Cell culture and FASN inhibition
PC-3 human prostate, MCF-7 human breast and SKOV-3 human ovarian cancer cells were routinely cultured in DMEM/F12 (Gibco, NY, USA) supplemented with 10% heat-inactivated FBS (Hyclone, UT, USA) and 100 U/mL penicillin 100 μg/mL streptomycin, 0.25 μg/mL amphotericin (Gibco, NY, USA) and 2 mM L-glutamine (Cellgro, VA, USA) at 37 °C in 5% CO2. For all FASN inhibition studies, FBS was lowered to 5% (in order to limit the amount of available extra-cellular FA) and glucose in the medium was reduced by half to 8.76 mM (financial reasons). To inhibit FASN, PC-3 cells were incubated for 24 h and 48 h with 30 μM Orlistat (treated) or with carrier dimethylsulfoxide (DMSO) at 0.5% v/v (control cells). MCF-7 and SKOV-3 cells were incubated for 48 h with 30 μM Orlistat (treated) or with DMSO at 0.5% v/v (control cells). MCF-7 cells were also treated with 30 μM cerulenin (treated) or with DMSO at 0.5% v/v (control cells).
FASN activity assay
FASN activity was determined as previously described (29
). Briefly, ~5 × 106
cells were trypsinized, washed in PBS and frozen at −80 °C. Cells were resuspended in 1 mL of lysis buffer containing 1 mM EDTA, 150 mM NaCl, 100 μg/mL PMSF and 50 mM Tris-HCl at pH 7.3, subjected to vortex for 30 s at 0 °C and disrupted at 0 °C by ultrasonication for 10 periods of 1 s. The lysates were centrifuged (16,000 × g
, 15 min) and the supernatant stored at −80 °C and assayed within 1 week. A sample was taken to measure protein content (Bio-Rad DC
protein assay, Bio-Rad Laboratories, CA, USA). Lyophilized supernatant (~ 5 μg) was added to a mixture of 200 mM potassium phosphate buffer (pH 6.6), 1 mM dithiothreitol, 1 mM EDTA, 0.24 mM NADPH and 30 μM acetyl-CoA in 0.2 mL reaction volume. After monitoring at 340 nm (Beckman Coulter DU800 UV/Visible spectrophotometer, CA, USA) at room temperature for 3 min to measure background NADPH oxidation, 50 μM of malonyl-CoA was added and the reaction mixture assayed for 10 min. Data was analyzed using Beckman Coulter DU800 System and Applications Software.
Cell cycle analysis
Flow cytometry was used to determine the effect of FASN inhibition on cell cycle as follows. ~ 2 × 106 PC-3 and MCF-7 trypsinized cells were fixed in 95% ice-cold ethanol and then stained with a PBS solution containing 40 μg/mL propidium iodide (PI) (Sigma-Aldrich, MO, USA) and 100 μg/mL RNase A (Sigma-Aldrich, MO, USA). Cells were analyzed using a BD FACSCalibur (Becton Dickinson, CA, USA) instrument using a 488 nm excitation wavelength. Cells of uniform width were gated and PI intensities were plotted and analyzed using ModFit LT (version 3.1) software (Topsham, ME, USA). In the case of PC-3 cells, the integration values of the two major G1 populations (diploid (2n) and aneuploid (2.8n)) within the culture were summed to derive the final cell cycle distribution results. The values for their corresponding G2 (4n and 5.6n) and S phase populations were also combined.
PC-3 and MCF-7 cells (1×105) were plated in 4 mm2 wells, incubated overnight, and treated with 30 μM Orlistat for 3, 24, and 48 hours. At each time-point, the media was removed and transferred to a 1.5mL tube to preserve any floating cells. The cell monolayers were then washed once with PBS, trypsinized, resuspended in 0.5 mL PBS and transferred to the corresponding media tubes. The cell suspensions were then centrifuged at 1000×g for 5 minutes, washed once with PBS and lysed using 100 μL of lysis buffer (50 mM HEPES, pH 7.4, 0.1% CHAPS, 5 mM DTT, 0.1 mM EDTA). The lysates were centrifuged at 10,000×g for 10 minutes, and stored at −20°C. Caspase-3 activity was measured by incubating 10 μL of each lysate with 30 μM Ac-DEVD-AMC substrate (Biomol, Plymouth Meeting, PA) in assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 1 mM EDTA, 10% glycerol) in a 100 μL total volume for 1 hour at 37°C. Cleavage of the substrate by Caspase-3 was measured using a Safire plate reader (Tecan Group, Switzerland) at excitation and emission wavelengths of 360nm and 460nm, respectively.
Cell extraction, MRS acquisition and analysis
For MRS studies, glucose in the medium (8.76 mM) was replaced by equal concentrations of 1-13
C-glucose and unlabeled glucose (to monitor FA synthesis and glycolysis) and choline in the medium was replaced with 1,2-13
C-choline at its normal concentration of 64.1 μM (to monitor choline metabolism). PC-3, MCF-7 or SKOV-3 cells (logarithmic growth) were cultured with Orlistat (treated) or DMSO (control) for 24 h and/or 48 h, replenishing medium every 24 h. Spent medium was saved. To monitor cell metabolism, ~ 5 × 107
cells were extracted using the dual-phase extraction method (25
). Briefly, cells were rinsed with ice-cold saline, fixed in 10 mL of ice-cold methanol and scraped from the culture flask surface. Alternatively, cells were trypsinized, centrifuged and the cell pellet combined with 10 mL of ice-cold methanol (results from both methods were indistinguishable). Following vortexing, 10 mL of ice-cold chloroform was added followed by 10 mL of ice-cold de-ionized water. After phase separation and solvent removal, cellular proteins were collected and stored at −80 °C until measurement (see below). To acquire 1
H and 13
C spectra, the aqueous fraction was reconstituted in 500 μL of deuterium oxide (D2
O, aqueous phase) and the lipid fraction was reconstituted in deuterated chloroform (CDCl3
, lipid phase). To acquire aqueous 31
P MR measurements, 25 μL of EDTA in D2
O were further added to a final concentration of 5 mM. To acquire lipid 31
P MR measurements, CDCl3
was evaporated and the precipitate re-suspended in 500 μL of a 2:1 mixture of CDCl3
and 60 mM methanolic EDTA adjusted to pH 7.3 with CsOH.
MR spectra were acquired on an Avance DRX500 Bruker spectrometer (Bruker Biospin, Germany). 1H spectra: 20 ppm spectral width, 30° flip angle and 5 second repetition time with water suppression. 13C spectra: 240 ppm spectral width, 30° flip angle and 3.5 second repetition time with broad-band proton decoupling. 31P spectra: 60 ppm spectral width, (aqueous 31P) or 35 ppm (lipid 31P), 30° flip angle, 4.5 second repetition time and broad-band proton decoupling. Data were analyzed using Bruker Topspin 2.0.a. software. Changes in metabolite levels were determined relative to matched controls. Absolute metabolite quantification was obtained by determining the appropriate peak area, normalizing to an external reference (tetramethylsilane (TMS), 1H MRS; CDCl3, 13C MRS; methylenediphosphonic acid (MDPA), 31P MRS), correcting for saturation and nuclear Overhauser effects (NOE) and normalizing to cellular protein. In quantifying the product of de novo FA synthesis (13C MR spectra) only the methylene carbons at 29–30 ppm were used and it was further assumed that FA observed represented only palmitate. From 1H MR spectra of lipid extracts only methylene protons at 1.2–1.3 ppm were used to quantify the total pool of FA and all FA were assumed to be palmitate.
To quantify the cellular proteins that precipitated during metabolite extraction, the pellet was re-suspended in 1 mL of 1 M NaOH by incubating for 1 h at 50 °C. A sample was then taken for measurement of protein content (Bio-Rad assay, Bio-Rad Laboratories).
Choline kinase assay
Choline kinase (ChoK) activity was determined as described (31
). Following incubation with DMSO or Orlistat for 48 h, ~ 15 × 106
PC-3 cells were trypsinized, washed in ice-cold PBS and re-suspended in 400 μL of Tris-HCl (pH 8.0) containing 10 mM dithiothreitol and 1 mM EDTA in D2
O. Cells were homogenized for 30 s at 0 °C and were disrupted at 0 °C by ultrasonication for 10 periods of 1 second. Cell lysates were centrifuged (16,000 × g
, 30 min) and the particle-free supernatant transferred to a NMR tube. Initial PCho levels were measured by 1
H MRS as described above. ChoK activity was assayed by monitoring temporal accumulation of PCho by MRS following addition of choline chloride, ATP and Mg2+
in Tris-HCl buffer (final concentrations: 5 mM choline chloride, 10 mM ATP, 10 mM MgCl2
). ChoK activity was determined from a straight line fit to plots of PCho as nmol/mg protein versus time following addition of all substrates.
The effect of Orlistat on ChoK activity was also monitored after only 1 h of incubation. Cells were incubated for 47 h with DMSO alone, then Orlistat added during the last hour of incubation, creating a treated group. Cells were then lysed and ChoK activity assayed as before. To detect any direct interaction between Orlistat and ChoK, cells were incubated for 47 h with DMSO alone. Following extraction, half the cell lysates were exposed to Orlistat for 1 h at room temperature, creating a treated group, and ChoK activity assayed as before.
Quantitative PCR (q PCR) Analysis
PC-3 cells (2.5 × 105) were seeded in 10 cm2 dishes for 24 hours before treating with 30 μM Orlistat. RNA from treated and control cells was extracted cells using TRIZOL (Invitrogen, Carlsbad, CA) and standardized to a concentration of 50 ng/mL. cDNA was produced using a Transcriptor First Strand synthesis kit (Roche, Mannheim, Germany) according to manufacturer’s instruction. The expression of phosphocholine cytidylyltransferase (CCT) and ChoK following treatment was measured by quantitative PCR (qPCR) using an ABI 7500 instrument (Applied Biosystems, Foster City, CA). PCR primers were designed based on GenBank sequences NM_005017 (CCT; Forward 5′-TGTTCAGCCAAGGTCAATGCAAGG-3′ and Reverse 5′-TTCTCGTTCATCACCGTGAAGCCT -3′) and NM_001277 (choline kinase; Forward 5′-TATCTTGTTGCTGGAAGGCCGAGA-3′ and Reverse 5′-TGGGCGTAGTCCATGTACCCAAAT-3′). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous standard and amplified using primers 5′-CCATGGAGAAGGCTGGGG-3′ (Forward) and 5′-CAAAGTTGTCATGGATGACC-3′ (Reverse). Expression levels of CCT and choline kinase were first normalized to endogenous GAPDH levels (ΔCt) and subsequently to the untreated control of each group (ΔΔCt). The results were expressed as percentages of relevant control using the equation 2ΔΔCt.
Western blot analysis
PC-3 and MCF-7 cells were treated with DMSO or Orlistat (30μM) for 3 hrs, 24 hrs and 48 hrs, then lysed using cell lysis buffer containing 1% NP40, 1% SDS and 1 μL/mL protease inhibitor cocktail set III (Calbiochem, La Jolla, CA). Lysates were centrifuged at 12,000 rpm for 10 minutes at 4°C, the protein supernatant was collected and total protein concentrations were determined using Bio-Rad DC protein assay reagents (Bio-Rad, Hercules, CA). Proteins were separated by SDS-PAGE using 10% gels and transferred electrophoretically to 0.45 μm nitrocellulose membranes. Membranes were blocked in blocking buffer containing 5% nonfat dry milk in TBS (pH 7.6) and 0.1% Tween 20 and incubated overnight at 4°C with primary antibodies as follows: Akt (1:1,000; Cell Signaling Technology, Danvers, MA), Phospho-Akt (P-Akt, 1:1,000; Cell Signaling Technology and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5,000; Stressgen). This was followed by 1-hour incubation with horse radish peroxidase–conjugated secondary anti-rabbit (Cell Signaling Technology) and anti-mouse (Cell Signaling Technology) antibodies at dilutions of 1:1,000 and 1:2,000 respectively. Membranes were washed with enhanced chemiluminescence reagents (LumiGLO & Peroxide, Cell Signaling Technology) for 1 minute and exposed to Hyperfilm (Amersham Biosciences, Piscataway, NJ), which was developed on a Konica SRX-101 automatic developer (Konica, Tokyo, Japan). The intensity of individual bands of interest was quantified using Image J software (National Institutes of Health, Bethesda, MD) and normalized to the intensity of glyceraldehyde-3-phosphate dehydrogenase (loading control) and Akt and P-Akt protein expressions compared between DMSO and Orlistat treated groups.
Unless otherwise stated, experiments were repeated 3–5 times. Results are expressed as mean ± SD. Two-tailed unpaired Student’s t tests were performed to assess the statistical significance of results. P value < 0.05 was considered significant.