Apolipoprotein E and colon cancer

Apolipoprotein E and colon cancer

European Journal of Internal Medicine 13 (2002) 37–43 www.elsevier.com / locate / ejim Original article Apolipoprotein E and colon cancer Expression...

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European Journal of Internal Medicine 13 (2002) 37–43 www.elsevier.com / locate / ejim

Original article

Apolipoprotein E and colon cancer Expression in normal and malignant human intestine and effect on cultured human colonic adenocarcinoma cells b ¨ Mari Niemi a,e , *, Tomi Hakkinen , Tuomo J. Karttunen c , Sinikka Eskelinen c,e , Kari Kervinen a,e , a,e b ¨ ¨ d , Seppo Yla-Herttuala ¨ Markku J. Savolainen , Juhani Lehtola a , Jyrki Makela , Y. a,e ¨ Antero Kesaniemi a

Department of Internal Medicine, University of Oulu, Kajaanintie 50, FIN-90220 Oulu, Finland b A.I. Virtanen Institute, University of Kuopio, Kuopio, Finland c Department of Pathology, University of Oulu, Oulu, Finland d Department of Surgery, University of Oulu, Oulu, Finland e Biocenter Oulu, University of Oulu, Oulu, Finland

Received 23 October 2000; received in revised form 29 May 2001; accepted 10 September 2001

Abstract Background: Apolipoprotein E (apo E) is a key regulatory protein in lipoprotein metabolism and it is also a potent inhibitor of cell proliferation. Although genetic alterations of apo E affect enterohepatic cholesterol transport and, presumably, the risk of colon carcinoma, the expression and potential functions of apo E in the human intestine are poorly known. Methods: The localization of apo E in normal and malignant gastrointestinal tract was studied using immunohistochemistry and in situ hybridization. The effect of apo E3 on cell polarity and the distribution of b-catenin was examined in HT29 human colon adenocarcinoma cell lines. Results: Both apo E protein and mRNA were present throughout human intestine. The macrophages in the superficial lamina propria of normal colon were more strongly positive for apo E than those in the small intestine, where the most positively stained cells were dendritic cells and macrophages in the germinal centers of lymphoid follicles. In carcinomas, intensely positive macrophages surrounded the tumor area. In cultured undifferentiated HT29 cells, treatment with apo E improved cell polarity and translocated b-catenin from the cytoplasm to cell–cell adhesion sites. Conclusions: Mononuclear phagocytes and endocrine cells are the main source of apo E in the gastrointestinal tract. We hypothesize that macrophage-derived apo E may modulate epithelial integrity and thus contribute to cell growth.  2002 Elsevier Science B.V. All rights reserved. Keywords: Apolipoprotein E; Colon cancer; b-catenin; Cell polarity; Cell–cell adhesion

1. Introduction Apolipoprotein E (apo E) is an arginine-rich plasma glycoprotein that mediates the cellular uptake of lipoproteins by binding to the low-density lipoprotein receptor (LDLR) and the low-density lipoprotein receptor-related *Corresponding author. Tel.: 1358-8-315-4570; fax: 1358-8-3154543. E-mail address: [email protected] (M. Niemi).

protein (LRP) [1,2]. Apo E has an important role in lipid transport [3], and numerous studies have shown that defective expression of apo E (either absent expression or expression of variant forms) is associated with altered plasma lipid levels [4,5] and premature atherosclerosis [6,7]. Apo E binds with very high affinity to heparin and proteoglycans [8–10] and inhibits the proliferation of several cell types, including lymphocytes [8], endothelial cells [11], smooth muscle cells [12], and tumor cells [11,13]. Despite intensive research, the antiproliferative

0953-6205 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0953-6205( 01 )00191-1

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mechanism of apo E is still largely unknown. Some recent findings suggest that apo E contributes to apoptotic cell death [14], possibly by association with amyloid-beta. Apo E is synthesized in many tissues, including the liver, adrenal gland, kidney, lung, spleen, testes, ovary, and brain [15,16], but some animal studies have suggested that it is not synthesized in the intestine [17]. However, genetic variations of apo E may modulate enterohepatic cholesterol transport [18], cholesterol absorption efficiency [19] and, presumably, the risk of colon carcinoma [20], suggesting that apo E has a physiological role in the intestine. One of the key features of differentiated epithelial cells is their polarized structure. Generation of the polarized phenotype is a multi-stage process that requires extracellular cues in the form of cell–cell and cell–matrix contacts and the reorganization of cell surface proteins and cytoplasmic proteins. Once established, the phenotype must be maintained by the segregation and retention of specific proteins and lipids in distinct plasma membrane domains [21]. Cell adhesion plays a central role in cell motility, growth, differentiation, and survival [22]. The adhesion and the development of cell surface polarity are regulated by calcium-dependent cell adhesion proteins called cadherins. Cadherin function is modulated by a class of proteins called catenins that regulate cadherin function in cell–cell adhesion [23]. Apart from their direct role as physical linkers of the actin cytoskeleton to cadherins, catenins also play a central role in signal transduction and the regulation of gene expression. Free pools of b-catenin in the cytoplasm may enter the nucleus and induce a transcription process [24]. Detachment of cell–cell adhesion is indispensable for the first step of invasion and metastasis of cancer [25], and it is also needed for the induction of endothelial cell proliferation and angiogenesis [26]. Previous studies on the effects of apo E on cell spreading [11] and neurite outgrowth [27] suggest that the antiproliferative effect of apo E may result from binding to cell surface or matrix heparan sulfate proteoglycans and, thus, have an effect on cellular interactions. Some preliminary reports have also suggested that apo E may enhance microtubule formation and thus enhance cell polarity [28]. The purpose of the present study was to investigate the expression of apo E in the human gastrointestinal tract and to further examine its potential functions in cell regulation. The following questions were addressed: (1) is apo E produced in normal human intestine? (2) Is apo E production altered in colon tumors? (3) What is the effect of apo E on intestinal carcinoma cells?

2. Materials and methods

2.1. Patients Ten specimens of histologically normal human esoph-

agus, stomach, duodenum, ileum and proximal, transversal, and distal colon were obtained by endoscopic biopsy performed for diagnostic reasons. Samples of carcinomatous and normal-appearing intestinal mucosa were obtained from a total of 14 patients admitted to the Oulu University Hospital for bowel resection. Nineteen of the patients were men and five women, and their ages ranged from 25 to 85 years (mean 55 years). Their cancer distribution was as follows: stomach 4, pancreas 2, papilla of Vater 1, jejunum 3, and colon 4. The histological classification [29] for all of the samples was performed by pathologists with special competence in gastrointestinal pathology at the Department of Pathology, Oulu University Hospital. Patients undergoing chemotherapy and patients with residual cancer were excluded from the study. All patients gave informed consent for the investigation, which was approved by the Ethical Committee of the University of Oulu.

2.2. In situ hybridization and immunohistochemistry The samples for in situ hybridization and immunocytochemistry studies were fixed in 4% buffered formaldehyde, sectioned (5 mm), and deparaffinized, as described elsewhere [30]. Nucleotides 582–872 of human apo E [31] in pBluescript SK (Stratagene, La Jolla, CA, USA) were used for apo E antisense and sense riboprobe synthesis, using T7 and T3 polymerases with 35 S-UTP (NEN Life Science Products, Boston, MA, USA), as described previously [30]. In situ hybridizations were performed on pretreated sections using 6310 6 cpm / ml of the labeled probe. The final wash after the hybridization was at 54 8C in 0.1% SSC for 30 min. The sample slides were dipped in Kodak NTB-2 nuclear track emulsion (Eastman-Kodak, Rochester, NY, USA) and exposed for 4 weeks. Nonhybridizing sense probes were used as controls. Immunostainings were carried out with the following antibodies: goat polyclonal antibody against human apo E (Genzyme, West Malling, Kent, UK), mouse monoclonal antibody (mAb) against apoB-100 (MB47) [32], mouse mAbs against human macrophages (KP1 and PGM-1, DAKO, Copenhagen, Denmark), mAb against chromogranin A to detect endocrine cells in the epithelium (DAKO), and mouse mAb against bovine and human LDL receptor (clone C7 / RPN 537; Amersham, New Hampshire, UK). Anti-goat and anti-mouse IgG biotin conjugates (diluted 1:400, DAKO) and avidin–biotin–horseradish peroxidase system (Zymed, San Francisco, CA, USA) were used for signal detection. Peroxidase reaction was performed using 3,39diamino-benzidine (Sigma Chemical Co., St. Louis, MO, USA) or 3-aminoethyl-carbazole (AEC Single SolutionE chromogen, Zymed) as a substrate and counterstained with hematoxylin. Irrelevant class- and species-matched immunoglobulins and incubations without the primary antibody were used as controls for the

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immunostainings. For the identification of apo E-positive cells, we used a double immunostaining procedure on the same sections. AEC Single SolutionE chromogen (Zymed), which produces a red stain, was used in the apo E immunostaining. The Double Stain EnhancerE (Zymed) was then added to separate the two chromogenic reactions, and the Vector SG substrate kit for peroxidaseE (Vector Laboratories Inc., Burlingame, CA, USA), which yields a blue–gray stain, was used in the macrophage and endocrine cell immunostaining. Micrographs were produced using a digital camera (SenSys KAF1400-G2, Photometrics Ltd., Tucson, AZ, USA), digital image-processing software (Image-Pro Plus, Media Cybernetics, Silver Spring, MD, USA), and a sublimation printer (Kodak DS 8650, Eastman-Kodak).

2.3. Cell culture HT29 cells (kindly provided by Dr. Jan Willem Kok, Groningen, The Netherlands), which were recently shown to express functional LDL receptors [33], were cultured as previously described [34] in DMEM supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA), 2 mM glutamine, and antibiotics (penicillin, 100 units / ml; streptomycin sulphate, 100 mg / ml; amphotericin B, 0.25 mg / ml) in a humidified atmosphere containing 5% CO 2 at 37 8C. The media were changed every 2–3 days and cells were used for the experiments in the passages 5–15. The cells were grown on glass coverslips for immunofluorescence microscopy, and sparse monolayers were used for experiments. The cells were serum-starved for 24 h and human recombinant apo E3 (Biodesign, Denmark, Copenhagen), with biological properties indistinguishable from those of the native protein [35] was then added to the culture to a final concentration of 0.5 mM. After 24 h of incubation, the cells were washed with Hank’s balanced salt solution (Gibco) and rapidly fixed.

2.4. Immunostaining and fluorescence microscopy for HT29 cells For staining with monoclonal anti-b-catenin (Zymed), cells were grown on glass coverslips and fixed with 4% formaldehyde in a cytoskeleton-stabilizing buffer (100 mM PIPES, 4 mM EDTA, 2 mM MgCl 2 , pH 6.8) containing 0.1% Triton X-100. After several washes with phosphatebuffered saline (PBS; 145 mM NaCl, 10 mM phosphate, pH 7.4), the cells were post-fixed with cold methanol (220 8C) for 5 min. After repeated washings with PBS, the cells were incubated with 10% FCS to saturate nonspecific, protein-binding sites. This was followed by incubation with the primary antibody at 14 8C for 30 min and then with Texas Red-conjugated goat anti-mouse antibody (Molecular Probes Eugene, OR, USA). The cells were then mounted in Shandon mounting liquid (Immu-

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Mount, Pittsburgh, PA, USA), and viewed under a Zeiss 405M microscope. Kodak TMAX 3200 ASA film was used for photography.

3. Results

3.1. Apo E expression in human intestine Both immunoreactive apo E protein and apo E mRNA were present throughout the stomach, small intestine, and colon. The phagocytes of lamina propria were positive for apo E (Fig. 1A, C, D and I), but there was some variation in the number of positive cells and in the staining intensity. The colonic macrophages in the superficial lamina propria were more strongly positive for apo E than those in the small intestine, where most positively stained cells were dendritic cells and macrophages in the follicular centers of lymphoid nodules (Fig. 1E, G, and H). Of the epithelial cells, gastric chief cells, some areas of intestinal metaplasia and a subpopulation of granulated endocrine cells were also positive (Fig. 1J, K, and L). The solid carcinomas contained intensely positive macrophages lining the tumor area (Fig. 1M and N). Apo E colocalized with KP1 and chromogranin A antibodies (Fig. 1I and J). Control immunostainings with irrelevant class- and speciesmatched immunostainings and in situ hybridizations with control oligonucleotide probes were negative (data not shown).

3.2. Effect of apo E on the HT29 human colon adenocarcinoma cell line The organization of b-catenin was analyzed by immunofluorescence microscopy. In apo E-treated, undifferentiated HT29 G1 cells, b-catenin (Fig. 2B) appeared as an intense line in the areas of cell-to-cell interaction, whereas in control cells cultured without apo E, the distribution of b-catenin was weaker and more diffuse (Fig. 2D). The polarity marked by the formation of lateral cell–cell contacts seemed to be enhanced in apo E-treated HT29 G1 cells. There was no clear effect of apo E on differentiated HT 29 G1 reversed cell line (data not shown), where the cell morphology was well differentiated in both apo E-treated and control cells.

4. Discussion We report here that macrophages and endocrine cells are the main source of apolipoprotein E in the gastrointestinal tract. To our knowledge, this is the first study on apo E location and expression in the human intestine, and it raises the important question of a possible physiological role of apo E there. Apo E is synthesized by a wide variety of peripheral cells, including macrophages [9,17], and such

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Fig. 1. (A–L) Expression of apo E protein and mRNA in normal intestinal mucosa. Apo E is present in macrophages in the lamina propria of the colon (A, C, D, I; black arrows), in the follicular centers of the small intestine (E, G, H; white arrows), and in endocrine cells at the crypt base (J, K, L; black arrowheads). (A) Immunostaining for apo E in colonic mucosa (antibody dilution 1:100). (B) Non-immune control for apo E immunostaining (first antibody omitted). (C, D) In situ hybridization with a 35 S-labeled antisense riboprobe for apo E mRNA in the colon. C was photographed using polarized light epiluminescence and D is a phase contrast micrograph from a corresponding region showing the cell morphology. (E) Immunostaining for apo E in follicular centers of the small intestine. Apo E is present in macrophages and dendritic cells. (F) Non-immune control (first antibody omitted). (G, H) In situ hybridization with a 35 S-labeled antisense riboprobe for apo E mRNA in the small intestine. G was photographed using polarized light epiluminescence and H is a phase contrast micrograph from a corresponding region. (I) Double immunostaining for apo E (red staining) and macrophage antibody KP1 (blue staining). The cells in colonic lamina propria show cytoplasmic staining for both antigens (black arrows). (J) Double immunostaining for apo E (red staining) and endocrine cell antibody anti-chromogranin A (blue staining). A subpopulation of epithelial cells show cytoplasmic staining for both antigens (black arrowheads). (K, L) In situ hybridization for apo E mRNA in the small intestine. K was photographed using polarized light epiluminescence and H is a phase contrast micrograph from the same region. (M, N) Expression of apo E in colon cancer. The tumor area is lined with apo E-positive cells. (M) In situ hybridization for apo E mRNA. M was photographed using polarized light epiluminescence and P is a phase contrast micrograph from the same region. Magnification 3200, except I, J 3400.

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Fig. 2. Phase contrast and immunofluorescence micrographs of HT29 G1 cells incubated for 24 h with human recombinant apo E. Phase contrast micrograph showing the morphology of the cells (A) and immunofluorescence micrograph showing the localization of b-catenin (B). Apo E-treated cells appeared columnar in shape and b-catenin appeared as an intense line in the areas of cell-to-cell interaction, whereas the cell morphology in untreated cells (C and D) is less regular and the distribution of b-catenin is weaker and more diffuse. Bar510 mm.

production by extrahepatic cells has raised questions regarding potential roles of apo E in peripheral tissues. Expression of apo E in macrophages facilitates cholesterol efflux from cholesterol-loaded macrophages in exogenous acceptors (e.g. HDL 3 ) [36]. Transgenic mice overexpressing apo E in the intestine have markedly reduced plasma cholesterol levels and post-prandial hypertriglyceridemia [37]. Apo E synthesized in the intestine may thus influence cellular cholesterol homeostasis and cholesterol absorption. The growth and behavior of a malignant tumor is governed by the interaction of macrophages, lymphocytes, mast cells, and endothelium. Apo E has been shown to have potent effects on lymphocyte function, suppressing the production of interleukin-2 and inhibiting lymphocyte proliferation [8], and macrophage-derived apo E could thereby modulate the mucosal immune function. Apo E is capable of inhibiting the growth of several tumor cell lines [11] including breast carcinoma cells, melanoma cells, and Kaposi’s sarcoma cells [13]. The underlying mechanisms are not known, but due to its high affinity to heparan sulfate proteoglycans and numerous receptors (e.g. LDL receptor and LRP), apo E may affect

cellular homeostasis in many ways. The ability of carcinomas to invade and metastasize largely depends on the degree of epithelial differentiation within the tumors. Ecadherin-mediated cell–cell adhesion has been shown to prevent invasiveness of human carcinoma cells [38], and mechanisms that lead to a loss of cell–cell adhesion (mutation, loss of catenin expression, alterations in phosphorylation) are thought to contribute to a more metastatic phenotype [39]. As our results suggest that apo E may enhance cell polarity, we hypothesize that apo E may improve the assembly of intercellular junctions as alterations in b-catenin distribution and thus induce contactdependent growth inhibition of cell growth and migration. In addition to its function in cell adhesion, b-catenin is highly homologous to Drosophila armadillo and belongs to the armadillo family [40]. Armadillo in Drosophila and b-catenin in Xenopus have been shown to play a role in the transduction of transmembrane signals initiated by the extracellular glycoprotein Wg / Wnt that regulates cell growth, differentiation, and fate [41]. The accumulation of cytoplasmic b-catenin is an important step in its nuclear translocation and complex formation with transcription

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factors, LEF / TCF. This process can be inhibited by linking b-catenin to the cadherin complex at cell–cell adhesion sites or by complexing it in the cytoplasm with GSK, APC, and axin [24]. It seems that apo E may also be one factor affecting the Wg / Wnt signaling pathway. The integrity of vascular endothelium is mainly dependent upon the organization of interendothelial adherent junctions. Adherent junctions are expected to play a major role not only in vessel permeability, but also in endothelial surface polarity [23]. The inhibitory effects of apo E on endothelial cell proliferation, motility, and adhesion [11] suggest a role for apo E as an antiangiogenic factor and further suggest that apo E may be useful in inhibiting pathological neovascularization. The potential effects of apo E in the intestine include modulation of cellular cholesterol balance, availability of growth factors, and mucosal immune response or improvement of epithelial and endothelial cell integrity. The effect of apo E on cell differentiation provides a new approach to the study of the physiological functions of apo E in cellular homeostasis and may be an important factor explaining the different associations of apo E polymorphisms with such complex diseases as atherosclerosis, Alzheimer’s disease, and colon cancer.

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Acknowledgements This work was supported by the Medical Council of the Academy of Finland and the Cancer Society of Northern Finland. The skillful technical assistance of Ms. Saija ¨ Kortetjarvi, Ms. Liisa Mannermaa, Ms. Leena Ukkola, Ms. Erja Tomperi, Ms. Marja Tolppanen, and the staff of the Gastroenterological unit of Oulu University Hospital is greatly appreciated.

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