D-Amino Acid Analogues of the Antimicrobial Peptide CDT Exhibit Anti- Cancer Properties in A549, a Human Lung Adenocarcinoma Cell Line

Evans, Hedeel Guy; Guthrie, Jeffrey; Jujjavarapu, Murali; Hendrickson, Nathan; Eitel, Anna R.; Park, Yeji; Garvey, Jennifer; Newman, Rebecca; Esckilsen, Daniel; Heyl, Deborah L.

doi:10.2174/0929866524666170621093647

Cited by 17 publications

(24 citation statements)

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“…At a final concentration of 100 mg/L, tachyplesin caused lysis of approximately 2% of hemocytes ( Figure 2A ), as well as a decline in the cell viability of 293FT human embryonic kidney cells ( Figure 2C ) and a sharp decline in the cell viability of L919 murine fibroblasts ( Figure 2B ), a cell line commonly used for toxicity testing. Interestingly, tumor cells such as A549 adenocarcinomic human alveolar basal epithelial cells seem to be more resistant to tachyplesin ( Figure 2D ), which made the reports of its anticancer effects somewhat controversial ( Evans et al, 2017 ; Li et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

Tachyplesin Causes Membrane Instability That Kills Multidrug-Resistant Bacteria by Inhibiting the 3-Ketoacyl Carrier Protein Reductase FabG

Liu¹,

Qi²,

Shu³

et al. 2018

Front. Microbiol.

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Tachyplesin is a type of cationic β-hairpin antimicrobial peptide discovered in horseshoe crab approximately 30 years ago that is well known for both its potential antimicrobial activities against multidrug-resistant bacteria and its cytotoxicity to mammalian cells. Though its physical interactions with artificial membranes have been well studied, details of its physiological mechanism of action the physiological consequences of its action remain limited. By using the DNA-binding fluorescent dye propidium iodide to monitor membrane integrity, confocal microscopy to assess the intracellular location of FITC-tagged tachyplesin, and RNA sequencing of the differentially expressed genes in four Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa) treated with lethal or sublethal concentrations of tachyplesin, we found that compared with levofloxacin-treated bacteria, tachyplesin-treated bacteria showed significant effects on the pathways underlying unsaturated fatty acid biosynthesis. Notably, RNA levels of the conserved and essential 3-ketoacyl carrier protein reductase in this pathway (gene FabG) were elevated in all of the four bacteria after tachyplesin treatment. In vitro tests including surface plasmon resonance and enzyme activity assays showed that tachyplesin could bind and inhibit 3-ketoacyl carrier protein reductase, which was consistent with molecular docking prediction results. As unsaturated fatty acids are important for membrane fluidity, our results provided one possible mechanism to explain how tachyplesin kills bacteria and causes cytotoxicity by targeting membranes, which may be helpful for designing more specific and safer antibiotics based on the function of tachyplesin.

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Section: Discussionmentioning

confidence: 99%

Tachyplesin Causes Membrane Instability That Kills Multidrug-Resistant Bacteria by Inhibiting the 3-Ketoacyl Carrier Protein Reductase FabG

Liu¹,

Qi²,

Shu³

et al. 2018

Front. Microbiol.

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“…MTT assay. The MTT reduction assay, used to measure cell viability, was purchased from Sigma-Aldrich and used as we described earlier 51,80 . Cells were seeded in 96-well plates as indicated in 200 μL 10% FBS-supplemented media per well as described above and maintained overnight at 95% humidity and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…Apoptosis assays. For the caspase 3 (cleaved) colorimetric assays, activated (cleaved) caspase 3 and tubulin, were simultaneously measured in triplicate in whole cells by a colorimetric in cell ELISA assay (ThermoFisher) using 96-well microplates as we previously described 80,81 . Cells were plated per well and incubated overnight at 37 °C in 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

IGFBP-3 Blocks Hyaluronan-CD44 Signaling, Leading to Increased Acetylcholinesterase Levels in A549 Cell Media and Apoptosis in a p53-Dependent Manner

Price

Muterspaugh

Clegg

et al. 2020

Sci Rep

105

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Insulin-like growth factor binding protein-3 (IGFBP-3) belongs to a family of six IGF binding proteins. We previously found that IGFBP-3 exerts its cytotoxic effects on A549 (p53 wild-type) cell survival through a mechanism that depends on hyaluronan-CD44 interactions. To shed light on the mechanism employed, we used CD44-negative normal human lung cells (HFL1), A549, and H1299 (p53-null) lung cancer cells. A synthetic IGFBP-3 peptide ( 215 -KKGfYKKKQcRpSKGRKR-232 ) but not the mutant (K228AR230A), was able to bind hyaluronan more efficiently than the analogous sequences from the other IGFBPs. In a manner comparable to that of the IGFBP-3 protein, the peptide blocked hyaluronan-CD44 signaling, and more effectively inhibited viability of A549 cells than viability of either H1299 or HFL1 cell lines. Treatment with the IGFBP-3 protein or its peptide resulted in increased acetylcholinesterase concentration and activity in the A549 cell media but not in the media of either HFL1 or H1299, an effect that correlated with increased apoptosis and decreased cell viability. These effects were diminished upon the same treatment of A549 cells transfected with either p53 siRNA or acetylcholinesterase siRNA. Taken together, our results show that IGFBP-3 or its peptide blocks hyaluronan-CD44 signaling via a mechanism that depends on both p53 and acetylcholinesterase.Lung cancer is a devastating human disease and among the most common causes of cancer deaths worldwide 1,2 . Of all cases of the disease, non-small cell lung cancer (NSCLC) accounts for approximately 85% 3 .CD44 is a type 1 transmembrane cell-surface glycoprotein with tumor promoting functions in many types of cancer cells 4-7 . It is the main cell surface receptor for hyaluronan (HA) 5-9 . Found on the extracellular side of the cell membrane is the CD44 globular HA-binding domain (HABD) 9,10 shown previously to bind HA as a globular water-soluble protein 11 . CD44 is encoded by a single gene 5,6,12 and many different variant isoforms (CD44v) are generated by alternative splicing that yield different patterns of amino acid insertion into the stalk domain of CD44 with the smallest being the standard CD44 (CD44s) 5,13-15 . Residues 32-123 in the N-terminal domain of CD44, common to both CD44s and CD44v isoforms, contain the HA-binding motif 16 . Assessment of CD44 expression in human lung cancer cell lines 17 , including A549 and H1299 used in this study, showed that the predominant isoform expressed is CD44s 18 . Being a common marker for tumor-initiating cells/cancer stem cells in human carcinomas, CD44 has gained much attention in the cancer literature 14 . HA-CD44 binding is known to modulate numerous downstream signaling cascades, such as the ERK1/2/MAPK and PI3K/Akt pathways, leading to tumor cell proliferation, survival, chemoresistance, and invasiveness 5,7,12,19 .HA is a non-sulfated, anionic glycosaminoglycan 5,16,20,21 polymer composed of the disaccharide sequence (D-glucuronic acid and D-N-acetylglucosamine) without known post-synthetic modification 6,22-24 ...

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“…The liposome dye leakage assay was performed as we reported previously [67] to determine whether the peptides had any membrane-damaging properties. Lipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA), and carboxyfluorescein was purchased from Sigma-Aldrich.…”

Section: Liposome Dye Leakagementioning

confidence: 99%

“…Lipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA), and carboxyfluorescein was purchased from Sigma-Aldrich. Carboxyfluorescein-encapsulated liposome vesicles were created and tested as previously described [67]. Briefly, a 5 mg mixture of 3 : 1 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC, a zwitterionic lipid)/1-Palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (POPG, a negatively charged lipid) was weighed and dissolved in 2 mL chloroform; the chloroform was evaporated using nitrogen, leaving a thin lipid film which was dried in a vacuum desiccator overnight.…”

Section: Liposome Dye Leakagementioning

confidence: 99%

Biochemical determinants of the IGFBP‐3–hyaluronan interaction

et al. 2020

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IGFBP‐3, the most abundant IGFBP and the main carrier of insulin‐like growth factor I (IGF‐I) in the circulation, can bind IGF‐1 with high affinity, which attenuates IGF/IGF‐IR interactions, thereby resulting in antiproliferative effects. The C‐terminal domain of insulin‐like growth factor‐binding protein‐3 (IGFBP‐3) is known to contain an 18‐basic amino acid motif capable of interacting with either humanin (HN) or hyaluronan (HA). We previously showed that the 18‐amino acid IGFBP‐3 peptide is capable of binding either HA or HN with comparable affinities to the full‐length IGFBP‐3 protein and that IGFBP‐3 can compete with the HA receptor, CD44, for binding HA. Blocking the interaction between HA and CD44 reduced viability of A549 human lung cancer cells. In this study, we set out to better characterize IGFBP‐3‐HA interactions. We show that both stereochemistry and amino acid identity are important determinants of the interaction between the IGFBP‐3 peptide and HA and for the peptide's ability to exert its cytotoxic effects. Binding of IGFBP‐3 to either HA or HN was unaffected by glycosylation or reduction of IGFBP‐3, suggesting that the basic 18‐amino acid residue sequence of IGFBP‐3 remains accessible for interaction with either HN or HA upon glycosylation or reduction of the full‐length protein. Removing N‐linked oligosaccharides from CD44 increased its ability to compete with IGFBP‐3 for binding HA, while reduction of CD44 rendered the protein relatively ineffective at blocking IGFBP‐3‐HA interactions. We conclude that both deglycosylation and disulfide bond formation are important for CD44 to compete with IGFBP‐3 for binding HA.

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D-Amino Acid Analogues of the Antimicrobial Peptide CDT Exhibit Anti- Cancer Properties in A549, a Human Lung Adenocarcinoma Cell Line

Abstract: D-CDT is both an effective AMP and ACP, and likely exerts its anticancer effects through both membranolytic as well as an HA-mediated mechanism.

Cited by 17 publications

References 0 publications

Tachyplesin Causes Membrane Instability That Kills Multidrug-Resistant Bacteria by Inhibiting the 3-Ketoacyl Carrier Protein Reductase FabG

Tachyplesin Causes Membrane Instability That Kills Multidrug-Resistant Bacteria by Inhibiting the 3-Ketoacyl Carrier Protein Reductase FabG

IGFBP-3 Blocks Hyaluronan-CD44 Signaling, Leading to Increased Acetylcholinesterase Levels in A549 Cell Media and Apoptosis in a p53-Dependent Manner

Biochemical determinants of the IGFBP‐3–hyaluronan interaction

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