A Novel Arylurea Fatty Acid That Targets the Mitochondrion and Depletes Cardiolipin To Promote Killing of Breast Cancer Cells.

Cancer cell mitochondria are promising anticancer drug targets because they control cell death and are structurally and functionally different from normal cell mitochondria. We synthesized arylurea fatty acids and found that the analogue 16-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)hexadecanoic acid (13b) decreased proliferation and activated apoptosis in MDA-MB-231 breast cancer cells in vitro and in vivo. In mechanistic studies 13b emerged as the prototype of a novel class of mitochondrion-targeted agents that deplete cardiolipin and promote cancer cell death.

A novel synthetic analogue of ω-3 17,18-epoxyeicosatetraenoic acid activates TNF receptor-1/ASK1/JNK signaling to promote apoptosis in human breast cancer cells.

A saturated analog of the cytochrome P450-mediated ω-3-17,18-epoxide of ω-3-eicosapentaenoic acid (C20E) activated apoptosis in human triple-negative MDA-MB-231 breast cancer cells. This study evaluated the apoptotic mechanism of C20E. Increased cytosolic cytochrome c expression and altered expression of pro- and antiapoptotic B-cell lymphoma-2 proteins indicated activation of the mitochondrial pathway. Caspase-3 activation by C20E was prevented by pharmacological inhibition and silencing of the JNK and p38 MAP kinases (MAPK), upstream MAPK kinases MKK4 and MKK7, and the upstream MAPK kinase kinase apoptosis signal-regulating kinase 1 (ASK1). Silencing of the death receptor TNF receptor 1 (TNFR1), but not Fas, DR4, or DR5, and the adapters TRADD and TNF receptor-associated factor 2, but not Fas-associated death domain, prevented C20E-mediated apoptosis. B-cell lymphoma-2 homology 3-interacting domain death agonist (Bid) cleavage by JNK/p38 MAPK linked the extrinsic and mitochondrial pathways of apoptosis. In further studies, an antibody against the extracellular domain of TNFR1 prevented apoptosis by TNF-α but not C20E. These findings suggest that C20E acts intracellularly at TNFR1 to activate ASK1-MKK4/7-JNK/p38 MAPK signaling and to promote Bid-dependent mitochondrial disruption and apoptosis. In in vivo studies, tumors isolated from C20E-treated nu/nu mice carrying MDA-MB-231 xenografts showed increased TUNEL staining and decreased Ki67 staining, reflecting increased apoptosis and decreased proliferation, respectively. ω-3-Epoxy fatty acids like C20E could be incorporated into treatments for triple-negative breast cancers.-Dyari, H. R. E., Rawling, T., Chen, Y., Sudarmana, W., Bourget, K., Dwyer, J. M., Allison, S. E., Murray, M. A novel synthetic analogue of ω-3 17,18-epoxyeicosatetraenoic acid activates TNF receptor-1/ASK1/JNK signaling to promote apoptosis in human breast cancer cells.

Activation of ALDH1A1 in MDA-MB-468 breast cancer cells that over-express CYP2J2 protects against paclitaxel-dependent cell death mediated by reactive oxygen species.

Cytochrome P450 2J2 (CYP2J2) expression is elevated in breast and other tumours, and is known to be protective against cytotoxic agents that may be used in cancer chemotherapy. This study evaluated the mechanisms by which MDA-MB-468 breast cancer cells that stably expressed CYP2J2 (MDA-2J2 cells) were protected against killing by the anti-cancer agent paclitaxel. Compared to control cells caspase-3/7 activation by paclitaxel was lower in MDA-2J2 cells, while cell proliferation and colony formation following paclitaxel treatment were increased. Basal lipid peroxidation was lower in MDA-2J2 cells than in control cells, and the paclitaxel-mediated increase in peroxidation was attenuated. The mitochondrial complex III inhibitor antimycin A modulated basal and paclitaxel-activated reactive oxygen species (ROS) formation in control cells; paclitaxel-activated ROS production was also modulated by the NADPH oxidase inhibitor diphenyleneiodonium. Paclitaxel increased the formation of protein adducts by the reactive aldehyde 4-hydroxynonenal that is produced by lipid peroxidation; adduct formation was attenuated in MDA-2J2 cells. ALDH1A1 expression and activity was strongly upregulated in MDA-2J2 cells that was attributed to CYP2J2-derived 14,15-epoxyeicosatrienoic acid (14,15-EET); the 8,9- and 11,12-EET regioisomers did not activate ALDH1A1 expression. Silencing of ALDH1A1 restored the sensitivity of MDA-2J2 cells to paclitaxel, as indicated by a more pronounced decrease in proliferation, and greater increases in caspase activity and formation of ROS to levels comparable with control cells. Similar findings were observed with doxorubicin, sorafenib and staurosporine, that also promoted ROS-mediated cell death that was attenuated in MDA-2J2 cells and reversed by ALDH1A1 gene silencing. These findings implicate ALDH1A1 as an important gene that is activated in MDA-MB-468-derived cells that contain high levels of CYP2J2. ALDH1A1 modulates the production of ROS by anti-cancer agents such as paclitaxel and diminishes their efficacy. Future approaches could adapt this information to facilitate the targeting of ALDH1A1 to promote the efficacy of ROS-generating cytotoxic agents and enhance the treatment of breast cancer.

Activation of the pro-migratory bone morphogenetic protein receptor 1B gene in human MDA-MB-468 triple-negative breast cancer cells that over-express CYP2J2.

Secondary metastases are the leading cause of mortality in patients with breast cancer. Cytochrome P450 (CYP) 2J2 (CYP2J2) is upregulated in many human tumors and generates epoxyeicosanoids from arachidonic acid that promote tumorigenesis and metastasis, but at present there is little information on the genes that mediate these actions. In this study MDA-MB-468 breast cancer cells were stably transfected with CYP2J2 (MDA-2J2 cells) and Affymetrix microarray profiling was undertaken. We identified 182 genes that were differentially expressed in MDA-2J2 cells relative to control (MDA-CTL) cells (log[fold of control] ≥2). From gene ontology pathway analysis bone morphogenetic protein (BMP) receptor 1B (BMPR1B) emerged as an important upregulated gene in MDA-2J2 cells. Addition of the BMPR1B ligand BMP2 stimulated the migration of MDA-2J2 cells, but not MDA-CTL cells, from 3D-matrigel droplets. Migration of MDA-2J2 cells was prevented by the BMPR antagonist dorsomorphin. These findings indicate that over-expression of CYP2J2 in MDA-MB-468-derived breast cancer cells activates BMPR1B expression that may contribute to increased migration. Targeting BMPR1B may be a novel approach to inhibit the metastatic activity of breast cancers that contain high levels of CYP2J2.

Pro-migratory actions of the prostacyclin receptor in human breast cancer cells that over-express cyclooxygenase-2.

Metastasis is the major cause of death in cancer patients. Elevated expression of cyclooxygenase-2 (COX-2) is observed in many human cancers and over-production of downstream prostaglandins (PGs) has been shown to stimulate metastasis. A role for increased PGE2 production has been proposed, but whether other PGs contribute is currently unclear. In this study the pro-migratory actions of individual PGs were evaluated in MDA-MB-468 breast cancer cells that stably over-expressed COX-2 (MDA-COX-2 cells); cell migration was quantified using 3D-matrigel droplet assays. Inhibition of the prostacyclin and PGE synthases, but not alternate prostanoid synthases, prevented the increase in MDA-COX-2 cell migration produced by arachidonic acid (AA); direct treatment of cells with the stable prostacyclin analogue cicaprost also promoted migration. Pharmacological antagonism and knockdown of the IP receptor decreased cell migration, while antagonists of the alternate DP, EP2, FP, and TP prostanoid receptors were inactive. In support of these findings, activation of the IP receptor also enhanced migration in the MDA-MB-468, MDA-MB-231 and A549 cell lines, and IP receptor knock-down in MDA-COX-2 cells decreased the expression of a number of pro-migratory genes. In further studies, the prostacyclin/IP receptor and PGE2/EP4 receptor pathways were found to be functionally independent and the inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK) selectively impaired the IP-receptor-dependent migration in MDA-COX-2 cells. Taken together, the prostacyclin/IP/PI3K-p38 MAPK axis has emerged as a novel pro-migratory pathway in breast cancer cells that over-express COX-2. This information could be utilized in novel treatment strategies to minimize tumor metastasis.

Identification of the docking site between a type III secretion system ATPase and a chaperone for effector cargo.

A number of Gram-negative pathogens utilize type III secretion systems (T3SSs) to inject bacterial effector proteins into the host. An important component of T3SSs is a conserved ATPase that captures chaperone-effector complexes and energizes their dissociation to facilitate effector translocation. To date, there has been limited work characterizing the chaperone-T3SS ATPase interaction despite it being a fundamental aspect of T3SS function. In this study, we present the 2.1 Å resolution crystal structure of the Salmonella enterica SPI-2-encoded ATPase, SsaN. Our structure revealed a local and functionally important novel feature in helix 10 that we used to define the interaction domain relevant to chaperone binding. We modeled the interaction between the multicargo chaperone, SrcA, and SsaN and validated this model using mutagenesis to identify the residues on both the chaperone and ATPase that mediate the interaction. Finally, we quantified the benefit of this molecular interaction on bacterial fitness in vivo using chromosomal exchange of wild-type ssaN with mutants that retain ATPase activity but no longer capture the chaperone. Our findings provide insight into chaperone recognition by T3SS ATPases and demonstrate the importance of the chaperone-T3SS ATPase interaction for the pathogenesis of Salmonella.

Lipid analogues as potential drugs for the regulation of mitochondrial cell death.

The mitochondrion plays an important role in the production of energy as ATP, the regulation of cell viability and apoptosis, and the biosynthesis of major structural and regulatory molecules, such as lipids. During ATP production, reactive oxygen species are generated that alter the intracellular redox state and activate apoptosis. Mitochondrial dysfunction is a well-recognized component of the pathogenesis of diseases such as cancer. Understanding mitochondrial function, and how this is dysregulated in disease, offers the opportunity for the development of drug molecules to specifically target such defects. Altered energy metabolism in cancer, in which ATP production occurs largely by glycolysis, rather than by oxidative phosphorylation, is attributable in part to the up-regulation of cell survival signalling cascades. These pathways also regulate the balance between pro- and anti-apoptotic factors that may determine the rate of cell death and proliferation. A number of anti-cancer drugs have been developed that target these factors and one of the most promising groups of agents in this regard are the lipid-based molecules that act directly or indirectly at the mitochondrion. These molecules have emerged in part from an understanding of the mitochondrial actions of naturally occurring fatty acids. Some of these agents have already entered clinical trials because they specifically target known mitochondrial defects in the cancer cell.

The SseC translocon component in Salmonella enterica serovar Typhimurium is chaperoned by SscA.

BACKGROUND: Salmonella enterica is a causative agent of foodborne gastroenteritis and the systemic disease known as typhoid fever. This bacterium uses two type three secretion systems (T3SSs) to translocate protein effectors into host cells to manipulate cellular function. Salmonella pathogenicity island (SPI)-2 encodes a T3SS required for intracellular survival of the pathogen. Genes in SPI-2 include apparatus components, secreted effectors and chaperones that bind to secreted cargo to coordinate their release from the bacterial cell. Although the effector repertoire secreted by the SPI-2 T3SS is large, only three virulence-associated chaperones have been characterized. RESULTS: Here we report that SscA is the chaperone for the SseC translocon component. We show that SscA and SseC interact in bacterial cells and that deletion of sscA results in a loss of SseC secretion, which compromises intracellular replication and leads to a loss of competitive fitness in mice. CONCLUSIONS: This work completes the characterization of the chaperone complement within SPI-2 and identifies SscA as the chaperone for the SseC translocon.

Degradation of MAC13243 and studies of the interaction of resulting thiourea compounds with the lipoprotein targeting chaperone LolA.

The discovery of novel small molecules that function as antibacterial agents or cellular probes of biology is hindered by our limited understanding of bacterial physiology and our ability to assign mechanism of action. We previously employed a chemical genomic strategy to identify a novel small molecule, MAC13243, as a likely inhibitor of the bacterial lipoprotein targeting chaperone, LolA. Here, we report on the degradation of MAC13243 into the active species, S-(4-chlorobenzyl)isothiourea. Analogs of this compound (e.g., A22) have previously been characterized as inhibitors of the bacterial actin-like protein, MreB. Herein, we demonstrate that the antibacterial activity of MAC13243 and the thiourea compounds are similar; these activities are suppressed or sensitized in response to increases or decreases of LolA copy number, respectively. We provide STD NMR data which confirms a physical interaction between LolA and the thiourea degradation product of MAC13243, with a Kd of ~150 µM. Taken together, we conclude that the thiourea series of compounds share a similar cellular mechanism that includes interaction with LolA in addition to the well-characterized target MreB.

Inhibition of WTA synthesis blocks the cooperative action of PBPs and sensitizes MRSA to ß-lactams.

Rising drug resistance is limiting treatment options for infections by methicillin-resistant Staphylococcus aureus (MRSA). Herein we provide new evidence that wall teichoic acid (WTA) biogenesis is a remarkable antibacterial target with the capacity to destabilize the cooperative action of penicillin-binding proteins (PBPs) that underlie ß-lactam resistance in MRSA. Deletion of gene tarO, encoding the first step of WTA synthesis, resulted in the restoration of sensitivity of MRSA to a unique profile of ß-lactam antibiotics with a known selectivity for penicillin binding protein 2 (PBP2). Of these, cefuroxime was used as a probe to screen for previously approved drugs with a cryptic capacity to potentiate its activity against MRSA. Ticlopidine, the antiplatelet drug Ticlid, strongly potentiated cefuroxime, and this synergy was abolished in strains lacking tarO. The combination was also effective in a Galleria mellonella model of infection. Using both genetic and biochemical strategies, we determined the molecular target of ticlopidine as the N-acetylglucosamine-1-phosphate transferase encoded in gene tarO and provide evidence that WTA biogenesis represents an Achilles heel supporting the cooperative function of PBP2 and PBP4 in creating highly cross-linked muropeptides in the peptidoglycan of S. aureus. This approach represents a new paradigm to tackle MRSA infection.

Novel repressor of Escherichia coli O157:H7 motility encoded in the putative fimbrial cluster OI-1.

Escherichia coli O157:H7 is a gastrointestinal pathogen that has become a serious public health concern, as it is associated with outbreaks and severe diseases such as hemolytic-uremic syndrome. The molecular basis of its greater virulence than that of other serotypes is not completely known. OI-1 is a putative fimbria-encoding genomic island that is found almost exclusively in O157:H7 Shiga toxin-producing E. coli strains and may be associated with the enhanced pathogenesis of these strains. In this study, we identified and characterized a novel repressor of flagellar synthesis encoded by OI-1. We showed that deletion of Z0021 increased the motility of E. coli O157:H7, which correlated with an increase in flagellin production and enhanced assembly of flagella on the cell surface. In contrast, overexpression of Z0021 inhibited motility. We demonstrated that Z0021 exerted its regulatory effects downstream of the transcription and translation of flhDC but prior to the activation of class II/III promoters. Furthermore, the master regulator of flagellar synthesis, FlhD(4)C(2), was shown to be a high-copy suppressor of the nonmotile phenotype associated with elevated levels of Z0021--a finding consistent with Z0021-FlhD(4)C(2) being a potential regulatory complex. This work provides insight into the mechanism by which Z0021, which we have named fmrA, represses flagellar synthesis and is the first report of a fimbrial-operon-encoded inhibitor of motility in E. coli O157:H7.

Studies of the genetics, function, and kinetic mechanism of TagE, the wall teichoic acid glycosyltransferase in Bacillus subtilis 168.

The biosynthetic enzymes involved in wall teichoic acid biogenesis in gram-positive bacteria have been the subject of renewed investigation in recent years with the benefit of modern tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old experiments in the model gram-positive bacterium, Bacillus subtilis 168, using phage-resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall teichoic acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we showed that deletion of tagE resulted in the loss of α-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also reported the first kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity of TagE using a wide variety of acceptor substrates and found that the enzyme had a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognized its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies of the mechanism of wall teichoic acid glycosylation, a largely uncharted aspect of wall teichoic acid biogenesis.

The impact of Dorothea E. Orem's life and work: an interview with Orem scholars.

This column serves as a tribute to Dorothea Orem's work and focuses on her leadership in the development of nursing science and nursing theory. Nurse scholars give a picture of Orem's contributions over her life and reflections about the future of nursing and healthcare.

Self-care requirements for activity and rest: an Orem nursing focus.

According to Orem's self-care deficit nursing theory, helping people to maintain a balance between activity and rest (a universal self-care requisite) is a legitimate concern of nursing. The meaning of activity and rest, the requirements and potential measures for meeting this self-care requisite, and factors that might influence the process are explored. Criteria for determining a need for nursing, guides for a nursing clinical assessment, and guides for nursing action are suggested as potential ways to assist persons to meet the action demands associated with this self-care requisite.