Hyaluronidases and their inhibitors in the serum of colorectal carcinoma patients

Background: Colorectal cancer is the third most commonly diagnosed type of cancer. Hyaluronan is involved in this malignancy. Moreover, hyaluronidases – its degrading enzymes - display controversial role regarding their involvement in tumor development. Hyal-1 is the major tumor derived hyaluronidase. The aim of the study was the determination and evaluation of hyaluronidase levels in serum of colorectal cancer patients, before and after surgery, with a view to assessing its potential role as a tumor marker for recurrence.Methods and Results:By zymography and western blotting, it was confirmed that Hyal-1 was the only hyaluronidase present in samples. Quantification of its activity indicated a statistically significant decrease in samples seven days postoperatively, compared with the respective ones before surgery. Hyal-1 levels before surgery were significantly reduced in comparison with normal samples and samples one year postoperatively. Hyaluronidase inhibitor activity was demonstrated in mild alkaline conditions via reverse zymography. A statistically significant increase was observed in samples seven days postoperatively, when compared with samples before surgery. Hyal-1 levels in sera of colorectal cancer patients were lower than those of normal population, possibly because of the local accumulation of the enzyme in tumor microenvironment.Conclusions:A gradual elevation up to one year postoperatively to reach normal levels might indicate a role of Hyal-1 levels in cancer.Εισαγωγή – Σκοπός: Ο καρκίνος του παχέος εντέρου είναι ο τρίτος σε συχνότητα τύπος καρκίνου. Σύμφωνα με μελέτες, το υαλουρονικό οξύ φαίνεται να έχει σημαντικό ρόλο στην ανάπτυξη της συγκεκριμένης κακοήθειας. Επιπλέον, οι υαλουρονιδάσες, τα ένζυμα δηλαδή που είναι υπεύθυνα για τον καταβολισμό του υαλουρονικού οξέως έχουν ασαφή ρόλο στην εξέλιξη του καρκίνου. Ο σκόπος της παρούσας μελέτης ήταν να καθοριστούν και να αξιολογηθούν τα επίπεδα της υαλουρονιδάσης στον ορό ασθενών με καρκίνο του παχέος εντέρου, τόσο πριν όσο και μετά τη χειρουργική αντιμετώπιση. Απώτερος στόχος της μελέτης ήταν να αξιολογηθεί το κατά πόσο τα επίπεδα της υαλουρονιδάσης στον ορό θα μπορούσαν να αποτελέσουν βιοχημικό καρκινικό δείκτη της νόσου και των υποτροπών της .Υλικό – Μέθοδος :Πρόκειται για μια προοπτική, τυχαιοποποιημένη, διπλή τυφλή μελέτη. Το βιολογικό υλικό ήταν δείγματα αίματος που ελήφθησαν από 15 ασθενείς με καρκίνο του παχέος εντέρου σε διάφορα στάδια. Τα δείγματα ελήφθησαν προεγχειρητικά και μετεγχειρητικά σε τακτά χρονικά διαστήματα και συγκρίθηκαν με δείγματα από τον ορό υγιών δοτών. Οι μέθοδοι που χρησιμοποιήθηκαν ήταν ζυμογράφημα, ανάστροφο ζυμογράφημα και ανοσοαποτύπωσηΑποτελέσματα :Το ζυμογράφημα και η ανοσοαποτύπωση επιβεβαίωσαν ότι η μόνη υαλουρονιδάση που βρέθηκε στον ορό ήταν η Hyal-1. Τα επίπεδά της παρουσίασαν σημαντική μείωση μια εβδομάδα μετεγχειρητικά σε σύγκριση με τα προεγχειρητικά επίπεδα. Τα επίπεδα της Hyal-1 των ασθενών με καρκίνο προεγχειρητικά ήταν σημαντικά μικρότερα σε σχέση με τα επίπεδα των υγιών δοτών, καθώς και σε σχέση με τα επίπεδα των ίδιων των ασθενών με καρκίνο ένα χρόνο μετεγχειρητικά. Μέσω ανάστροφου ζυμογραφήματος βρέθηκε η128δραστικότητα ενός αναστολέα της υαλουρονιδάσης, ενεργού σε ήπια αλκαλικό pH. Τα επίπεδα του αναστολέα της υαλουρονιδάσης αυξήθηκαν στατιστικώς σημαντικά (p<0,01) μια εβδομάδα μετεγχειρητικά σε σχέση με τα προεγχειρητικά επίπεδα. Τα επίπεδα της Hyal-1 στον ορό ασθενών με καρκίνο του παχέος εντέρου, προεγχειρητικά, ήταν μικρότερα από τα αντίστοιχα υγιών δοτών, πιθανώς εξαιτίας της τοπικής άθροισης του αναστολέα της υαλουρονιδάσης στο μικροπεριβάλλον του όγκου.Συμπεράσματα :Η σταδιακή αύξηση των επιπέδων της Hyal-1 στον ορό ασθενών με καρκίνο του παχέος εντέρου, μέχρι και ένα χρόνο μετεγχειρητικά, όπου φτάνουν τα επίπεδα του υγιούς πληθυσμού, μπορεί να υποδεικνύει ότι η Hyal-1 έχει σημαντικό ρόλο στον καρκίνο

Involvement of hyaluronidases in colorectal cancer

Abstract Background Hyaluronidases belong to a class of enzymes that degrade, predominantly, hyaluronan. These enzymes are known to be involved in physiological and pathological processes, such as tumor growth, infiltration and angiogenesis, but their exact role in tumor promotion or suppression is not clear yet. Advanced colorectal cancer is associated with elevated amounts of hyaluronan of varying size. The aim of the present study was therefore to illuminate the importance of hyaluronidases in colon carcinoma progression. Methods The patients' samples (macroscopically normal and cancerous) were subjected to sequential extraction with PBS, 4 M GdnHCl and 4 M GdnHCl - 1% Triton X-100. The presence of the various hyaluronidases in the extracts was examined by zymography and western blotting. Their expression was also examined by RT-PCR. Results Among hyaluronidases examined, Hyal-1, -2, -3 and PH-20 were detected. Their activity was higher in cancerous samples. Hyal-1 and Hyal-2 were overexpressed in cancerous samples, especially in advanced stages of cancer. Both isoforms were mainly extracted with PBS. Hyal-3 was observed only in the third extract of advanced stages of cancer. PH-20 was abundant in all three extracts of all stages of cancer. The expression of only Hyal-1 and PH-20 was verified by RT-PCR. Conclusion A high association of hyaluronidases in colorectal cancer was observed. Each hyaluronidase presented different tissue distribution, which indicated the implication of certain isoforms in certain cancer stages. The results provided new evidence on the mechanisms involved in the progression of colorectal cancer.</p

ADAMTS expression in colorectal cancer.

ADAMTSs are a family of secreted proteinases that share the metalloproteinase domain with matrix metalloproteinases (MMPs). By acting on a large panel of extracellular substrates, they control several cell functions such as fusion, adhesion, proliferation and migration. Through their thrombospondin motifs they also possess anti-angiogenic properties. We investigated whether ADAMTSs participate in colorectal cancer progression and invasion. Their expression was investigated at both mRNA and protein levels. Using RT-PCR, the expression of ADAMTS-1, -4, -5 and ADAMTS-20 was estimated in colorectal tumors of different cancer stage and anatomic site and 3 cell lines of different aggressiveness. An overexpression of ADAMTS-4 and -5 was observed, especially in tissue samples, whereas ADAMTS-1 and -20 were found to be down-regulated. Western blot analysis further supported the RT-PCR findings, revealing in addition the degradation of ADAMTS-1 and -20 in cancer. In situ expression and localization of ADAMTS-1, -4, -5 and -20 was also investigated by immunohistochemical analysis. Our data suggest a positive correlation between ADAMTS-4 and -5 expression and cancer progression, in contrast with the anti-angiogenic members of the family, ADAMTS-1 and -20, which were found to be down-regulated. Our findings support the notion that overexpression of ADAMTS-4 and ADAMTS-5 in colorectal cancer might be a possible invasive mechanism of cancer cells in order to degrade proteoglycans of ECM

Expression of ADAMTSs in healthy and cancerous colon tissues.

<p>Semi-quantitative RT-PCR analysis of (A) ADAMTS-1, (C) ADAMTS-4, (E) ADAMTS-5 and (G) ADAMTS-20. Values are the mean GAPDH-normalized ± S.D. Protein distribution of (B) ADAMTS-1, (D) ADAMTS-4, (F) ADAMTS-5 and (H) ADAMTS-20. *<i>P</i>≤0.05; statistically significant differences compared to healthy tissues. **<i>P</i>≤0.05; statistically significant differences compared to stage A. ***<i>P</i>≤0.05; statistically significant differences compared to stage B. Healthy Cecum, Rectum and Sigmoid colon tissues are shown; <i>A-C</i>: Duke’s CRC stages. Arrows indicate the migration of molecular mass markers. PBS, GdnHCl and Triton are the sequential extracting solutions used (see text).</p

Nucleotide sequence of the primers and Hybridization Temperature for each primer set used in RT-PCR experiments.

<p>Nucleotide sequence of the primers and Hybridization Temperature for each primer set used in RT-PCR experiments.</p

Immunohistochemical localization of ADAMTS-4 and ADAMTS-5 in healthy and in cancerous colon.

<p>Healthy colon tissue and cancerous colon of stage A, B, C and D, stained for ADAMTS-4 (A) and ADAMTS-5 (B). (A, magnification; x4, anatomic site; cecum, magnification; x10, anatomic site; cecum, magnification; x20, anatomic site; cecum, magnification; x4, anatomic site; rectum, magnification; x20, anatomic site; rectum and B, magnification; x4, anatomic site; cecum, magnification; x10, anatomic site; cecum, magnification; x20, anatomic site; cecum, magnification; x4, anatomic site; rectum, magnification; x10, anatomic site; rectum), stained for ADAMTS-5. Insets of A show the Mucosa and Submucosa layer on the healthy colon (magnification; x10) and the cytoplasmic localization of ADAMTS-4 in cancer cells of stage C and D (magnification; x20 and x40, respectively). Insets of B show the Mucosa and Submucosa layer on the healthy colon (magnification; x10) and the cytoplasmic localization of ADAMTS-5 in cancer cells of stage C (magnification; x20). Arrows in healthy colon show the expression of ADAMTS in the muscle tissue. Arrows in cancerous colon show the expression of ADAMTS in stroma, while arrowheads show the expression of ADAMTS in cancer cells.</p

Immunohistochemical localization of ADAMTS-1 and ADAMTS-20 in healthy and in cancerous colon.

<p>Healthy colon tissue and cancerous colon of stage B and C, stained for ADAMTS-1 (A) and ADAMTS-20 (B). (A, magnification; x4, anatomic site; cecum, magnification; x4, anatomic site; cecum, magnification; x4, anatomic site; rectum and B, magnification; x4, anatomic site; cecum, magnification; x20, anatomic site; cecum, magnification; x20, anatomic site; rectum). Insets of A and B (magnification of all; x10) show the Mucosa and the Submucosa layer. Arrows in healthy colon show the expression of ADAMTS in the muscle tissue. Arrows in cancerous colon show the expression of ADAMTS in stroma, while arrowheads show the expression of ADAMTS in cancer cells.</p