Effects of α-lactalbumin and sulindac on primary and metastatic human colon cancer cell lines

Cell lines and cell cultivation

The COLO-320 primary (HTL95027) and COLO-741 metastatic (HTL95008) human colon carcinoma cell lines were purchased from Interlab Cell Line Collection (Cenova, Italy) and were grown in RPMI-1640 (F1213, Biochrom, Berlin, Germany) medium containing 10% fetal bovine serum (FBS, S0113, Biochrom, Berlin, Germany), 1% L-glutamine (K0283, Biochrom, Berlin, Germany), and 1% penicillin/streptomycin (A2213, Biochrom, Berlin, Germany). The cell lines were maintained in a humidified atmosphere at 37°C in 5% CO₂. The medium was changed every other day and the cells passaged every 10 days. Cells were microscopically examined using an inverted microscope (IX71, Olympus, Japan) and photomicrographs were obtained.

Cytotoxicity assay

The cytotoxicities of α-lactalbumin (L6634, Sigma, St. Louis, MI, USA) and sulindac (S3131, Sigma, St. Louis, MI, USA) were measured using 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT, M5655, Sigma, Steinheim, Germany). COLO-320 and COLO-741 cells were passaged into 96-well (2.5×10⁵ cells/well) plates (Corning, Darmstadt, Germany) and seeded for 24 h before drug treatment. After incubating, cells were treated with an increasing concentration of α-lactalbumin (0 μM, 0.1 μM, 1 μM, 10 μM, 100 μM) and sulindac (0 μM, 1 μM, 10 μM, 100 μM, 1000 μM) for 24, 48 and 72 h. Drug applications were repeated every day due to the half-live of drugs being less than 24 h. Medium was then removed and 100 μL of fresh medium containing 10 μL MTT (5 mg/mL in distiled water) were added and incubated at 37°C for 4 h. MTT containing medium was then decanted and 100 μL of dimethyl sulphoxide (DMSO, A3672, AppliChem, Darmstadt, Germany) was added to each well, and absorbance was measured at a wavelength of 570 nm using an UV visible spectrophotometer multiplate reader (Versa Max, Molecular Device, USA) [20]. The 50% lethal doses (IC₅₀) of drugs were calculated using measurements of absorbance by GraphPad (GraphPad Software, San Diego, CA, USA).

Drug treatments

COLO-320 and COLO-741 cell lines were placed in 12-well plates at a density of 2.5×10⁵ cells/per well and allowed to grow under routine conditions for 24 h. Both two types of tumor cells were randomly divided into four groups which were the control (no drug treatment), α-lactalbumin (IC₅₀ dose, 0.250 μM), sulindac (IC₅₀ dose, 18.75 μM) and a combination of both drugs. Cells were treated with drugs for 24, 48, 72 h. Drug applications were repeated every day due to the half-live of drugs being less than 24 h.

Transmission electron microscopy (TEM)

The cells were fixed in 2.5% buffered glutaraldehyde +2% paraformaldehyde in 0.1 M sodium phosphate buffer (16210, Electron Microscoby Sciences, Hatfield, PA, USA). The following day, cells were postfixed with 1% Osmium tetroxide (R1015, Agar Scientific, Essex, UK) at +4°C in the dark. After washing in buffer solution, cells were embedded into 2% agar (A 2114, AppliChem, Darmstadt, Germany) and were washed in buffer solution. Cells in agar were passed through 70% and 100% alcohol series (1 11727, Merk, Darmstadt, Germany) and were embedded into epon (2660, SPI-CHEM, West Chester, PA, USA) blocks and ultramicrotome (EM UC6, Leica, Vienna, Austria) was used for sections. The 50- to 100-nm thickness sections were examined with an EFTEM (Carl Zeiss Libra 120, Germany).

In Situ Cell Death Detection assay for apoptosis

Terminal deoxynucleotidyltransferase-biotin nick end-labelling (TUNEL) method was carried out to detect DNA fragmentation using a commercial in situ apoptosis detection kit (11 684 817 910, Roche, Mannheim, Germany). Cells were cultured in 12-well plates, after washing with phosphate buffered saline (PBS) (DMR E404-01, Diagnostic Biosystems, Pleasanton, CA, USA) twice and were incubated with 0.1% Triton X-100 (A4975, AppliChem, Darmstadt, Germany) for 15 min. The cells were again then rinsed with PBS twice. The samples were incubated with Terminal deoxynucleotidyl transferase (TdT)-enzyme for 1 h at 37°C in a humidified atmosphere. They were then washed with PBS three times and viewed using an IX71 inverted-florescence-phase microscope (Olympus, Japan) and photographed. As a negative staining control for TUNEL, TdT was omitted during the tailing of reactions. Staining was examined independently by the two authors, who both had no prior information regarding the samples. TUNEL positive cells were counted randomly from chosen fields (per sample) and data was presented as a percentage of the total amount of cells present. Cells in areas with necrosis or poor morphology were not analyzed [20].

Immunocytochemistry

After drug treatment, the medium was decanted and cells were rinsed with sterilized PBS and fixed with 4% paraformaldehyde in PBS at +4°C for 30 min. Following the fixation process they were washed with PBS three times for 5 min and permeabilized with 0.1% Triton X-100 (A4975, AppliChem, Darmstadt, Germany) in PBS at +4°C for 15 min. Endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide (H₂O₂, 1 08600, Merck, Darmstadt, Germany) for 5 min at room temperature. Cells were then washed with PBS and incubated with primary antibodies: anti- cytochrome-c (sc-13156, Santa Cruz, Heidelberg, Germany), anti-caspase 3 (C 5737, Sigma, St. Louis, MI, USA), and anti-caspase 9 (C 4356, Sigma, St. Louis, MI, USA) at +4°C overnight. After removal of the primary antibodies, cells were washed with PBS and incubated with biotinylated secondary antibodies and peroxidase-conjugated streptavidin using the protocol from a HISTOSTAIN kit (85-9043, Zymed, Carlsbad, CA, USA). Sample were then incubated with diaminobenzidine/hydrogen peoxide (K 3467, DakoCytomation, Carpinteria, CA, USA) and mounted with mounting medium (K002, DBS, Pleasanton, CA, USA). Stained samples were observed using a light microscope (IX71 inverted-florescence-phase microscope) (Olympus, Japan) and photographed [21]. The staining procedure was repeated three times and immunocytochemical staining was evaluated as weak (+), moderate (++) and strong (+++), respectively, in three different microscopic areas cells were counted for each intensity. The respective score was then calculated using the formula H-Score=Pi (intensity of staining +1). Pi is the percentage of stained cells for each intensity, varying from 0% to 100%. The H-score was evaluated by at least two investigators independently [22].

Statistical analysis

The data was statistically expressed as mean±SD values and were analyzed using repeated-measures ANOVA. The Tukey-Kramer multiple comparisons test was used to determine differences amongst the mean values. Values for p≤0.05 were considered significant.

Cell viability and cytotoxicity

Cell viability and proliferation were determined using an MTT assay, in the presence of α-lactalbumin and sulindac. COLO-320 and COLO-741 cells were treated with α-lactalbumin at concentrations of 0 μM, 0.1 μM, 1 μM, 10 μM, 100 μM and sulindac at concentrations ranging from 0 μM to 1000 μM for 24, 48 and 72 h (Figure 1). Starting at 10 μM, sulindac significantly increased cell death (***p<0.001), and reaching 100% cell death at 1000 μM and at a IC₅₀ dose of 18.75 μM. α-Lactalbumin caused 85% cell death at a concentrations of 10 and 100 μM (***p<0.001) and its IC₅₀ dose was 0.250 μM.

Figure 1:

Survival of COLO-320 primary human colon carcinoma cells and COLO-741 metastatic human colon carcinoma cells exposed with α-lactalbumin (IC50: 0.250 μM) (A) and sulindac (IC50: 18.75 μM) (B) for 72 h was analyzed using MTT assay.

Percentage of cell survival was evaluated using One-way ANOVA with Tukey-Kramer Multiple Comparisons Test (*p<0.05, **p<0.01, ***p<0.001).

Transmission electron microscopy (TEM)

After treatment with α-lactalbumin, sulindac and/or a combination of the two drugs for 24, 48 and 72 h, transmission electron microscopic studies were conducted. The findings are presented in Figure 2. In the control group, COLO-320 and COLO-741 cells had normal nuclei and organelles which revealed a regular shape, integrated nuclear membrane and smooth surface. In the case where treatment was with both drugs, chromosomal condensation and partial cell shrinkage were observed. α-Lactalbumin and sulindac exposed COLO-320 cells exhibited more cell damage compared with COLO-741 cells. There were no significant differences between 24, 48 and 72 h applications of α-lactalbumin and sulindac in both cell lines.

Figure 2:

Findings of TEM studies.

Electron micrographs of COLO-320 primary human colon carcinoma cells (A: control, B: 0.250 μM α-lactalbumin, C: 18.75 μM sulindac, D: combination of IC50 doses) and COLO-741 metastatic human colon carcinoma cells (E: control, F: 0.250 μM α-lactalbumin, G: 18.75 μM sulindac, H: combination of IC50 doses) were exposed to the drugs for 72 h (arrow heads: nuclei, asterisk: cytoplasm). Scale bars: 0.2 μm.

In Situ Cell Death Detection and immunocytochemistry assay

The apoptotic effects of α-lactalbumin and sulindac on COLO-320 and COLO-741 cells and distribution of caspase-3, caspase-9 and cytochrome-c were observed using immunocytochemistry and TUNEL assays.

The percentage of apoptotic cells is presented in Figure 3. The number of TUNEL positive cells increased in all treated groups especially in the COLO-320 cell line compared to COLO-741 cells. α-Lactalbumin and sulindac caused cell death of COLO-320 after application for 24, 48 and 72 h compared to control group (***p<0.001) (Figures 3 and 4). The number of TUNEL positive cells in the combination group were at 24, 48 and 72 h were greater than the control (***p<0.001), α-lactalbumin and sulindac groups (**p<0.01). The difference between treatments with α-lactalbumin and sulindac were not statistically significant (p>0.05). In the group of COLO-741 cells, apoptotic cells increased following the administration of the two drugs independently and in combination, whereas apoptotic cells were observed rarely in the control group (***p<0.001). The increase of apoptotic cells in the combination group was statistically significant (**p<0.01) (Figure 3). The combination of the IC₅₀ doses of two drugs had the most toxic effect on COLO-320 cells (Figures 4 and 5).

Figure 3:

In Situ Cell Death Detection of COLO-320 primary human colon carcinoma cells (A) and COLO-741 metastatic human colon carcinoma cells (B) after applications of α-lactalbumin (0.250 μM), sulindac (18.75 μM) and combination of the IC50 doses of two drugs for 24, 48 and 72 h.

Percentage of apoptotic cells was evaluated for each drug (*p<0.05, **p<0.01, ***p<0.001).

Figure 4:

In Situ Cell Death Detection of COLO-320 primary human colon carcinoma cells after application with α-lactalbumin (0.250 μM), sulindac (18.75 μM) and the combination of of IC50 doses of both drugs.

Arrows: Apoptotic cells. Scale bars: 20 μm.

Figure 5:

In Situ Cell Death Detection of COLO-741 metastatic human colon carcinoma cells after application of α-lactalbumin (0.250 μM), sulindac (18.75 μM) and the combination of of IC50 doses of both drugs.

Arrows: Apoptotic cells. Scale bars: 20 μm.

After investigation of immunocytochemical stainings, the localization of caspase-3, caspase-9 and cytochrome-c were identified in the cytoplasm, especially caspase-3 was located near the nucleus. Immunoreactivities of caspase-3, caspase-9 and cytochrome-c were detected in all treated groups, these immunoreactivities increased in groups which were treated with both drugs compared with the control groups. There were no statistically significant differences between 24, 48 and 72 h applications (p>0.05) (Figure 6). The caspase-3 stained cells were rare in the control groups of both two cell lines. The caspase-3 immunoreactivity increased following the combined application of the IC50 doses of two drugs on COLO-320 cells (***p<0.001) (Figure 6A). The number of cytochrome-c showing cyptoplasmic staining of cells increased in the α-lactalbumin and sulindac treated COLO-320 cells compared with control group (***p<0.001) and also caspase-9 stained cells were enhanced in the treated groups (***p<0.001). In the COLO-741 cells, combination of IC50 doses of α-lactalbumin and sulindac administration caused a significant increase in the immunoreactivity of cytochrome-c when compared with the control group (***p<0.001) (Figure 6F). The immunoreactivities of caspase-3 and caspase-9 were similar in all treated groups of COLO-741 cells and significant difference was not found between the α-lactalbumin and sulindac applications (p>0.05). Immunoreactivities of caspase-3, caspase-9 and cytochrome-c were the greatest in the treated groups of COLO-320 cells in comparison to the COLO-741 cells (Figure 6D, E).

Figure 6:

The H-Score analysis of immunocytochemical staining of caspase-3, caspase-9 and cytochrome-c in COLO-320 primary human colon carcinoma cells (A–C) and and COLO-741 metastatic human colon carcinoma cells (D–F) after application with α-lactalbumin (0.250 μM), sulindac (18.75 μM) and the combination of of IC50 doses of both drugs for 24, 48 and 72 h (*p<0.05, **p<0.01, ***p<0.001).

α-Lactalbumin and sulindac had significantly more toxic effects on COLO-320 cells than on COLO-741 cells. We found a positive relationship between the number of apoptotic cells and immunoreactivities of caspase-3, caspase-9 and cytochrome-c.

Limitation of this study

In this study, we aimed to investigate the activity of both substances in IC₅₀ doses. We investigated their IC₅₀ dose activity separately and compared them with combinations of these doses. The combinations of different concentrations were not evaluated. Some figures have less clarity for interpretation.