Human β-defensin 3 contains an oncolytic motif that binds PI(4,5)P2 to mediate tumour cell permeabilisation

Phan, Thanh Kha; Lay, Fung T.; Poon, Ivan K. H.; Hinds, Mark G.; Kvansakul, Marc; Hulett, Mark D.

doi:10.18632/oncotarget.6520

Cited by 45 publications

(77 citation statements)

References 84 publications

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“…Some studies indicated that defensin promoted cancer growth (21,22), whereas other studies showed that defensin is cytotoxic (16,17,(23)(24)(25)(26). Our results suggest that the cytotoxic effect is specific to hBD3 and its mouse homolog, Defb14, among members of the defensin family.…”

Section: Figure 3 Time Dependency Of Cellular Damage Induced By Humamentioning

confidence: 60%

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In Vitro and In Vivo Anticancer Activity of Human β-Defensin-3 and Its Mouse Homolog

Hanaoka

Yamaguchi

Yamamoto

et al. 2016

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Abstract. Background Antimicrobial peptides have emerged as a part of the host defense mechanism in both animals and plants (1, 2). Specifically, defensins and cathelicidins are antimicrobial peptides found in humans (3). In the defensin family, there are α-defensin and β-defensin subfamilies, which conserve three specific disulfide pairings. These are produced by leukocytes and various types of epithelial cells constitutively, or in response to microbial signals and inflammatory cytokines (4-6).Human β-defensin-3 (hBD3) was first isolated from human skin (7) and its expression was detected in many tissues, including airway epithelial cells (8,9). In contrast to other defensin isoforms, the prominent features of hBD3 antimicrobial activity are its salt-independency and its broad antimicrobial spectrum (7, 10, 11).While antimicrobial peptides have attracted attention as potential molecules to treat cancer (12), previous observations on the anticancer effects of hBD3 have not been consistent. Some studies hypothesized that hBD3 can be oncogenic because hBD3 is frequently overexpressed in oral squamous cell carcinomas (13-15). Winter et al. indicated that hBD3 inhibited colon cancer-cell migration, although not cell proliferation (16). Moreover, Phan et al. indicated that hBD3 exhibits anticancer effects on various tumor cells through cell permeabilization (17).Here, we investigated the anticancer activities of various isoforms of β-defensin on lung cancer cells, and confirmed that this anticancer activity is also seen in animal models of cancer. Materials and MethodsReagents. Synthetic hBD1, hBD2, hBD3 and human neutrophil peptide-1 (HNP1) peptides were purchased from the PEPTIDE institute (Minoh, Japan), and were dissolved in 0.001% acetic acid to give a final concentration of 2 mg/ml.We predicted the mature Defb14 peptide spanning 45 COOHterminal amino acids as a mouse homolog of hBD3, identical to the predicted mature peptide of Defb14 from a previous report (18). At the Peptide Institute (Minoh, Japan), we chemically synthesized mature Defb14 peptide (19,20). The synthetic peptide was airoxidized to form three disulfide bonds. The material was eluted as a 5999 *These Authors contributed equally to this study.Correspondence to: Yasuhiro Yamaguchi, Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan. Tel: +81 358008652, Fax: +81 358006530, e-mail: yamayasu-tky@umin.ac.jp Key Words: Antimicrobial peptide, cytotoxicity, membrane damage. ANTICANCER RESEARCH 36: 5999-6004 (2016) single peak on reverse-phase high-performance liquid chromatography and confirmed by mass spectroscopy. It was lyophilized and dissolved in 0.001% acetic acid to give a final concentration of 2 mg/ml.Cell culture. A549 human lung carcinoma cells were obtained from the Cell Resource Center for Biomedical Research, Tohoku University (Sendai-shi, Japan). Lewis lung carcinoma (LLC) cells were obtained from RIKEN BRC (Tsukuba-shi, Japan). The cells were routine...

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Section: Figure 3 Time Dependency Of Cellular Damage Induced By Humamentioning

confidence: 60%

“…Winter et al indicated that hBD3 inhibited colon cancer-cell migration, although not cell proliferation (16). Moreover, Phan et al indicated that hBD3 exhibits anticancer effects on various tumor cells through cell permeabilization (17).…”

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confidence: 99%

In Vitro and In Vivo Anticancer Activity of Human β-Defensin-3 and Its Mouse Homolog

Hanaoka

Yamaguchi

Yamamoto

et al. 2016

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“…Moreover, substantial inhibition of HBD‐3‐induced TNF and IL‐6 release was also observed upon treatment of HBD‐3 with synthetic PI(4,5)P 2 and other PIPs, but not PA, or the incubation of monocytes with PI(4,5)P 2 ‐sequestering neomycin (Figure ). In our previous report, HBD‐3 was demonstrated to internalize and directly bind to cellular PI(4,5)P 2 , while the HBD‐3 mutants, synthetic PIPs and neomycin were shown to impede this interaction (and PI(4,5)P 2 ‐mediated cancer cell membrane permeabilization by HBD‐3) . The HBD‐3:PI(4,5)P 2 interaction is not simply based on “non‐specific” electrostatic forces, as it appears to be structurally‐dependent.…”

Section: Discussionmentioning

confidence: 96%

“…Interestingly, HBD‐3 internalization into leukocytes has been consistently reported, although its relevance in immunomodulation is currently unknown . In our previous report,HBD‐3 was demonstrated to internalize into mammalian cells and bind to phosphatidylinositol 4,5‐bisphosphate [PI(4,5)P 2 ] at the inner leaflet through its cationic β2‐β3 loop (K 32 CSTRGRK 39 ), with K32 and K39 being particularly important for PI(4,5)P 2 interaction. PI(4,5)P 2 and other phosphorylated derivatives of phosphatidylinositol, which are collectively known as phosphoinositides (PIPs), are minor membrane phospholipids but serve important roles in diverse cellular processes.…”

Section: Introductionmentioning

confidence: 99%

Importance of phosphoinositide binding by human β‐defensin 3 for Akt‐dependent cytokine induction

2017

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Host defense peptides (HDPs) are well-characterized for their antimicrobial activities but also variously display potent immunomodulatory effects. Human β-defensin 3 (HBD-3) belongs to a well-known HDP family known as defensins and is able to induce leukocyte chemotactic recruitment, leukocyte activation/maturation, proinflammatory cytokine release, and co-stimulatory marker expression. HBD-3-stimulated cytokine induction is NF-κB-dependent and was initially suggested to act via G protein-coupled C-C chemokine receptor phospholipase C (PLC) and/or Toll-like receptor signaling. Subsequent pharmacological inhibition, however, revealed that NF-κB activation by HBD-3 is receptor-independent and instead involves the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) pathway, the mechanism of which remains undetermined. Recently, we have shown that HBD-3 can enter mammalian cells and bind to inner membrane phosphoinositide 4,5-bisphosphate [PI(4,5)P], an important second lipid messenger of PLC and PI3K-Akt pathways. In this study, we report that the interaction of HBD-3 with PI(4,5)P is important for PI3K-Akt-NF-κΒ-mediated induction of tumor necrosis factor and interleukin-6. These data provide insights into the mechanism of immunomodulation by HBD-3, and more generally, highlight the complex multifaceted signaling roles of HDPs in innate defense. Furthermore, it is suggested that the proposed mode of action may be conserved in other HDPs.

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“…LPA, lysophosphatidic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; pPI(3)P, phosphatidylinositol 3-phosphate; PI(4)P, phosphatidylinositol 4-phosphate; PI(5)P, phosphatidylinositol 5-phosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine 1-phosphate; PI(3,4)P 2 , phosphatidylinositol 3,4-bisphosphate; PI(3,5)P 2 , phosphatidylinositol 3,5-bisphosphate; PI(4,5)P 2 , phosphatidylinositol 4,5-bisphosphate; PI(3,4,5)P 3 , phosphatidylinositol 3,4,5-trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine; blue blank, lipid-negative control. (23) and blocks the antitumor activity of NaD1 and other defensins that bind to PI(4,5)P 2 (11,17). To determine whether neomycin could block the antifungal activity of NaD1, we employed the model yeast S. cerevisiae, as the antifungal mechanism of action of NaD1 is conserved between filamentous fungi and yeast (21,24).…”

Section: Figmentioning

confidence: 99%

Nicotiana alata Defensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant

Bleackley

Payne

Hayes

et al. 2016

Antimicrob Agents Chemother

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bThe plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized ␣-␤ motif which consists of an ␣ helix and a triple-stranded ␤-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P 2 binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P 2 , suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants.

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Human β-defensin 3 contains an oncolytic motif that binds PI(4,5)P2 to mediate tumour cell permeabilisation

Cited by 45 publications

References 84 publications

In Vitro and In Vivo Anticancer Activity of Human β-Defensin-3 and Its Mouse Homolog

In Vitro and In Vivo Anticancer Activity of Human β-Defensin-3 and Its Mouse Homolog

Importance of phosphoinositide binding by human β‐defensin 3 for Akt‐dependent cytokine induction

Nicotiana alata Defensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant

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