Metabolic characterization of invaded cells of the pancreatic cancer cell line, PANC-1

Cell culture and reagents

The human pancreatic cancer cell line, PANC-1 was purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS, HyClone Laboratories, GE Healthcare, Logan, UT, USA), 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin (Gibco, Gaithersburg, MD, USA) in a humidified atmosphere with 5% CO₂ at 37°C. SUM149, a human inflammatory ductal carcinoma cell line, was purchased from Asterand Bioscience (Ann Arbor, MI, USA) and grown in Ham's F-12 medium (Gibco) supplemented with 5% FBS (GE Healthcare), 5 μg/mL insulin (Sigma Aldrich, St. Louis, MO, USA), 1 μg/mL hydrocortisone (Sigma Aldrich), and 100 U/mL penicillin/streptomycin (Gibco). L-buthionine-sulfoximine (BSO), an inhibitor of GSH biosynthesis, was purchased from Sigma Aldrich. Cells in logarithmic growth phase seeded at an appropriate density were used for all experiments.

Real time imaging of the invading cells from the 3D spheroid

Cells were seeded into a 96-well ultra-low attachment plate (Corning, NY, USA, 1 × 10⁴ cells/ well) with 100 μL DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin, and spheroid formation was proceeded with by incubating the plate in a 5% CO₂ incubator at 37°C for 72 h. Half of the media, 50 μL, was carefully removed, and 50 μL of matrigel with concentration of 6 mg/mL protein (BD Biosciences, Franklin Lakes, NJ, USA) was poured into each well so that the final concentration of matrigel was 3 mg/mL; this was solidified for 2 h in the CO₂ incubator. The plate was then centrifuged at 100 g for 3 min, and movies of the real time imaging of the spheroid embedded in the matrigel were captured using IncuCyte Zoom (Essen BioScience Inc., Ann Arbor, MI, USA).

Preparing the invaded cells and whole cultured cells

To prepare the invaded cells, Boyden chamber transwell invasion assays were performed as described previously.6, 16 Briefly, cells were trypsinized and viable cell numbers were counted with trypan blue. Cells were then separated into two sets; one of them was for the collection of whole cultured cells, the other set is for preparing the invaded cells, respectively. For the collection of whole cultured cells, cells were suspended into serum-added DMEM, and 1 × 10⁶ cells were seeded on the 10 cm culture dish. For collecting the invaded cells, cells were suspended into serum-free DMEM containing 0.35% BSA, and 1 × 10⁶ cells were seeded into the upper well of the transwell chamber (the 24 mm transwell insert diameter with a pore size of 8 μm, Corning) coated with 21 μL matrigel (3 mg/mL concentration); 90 transwells were used for each experiment. DMEM supplemented with 10% fetal bovine serum was added to the lower well as a chemoattractant. After incubation for 24 h from the time of cell seeding on the matrigel, the non-invasive cells remaining on the matrigel-coated side were wiped off with a cotton swab, and the cells that reached the undersurface of transwell membrane were collected by incubating the cells with accutase (Innovative Cell Technologies, San Diego, CA, USA) for 30 min at room temperature. Invaded cells, which were collected from thirty transwells, were pooled together so that three sets of invaded cell groups were made, and we used those three sets to test reproducibility of metabolites analysis. At the same time point with the collection of the invaded cells, the whole cultured cells were also collected with accutase for 30 min incubation at room temperature. Collected cells were suspended in DMEM and used for the metabolome analysis.

Sample preparation for the metabolome analysis

The invaded cells and the whole cultured cells (1 × 10⁵ cells/sample for the invaded cells, and 1 × 10⁶ cells/sample for the whole cultured cells, respectively) were used for the extraction of intracellular metabolites. Cells were collected by centrifugation at 1000 g for 5 min at room temperature and washed twice with 5% mannitol solution. Cells were then treated with methanol to inactivate enzymes. Cell extract was treated with milliQ containing internal standards (Human Metabolome Technologies, Inc., Tsuruoka, Yamagata, Japan). The extract was centrifuged at 2300 g and 4°C for 5 min, and the aqueous layer was filtrated through a Millipore 5-kDa cutoff filter (Merck Millipore, Billerica, MS, USA) at 9100 g and 4°C for 120 min. The concentrated filtrate was then re-suspended into 50 μL of milliQ for the CE-MS analysis.

Metabolome analysis by CE-TOFMS

CE-TOFMS was performed using an Agilent CE Capillary Electrophoresis System with an Agilent 6210 Time of Flight mass spectrometer, Agilent 1100 isocratic HPLC pump, Agilent G1603A CE-MS adapter kit, and Agilent G1607A CE-ESI-MS sprayer kit (Agilent Technologies, Waldbronn, Germany), as described in the previous papers.17-20 The systems were controlled by the Agilent G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies). The metabolites were analyzed by using a fused silica capillary with the electrophoresis buffer (Human Metabolome Technologies) as the electrolyte. The sample was injected at a pressure of 50 mbar for 10 s in cation analysis and 25 s in anion analysis. The spectrometer was scanned from m/z 50 to 1000.

Metabolome data analysis

Raw data measured by CE-TOFMS was analyzed using the MasterHands software (Keio University, Tsuruoka, Japan), and peak information including m/z, migration time for CE-TOFMS measurement (MT), and peak area were obtained.21 Signal peaks corresponding to isotopomers, adduct ions, and other product ions of known metabolites were excluded, and the remaining peaks were annotated with the putative metabolites from the HMT metabolite database (Human Metabolome Technologies) based on their MTs and m/z values determined by TOFMS. The tolerance range for the peak annotation was configured at ±0.5 min for MT, and ±10 ppm for m/z. In addition, peak areas were normalized against those of the internal standards, and the resultant relative area values were normalized by the sample amount. Principal component analysis (PCA) were performed by the software, SampleStat (Human Metabolome Technologies).

Calculation of energy charge

Adenylate and guanylate energy charges (AEC and GEC, respectively) were calculated using the absolute concentrations of ATP, ADP, and AMP, or GTP, GDP, and GMP as follows:22

GSH and GSSG measurement

Invaded PANC-1 cells or the whole cultured PANC-1 cells were plated as 7000 cells per well in black 96-well plates, and triplicate samples were prepared for each assay. Cells were allowed to attach overnight in CO₂ incubator at 37°C; then, growth media were removed, and cells were washed with PBS. Cellular concentrations of GSH and GSSG were quantified using the GSH/GSSG-Glo Assay kit, according to the manufacturer's protocol (Promega, Madison, WI, USA).23 Luminescence was measured with a plate reader (PerkinElmer, MA, USA). Concentrations of total glutathione and GSSG were calculated against concurrently run standards provided in the kit. To calculate GSH/GSSG ratios, the following formula was used:

since each molecule of oxidized glutathione is metabolized into two molecules of reduced glutathione on breakage of the disulfide bond.23

Cell viability assays

Invaded PANC-1 cells or the whole cultured PANC-1 cells were plated as 7000 cells per well in black 96-well plates and allowed to attach overnight in CO₂ incubator at 37°C. The next day, appropriate concentrations of hydrogen peroxide (H₂O₂) were added with or without the addition of 50 mM N-acetylcysteine (NAC) solution pre-adjusted with NaOH to a pH of 7. Cells were further incubated for 24 h in a CO₂ incubator, and cell viability was measured with CellTiter blue, according to the manufacture's protocol (Promega).

Statistical analysis

Statistical analyses were performed using unpaired Student's t-test. All of the statistical analyses were performed using a two-sided test. A P < 0.05 was considered significant.

Real time-imaging of invading cells from the 3D spheroid embedded in matrigel

Before collecting the invaded PANC-1 cells from the underneath of the transwell chamber, we first checked the cells exhibiting invasive potential within the whole cultured PANC-1. The PANC-1 spheroid was embedded in matrigel of the same rigidity as that used in the invasion assay, and the cells from the spheroid invading into the matrigel were observed with real-time imaging. As in Movie S1 and Figure 1(a), we confirmed that the invading PANC-1 cell clearly moved apart from the spheroid, which is distinct from the spheroid population.

Metabolomics profile of invaded PANC-1 cells compared with the whole cultured PANC-1

Invaded PANC-1 cells and the whole cultured PANC-1 were collected (Fig. S1), and metabolites in these cells were analyzed by CE-TOFMS. PCA indicated that the metabolomics profile of the invaded cells was very distinct from those of the whole cultured PANC-1 (Fig. S1b). Among the metabolites whose concentrations were detected, the levels of 43 metabolites were significantly decreased in the invaded PANC-1 compared with the whole cultured cells; these were 18 amino acids, three glycolytic intermediate metabolites, three TCA cycle intermediates, and nine compounds produced during nucleic acids metabolism (Table 1). In contrast, the levels of only six metabolites were significantly increased in the invaded cells compared with the whole cultured cells, including two glycolytic metabolites, 3-Phoshoglyceric acid (3PG) and phosphoenolpyruvic acid (PEP), and four intermediates from nucleic acids metabolism, IMP, GMP, AMP, and UMP (Table 2).

The 20 amino acids can be divided into two groups, essential or non-essential amino acids. Most of the amino acids, 18 out of the 20 amino acids, were reduced in the invaded cells (Table 1), and we further divided those amino acids into essential or non-essential amino acid groups and calculated the total amounts for each group. The results showed that both levels, essential and non-essential, were similarly reduced in the invaded cells compared with the whole cultured cells (Fig. 2a).

On the other hand, the glycolytic intermediates of the invaded PANC-1 showed both reductions and increases in their concentrations compared with those of whole cultured cells; the level of G6P was reduced in the invaded cells, whereas the downstream metabolites, 3PG and PEP, were both increased (Fig. 2b). In addition, lactate levels were decreased in the invaded cells, suggesting that lactate fermentation is less activated in the invaded cells (Fig. 2b).

For the TCA cycle, citric acid, succinic acid, and malic acid were all reduced in the invaded cells (Fig. 2c); thus, those metabolites may have been used more than they were produced in the invaded cells, or they were simply consumed less in the invaded cells.

Nucleic acid metabolism and the energy charge of PANC-1 invaded cells compared with the whole cultured PANC-1

For the nucleic acid metabolism, significantly higher concentrations of nucleoside monophosphates, AMP, GMP, and UMP among other purine or pyrimidine nucleotides, were found in the invaded cells compared with the whole cultured PANC-1 (Fig. 3a,b). In contrast, most of other nucleoside di- or triphosphates, ADP, ATP, GDP, GTP, CDP, CTP, UDP, and UTP, were reduced in invaded cells (Fig. 3a,b).

From the concentrations of adenylate and guanylate nucleotides, the AEC and GEC were calculated using the formula indicated in the Materials and Methods.22 Interestingly, both the AEC and GEC were significantly lower in the invaded cells compared with the whole cultured cells, at 0.69 for the invaded cells and 0.82 for the whole cultured cells, respectively (Table 3), indicating that the invading cells consumed more ATP and GTP. Accordingly, this suggests the ATP-generating pathways were stimulated in the invaded cells, as was indicated in a recent report.22

Arginine metabolism of the PANC-1 invaded cells compared with the whole cultured PANC-1

We recently demonstrated that invaded PANC-1 cells exhibited NO production and the NOS-NO-PI3K-AKT2-GIRDIN pathway was activated compared with the whole cultured PANC-1,6 suggesting that the invaded cell population may have a unique metabolic profile relating to NO production. Arginine is a substrate for the NO-producing enzyme, NOS; NOS converts arginine to citrulline with the concomitant production of NO.24 However, arginine is also metabolized to be ornithine via another enzyme, arginase, without the production of NO. Interestingly, the ratio of citrulline to ornithine, an indicator of the consumption of arginine by NOS versus arginase,24 was significantly greater in the invaded PANC-1 compared with the whole cultured cells, at 1.11 and 0.33, respectively (Fig. 4a). In addition, the ratio of citrulline to arginine, reflecting NOS activity,24 was increased in the invaded cells (Fig. 4b), whereas the ratio of ornithine to arginine, indicating the arginase activity,24 showed no significant difference between the invaded cells and the whole cultured cells (Fig. 4c). Thus, in the invaded PANC-1, the utilization of arginine by NOS was more activated than the consumption by arginine, which may be related to our recent findings of higher production of NO in the invaded cells.6

GSH/GSSG ratio for estimating the cellular oxidative stress

Glutathione is one of the major factors involved in antioxidation.25 Intracellular glutathione usually exists in its reduced form (GSH), and GSH is converted into the oxidized form (GSSG) by stimulation with oxidative stress. When cells are exposed to oxidative stress, GSSG are accumulated, and the ratio of GSH to GSSG will decrease. Therefore, the GSH/GSSG ratio is a useful indicator of oxidative stress in cells and has been used as the index of intracellular oxidative stress.25 We used GSH and GSSG concentrations measured by CE-TOFMS as well as the GSH/GSSG-Glo Assay kit, and calculated the GSH/GSSG ratio shown in Figure 5 and S1, respectively. We found the GSH/GSSG ratio was significantly lower in the invaded cells compared with the whole cultured PANC-1 (Fig. 5, S2). This was also true in the other cell line, SUM149; the GSH/GSSG ratio in invaded SUM149 was lower than that of the whole cultured SUM149 (Fig. S3).

Invaded PANC-1 cells were more resistant to oxidative stress than the whole cultured PANC-1

From our results, the GSH/GSSG ratio was significantly lower in the invaded cells compared with the whole cultured cells, indicating that the invaded cells may have experienced higher oxidative stress than the whole cultured cells. Greater production of oxidants results in higher oxidative stress to cells, which then impairs cellular functions.26 Thus, we next compared the sensitivity of invaded or whole cultured PANC-1 cells to oxidative stress by performing the cell surviving assay with the addition of H₂O₂ to the culture media with or without antioxidant, NAC, treatment. The results showed that the survival fractions of invaded cells after exposure to 450 or 500 μM H₂O₂ were significantly higher than those of whole cultured cells (Fig. 6a,b), suggested that invaded PANC-1 cells were more resistant to oxidative stress than the whole cultured PANC-1.

Reduction of GSH levels inhibited PANC-1 invasion

So far, the invaded cells are suggested to experience higher oxidative stress but also greater resistance to it. In addition, we showed the invaded cells consumed more amount of GSH than the whole cultured cells in Figure 5(a), indicated that intercellular GSH may have an important role in invaded cells to withstand the higher oxidative stress. De novo GSH synthesis is one of the important factors maintaining the intracellular GSH level.27 Among the enzymes involved in the GSH synthesis, γ-glutamylcysteine synthetase (γ-GCS) is the rate-limiting enzyme. In order to clarify the role of GSH in PANC-1 invasion, we next performed the invasion assay with using an irreversible inhibitor of γ-GCS, L-buthionine-sulfoximine (BSO).28 Treatment of PANC-1 cells with 0.3, 1, 2, 3, or 4 μM BSO for 24 h clearly reduced the intercellular GSH content in a dose-dependent manner (Fig. 6c). Interestingly, although 0.3 μM BSO or 1 μM BSO diminished only 8% or 11% of cell numbers (Fig. 6d), 0.3 μM BSO or 1 μM BSO clearly inhibited PANC-1 invasion; 23%, or 46% reduction, respectively (Fig. 6e). Thus, it is indicated that intracellular GSH has role in PANC-1invasion.