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Characterization of Normal Tissues and Metastatic Adenocarcinoma of the Lungs using Lectin Histochemistry
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Ourlad Alzeus G. Tantengco

Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City, Philippines, 1101

ABSTRACT Lectin Histochemistry shows that lectin can be utilized as specific markers for cell population. Several lectins were studied in this paper including Helix pomatia agglutinin (HPA), which is used as primary marker-based predictor of prognosis on various adenocarcinoma cells. It was observed that expression of HPA, PHA-L and UEA-I are strongly associated with metastatic spread in several cells of the primary tumor and to their lymph node metastases. Detection of lectin binding using the simple avidin-biotin method

is advantageous because of its availability, speed, suitability for Western blotting.

By the virtue of studying the pattern of glycosylation and glycoprofiles of cells and tissues, better understanding regarding disease mechanisms can be achieved. Disruption of cellular

glyscosylation mechanisms can result in changes in the extension of oligosaccharide chains; this is associated with tumor progression and metastasis. INTRODUCTION The respiratory system which macrophages, alveolar epithelial cells

(pneumocytes), mast cells and the capillary network. Observation of migratory cell population or changes in the normal number of resident cell population is vital in understanding the dynamics of various diseases inside the organ system.

includes the lungs is a branching system of tubes that link the site of gas exchange with the external environment. It provides a conduit to ensure uninterrupted supply of air but it also serves as passageway for toxins, chemical substances and pathogens.

(Barkhordari et. al., 2004) The study of glycan in cells and tissues is attracting greater attention because of its use for characterization of cell population. By the virtue of studying the

Understanding the normal histology of the lungs allow us to observe the structures and function of the resident cell populations of pulmonary tissues such as alveolar

pattern of glycosylation and glycoprofiles of cells and tissues, better understanding regarding disease mechanisms can be Immunohistology of Normal Lungs Lectins can be used as reliable markers for identification of the cells and studying subtle differences in sugar

achieved. Lectins, a group of specific glycoproteins present in animal as well as plant cells, are used in analysis of glycans. The worldwide incidence of

sequences of certain cell population. Lectin Histochemistry shows that lectin can be utilized as specific markers for cell

adenocarcinoma of the lung is rising. The classification of primary lung carcinomas is probably one of the least precise among all the solid tumors (Laack et. al., 2002). A carbohydrate rich coat covers the outer surface of mammalian cells. This coat is called glycocalyx and is involved in cell to cell and cell to matrix interaction which is important in tumor metastasis. Changes in the glycosylation of this carbohydrate rich coat are associated with metastatic potential of tumors and therefore have potential as markers for lung cancer prognosis. Since lectins can detect protein these changes in

population. In order to achieve better characterization of cell populations using lectins, it was improved with

immunohistology of the cells (Barkhordari et. al., 2004). Lectins used in immunohistology of the normal lungs are as follows: Galanthus nivalis (GNA) and (NPA) for Narcissus alveolar esculentum

pseudonarcissus macrophages,

Lycopersicon

(LEA), Phytolacca americana (PAA) and Triticum vulgaris-succinylated (sWGA) for type I pneumocytes, Hippeastrum hybrid (HHA) and MPA for type II pneumocytes, Psophocarpus tetragonolobus (PTL-II),

membrane

glycosylation,

comprehensive lectin-binding analysis of adenocarcinomas undertaken to of the lung possible was new

Phaseolus vulgaris leukoagglutinin (l-PHA) and Anguilla anguilla (AAA) for endothelial cells and Dolichos biflorus (DBA) for bronchial epithelium. The universal staining of type I pneumocytes by LEA, PAA and sWGA, but patchy staining by Arachis hypogaea (AHA), Sambucus nigra (SNA) and Maackia amurensis (MAA) also

identify

prognostic markers. (Thom et. al., 2007) The aim of current researches is to analyze the glycoconjugate expression of normal lung and adenocarcinomas of the lung by using several lectins to define a prognostic marker for cancer.

indicated not only the presence of an abundant set of the related glycoforms in these cells in normal lung, but also the possibility of two distinct populations or a pattern of differentiation which is

L-binding

oligosaccharides

in

oral

squamous-cell carcinoma and its regional lymph node metastases. Furthermore, the lectin Ulex europaeus agglutinin-I (UEA-I) was investigated in breast cancer where its binding related to disease-free interval and survival. It has also been established as an excellent marker for human endothelial cells (Laack et. al., 2002).

morphologically unapparent (Barkhordari et. al., 2004). Lectin Immunohistology of normal lungs helps in decoding the patterns of glycosylation of cells which is helpful in understanding the mechanisms of diseases such as metastasis of adenocarcinoma.

Labelling Methods for Detection of Lectin Binding Current studies have used the

Labeled Lectins for Histochemical Detection of Metastasis Helix pomatia agglutinin (HPA) binding was the primary marker-based predictor of prognosis cells such on as various breast,

complex multi-layered method to detect lectin binding specifically for HPA. The tissue specimen is incubated with

unconjugated HPA which recognizes the GalNAc residues on cellular glycoproteins. Multiple layering starts with a polyclonal rabbit antibody directed against HPA, then a biotin labeled secondary anti-rabbit antibody and the final layering is through

adenocarcinoma

stomach, ovary, esophagus, colorectum, thyroid and prostate. HPA has been used to define the metastatic phenotype of these tumors. However, HPA has not been the only lectin that has provided useful prognostic information. Phaseolus vulgaris

incorporation of horseradish peroxidase labeled avidin or streptavidin (Brooks & Wilkinson, 2003). The advantage of this kind of method is its high specificity in labeling therefore eliminating possible problems concerning sterical intereference of reporter molecule with carbohydrate combining site of lectins. The disadvantage of this method is the

leucoagglutinin (PHA-L) has been reported by several studies as an effective marker that detects the tumor development in breast and colon cancer. There is also a significant correlation between the expression of PHA-

availability of the polyclonal antibody and the length of time consumed. This method is also unsuitable for Western blotting because the reagents are cross reactive with

of normal cells. Disruption of cellular glyscosylation mechanisms can result in changes in the extension of oligosaccharide chains; this is associated with tumor progression and metastasis. (Brooks & Wilkinson, 2003) It was observed that expression of HPA, PHA-L and UEA-I are strongly associated with metastatic spread in several cells of the primary tumor and to their lymph node metastases. Using univariate analysis, it was found out that these lectins bind to the primary tumors and therefore can be used as prognostic markers for overall and relapse-free survival after the tumor was

antibodies used in multiple layering. Due to these disadvantages,

researchers resorted to a simpler detection method using commercially widely-

available reagents such as simple avidinbiotin method. Biotin-labeled HPA detected with an advitin-biotin complex (ABC) method has been used as prognostic and biological marker in breast cancer and animal models of human melanoma.

Lectin

Histochemistry

of

Resected

surgically

removed.

However,

upon

Adenocarcinoma Lung The classical way of assessing the tumor spread is through anatomical

application of multivariate analysis only HPA binding remained as a significant independent prognostic indicator.

description of tumor. This classical TNM method is disadvantageous because this will not give any understanding of the metastatic capability of the tumor. Several researches about adenocarcinoma other than lung, the lectin from the Roman snail, Helix pomatia agglutinin (HPA), has been used to define the metastatic phenotype of these tumors. HPA recognizes oligosaccharides with a terminal -glycosidically-linked Nacetylgalactosamine (-GalNAc), an Lung Cancer-Specific Lectin According to current researches

reagarding lectin Histochemistry of the lungs, specific lectins for lung cancer cases are not yet worked out widely. Recent studies show 159 cases of lung cancer that were investigated for their capacity to bind Viscum album L. (VAA) and galectin-1 and for Lewis antigen reactivity. VVA has a high affinity for the disaccharide GalNAc1 and 3Gal1-. VAA showed approximately

uncommon terminal residue in the glycans

70% moderate to strong binding to lung tumour. Adenocarcinomas and

detectable by the lectin HPA and other lectins, is one of the most frequently reported glycosylation changes in

branchioalveolar carcinomas were more frequently labelled than squamous

malignancy. Deviation from the lectin labelling and expression present in the normal tissues is also one of the changes in malignancy. Expression of several lectins

carcinomas. There is no direct correlation between the presence on VAA binding capacity and survival whereas better

prognosis has been observed due to the presence of the galectin-1 binding sites (Sherwani et. al., 2003). Galectins are a family of proteins that includes calcium independent galactoside binding lectins. 84% of the lung tumours showed HPA binding (Laack et. al., 2002).

including HPA, PHA-L and UEA-I are strongly associated with metastatic spread in several cells of the primary tumor and to their lymph node metastases. Viscum album
L. (VAA) and galectin-1 also provided useful

prognostic

information

in

tumor

However, the molecular basis why HPA binds preferentially to metastasizing

development. However, upon application of multivariate analysis only HPA binding remained as a significant independent prognostic indicator for adenocarcinoma of the lungs. Several methods of labeling lectin binding were also presented here including the multilayering method and the use of simple avidin-biotin method for detection of HPA binding. Avidin-biotin method is simple and advantageous because it does not require access to polyclonal antibodies against the lectin.

adenocarcinomas has not been elucidated completely. The precise structure of the

HPA binding oligosaccharide associated with metastasis has not been identified yet, however, a monosialylated oligosaccharide of 4.58 glucose units termed HPA gly-1 has been identified in HPA-positive breast cancers, whose functional role has to be elucidated in future studies.

CONCLUSION Lectins are used in characterization of the glycoconjugate content of normal and malignant tissues of the lungs. The aberrant expression of GalNAc oligosaccharides, as

The use of lectin Histochemistry is of prognostic significance in many human cancer types, and, as it is associated with the presence of metastases in lymph nodes.

REFERENCES Barkhordari, Abolfazl of et. al. Lectin Lung.

Histochemistry

Normal

Journal of Molecular Histology 35: 147156. Kluwer Academic Publishers, Netherlands. 2004

Brooks, Susan A & Diane Wilkinson. Validation of a simple avidin-biotin detection method for Helix pomatia lectin (HPA) binding as a prognostic marker in cancer. Acta Histochemica. 105(3) 205212 (2003)

Laack, Eckart et. al. Lectin Histochemistry of Resected Adenocarcinoma of the Lung. American Journal of Pathology, Vol. 160, No. 3, March 2002

Thom, Ina et. al. Lectin histochemistry of metastatic adenocarcinomas of the lung. Lung Cancer Volume 56, Issue 3, June 2007, Pages 391397 Sherwani, Asma Farhat et. al.

Characterization of Lectins and Their Specificity in Carcinomas An

Appraisal. Indian Journal of Clinical Biochemistry, 2003, 18 (2) 169-180