Materials and Methods

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Chapter 3

Materials and Methods

3.1. Materials

3.1.1 Materials used

All the compounds used were solubilized in the appropriate solvent (DMSO or EtOH). Molecules tested during my work of thesis were synthesized in Pisa – Department of Pharmacy, from the research group of prof.ssa Clementina Manera, while all the not radiolabeled compounds (2-AG, AEA, AA, 2-OG, WIN 55,212-2, AM-630, SR141716A, SR144528, CP-55,940, JZL-184, MAFP, URB-597, KT-172, Orlistat, WW70, UCM-707 and OMDM-2) were purchased from Cayman Chemicals (Ann Arboe, MI, USA).

[Ethanolamine-1-3H]-anandamide (3H-AEA) (40–60 Ci/mmol) and [1,2,3-3 H]-2-arachidonyl glycerol (20–40 Ci/mmol) were obtained from American Radiolabeled Chemicals Inc. (Saint Louis, MO, USA), while the radioligands [35S]GTPγS (1250 Ci/mmol) and [3H]-CP-55 940 (168 Ci/mmol) were obtained from PerkinElmer Life Sciences (Waltham, MA, USA). Scintillation liquid and Tri-Carb 2100 TR scintillation counter were obtained from PerkinElmer Life Science as well. Other substances, such as PBS (Phosphate Buffer Saline) and BSA (Bovine Serum Albumin) were purchased from Sigma-Aldrich, Saint Louis, MO, USA.

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During the period of my thesis I cultured and used two different type of cell lines: Chinese Hamster Ovary cells (CHO-cells) and Human Leukemic monocytic lymphoma U937 cells (U937 cells). Murine microglial cell line BV-2 homogenate and pig brain homogenate were kindly provided from other members of the laboratory.

CHO-Cells: Chinese Hamster Ovary cells are a laboratory-cultured cell line derived

from the ovaries of Chinese hamsters, commonly used in biology and in medical and pharmaceutical research. In 1957, T. Puck obtained a Chinese hamster from a lab at the Boston Cancer Research Foundation, where he used it to derive the original CHO cell line. Since then, CHO cells have been found useful for many research purposes. The shape appears elongated under the microscope and the cells grow attached to the flask, due to this was necessary a pre-treatment with either a scraper or Trypsin treatment to detach them. hCB1r- or hCB2r-expressing CHO-K1 cells (Raduner et

al., 2006) were purchased from American Type Culture Collection (Manassas, VA,

USA) and were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 g/mL fungizone (amphotericin B), 100 units/mL penicillin, 100 g/mL streptomycin, 2 mM L-glutamine and supplemented with 400 mg/mL G-418 (all from Sigma-Aldrich, Saint Louis, MO, USA). Cells were grown in a humidified incubator at 37°C and 5% CO2.

Human Leukemia monocytic lymphoma U937 cells: they were isolated from

the histiocytic lymphoma of a 37-year-old male patient and represent an important cell line to study the behavior and differentiation of monocytes. The shape is rounded and, differently to CHO-cells, they grow in solution therefore was not necessary any treatment to detach them. Human leukemia monocytic lymphoma U937 cells were purchased from American Type Culture Collection (Manassas, VA, USA) and were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 g/mL

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fungizone (amphotericin B), 100 units/mL penicillin, 100 g/mL streptomycin and 2 mM L-glutamine (all from Sigma-Aldrich, Saint Louis, MO, USA). Cells were grown in a humidified incubator at 37°C and 5% CO2.

a b

Fig. 3.1 CHO-cells (a) and U937 cells (b) pictures captured under the microscope

3.2. Methods

3.2.1 Defrost and maintenance of cell culture

Defrost is necessary to bring back the cells in an active metabolic condition after they have been maintained under liquid nitrogen for a certain period. The Eppendorf tube is immediately placed at 37°C in water-bath and after a while everything is moved in a sterile 15 mL vial, in which 5-6 mL of 37°C medium were previously added inside. The entire volume is mixed with the pipette (up and down) and then the vial is centrifuged for 5 min. at 1100 rpm. The supernatant is removed and the pellet is resuspended in few mL of complete medium. Then the suspension is moved into a

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flask and the medium is filled up to the final volume. At last, the flask is placed inside the incubator (37°C, 5% CO2).

The cell line is kept in exponential phase of growth in flask T75 inside the incubator. Cells are checked every day: the medium, that contains the metabolism discards, is a perfect indicator of cell line health and condition, indeed appears in red when is completely empty of metabolites and changes colour (yellow/orange) as a result of changing of pH after accumulation of debrides and discard materials. When the flask appears like that, it means that old medium need to be replaced from fresh.

When cells reach a 70-80% of confluence, a further increase could cause a multi-layer stratification. In order to avoid this situation, the old medium is discarded and the surface of the flask is washed with few mL of either PBS or new medium. After that, cells are detached with either a scraper or a trypsin treatment and are collected in 5-6 mL of new medium (in case of Trypsin treatment, 1 mL of Trypsin-EDTA diluted with 1 mL of PBS are added and the flask is kept into the incubator for 5 min. After the incubation time 3-4 mL of new medium are added to inactivate the Trypsin and everything is moved into a 15 mL vial and centrifuged for 5 min. at 1100 rpm. The supernatant is discarded and the pellet is resuspended in new medium). In order to reduce the confluence the entire suspension can be split in two new different flasks T75 or just few mL can be added to a new flask, whereas the rest of the suspension is discarded. Sometimes may be useful to wash the pellet with PBS before re-suspend it in new fresh medium.

If the cells are not attached to the flask (U937 cell line), the old medium is required and replaced after centrifugation with fresh one. In this case it is particularly important to wash the pellet with PBS.

3.2.2 CB1r/CB2r membranes preparation

Membranes were obtained from CHO-Cells overexpressing human recombinant CB1r and CB2r and cultured in the laboratory. Pellets preserved in -20°C fridge were put in ice and 20 mL of Homogenization buffer were added inside the vial (250 mM sucrose, 1mM EDTA, 10 mM Tris-HCl, pH 7.2 plus protease inhibitors), and the

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pellet was resuspended. Afterwards the pellet was homogenized with the homogenizer (Politron 3000) for 30-45 seconds at maximum speed and after that the vial was left for 5 minutes in the sonicator (at cold temperature) in order to help the desegregation of the cell material. The vial was centrifuged at 4 °C and 500 rpm for 10 minutes and the supernatant placed in a new vial kept in ice, whereas the pellet discarded (it is possible to repeat this procedure using the discarding pellet in order to increase the final amount of membranes). The supernatant was split in ultra-centrifuged vials taking care of reaching the same weight in all the vials (otherwise just buffer is added). The vials were put into the rotor (TFT 65.13) and the machine was set (4 °C and 34000 rpm for 1 hour). Afterwards the vials were put in ice again and the homogenization buffer replaced with PBS in order to wash the membranes (the ultra-centrifuge procedure was repeated with the same setting and the PBS discarded at the end of the process). The membranes were removed from the wall of the vial and resuspended in Tris-HCl 50 mM inside an Eppendorf tube, eventually using a needle with the syringe to simplify the solubilization. The last step is the quantification of membranes.

3.2.3 Quantification of membranes with method of Biureto

Membranes quantification was performed with method of Biureto. This method is based on the interaction between an alkaline solution of CuSO4 and tartrate (to keep

Cu2+ in solution) with peptides. When Cu2+ binds peptides appears a coloration purple-blue and the intensity is linked with the amount of peptides.

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Fig 3.2. Application of method of Biureto: the purple solution means that peptides are

present.

First of all, a concentration of 2 mg/mL of BSA (Bovine Albumin Serum) in Tris-HCl was prepared and diluted eight times in order to have a linear decrease of the concentration, with the last one in presence of TRIS-HCl alone (Blank). The Working Reagent was prepared mixing 50 parts of Reagent A with 1 part of Reagent B (directly from the kit). 25 µL of each protein solution, included the unknown membranes as well, were added in the 96 well-plate in duplicate, with 200 µL of Working Reagent. The 96 well-plate was kept shaking for 10 minutes and then incubated at 37°C for 30 minutes. Absorbance was read at 540 nm and BSA values were used to draw a reference straight line.

3.2.4 [3H]CP-55,940 competition binding Assay

Binding procedure was performed using 20 µg of CB1r/CB2r membranes generated in laboratory (see above) per sample in 300 µL of assay buffer (50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl2, 0.5% fatty acid free BSA, pH 7.4). 3 µL of ligand or

control were added (finally 1% of solvent), and (R)-WIN55,212 [10 µM] was used to determine the nonspecific bound. Immediately afterwards [3H]CP-55,940 [0,5 nM] was added and samples were shaken with the vortex before insert them into the

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heating block for 90-120 minutes at 30°C. Simultaneously, 100 µL of PEI (poly ethyleneimine) solution (0,5 g dissolved in 100 mL di-distillated H20) were added to

each well of the GF/B filter-plate and incubated for 90-120 minutes. After the incubation time, the plate was put on the vacuum and washed 2x with 150 µL assay buffer. Samples were loaded onto the filter and each well was washed 12x with 150 µL with assay buffer. The plate was dried at room temperature or under a drier flow. Next, a transparent foil was attached on the bottom and 45 µL of MicroScint20 were added, than the top was closed with another foil as well. Plate was kept shaking for 20 minutes and then the radioactivity signal was detected (cpm).

Results were analyzed with GraphPad Prism 5® and expressed as bound of [3 H]CP-55,940 (% of vehicle control). IC50 values and Confidence Intervals were

standardized with Cheng-Prusoff equation: Ki = IC50 / 1 + [L] / Kd.

3.2.5 [35S]GTPγS binding assay

[35S]GTPγS binding was performed using 5 µg of in house generated CB1r/CB2r membranes per sample in 200 µL of assay buffer (50 mM Tris-HCl, 0.2 mM EGTA, 3 mM MgCl2, 100 mM NaCl, 0.5% fatty acid free BSA, pH 7.4). In a vial were

mixed the totality of membranes, assay buffer, GDP [10 µM] (guanosine diphosphate) and hot GTP [0.1 nM] – guanosine triphosphate radiolabeled. The concentration of hot GTP was increased after three months, corresponding at the decay half-live of [35S]. The solution was split in silanized vials and kept in ice until the binding reaction was started by adding tested compounds, vehicle and controls. Nonspecific bound was measured in presence of cold GTPγS [10 µM] (not radiolabeled). Tubes were shaken and incubated at 30°C for 90 minutes (when an antagonist was added to compete with the ligand, the antagonist was always pre-incubated for 30 minutes at 30 °C before the other compounds). Afterwards, vials were exposed to a brief spin to pick down all the solvent condensed on the cap and immediately put in ice to stop the reaction. An aliquot of 185 µL of each sample was transfected on GF/B 96 well-plate, previously pre-soaked with ice-cold washing buffer (50 mM Tris-HCl, pH 7.4 and 0.1% fatty acid free BSA). Filters were washed

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6x with 185 µL of cold washing buffer and dried at room temperature or under drier flow. Next, a transparent foil was attached on the bottom and 45 µL of MicroScint20 were added, than the top was closed with another foil as well. The plate was kept shaking for 20 minutes and the radioactivity signal was detected (cpm). Results were analyzed with GraphPad Prism 5® and expressed as bound of [35S]GTPγS (% of vehicle control).

3.2.6 MAGL activity inhibition assay

Enzyme activity inhibition was conducted in silanized tubes using 100 µg of pig brain homogenate per sample in 200 µL of assay buffer (10 mM Tris HCl, 1 mM EDTA, 0.1 % BSA, pH 8.0). The final quantity of brain homogenate and buffer were mixed in a single vial and split in tubes. 2 µL of tested compounds (1% solvent) or vehicle were added and tubes were pre-incubated for 30-45 minutes at 37 °C.

Inhibitors JZL-184 [10 µM] and Methylarachidonylfluorophosphonate – MAFP [10 µM] were used as positive controls. JZL-184 was used in presence of URB-597 [1 µM] to minimize the effect of other endocannabinoid enzymes in the reaction matrix. This trick was not necessary for MAFP considering the lower selectivity for MAGL compared with the other endocannabinoid enzymes. Hot 2-OG [0,250 µM] mixed with cold 2-OG [1 µM] were added in the tubes and incubated for 15 minutes, at 37°C and 450 rpm. The reaction was stopped with 400 µL of ice-cold mix chloroform-methanol (1:1 v/v) and then the tubes were shaken and centrifuged for 10 minutes, at 0 °C and 10'000 rpm. The aqueous phase (the upper phase) was collected in scintillation vials (200 µL aliquot) and 3 ml of scintillation liquid were added in each vial. After shaking time, signal was detected with TRICARB 2100TR scintillation counter and results were analyzed with GraphPad Prism 5® and expressed as [3H]2-OG hydrolysis (% of vehicle).

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Enzyme activity inhibition was conducted in silanized tubes using 100µg of BV2 cell line homogenate per sample in 200 µL of assay buffer (10 mM Tris HCl, 1 mM EDTA, 0.1 % BSA, pH 8.0). The final quantity of brain homogenate and buffer were mixed in a single vial and split in the tubes. 2 µL of tested compounds (1% solvent) or vehicle were added and tubes were pre-incubated for 30-45 minutes at 37 °C. KT-172 [1 µM], Orlistat [20 µM], WW70 [10 µM], and the mixture Orlistat + WW70 were used as positive controls. URB-597 [1 µM] was added to each sample to inhibit the effect of FAAH. Hot 2-OG [0,250 µM] mixed with cold 2-OG [1 µM] were added in the tubes and incubated for 15 minutes, at 37 °C and 450 rpm. The reaction was stopped with 400 µL of ice-cold mix chloroform-methanol (1:1 v/v) and then tubes were shaken and centrifuged for 10 minutes, at 0 °C and 10'000 rpm. The aqueous phase (the upper phase) was collected in scintillation vials (200 µL aliquot) and 3 ml of scintillation liquid were added in each vial. After shaking time, signal was detected with TRICARB 2100TR scintillation counter and results were expressed as [3H]2-OG hydrolysis (% of vehicle).

3.2.8 FAAH activity inhibition assay

Enzyme activity inhibition was conducted in silanized tubes using 100 µg of mouse/pig brain homogenate per sample in 200 µL of assay buffer (10 mM Tris-HCl, 1 mM EDTA, 0.1 % BSA, pH 8.0). The final quantity of brain homogenate and buffer were mixed in a single vial and split in the tubes. 2 µL of tested compounds (1% solvent) or vehicle were added and tubes were pre-incubated for 30-45 minutes at 37 °C. Inhibitor URB-597 [1 µM] was used as positive control. Hot AEA [0,167 µM] mixed with cold AEA [144 µM] were added in the tubes and incubated for 15 minutes, at 37 °C and 450 rpm. The reaction was stopped with 400 µL of ice-cold mix chloroform-methanol (1:1 v/v) and then tubes were shaken and centrifuged for 10 minutes, at 0 °C and 10'000 rpm. The aqueous phase (the upper phase) was collected in scintillation vials (200 µL aliquot) and 3 ml of scintillation liquid were

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added in each vial. After shaking time, signal was detected with TRICARB 2100TR scintillation counter and results were analyzed with GraphPad Prism 5® and expressed as [3H]AEA hydrolysis (% of vehicle).

3.2.9 Selective COX-2 inhibition assay

A Greiner Bio One 384 plate was used for this assay. On the bottom of each well were added 37.5 µL of COX-2 analyze buffer (1 mM Tris-HCL, pH 8), 2.5 µL Heme [1 µM final concentration] and 2.5 µL of ADHP (10-acetyl-3,7-dihydroxyphenoxazine) obtained from the Kit (Cayman Chemicals, Ann Arbor, MI, USA). Then, 2.5 µL of inhibitor or vehicle were added and 2.5 µL of COX-2 run solution (diluted 1:12 of stock solution, obtained from the kit) were added as well (Cayman Chemicals, Ann Arbor, MI, USA). For the blank the 2.5 µL of inhibitor/vehicle were replaced from buffer. The plate was kept shacking for 15-20 min. (pre-incubation time) and after 2.5 µL of AA or 2-AG [10 µM in final concentration] were added to each well. Immediately the signal was detected with FarCyte (Fluorescence Polarization Exc. 535nm / 585 nM), cycling mode, with at least 10 cycles (Gain = 50; Z = 8083; CyclinG: with the lowest interval). Results were expressed as COX-2 activity (% of vehicle).

3.2.10 AEA uptake inhibition assay

Human U-937 leukemic cells were cultured and resuspended to cell density of 2x106 cells/mL in RPMI non complete at 37 °C. Experiment was carried in silanized tubes and 250 µL of cell suspension were split in each one (0.5 million of cells per sample), and pre-incubated for 30 minutes at 37 °C with 2.5 µL of tested compounds or vehicle. UCM-707 and/or OMDM-2 were used as positive controls at concentration of 10 µM. A mixture of hot AEA [0.5 nM] / cold AEA [99.5 nM] was added and immediately incubated for 5 minutes at 37 °C. Afterwards samples were loaded onto GF/C filter pre-soaked with 0.25 % BSA and washed 3x with 100 µL of

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PBS 0.1% BSA. The plate was dried with paper and/or drier flow and the bottom was covered with a transparent foil. Then, 45 µL MicroScint20 were added to each sample and the top closed with a foil as well. The plate was kept shaking for 20 minutes and then the radioactivity was detected. The signal was expressed as [3H]AEA uptake (% of vehicle).

3.2.11 2-AG uptake inhibition assay

The uptake of [1,2,3-3H]-2-arachidonyl glycerol in intact cells was performed using U937 cells. 106 cells were re-suspended in 500 mL of serum-free medium in silanized plastic tubes and pre-incubated with different concentrations of a-amyrin, b-amyrin or JZL-184 for 30 min at 37°C. Then, a mixture of 2-AG/[3H]-2-AG to a final concentration of 1 mM, was added to the cells and incubated for 5 min at 37°C. The uptake process was stopped by placing the tubes on ice and rapidly centrifuging them at 800 rpm for 5 min at 4°C. The supernatant was collected and transferred into 1 mL of a methanol : chloroform mixture 1:1 (v/v), while the pellet was re-suspended in ice-cold PBS plus 1% of fatty acid-free BSA and centrifuged at 800 rpm for 5 min at 4°C (washing step). The washing solution was discarded while the cell pellet was re-suspended in 250 mL of ice-cold PBS and transferred into 500 mL of a methanol : chloroform mixture 1:1 (v/v), vortexed vigorously, sonicated on ice-cold water bath for 5 min and finally centrifuged at 10 000 rpm for 10 min at 4°C. The aqueous phase was pooled with the aqueous phase extracted from the supernatant and transferred in a scintillation tube while the lipophilic phase was transferred in a separated tube. The radioactivity associated with the intracellular [3H]-2-AG or the hydrolysis product [3H]-glycerol was measured by adding 3 mL of scintillation liquid using a Tri-Carb 2100 TR scintillation counter. Results were expressed as [3 H]-glycerol formation (or [3H]-2-AG intracellular accumulation), relative to that in vehicle-treated samples (=100%).