Cell culture

CaCo-2 human colorectal carcinoma cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) from Gibco BRL Life Technologies Italia Srl (S Giuliano Milanese, Milano, Italy) containing 862 mg/l l-alanyl-l-glutamine (Glutamax-I), 4500 mg/l glucose, and 110 mg/l sodium pyruvate, supplemented with 20% fetal bovine serum (Gibco BRL, Life Technologies Italia Srl), and antibiotics/antimycotics (100 units/ml penicillin, 0.1 mg/ml streptomycin, and 250 ng/ml amphotericin B from Sigma Aldrich Italia Srl, Milano, Italy). Cells were cultured in 25 cm² flasks at 37°C in a humidified atmosphere of 5% CO₂ in air and five days after plating (about 70% confluency) were used for the different experiments. At the end of the treatments, CaCo-2 cells were harvested by trypsinisation with trypsin-EDTA (2.5 g/l—1 g/l in 0.9% sodium chloride) (Sigma Aldrich Italia Srl) and resuspended at 200 000 cells/ml in serum and phenol red free DMEM. A total of 80 000 cells were collected by cytocentrifugation at 30 g for seven minutes (Cytospin I; Shandon Inc., Pittsburgh, USA).

Tumour bearing patients

Fifteen adult patients (eight women, seven men) affected by colon adenocarcinoma were recruited from San Luigi Gonzaga Hospital (Orbassano, Torino, Italy). Mean age of the patients was 60 (10) years.

The different stages of tumour malignancy were defined both in terms of the TMN classification system (T2, five cases; T3, eight cases; T4, two cases) and the histological grading of atypia (G2, 11 cases; G3, four cases).

Tissue sample processing

The surgically removed segment of colon of each patient was divided into two sections: one zone directly involved in the tumour mass and another distal from the tumour site (normal tissue). Part of the tumour biopsy was embedded in paraffin and part was directly frozen in liquid nitrogen and stored at –80°C until use.

Biochemical markers of lipid peroxidation

The presence of oxidative damage was assessed in 10% (w/v) tissue homogenates prepared from biopsies in 0.1 M Tris HCl buffer (pH 7.4) using a Polytron apparatus (Kinematika GmbH, Luzerna, Switzerland): protein-aldehyde adducts were determined in homogenates as fluorescence exhibited by interaction between protein amino functions and malonaldehyde (MDA) or HNE at wavelengths of 390/460 nm and 355/460 nm, excitation/emission respectively.^{17, 18}

MDA levels were also evaluated in 10% (w/v) tissue homogenate specimens using the thiobarbituric acid (TBA) reaction¹⁹: homogenates were incubated in a thermostatic shaking bath at 37°C for one hour before evaluation.

Western blot analysis of TGF-β1 tissue levels

Tissues were homogenised and after evaluation of protein content by Lowry assay,²⁰ samples were normalised—that is, adjusted to achieve an equal amount of protein. For immunoblot analyses, 40 μg of total protein were denatured in Laemmli buffer,²¹ loaded on the same 12% polyacrylamide sodium dodecyl sulphate gel and subjected to electrophoresis under reducing conditions. Comassie blue staining was used to verify appropriate protein normalisation in the electrophoretic run. Proteins were transferred onto nitrocellulose membranes (Hybond ECL; Amersham Pharmacia Biotech GmbH, Germany). Unspecific binding was blocked with 5% (w/v) non-fat dried milk in Tris sodium buffer (TBS)-Tween (50 mM Tris HCl, pH 7.4, containing 200 mM NaCl, 0.05% v/v Tween 20). Blotted membranes were incubated with a polyclonal TGF-β1 primary antibody (Santa Cruz Biotechnology, Santa Cruz, California, USA) followed by incubation with peroxidase conjugated polyclonal antirabbit secondary antibodies (Santa Cruz Biotechnology). The immunoblots were developed with the ECL detection system (Amersham Pharmacia Biotech GmbH, Germany) following the manufacturer's directions.

Immunocytohistochemical receptor analysis

TGF-β1 receptors were visualised both on the CaCo-2 cell line and on tissue sections from patients affected by colon adenocarcinoma.

CaCo-2 cells were plated and harvested (see cell culture above). After cytocentrifugation the slides were fixed in paraformaldehyde solution (4% in phosphate buffered saline (PBS), pH 7.4) for 10 minutes and permeabilised with 0.1% Triton X-100 in 0.1% sodium citrate for five minutes at 4°C. Paraffin was removed from the embedded tissue sections through consecutive passages with pure xylol, 99% and 95% ethanol, and water. For antigen retrieval the samples were sunk in 10 mM sodium citrate buffer (pH 6.0) and heated in a microwave oven (for three minutes at 750 W and twice for two minutes at 350 W).

In both CaCo-2 cells and tissue specimens, endogenous peroxidase was inhibited by incubation in 0.6% H₂O₂ for 25 minutes. After preincubation with bovine serum albumin in TBS (0.1 % w/v) containing 30% normal porcine serum (v/v) for 20 minutes at room temperature to block non-specific binding, slides were incubated in the presence of the following polyclonal rabbit antibody: 4 μg/ml anti-TGF-β1-RI (L-21) or 8 μg/ml anti-TGF-β1-RII (T-19) (Santa Cruz Biotechnology). Incubation was performed in a humidified chamber at 4°C overnight. Negative controls were incubated overnight in the presence of 30% normal porcine serum (v/v) alone.

In order to block endogenous avidin and biotin, slides were preincubated for 10 minutes with 0.005% avidin and for 10 minutes with 0.0005% biotin (Dako Biotin Blocking System; Dako SpA, Milano, Italy). After this treatment samples were incubated with polyclonal pig antirabbit biotinylated immunoglobulins (1:200 dilution v/v) (Dako SpA), and then with biotin-avidin-peroxidase reagent (ABC complex-HRP; Dako SpA) at room temperature for 30 minutes. The bound immunocomplex was visualised by incubation with 0.6% 3,3`-diaminobenzidine tetrahydrochloride in TBS containing 0.003% H₂O₂ for 5–10 minutes. All incubations were preceded by three washes with TBS (Tris NaCl buffer, 50–150 mM, pH 7.6) for five minutes.

Sections were lightly counterstained with Mayer haematoxylin.

Morphological detection of apoptosis induced by TGF-β1

To induce apoptosis, five days after plating (about 70% confluency) CaCo-2 cells were treated with two repeated doses of TGF-β1 (5 ng/ml) (Boehringer Mannheim, Roche Diagnostics, Monza, Milano, Italy) at time zero and after 24 hours.

Twenty four hours after the second TGF-β1 treatment, cells were harvested from tissue flasks and cytocentrifuged at 30 g for seven minutes (to obtain 80 000 cells/slide).

To detect nuclear staining by 4,6-diamidino-2-phenylindole (DAPI), after collection by cytocentrifugation slides were fixed in ethanol:ether 1:1 (v:v) for 10 minutes, incubated at 37°C for 10 minutes in DAPI solution (1 μg/ml of absolute methanol, final concentration), and washed with PBS and 96% ethanol. Analysis of nuclear morphology changes was conducted using a fluorescent microscope with an ultraviolet filter, 630× magnification.

Nuclear fragmentation was also detected by the TdT mediated dUTP nick end labelling (TUNEL) technique. Cells were fixed in paraformaldehyde solution (4% in PBS, pH 7.4) for 10 minutes and permeabilised with 0.1% Triton X-100 in 0.1% sodium citrate for two minutes at 4°C. The staining reaction was performed using the “in situ cell death detection kit” from Boehringer Mannheim (Roche Diagnostics SpA, Monza, Italy) and visualised by fluorescein at 488 nm excitation with 505 nm long pass filter emission using a laser scanning confocal microscope (LSCM Zeiss, Germany). The objective used was 40×, the dimensions of the optical sections were 325 μm×325 μ m×10 μm, and the unit scale was 0.64 μm² .

All slides were sealed with Vectashield H-1000 mounting medium (Vector Lab. Inc., Burlingame, California, USA).

Statistical analyses

Data are mean (SD). The statistical significance of differences between experimental groups was assessed using the Student's t test.

TGF-β1 induced apoptosis in human colon cancer CaCo-2 cell line

The immunocytochemical semiquantitative analysis for the TGF-β receptor system in the CaCo-2 cell line performed in our laboratory showed low levels of both TGF-β1-RI and TGF-β1-RII receptors: only 30% of the randomly examined cancer cells were immunostained when antibodies against type I receptor were employed while about 60% were immunostained for TGF-β1-RII receptors (data not shown). Of note, such pronounced TGF-β1 receptor deficiency was not sufficient to abolish the proapoptotic effect of the cytokine. In fact, treatment of CaCo-2 cells with an appropriate concentration of TGF-β1 (two doses of 5 ng/ml) for 48 hours led to marked induction in programmed death, as detected both by DAPI staining of apoptotic bodies (data not shown) and TUNEL, analysed by confocal microscopy (fig 1⇓).

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Figure 1

Characterisation of apoptosis in the CaCo-2 cell line using the TdT mediated dUTP nick end labelling (TUNEL) test. (A) Untreated CaCo-2 cells; (B) CaCo-2 cells treated with 10 ng/ml transforming growth factor β1 (TGF-β1) for 48 hours. Fluorescence was detected by a laser scanning confocal microscope (Zeiss): the laser band of excitation was 488 nm with a 505 nm long pass filter emission; the lens used was a plan neofluar 40×/0.75. The image dimension was 325 μm×325 μm×10 μm, and the unit scale used was 0.64 μm². The image was elaborated using a LSCM 510 Image Examiner Program (Zeiss). Exciting light intensity, black level, and photomultiplier gain were adjusted on control specimens; the same setting was employed for scanning of experimental material.

Type I and type II receptor proteins in normal and transformed colon mucosa

Following histopathological and clinical characterisation, the 15 different human colon adenocarcinomas were divided into subgroups according to their TNM classification and degree of dedifferentiation (G1, G2, G3). Five tumours were classified as T2 (G2), six as T3 (G2), two as T3 (G3), and two tumours as T4 (G3). From the paraffin blocks of the individual tumours, 5 μm slices were cut for receptor immunostaining using the scattered portions of colon mucosa surrounding the neoplasia as the internal reference. Figure 2⇓ shows a representative immunostaining of TGF-β1 receptors in tumour tissue and surrounding normal colon mucosa from one of the 15 cancer patients (reported as CP in table 1⇓). Both type I and type II receptors had a homogenous distribution in normal colon epithelial cells but type II staining was definitely stronger and also extended to the stroma (fig 2A, B⇓). In the adenocarcinoma cells from this clinical case however type I receptors were dramatically reduced in number, and only scattered cells exhibited a fine granular positive reaction (fig 2C, E⇓). In contrast, expression of type II receptors in cancer tissue appeared to be similar to that of normal mucosa (fig 2D, F⇓). Immunohistochemical findings of all other patients are summarised in table 1⇓. Reduced positivity for TGF-β1-RI versus the internal control was evident in tumour samples from seven of 15 patients, independent of the degree of tumour cell growth and differentiation. In contrast, only three cases of all tumours examined showed a low content of type II receptor (TGF-β1-RII).

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Figure 2

Immunohistochemical analysis of types I and II transforming growth factor β1 (TGF-β1) receptors in colon cancer versus normal mucosa in a representative patient (reported as CP in table 1⇑). Receptors were visualised using an appropriate purified rabbit polyclonal antibody; colour was developed as described in materials and methods. Normal colon mucosa: expression of type I (A) and type II (B) TGF-β1 receptors (100× magnification). Colon cancer: expression of type I (C, 40×; E, 250×) and type II (D, 40×; F, 250×) TGF-β1 receptors.

Lipid peroxidation markers in human colon adenocarcinoma

Steady state levels of free MDA, the most commonly employed index of membrane lipid peroxidation, showed a decreasing trend in tumours compared with the surrounding area (normal colon tissue) in T2 tumours, and a statistically significant reduction in T3 tumours, while in the highly dedifferentiated and advanced T4 tumours the amount of tissue aldehyde was again within the control range. Also, MDA bound to total tissue protein showed a statistically significant decrease only in T3 tumour specimens. Detection of another much more reactive aldehyde stemming from membrane oxidation, HNE, appeared to be a more sensitive marker. In fact, HNE-protein adducts were found to be significantly reduced in all T2 and T3 tumours (table 2⇓). As in the case of free MDA, the aldehyde-tissue protein adducts detected in the two T4/G3 tumour specimens showed values within the control range.

TGF-β1 cytokine level in human colon cancer tissue

The actual amount of cytokine present in colon adenocarcinoma tissue at different stages of growth and differentiation was determined by the western technique applied on immunoblotted samples. As an internal positive control, a sample was taken from Crohn's disease mucosa which was characterised by an MDA level of 1.25 nmol/mg protein—that is, about twofold the mean value obtained from normal tissue samples, reported in table 2⇑. As shown by a representative immunoblot in fig 3⇓, the majority of the specimens were examined together in a single gel for TGF-β1 level. Direct densitometric analysis of the immunoblots was made possible by actual deposition of very similar amounts of the sample's protein, as proved by protein staining with Comassie blue. In T2 and T3 colon cancers, almost all with G2 differentiation, TGF-β1 content was without exception dramatically lower than that detected in surrounding normal colon mucosa. In agreement with the corresponding extent of lipid peroxidation, the decrease in TGF-β1 protein level appeared to be more intense in T3 than in T2 tumours.

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Figure 3

Western blot analysis of transforming growth factor β1 (TGF-β1) protein in patients affected by colon adenocarcinoma at different stages of malignancy. A representative immunoblot. Double protein aliquots (40 μl each) were taken from biopsy specimens after homogenisation. One aliquot was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Comassie blue to verify actual protein normalisation (A). The second protein aliquot was also separated by SDS-PAGE but this time processed with a TGF-β1 polyclonal antibody (B). Densitometric analysis of the cytokine level of the single specimens is reported in terms of arbitrary densitometric units (ADU) (C). Samples: 1, colon mucosa from Crohn's disease patient; 2–4, normal colon tissue; 5–9, mucosa from colon adenocarcinoma at T2 (G2) stage; 10–17, mucosa from colon adenocarcinoma at stage T3 (G2); 18–19, mucosa from colon adenocarcinoma at stage T4 (G3).

As in the case of lipid peroxidation markers, the two T4 tumour samples, both with G3 grading, showed a cytokine level similar to control mucosa while the positive control, Crohn's mucosa, showed a net increase in both TGF-β1 and lipid oxidation levels.