3.1 Culturing of cell lines
A549, H460, H292 and H1437 human NSCLC cell lines were cultured to study KRAS-induced tumorigenesis and the role of GSTP1 in KRAS induced transformation. A549 (KRASG12D mutation, high KRAS activity) cells where cultured in DMEM/F12 ([+] L-Glutamine and [+] 15 mM Hepes) and H460 (KRASQ61H mutation, low KRAS activity), H292 and H1437 (no KRAS mutation) in RPMI 1640 ([+] L-Glutamine) (Life Technologies, Grand Island, NY) with 10% fetal bovine serum (Life Technologies), 50 U/ml Penicillin and 50 U/ml streptomycin (Life Technologies) in a humidified 37˚C, 5% CO2 incubator.
Human tumor tissue samples were obtained from adult donors undergoing biopsy surgery at the University of Vermont Medical Center. Tissues of these patients were treated with RNase and elastase to disrupt tumor tissue and obtain cells. The cell suspension was plated on collagen coated 35mm dishes and cultured in DMEM/F12 ([+] L-Glutamine and [+] 15 mM Hepes) ) in a humidified 37˚C, 5% CO2 incubator.
3.2 Cell treatment
For cell experiments, a cell density of either 1x104, 5x104 2x105 per 35 mm cell culture dish (Falcon) was used. For TLK treatment cells were cultured during 24 hours with a retreatment of TLK-117 6 hours prior to the harvest. Dependent on the experiment, cells were incubated with the GSTP1 inhibitor 117, which was dissolved in RPMI 1640 or DMEM/F12 medium with 0,02% DMSO. TLK-117 is made in house (Department of Pathology and Laboratory Medicine, Janssen-Heiniger lab, UVM) following the protocol described by Anathy et al. [35]. Three different TLK-117 concentrations were used, 50µM, 100µM and 200µM.
3.3 Animals
For animal experiments eight-week-old C57B/6NJ mice were used. CCSP-rtTA/tetON-Cremice (University of Vermont, Burlington, VT) and LoxP-Stop-LoxP/KRASG12D mice (The Jackson Laboratory, Bar Harbor, ME) were bread in house. Oncogenic KRASG12D expression, specific in lung epithelial cells was achieved by breeding mice expressing CCSPrtTA/tetON-Cre with mice that express the LoxP-Stop-LoxP system and the KRASG12D oncogene (fig. 5). You can find more
information about the mechanism in the appendix 2. Eight-week-old triple and double transgenic mice expressing either the CCSPrtTA/tetON-Cre/LSL-KRASG12D or CCSPrtTA/tetON-Cre/KRASWT were fed food with 6g/kg Dox (Purina Diet Tech, St. Louis, MO, USA). Double transgenic mice expressing KRASWT were used as controls.
One group of the double and triple transgenic mice were treated (every three day’s) with TLK-117 the other group with the vehicle as control, starting 14 days after the first administration of Adenovirus expressing Cre recombinase (described below). The Institutional Animal Care and Use Committee (IACUC) at the University of Vermont approved each animal experiment for this study.
3.4 Microsprayer Instillation of Adenovirus Cre
We also induced KRAS mediated tumorigenesis in mice lacking either the CCSP rTetA or the Tet-OpCre transgenes. In these mice we introduce the Adenovirus expressing Cre recombinase. Like in the triple transgenic mice, these mice will express the KRASG12D gene due to the presents of the LoxP-Stop-LoxP sequence that gets terminated due to the presence of the Cre recombinase. We administred the Aenovirus Cre (AdCre) by micro sprayer instillation (Vector BioLabs, Malvern, PA), LSL-KRASG12D and KRASWT expressing mice were anesthetized as described above. Tongues were retracted, and an otoscope with a specula and light source was gently maneuvered into place to make the trachea clearly visible. Then A Microsprayer (Penn Century) was placed into the trachea to deliver 50 µL AdCre (1x108pfu/mouse) aerosolized directly into the trachea and subsequently the lungs. The instruments were then removed; the mice were then briefly rotated and allowed to take 3 deep breaths in the left and right lateral decubitus position. Mice recovered from anesthesia within 30 to 60 seconds, and were monitored periodically thereafter.
Figure 5 Triple transgenic CCSP-rtTA/tetON-Cre LSL-KRASG12D mouse.
3.5 Oropharyngeal Instillation of TLK-117
To anesthetize experimental mice, mice were placed in a Plexiglas induction chamber and anesthetized with 5% isoflurane. Once breathing slowed down to a rate less than one breath per second, mice were extracted from the chamber and positioned on a 60° incline board. Their tongues were retracted, and 50 µl of solution was delivered into the distal part of the oropharynx. The tongue was kept in a retracted position to prevent swallowing, until the fluid had been fully aspirated. Mice were then briefly rotated and allowed to take 3 deep breaths in the left and right lateral decubitus position. Mice recovered from anesthesia within 30 to 60 seconds, and were monitored periodically thereafter. Agents to be delivered were 25 mg/kg TLK-117 (in 1,5M Tris-HCl and 0,02% DMSO) and vehicle (in 1,5M Tris-HCl 0,02 % DMSO) as a control.
3.6 Lung harvest and bronchoalveolar lavage
Mice were anesthetized by an IP injection with 0.1 ml Pentobarbital (stock 50 µg/ml). Upon effect where they are immobile and the heart rate has stopped, a cervical dislocation was performed as a secondary method. A with ethanol dampened gauze was used to wipe the fur on and around future cut area. An incision was made in the skin above the trachea. Afterwards the trachea was exposed, and freed from surrounding fascia. The trachea was than raised with forceps and a 4cm long suture was placed underneath it. After this a tiny cut was made, at the top of trachea. The suture was tide around the cannula, to assure sure that the cannula stays in place. A syringe filled with PBS was inserted into the cannula. The lung of the mouse is slowly filled up with 1 ml sterile PBS (Life Technologies) and the chest was gentle massaged to ensure that a fair amount of cells were collected. Next the just inserted PBS was slowly pulled out back into the syringe with an total end volume of 0.7-0.8 ml. The syringe was carefully removed and the cannula without removing it from the trachea. The cell suspension is stored on ice or in the -80˚C. The rib cage is opened up, to harvest the lung lobes. For this an incision a long the sternum was made and the rib cage was opened just above the trachea. Ribs were grabbed with forceps and separate, to open up the rib cage. Cutting through the bone above the trachea exposed the rest of the trachea. A cut was made above the suture and the lung was gently lifted up by the trachea. Fine tip scissors were placed flat underneath the trachea and the lung dissection finished by cutting out the lobes and all attached tissue around the trachea. A new syringe was inserted in the cannula to inflate the lung, to ensure that the lung wasn’t punctured during the procedure. Lung lobes
where separated from each other by sutures. Holding the suture ends, each lobe was cut off carefully.
The Lobes were afterwords embedded in paraffin for histological analysis.
3.7 GSH-Immunoprecipitation
S- glutathionylated proteins were immuno-precipitated (IP) using an anti-glutathione (GSH) antibody (Virogen, Watertown, MA) followed by Western Blotting of PKM2 (Cell Signaling, Baverly, MA). For this procedure cells were harvested using the GSH IP lysis buffer. This buffer contains: 50 mM Tris-HCl (pH = 7.4), 150 mM NaCl, 1% NP-40, 0.2% SDS, 0.2% CHAPS, 20 mM N-ethylmaleimide (NEM), 100 uM DTPA, 100 uM Neocuproine, 1% Protease inhibitors, 1% Phosphatase inhibitors (Sigma- Aldrich Co.). Cells were plated at a density of 2x105 cells and harvested after a 24-hour treatment with 50µM TLK-117. Protein concentration was determined and a total of 200 µg protein was used for the IP procedure. As a control one protein sample (200µg of protein) was incubated with DTT (final concentration of 50mM) (Sigma- Aldrich Co.), for 1 hour at 4 ˚C. IP- samples were over night incubated with 2 µl GSH primary antibody at 4˚C. In the next step Protein G Agarose beads (50-60 µl beads for each sample), were prepared by washing and centrifugation at 20,000xg for 30s. GSH IP lysis buffer was subsequently added; this step was repeated for a total of 3 times. After the preparation, beads were added to each sample tube. Sample tubes than were incubated for 2 hours on a rotator at 4˚C and centrifuged at 14,000 xg for 30 sec. Supernatant was aspirated and 500 µl GSH IP lysis buffer was added, the samples were spun down and the supernatant was removed, this was repeated once. Samples were prepared for SDS-Page and western blotting. Therefore 20 µl of GSH IP lysis buffer and 7µl of 4x leamli buffer were added to the protein bound beads. Cell lysate samples were separated by SDS-Page for Western blotting. Therefore a Bio-Rad Mini-PROTEAN®
Electrophoresis System (Bio-Rad) was used. Proteins were transferred on a PVDF membrane for 1 hour at 100 V. To identify the impact of TLK-117 on S- glutathionylation of PKM2, the PKM2 specific primary antibody, was used. A secondary peroxidase labeled rabbit antibody (GE, Pittsburg, PA, USA) was used for detection. The antibody was visualized by enhanced chemiluminescence with LumiGLO chemiluminescent substrates (Thermo Scientific Inc.)
3.8 Protein Harvest and analysis (whole cell lysate)
For protein experiments cells were plated at a density of 2x105 cells per 35 mm cell culture dish (Falcon by Fisher Scientific). Cells were treated with TLK-117 or transfected with GSTP1 siRNA
dependent on the experiment. For the protein harvest a whole cell lysate extract lysis buffer was used.
This buffer contains: 20 mM tris pH 7.4, 150 mM NaCl, 1 % Nonidet (NP-40), ddH2O, 1mM DTT (Sigma- Aldrich Co, St. Louis, MO, USA), Phosphatase inhibitor cocktail and a Protease inhibitor cocktail. For each 35 mm dish 200µl of lysis buffer was added and cells were scraped with a cell scraper (Sarstedt, Newton, NC, USA). Lysates were transferred to eppendorf tupes (Eppendorf, Hauppauge, NY, USA). After a 30-minute incubation on ice, cells were centrifuged for 30 minutes by 14000 rpm at 4˚C. After the spin 150 µl of the supernatant was transferred to a new tube and 50 µl of 4x laemli buffer was added. To assess protein concentration a detergent compatible protein assay was used (Bio-Rad, Hercules, CA, USA), following the manufacturer’s instructions.
3.9 Lung tissue homogenization and Western blotting
Lung tissues for the protein analysis were prepared by mincing lung tissue in liquid nitrogen to powder. Lung powder was added into the cold RIPA buffer immediately followed by freeze- thawing samples at -80˚C. RIPA buffer contains: 20mM Tris-HCl (pH=7,4), 130mM NaCl, 1mM EDTA, 1% NP-40, 0.05% Natriumdioxycholate and 0,1% SDS. After the freeze-thaw step, lung lysates were centrifuged for 30 min at 14,000 g. A portion of the supernatant was saved for protein determination, before the addition of Laemli sample buffer. Total protein, was assessed by the Bio-Rad DC Protein Assay kit (Bio-Rad). Total G6PD, PFKP, HKI, HKII, PKM2, PFKFB3, GSTP1 and β-actin protein abundance was evaluated by Western blotting.
3.10 Lactate assay
For the lactate assay cells were plated at a density of 2x105 cells and harvested after a 24-hour treatment with 50µM TLK-117. To determine intracellular and extracellular lactate in those cells, 500µl cell culture medium was collected and cells were washed wit PBS. After the washing step, 200µl RIPA buffer was added to the cell culture dishes and the scraped cells were transferred to eppendorf tubes.
In the next step 200 µl of cell culture medium and/or cell lysates were transferred to Amicon Ultra -0.5 ml centrifugal filters with a molecular weight cut-off of 10 kD (EMD Millipore, Billerica, MA, USA).
Samples were centrifuged at 10000xg for 30 min. Supernatant was collected to perform the lactate assay using the L-Lactate Assay Kit II (Eton Biosciences, Charlestown, MA, USA), following the manufacturer’s instructions.
3.11 GST-activity assay
To assess GST activity in A549 and H460 cell samples, cells were either treated with TLK-117 or transfected with GSTP1 siRNA. Cells were plated at a density of 2x105 cells and harvested after at the indicated time points. Cells were harvested by washing cells twice with PBS and scraping them by adding 200µl GSH lysis buffer consisting of 100mM Potassium Phosphate Monobasic, 100mM EDTA, 1 mM GSH (added fresh prior to experimental use), pH 6.5 (Sigma- Aldrich Co.). Samples were centrifuged at 10,000x g for 15 min at 4°C. The supernatant was collected and use for the assay.
Before starting the procedure protein concentration of the cell samples is determined. Samples were prepared in a final volume of 100uL with GST Assay buffer (for duplicate) including a blank sample containing GST Assay buffer alone. GSH was added to each sample at a final concentration of 5 mM.
To detect GST activity a CDNB substrate mix was used. This substrate mix, which consists of 1 mM CDNB in GST Assay buffer (+GSH), was added to each 50µl sample, to initiate the reaction of the GST enzyme with the CDNB substrate. The assay was carried out in a 96 well plate (Bio-Rad), reading the absorbance once every minute at 340 nm using a plate reader to obtain at least 10 time points.
3.12 Migration/Invasion assays
To examine cell migration and determine whether TLK-117 can affect growth and migration a Scratch assays was carried out. For the Scratch assay cells were plated at a density of 2x105 cells per dish. Confluent A549, H460, H292, H1437 and HLCB1 cell monolayers were scratched using a sterile 200µl pipette tip, and the closure of the scratched areas were monitored for at least 72 hours, dependent on the closure rate. Scratches were analyzed by microscopy. For a better visualization of the scratchs, cells were stained with the May Grunewald- Giemsa staining (Sigma- Aldrich Co
3.13 Quantification of gene expression by real-time quantitative reverse transcriptase PCR
To assess gene expression in CCSPrtTA/tetON-Cre/LSL-KRASG12D (KRASG12D) or CCSPrtTA/tetON-Cre/KRASWt (KRASWt) mice lung lobes were harvested and RNA was isolated with the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. cDNA was synthesized from 1 ug of RNA using oligo dT-primers and M-MLV Reverse Transctiptase (Promega BioSciences, LLC, San Luis Obispo, CA, USA). Quantitative PCR using SYBR Green
(Bio-Rad, Hercules, CA) was carries out, to assess gene PKM2, H6PD, G6PDX, HKII, HKIII, Glut1, Glut2, Glut3, PFKF, PFKL, LDHA, LDHB PDHKI and PFKFB3 mRNA expression. The Quantitative PCR
experiments were performed on the C1000 Thermal Cycler (CFX96 real-time system) (Bio-Rad). The following PCR program was used: Pre-incubation for 3 minutes at 95 °C followed by 40 cycles of denaturation at 95 °C for 10 seconds, annealing at 50 °C for 10 seconds and amplification at 72 °C for 30 seconds. Followed by a melting curve at 95 °C for 10 seconds 65 °C for 5 seconds and 95˚C for 5 sec. Primer sets used in this study are listed in the appendix. Expression values were normalized to the housekeeping gene beta-actin and calculated with the ddCt method.
3.14 Assessment of cancer development and tumor growth
Lungs harvested form mice were inflated to 25 cm H2O and fixated with 4% paraformaldehyde in PBS, followed by paraffin imbedding. Tissue blocks were cut into 5-µm sections and mounted to slides. Tissue histopathology and GSTP1 levels were assessed by a Hematoxylin and Eosin staining and immunohistochemistry staining (IHC) for GSTP1 and protein s-glutathionylation (pssg-staining).
3.15 GSTP1 immunohistochemistry
GSTP1 staining was performed on harvested lung sections after antigen retrieval during an 20-minute incubation in 0.01 M sodium citrate (pH 6.0) at 95˚C. Tissue slides were blocked with normal goat serum (Vectastein ABC-AP Kit, Vector laboratories, Burlingame, CA, USA) for 30 minutes accompanied by over night incubation with GSTP1 monoclonal rabbit antibody against GSTP1 (1:1000 dilution) at 4˚C. Biotinylated universal antibody was added and slides were incubated for 30 min at room temperature. Additionally slides were incubated with avidin-biotin complex-alkaline phosphatase (Vector Laboratories) for another 30 min at room temperature. Following slides were rinsed in distilled water and the substrate Vector Red (Vector Laboratories) was added and incubated for 20 minutes. After a color change in tissue to an intense red, slides were counter stained with Mayer’s hematoxylin. Tissue slides were mounted and cover slipped. Images were acquired on a EVOS xl Core microscope (Life Technologies) and analyzed using a 10x magnification.
3.16 Immunofluorescence staining for S-Glutathionylation
To determine protein s-glutathionylation in human lung tumor and normal tissue, 106 TMA’s obtained from biopsies performed in the University of Vermont Medical Center, were stained with the
pssg-staining. Tissue blocks were cut into 5-µm sections and mounted to slides and prior to the staining deparafinized. To deglutathionylate and label S-glutathionylated proteins, free thiol groups were blocked with 40mM NEM in HEM (HEN (25mM Hepes, 0.1mM EDTA, 0.01mM Neocuproine) and 1 M NEM in DMSO), with 1% Triton and HRP Streptavidin (dilution 1:2000), for 1 hour on room
temperature. After blocking, slides were washed three times for 5 minutes with PBS. The tissues were deglutathionylated by adding the GRX reaction mix (137 mM Tris-HCl pH=7.5 (Sigma), 0.1875 mM EDTA pH= 8.0 (Sigma), 50 mM NaCl (Sigma) and 100mM stocks of GSH and NADPH reduced (Sigma), to prepare the reaction mix 10 µl/ml of these stock solution was added). Slides were washed three times for 5 minutes with PBS. The newly exposed thiol groups were labeled with 1mM MBP in HEN buffer for 1 hour at room temperature. Slides were washed again 3 times for 5 minutes is PBS.
Streptavidin conjugated Alexaflour-647 (diluted 1:400 with PBS) was added and slides were incubated at room temperature for 1 hour. After a 1-hour incubation slides were washed with PBS as described before. The tissues were counter stained with DAPi (diluted 1:4000 in PBS) for 10 minutes at room temperature. Tissue slides were washed, mounted and cover slipped. Images were acquired on a Zeiss CSM 510 META Laser scanning Imaging System microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA) and analyzed using a 10x magnification.
3.17 Statistical analysis
Statistical analyses were performed using Graphpad Prism software (GraphPad, San Diego, Ca, USA) using ONE-Way ANOVA and the student T-test for multiple comparisons. All experiments were conducted at least two times and data is presented as mean values plus the standard error of the mean.