Expression profiles of metallothionein-I/II and megalin/LRP-2 in uterine cervical squamous lesions

Abstract

Metallothioneins (MTs) are phylogenetically old cysteine-rich proteins, which are implicated in a variety of physiological and pathological processes. Their growth-regulating, anti-apoptotic and anti-inflammatory functions have been attributed not only to intracellular free radical scavenging and to zinc and copper regulation but also to the ability of secreted MT to bind on surface lipoprotein receptor-megalin/LRP2, which enables the endocytosis of MT-I/II and a wide range of other functionally distinct ligands. In the present study, we analysed the expression pattern of both proteins in 55 cases of premalignant transformation of cervical squamous cells, i.e. in low- and high-grade squamous intraepithelial lesion (LSIL and HSIL). The data showed that in LSIL (cervical intraepithelial neoplasia CIN1; N = 25) MTs were present only in basal and parabasal cells and that megalin was only weakly expressed. In HSIL (CIN2; N = 15 and CIN 3/carcinoma in situ; N = 15), however, overexpression and co-localization of MT with megalin were found in the entire hyperplastic epithelium. Moreover, megalin immunoreactivity appeared on the glandular epithelium and vascular endothelium, as well as on lymphatic cells in stroma. Besides, multiple megalin-positive cells expressed phosphorylated Akt1, implying that MT- and/or megalin-dependent prosurvival signal transduction pathways might contribute to the development of severe cervical dysplasia. The data emphasize the diagnostic power of combined MT/megalin analysis in pre-cancer screening.

Keywords: Akt1/protein kinase B phosphorylation; Biomarkers; CIN lesions, low-density lipoprotein receptor–related protein-2; Metallothionein-I/II; Tumour microenvironment.

Fig. 1

MT-I/II immunoreactivity in low- and high-grade squamous intraepithelial lesions. (A) Representative immunohistochemical pictures show staining with anti-MT-I/II antibody in paraffin-embedded sections of the cervical tissue samples, classified as normal cervix (a, b), LSIL (CIN1) (c, d) and HSIL, subdivided as CIN2 (e–h) and CIN3/CIS (i, j). (B) Negative (isotype-matched) control—staining of cervical tissue by mouse irrelevant IgG1 kappa immunoglobulin (a, b); Positive control—staining of murine hepatocytes after 1/3 partial hepatectomy by anti-MT-I/II antibodies (c, d) [28]. Scale bars 50 μm (A a–j; B a–c) and 20 μm (B d). (C) MT-I/II immunoreactivity in different SIL/CIN categories (expressed as total immunoreactive score (IRS) in squamous epithelium, in specific cell compartment and in mononuclear lymphatic cells (MNLC). IRS was calculated by taking the product of staining intensity (ranged from 0 to 3) and percentage positivity (ranged from 1 to 4). Values are expressed as mean ± SE. *p < 0.05, **p < 0.01 and ***p < 0.001 in comparison with basal layer in the intact cervix; #p < 0.05, ##p < 0.01 comparison of the basal and superficial layer in each group

Fig. 2

Megalin immunoreactivity in low- and high-grade squamous intraepithelial lesions. (A) Representative immunohistochemical pictures show staining with anti-megalin antibody in paraffin-embedded sections of the cervical tissue samples, classified as normal cervix (a–d), LSIL (CIN1) (c–h) and HSIL, subdivided as CIN2 (i–l) and CIN3/CIS (m–p). (B) Negative (isotype-matched) control—staining of cervical tissue by mouse irrelevant IgG1 kappa immunoglobulin (a, b); Positive control—staining of choroid plexus in cuprizone-treated mice by anti-megalin antibody [14]. Scale bars 100 μm (A a, e, i, m); 50 μm (all other pictures). (C) Megalin immunoreactivity in different SIL/CIN categories (expressed as total immunoreactive score (IRS) in squamous epithelium, in specific cell compartment, in mononuclear lymphatic cells (MNLC) and in glandular epithelium IRS was calculated by taking the product of staining intensity (ranged from 0 to 3) and percentage positivity (ranged from 1 to 4). Values are expressed as mean ± SE. ***p < 0.001 in comparison with findings in intact cervix

Fig. 3

MT-I/II and megalin expression and co-expression in HSIL (CIN2). Cells expressing MTs and megalin were detected by the use of anti-MT-I + II (red staining) and anti-megalin antibodies (green staining) in paraffin-embedded sections of the cervical tissue samples, classified as HSIL/CIN2 lesions. Blue marks DAPI staining of nuclei and yellow marks the overlapping of MT-I + II with megalin. Representative images show findings in squamous epithelium (a–i); in glandular epithelium and vascular endothelium (j–l) and in lymphatic infiltrates in stroma (m–o). Scale bars: 100 μm (a–c) and 50 μm (d–o)

Fig. 4

(A) MT-I/II and megalin expression and co-expression in HSIL (CIN3/CIS). Cells expressing MTs and megalin were detected by the use of anti-MT-I + II (red staining) and anti-megalin antibodies (green staining) in paraffin-embedded sections of the cervical tissue samples, classified as CIN3/CIS. Blue marks DAPI staining of nuclei and yellow marks the overlapping of MT-I + II with megalin. Representative images show findings in squamous epithelium (a–i); in glandular epithelium and lymphatic infiltrates in stroma (j–o). Scale bars: 100 μm (a–c) and 50 μm (d–o). B) MT-I/II and CD3 expression in stroma of CIN3/CIS. Representative images show the presence of T lymphocytes (green staining) in the vicinity of MT-I/II-positive cells (red staining). Scale bars 50 μm

Fig. 5

Megalin-positive cells express phosphorylated Akt-1/protein kinase B. Cells expressing megalin (green staining) and phospho-AKT1/protein kinase B (pAkt1) (red staining) were detected by the use of anti-megalin and anti-pAkt1 (phospho-Thr308) antibodies in paraffin-embedded sections of the cervical tissue samples, classified as HSIL (CIN2) (A) or CIN3/CIS (B). Blue marks DAPI staining of nuclei and yellow marks the overlapping of megalin with pAkt1. Scale bars: 50 μm (A a, b; B a–c) and 20 μm (A c)