Cell culture and electrical stimulation set up
Rat glioma C6 (Cat# CCL-107), prostate tumor (PC-3, Cat# CRL-1435), lung tumor (H1299 Cat# CRL-5803), fibrosarcoma (H1080 Cat# CRL-12012) and ovarian cancer (SKOV-3 Cat# HTB-77) cell lines were purchased from ATCC and characterized for MDR1 expression. Cells were initially expanded in Dulbecco's modified essential medium (DMEM-F12) supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS), 100 units of penicillin G sodium per ml, and 100 μg of streptomycin sulfate per ml. All cells were maintained at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air. The cells were seeded into pre-coated (3 μg/cm2 Poly-d-Lysine) 24 well plates with average seeding density being 1 × 104 cells/cm2 in every experiment. Well plates were engineered to accommodate stainless steel electrodes, which were connected to a computer controlled waveform generator via a programmable I/O board. We used a dedicated program to generate and monitor the electrical stimulation protocol. Cultures were stimulated for 3 days after the initial cell seeding. Media samples were taken on a daily basis and processed for AK measurements. Cellular growth was monitored every day by inspection with phase contrast microscopy.
Choice of the electrical stimulation paradigms
Cells undergoing stimulation protocols were exposed to 50 Hz AC (7.5 μA 32 cycles/pulse, 10 sec interval between pulses) for three days. The stimulation paradigms presented herein (frequency, intensity interstimulus interval) originate from a previous study [see Additional file 1] aimed at demonstrating the cytostatic effect promoted by the exposure to AC current of tumor cell lines. We shown that cells exposed to AC stimulation at 10 Hz, ≈ 10 μA for 2 to 5 days grew at a rate similar to non-stimulated controls. In contrast, stimulation at 25 – 100 Hz caused a pronounced decrease in cell proliferation as early as three days after stimulation. The effects persisted and amplified with prolonged exposure to electric pulses. However, while stimulation up to 50 Hz decreased cell number through a direct effect on cell cycle, at frequencies greater than 50 Hz and or intensities greater than 15 μA the effect on cell proliferation significantly overlapped with a cytotoxic one on both tumors and normal cells. In view of a possible clinical application of this novel approach, limitation of peripheral "normal" cells damage due to a possible exposure to the electric field was vital to our project and was as well an element of distinction from the classical electrochemical protocol.
Cell isolation, characterization and primary culture
MDR1 over-expressing astrocyte cultures were established from human cerebral cortical tissue of patients undergoing temporal lobectomies to relieve medically intractable seizures. Brain resections were collected in an ice-cold artificial cerebrospinal fluid solution bubbled with 5% CO2and 95% O2. This solution consisted of (in mM): 120 NaCl, 3.1 KCl, 3 MgCl2, 1 CaCl2, 1.25 KH2PO4, 26 NaHCO3, 10 dextrose. Briefly, tissue was homogenized for 20 min. at 37°C after gentle trituration and incubation in phosphate buffer saline (PBS) containing trypsin (0.2%)/DNase (1 mg/ml, Sigma-Aldrich, MO, USA). After centrifugation (200 g for 5 min.) and filtration through 70 μm nylon sieve, cells were seeded in appropriated poly-D-lysine coated flask. The culture medium consisted of Dulbecco's modified essential medium (DMEM) supplemented with 10% FBS and 2 mM glutamine, 100 U/ml penicillin G sodium and 100 μg/ml streptomycin sulfate. Immunological characterization was performed with rabbit polyclonal antibodies that recognize the glial marker GFAP (Dako Corporation, Carpentaria, CA, USA) and with human anti-P-Glycoprotein polyclonal antibody (1:100, Calbiochem-Novabiochem Corporation, San Diego, CA, USA) to assess for MDR1 expression. Normal astrocytes from ScienCell (Cat# 1810), were used as control (Normal Astro).
Current density calculation
The peak surface current density was calculated by an automated 2-D finite-elements approximation of the system. In the frequency range of interest, the system can be considered purely resistive due to the lack of inductive or capacitive effects, thus allowing the use of a simplified model. Each well or Petri dish was divided into 360 sub-elements, and Kirchhoff's laws applied according to: ∑I = 0 (for every node) and ∑V = 0 (for every closed loop). The resulting current, divided by the element area, allowed the calculation of the surface current density.
Adenylate Kinase measurement
Detection of cytotoxicity and cytolysis was assessed by measurement of AK release. Media samples were taken before and after the experiment. The measurements were performed by the use of the ToxiLight™ HS kit (Cambrex Bio Science Rockland, Inc.). The assays were conducted at ambient temperature (18–22°C) following the procedure described by the manufacturer. In brief, the assay method involves the release the AK into the surrounding matrix whenever cell damage occurs and the integrity of the plasma membrane is compromised. The AK enzyme following the introduction of an excess of ADP then generates ATP. Luciferase/luciferin is added to the sample, light is emitted in the presence of the ATP and the photon emission is measured using a luminometer.
MDR1 immunohistochemical detection and distribution
To investigate the expression of MDR1 protein and its localization, cells were cultured on poly-d-lysine pre-coated slides. The slides were positioned in the electrified well plate and stimulated for 3 days. Cells were fixed in 4% formaldehyde at room temperature for 20 minutes and then washed three times with 1× PBS. Blocking was performed at room temperature for 1 h with: 0.3% Triton-X, 3% bovine serum albumin, 3% normal goat serum and 1× TBS. The primary antibody used was Anti-P-Glycoprotein (C494) hamster and human (mouse) monoclonal (1:40; Calbiochem, San Diego, California). The secondary antibody used was Fluorescein (FITC)-conjugated affinipure donkey anti-mouse IgG (1:200; Jackson Immunoresearch Laboratories, West Grove, Pennsylvania). Cellular distribution of MDR1 was assessed with the use of a 35-mm camera mounted on a fluorescent microscope unit (Leica Leitz DM-RXE) and interfaced to a PC (using Qcapture software – Quantitative Imaging Company). The images were analyzed by Phoretix 2D Image Analysis Software. Data were further analyzed using Origin Lab 7 software. Experiments were performed in triplicate.
Doxorubicin uptake measurement, toxicity and cell viability
Parallel 24 well plate cultures (stimulated; pre-treated with the MDR1 blocker XR9576 [20 nM] and a control) were used in triplicates per each tumor cell type. Cells were exposed to different concentrations (1, 2, 4 and 8 μM) of doxorubicin for 3 hours. Media samples were collected before and after the exposure to doxorubicin to assess for AK release. Media containing doxorubicin was then washed out and replaced with cold media and kept at 4°C in order to inhibit MDR1 activity [16, 17]. Cells were then exposed to Calcein (Calcein AM Fluorogenic Esterase Substrate Cell Viability and Cytotoxicity kit -Invitrogen, Carlsbad – CA, USA) according to the manufacturer protocols and specifications to assess for cell viability. In the absence of efflux activity, free calcein accumulates within the cell resulting in a 100- to 500-fold increase in the intracellular concentration of the dye and bright fluorescence exhibited by living target cells. Cell viability and quantification of the intracellular levels of doxorubicin were assessed by measurement of fluorescence intensity and compared to parallel XR9576-treated cultures and controls. Data were analyzed by Phoretix 2D Image Analysis Software and Origin Lab 7. The final experiment with normal and MDR1 over-expressing astrocytes was performed by increasing the concentration of XR9576 to 1 μM.
Isolation of cellular fractions
Extraction of proteins from stimulated and control cell cultures were performed by the use of ProteoExtract Subcellular Proteome Extraction Kit (EMD Biosciences San Diego, CA Cat# 539790) designed for fast and reproducible extraction of subcellular proteomes from mammalian tissue and adherent and suspension-grown cells. In the specific case of adherent cells, the procedure is performed directly in the tissue culture dish without the need for cell removal. Cells or the parts of the cells remain attached to the plate during sequential extraction of subcellular compartments until the appropriate extraction reagent is used. Thus, the early destruction of the cellular structure by enzymatic or mechanical detachment of cells from the tissue culture plate and any mixing of different subcellular compartments is prevented. The assay was performed following the procedure described by the manufacturer. The stepwise extraction delivers four distinct protein fractions from one sample: 1) Cytosolic protein fraction; 2) Membrane/organelle protein fraction; 3) Nuclear protein fraction; 4) Cytoskeletal protein fraction. Proteins were obtained in the native state and processed for Western blot and protein analysis.
Western Blot Analysis
Proteins differentially extracted from stimulated and control cells were re-dissolved in RIPA buffer containing protease inhibitors (0.17 mg/mL PMSF, 2 μg/mL leupeptin, and 0.7 μg/mL aprotinin). Prior to electrophoresis, protein extracts were denatured by heating at 100°C for 5 minutes in a running buffer solution containing RIPA, β-mercaptoethanol, and bromophenol blue tracking dye. 15 μg proteins were loaded in each lane. Duplicate acrylamide gels (12%, precast gels; Bio-Rad Labs, Hercules, CA) were run for 2.5–3 hrs at constant voltage (80 V) until the bromophenol blue tracking dye migrated to the bottom edge of the gels. Proteins were then transferred onto a blot of PVDF using constant current (40 mA) overnight at 4°C. Proteins were probed overnight at 4°C with primary MDR1 mouse anti-human antibody (1:100; Calbiochem Clone C494, San Diego, CA). Blots were washed and treated with rabbit anti-mouse IgG HRP conjugated secondary antibody (1:5000; Dako Corp., Carpinteria, CA). To ensure that the same amount of total protein was electroblotted, PVDF membranes were incubated for 20 minutes at 37°C in a "stripping buffer" (Restore Western Blot Stripping Buffer, Pierce, Rockford, IL). Non-specific binding blocking was performed as described above; membranes were reprobed with monoclonal anti β-Actin antibody (1:10,000, Clone AC-15, Sigma-Aldrich, St. Louis, MO). Protein bands were analyzed by Phoretix 2D Image Analysis Software and Origin Lab 7.