Expression and localisation of kinin receptors in colorectal polyps

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Abstract

Kinins increase vascular permeability as well as mitogenesis and proliferation, hence they have a potential to promote neoplasmatic transformation. In the present study we investigated the expression profile and localization of kinin B1 and B2 receptors in colorectal polyps. The biopsy samples from various polyps were obtained during endoscopy in tubular (n = 18), villous (n = 15) and hyperplasic polyps (n = 15). The expression of genes encoding B1 and B2 was estimated by QRT-PCR TaqMan analysis. In second series B1 and B2 receptors were visualized by immunohistochemical staining in tissue specimens from colonic polyps and adjacent normal tissue. We found the highest expression of gene encoding B1 in tubular adenomas (1891 number of copies mRNA/μg total RNA ± 312 SE) which is significantly higher as compared with controls (683 ± 197 SE, p < 0.013). In contrast, the expression of gene for B2 was significantly increased in hyperplastic polyps (3852 ± 936 SE) as compared with controls (843 ± 263 SE, p < 0.0016). In normal colon a well as in hyperplasic polyps B1 and B2 receptors were immunohistochemically localized in enterocytes, however in hyperplastic polyps the intensity of staining was more prominent for B2 comparing to the control group. In contrast, in tubular adenomas staining reaction for B1 was more intense than in control samples. Increased level of B1 in adenoma suggests that kinins may play a role in abnormal cellular transformation; whereas higher B2 level in hyperplasic polyp suggests its protective role. Our data may indicate that the overall effect of kinins on cellular proliferation depends on the relative level of B1 and B2 receptor expression.

Introduction

Colorectal polyps are a heterogeneous family different in the genetic background, behaviour pattern and clinical approach. The most important group is adenoma; begin neoplasm that tends to evolve into colonic cancer. This process is typical for almost 95% of all colorectal cancers [1]. In contrast, the largest group of colorectal polyps is hyperplastic, one that have no neoplastic potential. Although adenomas and hyperplastic polyps are histopatologically different both may be considered as a pathological growth of colonic epithelium. Among factors, which may influence cellular growth and proliferation are kinins, endogenous peptides generated by cleavage protein kininogen by enzyme kallikrein. Kinins may increase vascular permeability [2], stimulate angiogenesis [3], and cellular proliferation [4], [5].

There are two types of kallikrein; plasma kallikrein and tissue kallikrein. Tissue kallikrein [TK]-serine protease, generates kinins by proteolysis of low molecular weight kininogen, whereas plasma kallikrein cleaves high molecular weight kininogen to generate kinins. TK releases lysyl-bradykinin (itself a potent kinin), which may be converted to bradykinin [BK]. BK has a short half time of about 30 s, and then it is metabolized by kininase I to des-Arg-BK, also an active form, which has much longer half time of about 2 h. Kininase II [angiotensin converting enzyme — ACE] finally metabolizes kinins to their inactive forms [6].

Kinins act via two receptors; the constitutive B2 type and inducible B1 type. B1 receptor is sensitive mainly to the kinin active metabolites as des-arg9-BK and des-arg10kallidin, whereas B2 receptor is more sensitive to BK and kalidin. In physiological conditions, B2 receptor predominates in tissues whereas B1 receptor seems to be expressed in pathological conditions such as neoplasm, inflammation, and tissue injury. Kinin receptor profile may change in various pathological states. This indicates that total effect of kallikrein–kinin system is in part regulated by relative levels of kinin receptors [7].

The role of kallikrein–kinin system in cancer is recently under investigation. There are strong evidences supporting the important role of kinins in angiogenesis [3], a key factor for tumour growth and cancer metastases [8]. For example, in vitro data indicate that addition of Captopril–ACE inhibitor, and subsequent accumulation of kinins in a tumour tissue, lead to angiogenesis stimulation and in turn accelerate tumour growth [9]. Recently some authors using immunohistochemical methods documented different kinin receptors profile in human esophageal [10], and gastric cancer tissues [11], but they not investigate genes expression. Until now kinins and kinin receptors has not been investigated in human colorectal polyps and cancer. In the present study we evaluate the kinin receptors expression and profile in human adenomas and hyperplastic polyps using real time RT-PCR and immunohistochemical technique.

Section snippets

Patients and controls

Patients were studied in accordance with the protocol approved by the Institutional Committee on Human Subjects of the Medical University of Silesia. All patients gave written, informed consent. Consecutive 41 patients were examined. There were 19 women and 22 men, mean age 59 years. The including criteria to the study were the presence of adenoma(s) and/or hyperplastic polyp(s) in the colon. There were 48 polyps found in those patients. The biopsy samples from polyps were obtained during

Statistical analysis

We searched for significant differences in gene expression (BDKRB1 and BDKRB2 subsequently) between each of polyps group (tubular adenoma, villous/tubulo–villous adenoma, hyperplastic polyps) as well as controls (normal tissue). The significance of the difference between medians of groups was determined by Wilcoxon test. All values were expressed as median ± SE.

B1 and B2 expression

For the first time we documented transcriptional activity of B1 and B2 receptor genes in adenomas and hyperplastic polyps. The transcriptional activity showed different profile in colorectal polyps and in controls as presented in Table 1. We found the highest expression of genes encoding B1 in tubular adenomas (1891 ± 312 SE), which is significantly higher as compared with controls (683 ± 197 SE, p < 0.013). We also found higher level of B1 receptors gene expression in villous adenomas (1398 ± 522 SE),

Discussion

In the present study for the first time, we documented changes in the expression profile of kinin receptors in colorectal adenomas and hyperplastic polyps. This profile seems to be related to histological type of the polyp, thus transcriptional activity of kinin receptors genes in examined polyps is in accordance with kinin receptors protein pattern.

A presence of the kallikrein–kinin system components in colonic tissue has been already documented. In our recent study we visualized

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