Lewis(y) antigen-mediated positive feedback loop induces and promotes chemotherapeutic resistance in ovarian cancer.

The present study aimed to investigate the association between Lewis(y) antigen and chemoresistance in ovarian cancer and to elucidate the underlying molecular mechanisms. Lewis(y) expression in chemoresistant ovarian cancer tissues and cells was detected by immunohistochemistry. α1,2­fucosyltransferase (FUT1) expression in different ovarian cancer chemotherapy-resistant cells was analyzed by reverse transcription-quantitative PCR (RT-qPCR). Genes differentially expressed in the chemoresistant and sensitive groups were screened using a gene chip followed by validation using RT-qPCR and western blot analysis. We found that Lewis(y) and FUT1 expression in ovarian cancer cells was significantly increased following the induction of drug resistance. The positive expression rate and intensity of Lewis(y) in ovarian cancer chemoresistant tissues were also significantly higher than those in the sensitive group. Compared with the non-resistant cell lines, the differentially expressed genes were mainly enriched in the terms related to the transmembrane receptor protein tyrosine kinase signaling pathway and positive regulation of cell proliferation. Interaction network analysis predicted genes participating in the regulation of apoptotic processes. The highly differential expression of Annexin A4 (ANXA4), BCL2 interacting killer (BIK), transmembrane 4 L six family member 4 (TM4SF4) and pleckstrin homology-like domain family A member 1 (PHLDA1) was validated using RT-qPCR in ovarian cancer cell lines. Finally, ANXA4 expression was increased at both the mRNA and protein level in the drug­resistant cells, and in addition, ANXA4 contained a Lewis(y) structure. The expression of Bcl-2 and other anti-apoptotic proteins increased with the increase of Lewis(y) expression. After blocking Lewis(y) using an antibody, the expression of the involved signaling pathway and apoptosis-related proteins decreased significantly. These findings provide strong evidence that Lewis(y) is a component of the structure of the ANXA4 membrane protein. Its overexpression can abnormally activate signaling pathways and regulate the expression of a number of factors, forming a positive feedback loop to induce the chemoresistance of ovarian cancer cells, and ultimately promoting the progression of ovarian cancer.

Targeting pathogenic postischemic self-recognition by natural IgM to protect against posttransplantation cardiac reperfusion injury.

BACKGROUND: Natural IgM antibodies represent a class of innate pattern recognition receptors that recognize danger-associated molecular patterns expressed on stressed or dying cells. They play important roles in tissue homeostasis by disposing of prenecrotic cells and suppressing inflammation. However, ischemic insult leads to a pathogenic level of IgM binding and complement activation, resulting in inflammation and injury. We investigate the role of self-reactive IgM in the unique setting of transplantation where the donor organ undergoes both cold and warm ischemia and global ischemic insult. METHODS AND RESULTS: By transplanting hearts from wild-type donor mice into antibody-deficient mice reconstituted with specific self-reactive IgM monoclonal antibodies, we identified neoepitopes expressed after transplantation and demonstrated a key role for IgM recognition of these epitopes in graft injury. With this information, we developed and characterized a therapeutic strategy that exploited the postischemia recognition system of natural antibodies. On the basis of neoepitope identification, we constructed an anti-annexin IV single-chain antibody (scFv) and an scFv linked to Crry, an inhibitor of C3 activation (scFv-Crry). In an allograft transplantation model in which recipients contain a full natural antibody repertoire, both constructs blocked graft IgM binding and complement activation and significantly reduced graft inflammation and injury. Furthermore, scFv-Crry specifically targeted to the transplanted heart and, unlike complement deficiency, did not affect immunity to infection, an important consideration for immunosuppressed transplant recipients. CONCLUSIONS: We identified pathophysiologically important epitopes expressed within the heart after transplantation and described a novel translatable strategy for targeted complement inhibition that has several advantages over currently available approaches.

Suppression of MIP-2 or IL-8 production by annexins A1 and A4 during coculturing of macrophages with late apoptotic human peripheral blood neutrophils.

Annexin A1 (ANXA1) is a well-known anti-inflammatory protein that is expressed on the surface of apoptotic cells. Annexin A4 (ANXA4) is also recruited from the nucleus to the cytoplasm in apoptotic cells, although it is not known whether or not ANXA4 is expressed on the surface of apoptotic cells. In this study, we obtained rabbit anti-human ANXA1 and ANXA4 antibodies, and then examined whether or not ANXA1 and ANXA4 are expressed on the surface of early and late human apoptotic cells. ANXA1 and, to a lesser extent, ANXA4 were detected on late but not early apoptotic HeLa cells, whereas ANXA1 and a small amount of ANXA4 were detected on both early and late apoptotic human neutrophils. We then examined the effects of the anti-human ANXA1 and ANXA4 antibodies on the mouse or human macrophage response to human apoptotic cells. Upon coculturing of mouse or human macrophages with late apoptotic human neutrophils, anti-human ANXA1 antibodies and, to a lesser extent, anti-human ANXA4 antibodies increased MIP-2 or IL-8 production significantly, suggesting that ANXA1 and ANXA4 suppress MIP-2 or IL-8 production by macrophages in response to late apoptotic human neutrophils.

Detection of annexin A autoantibodies in sera from colorectal cancer patients.

BACKGROUND: Annexins (ANXAs) belong to a superfamily of closely related calcium and membrane-binding proteins and are overexpressed in some carcinomas. The overexpression of ANXAs presumably induces an autoantibody response, but this has not been demonstrated for sera from colorectal cancer (CRC) patients. STUDY: We examined serum samples from 220 CRC patients and 216 healthy volunteers to evaluate the ANXA autoantibody response in patients by using an enzyme-linked immunosorbent assay with recombinant ANXA A4 as the antigen. RESULTS: The sensitivity of the anti-ANXA response from CRC patient sera was about 85% and the specificity was about 61.6%. When a cut-off value of 5.0 ng/mL was chosen for carcinoembryonic antigen (CEA) in the same sera samples, the sensitivity and specificity values were 43.2% and 85.2%, respectively. Combined detection using ANXA autoantibodies and CEA produced better sensitivity (71.8%) and specificity (79.2%) compared with CEA sensitivity (43.2%) and anti-ANXA specificity (61.6%). The area under a receiver operating characteristic curve was 0.794 for ANXA autoantibodies, 0.666 for CEA, and 0.84 for both markers together. Importantly, in comparison to CEA (27.5% seropositivity), ANXA autoantibody showed a remarkable change (81.3% seropositivity) at the early stage of CRC. CONCLUSIONS: Measurement of ANXA autoantibody levels may provide an alternative detection indicator for CRC, particularly among early-stage patients.

Pathogenic natural antibodies recognizing annexin IV are required to develop intestinal ischemia-reperfusion injury.

Intestinal ischemia-reperfusion (IR) injury is initiated when natural IgM Abs recognize neo-epitopes that are revealed on ischemic cells. The target molecules and mechanisms whereby these neo-epitopes become accessible to recognition are not well understood. Proposing that isolated intestinal epithelial cells (IEC) may carry IR-related neo-epitopes, we used in vitro IEC binding assays to screen hybridomas created from B cells of unmanipulated wild-type C57BL/6 mice. We identified a novel IgM mAb (mAb B4) that reacted with the surface of IEC by flow cytometric analysis and was alone capable of causing complement activation, neutrophil recruitment and intestinal injury in otherwise IR-resistant Rag1(-/-) mice. mAb B4 was found to specifically recognize mouse annexin IV. Preinjection of recombinant annexin IV blocked IR injury in wild-type C57BL/6 mice, demonstrating the requirement for recognition of this protein to develop IR injury in the context of a complex natural Ab repertoire. Humans were also found to exhibit IgM natural Abs that recognize annexin IV. These data in toto identify annexin IV as a key ischemia-related target Ag that is recognized by natural Abs in a pathologic process required in vivo to develop intestinal IR injury.

Detection of annexin IV in bovine retinal rods.

The fraction of proteins capable of binding to photoreceptor membranes in a Ca2+-dependent manner was isolated from bovine rod outer segments. One of these proteins with apparent molecular mass of 32 kD (p32) was purified to homogeneity and identified as annexin IV (endonexin) by MALDI-TOF mass-spectrometry. In immunoblot, annexin IV purified from bovine rod outer segments cross-reacted with antibodies against annexin IV from bovine liver. This is the first detection of annexin IV in vertebrate retina.

Alteration of annexin IV expression in alcoholics.

Western blot analysis was performed by using a specific antibody to measure annexin IV in human postmortem brain samples from alcoholic subjects. The analysis showed a significantly augmented expression in the hippocampus compared with controls, whereas the expression in the frontal cortex was equivalent in both groups. Annexin IV expression in the occipital cortex tended to increase in alcoholics. It was shown further that autoantibodies to annexin IV were increased significantly in alcoholic patients compared with controls. Thus, annexin IV may become a novel biological marker for alcoholics.

Detection of anti-annexin IV and V antibodies in patients with antiphospholipid syndrome and systemic lupus erythematosus.

OBJECTIVE: Annexins (Anx) are a family of structurally related proteins that bind to phospholipids in a calcium dependent manner. It has been reported that antibodies to Anx V, which acts as an antithrombotic protein, are associated with thrombosis in systemic lupus erythematosus (SLE) and/or antiphospholipid syndrome (APS). Homology between the primary structures of Anx IV and Anx V is the highest among members of the Anx family. We investigated whether anti-Anx IV autoantibodies can be detected in the sera of patients with SLE and/or APS. METHODS: Seventy-four patients with SLE/APS were divided into 3 groups: Group A, patients with SLE but no clinical or serological features of APS; Group B, patients with SLE having only serological signs of APS; and Group C, patients with clinical symptoms and serological signs of APS. Anx IV and Anx V were prepared by recombinant technique. Anti-Anx IV, Anx V, cardiolipin (CL), and CL beta2-glycoprotein I were detected by ELISA. RESULTS: Anti-Anx IV was found in 15.4% of Group A, 20.0% of Group B, and 21.7% of Group C. Anti-Anx V was found in 3.8% of Group A, 28.0% of Group B, and 30.4% of Group C. Significant correlations were noted between anti-Anx IV titer and anti-Anx V titer (p<0.001), and between anti-Anx IV titer and aCL titer (p<0.01). CONCLUSION: Anti-Anx IV and V antibodies were characterized in the sera of patients with SLE/APS. Significantly higher frequency of arterial or venous thrombosis was found in patients with anti-Anx V.

Characterization of human p33/41 (annexin IV), a Ca2+ dependent carbohydrate-binding protein with monoclonal anti-annexin IV antibodies, AS11 and AS17.

p33/41 (annexin IV) is a member of the family of Ca(2+)-dependent phospholipid binding proteins known as annexins. We previously described that bovine kidney p33/41 (annexin IV) has Ca(2+)-dependent carbohydrate binding activity. In this study, we purified human p33/41 (annexin IV) from the HT29, human colon adenocarcinoma cell line, as well as the bovine kidney annexin by affinity chromatography. Then, we prepared recombinant human p33/41 (annexin IV) expressed in Escherichia coli. The apparent size and the Ca(2+)-dependent carbohydrate binding properties of purified recombinant p33/41 (annexin IV) were indistinguishable from those of the bovine kidney protein. We also performed inhibition assays of carbohydrate binding and of phosphatidylserine/phosphatidylcholine liposome binding of recombinant p33/41 (annexin IV) with anti-p33/41 monoclonal antibodies (AS11 and AS17). We determined the epitopes recognized by the monoclonal antibodies by Western blot analysis using deleted-recombinant p33/41 (annexin IV). The monoclonal antibodies recognized domain 1 and/or 2 of p33/41 (annexin IV). The results of the inhibition assays and the determination of the epitope showed that a carbohydrate binding site is located at domains 3 and 4 of p33/41 (annexin IV) and on the cell surface.

Annexin IV inhibits calmodulin-dependent protein kinase II-activated chloride conductance. A novel mechanism for ion channel regulation.

Ca(2+)-activated Cl- current (ICl,Ca) in colonic T84 cells is inhibited by the specific peptide inhibitor of Ca2+/calmodulin-dependent kinase II (CaM KII). Annexin IV, a Ca(2+)-dependent phospholipid binding protein also inhibits Ca(2+)-dependent anion current activation (Kaetzel, M.A., Chan, H.-C., Dubinsky, W.P., Dedman, J.R., and Nelson, D.J. (1994) J. Biol. Chem. 269, 5297-5302). Intracellular injection of antibodies against annexin IV enhances current activation; this activation is inhibited by the peptide inhibitor of CaM KII. Intracellular application of autonomously active CaM KII in the presence of ATP induced a Cl- current similar to that activated by the Ca2+ ionophore A23187. Current activation by the exogenous kinase was completely inhibited in the presence of purified annexin IV. In vitro, annexin IV does not inhibit CaM KII activity nor does it act as a substrate for CaM KII. Thus, it appears that annexin IV inhibits phosphorylation-dependent anion conductance activation by preventing CaM KII-ion channel interaction rather than by direct interaction with the enzyme itself. These findings suggest a novel mechanism by which Ca(2+)-dependent membrane binding proteins, cytoplasmic kinases, and ion channels interact to regulate membrane conductance. The characterization of unique channel regulatory pathways in Cl- transporting epithelia may identify potential avenues of alternate therapy to compensate for the loss of functional Cl- channels in the disease of cystic fibrosis.