Evaluation of serum and tissue levels of VAP-1 in colorectal cancer

Abstract

Background: The endothelial adhesion molecule, vascular adhesion protein-1 (VAP-1, AOC3) promotes lymphocyte recruitment to tumours, although the contribution that VAP-1 makes to lymphocyte recruitment in human colorectal cancer (CRC) is unknown. VAP-1 exists in circulating soluble form (sVAP-1). A previous study demonstrated elevated sVAP-1 levels in CRC patients. The aim of this study was to confirm this finding and study the differences in tissue VAP-1 expression between CRC and healthy tissues.

Methods: sVAP-1 levels were measured in the serum of 31 patients with CRC and 31 age- and sex-matched controls. Tissue VAP-1 levels were measured by immunohistochemistry, quantitative real-time PCR and Western blotting.

Results: The mean sVAP-1 level ± SD was significantly lower in the CRC group compared with the control group (399 ± 138 ng/ml versus 510 ± 142 ng/ml, P = 0.003). Tissue VAP-1 protein and mRNA levels were significantly lower in CRC compared with normal colon tissue. VAP-1 immunostaining was practically absent from CRC.

Conclusions: VAP-1 is downregulated in human CRC and although the molecular basis of this down regulation is not yet known, we suggest it may be part of a mechanism used by the tumour to prevent the recruitment of anti-tumour immune cells. Our data contradicts the findings of others with regard sVAP-1 levels in patients with CRC. Possible reasons for this are discussed.

Fig. 1

Serum VAP-1 (sVAP-1) levels measured in patients with CRC and controls. The bars indicate the mean ± one standard deviation. Differences between groups were tested for statistical significance using a two-tailed unpaired t-test

Fig. 2

a VAP-1 protein expression by Western blot for 8 normal colon (NC) and matched CRC samples. The vertical black line indicates separate gels. b Protein band densitometry of VAP-1/GUSB shows a statistically significant reduction in VAP-1 protein expression in CRC tissue compared with matched colon. c VAP-1 gene expression by 8 normal colon and CRC samples shows a statistically significant reduction in VAP-1 mRNA expression in CRC tissues compared with matched colon, relative to GUSB and IPO8 expression. The bars indicate the mean ± one standard deviation. Differences between groups were tested for statistical significance using a two-tailed unpaired t-test

Fig. 3

Paraffin-embedded sections from 3 matched normal colon (NC) and CRC samples demonstrating positive VAP-1 staining in the lamina propria and submucosa of the colon with only weak staining in CRC stroma. Magnification x200. VAP-1 staining is brown-red, haematoxylin staining is blue. MM = muscularis mucosae; SM = submucosal; LP = lamina propria; St = tumour stroma; Epi = tumour epithelium

Fig. 4

VAP-1 expression in flanking normal colon (NC) tissue abruptly disappears at the border with CRC tissue. Dot-dash line marks the border between NC and CRC. Magnification x200. VAP-1 staining is brown-red, haematoxylin staining is blue

Fig. 5

Dual-colour immunofluorescent staining of sections of normal colon tissue and cultured colonic endothelial cells. Magnification x630. a VAP-1 staining is present in SMA+ cells of vessels in sections of colonic mucosa. b VAP-1 staining does not co-localise with CD31+ endothelial staining of vessels in sections of colonic mucosa. Right-most images are digitally magnified x3. Yellow staining represents autofluorescence from erythrocytes. c Positive CD31 staining (left) and absent VAP-1 staining (right) of cultured colonic endothelial cells