The human epithelial serous cystadenocarcinoma cell line SKOV3 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were grown in Dulbecco’s-modified Eagle’s medium (DMEM, Gibco, USA) containing 10% fetal calf serum (Hyclone, USA), penicillin (107 U/l) and streptomycin (10 mg/l) at 37°C in a humidified chamber containing 5% CO₂. Orlistat was dissolved in dimethyl sulphoxide (DMSO). When the cells reached 50–70% confluency, the medium was replaced by a fresh culture medium containing Orlistat. Control cells were cultured in a medium containing an equal amount of DMSO instead of Orlistat. For 2-DE analysis, SKOV3 cells were treated with 20 mM Orlistat for 4 days and the media were changed every day. Cells were washed twice by centrifugation in phosphate buffered saline (PBS) and transferred to sterile plastic tubes for storage at −80°C prior to use.

Cell growth and viability were assessed using an MTT cell proliferation kit (Roche Applied Science). The cells were seeded on 96-well microplates at 2.0x10³/well. At 48, 72 and 96 h, the cells were treated with different concentrations of Orlistat and incubated at 37°C in 5% CO₂. The cells were subsequently incubated with 10 μl of MTT for 4 h, then the media were removed and 150 μl DMSO were added. We put the plate in a shaker before reading absorbance at 490-nm using a microplate reader (3550-UV, Bio-Rad, USA), after 20 min of incubation. The procedure was repeated three times with similar results. The following formula was used to calculate the inhibition rate of SKOV3 cell proliferation: (1-experimental group OD value/negative control OD value) x 100%. Media-only treated (untreated) cells were considered as the negative control group.

Cells (1.3x10⁸) were lysed in 1 ml lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 0.2% pH 3.0–10.0 ampholyte, Bio-Rad, USA) containing protease inhibitor cocktail 8340 (Sigma, St. Louis, MO, USA). Samples were then kept on ice and sonicated for six cycles of 10 sec, with each cycle consisting of 5 sec sonication, followed by a 10 sec break. After centrifugation at 14,000 rpm for 1 h at 4°C, the supernatant was collected and the protein concentrations determined using the DC Protein Assay Kit (Bio-Rad). Protein samples (3 mg) were applied to an immobilized pH gradient (IPG) strip (17 cm, pH 3.0–10.0 NL, Bio-Rad) using a passive rehydration method. After 12–16 h of rehydration, the strips were transferred to an isoelectric focusing (IEF) cell (Bio-Rad) and focused for a total of 60,000 Vh. The second dimension was performed using 12% equilibration. The gels were stained using CBB R-250 (Merck, Germany) and scanned with a Bio-Rad GS-800 scanner. Triplicate samples were analyzed at each time point of treatment to ensure the reproducibility of analyses. The maps were analyzed by PDQuest software Version 6.1 (Bio-Rad). Each gel spot was normalized as a percentage of the total quantity of all spots in that gel and evaluated in terms of OD. Only those spots that changed consistently and significantly (>2.0-fold) were selected for MS analysis.

In-gel digestion of proteins was carried out using MS-grade Trypsin Gold (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Briefly, spots were cut out of the gel (1–2 mm diameter) using a razor blade, and destained twice with 100 mM NH₄HCO₃/50% acetonitrile (ACN) at 37°C for 45 min in each treatment. Following dehydration and drying, the gels were pre-incubated in 10–20 μl trypsin solution for 1 h. Samples were then added in adequate digestion buffer (40 mM NH₄HCO₃/10% ACN) to cover the gels and incubated overnight at 37°C. Tryptic digests were extracted using MiliQ water initially, followed by extraction twice with 50% ACN/5% trifluoroacetic acid (TFA) for 1 h each time. The combined extracts were dried in a vacuum concentrator at room temperature. The samples were then subjected to MS analysis.

Mass spectra were acquired using a quadrupole time-of-flight (Q-TOF) mass spectrometer (Micromass, Manchester, UK) with a matrix-assisted laser desorption ionization (MALDI) source (Micromass). Tryptic digests were dissolved in 5 μl of 70% ACN/0.1% TFA, and then 1 μl of the digestion was mixed with 1 μl saturated α-cyano-4-hydroxy-cinnamic acid (CHCA) in 50% ACN/0.5% TFA and spotted onto a 96-well target plate. MS/MS was performed in a data-dependent mode in which the top ten most abundant ions for each MS scan were selected for MS/MS analysis. The MS/MS data were acquired and processed using the MassLynx software (Micromass) and MASCOT was used to search the database. Database searches were carried out using the following parameters: database, Swiss-Prot; taxonomy, Homo sapien; enzyme, trypsin; and allowance of one missed cleavage. Carbamidomethylation was selected as a fixed modification and oxidation of methionine was allowed to be variable. The peptide and fragment mass tolerance were at 1 and 0.2 Da, respectively. The data format selected was Micromass PKL and the instrument selected was MALDI-Q-TOF. Proteins with probability-based MOWSE scores exceeding their threshold (P<0.05) were considered to be positively identified.

Collected cells were lysed in RIPA buffer (50 mM Tris-base, 1.0 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF) to extract all the proteins and quantified by the DC protein assay Kit (Bio-Rad). Samples were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were blocked overnight with PBS containing 0.1% Tween 20 in 5% skimmed milk at 4°C, and subsequently probed by the primary antibodies: rabbit anti-PKM2 (diluted 1:500, Abcam, UK). Blots were incubated with secondary antibody conjugated to horseradish peroxidase for 2 h at room temperature. Target proteins were detected by enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, USA), and β-actin was used as an internal control.

All quantitative data are recorded as the means ± SD. Comparisons between two groups were performed by Student’s t-test. Differences among multiple groups were assessed by one-way ANOVA analysis. Relevance analysis of ordinal data was performed by cross χ² test. A statistically significant difference was defined as p<0.05.

The proliferation activity of Orlistat-treated SKOV3 cells was examined using the MTT assays. MTT results showed that the proliferation activity was suppressed by Orlistat in both a dose- and duration-dependent manner, and the proliferation ratio was decreased to 60% of the control value 96 h after treatment with Orlistat when the drug concentration was 20 mM, as shown in Fig. 1.

To explore the molecular mechanisms underlying the Orlistat-induced antitumor activity of SKOV3 cells, 2-DE based proteomics was used to profile differentially expressed proteins in SKOV3 cells treated with or without Orlistat. Image analysis was performed using PDQuest 7.1 software. Representative 2-DE maps are shown in Fig. 2. Approximately 1000–1100 protein spots were detected by CBB R-250 staining in a single 2-DE gel. Each protein spot was normalized as a percentage of the total intensity of all spots in the gel. By comparing 2-DE patterns, differentially expressed proteins were defined as statistically meaningful (p<0.05) based on both of the following two criteria: i) intensity alterations of >2.0-fold (t-test, p<0.05) and ii) observed in at least 3 individual experiments. According to these criteria, a total of 111 spots were selected and analyzed using MALDI-Q-TOF tandem mass spectrometry. A total of 71 proteins from the 111 spots were identified (Fig. 2). As different isoforms of a protein might have distinct functions, each isoform/spot was considered to be a single protein for analysis in our study.

In total, 71 spots with differential expression levels were subjected to MS/MS analysis. The MS/MS data were queried using the search algorithm MASCOT against the Expasy protein sequence database. Proteins were identified based on a number of criteria including PI, MW, the number of matched-peptides and MOWSE score (Table I and II).

The identified proteins were divided into various groups based on their biological functions and subcellular localization. This implicated roles in metabolism (32%), protein folding (8%), translation (5%), protein modification (4%), cell proliferation (15%), apoptosis (10%), signal transduction (14%) and cell cytoskeleton (12%). The proteins were found to be located in the cytoplasm (57%), nucleus (11%), mitochondrion (15%), cell membrane (10%) or were secreted (12%) (Fig. 2). For a macroscopic presentation, cluster maps and protein interaction and function networks were generated using Cluster or the KEGG-based software tool Cytoscape, respectively. Twenty-three proteins, accounting for 32% of the proteins identified, were found to be associated with metabolism regulation. The metabolism-regulating proteins were grouped in different clusters. Pyruvate kinase isozymes M1/M2 were found to show one of the most significant differences in expression between SKOV3 cells treated with or without Orlistat. It was downregulated more than 10-fold in SKOV3 cells treated with Orlistat compared to those without Orlistat, and MS/MS analysis revealed 15 matched peptides with 36% sequence coverage and a MOWSE score of 309 (Fig. 3).

The expression level of PKM2 was further validated by western blotting. Consistent with the observations in 2-DE analysis, PKM2 was downregulated in the Orlistat-treated SKOV3 cells compared with the parental SKOV3 cells. A similar change in the expression level of FASN was detected in SKOV3 cells treated with Orlistat (Fig. 5).