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Expression of the acute phase protein haptoglobin in human lung cancer and tumor-free lung tissues
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Pathology – Research and Practice 205 (2009) 639–647 www.elsevier.de/prp
ORIGINAL ARTICLE
Expression of the acute phase protein haptoglobin in human lung cancer and tumor-free lung tissues Mahdi Abdullaha,1, Holger Schultza,1, Daniel Ka¨hlera,, Detlev Branscheidb, Klaus Dalhoffc, Peter Zabelc,d, Ekkehard Vollmera, Torsten Goldmanna a
Research Center Borstel, Clinical and Experimental Pathology, Parkallee 3a, D-23845 Borstel, Germany Hospital Großhansdorf, Department of Thoracic Surgery, Wo¨hrendamm 80, D-22927 Großhansdorf, Germany c University of Lu¨beck, Medical Hospital III, Ratzeburger Allee 160, D-23562 Lu¨beck, Germany d Research Center Borstel, Department of Clinical Medicine, Parkallee 35, D-23845 Borstel, Germany b
Received 18 November 2008; received in revised form 26 March 2009; accepted 9 April 2009
Abstract Besides its main function, i.e., the binding of free hemoglobin and prevention of oxidative stress, the acute phase protein haptoglobin acts as a potent immunoreactive modulator. As part of an investigation that aimed at illuminating the role of acute phase proteins in the local defense of the lungs, this study is the first to describe the expression and synthesis of haptoglobin in human lung tissues and lung tumors. Prompted by the results obtained from a transcription array study, we analyzed 115 lung (cancer) specimens using immunohistochemistry. Thirty-seven specimens were subjected to mRNA-in situ hybridization. 40.4% of the adenocarcinomas showed distinct granular and perinuclear staining of the tumor cells. By contrast, only 4.8% of the squamous cell carcinomas showed haptoglobin within tumor cells, but 19% displayed haptoglobin expressing alveolar epithelial cells type II surrounding the tumor. One small cell lung cancer displayed haptoglobin expression. In tumor-free lungs, we located haptoglobin in alveolar macrophages, alveolar epithelial cells type II, and bronchiolar cells. In situ hybridization verified the results of immunohistochemistry. The results were further verified by RT-PCR and Western blot compared to liver tissues, which both showed comparable amounts of haptoglobin mRNA and protein in NSCLC and in liver, while tumor-free lung tissues showed lower expression. Due to the known immunomodulatory effects of haptoglobin, its broad expression and synthesis within human lung tissues strongly suggests a function as a fundamental pulmonary local defense element. r 2009 Elsevier GmbH. All rights reserved. Keywords: Haptoglobin; Tissue; NSCLC; Acute phase protein; Immune response
Corresponding author.
E-mail addresses: [email protected] (M. Abdullah), [email protected] (H. Schultz), [email protected] (D. Ka¨hler), [email protected] (D. Branscheid), [email protected] (K. Dalhoff), [email protected] (P. Zabel), [email protected] (E. Vollmer), [email protected] (T. Goldmann). 1 M.A. and H.S. contributed equally to this work. 0344-0338/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2009.04.007
Introduction Haptoglobin (Hp) is an acute phase plasma glycoprotein (with a concentration of 0.45–3 mg per ml blood plasma) composed of a1, a2, and b 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
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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.
Materials and methods 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 without cultivation. Sample 6: tumor-free lung 16 h medium without stimulation. Sample 7: tumor-free lung 16 h stimulation with inactive grass pollen allergen. Sample 8: tumor-free lung 16 h stimulation with active grass pollen allergen. After HOPE-fixation, total RNA was extracted as described previously [19] and submitted to Imagenes (Berlin, Germany) for analysis using an Agilent 44 k transcription array.
Patients/tissues Patients had a mean age of 65 years (range from 45 to 85 years; 61 males, 48 females). A hundred and fifteen tissue samples from these patients were investigated. Pathological characterization defined 47 adenocarcinomas, 42 squamous cell carcinomas, 13 small cell lung cancers, and 13 normal lungs. After lobectomy/ pneumonectomy, tissues were fixed with formalin and embedded in paraffin. For mRNA analysis, 37 tissues were fixed using the HOPE technique and mounted into a TMA [35].
Tissue microarrays (TMAs) All specimens were placed in TMAs as described previously [35]. Sections from these paraffin blocks were mounted on superfrost+ slides (Menzel-Gla¨ser, Germany) for treatment. One TMA containing only HOPE-fixed material was produced for ISH with 20 lung specimens and 17 lung cancer tissues (6 squamous cell carcinomas/10 adenocarcinomas, 1 pleomorphic
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carcinoma). Small cell carcinomas were investigated using single sections.
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then photographed under ultraviolet radiation. The PCR products were sequenced using an ABI 377 sequencer and cycle sequencing [18].
IHC ISH For the immunohistochemical stainings, we used TMA sections as described above [35]. The tissues were deparaffinized by xylene incubation (two times 10 min). Rehydration of the tissues was carried out in a graded ethanol series for two minutes in each step (two times 100%, 2 times 96%, 90%, 80%, 70%; two times aqua dest). For antigen retrieval, slides were cooked in citric buffer (pH 6) for 35 min, and endogenous peroxidases were blocked by incubation of 3% H2O2 solution (10 min). Primary antibody (mouse anti-human; monoclonal anti-haptoglobin antibody; clone HG-36, dilution 1:100; Abcam, UK) was applied for 50 min at room temperature. After washing (Tris saline buffer, 2 min, twice), the detection system (ZytochemPlus HRP polymer kit, Zytomed System, Berlin) was incubated for 30 min, and color reaction was performed using aminoethylcarbazole (AEC) as chromogen (3–15 min) [40]. Slides were counterstained using Mayer’s hemalum and covered with Kayser’s glycerine gelatine. Negative controls were included in each staining series, as well as positive reference sections from human liver to ensure even results.
RT-PCR Total RNA was extracted from 13 human HOPEfixed lung cancer tissues, two healthy lung tissues, and one liver sample according to the manufacturer’s recommendations (RNeasy Mini kit, Qiagen, Germany). After destroying residual DNA by DNase (Gibco, Germany) treatment, cDNA was synthesized by reverse transcription (Superscript II, Invitrogen, Germany) using 1 mg of total RNA template and special oligodT15 primers to enhance reverse mRNA transcription as described previously [19]. PCR was performed, targeting a 341 bp fragment of the human haptoglobin gene using the following primer sequences: Hp forward 50 -AGGCATTATGAAGGCAGCAC-30 ; Hp reverse primer 30 -CTTCCAGGCTGAAATCTTGC-50 . In parallel, specific primers for the house keeping gene glyceraldehde3-phosphate-dehydrogenase (GAPDH; forward primer 50 -AGAACGGGAAGCTTGTCATC-30 ; reverse primer 30 -TGCTGATGATCTTGAGGCTG-50 ) were designed, targeting an amplicon of 257 bp as control. 40 PCR cycles were set as follows: 94 1C for 1 min, 58 1C for 1 min, and 72 1C for 1 min. Finally, one additional cycle at 72 1C for 15 min was programmed. PCR products and a molecular weight marker (pBr322-Msp1) were separated through a 2.4% concentrated ethidium bromide containing (10 ml) agarose gel by electrophoresis, and
Haptoglobin mRNA targeting was performed using a TMA containing 20 healthy lung tissues and 17 cancerous tissues (10 adenocarcinomas, 6 squamous cell carcinomas, and 1 pleomorphic carcinoma). Double stranded, digoxigenin (DIG)-labeled DNA-probes were synthesized as previously described using the PCR products from above. Concentrations of the labeled probe solutions were determined by comparison with several differently concentrated DIG-labeled control DNA spots on a positively charged nylon membrane. In situ hybridization was carried out as previously described using a probe concentration of 2 ng/ml [19,38].
SDS–PAGE and Western blotting Two NSCLC specimens with corresponding tumorfree tissues and two specimens from liver were analyzed using Western blot. The procedure was performed as described previously, with slight modifications [45]. Samples of HOPE-fixed, paraffin-embedded lung, lung cancer, and liver tissue were deparaffinized twice with xylene treatment (1 ml; 10 min) and one step of ethanol treatment (1 ml; 10 min) with respective removal of the supernatants before drying in a vacuum centrifuge (1 h; Speed Vac 110; Savant, Germany). Proteins were extracted at 4 1C overnight using a lysis buffer containing 7 M urea; 2 M thiourea; 2% IGEPAL; 1% Triton-X; 100 mM dithiotreitol (DTT); 5 mM PMSF; 4% CHAPS; 0.5 mM Tris and lysates were denatured (100 1C; 5 min). Protein pellets were resuspended in 5 ml of sample buffer (LDS sample buffer, Invitrogen, Germany). These lysates were subjected to SDS–PAGE (4% stacking gel; 12% resolving gel; Novex minicell, Invitrogen, Germany). Western blotting was performed on a nitrocellulose membrane using the iblot gel transfer system (Invitrogen, Germany) according to the instructions of the manufacturer. Membrane was blocked for 2 h using DIG wash and block buffer (Boehringer Mannheim, Germany). Primary antibody (from above, Section ISH) was incubated in a dilution of 1:100 for 90 min, and the membrane was washed three times in Tris-buffer pH 7.4 (10 min each). Secondary antibody (goat-antimouse, conjugated to alkaline phosphatase; Dianova, Hamburg, Germany) was applied for 1 h, dilution 1:10000, and washed twice, followed by incubation in Tris-buffer at pH 9.5. Color reaction was then achieved using NBT/BCIP as a substrate for the alkaline phosphatase
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(Boehringer Mannheim, Germany) according to the manufacturer’s protocol.
type of tissue sample investigated. Negative controls showed no signal.
RT-PCR
Results 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 tumorfree. 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 Figs. 2 and 3. Among lung cancers, immunohistochemical Hp signals appeared mostly in adenocarcinomas (19/47 cases Hp-positive, 40.4%), showing perinuclear and granular staining of tumor cells (Fig. 2A). In four cases, AEC II cells were positive (8.5%); staining of alveolar macrophages was observed in one case (2.1%). Among the squamous cell carcinoma tissues, 2 of 42 specimens were Hp-positive in tumor cells (4.8%); however, 8 cases (19%) showed stained cells of tumor surrounding AEC II cells (Fig. 2B). Small cell carcinoma staining revealed that 2 of 13 tumors were positive for Hp protein (15.4%; Fig. 2C). Concerning the 13 cases of tumor-free lungs, AEC II cells were found to be positive in four cases (Fig. 3A), alveolar macrophages in all 13 cases (Fig. 3B), and four bronchi were stained (Fig. 3C). Fig. 4 shows positive cases in relation to the total number and the
RT-PCR was performed to additionally address transcripts of Hp. Thirteen NSCLC tissues, 2 corresponding lung tissues, and 1 liver sample were analyzed using Hp targeting RT-PCR. We found Hp mRNA in all NSCLC and lung tissues and, as expected, Hp in the liver. Negative control lanes showed no bands (Fig. 6). This verifies the IHC results and proves transcription of the Hp gene in human lung tissues and lung malignancies (Figs. 5 and 6).
ISH We additionally analyzed 17 carcinomas by in situ hybridization to detect cellular transcripts of the haptoglobin mRNA within the tissues. In all adenocarcinomas and in four of six squamous cell carcinomas, signals were generated within the tumor cells. Alveolar macrophages, AEC II cells, and epithelial cells of the bronchi also displayed signals of Hp transcripts (Fig. 7). Negative controls showed no signals.
Western blot Western blot analysis was performed to compare the amounts of Hp-protein from NSCLC, tumor-free lungs, and liver. As depicted in Fig. 8, we found comparable amounts of Hp in NSCLCs compared to the liver tissues. Tumor-free lungs showed comparably weaker signals. All signals had a molecular weight of around 40 kDa (the size of a single b-chain of haptoglobin).
Discussion
Fig. 1. Hp transcription array data of eight different tissue samples in a respective comparison with housekeeping gene GAPDH for tumor+medium (1), tumor+chemotherapeutics+medium (2), lung+medium (3), lung+Haemophilus influencae+medium (4), lung HOPE-fixed uncultured (5), lung+medium uncultured (6), lung+inactive grass pollen allergen (7), lung+active grass pollen allergen (8).
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 lung tissues is observed for the first time. Cellular localization observable by granular staining was either perinuclear (adenocarcinomas and bronchi), cytoplasmic (small cell carcinomas and macrophages) or, in case of squamous cell carcinomas, mostly apparent in areas around the tumor (AEC II cells). Therefore, it is possible that Hp as
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Fig. 2. Immunohistochemical detection of haptoglobin in different subtypes of lung cancer employing adenocarcinomas (A), squamous cell carcinomas (B), and small cell carcinomas (upper panel 400 , lower panel 800 ).
Fig. 3. Immunohistochemical detection of haptoglobin in different non-malignant cell types of human lung tissues: AEC II cells (A), alveolar macrophages (B), and bronchi (C). (upper panel 400 , lower panel 800 ).
an acute phase protein is involved in a variety of immunoreactive processes. Inflammation might also be a relevant factor in immunoreactions during lung cancer genesis. Our results reveal that in cases of squamous cell carcinomas, no frequent expression of haptoglobin could be observed in tumor cells. However, the tumor surrounding AEC II cells and bronchile epithelial cells were frequently stained with high intensity. This was also observed in tumor-free lungs. By contrast, the tumor cells of adenocarcinomas, to a large degree, revealed intense perinuclear and granular staining. This phenomenon could be due to the different cell types from which the carcinomas derive [47].
There is a variation of expression concerning tumor subtypes (about 40.4% of adenocarcinomas, 4.8% of squamous cell carcinomas, and 15.4% of small cell carcinomas are Hp-positive), which may be caused by different haptoglobin phenotypes or Hp polymorphisms associated with tumor types. Many studies conducted worldwide suggest that Hp1-1 and Hp1-2 are more common than Hp2-2 [3,6,9,10,23,44], and that the geographical distribution is important due to differences in gene sequences/frequencies concerning haptoglobin, such as the absence of haptoglobin in Caucasian people or the low Hp1 allele frequency in Southeast Asia [29]. This leads to the speculation that most cases of adenocarcinomas might belong to Hp type 1-1 or 1-2,
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Fig. 4. Expression of haptoglobin in different tumor-entities and tumor-free lung tissues determined by immunohistochemistry. Left bar: number of cases analyzed, right bar: number of positive cases for adenocarcinomas (A), squamous cell carcinomas (B), small cell carcinomas (C), and tumor-free lung tissues (D).
Fig. 5. Results of RT-PCR targeting Hp-transcripts in 10 specimens of NSCLC. Products were separated on agarose gels and stained with ethidiumbromide. Panel A shows the 341 bp fragment of Hp; Panel B the 257 bp fragment of GAPDH. Molecular weight marker: M ¼ pBr322/Msp1.
Fig. 6. RT-PCR targeting haptoglobin (lower bands) and GAPDH (upper bands) for healthy lung (lu), lung tumor (tu); liver (li) and negative control (neg).
while squamous- or small cell carcinomas might belong to a patient carrying Hp type 2-2. Further studies are necessary to understand the relations between Hp production of tissues and correlating cancer types. Our results are also in agreement with Beckman et al. [9], who reported that a group of patients with squamous cell carcinomas and another one with small cell carcinomas showed no significant difference in Hp concentration in the plasma compared to a control group. However, among patients with pulmonary adenocarcinomas, the frequency of haptoglobin
type 2-2 is lower compared to the control group, with a corresponding increase in the Hp 1-1 and Hp 1-2 type [12]. Another important finding related to the functions of Hp in cancer is the recent demonstration of haptoglobin as a natural inhibitor of collagen degradation, and it is locally expressed by fibroblasts in the arterial wall [25,42]. The Hp-glycoprotein in this environment is crucial for cell migration processes and arterial restructuring like angiogenesis. Moreover, collagen turnover is important in many physiological processes, such as tumor cell invasion, wound healing, cell growth, metastasis, and enhancement of collagen degradation. This is related to a severe tissue destruction and malfunction, which is often encountered in pathological processes, such as arthritis and metastasis. Thus, the high expression of Hp in adenocarcinomas may be correlated with their comparably high potential to metastasize. This reveals an importance of haptoglobin for functions in the extracellular matrix and during cell migration, and suggests another role for this polypeptide in cancers [24,43,46]. In general, our results are correlated with those of other groups which showed that increased levels of haptoglobin synthesis appear during lung cancer genesis, and that higher amounts of Hp can be observed in the blood plasma of those patients. The findings of these groups could affirm the Hp’s capability of neutralizing super oxidative products, and suggest Hp as a potential serum tumor biomarker candidate for lung cancers. We are in accordance with this presumption due to our finding of Hp also in tumor tissues. The increase in haptoglobin expression in tissues and serum may enable evasion and surveillance mechanisms of tumor cells [8,11,20,21,26,31,39,41]. Another recent study has reported that native haptoglobin interferes with the activated human neutrophil function by blocking its receptor-mediated Ca2+ and haptoglobin influx from normal human blood plasma [33]. This interferes again with macrophageand lymphocyte-activity during mitogenesis [7,32,34]. Therefore, it is suggested that Hp may act as a natural antagonist for receptor ligand activation of the immune system. Arredouani et al. [2] further described the role of haptoglobin affecting the immune system, showing that haptoglobin directly affects T-cells and suppresses T-helper-cells through downregulation of the cytokine production and associated mechanisms. However, it is not clear whether this is an indirect phenomenon caused by modulation of antigen-presenting cell (APC) function or whether haptoglobin acts directly on T cells [2]. Seder et al. [41] showed that passive removal of Hp out of the plasma of cancer patients restores the immune response at least temporarily. In conclusion, our findings are the first to demonstrate strong expression of haptoglobin in lung cancer
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Fig. 7. Results of in situ hybridization targeting haptoglobin transcripts using a 341 bp digoxigenated, double stranded DNA-probe on adenocarcinomas (A, 400 ), squamous cell carcinomas (B, 400 ), alveolar macrophages (C, 100 ), bronchi (D, 400 ), and AEC II cells (E1 100 ; E2 400 ).
Fig. 8. Western immunoblot targeting haptoglobin in tumorfree lung (lu), tumor (tu), and liver (li, lih) (lih for liver high protein concentration).
tissues, and more often in adenocarcinomas than in squamous cell carcinomas or small cell carcinomas. There is also an increased amount of haptoglobin mRNA in tumor tissues and tumor surrounding cells, shown by positive ISH and RT-PCR. This detection is correlated with the expression level of the protein, which was observed in IHC staining. Hence, the ability of Hp to affect immune responses could play an important role in lung carcinogenesis, including tumor evasion from immunological attacks [39]. Modulation of Hp expression and its involvement in immunoregulatory events is currently investigated using a novel human ex vivo model [19,28]. Hp expression in tumor-free lung tissues was located in alveolar macrophages, AEC II and bronchi. Taken together, our results strongly suggest a role of Hp in the
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local immune defense of the lung and might also be important in different inflammatory processes. Appropriate studies are being conducted to further elucidate these aspects.
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. Dro¨mann 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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Pathology – Research and Practice 205 (2009) 639–647 www.elsevier.de/prp
ORIGINAL ARTICLE
Expression of the acute phase protein haptoglobin in human lung cancer and tumor-free lung tissues Mahdi Abdullaha,1, Holger Schultza,1, Daniel Ka¨hlera,, Detlev Branscheidb, Klaus Dalhoffc, Peter Zabelc,d, Ekkehard Vollmera, Torsten Goldmanna a
Research Center Borstel, Clinical and Experimental Pathology, Parkallee 3a, D-23845 Borstel, Germany Hospital Großhansdorf, Department of Thoracic Surgery, Wo¨hrendamm 80, D-22927 Großhansdorf, Germany c University of Lu¨beck, Medical Hospital III, Ratzeburger Allee 160, D-23562 Lu¨beck, Germany d Research Center Borstel, Department of Clinical Medicine, Parkallee 35, D-23845 Borstel, Germany b
Received 18 November 2008; received in revised form 26 March 2009; accepted 9 April 2009
Abstract Besides its main function, i.e., the binding of free hemoglobin and prevention of oxidative stress, the acute phase protein haptoglobin acts as a potent immunoreactive modulator. As part of an investigation that aimed at illuminating the role of acute phase proteins in the local defense of the lungs, this study is the first to describe the expression and synthesis of haptoglobin in human lung tissues and lung tumors. Prompted by the results obtained from a transcription array study, we analyzed 115 lung (cancer) specimens using immunohistochemistry. Thirty-seven specimens were subjected to mRNA-in situ hybridization. 40.4% of the adenocarcinomas showed distinct granular and perinuclear staining of the tumor cells. By contrast, only 4.8% of the squamous cell carcinomas showed haptoglobin within tumor cells, but 19% displayed haptoglobin expressing alveolar epithelial cells type II surrounding the tumor. One small cell lung cancer displayed haptoglobin expression. In tumor-free lungs, we located haptoglobin in alveolar macrophages, alveolar epithelial cells type II, and bronchiolar cells. In situ hybridization verified the results of immunohistochemistry. The results were further verified by RT-PCR and Western blot compared to liver tissues, which both showed comparable amounts of haptoglobin mRNA and protein in NSCLC and in liver, while tumor-free lung tissues showed lower expression. Due to the known immunomodulatory effects of haptoglobin, its broad expression and synthesis within human lung tissues strongly suggests a function as a fundamental pulmonary local defense element. r 2009 Elsevier GmbH. All rights reserved. Keywords: Haptoglobin; Tissue; NSCLC; Acute phase protein; Immune response
Corresponding author.
E-mail addresses: [email protected] (M. Abdullah), [email protected] (H. Schultz), [email protected] (D. Ka¨hler), [email protected] (D. Branscheid), [email protected] (K. Dalhoff), [email protected] (P. Zabel), [email protected] (E. Vollmer), [email protected] (T. Goldmann). 1 M.A. and H.S. contributed equally to this work. 0344-0338/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2009.04.007
Introduction Haptoglobin (Hp) is an acute phase plasma glycoprotein (with a concentration of 0.45–3 mg per ml blood plasma) composed of a1, a2, and b 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
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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.
Materials and methods 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 without cultivation. Sample 6: tumor-free lung 16 h medium without stimulation. Sample 7: tumor-free lung 16 h stimulation with inactive grass pollen allergen. Sample 8: tumor-free lung 16 h stimulation with active grass pollen allergen. After HOPE-fixation, total RNA was extracted as described previously [19] and submitted to Imagenes (Berlin, Germany) for analysis using an Agilent 44 k transcription array.
Patients/tissues Patients had a mean age of 65 years (range from 45 to 85 years; 61 males, 48 females). A hundred and fifteen tissue samples from these patients were investigated. Pathological characterization defined 47 adenocarcinomas, 42 squamous cell carcinomas, 13 small cell lung cancers, and 13 normal lungs. After lobectomy/ pneumonectomy, tissues were fixed with formalin and embedded in paraffin. For mRNA analysis, 37 tissues were fixed using the HOPE technique and mounted into a TMA [35].
Tissue microarrays (TMAs) All specimens were placed in TMAs as described previously [35]. Sections from these paraffin blocks were mounted on superfrost+ slides (Menzel-Gla¨ser, Germany) for treatment. One TMA containing only HOPE-fixed material was produced for ISH with 20 lung specimens and 17 lung cancer tissues (6 squamous cell carcinomas/10 adenocarcinomas, 1 pleomorphic
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carcinoma). Small cell carcinomas were investigated using single sections.
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then photographed under ultraviolet radiation. The PCR products were sequenced using an ABI 377 sequencer and cycle sequencing [18].
IHC ISH For the immunohistochemical stainings, we used TMA sections as described above [35]. The tissues were deparaffinized by xylene incubation (two times 10 min). Rehydration of the tissues was carried out in a graded ethanol series for two minutes in each step (two times 100%, 2 times 96%, 90%, 80%, 70%; two times aqua dest). For antigen retrieval, slides were cooked in citric buffer (pH 6) for 35 min, and endogenous peroxidases were blocked by incubation of 3% H2O2 solution (10 min). Primary antibody (mouse anti-human; monoclonal anti-haptoglobin antibody; clone HG-36, dilution 1:100; Abcam, UK) was applied for 50 min at room temperature. After washing (Tris saline buffer, 2 min, twice), the detection system (ZytochemPlus HRP polymer kit, Zytomed System, Berlin) was incubated for 30 min, and color reaction was performed using aminoethylcarbazole (AEC) as chromogen (3–15 min) [40]. Slides were counterstained using Mayer’s hemalum and covered with Kayser’s glycerine gelatine. Negative controls were included in each staining series, as well as positive reference sections from human liver to ensure even results.
RT-PCR Total RNA was extracted from 13 human HOPEfixed lung cancer tissues, two healthy lung tissues, and one liver sample according to the manufacturer’s recommendations (RNeasy Mini kit, Qiagen, Germany). After destroying residual DNA by DNase (Gibco, Germany) treatment, cDNA was synthesized by reverse transcription (Superscript II, Invitrogen, Germany) using 1 mg of total RNA template and special oligodT15 primers to enhance reverse mRNA transcription as described previously [19]. PCR was performed, targeting a 341 bp fragment of the human haptoglobin gene using the following primer sequences: Hp forward 50 -AGGCATTATGAAGGCAGCAC-30 ; Hp reverse primer 30 -CTTCCAGGCTGAAATCTTGC-50 . In parallel, specific primers for the house keeping gene glyceraldehde3-phosphate-dehydrogenase (GAPDH; forward primer 50 -AGAACGGGAAGCTTGTCATC-30 ; reverse primer 30 -TGCTGATGATCTTGAGGCTG-50 ) were designed, targeting an amplicon of 257 bp as control. 40 PCR cycles were set as follows: 94 1C for 1 min, 58 1C for 1 min, and 72 1C for 1 min. Finally, one additional cycle at 72 1C for 15 min was programmed. PCR products and a molecular weight marker (pBr322-Msp1) were separated through a 2.4% concentrated ethidium bromide containing (10 ml) agarose gel by electrophoresis, and
Haptoglobin mRNA targeting was performed using a TMA containing 20 healthy lung tissues and 17 cancerous tissues (10 adenocarcinomas, 6 squamous cell carcinomas, and 1 pleomorphic carcinoma). Double stranded, digoxigenin (DIG)-labeled DNA-probes were synthesized as previously described using the PCR products from above. Concentrations of the labeled probe solutions were determined by comparison with several differently concentrated DIG-labeled control DNA spots on a positively charged nylon membrane. In situ hybridization was carried out as previously described using a probe concentration of 2 ng/ml [19,38].
SDS–PAGE and Western blotting Two NSCLC specimens with corresponding tumorfree tissues and two specimens from liver were analyzed using Western blot. The procedure was performed as described previously, with slight modifications [45]. Samples of HOPE-fixed, paraffin-embedded lung, lung cancer, and liver tissue were deparaffinized twice with xylene treatment (1 ml; 10 min) and one step of ethanol treatment (1 ml; 10 min) with respective removal of the supernatants before drying in a vacuum centrifuge (1 h; Speed Vac 110; Savant, Germany). Proteins were extracted at 4 1C overnight using a lysis buffer containing 7 M urea; 2 M thiourea; 2% IGEPAL; 1% Triton-X; 100 mM dithiotreitol (DTT); 5 mM PMSF; 4% CHAPS; 0.5 mM Tris and lysates were denatured (100 1C; 5 min). Protein pellets were resuspended in 5 ml of sample buffer (LDS sample buffer, Invitrogen, Germany). These lysates were subjected to SDS–PAGE (4% stacking gel; 12% resolving gel; Novex minicell, Invitrogen, Germany). Western blotting was performed on a nitrocellulose membrane using the iblot gel transfer system (Invitrogen, Germany) according to the instructions of the manufacturer. Membrane was blocked for 2 h using DIG wash and block buffer (Boehringer Mannheim, Germany). Primary antibody (from above, Section ISH) was incubated in a dilution of 1:100 for 90 min, and the membrane was washed three times in Tris-buffer pH 7.4 (10 min each). Secondary antibody (goat-antimouse, conjugated to alkaline phosphatase; Dianova, Hamburg, Germany) was applied for 1 h, dilution 1:10000, and washed twice, followed by incubation in Tris-buffer at pH 9.5. Color reaction was then achieved using NBT/BCIP as a substrate for the alkaline phosphatase
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(Boehringer Mannheim, Germany) according to the manufacturer’s protocol.
type of tissue sample investigated. Negative controls showed no signal.
RT-PCR
Results 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 tumorfree. 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 Figs. 2 and 3. Among lung cancers, immunohistochemical Hp signals appeared mostly in adenocarcinomas (19/47 cases Hp-positive, 40.4%), showing perinuclear and granular staining of tumor cells (Fig. 2A). In four cases, AEC II cells were positive (8.5%); staining of alveolar macrophages was observed in one case (2.1%). Among the squamous cell carcinoma tissues, 2 of 42 specimens were Hp-positive in tumor cells (4.8%); however, 8 cases (19%) showed stained cells of tumor surrounding AEC II cells (Fig. 2B). Small cell carcinoma staining revealed that 2 of 13 tumors were positive for Hp protein (15.4%; Fig. 2C). Concerning the 13 cases of tumor-free lungs, AEC II cells were found to be positive in four cases (Fig. 3A), alveolar macrophages in all 13 cases (Fig. 3B), and four bronchi were stained (Fig. 3C). Fig. 4 shows positive cases in relation to the total number and the
RT-PCR was performed to additionally address transcripts of Hp. Thirteen NSCLC tissues, 2 corresponding lung tissues, and 1 liver sample were analyzed using Hp targeting RT-PCR. We found Hp mRNA in all NSCLC and lung tissues and, as expected, Hp in the liver. Negative control lanes showed no bands (Fig. 6). This verifies the IHC results and proves transcription of the Hp gene in human lung tissues and lung malignancies (Figs. 5 and 6).
ISH We additionally analyzed 17 carcinomas by in situ hybridization to detect cellular transcripts of the haptoglobin mRNA within the tissues. In all adenocarcinomas and in four of six squamous cell carcinomas, signals were generated within the tumor cells. Alveolar macrophages, AEC II cells, and epithelial cells of the bronchi also displayed signals of Hp transcripts (Fig. 7). Negative controls showed no signals.
Western blot Western blot analysis was performed to compare the amounts of Hp-protein from NSCLC, tumor-free lungs, and liver. As depicted in Fig. 8, we found comparable amounts of Hp in NSCLCs compared to the liver tissues. Tumor-free lungs showed comparably weaker signals. All signals had a molecular weight of around 40 kDa (the size of a single b-chain of haptoglobin).
Discussion
Fig. 1. Hp transcription array data of eight different tissue samples in a respective comparison with housekeeping gene GAPDH for tumor+medium (1), tumor+chemotherapeutics+medium (2), lung+medium (3), lung+Haemophilus influencae+medium (4), lung HOPE-fixed uncultured (5), lung+medium uncultured (6), lung+inactive grass pollen allergen (7), lung+active grass pollen allergen (8).
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 lung tissues is observed for the first time. Cellular localization observable by granular staining was either perinuclear (adenocarcinomas and bronchi), cytoplasmic (small cell carcinomas and macrophages) or, in case of squamous cell carcinomas, mostly apparent in areas around the tumor (AEC II cells). Therefore, it is possible that Hp as
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Fig. 2. Immunohistochemical detection of haptoglobin in different subtypes of lung cancer employing adenocarcinomas (A), squamous cell carcinomas (B), and small cell carcinomas (upper panel 400 , lower panel 800 ).
Fig. 3. Immunohistochemical detection of haptoglobin in different non-malignant cell types of human lung tissues: AEC II cells (A), alveolar macrophages (B), and bronchi (C). (upper panel 400 , lower panel 800 ).
an acute phase protein is involved in a variety of immunoreactive processes. Inflammation might also be a relevant factor in immunoreactions during lung cancer genesis. Our results reveal that in cases of squamous cell carcinomas, no frequent expression of haptoglobin could be observed in tumor cells. However, the tumor surrounding AEC II cells and bronchile epithelial cells were frequently stained with high intensity. This was also observed in tumor-free lungs. By contrast, the tumor cells of adenocarcinomas, to a large degree, revealed intense perinuclear and granular staining. This phenomenon could be due to the different cell types from which the carcinomas derive [47].
There is a variation of expression concerning tumor subtypes (about 40.4% of adenocarcinomas, 4.8% of squamous cell carcinomas, and 15.4% of small cell carcinomas are Hp-positive), which may be caused by different haptoglobin phenotypes or Hp polymorphisms associated with tumor types. Many studies conducted worldwide suggest that Hp1-1 and Hp1-2 are more common than Hp2-2 [3,6,9,10,23,44], and that the geographical distribution is important due to differences in gene sequences/frequencies concerning haptoglobin, such as the absence of haptoglobin in Caucasian people or the low Hp1 allele frequency in Southeast Asia [29]. This leads to the speculation that most cases of adenocarcinomas might belong to Hp type 1-1 or 1-2,
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Fig. 4. Expression of haptoglobin in different tumor-entities and tumor-free lung tissues determined by immunohistochemistry. Left bar: number of cases analyzed, right bar: number of positive cases for adenocarcinomas (A), squamous cell carcinomas (B), small cell carcinomas (C), and tumor-free lung tissues (D).
Fig. 5. Results of RT-PCR targeting Hp-transcripts in 10 specimens of NSCLC. Products were separated on agarose gels and stained with ethidiumbromide. Panel A shows the 341 bp fragment of Hp; Panel B the 257 bp fragment of GAPDH. Molecular weight marker: M ¼ pBr322/Msp1.
Fig. 6. RT-PCR targeting haptoglobin (lower bands) and GAPDH (upper bands) for healthy lung (lu), lung tumor (tu); liver (li) and negative control (neg).
while squamous- or small cell carcinomas might belong to a patient carrying Hp type 2-2. Further studies are necessary to understand the relations between Hp production of tissues and correlating cancer types. Our results are also in agreement with Beckman et al. [9], who reported that a group of patients with squamous cell carcinomas and another one with small cell carcinomas showed no significant difference in Hp concentration in the plasma compared to a control group. However, among patients with pulmonary adenocarcinomas, the frequency of haptoglobin
type 2-2 is lower compared to the control group, with a corresponding increase in the Hp 1-1 and Hp 1-2 type [12]. Another important finding related to the functions of Hp in cancer is the recent demonstration of haptoglobin as a natural inhibitor of collagen degradation, and it is locally expressed by fibroblasts in the arterial wall [25,42]. The Hp-glycoprotein in this environment is crucial for cell migration processes and arterial restructuring like angiogenesis. Moreover, collagen turnover is important in many physiological processes, such as tumor cell invasion, wound healing, cell growth, metastasis, and enhancement of collagen degradation. This is related to a severe tissue destruction and malfunction, which is often encountered in pathological processes, such as arthritis and metastasis. Thus, the high expression of Hp in adenocarcinomas may be correlated with their comparably high potential to metastasize. This reveals an importance of haptoglobin for functions in the extracellular matrix and during cell migration, and suggests another role for this polypeptide in cancers [24,43,46]. In general, our results are correlated with those of other groups which showed that increased levels of haptoglobin synthesis appear during lung cancer genesis, and that higher amounts of Hp can be observed in the blood plasma of those patients. The findings of these groups could affirm the Hp’s capability of neutralizing super oxidative products, and suggest Hp as a potential serum tumor biomarker candidate for lung cancers. We are in accordance with this presumption due to our finding of Hp also in tumor tissues. The increase in haptoglobin expression in tissues and serum may enable evasion and surveillance mechanisms of tumor cells [8,11,20,21,26,31,39,41]. Another recent study has reported that native haptoglobin interferes with the activated human neutrophil function by blocking its receptor-mediated Ca2+ and haptoglobin influx from normal human blood plasma [33]. This interferes again with macrophageand lymphocyte-activity during mitogenesis [7,32,34]. Therefore, it is suggested that Hp may act as a natural antagonist for receptor ligand activation of the immune system. Arredouani et al. [2] further described the role of haptoglobin affecting the immune system, showing that haptoglobin directly affects T-cells and suppresses T-helper-cells through downregulation of the cytokine production and associated mechanisms. However, it is not clear whether this is an indirect phenomenon caused by modulation of antigen-presenting cell (APC) function or whether haptoglobin acts directly on T cells [2]. Seder et al. [41] showed that passive removal of Hp out of the plasma of cancer patients restores the immune response at least temporarily. In conclusion, our findings are the first to demonstrate strong expression of haptoglobin in lung cancer
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Fig. 7. Results of in situ hybridization targeting haptoglobin transcripts using a 341 bp digoxigenated, double stranded DNA-probe on adenocarcinomas (A, 400 ), squamous cell carcinomas (B, 400 ), alveolar macrophages (C, 100 ), bronchi (D, 400 ), and AEC II cells (E1 100 ; E2 400 ).
Fig. 8. Western immunoblot targeting haptoglobin in tumorfree lung (lu), tumor (tu), and liver (li, lih) (lih for liver high protein concentration).
tissues, and more often in adenocarcinomas than in squamous cell carcinomas or small cell carcinomas. There is also an increased amount of haptoglobin mRNA in tumor tissues and tumor surrounding cells, shown by positive ISH and RT-PCR. This detection is correlated with the expression level of the protein, which was observed in IHC staining. Hence, the ability of Hp to affect immune responses could play an important role in lung carcinogenesis, including tumor evasion from immunological attacks [39]. Modulation of Hp expression and its involvement in immunoregulatory events is currently investigated using a novel human ex vivo model [19,28]. Hp expression in tumor-free lung tissues was located in alveolar macrophages, AEC II and bronchi. Taken together, our results strongly suggest a role of Hp in the
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local immune defense of the lung and might also be important in different inflammatory processes. Appropriate studies are being conducted to further elucidate these aspects.
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. Dro¨mann 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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