Human neutrophil peptides 1,2 and 3 (HNP 1–3): elevated serum levels in colorectal cancer and novel marker of lymphatic and hepatic metastasis

Abstract

Objective. Biological levels of localized colon tumors are needed for the diagnosis and prevention of colon cancer. Numerous biomarker studies are produced currently through research. We investigated some peptides with the aim of identifying biomarkers for colorectal cancer and lymphatic and hepatic metastasis. Materials and methods. A total of 100 patients with histologically proven colorectal cancer and 60 normal volunteers were included in this study. Preoperative serum was collected for analysis. Human neutrophil peptides levels (HNP) were assayed by solid phase RIA (Radio Immun Assay) based on monoclonal antibodies. Results. The HNP-1, -2 and -3—also known as α-defensin-1, -2 and -3—are present in elevated concentrations in the plasma from colorectal cancer patients. Conclusion. Preoperative high plasma HNP 1–3 levels are associated with colorectal cancer. The HNP 1–3 levels may procure information on patients with lymph node or hepatic metastasis.

Keywords

colorectal cancer human neutrophil peptides lymph node metastasis hepatic metastasis

Introduction

Colorectal cancer is one of the most common cancers worldwide, and it is the third most diagnosed cancer in the world. The basis for colon cancer development is considered to typically involve the growth of adenomatous polyps into carcinoma.^1–3 Individuals with a history of adenomatous polyps have a higher risk of subsequent cancer.⁴ Rescission of adenomatous polyps results in a decreased incidence of colon cancer.⁵ The basis for chasing screening for colon cancer is to determine early-stage cancer as well as the adenomatous polyps, which may help decrease the risk of the disease.

Several methods are available for the detection of colon cancer. These include fecal occult blood testing, flexible sigmoidoscopy, double contrast barium enema, as well as colonoscopy.⁶ However, these tests are not widely used by universal public due to the damages and risk of complications and discomfort. For example, although the fecal occult blood test is noninvasive, it is not sensitive for the determination of adenomatous polyps. Fecal occult blood test may generate false-positive results because blood may be offer in the stead for other reasons, such as hemorrhoidal bleeding. Although colonoscopy results are highly sensitive,⁷ this screening method is invasive. A noninvasive biological marker would be of great benefit.

Human neutrophil peptides-1, -2 and -3 (HNP 1, -2 and -3), also known as α-defensin-1, -2 and -3, is up-regulated in the tumor microenvironment compared to normal colon tissue. We investigate this serum peptide with the aim of identifying specific biomarkers for colorectal cancer and novel marker for lymphatic and hepatic metastasis, discussing the potential effects of HNP 1–3 in the tumor tissue and determining its clinicopathological importance.

Materials and methods

Patients

A total of 100 patients who underwent colonoscopy or resection of colorectal carcinoma at our institution between May 2005 and May 2010 were enrolled for this study. The locations of the tumors and distant metastases were determined in all patients by barium enemas, colonoscopy and computerized tomography, and magnetic resonance imaging was performed to investigate local invasion and lateral lymph node metastasis for rectal cancer patients.

The primary lesion was located in the rectum in 43 patients and the colon in 67 patients. All patients were classified according to the TNM (TNM classification of malignant tumors) classification using resected specimens. Stage III and IV patients received fluorouracil-based chemotherapy, whereas stage I and II patients received no adjuvant therapy postoperatively. All patients received no neo adjuvant chemotherapy. Furthermore, all of the rectal cancer patients did not receive neo adjuvant chemoradiotherapy. The clinicopathological parameters studied for prognostic value were serosal invasion, vessel involvement, lymphatic invasion, lymph node metastases, distant metastases including liver and TNM classification.

All biological samples were obtained by trained staff at Haseki Education and Research Hospital, Istanbul, and Yüzüncü Yıl University Hospital, Van. Written consent was obtained from all donors. Permission for the study was obtained from the Haseki Ethical Committee.

Plasma samples were obtained from 100 patients before surgery. Healthy samples obtained from 60 normal healthy volunteers were used as controls. The absence of disease such as infection and asymptomatic early adenocarcinoma and adenoma was assessed by clinical history, physical examination, routine biochemical tests, including liver and renal function tests, and colonoscopy.

Plasma samples were allowed to clot and were stored at –80°C until use. Plasma HNP 1–3 levels were determined by solid-phase enzyme-linked immunosorbent assay (Cell Sciences, catalog no: HK317). The absorbance at 450 nm was measured with a spectrophotometer (human HNP 1–3; range 41–10,000 pg/ml). Measurements were implemented in duplicate and mean of the original and duplicate was presented.

Statistical Analysis

Data were presented as mean ± SD. Comparisons were performed with the non-parametric Mann-Whitney U test for continual variables and with the χ² test for categorical data. Differences between the two groups were determined using the log-rank test. Logistic regression analysis was used to determine the independent effect of factors on lymph node or hepatic metastasis as the final outcome. Two-sided P values < 0.05 were considered statistically significant.

Results

The patients included 48 men and 52 women with a mean age of 56.4 years. The primary lesion was located in the rectum in 43 patients, sigmoid colon in 24 patients, ascending colon in 28 patients, transverse colon in 2 patients, and descending colon in 3 patients. In all 38 patients were diagnosed as having synchronous liver metastasis. Tumor resection was performed on all patients, and the simultaneous partial hepatectomy for liver metastasis was performed on 11 patients. No perioperative mortality was observed among these patients. In all 20 patients had a poorly differentiated adenocarcinoma, whereas in 80 patients, the adenocarcinoma was well or moderately differentiated. There were 18 patients with stage I (T1-2 N0M0), 30 patients with stage II (T3-4 N0M0) and 32 patients with stage III (TX N1-2MO) disease. Twenty patients with distant metastases were classified as having stage IV (TX NXM1) disease.

Plasma HNP 1–3 levels were analysed in 100 colorectal cancer patients and 60 normal controls. There were no age or gender differences between colorectal cancer patients and controls. The plasma concentrations of HNP 1–3 in patients ranged from 100 to 4045 pg/ml. The mean concentrations of plasma HNP 1–3 in patients were significantly higher than that in normal volunteers (3954 ± 678 pg/ml vs. 68 ± 22 pg/ml, p = 0.0001).

The plasma HNP 1–3 levels were decreased in accordance to the stage of classification.

Table 1 shows the relationship between serum HNP 1–3 levels and clinicopathological variables in all patients. Serum HNP 1–3 were associated with factors that reflected disease development, such as venous invasion (p = 0.004), lymph node involvement (p = 0.0037) and distant metastases (p = 0.041), including liver metastases (p = 0.0146). In addition, serum HNP 1–3 levels reduced significantly in accordance with the TNM classification (p = 0.0352).

Table 1.

Relationships Between HNP 1-3 Levels and Clinicopathological Factors.

Variable	n	HNP 1	HNP 2	HNP 3	p
Gender
Male	48	5 ± 2	2 ± 2	6 ± 1
Female	52	5 ± 1	3 ± 1	6 ± 2
Age (y)
< 55	45	6 ± 2	4 ± 1	6 ± 2
≥ 55	55	4 ± 2	3 ± 1	4 ± 2	0.001
Serosal invasion
Negative	38	5 ± 2	4 ± 1	5 ± 2
Positive	62	5 ± 1	4 ± 2	5 ± 1	0.872
Lymphatic duct invasion
Negative	32	6 ± 1	5 ± 1	6 ± 2
Positive	67	4 ± 1	4 ± 1	4 ± 1	0.001
Venous invasion
Negative	25	6 ± 2	6 ± 1	6 ± 2
Positive	75	4 ± 1	4 ± 1	4 ± 1	0.001
Lymph node metastasis
N0	35	7 ± 1	6 ± 1	7 ± 1
N1	65	4 ± 1	4 ± 1	4 ± 2	0.001
Liver metastasis
H0	70	7 ± 2	6 ± 1	7 ± 1
H1	30	4 ± 1	3 ± 2	4 ± 2	0.001
Distant metastasis
M0	47	7 ± 1	5 ± 2	7 ± 2
M1	53	3 ± 2	3 ± 2	4 ± 1	0.001
TNM classification
I	18	4 ± 1	3 ± 2	4 ± 2
II	30	7 ± 1	6 ± 1	7 ± 2
III	32	6 ± 2	6 ± 2	6 ± 2
IV	20	4 ± 1	3 ± 1	4 ± 1	0.001

To examine the predictive value of serum HNP 1–3 for different clinicopathological characteristics, we conducted a χ² test. We described elevated serum HNP 1–3 levels according to the best predictive values calculated by ROC (Reactive Operating Curative System) analysis for lymph node metastasis and liver metastasis (Table 2 ). Clinical parameters for significantly presuming lymph node pertinent were serosal invasion, lymphatic duct invasion, and HNP 1–3 levels were below the cutoff value for clinical parameters (Table 3 ).

Table 2.

Correlations between lymph node invasion and clinical parameters or HNP 1–3 concentrations

	Lypmh node invasion		p
	(−)	(+)	p
Serosal invasion
(+)	20	19	0.0348
(−)	15	16	0.0348
Lypmhatic duct invasion
(+)	12	30	0.0089
(−)	18	6	0.0089
Venous invasion
(+)	23	30	0.093
(−)	12	11	0.093
HNP 1–3 (7)
< Cutoff	25	10	0.0019
> Cutoff	50	65	0.0019

Table 3.

Correlations between hepatic metastasis and clinical parameters or HNP 1–3 concentrations

	Hepatic metastasis		p
	(−)	(+)	p
Serosal invasion
(+)	16	20	0.2147
(−)	35	11	0.2147
Lypmhatic duct invasion
(+)	14	19	0.7845
(−)	20	5	0.7845
Venous invasion
(+)	28	27	0.0142
(−)	20	1	0.0142
Lypmh node metastasis
(+)	20	21	0.0747
(−)	17	8	0.0747
HNP 1–3 (5)
> Cutoff	58	3	0.0049
< Cutoff	49	104	0.0049

Discussion

Evaluating the concentration of HNP 1–3 in blood has been proved. Increased concentrations of plasma HNP 1–3 following infection (bacterial/non-bacterial infection and pulmonary tuberculosis) has been found in patients with septicemia or bacterial meningitis.^8,9 The HNP 1–3 have been found in urine of patients with transitional cell carcinoma of the bladder¹⁰ and HNP-1 has been found in the salvia of patients with oral carcinomas.¹¹ Consequently, HNP 1–3 are found in exaggerated amounts in tears after ocular surface surgery.¹² Our study is the first to demonstrate increased concentrations of HNP 1–3 in blood following tumor growth and its associationwith clinicopathological variables.

Previous studies indicated that increased HNP 1–3 expression in tumors is mainly produced from tumor-invading eosinophils¹³ and neutrophils.^14,15 However, it has been demonstrated that the exaggerated amounts of HNP 1–3 observed in urine from bladder cancer patients were often produced by the actual bladder cancer cells and that highly invasive bladder cancer cells affected more HNP 1–3 than less invasive ones.¹⁰ We cannot say whether the peptides are fabricated by the colon cancer cells or by tumor-infiltrating neutrophils. In the case of active inflammatory bowel disease, it is unclear whether epithelial expression of HNP 1–3 is induced by the inflammatory condition or whether the peptides are released by coterminous neutrophils and are taken up by the epithelial cells.¹⁶ Here, it is believed that the peptides provide protection against microbial invasion when the mucosal barrier is damaged (such as is the case in inflammatory bowel diseases). The HNP 1–3 are known to stimulate bronchial epithelial cells to up-regulate the production of interleukin-8,¹⁷ an intense neutrophil chemotactic factor. Thus, the up-regulated expression of HNP 1–3 in tumors may mainly be due to the invading immune cells but could be leaded by HNP 1–3 producing cancer cells.

We avowed the increased concentrations of HNP 1–3 in colon cancer and clinicopathological features. One study showed that HNP 1–3 mediates lysis of tumors in a concentration-dependent manner.¹⁸ This is in agreement with another study which shows that only comparatively high concentrations of HNP 1–3 are cytotoxic for human monocytes, whereas lower concentration of HNP-1 increases TNF-α production by monocytes.¹⁹

The high concentration of HNP 1–3 observed in tumors and the observation that HNP 1–3 are competent of lysing mammalian cells may conduct to the conclusion that the peptides course to the profit of the crew by killing tumor cells. However, in one study HNP 1–3 were found to bind to HLA (Human leukocyte antigen)-Class II molecules and were competent in decreasing the proliferation of a HLA-DR-restricted T-cell line after stimulation.²⁰ Defensins also regulate the systemic immune response. Through relation with the chemokine receptor CCR6, β-defensins amends dendritic and T cells.^21–23 Up-regulated immune responses are recognized to stimulate tumor proliferation; immune cells are amended by tumors to employ their pro-angiogenic and pro-metastatic effects.^24,25 Whether high concentrations of HNP 1–3 in the tumor limits the tumor growth or on the contrary stimulate tumor proliferation is unclear; we demonstrated that HNP 1–3 are expressed in various stages of carcinogenesis. Van den Broek and colleagues showed that serum HNP 1–3 levels increased in patients with colorectal cancer.²⁶

Our results showed that the increase in HNP 1–3 is associated with tumor growth and proliferation. We suggested that the HNP 1–3 concentrations may assist as biological markers for colon cancer with introduced diagnostic implement.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.

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