an AUROC value of approximately 0.86, based on levels of serum biomarkers. We validated the findings in 2 separate sets of patients with biopsy-proven NAFLD. The algorithm can be used non-invasively to determine risk of advanced fibrosis in patients with NAFLD.

Exploring the interaction of anti-androgen drug-bicalutamide with human alpha-2-macroglobulin: A biophysical investigation.

Bicalutamide (BCT), a drug used in the treatment of prostate cancer, antagonises the actions of androgens, at the receptor level, thereby inhibiting the growth of prostate tumours. Alpha-2-macroglobulin (α2M), a pan-proteinase inhibitor, inhibits proteinase, regardless of specificity and catalytic mechanism. α2M is deficient in patients of advanced prostate cancer with bone metastases. Our studies explored the interaction of BCT with α2M and analysed the BCT induced structural alteration to the α2M. The result suggests that BCT decreases the antiproteolytic potential and causes structural and functional change in human α2M. UV-visible absorption spectroscopy confirms the formation of α2M-BCT complex. Fluorescence analysis shows significant quenching in fluorescence intensity of α2M upon binding with BCT. Synchronous fluorescence result suggests the interaction of BCT with α2M changed the microenvironment around tyrosine residues. Secondary structure of α2M also undergoes a slight change upon complexation with the drug as evident by shift in negative ellipticity in far UV CD spectroscopy. FTIR results confirm the alteration in secondary structure of α2M upon drug interaction. Molecular docking studies show that BCT bind to a monomer of α2M primarily through hydrophobic force. Thermodynamics parameters were determined by isothermal titration calorimetry found that the binding was exothermic in nature.

PSA-alpha-2-macroglobulin complex is enzymatically active in the serum of patients with advanced prostate cancer and can degrade circulating peptide hormones.

BACKGROUND: Prostate cancer cells produce high levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the tumor microenvironment but is presumed to be enzymatically inactive in the blood due to complex formation with serum protease inhibitors α-1-antichymotrypsin and α-2-macroglobulin (A2M). PSA-A2M complexes cannot be measured by standard ELISA assays and are also rapidly cleared from the circulation. Thus the exact magnitude of PSA production by prostate cancer cells is not easily measured. The PSA complexed to A2M is unable to cleave proteins but maintains the ability to cleave small peptide substrates. Thus, in advanced prostate cancer, sufficient PSA-A2M may be in circulation to effect total A2M levels, levels of cytokines bound to A2M and hydrolyze small circulating peptide hormones. METHODS: Total A2M levels in men with advanced prostate cancer and PSA levels above 1000 ng/mL were measured by ELISA and compared to controls. Additional ELISA assays were used to measure levels of IL-6 and TGF-beta which can bind to A2M. The ability of PSA-A2M complexes to hydrolyze protein and peptide substrates was analyzed ± PSA inhibitor. Enzymatic activity of PSA-A2M in serum of men with high PSA levels was also assayed. RESULTS: Serum A2M levels are inversely correlated with PSA levels in men with advanced prostate cancer. Il-6 Levels are significantly elevated in men with PSA >1000 ng/mL compared to controls with PSA <0.1 ng/mL. PSA-A2M complex in serum of men with PSA levels >1000 ng/mL can hydrolyze small fluorescently labeled peptide substrates but not large proteins that are PSA substrates. PSA can hydrolyze small peptide hormones like PTHrP and osteocalcin. PSA complexed to A2M retains the ability to degrade PTHrP. CONCLUSIONS: In advanced prostate cancer with PSA levels >1000 ng/mL, sufficient PSA-A2M is present in circulation to produce enzymatic activity against circulating small peptide hormones. Sufficient PSA is produced in advanced prostate cancer to alter total A2M levels, which can potentially alter levels of a variety of growth factors such as IL-6, TGF-beta, basic FGF, and PDGF. Alterations in levels of these cytokines and proteolytic degradation of small peptide hormones may have profound effect on host-cancer interaction.

Ovostatin 2 knockdown significantly inhibits the growth, migration, and tumorigenicity of cutaneous malignant melanoma cells.

BACKGROUND: We previously identified ovostatin 2 (OVOS2) as a new candidate gene for cutaneous malignant melanoma (CMM) in a Chinese population. In this study, we aimed to investigate the exact role of OVOS2 in cell proliferation, invasion, and tumorigenesis of melanoma A375 cells. METHODS: The downregulation of OVOS2 expression was performed using lentiviral vectors with specific shRNA. The effects of OVOS2 expression on cell proliferation, cell cycle, cell migration, cell invasion, and potential of tumorigenesis were further investigated. RESULTS: The downregulation of OVOS2 significantly suppressed the proliferation of A375 cells and led to a G2/M phase block. The transwell cell migration assay showed that the reduced expression of OVOS2 also significantly inhibited the transmigration of A375 cells. The western blot results showed downregulated expression of p-FAK, p-AKT, and p-ERK. This was accompanied by the upregulated epithelial phenotypes E-cadherin and ß-catenin, and downregulated expression of mesenchymal phenotype N-cadherin after OVOS2 knockdown. The transplantation tumor experiment in BALB/C nude mouse showed that after an observation period of 32 days, the growth speed and weight of the transplanted tumors were significantly suppressed in the BALB/c nude mice subcutaneously injected with OVOS2 knocked-down A375 cells. CONCLUSION: The inhibition of OVOS2 had significant suppressive effects on the proliferation, motility, and migration capabilities of A375 cells, suggesting a crucial promotive role of OVOS2 in the pathogenesis and progression of CMM. The involved mechanisms are at least partly associated with the overactivation of FAK/MAPK/ERK and FAK/PI3K/AKT signals.