Human Burkitt lymphoma (Daudi, Raji, Ramos), human follicular lymphoma (DoHH2), human colon adenocarcinoma (LoVo), and human embryonic kidney (HEK293T) cell lines were purchased from the American Tissue Culture Collection (Manassas, VA). Cells (Daudi, Raji, Ramos, DoHH2, and HEK293T) were cultured in RPMI 1640 or Dulbecco modified Eagle medium/F-12 medium (LoVo) supplemented with 10% heat-inactivated fetal bovine serum, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B (all from Invitrogen, Carlsbad, CA). Cells were cultured at 37°C in a fully humidified atmosphere of 5% CO2 and were passaged approximately every other day.
The following statins were used: atorvastatin (Pfizer Pharmaceuticals, Inc, Groton, CT), cerivastatin (Bayer Corp, West Haven, CT), fluvastatin (Novartis Pharma AG, Basel, Switzerland), lovastatin, and simvastatin (both from Merck, Sharp & Dohme Res. Lab., Rahway, NJ). Lovastatin and simvastatin were obtained in the inactive lactone form. They were converted to the active form by dissolving in ethanol, heating for 2 hours at 50°C in 0.1N NaOH and neutralizing with HCl. Distilled water was added to obtain the final stock concentration of 10 mM. Stock solution was aliquoted and stored frozen (-20°C). Mevalonic acid (MA), farnesyl pyrophosphate (FPP), methylβ-cyclodextrin (MβCD), and water-soluble cholesterol were purchased from Sigma (St Louis, MO). Farnesyltransferase inhibitor L-744,832 was obtained from Merck KGaA (Darmstadt, Germany); 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-deoxyglucose (6-NBDG) was from Invitrogen and was dissolved in dimethyl sulfoxide to 10-mM stock concentration; 4,6-ethylidine-d-glucose (ETDG) was purchased from Sigma.
For flow cytometry studies, 5 x 105 cells were resuspended in 300 µl of phosphate-buffered saline (PBS) and incubated with 300 µM 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG) or with anti-hemagglutinin (HA) phycoerythrin (PE)-conjugated monoclonal antibody (Miltenyi Biotec, Bergischgladbach, Germany; 1:10 dilution) or PE-conjugated immunoglobulin G1 (Beckman Coulter, Miami, FL; 1:10 dilution), which served as an isotypic control, for 30 minutes at room temperature in dark. As a control of glucose uptake, 200mMETDG was used. Before analysis, cells were washed twice with PBS and resuspended in 300 µl PBS. Additional staining with 2.5 µg/ml propidium iodide (Sigma-Aldrich) was performed to distinguish dead cells. The cells were analyzed on FACS Scan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using CellQuest Pro Software Version 5.2. 6-NBDG uptake was identified by gating the viable (propidium iodide [PI].negative) cells. The mean fluorescence intensity (MFI) served as a measure for 6-NBDG uptake on a per-cell basis.
Radioisotope Measurement of Glucose Uptake
For the evaluation of [1,2-3
-glucose (2-DOG; Perkin-Elmer, Waltham, MA) uptake, the protocol of Kaliman et al. [22
] was used with minor modifications. Briefly, 3 x 105
cells resuspended in 300 µl of transport solution (20 mM HEPES, 150 mM NaCl, 5 mM KCl, 5 mM MgSO4
, 1.2 mM KH2
, 25 mM CaCl2
, 2 mM pyruvate, pH 7.4) were incubated for indicated time with 1.5 µl of 2-DOG (8.0 mCi/ml radionuclide concentration). 2-DOG uptake was stopped by adding 50 mM d
-glucose solution in PBS at 4°C. After a three-time wash in PBS, the cells were lysed with 0.1 M NaOH + 0.1% SDS solution and left overnight at 4°C to enable proper cell lysis. To evaluate the nonspecific uptake of isotope-labeled deoxy-d
-glucose (time t0
), a stop solution (50 mM of d
-glucose in PBS) was added to the cellular suspension instead of the PBS solution. The following day, the lysates were analyzed in a scintillation counter (Wallac, Gaithersburg, MD). All experimental groups were performed in duplicates. The values presented net uptake per 3 x 105
cells of label, which is the total label accumulated at given time minus the radioactivity bound to the surface of the cells (time t0
Cell Cycle Analysis
For the cell cycle analysis, 5 x 105 cells were fixed for 1 hour in ice-cold 70% ethanol, washed twice with PBS, digested for 10 minutes with 100 µg/ml RNAse A (Qiagen, Chatsworth, CA) and stained with 5 µg/ml propidium iodide (Sigma). Next, the cells were analyzed with FACS Scan flow cytometer using CellQuest Pro Software Version 5.2. G1, S, and G2/M phases of the cell cycle were distinguished according to the cellular DNA content (≤2n; (2n–4n), ≥4n, respectively).
Lactate Concentration Measurements
Lactate concentration in cell culture supernatants was estimated electrochemically using lactate oxidase method on ABL Radiometer ABL 800 FLEX blood gas analyzer (Radiometer Medical ApS, Brønshøj, Denmark). Values obtained were normalized for total cellular protein concentration.
Western Blot Analysis
Control or drug-treated cells were washed twice with PBS, pelleted, and lysed with 25 mM HEPES, 0.3 M NaCl, 1.5 mM MgCl2, 20 mM β-glycerol-phosphate, 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 1% Triton X-100, and 10% glycerol-containing buffer supplemented with Complete protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Protein concentration was measured using Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Equal amounts of whole-cell proteins were separated on 10% SDS.polyacrylamide gel, transferred onto Protran nitrocellulose membranes (Schleicher & Schuell BioScience, Keene, NH), blocked with Tris-buffered saline (pH 7.4) and 0.05% Tween 20 supplemented with 5% bovine serum albumin. Anti-GLUT1 (Millipore, Temecula, CA) rabbit polyclonal antibody at 1:1000 dilution was used for overnight incubation. After extensive washing with Tris-buffered saline (pH 7.4) and 0.05% Tween 20, the membranes were incubated for 45 minutes with antirabbit HRP-coupled secondary antibodies (Jackson Immuno Research, West Grove, PA). The chemiluminescence reaction for horseradish peroxidase (HRP) was developed using self-made chemiluminescence reagent (100 mM Tris pH 8.0, 1.25 mM luminol, 0.2 mM coumaric acid, 0.006% hydrogen peroxide) and visualized with Stella 8300 bioimager (Raytest, Straubenhardt, Germany). The blots were stripped in 0.1 M glycine (pH 2.6) and reprobed with anti-actin-HRP conjugated rabbit polyconal antibody (Sigma) at 1:50,000 dilution for 45 minutes. Densitometric analysis was performed using ImageQuant 5.2 software (Amersham Bioscience, Piscataway, NJ).
Real-time Polymerase Chain Reaction
A total of 1 x 106 cells were washed twice with PBS, pelleted, and treated with 1 ml of TRIzol reagent (Invitrogen) to extract total RNA according to the manufacturer's protocol. RNA concentration was measured with Bio-Rad spectrophotometer. The first-strand complementary DNA synthesis containing 100 ng of total RNA was primed with oligo(dT) using Native AMV Reverse Transcriptase (EurX, Gdansk, Poland). The primers for GLUT1, GLUT2, GLUT3, GLUT4, and β2-microglobulin (B2M, reference gene) were custom designed (). Quantitative real-time polymerase chain reaction (RT-PCR) amplification reactions were performed in the LightCycler II 480 Real-time PCR System (Roche, Mannheim, Germany), using the LightCycler FastStart DNA Master SYBR Green I Kit (Roche). Data were analyzed using the LightCycler 480 Software 1.5 (Roche). The fold change for each gene relative to the reference gene (B2M) was calculated using user-noninfluent, second-derivative method. The specificity of all the reactions was confirmed through analysis of the PCR product's melting profile, obtained by dissociation of the DNA present after the amplification step. The primers and reaction parameters were validated and optimized according to the MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines. In addition, reaction specificity was also confirmed by agarose gel electrophoresis in which the PCR products were of expected size.
Primers Used for Quantitative RT-PCR.
Generation of Raji and HEK293T Cells Stably Expressing HA-GLUT1
A pCIS2 vector expressing human GLUT1 gene with an HA tag built into the first extracellular loop (hereafter called HA-GLUT1) was a generous gift from Dr Samuel W. Cushman from the National Institutes of Health, Baltimore, MD. The HA-GLUT1 gene was cloned into the pLVX-IRES-Puro vector (Clontech Laboratories, Inc, Mountain View, CA) enabling its lentiviral expression. Next, HEK293T cells were cotransfected with pLVX-HA-GLUT1-IRES-Puro, envelope (pMD2.G), and packaging (pPAX2) vectors using GeneJuice reagent (Calbiochem, San Diego, CA) as a DNA carrier. pMD2.G and pPAX2 plasmids were obtained from Prof Didier Trono (École polytechnique fédérale de Lausanne, Switzerland). Lentivirus-containing supernatants were collected 72 hours after transfection and added to the culture of Raji or HEK293T cells. Positive clones were selected with puromycin (Sigma) and evaluated with flow cytometry using anti-HA-PE antibodies as described previously.
Total Cellular Cholesterol Measurements
Cholesterol concentration was quantified in the Amplex Red cholesterol assay (Invitrogen) in whole-cell Raji lysates. Briefly, samples containing 2.5 x 105 cells were diluted in reaction buffer composed of 300 µM Amplex Red, 2 U/ml cholesterol oxidase, 2 U/ml cholesterol esterase, and 2 U/ml HRP. The samples were incubated at 37°C for 30 minutes in the dark, and fluorescence was measured at Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) equipped with 560-nm excitation and 590-nm emission filters. Cholesterol values were calculated according to the cholesterol standard curve and normalized to the protein content as measured with Bio-Rad Protein Assay (Bio-Rad).
G-actin to F-actin Evaluation
Determination of depolymerized (free globular—G) to polymerized (filamentous—F) actin was performed using a G-actin/F-actin in vivo assay kit (Cytoskeleton, Denver, CO) according to the manufacturer's protocol. Briefly, 5 x 105 cells were lysed in F-actin stabilization buffer supplemented with Complete protease inhibitor cocktail (Roche Diagnostics). Next, G-fraction was separated from F-fraction with ultra-centrifugation (100,000g for 1 hour at 37°C; Beckman Coulter). Supernatants (G-actin) and pellets (F-actin) were separated electrophoretically in 10% SDS-PAGE gel, blotted onto nitrocellulose membrane (Schleicher & Schuell BioScience), blocked with Tris-buffered saline (pH 7.4) and 0.05% Tween 20, and supplemented with 5% nonfat milk. For β-actin detection, HRP-coupled rabbit polyclonal antibody (Sigma) was used at 1:100,000 dilution for 1 hour of incubation at room temperature. The reaction was developed with homemade chemiluminescence reagent solution (100 mM Tris pH 8.0, 0.2 mM coumaric acid, 1.25 mM luminol [from Sigma-Aldrich], and 0.006% H2O2) using Stella bioimager (Raytest).
Membrane Protein Biotinylation
Control- and drug-treated cells (1 x 107 per each experimental group) washed twice with ice-cold PBS were surface-labeled with 2 mM disulfide-cleavable biotin (EZ-link sulfo-N-hydroxysuccinimido [NHS]-biotin [Thermo Scientific, Rockford, IL]) dissolved in biotinylation buffer (10 mM triethanolamine, 2 mM CaCl2, 150 mM NaCl, pH 7.5) for 30 minutes with gentle agitation on ice. The cells were next washed twice in PBS with 100 mM glycine followed by two washes with PBS only and lysed in lysis buffer (10% glycerol, 1.0% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM HEPES, pH 7.4) enriched with protease inhibitors cocktail Complete protease inhibitor cocktail (Roche Diagnostics). Next, samples containing equal amounts of total cellular proteins (300 µg) were incubated overnight at 4°C on a rotary wheel with 100 µl of immobilized NeutrAvidin agarose resin (Thermo Scientific)—50% slurry of—to separate the biotinylated surface proteins from nonbiotinylated intracellular proteins. Next, neutravidin-coated agarose beads were spun at 400g for 4 minutes, and the supernatant (nonbiotinylated fraction) was collected and used subsequently as a control of protein loading. The beads were washed thrice in a beads wash buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100) and boiled in Laemmli sample buffer for 10 minutes at 95°C with gentle shaking that releases biotinylated proteins from their binding with neutravidin. Membrane (biotinylated) and cytosolic proteins were subsequently analyzed with Western blot using anti-GLUT1 (1:1000 dilution; Millipore), anti-intercellular adhesion molecule 1 (ICAM, 1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) or anti-actin HRP-conjugated (1:50,000 dilution; Sigma) antibodies as described in the Western Blot Analysis section.
Total cholesterol concentration in serum of hypercholesterolemic patients on days 0, 3, and 21 of atorvastatin treatment was estimated using a standard cholesterol oxidase method with Cobas Integra 800 analyzer (Roche). Approval for the study was obtained from the institutional review board of the Medical University of Warsaw and was conducted according to the Declaration of Helsinki. Each patient signed a written informed consent for the procedures.
Leukocyte Isolation from Blood
Peripheral blood sample (5 ml) of patients was collected before (day 0), on the third day (day 3), or after 3 weeks (day 21) of atorvastatin (20 mg/day) treatment. The blood was diluted twice with PBS. Next, 3 ml of Histopaque-1077 (Sigma-Aldrich) was pipetted into conical centrifuge tubes. Ten milliliters of diluted peripheral blood was slowly layered on the top of Histopaque layer. Next, tubes were centrifuged (400g for 30 minutes at 21°C) without brake. The buffy coats were isolated and washed twice with PBS. The leukocyte pellet was resuspended in 3 ml of PBS and counted in a Brüker chamber using Türk dye. Peripheral blood leukocytes were used to determine 2-DOG uptake according to the protocol described in the Radioisotope measurement of glucose uptake section of the Materials and Methods. Approval for the study was obtained from the institutional review board of the Medical University of Warsaw and was conducted according to the Declaration of Helsinki. Each patient gave a written informed consent for the procedures.
Positron Emission Tomography/Computed Tomography
Whole-body PET/CT examinations in three-dimensional mode (2 minutes per bed position) were performed on PET/CT scanner Biograph 64 TruePoint (Siemens Medical Solutions, Erlangen, Germany). The PET studies were performed 55 to 65 minutes after injection of 350 to 360 MBq 18F-FDG. All patients were requested to drink 1.5 L of water-equivalent oral contrast dispersion. Immediately before the PET/CT examination, patients were requested to empty their bladder. Patients were positioned head first supine on the common patient handling system with the arms raised in according with standard CT practice. Coaxial whole-body imaging ranges were defined on the Topogram, covering the area from the skull to the upper thighs (2 minutes, six to seven PET bed positions depending on the size of the patient—one PET bed have approximately 15 cm). CT was performed in spiral mode using a continuous acquisition at 120 kV, 170 mAs, 2-mm slice width, and pitch of 0.8. Emission data were reconstructed with attenuation correction based on low-dose CT. PET emission data were reconstructed (reconstruction method—TrueX) using an attenuation-weighted approach on 168 x 168 matrices and with a 4-mm Gaussian postreconstruction filtering (two iterations, 14 subsets). The PET/CT images were assessed using Multimodality Work Station Syngo (TrueD) by Siemens. All pathologic foci of 18F-FDG uptake were included in the evaluation. Uptake in organs was evaluated assuming a uniform distribution in the organ of interest. Regions of interest (ROIs) were drawn around each foci. Maximum standardized uptake values (SUVmax) were calculated in all ROIs. CT data were used for allocation of regions with increased uptake of the radiopharmaceutical to specific morphologic structures. The study was approved by the ethical committee of Medical University of Warsaw. All patients have given an informed consent.