Nitric oxide-mediated apoptosis in rat macrophages subjected to Shiga toxin 2 from Escherichia coli

Shiga toxin-producing Escherichia coli are important food-borne pathogens. The main factor conferring virulence on this bacterium is its capacity to secrete Shiga toxins (Stxs), which have been reported to induce apoptosis in several cell types. However, the mechanisms of this apoptosis have not yet been fully elucidated. In addition, Stxs have been shown to stimulate macrophages to produce nitric oxide (NO), a well-known apoptosis inductor.The aim of this study was to investigate the participation of NO in apoptosis of rat peritoneal macrophages induced by culture supernatants or Stx2 from E. coli. Peritoneal macrophages incubated in the presence of E. coli supernatants showed an increase in the amounts of apoptosis and NO production. Furthermore, inhibition of NO synthesis induced by addition of aminoguanidine (AG) was correlated with a reduction in the percentage of apoptotic cells, indicating participation of this metabolite in the apoptotic process. Similarly, treatment of cells with Stx2 induced an increase in NO production and amount of apoptosis, these changes being reversed by addition of AG. In summary, these data show that treatment with E. coli supernatants or Stx2 induces NO-mediated apoptosis of macrophages.     

Role of Shiga toxin 2 (Stx2)-binding protein,human serum amyloid P component (HuSAP), in Shiga toxin-producing Escherichia coli infections: assumption from in vitro and in vivo study using HuSAP and anti-Stx2 humanized monoclonal antibody TMA-15

Shiga toxin 2 (Stx2) is a major pathogenic factor in Shiga toxin-producing Escherichia coli (STEC) infections. Some factor that neutralizes Stx2 in vitro had been shown to be specifically present in human serum and we recently identified it as human serum amyloid P component (HuSAP). Here, we report the role of HuSAP in STEC infections. HuSAP could not rescue Stx2-challenged mice from death, and it instead reduced the efficacy of the Stx2-neutralizing humanized monoclonal antibody TMA-15 when a lower dose of TMA-15 was injected to the mice. By contrast, the efficacy of TMA-15 at a higher dose was uninfluenced by the presence of HuSAP. These findings suggest that HuSAP acts as a carrier protein of Stx2 rather than as a Stx2-neutralizing factor in the human circulation and that passive immune therapy with Stx2-neutralizing antibodies such as TMA-15 is useful to prevent severe complications associated with STEC infections even in the presence of HuSAP.     

Failure of multinucleated giant cell formation in k562 cells infected with newcastle disease virus and human parainfluenza type 2 virus

When K562 cells were infected with Newcastle disease virus (NDV) or human parainfluenza type 2 virus (hPIV-2), polykaryocyte formation could not be detected. Failure of multinucleated giant cell formation in K562 cells infected with either NDV or hPIV-2 is due to disturbance of the viral envelope-cell fusion step or to defect in the cell-cell fusion step, respectively. Especially, NDV completely replicated in K562 cells, and the hemagglutinin-neuraminidase and fusion proteins expressed on the cell surface of NDV-infected K562 cell were fully functional for fusion inducing activity. Therefore, the cell membranes of K562 cells are considered to be resistant to virus-induced cell fusion. Membrane fusion is regulated by many host factors including membrane fluidity, cytoskeletal systems, and fusion regulatory proteins system. An unknown regulatory mechanism of virus-induced cell fusion may function on the cell surface of K562 cells.     

The investigation of minoxidil-induced [Ca2+]i rises and non-Ca2+-triggered cell death in PC3 human prostate cancer cells

Minoxidil is clinically used to prevent hair loss. However, its effect on Ca²⁺ homeostasis in prostate cancer cells is unclear. This study explored the effect of minoxidil on cytosolic-free Ca²⁺ levels ([Ca²⁺]_i) and cell viability in PC3 human prostate cancer cells. Minoxidil at concentrations between 200 and 800?μM evoked [Ca²⁺]_i rises in a concentration-dependent manner. This Ca²⁺ signal was inhibited by 60% by removal of extracellular Ca²⁺. Minoxidil-induced Ca²⁺ influx was confirmed by Mn²⁺-induced quench of fura-2 fluorescence. Pre-treatment with the protein kinase C (PKC) inhibitor GF109203X, PKC activator phorbol 12-myristate 13 acetate (PMA), nifedipine and SKF96365 inhibited minoxidil-induced Ca²⁺ signal in Ca²⁺ containing medium by 60%. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-ditert-butylhydroquinone (BHQ) in Ca²⁺-free medium abolished minoxidil-induced [Ca²⁺]_i rises. Conversely, treatment with minoxidil abolished BHQ-induced [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 abolished minoxidil-evoked [Ca²⁺]_i rises. Overnight treatment with minoxidil killed cells at concentrations of 200–600?μM in a concentration-dependent fashion. Chelation of cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not prevent minoxidil’s cytotoxicity. Together, in PC3 cells, minoxidil induced [Ca²⁺]_i rises that involved Ca²⁺ entry through PKC-regulated store-operated Ca²⁺ channels and PLC-dependent Ca²⁺ release from the endoplasmic reticulum. Minoxidil-induced cytotoxicity in a Ca²⁺-independent manner.     

Bradykinin‐mediated Ca2+ signalling regulates cell growth and mobility in human cardiac c‐Kit+ progenitor cells

Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c‐Kit⁺ progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin‐mediated Ca²⁺ signalling participates in regulating cellular functions in cultured human cardiac c‐Kit⁺ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca²⁺ () by triggering a transient Ca²⁺ release from ER IP3Rs followed by sustained Ca²⁺ influx through store‐operated Ca²⁺ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca²⁺ release and Ca²⁺ influx. It is interesting to note that the bradykinin‐induced cell cycle progression and migration were not observed in cells with siRNA‐silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin‐induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin‐mediated increase in free via ER‐IP3R3 Ca²⁺ release followed by Ca²⁺ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c‐Kit⁺ progenitor cells.     

F2L, a peptide derived from heme-binding protein, inhibits LL-37-induced cell proliferation and tube formation in human umbilical vein endothelial cells

F2L, a peptide derived from heme-binding protein, was originally identified as an endogenous ligand for formyl peptide receptor-like (FPRL)2. Previously, we reported that F2L inhibits FPR and FPRL1-mediated signaling in neutrophils. Since endothelial cells express functional FPRL1, we examined the effect of F2L on LL-37 (an FPRL1 agonist)-induced signaling in human umbilical vein endothelial cells (HUVECs). F2L stimulated the chemotactic migration in HUVECs. However, F2L inhibited FPRL1 activity, resulting in the inhibition of cell proliferation and tube formation induced by LL-37 in HUVECs. We suggest that F2L will potentially be useful in the study of FPRL1 signaling and the development of drugs to treat diseases involving the FPRL1 in the vascular system.     

Actin cross-linking domain of Aeromonas hydrophila repeat in toxin A (RtxA) induces host cell rounding and apoptosis

The repeat in toxin (Rtx) of an environmental isolate ATCC 7966 of Aeromonas hydrophila consists of six genes (rtxACHBDE) organized in an operon similar to the gene organization found for the Rtx of the Vibrio species. The first gene in this operon (rtxA) encodes an exotoxin in vibrios, while other genes code for proteins needed for proper activation of RtxA and in secretion of this toxin from Vibrio cholerae. However, the RtxA of ATCC 7966, as well as from the clinical isolate SSU of A. hydrophila, was exclusively expressed and produced during co-infection of this pathogen with the host, e.g., HeLa cells, indicating that rtxA gene expression required host cell contact. Within the RtxA, an actin cross-linking domain (ACD) exists and to investigate the functionality of this domain, several truncated versions of ACD were generated to discern its minimal biological active region. Such genetically modified genes encoding ACD, which were truncated on either the NH(2) or the COOH terminal, as well as on both ends, were expressed from a bidirectional promoter of the pBI-enhanced green fluorescent protein (EGFP) vector in a HeLa-Tet-Off cell system. We demonstrated that only the full-length ACD of RtxA from A. hydrophila catalyzed the covalent cross-linking of the host cellular actin, whereas the ACD truncated on the NH(2), COOH or both ends did not exhibit such actin cross-linking characteristics. Further, we showed that the full-length ACD of A. hydrophila RtxA disrupted the actin cytoskeleton of HeLa cells, resulting in their rounding phenotype. Finally, our data provided evidence that the full-length ACD of RtxA induced host cell apoptosis. Our study is the first to report that A. hydrophila possesses a functional RtxA having an ACD that contributes to the host cell apoptosis, and hence could represent a potential virulence factor of this emerging human pathogen.     

Potential roles and targeted therapy of the CXCLs/CXCR2 axis in cancer and inflammatory diseases

The chemokine receptor CXCR2 and its ligands are implicated in the progression of tumours and various inflammatory diseases. Activation of the CXCLs/CXCR2 axis activates multiple signalling pathways, including the PI3K, p38/ERK, and JAK pathways, and regulates cell survival and migration. The CXCLs/CXCR2 axis plays a vital role in the tumour microenvironment and in recruiting neutrophils to inflammatory sites. Extensive infiltration of neutrophils during chronic inflammation is one of the most important pathogenic factors in various inflammatory diseases. Chronic inflammation is considered to be closely correlated with initiation of cancer. In addition, immunosuppressive effects of myeloid-derived suppressor cells (MDSCs) against T cells attenuate the anti-tumour effects of T cells and promote tumour invasion and metastasis. Over the last several decades, many therapeutic strategies targeting CXCR2 have shown promising results and entered clinical trials. In this review, we focus on the features and functions of the CXCLs/CXCR2 axis and highlight its role in cancer and inflammatory diseases. We also discuss its potential use in targeted therapies.