Fig 1.
Immunostaining for HPSE and HPSE2 in malignant and benign thyroid samples.
Left panel represents malignant thyroid tissues (PTC, papillary thyroid carcinoma; FVPTC, follicular variant of papillary thyroid carcinoma and FC, follicular carcinoma), whilst the middle panel represents benign thyroid tissues (HN, hyperplasic nodule, NG nodular goiter and normal adjacent thyroid tissue). Immunohistochemistry obtained using optical microscopy, X400. Right Panel (Transition area) demonstrates different pattern of HPSE2 labeling, showing adjacent normal thyroid tissue (arrow head) and follicular carcinoma (arrow) in the same photomicrography. Immunohistochemistry was obtained using light microscopy, X200. Legend: * represent colloidal areas in each photomicrography.
Fig 2.
HPSE and HPSE2 quantitative RT-PCR analysis.
The values are represented by box plots and demonstrate the high expression of HPSE2 in DTC (**P = 0.001, Mann-Whitney test) and also of HPSE but with just a statistical trend (*P = 0.067, Mann-Whitney test); (N = 27).
Table 1.
Heparanase enzymatic assay in DTC and benign thyroid lesions (N = 27).
Fig 3.
Proteins and heparan sulfate expression in thyroid tissues.
(A), Expression of heparanase-2 isoforms (HPSE2a, HPSE2b and HPSE2c); heparanase active enzyme (50 kDa) and pro-enzyme (65 kDa); heparan sulfate (HS). The samples were analyzed on a single sample of normal thyroid sample (“Normal”), and papillary thyroid carcinoma (“PTC”). (B), The values indicate relative expression obtained by the ratio of proteins and HS corrected by beta-actin expression.
Fig 4.
Immunostaining for HPSE2 in malignant and benign FNAB samples.
Left panel represents malignant thyroid tissues (two cases of PTC) and the right panel represents benign thyroid tissues. Immunocytochemistry obtained using light microscopy, X400, showed a different pattern of HPSE2 staining: high expression on follicular cells of PTC and none expression on benign lesions and also a negative colloid expression on PTC (*) and positive on nodular goiter.
Table 2.
HPSE2 patterns of immunostaining in DTC and benign lesions.
Fig 5.
Hypothetical scheme to explain herapanase physiology based on data of this study and also from Gengis-Velitski et al. [24], Levy-Adam et al. 2010 [25], Linke et al. 2002 [26], Zetzer et al. 2004 [27] and Nadav et al 2002 [17].
(I) In the normal thyroid follicle or benign lesions HPSE and HPSE2 are first targeted to the endoplasmic reticulum lumen (ER), shuttled to the Golgi apparatus (step 1), partially processed and activated in lysosomes (step 2), and both heparanases (HPSE and HPSE2) are highly secreted to colloid (step 3); HPSE is therefore partially internalized (step 4) and also activated in the lysosomes (step 5) and directed to the basolateral surface of follicular thyroid cells where it presents low activity (step 6). (II) In the differentiated thyroid carcinoma there is overexpression of both heparanases (step 1), increased processing and activation of HPSE in lysosomes (step 2) and low secretion to colloid (step 3) and follows that described in steps 4 and 5 below. An increased secretion of HPSE to basolateral surface and extracellular matrix occurs to degrade HSPG (step 6, large arrows). * Additional information: (Ia) low presence of latent HPSE and no presence of HPSE2 in the cytoplasm of normal / benign lesion follicular cell (Western blotting); (Ib) immunoexpression of both heparanases by immunohistochemistry: low positivity in follicular cells for HPSE and positive staining in colloid for HPSE2; (IIa) high immunostaining in neoplastic cells for HPSE2; (IIb) high immunostaining in neoplastic cells for HPSE and Western blotting demonstrating the presence of the active form (50 kDa). Legend: HPSE 65 kDa, ο; HPSE 50 kDa, οο; HPSE2, Δ.