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. 2002 Oct;83(5):239–245. doi: 10.1046/j.1365-2613.2002.00237.x

Serum and tissue level of insulin-like growth factor-I (IGF-I) and IGF-I binding proteins as an index of pancreatitis and pancreatic cancer

Ewa Karna *, Arkadiusz Surazynski *, Kazimierz Orłowski †, Joanna Łaszkiewicz †, Zbigniew Puchalski †, Piotr Nawrat *, Jerzy Pałka *
PMCID: PMC2517686  PMID: 12641820

Abstract

Previously we have found deregulation of collagen metabolism in human pancreatitis and pancreatic cancer tissues. Insulin-like growth factor-I (IGF-I) is known to stimulate collagen biosynthesis through interaction with IGF-I receptor. IGF-I binding proteins (BPs) regulate the activity of IGF-I. We investigated whether serum and tissue IGF-I and IGF-BPs as well as tissue IGF-I receptor expression may reflect disturbances of collagen metabolism in patients with pancreatitis and pancreatic cancer. In pancreatitis tissue, a significant increase in IGF-I and IGFBP-3 content was accompanied by a distinct increase in IGF-I receptor expression, compared to control pancreas tissue. In contrast, serum from patients with pancreatitis did not show significant increases in IGF-I and IGFBP-3 levels, however, significant increases in IGFBP-1 level (2.5 fold). Moreover, a distinct decrease in radioactive IGF-binding to the BPs, compared to control serum, was found. Pancreatic cancer tissue and serum of patients with pancreatic cancer showed significant increases in IGF-I, IGFBP-3 and IGFBP-1 content, accompanied by dramatic increases in IGF-I tissue receptor expression, compared to controls. In serum of patients with pancreatic cancer distinct increases in radioactive IGF-binding to 46 kDa BP, compared to control serum, were observed. The data suggest that disturbances in tissue collagen metabolism during pancreatic diseases may result from deregulation of IGF-I homeostasis and that elevated serum levels of IGF-I, IGFBP-3 and IGFBP-1 may serve as markers of pancreatic cancer

Keywords: chronic pancreatitis, collagen, IGF-I binding proteins, IGF-I receptor, insulin-like growth factor-I (IGF-I), pancreas, pancreatic cancer

Several human tumours are often associated with fibrosis. Accumulation of connective tissue around the growing tumour consists of various types of interstitial collagen, fibronectin, elastin and proteoglycans (Merrilees & Finlay 1985; Kao et al. 1986; Martinez-Hernandez 1988; Christensen 1990). Pancreatic diseases, such as chronic pancreatitis and pancreatic cancer are characterized by profound alterations in extracellular matrix (ECM) formation, composition and tissue deposition (Gress et al. 1998). Chronic inflammatory diseases of the pancreas are characterized by destruction of acinar cells and islet cells and replacement by connective tissue (Kennedy et al. 1987). Pancreatic cancer shows increase of interstitial connective tissue (Klöppel 1993; Gress et al. 1994; Gress et al. 1995), especially collagen. Collagen is essential not only for the maintenance of architecture and integrity of connective tissue, but also in interaction with cell surface receptors. It is known that the interaction between cells and ECM proteins, e.g. collagen, can regulate cellular gene expression, differentiation, cell growth (Bissel 1981; Carey 1991) and can play an important role in tumorigenicity and invasiveness (Ruoslahti 1992).

It has been postulated that the accumulating matrix components are products of host mesenchymal cells but not of the tumour cells (Merrilees et al. 1985; Kao et al. 1986). Tumour cells have been shown to secrete a variety of cytokines and growth factors (Dickson et al. 1987) known to stimulate collagen synthesis by fibroblasts (Bano et al. 1983). One of them is insulin-like growth factor-I (IGF-I), a multifunctional growth factor with potent growth, collagen- and proteoglycan-stimulating activity (Oyamada et al. 1990). The effects of IGF-I are regulated by a family of distinct IGF-I binding proteins, designated IGF BP-1 to -6 (Clemmons 1997; Kim et al. 1997). The different IGF BPs have been shown to restrict IGF tissue availability, regulate IGF transport to cells, and modulate IGF binding to membrane receptors (Le Roith et al. 1995).

A high molecular weight (150 kDa) binding protein complex (HMW-BPs) and low molecular weight (30–50 kDa) binding proteins (LMW-BPs) have been found. Under normal conditions most of the IGF-I circulates in adult human plasma in a form bound to HMW-BPs, which contain BP-3 as an active constituent. It has been postulated that HMW-BPs prolong the half-life of IGF-I and increase cell responsiveness to IGF-I stimulation (Clemmons 1998). The LMW-BPs (BPs 1 and 2 as well as BPs 4, 5 and 6) are present in plasma and tissues in concentrations sufficient to modify IGF-I action (Sara & Hall 1990; Lamson et al. 1991; Levitt et al. 1993; Le Roith et al. 1995; Clemmons 1997).

Since the content of extracellular collagen in pancreatitis and pancreatic cancer is different, we assumed that the changes might be due to deregulation of IGF-I activity in these tissues. Therefore the current study was undertaken to determine tissue and serum IGF-I, IGF-BPs content, tissue IGF-I receptor expression and ability of BPs to bind IGF-I in patients with pancreatitis and pancreatic cancer.

Materials and methods

Anti-human insulin-like growth factor-I receptor (IGF-I sR) antibody, alkaline phosphatase conjugated anti-goat IgG, bovine serum albumin (BSA), IGEPAL-CA 630 and Sigma-Fast BCIP/NBT reagent, and most other chemicals and buffers used were provided by Sigma Corporation (USA). IGF-I radioimmunoassay (RIA) kit was purchased from Incstar Corporation, USA. [125I]-IGF-I, Amerlex-M-Separation reagent was obtained from Amersham (UK). IGFBP-1 immunoradiometric assay (IRMA) and IGFBP-3 (RIA) kits were purchased from Diagnostic Systems Laboratories, Inc., USA. Nitrocellulose membrane (0.2 µm) and Tween-20 were received from Bio-Rad Laboratories (USA).

Histology

The samples of pancreatic tissues obtained following pancreas resection were placed in cold-ice 0.05 mol/L Tris-HCl, pH 7.6 buffer and extensively perfused with the same buffer. On the day of resection, matched sera were prepared. The materials (pancreatic tissues and sera) were stored at −70 °C, until assay. Ten human pancreatic carcinomas, 10 samples of pancreatitis tissue and 10 representative samples of normal pancreas, obtained from the margin of surgically resected tumours were selected from the multi-files of the Department of Pathology, Medical Academy in Białystok, Poland. The histological type of pancreatitis and pancreatic carcinoma was determined according to the classification of Becker and Stömmer (Becker & Stömmer 1997). Pancreatitis was recognized according to the criteria of Ammann (Ammann 1997).

Preparation of tissue extracts

The tissue homogenates (20% w/v) were prepared in 0.05 mol/L Tris-HCl, pH 7.6 with the use of a knife homogenizer (Polytron) and subsequently were sonicated at 0 °C. Homogenates were centrifuged at 16 000 g for 30 min at 4 °C. Supernatant (tissue extract) was used for assays.

Separation of IGF-I from BPs

At acidic pH the IGF-BPs complex dissociates releasing free IGF-I, which may be determined by radioimmunoassay (RIA). This procedure has been described in detail in a previous paper (Pałka et al. 1989). Briefly, 250 µL of tissue extract or 25 µL of serum in 1 m acetic acid were submitted to gel filtration on Bio-gel P-60 (100–200 mesh) column (1 × 40 cm) and eluted with 1 m acetic acid. Fractions of 1 mL were collected. To determine the position of IGF-I in the eluate the 125I-labelled IGF-I was added to 250 µL of tissue extract or 25 µL of serum and chromatographed as described above.

Fractions of eluate, containing IGF-I were pooled, evaporated to dryness, and redissolved in 0.5 mL of assay buffer, consisting of 0.03 m sodium phosphate, 0.2 mg/mL protamine sulphate, 0.02% sodium azide, 0.01 m EDTA and 0.25% BSA. Aliquots were submitted to radioimmunoassay for IGF-I, as described below.

Fractions of eluate containing BPs were pooled, evaporated, redissolved in 0.5 mL of 0.1 m Tris-HCl buffer pH 7.6 and submitted to specific BP-assays.

RIA for IGF-I

The assay was performed according to a slightly modified protocol provided by Amersham with the Somatomedin C reagent pack for radioimmunoassay (code IM 1721). The reaction mixture contained 100 µL of assay buffer, supplemented with either unlabelled IGF-I (0.05–3.2 ng) or the pooled low molecular weight fractions from the Bio-gel P-60 column (20–40 µL diluted with assay buffer 1 : 10) and 100 µL of antibody diluted 1 : 4000 with assay buffer. Samples were incubated for 30 min at room temperature and then 100 µL of [125I]-IGF-I (about 15 000 cpm, specific activity about 74 TBq/mmol = 2000 Ci/mmol) was added. Incubation was continued for 48 h at 4 °C after which 500 µL of Amerlex-M second antibody reagent was added. The mixture was incubated for 10 min at room temperature with occasional mixing, centrifuged at 25 000 g for 10 min and the radioactivity of the sediment was determined. A standard competition curve was established using 0.05–3.2 ng of unlabelled IGF-I per tube. The radioactivity of control samples (containing 100 ng of unlabelled IGF-I) was subtracted from the radioactivity of the test samples to correct for non-specific binding.

Immunoradiometric assay (IRMA) for IGFBP-1

The assay was performed according to a protocol provided by Diagnostic Systems Laboratories Inc. with the Active Total IGFBP-1 Coated-Tube Immunoradiometric Assay Kit (code DSL-7800). The assay is based on the procedure of Miles et al. (1974). The reaction mixtures contained 10 µL or 25 µL samples (1 : 10 diluted pooled fraction containing BPs from serum or tissue extracts).

Radioimmunoassay (RIA) for IGFBP-3

The assay was performed according to protocol provided by Diagnostic Systems Laboratories Inc with the IGFBP-3 Radioimmunoassay Kit (code DSL-6700). The reaction mixtures contained 100 µL of samples (1 : 100 diluted pooled fraction containing BPs from serum or tissue extracts).

Radioactivity assay

Radioactivity was measured with the use of a Mini-gamma 1275 counter (LKB Wallac).

Sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE)

Slab SDS/PAGE was performed according to the method of Laemmli (1970). Samples of BPs isolated from 2 µL of serum or 2 mg of tissue were subjected to electrophoresis on 10% SDS-PAGE gels. The following Bio-Rad's unstained low molecular weight standards were used: IgG (150 kDa), BSA (69 kDa), ovalbumin (46 kDa) and carbonic anhydrase (30 kDa). After SDS-PAGE, the gels were allowed to equilibrate in a mixture of 0.025 m Tris and 0.2 m glycine in 20% (v/v) methanol for 5 min. The protein was transferred to 0.2 µm pore-sized nitrocellulose, at 100 mA for 1 h using a LKB 2117 Multiphor II electrophoresis unit. Nitrocellulose was blocked with 3% IGEPAL CA-630 in TBS for 30 min, then in 1% BSA in TBS for 2 h and finally in 0.1% Tween 20 in TBS for 10 min at room temperature and submitted to ligand binding.

Ligand blotting (125I-IGF-I binding to BPs)

125I-IGF-I binding to BPs on the membrane was carried out as described by Hosenlopp et al. (1986) with 200 000 cpm 125I-IGF-I in 10 mL of mixture containing 1% BSA, 0.1% Tween-20 in TBS. The membrane was washed twice with 20 mL of 0.1% Tween-20 in TBS, three times with 20 mL of TBS, dried for 1 h and exposed to Kodak X-Omat film for 4 weeks at −70 °C.

Western blot analysis

After SDS-PAGE, the gels were allowed to equilibrate for 5 min in 25 mmol/L Tris, 0.2 mol/L glycine in 20% (v/v) methanol. The protein was transferred to 0.2 µm pore-sized nitrocellulose at 100 mA for 1 h by using LKB 2117 Multiphor II electrophoresis. The nitrocellulose was incubating with anti-human insulin-like growth factor-I receptor at concentration 1 : 1000 in 5% dried milk in TBS-T (20 mmol/L Tris-HCl buffer, pH 7.4, containing 150 mmol/L NaCl and 0.05% Tween 20) for 1 h. Alkaline phosphatase conjugated antibody against goat IgG (whole molecule) was added at concentration 1 : 1000 in TBS-T. Incubation was continued for 30 min with slow shaking. Then nitrocellulose was washed with TBS-T (five times for 5 min) and submitted to Sigma-Fast BCIP/NBT reagent.

Statistical analysis

In all experiments the mean values for 10 assays ± standard deviations (SD) were calculated. The results were submitted to statistical analysis using Student's ‘t’ test, accepting P < 0.05 as significant.

Results

The concentration of IGF-I was measured in normal human pancreas, pancreatitis and pancreatic cancer tissues. The amounts of IGF-I in pancreatitis and pancreatic cancer tissues were higher then those in control tissue (Figure 1). Pancreatitis tissue contained about 142% of IGF-I (208 ± 33 ng/g) and pancreatic cancer tissue about 258% (377 ± 76 ng/g) in comparison to control pancreas (146 ± 48 ng/g).

Figure 1.

Figure 1

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The concentrations of IGF-I in control, pancreatitis and pancreatic cancer tissues. *P < 0.05; **P < 0.001.

Serum IGF-I levels were also different between the studied groups of patients (Figure 2). Serum of patients with pancreatitis contained about 115% of IGF-I (187 ± 41 ng/mL), and that from patients with pancreatic cancer 161% of IGF-I (261 ± 30 ng/mL), in comparison to control patients (162 ± 28 ng/mL).

Figure 2.

Figure 2

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The concentrations of IGF-I in control, pancreatitis and pancreatic cancer serum. *P < 0.01.

Western blot analysis, with specific antibody against human insulin-like growth factor-I receptor (IGF-I sR) showed that IGF receptor from the studied sources evoked identical electrophoretic mobility and reactivity with specific antibodies (Figure 3). An increase in the amount of receptor protein in the pancreatitis and a massive increase in the pancreatic cancer tissue extracts, compared to normal pancreas, was observed.

Figure 3.

Figure 3

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Western immunoblot analysis of IGF receptor from 10 polled homogenate extracts of control pancreas (lane 1), pancreatitis (lane 2) and pancreatic cancer (lane 3). The same amount of homogenate extract protein (20 µg) was run in each lane.

Since IGF-I activity depends on the level of IGF-I binding proteins (IGFBPs) we determined the level of BP-3 and BP-1 in the tissues and serum of studied patients. The concentration of BP-3 in pancreatitis and pancreatic cancer tissues was significantly increased, compared to control pancreas tissue (Figure 4a). In respect of BP-1, its concentration in pancreatitis tissue was not significantly altered, however, it was increased almost two-fold in pancreatic cancer tissue, compared to control (Figure 4b).

Figure 4.

Figure 4

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The concentrations of (a)BP-3 and (b)BP-1 in control, pancreatitis and pancreatic cancer tissues. Mean values from 10 samples ± SD are presented.

As can be seen from Figure 5(a), the concentration of BP-3 in serum of patients with pancreatic cancer was significantly increased, compared to control. In serum of patients with pancreatitis no significant difference in BP-3 level was found. A similar direction of changes was found in the serum level of BP-1. About a 2.5-fold increase in serum level of BP-1 was found in patients with pancreatitis and about a six-fold increase in serum of patients with pancreatic cancer (Figure 5b).

Figure 5.

Figure 5

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The concentrations of(a)BP-3 and (b)BP-1 in the serum of control patients and those with pancreatitis and pancreatic cancer. Mean values from 10 samples ± SD are presented.

The BPs of serum were analysed by ligand blotting. In this procedure, the BPs were submitted to SDS/PAGE and binding of 125I-IGF-I was carried out after blotting the BPs onto a nitrocellulose membrane. In the studied sera, three BP bands were detected (Figure 6). First, an IGFBP band of about 46 kDa BP (at the position corresponding to the acid-dissociated BP-3, derived from the 150 kDa complex), a second band (at the position corresponding to IGFBP-2), and a third one, 30 kDa BP (at the position corresponding to BP-1).

Figure 6.

Figure 6

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Ligand blot of BPs: 2 µL of 10 polled respective sera was applied on the gel. Pancreatic cancer serum (lane 1), pancreatitis (lane 2) and control pancreas (lane 3). The arrows indicate the positions of molecular weight standards.

In the serum of patients with pancreatic cancer (Figure 6, lane 1) a distinct increase in radioactive IGF-binding to 46 kDa BP and higher molecular weight material, compared to control serum (Figure 6, lane 3), was observed. No differences however, were observed in respect of BP-2 and BP-1.

In the serum of patients with pancreatitis, a distinct decrease in radioactive IGF-binding to all designated BPs (Figure 6, lane 2), compared to control serum (Figure 6, lane 3), was found.

Discussion

Previously we have found disregulation of collagen metabolism in pancreatitis and pancreatic cancer tissues (Pałka et al. 2002). However, the extent of collagen metabolism abnormalities in both tissues was different. At first we found a significant increase in collagen content in pancreatitis tissue, whereas in pancreatic cancer tissue the amount of this protein was not significantly elevated, compared to normal pancreas.

The data presented here provide support for the concept that disturbances in tissue collagen metabolism during pancreatic diseases may result from deregulation of IGF-I homeostasis. IGF-I is considered as a potent stimulator of collagen biosynthesis (Oyamada et al. 1990). In fact, serum from patients with pancreatitis and pancreatic cancer contains increased levels of IGF-I, compared to controls. Similar increases of IGF-I concentration were found in pancreatitis and pancreatic cancer tissues. Interestingly, the phenomenon was accompanied by increased expression of IGF-I receptor. Among the studied tissues the highest IGF-I level and IGF-I receptor expression was found in pancreatic cancer tissue. Over-expression of both ligand and IGF-I receptor might therefore contribute to stimulation of collagen gene expression and protein biosynthesis. However, in our previous studies we found an insignificant increase in collagen content in pancreatic cancer tissue (Pałka et al. 2002). It is possible that the discrepancy may result from the appearance in both serum and pancreatic cancer tissue of IGFBP-1, which is known to inhibit IGF-I dependent functions (Pałka et al. 1989; Oyamada et al. 1990; Peterkofsky et al. 1991). However, ligand blotting of BP-1 from serum of patients with pancreatic cancer showed no differences in IGF-I binding, compared to control serum. Instead, much IGF-I binding to BP-3 and some higher molecular weight material was observed. Although the high molecular weight material that binds IGF-I may represent tumour cell-derived glycosaminoglycans or other extracellular matrix degradation products (Clemmons 1997), its nature is not known at present. Whether the phenomenon may be responsible for the suppression of IGF-I activity remains to be explored. It cannot be excluded that other mechanisms may alter the IGF-I affinity for BPs. For instance, proteolytic cleavage of BPs (Rechler & Clemmons 1998) or their dephosphorylation (Rechler & Clemmons 1998) may result in a decrease of BP affinity for IGF-I. Another explanation for the discrepancy between increased IGF-I level and lack of increase in collagen content in pancreatic cancer is increased collagen degradation and decrease in prolidase activity in this tissue. Previously, we provided evidence for the increase in gelatinolytic activity and decrease in prolidase activity in pancreatic cancer tissue (Pałka et al. 2002). Prolidase [E.C. 3.4.13.9] is a cytosolic exopeptidase cleaving imidodipeptides with C-terminal proline (Endo et al. 1989; Myara et al. 1984). The primary biological function of the enzyme involves the metabolism of collagen degradation products (CDP) and the recycling of proline from imidodipeptides for collagen resynthesis. The efficiency of recycling of proline for collagen biosynthesis was found to be about 90% (Jackson et al. 1975). It is evident that an absence of prolidase will severely impede the efficient recycling of collagen proline (Goodman et al. 1968). On the other hand, enhanced liver prolidase activity was found during the fibrotic process (Myara et al. 1987). It suggest that prolidase providing proline for collagen biosynthesis may regulate turnover of collagen and may be a rate-limiting factor in the regulation of collagen production. Therefore despite the increase in IGF-I content in pancreatic cancer tissue, collagen biosynthesis is not stimulated.

The potential role of the above regulatory mechanisms in connective tissue pathobiochemistry of pancreatic cancer is supported by the data from patients with pancreatitis. As we reported previously, pancreatitis was accompanied by significant increase in collagen accumulation in this tissue (Pałka et al. 2002). Simultaneously, gelatinolytic activity (Pałka et al. 2002) as well BP-1, BP-3 levels and the IGF-I binding to these proteins that we found in the present study, were elevated to a much lesser extent than those found in pancreatic cancer patients.

Thus, in summary this data suggest that disturbances in tissue collagen metabolism during pancreatic diseases may result from deregulation of IGF-I homeostasis, and that elevated serum levels of IGF-I, IGFBP-3 and IGFBP-1 may serve as a marker of pancreatic cancer.

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