Human defensins as cancer biomarkers and antitumour molecules
Introduction
Mammalian defensins are endogenous peptides produced by certain leukocytes and epithelial cells. These peptides demonstrate antimicrobial, antiviral, toxin-neutralizing and immuno-modulatory properties. They are small, cationic, beta-sheet, tridisulphide peptides and, in humans, comprise genetically distinct alpha and beta subfamilies that evolved from a common ancestral gene [1]. Similar cysteine-rich antimicrobial peptides, also called defensins, are found in plants, fungi and invertebrates and share many structural features and activities with human α and β-defensins [2]. It is noticeable that neutrophils of mice lack defensins completely but contain cathelicidin peptides [3]. Neutrophils of sheep and pigs also lack defensins whereas those of cattle and chicken contain β and no α-defensins [2], [4]. Humans have lost the ability to produce a cyclic theta-defensin that is found only in nonhuman primates [1].
In humans, α-defensins are mainly packaged in azurophil granules of neutrophils or secreted by intestinal Paneth cells, while β-defensins are constitutively expressed in various mucosa and epithelial cells where they can be up-regulated in response to infectious and inflammatory stimuli. Humans produce six different α-defensins, including 4 peptides (HNP-1 to HNP-4) in neutrophils and 2 peptides (HD5 and HD6) in Paneth cells of the small bowel. β-defensins, which are phylogenically older, more basic and slightly longer (36 or more amino-acid residues), form a rapidly growing family. The first human β-defensin hBD1 was identified in 1995 [6]. Five other hBD peptides have been partly characterized in the following years and a recent analysis of the human genome identified almost 40 potential coding regions for β-defensins [4]. α-defensin and β-defensin families do not share DNA sequence similarity or disulfide topology but possess highly similar tertiary structures with comparable antimicrobial activity [2].
A deregulated expression and secretion of α- and β-defensins has been described in a number of tumour types [7], [8], [9], [10], [11], [12], [13]. Reasons for this deregulated expression and the role of defensins in oncogenesis remain poorly understood. As identification of α-defensins in tumours and biological fluids was mostly based on the use of surface enhanced laser desorption/ionisation time-of-flight (SELDI-TOF) mass spectrometry (MS), the method itself could artificially favour the detection of these small size peptides over other markers. It was also though initially that α-defensin expression in tumour tissues reflected infiltration by polymorphonuclear cells [8]. Actually, both HNP1–3 mRNA and the proteins were found overexpressed in human tumour cell lines [7] and microdissected fresh tumour cells [9] and α-defensins HNP1–3 secreted by tumour cells in biological fluids was tested as tumour biomarker [9], [14]. The role of β-defensins in human tumours is still poorly explored but recent data suggest that β-defensin-1 (HBD-1) could behave as a tumour suppressor gene [13], [15]. The present paper summarises current knowledge of defensins and discuss their versatile influence on tumour growth, their potential use as tumour biomarkers and their place in anticancer therapeutic strategies.
Section snippets
Human alpha- and beta-defensins
Human neutrophil α-defensins are termed “human neutrophil peptides” or “HNP”. There are four HNP (1, 2, 3 and 4) in which HNP-1, HNP-2 and HNP-3 represent the most abundant bactericidal factor compounds stored in the azurophilic granules (more than 30% of the total proteins in these granules; up to 5–7% of total cellular neutrophil proteins). Defensins are the major component of an oxygen-independent system used by scavenger cells to eliminate invading microorganisms.
Mature HNP1–3 are 3 kDa
Human defensin genes
Several defensin genes are organized into clusters within a two megabase region of human chromosome 8q23.1 (Fig. 1B). Variability in copy number has been documented for several of these genes [35], [36], [37]. Within the α-defensin cluster, DEFA1 (encoding HNP1) and DEFA3 (encoding HNP3) are consistently found variable in copy from four to fourteen per diploid genome. All other genes of this first cluster are invariant with two copies per diploid genome [38], [39]. About 10% of people lack DEFA3
Human defensins in biological fluids
Alpha-defensins secreted by neutrophils can be detected in biological fluids [52], [53], [54], [55]. The concentration of HNP1–3 in human plasma under normal physiological conditions is about 40 ng/ml, as measured by ELISA [52]. This concentration increases 2- to 4-fold in patients with an inflammatory syndrome and reaches micromolar concentrations in septic patients [53]. In the plasma, HNP1–3 bind unspecifically to high mass plasma proteins such as serum albumin, α2-macroglubulin and C1
Human α-defensins in innate immunity
Neutrophils are the first cells that are recruited to the sites of infection. At these inflammatory sites, they serve as professional phagocytes, which rapidly engulf and kill micro-organisms by oxygen-dependent and -independent mechanisms. Defensins exert their antimicrobial activity in distinct niches such as the phagolysosomes of human neutrophils and the lumen of the crypts of Lieberkühn where bacteria are exposed to high concentrations (1–10 mg/ml) of HNPs in appropriate environment [1],
Human α-defensins in adaptive immunity
In the presence of serum or physiological salt concentrations, the direct microbicidal activity of HNP1–3 is reduced [1], [45]. This observation suggested that these cationic peptides might exert non microbicidal functions and evidence accumulated that several human defensins could promote a local innate inflammatory response and a systemic adaptive response by serving as chemoattractants and activators of immune cells, linking innate and adaptive immunity [1], [59]. For example, HNP-2 and hBD2
Human defensins as cancer biomarkers
In the last years, several studies have detected defensins in malignant cells and biological fluids from patients with tumours. Except in older studies, most authors used SELDI-TOF-MS and confirmed their observations by additional techniques, mostly immunohistochemistry and sometimes RT-PCR and flow cytometry.
Using SELDI-TOF-MS, several groups have detected elevated HNP1–3 in plasma and tumour tissue of patients with colorectal cancer [9], [14]. The most demonstrative study was performed on
Defensins as therapeutic tools in cancer
Defensins have pharmacological potential, especially considering their strong antimicrobial and antiviral activity [94], e.g. as broad-spectrum topical microbicides. The cytotoxic effect of defensins on cancer cells, including both solid tumours and leukemias, has also stimulated interest in these peptides as possible leads for new cytotoxic drugs. Defensins could kill tumour cells by a unique mechanism that involves membrane lysis and DNA damage [1], [97], [98], [99], [100]. Recently, a
Conclusions
Proteomic analysis of tumours has identified an abnormally high expression of human α-defensins, mainly HNP1–3 that are normally found in neutrophils, in a variety of tumour cells. Immunostaining suggests that these peptides are expressed in the tumour cell cytoplasm and at the tumour cell surface, in association with HLA class II antigens. The molecular mechanisms that induce their overexpression and their role in oncogenesis remain to be clarified. Depending on the experimental conditions,
Acknowledgements
Our group is supported by the Ligue Nationale Contre le Cancer (labelled team), la Fondation de France, Committee against Leukemia, Centpoursanglavie association, INCa and ANR.
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