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1.
FASEB J ; 33(5): 6470-6482, 2019 05.
Article in English | MEDLINE | ID: mdl-30794440

ABSTRACT

Defensins are an extensive family of host defense peptides found ubiquitously across plant and animal species. In addition to protecting against infection by pathogenic microorganisms, some defensins are selectively cytotoxic toward tumor cells. As such, defensins have attracted interest as potential antimicrobial and anticancer therapeutics. The mechanism of defensin action against microbes and tumor cells appears to be conserved and involves the targeting and disruption of cellular membranes. This has been best defined for plant defensins, which upon binding specific phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid, form defensin-lipid oligomeric complexes that destabilize membranes, leading to cell lysis. In this study, to further define the anticancer and therapeutic properties of plant defensins, we have characterized a novel plant defensin, Nicotiana occidentalis defensin 173 (NoD173), from N. occidentalis. NoD173 at low micromolar concentrations selectively killed a panel of tumor cell lines over normal primary cells. To improve the anticancer activity of NoD173, we explored increasing cationicity by mutation, with NoD173 with the substitution of Q22 with lysine [NoD173(Q22K)], increasing the antitumor cell activity by 2-fold. NoD173 and the NoD173(Q22K) mutant exhibited only low levels of hemolytic activity, and both maintained activity against tumor cells in serum. The ability of NoD173 to inhibit solid tumor growth in vivo was tested in a mouse B16-F1 model, whereby injection of NoD173 into established subcutaneous tumors significantly inhibited tumor growth. Finally, we showed that NoD173 specifically targets PIP2 and determined by X-ray crystallography that a high-resolution structure of NoD173, which forms a conserved family-defining cysteine-stabilized-αß motif with a dimeric lipid-binding conformation, configured into an arch-shaped oligomer of 4 dimers. These data provide insights into the mechanism of how defensins target membranes to kill tumor cells and provide proof of concept that defensins are able to inhibit tumor growth in vivo.-Lay, F. T., Ryan, G. F., Caria, S., Phan, T. K., Veneer, P. K., White, J. A., Kvansakul, M., Hulett M. D. Structural and functional characterization of the membrane-permeabilizing activity of Nicotiana occidentalis defensin NoD173 and protein engineering to enhance oncolysis.


Subject(s)
Amino Acid Substitution , Antineoplastic Agents, Phytogenic , Defensins , Neoplasms , Nicotiana , Plant Proteins , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Cell Membrane Permeability/drug effects , Defensins/chemistry , Defensins/genetics , Defensins/pharmacology , HeLa Cells , Human Umbilical Vein Endothelial Cells , Humans , Mutation, Missense , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , PC-3 Cells , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/pharmacology , Protein Multimerization , Protein Structure, Quaternary , Structure-Activity Relationship , Nicotiana/chemistry , Nicotiana/genetics , U937 Cells
2.
Inorg Chem ; 55(6): 2830-9, 2016 Mar 21.
Article in English | MEDLINE | ID: mdl-26930516

ABSTRACT

In this paper we report on the use of [NO][BF4] to access tricationic tetrakis(pyridine)gold(III) from Au powder, a species inaccessible using the more traditional (tetrahydrothiophene)AuCl route. It is then demonstrated that this family of compounds can be used to access new terminal Au(III) hydroxides, a challenging class of compounds, and the first crystallographically characterized examples employing bidentate ligands. Finally, preliminary biological studies indicate good activity for derivatives featuring polydentate ligands against the HeLa and PC3 cell lines but also strong inhibition of primary HUVEC cells.


Subject(s)
Gold/chemistry , Inorganic Chemicals/pharmacology , Pyridines/chemistry , Human Umbilical Vein Endothelial Cells , Humans , Hydroxides/chemistry , Spectrophotometry, Ultraviolet
3.
Eur J Immunol ; 44(4): 1016-30, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24532362

ABSTRACT

Heparanase is a ß-d-endoglucuronidase that cleaves heparan sulphate, a key component of the ECM and basement membrane. The remodelling of the ECM by heparanase has been proposed to regulate both normal physiological and pathological processes, including wound healing, inflammation, tumour angiogenesis and cell migration. Heparanase is also known to exhibit non-enzymatic functions by regulating cell adhesion, cell signalling and differentiation. In this study, constitutive heparanase-deficient (Hpse(-/-) ) mice were generated on a C57BL/6 background using the Cre/loxP recombination system, with a complete lack of heparanase mRNA, protein and activity. Although heparanase has been implicated in embryogenesis and development, Hpse(-/-) mice are anatomically normal and fertile. Interestingly, consistent with the suggested function of heparanase in cell migration, the trafficking of dendritic cells from the skin to the draining lymph nodes was markedly reduced in Hpse(-/-) mice. Furthermore, the ability of Hpse(-/-) mice to generate an allergic inflammatory response in the airways, a process that requires dendritic cell migration, was also impaired. These findings establish an important role for heparanase in immunity and identify the enzyme as a potential target for regulation of an immune response.


Subject(s)
Cell Movement/immunology , Dendritic Cells/immunology , Glucuronidase/immunology , Pneumonia/immunology , Animals , Blotting, Western , Cell Movement/genetics , Cells, Cultured , Dendritic Cells/metabolism , Female , Flow Cytometry , Gene Expression/genetics , Gene Expression/immunology , Glucuronidase/deficiency , Glucuronidase/genetics , Lymph Nodes/immunology , Lymph Nodes/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Fluorescence , Pneumonia/genetics , Reverse Transcriptase Polymerase Chain Reaction , Skin/immunology , Skin/metabolism
4.
Cell Death Dis ; 15(2): 123, 2024 02 09.
Article in English | MEDLINE | ID: mdl-38336804

ABSTRACT

Discovery of new small molecules that can activate distinct programmed cell death pathway is of significant interest as a research tool and for the development of novel therapeutics for pathological conditions such as cancer and infectious diseases. The small molecule raptinal was discovered as a pro-apoptotic compound that can rapidly trigger apoptosis by promoting the release of cytochrome c from the mitochondria and subsequently activating the intrinsic apoptotic pathway. As raptinal is very effective at inducing apoptosis in a variety of different cell types in vitro and in vivo, it has been used in many studies investigating cell death as well as the clearance of dying cells. While examining raptinal as an apoptosis inducer, we unexpectedly identified that in addition to its pro-apoptotic activities, raptinal can also inhibit the activity of caspase-activated Pannexin 1 (PANX1), a ubiquitously expressed transmembrane channel that regulates many cell death-associated processes. By implementing numerous biochemical, cell biological and electrophysiological approaches, we discovered that raptinal can simultaneously induce apoptosis and inhibit PANX1 activity. Surprisingly, raptinal was found to inhibit cleavage-activated PANX1 via a mechanism distinct to other well-described PANX1 inhibitors such as carbenoxolone and trovafloxacin. Furthermore, raptinal also interfered with PANX1-regulated apoptotic processes including the release of the 'find-me' signal ATP, the formation of apoptotic cell-derived extracellular vesicles, as well as NLRP3 inflammasome activation. Taken together, these data identify raptinal as the first compound that can simultaneously induce apoptosis and inhibit PANX1 channels. This has broad implications for the use of raptinal in cell death studies as well as in the development new PANX1 inhibitors.


Subject(s)
Apoptosis , Connexins , Fluorenes , Adenosine Triphosphate/metabolism , Apoptosis/drug effects , Cell Death , Connexins/antagonists & inhibitors , Connexins/metabolism , Cyclopentanes/pharmacology
5.
Cancers (Basel) ; 15(11)2023 Jun 05.
Article in English | MEDLINE | ID: mdl-37297024

ABSTRACT

Breast cancer is the second most common human malignancy and is a major global health burden. Heparanase (HPSE) has been widely implicated in enhancing the development and progression of solid tumours, including breast cancer. In this study, the well-established spontaneous mammary tumour-developing MMTV-PyMT murine model was utilised to examine the role of HPSE in breast cancer establishment, progression, and metastasis. The use of HPSE-deficient MMTV-PyMT (MMTV-PyMTxHPSE-/-) mice addressed the lack of genetic ablation models to investigate the role of HPSE in mammary tumours. It was demonstrated that even though HPSE regulated mammary tumour angiogenesis, mammary tumour progression and metastasis were HPSE-independent. Furthermore, there was no evidence of compensatory action by matrix metalloproteinases (MMPs) in response to the lack of HPSE expression in the mammary tumours. These findings suggest that HPSE may not play a significant role in the mammary tumour development of MMTV-PyMT animals. Collectively, these observations may have implications in the clinical setting of breast cancer and therapy using HPSE inhibitors.

6.
Elife ; 3: e01808, 2014 Apr 01.
Article in English | MEDLINE | ID: mdl-24692446

ABSTRACT

Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique 'cationic grip' configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism. DOI: http://dx.doi.org/10.7554/eLife.01808.001.


Subject(s)
Defensins/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Cell Death , Crystallography, X-Ray , Defensins/chemistry , Defensins/genetics , Defensins/isolation & purification , Epithelial Cells/drug effects , Epithelial Cells/physiology , Fusarium/drug effects , Fusarium/physiology , HeLa Cells , Humans , Microbial Viability/drug effects , Mutagenesis, Site-Directed , Phosphatidylinositol 4,5-Diphosphate/chemistry , Protein Binding , Protein Multimerization , Nicotiana/chemistry
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