ORIGINAL ARTICLEExpression of the acute phase protein haptoglobin in human lung cancer and tumor-free lung tissues
Introduction
Haptoglobin (Hp) is an acute phase plasma glycoprotein (with a concentration of 0.45–3 mg per ml blood plasma) composed of α1, α2, and β polypeptide chains (their size being around 9, 19, and 40 kDa) in different combinations. Its main function is the binding of hemoglobin and prevention of oxidative stress. First reports on Hp date back to studies performed by Polonovski and Jayle in Paris in 1938 and 1940 [36], [37], describing Hp as a “plasma substance” interacting with hemoglobin (Hb).
The Hp gene is localized on chromosome locus 16q22 [17]. The primary function of Hp is to bind free Hb for protection against oxidative stress and to facilitate the Hb-uptake by Hb scavenger receptors like CD163 [27].
Hp originates from the liver. Elevated amounts of the protein in blood plasma have been observed in infection, inflammation, and various malignant diseases, including lung- and bladder cancer [9], [10], leukemia [30], breast cancer [3], esophageal squamous cell carcinoma [1], urogenital tumors [14], malignant lymphomas [15], and ovarian cancer [48]. To date, research over the past 60 years has revealed that Hp is a multifunctional protein involved in a variety of regulation processes, including immune responses [33], angiogenesis [25], prostaglandin synthesis [29], and reverse cholesterol transport [5].
We focused on one of the most frequent lethal diseases in humans: lung cancer [47]. To date, no details on molecular techniques such as immunohistochemistry (IHC), in situ hybridization (ISH), or reverse transcription polymerase chain reaction (RT-PCR) concerning the detection the Hp protein or mRNA have been provided to demonstrate the presence in the human lung and referring cancer types (mostly adeno-, squamous cell-, and small cell carcinomas).
The detection of a serum biomarker preferably indicating an early stage of the disease is helpful for immediate treatments and, therefore, for overall survival of patients. Previous reports have faced glycoprotein concentrations, such as Hp, in the blood plasma of patients with primary or secondary carcinoma of the lung for statistical studies. Increased Hp levels have been found in the serum of patients suffering from lung cancer; therefore, those studies have revealed that Hp is a potential serum biomarker candidate for lung cancer [4], [11], [16], [20], [21], [31].
Fedorovych et al. [16] showed that the function of the Hp–Hb complex in sera of lung cancer patients is to neutralize super oxidative products. Concerning the immunomodulating effects of acute phase proteins and their relevance in cancer, Samak et al. [22], [39] demonstrated that Hp acts as a non-specific blocking factor protecting tumors against immunological attacks.
In this study, we describe the cellular expression of Hp in 47 pulmonary adenocarcinomas, 42 squamous cell carcinomas, 13 small cell lung cancers, and 13 normal lung tissues using IHC. The results were verified by RT-PCR and transcription arrays. ISH was performed on a selected collective for localization of transcripts within the tissues, and Western blotting [28] was conducted to compare the amounts of pulmonary and hepatic Hp.
Section snippets
Transcription arrays
Two NSCLC specimens and five tumor-free lung tissues were subjected to ex vivo-cultivation with or without being stimulated, employing a recently described model [13], [28]. One sample of tumor-free lung was not subjected to cultivation.
Sample 1: NSCLC 16 h medium without stimulation.
Sample 2: NSCLC 16 h stimulated with carboplatin and gemzitabine.
Sample 3: tumor-free lung 16 h medium without stimulation.
Sample 4: tumor-free lung 16 h infected with Haemophilus influenzae.
Sample 5: tumor-free lung
Transcription array
The results of the analysis by transcription array are shown in Fig. 1. They were set in correlation to GAPDH. Transcripts of Hp were clearly detected in all specimens, irrespective of being NSCLC or tumor-free. The level of transcription was comparably high in all tissues analyzed, even exceeding the GAPDH level in the case of non-cultivated sample 5.
IHC
The results of IHC are displayed in Fig. 2, Fig. 3. Among lung cancers, immunohistochemical Hp signals appeared mostly in adenocarcinomas (19/47
Discussion
We showed that Hp is strongly expressed and synthesized in human lungs, with expression levels that can reach the levels of the liver in cases of NSCLC.
Hp appearance is related to a physiologically important modulation of the immune response. Considering the immunoactive properties of haptoglobin, it is conceivable that tumors produce Hp either innately or by inducing other parts of the organism, such as the liver, which is the main site of Hp expression [22], [39].
The expression of Hp in human
Acknowledgements
The authors thank Dr. Elvira Richter for sequencing the RT-PCR-products, Prof A. Petersen for providing grass pollen allergen and support with transcription arrays, and Dr. J. Rupp and Dr. D. Drömann for support with Haemophilus influenzae infection, as well as Jasmin Tiebach, Maria Lammers, and Tanja Zietz for their excellent technical support.
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M.A. and H.S. contributed equally to this work.