For immunohistochemical staining, we obtained formalin-fixed, paraffin-embedded tissue samples from 80 patients diagnosed with CRC at Chonnam National University Hospital, Gwangju, Korea. The histology of the tumors was analyzed, and the pathological stage was estimated according to the TNM score. Patients (stage I, n=20; stage I, n=20; stage III, n=20 and stage IV, n=20) were randomly selected from each of the stage categories. The specimens were fixed in 10% neutral-buffered formalin, embedded in paraffin and stained with hematoxylin and eosin. The present study was approved by the Institutional Review Board of Chonnam National University Hospital.

For immunohistochemical staining, tissue sections were deparaffinized, rehydrated and subjected to epitope retrieval. To block endogenous peroxidase activity, tissues were treated with Peroxidase-Blocking solution (Dako, Carpinteria, CA, USA) and incubated with a polyclonal rabbit anti-leptin antibody (A-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), which was diluted 1:100 using goat serum and incubated with the sections at room temperature for 1 h. After three 2-min washes with phosphate-buffered saline (PBS), the sections were incubated with a biotinylated goat anti-rabbit secondary antibody for 30 min (Dako). After three 2-min washes in PBS, horseradish peroxidase-streptavidin (Dako) was added to the sections for 30 min, followed by another three washes for 2 min in PBS. Reactions were detected using with 3,3′-diaminobenzidine substrate (Vector Laboratories, Burlington, ON, Canada) for 1 min, and the cells were counterstained using Mayer’s hematoxylin. Then slides were dehydrated following a standard procedure and sealed with coverslips.

Tissue specimens reacted with the anti-leptin antibody were examined at low and then at high magnification by two pathologists blinded to the identities of the samples. In cases of heterogeneous patterns in some sections, the classification was determined by the dominant pattern, and the intensity of stained cells was designated as negative, weak, moderate or strong.

We selected three human CRC cell lines (LS174T, HCT 116 and CaCo-2) since they express leptin at high levels. They were obtained from the Korean Cell Line Bank (KCLB) and cultured at 37°C in a humidified atmosphere containing 5% CO₂. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 4,500 mg/l glucose, 100 mg/l streptomycin and 2 mM L-glutamine supplemented with 10% fetal bovine serum (FBS) (both from Gibco Invitrogen Inc., USA). After 24 h of serum starvation, the culture media were replaced with serum-free media containing the indicated treatments. After a further 24 h incubation, 10 ng/ml human recombinant leptin (Sigma-Aldrich Corp., St. Louis, MO, USA) was added for different times.

Cells were grown to 70% confluence, trypsinized and plated in 100 cm² diameter culture dishes at a density of 1,000 cells/ml in serum-free DMEM containing 10 ng/ml human recombinant basic fibroblast growth factor, (bFGF) and 10 ng/ml human epidermal growth factor (hEGF) (both from R&D Systems, Minneapolis, MN, USA). A density of at least 1,000 cells/ml was established for forming tumorspheres/colonospheres (serving as an in vitro model of cancer stem cells). All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂. Cells were grown as suspension cultures for 1–2 weeks for tumorsphere formation. Colonies were counted in 10 randomly selected fields at ×10 magnification using an Olympus IX50 inverted microscope.

To extract proteins, cells were lysed with RIFA buffer (1 M Tris-HCl, 150 mM NaCl, 1% Triton X-100 and 2 mM EDTA) containing 1 mM phenylmethanesulfonyl fluoride (PMSF) and Halt™ protease inhibitor cocktail (Thermo, Rockford, IL, USA). The protein concentrations of the cell lysates were quantitated using the BCA™ protein assay (Thermo) with bovine serum albumin (BSA) as a standard. The lysates (25 μg protein) were subjected to electrophoresis on 10% SDS-polyacrylamide gels and then electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated for 1 h in blocking solution [5% BSA in TBS-Tween-20 buffer (TBST)] and sequentially blotted with primary antibodies at 4°C overnight. Antibodies against Janus kinase 2 (JAK2), phospho-JAK2, AKT, phospho-AKT, ERK, phospho-ERK, MAPK, phospho-MAPK and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). After rinsing in TBST, membranes were incubated with horseradish peroxidase (HRP)-labeled anti-rabbit or anti-mouse immunoglobulin secondary antibodies (1:2,000 dilution) (Cell Signaling Technology) at room temperature for 1 h. Enhanced chemiluminescence was used to detect the bands, which were visualized using a Fuji LAS-3000 image analyzer (Fuji Film, Tokyo, Japan).

The 96-well plates were prepared. Three human CRC cell lines were detached from the surfaces of culture flasks with 5 mM EDTA in PBS, resuspended in culture medium containing 0.02% BSA to 2.4×10⁵ cells/ml, and 100 μl of cell suspension was added to each well. All cells were assayed in quadruplicate. After incubation for 12 h at 37°C in an atmosphere containing 5% CO₂, the supernatant from each well was removed. After washing out non-adherent cells, adherent cells were incubated for 4 h in medium containing 500 μg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution. The absorbance of the reaction product was measured at 550 nm. Adherent cells were counted in three random area of each well.

Cell invasion assays were performed using Transwell filter chambers (8.0-μm pores) coated with 1% gelatin overnight and dried at room temperature. Human CRC cell lines were harvested, washed once in serum-free media, and seeded at 2×10⁵ cells in 120 μl of medium containing 0.2% BSA in the upper chamber. Then, 400 μl of 0.2% BSA medium containing 20 μg/ml of human plasma fibronectin (Calbiochem, La Jolla, CA, USA), a chemotactic factor, was loaded into the lower chamber. The Transwell apparatus was incubated for 24 h at 37°C. Cells that invaded the bottom surface of the upper chamber were fixed with 70% ethanol and stained with Diff-Quik solution (Sysmex, Kobe, Japan) following the manufacturer’s protocol. The non-invasive cells on the top surface were wiped off with cotton balls, and the stained cells on the bottom surface were counted in five selected fields (each 0.5 mm²) of six random squares using a hematocytometer placed on the stage of a light microscope. Results are expressed as the means ± standard error of the mean (SEM) of the number of cells/field of three individual experiments.

The statistical significance of differences between data sets was determined using the paired t-test. The λ² and Fisher’s exact test, where appropriate, were used to compare expression of leptin with various tumor stages. All reported p-values were two-sided and p≤0.05 was considered to indicate a statistically significant result. The Statistical Package for the Social Sciences (SPSS)/PC 20.0 (Chicago, IL, USA) was used to perform the calculations.

We determined the level of expression of leptin in 80 CRCs of different stages. Leptin was clearly expressed in the cytoplasm of the CRC cells. Leptin expression was ‘undetectable’ in 19/20 (95%) patients with stage I CRC and 5/20 (25%) patients with stage IV CRC. In contrast, leptin was ‘moderately to strongly’ expressed in 0/20 patients with stage I CRC and in 10/20 (50%) patients with stage IV CRC. Expression of leptin was significantly associated with tumor stage (Fig. 1, p<0.0001).

Cancer cells can be cultured in suspension to form spheres in serum-free replacement medium. To test whether human CRC cell lines could form spheres, CaCo-2, LS174T and HCT 116 cells were cultured in a suspension-culture system. Tumorsphere formation by CaCo-2, LS174T and HCT 116 cells was observed on day 5, accounting for 3.73±0.25, 2.80±0.26 and 3.13±0.32% of the total cell population by day 11, respectively. A greater number of large aggregates and colonies formed in the leptin-treated cultures relative to the controls (p<0.05). Leptin exposure increased the average colony size formed by each cell line (Fig. 2).

In the adhesion assays, leptin treatment for 12 h enhanced cell-cell adhesion of HCT 116 cells compared with the untreated HCT 116 cells (p=0.006); however, there was no statistically significant difference between the treated and untreated LS174T (p=0.140) and CaCo-2 cells (p=0.147, Fig. 3).

We next determined whether leptin treatment influences the invasiveness of the colon cancer cell lines The number of invading HCT 116 cells treated with leptin (10 ng/ml) was 574.3±562.5 when compared with the control value of 502.6±502.2 (Fig. 4A, p=0.178). The number of invading CaCo-2 cells treated with leptin (10 ng/ml) was 3,395.6±1571.2 when compared with the control value of 1,512.6±850.9 (Fig. 4B, p<0.001).

In the CRC CaCo-2 cells, leptin (10 ng/ml) stimulated the phosphorylation of JAK2 at the different treatment times. Increased phosphorylation of JAK2 was observed within 30 min after leptin treatment followed by a decline. In the LS174T and HCT 116 cells, leptin stimulated the phosphorylation of ERK. The levels of total (T) JAK2, ERK, MAPK and AKT were not altered in the three CRC cell lines after leptin treatment (Fig. 5).