Sixty-six fresh colon cancer tissues and matched normal tissues from patients were collected and fresh-frozen in liquid nitrogen after surgical resections performed at the First Affiliated Hospital of Guangxi Medical University (Guangxi, China) from 2009 to 2010. For the 66 fresh tissues, one-half of the tissue was snap-frozen immediately in liquid nitrogen and stored at −80°C and the other half was formalin-fixed and paraffin-embedded. Each case was reviewed by 2 experienced histopathologists who were blinded to disease status. The patients had not received chemotherapy or radiation therapy before tumor resection. The study was approved by the Institutional Ethics Committee of Guangxi Medical University under full consideration of the declaration on human rights of Helsinki.

Formalin-fixed, paraffin-embedded tissue blocks were serially sectioned at 4 μm. Sections were deparaffinized in xylene and rehydrated before analysis. Endogenous peroxidase was quenched with 3.0% hydrogen peroxide in methanol for 10 min, antigen retrieval was performed by microwave for 15 min and tissue sections were blocked with normal rabbit serum for 20 min. This was followed by incubation overnight at 4°C with mouse monoclonal FAK (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit polyclonal p-FAK (Abcam, Cambridge, UK) and mouse monoclonal SphK1 (Sigma-Aldrich, St Louis, MO, USA) primary antibody. Sections were washed with PBS and incubated with second antibody at room temperature for 30 min. After being washed with PBS, sections were stained by a streptavidin-peroxidase detection system. Antibody binding was visualized using diaminobenzidine as chromogen and counterstained with hematoxylin. Incubation with PBS instead of the primary antibody served as a negative control.

The degree of staining was estimated by semiquantitative evaluation and categorized by the extent and intensity of staining as follows: i) The extent of positive cells was scored as: 0, positive-staining cells <5%; 1, positive-staining cells 5–25%; 2, positive-staining cells 26–50%; 3, positive-staining cells 51–75%; 4, positive-staining cells >75%. ii) The intensity of staining was scored as: 0, achromatic; 1, light yellow; 2, yellow; 3, brown. The percentage of positive tumor cells and staining intensity were multiplied to produce a weighted score for each case. Cases with weighted scores ≤1 were defined as negative; otherwise as positive.

Human colon cancer LOVO cell line was obtained from ATCC. Cells were cultivated in DMEM high glucose medium (Invitrogen Co., Carlsbad, CA, USA) supplemented with 10% FBS (Gibco-BRL, Rockville, MD, USA) and antibiotics (100 U/ml penicillin plus 50 mM streptomycin, Invitrogen Co.) in an atmosphere of 5% CO₂ at 37°C.

In order to develop a new model system to better study effects of SphK1 on cell proliferation, apoptosis and invasion, LOVO cells were transfected with SphK1 DNA (SphK1 group), SphK1 shRNA (shRNA group) and SphK1 non-targeting shRNA control (NC group). In brief, 24 h before transfection, cells were cultivated in medium without antibiotics. Cells were seeded in 6-well plates(2×10⁵ cells/well). The shRNA, non-targeting shRNA control (NC) or DNA plasmid targeting SphK1 (Shanghai GenePharma Co., Ltd., Shanghai, China) were diluted in Opti-MEM^® (Gibco-BRL) Reduced Serum Medium without serum. Cells were transfected with 40 nmol shRNA oligomer, NC or 0.8 μg of DNA using Lipofectamine™ 2000 reagent (Life Technology Co., Carlsbad, CA, USA) according to the manufacturer’s instructions. Cells were harvested 48 h later for the following experiment.

Cells were cultured in a culture dish. Cells were transfected with SphK1 shRNA, NC, SphK1 DNA or treated with 50 μM DMS (Merck KGaA, Darmstadt, Germany) for 24 h (DMS group). Cells treated with equal amount of 0.9% NaCl instead of drugs served as the control group. Activation of SphK1 was measured as described previously with slight modification (18). Cells were resuspended in ice-cold 0.1 M phosphate buffer [(pH 7.4) containing 20% glycerol, 1 mM mercaptoethanol, 1 mM EDTA, phosphatase inhibitors 1 mM sodium orthovanadate, 15 mM sodium fluoride], protease inhibitors (10 μg/ml leupeptin, aprotinin, trypsin, chymotrypsin and 1 mM phenylmethylsulfonylfluoride) and 0.5 mM 4-deoxypyridoxine, were then disrupted by three 5-sec pulses with a Fisher 550 sonic dismembranator. Each sample containing 100 μg protein was assayed for sphingosine kinase activity by incubation with sphingosine and [γ-³²P]-ATP (Beijing Free Biotech. Co., Beijing, China) for 30 min at 37°C. Products were separated on TLC on Silica Gel G60 (Merck KGaA) using 1-butanol/methanol/acetic acid/water (80:20:10:20) and visualized by autoradiography. The radioactive spots corresponding to sphingosine phosphate were scraped and counted in a scintillation counter.

Cell viability was determined by colorimetric assay utilizing MTT (Sigma-Aldrich). In brief, non-transfected LOVO cells (DMS group), cells of SphK1, shRNA and NC group were seeded in 96-well plate (5×10³ per well) as different experimental groups. Cells of DMS group were incubated with 25, 50 and 100 μM DMS for 0, 6, 12 and 24 h. LOVO cells treated with equal amount of 0.9% NaCl instead of drugs served as the control group. Then 20 μl MTT solution (5 mg/ml) was added to each well and incubated at 37°C for 4 h. The solution was carefully removed, followed by addition of 150 μl dimethyl sulfoxide (DMSO, Invitrogen Co.) to each well to solubilize MTT. The absorbance (A) of sample was measured at 490 nm and the cell viability was expressed as A value of experimental cells/control cells ×100%. Triplicate measurements were done at each time-concentration.

The treatment of cells was the same as in the SphK1 activity assay. Cells were harvested and washed twice with PBS. The cells were resuspended in 500 μl binding buffer at a density of 1×10⁶ cells/ml, 1 μl Annexin V-FITC (Nanjing KeyGen Biotech. Co., Nanjing, China) was added to the sample and incubated for 20 min at room temperature in the dark, and then 5 μl propidium iodine (PI) buffer was added and incubated for 5 min at 4°C in the dark. Finally, samples were evaluated by flow cytometry and data were analyzed using CellQuest (Becton-Dickinson, San Jose, CA, USA).

The cell treatment was the same as in the SphK1 activity assay. Total-RNAs were extracted from tissue samples or cells using TRIzol reagent (Invitrogen Co., San Diego, CA, USA). First-strand cDNA was synthesized from 1 μg of total-RNA using oligo-dT primer and Moloney murine leukemia virus reverse transcriptase (Fermentas International Inc., Burlington, Canada) according to the instructions from the manufacturers. Then cDNA was amplified by using the following primers: SphK1, 5′-ATG CAC GAG GTG GTG AAC G-3′ (sense), 5′-GGA GGC AGG TGT CTT GGA AC-3′ (antisense), FAK: 5′-ACC TCA GCT AGT GAC GTA TGG-3′ (sense), 5′-CGG AGT CCC AGG ACA CTG TG-3′ (antisense), β-actin: 5′-AGC CAT GTA CGT AGC CAT CC-3′ (sense), 5′-CTC TCA GCT GTG GTG GTG AA-3′ (antisense). The PCR products were analyzed on 2% agarose gels and visualized by ethidium bromide staining. Quantitation of expression levels was achieved after adjustment for the expression levels of β-actin by densitometry. A 100-base pair DNA ladder was used as a molecular weight marker on each gel.

The treatment of cells was the same as in the SphK1 activity assay. Tissue samples or cells of different groups were lysed in lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM EDTA, 1 mM Na₃VO₄, 0.5 μg/ml leupeptin and 1 mM PMSF] and then the lysate was incubated on ice for 30 min and centrifuged at 12000 rpm for 10 min. The supernatant was collected and the content of total protein was evaluated by the Bradford colorimetry. After denaturation, 40 μg of protein from each sample was separated on a 12% SDS-PAGE and electroblotted onto nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% non-fat milk in TBST [10 mM Tris (pH 7.4), 100 mM NaCl, 0.5% Tween 20] for 2 h at room temperature. Membrane was subsequently incubated overnight at 4°C in 5% non-fat milk in TBST containing either mouse monoclonal β-actin, mouse monoclonal FAK, mouse monoclonal ICAM-1, rabbit polyclonal VCAM-1 (Santa Cruz Biotechnology, Inc.) or rabbit polyclonal p-FAK (Abcam) antibody, followed by 1 h incubation with peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.). The protein signals were visualized using Pierce enhanced chemiluminescence (ECL) reaction Western Blotting Substrate (Pierce Co., Rockford, IL, USA) and exposed to medical X-ray film.

The cell treatment was the same as in the SphK1 activity assay. Cell migration and invasion were evaluated in 24-well transwell chamber model. Briefly, the upper and lower culture compartments of each well were separated by polycarbonate membranes (8-μm pore size). To determine baseline migration ability, 1.0×10⁵ cells in 0.2 ml of complete medium containing 5% FBS were placed into the upper compartment of uncoated wells (Corning Costar Inc., Corning, NY, USA), and 0.6 ml of complete medium containing 10% FBS were placed into the lower compartment. In parallel, to assess the ability of the cells to penetrate a collagen matrix (invasiveness), the experiment was repeated using the upper compartment coated with 100 μl 5 μg/ml collagen matrix (Corning Costar Inc.). The transwell chambers were incubated for 24 h at 37°C in 95% air and 5% CO₂. Cell penetration through the porous membrane into the plate was detected by crystal violet staining, and then observed and photographed at ×400 magnification on a light microscope. Data were quantitated by cell count and expressed as average of cell number from 4 random fields. The cell number indicates the cell migration capability and invasiveness.

The cell treatment was the same as in the SphK1 activity assay. The cell culture supernatant was collected for ICAM-1 and VCAM-1 analysis using ELISA detection kits (Life Technology Co.) according to the instructions from the manufacturers. In this experiment, blank wells (no sample or HRP-conjugate reagent), standard wells and testing sample well were set, 50 μl diluted standard was added to standard wells, 40 μl sample dilution and 10 μl culture supernatants were added to test sample wells, mixing gently with shaking and incubated for 30 min at 37°C. Discard liquid and washed each well with diluted washing liquid for 5 times. Then 50 μl HRP-conjugate reagent was added to each well, except the blank wells. Mixing gently with shaking, incubated for 30 sec at 37°C. Liquid was discarded followed by 5 washes. Chromogen solution A and B (50 μl each) were added to the wells, gently mixing, and incubated for 10 min at 37°C. Then, 50 μl stop solution was added to each well to stop the reaction. After zero setting, the A value was measured at 450 nm. According to standard concentration and the corresponding A values, the standard curve linear regression equation was harvested, then the sample concentration was calculated according to the regression equation.

The significance between SphK1, FAK, p-FAK and clinicopathologic characteristics of the patients was assessed with the χ² test. The correlation between SphK1, FAK and p-FAK was calculated by the method of Pearson’s correlation coefficient. Data are presented as mean ± standard deviation (SD) and Student’s t-test were used to compare continuous variables among groups. A p-value of <0.05 was considered significant.

Immunohistochemistry staining for SphK1, FAK and p-FAK was revealed as yellow or brown color and present in the cytoplasm of colon cancer cells, and the staining of cancerous tissue was significantly stronger than that of matched normal tissue (Fig. 1). As shown in Table II, in 66 cancerous tissues, 48 (72.73%) showed positive staining for SphK1, 51 (77.27%) showed positive staining for FAK and 39 (59.09%) showed positive staining for p-FAK. In 66 matched normal tissues, 23 (34.85%) showed positive staining for SphK1, 15 (22.73%) showed positive staining for FAK and 11 (16.67%) showed positive staining for p-FAK, the positive staining rates were significantly lower than those of cancerous tissues (p<0.01). Moreover, there was a close correlation between the expression of SphK1 and FAK or p-FAK (Table I). The co-expression of SphK1, FAK and p-FAK significantly associated with histological grade, Dukes’ stage, lymph node metastasis and distant metastasis, but no correlation was found between SphK1, FAK, p-FAK expression and age or gender (Table II).

The expression of SphK1, FAK and p-FAK was also detected in these 66 colon cancer specimens and matched normal colonic tissues using western blot analysis. The density of SphK1, FAK and p-FAK (Tyr 397) expression in cancerous tissues was much stronger than that in matched normal tissues (Fig. 2). The western blot analysis results fit very well with those obtained by immunohistochemistry.

Fig. 3 shows that SphK1 DNA transfection dramatically enhanced the mRNA expression of SphK1, shRNA knockdown significantly decreased the mRNA expression of SphK1 and NC transfection had no significant effect on the mRNA expression of SphK1. As shown in Fig. 4, LOVO cells possessed a basal activity of SphK1 (26.16 pmol/mg/min), SphK1 DNA transfection significantly enhanced the activity of SphK1 (65.19 pmol/mg/min), SphK1 shRNA knockdown significantly suppressed the activity of SphK1 (6.28 pmol/mg/min) and NC had no significant effect on the activity of SphK1 (25.46 pmol/mg/min). Moreover, in cells treated with 50 μM DMS for 24 h, the SphK1 activity reduced to 5.31 pmol/mg/min.

Overexpression of SphK1 dramatically enhanced the cell proliferation, migration and invasiveness (Figs. 5A and 7), and significantly suppressed cell apoptosis (Fig. 6). Compared with control group, SphK1 shRNA significantly suppressed the cell proliferation, cell migration and invasiveness (Figs. 5A and 7), while significantly promoted cell apoptosis (Fig. 6). DMS suppressed cell proliferation in a time- and dose-dependent manner (Fig. 5B), DMS significantly promoted cell apoptosis and suppressed cell migration and invasiveness (Figs. 6 and 7). NC transfection had no significant effect on cell biological behavior.

Overexpression of SphK1 not only enhanced the mRNA expression of FAK, but also promoted the protein expression of FAK and p-FAK in tumor cells. In contrast, suppression of SphK1 by shRNA and DMS reduced the mRNA and protein expression of FAK (Fig. 8). These results suggest that SphK1 may activate the FAK pathway in tumor cells.

Fig. 9A shows that overexpression of SphK1 promoted the secretion of ICAM-1 and VCAM-1 in LOVO cells, in contrast, the secretion of ICAM-1 and VCAM-1 was reduced with the suppression of SphK1 by DMS and shRNA. In addition, overexpression of SphK1 enhanced the protein expression of ICAM-1 and VCAM-1, suppression of SphK1 significantly reduced the protein expression of ICAM-1 and VCAM-1 (Fig. 9B).