Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFκB Activity and Breast Cancer Stem Cells

The overall goals of this procedure are to demonstrate how the anti-inflammatory aspirin-fumarate prodrug GTCpFE is synthesized, and how it improves anti-NFkappaB and anti-cancer stem cell activity in breast cancer cells. This method can help answer key questions in the cancer therapeutics field by showing how we can repurpose or even improve anti-inflammatory drugs to target therapy resistant breast cancer stem cells. The main advantage of this technique is that we utilize the multidisciplinary approach to obtain and characterize the improved aspirin-fumarate prodrug.

Demonstrating the synthesis of the aspirin-fumarate prodrug GTCpFE will be Loruhama Delgado-Rivera, a graduate student in Dr.Gregory Thatcher's laboratory. Whereas the anti-NFkappaB activity of GTCpFE in breast cancer cells will be demonstrated by Gergana Georgieva, a pharmacy student researcher in Dr.Jonna Frasor's laboratory. Using a plastic plunger syringe, measure 0.81 milliliters of methanol and mix it in 10 milliliters of water in a round-bottom flask.

Cool the resulting mixture to zero degrees Celsius by placing the flask in an ice water bath. Add 2.48 milligrams of 4-hydroxybenzyl alcohol to the reaction mixture. Stir until the solution is clear.

Next, prepare a solution of O-acetylsalicyloyl chloride in anhydrous toluene by dissolving the solute in the solvent in a separate flask. Using a plastic plunger syringe add this solution to the 4-hydroxybenzyl alcohol mixture and leave the reaction stirring at zero degrees celsius. Monitor the reaction using thin-layer chromatography, or TLC, as described in the text protocol.

When the reaction is complete remove the ice water bath and allow the reaction to stir at room temperature for 20 hours. Filter the precipitate using a Buchner funnel with a fritted disc of medium porosity. Then, place the solid in a scintillation vial and leave the compound in vacuo overnight in a desiccator at room temperature.

Next, seal a thoroughly dried round-bottom flask with a septum and pierce it with a needle that is connected to a vacuum pump system. Inflate a balloon using argon gas and connect it to a plastic syringe with a needle. Insert the needle connected to the argon balloon through the septum sealing the round-bottom flask and remove the needle connected to the vacuum pump.

Then, add 4-hydroxymethyl-phenyl ester, of 2-acetyloxybenzoic acid, 4-dimethylaminopyridine and trimethylamine to a separate flask. Dissolve the chemicals in five milliliters of anhydrous tetrahydrofuran. With a plastic syringe attached to a needle add the solution to the sealed round-bottom flask.

Cool the mixture down to zero degrees Celsius by placing the flask in an ice water bath. To the previous mixture, add a solution of ethyl fumaroyl chloride and anhydrous tetrahydrofuran dropwise over a period of 10 minutes. Stir the resulting mixture for two to four hours monitoring the reaction by TLC as described in the protocol.

Extract the reaction mixture by adding it to a separatory funnel along with 100 milliliters of ethyl acetate and 50 milliliters of brine. Cap the funnel and shake it vigorously. Tilting the funnel to the side of the cap, slowly open the valve to allow air to escape.

Once the extraction is complete, remove the aqueous phase and repeat the extraction two more times. After removing the aqueous phase, dry the mixture by adding sodium sulfate to the organic phase until the solid does not clump up when the glassware is stirred. Using another Buchner funnel with a fritted disk filter the mixture to a round-bottom flask to remove the sodium sulfate.

Then, evaporate to dryness using a rotary evaporator with the temperature set at 40 degrees celsius. Next, prepare a column with silica gel and the appropriate solvent phase. Once the compound has been added, add a layer of sodium sulfate to protect the column as the solvent is added.

As the column runs, monitor the eluate by TLC. Spot every other test tube as they are collected from the column. When the product of interest dilutes, mix all the samples containing pure product into a large round-bottom flask.

Dry the products using a rotary evaporator with the bath temperature set at 40 degrees celsius. Perform cell culture and transfect the cells with DNA for the NFkappaB response element luciferase reporter assay as described in the text protocol. Then dissolve drug stock solutions of the GTCpFE or aspirin drugs in dimethyl sulfoxide at 1000x concentration.

After 16 hours of transfection and 1 microliter of vehicle or different drug stock solutions to each well. After adding the drugs, incubate the cells for two hours at 37 degrees Celsius. To activate the NFkappaB pathway, at the proinflammatory cytokine, TNFalpha, into each well for a final concentration of 10 nanograms per milliliter.

Also include a TNFalpha alone control. After incubating the cells for four hours at 37 degrees Celsius, aspirate the medium. Store the cells at minus 80 degrees Celsius.

Measure luciferase using a luciferase reporter assay system according the manufacturer's instructions. To perform the mammosphere assay, first prepare mammosphere medium as described in the text protocol. Next prepare singles cells of the MDA-MB-231 cell line by trypsin digestion of monolayer cultures and filter through mesh sieves.

After manually counting the single dissociated cells, plate them in 96 well ultra low attachment plates at a density of 400 cells per well. Then place the cells in the incubator at 37 degrees Celsius overnight. The next day, add different concentrations of GTCpFE to a final volume of 100 microliters.

Conduct every treatment in triplicate. After seven days of culture in the incubator at 37 degrees Celsius, acquire images via imaging software using an inverted microscope. Manually count the number of mammospheres greater than 75 microns in diameter.

To measure the CD44 high, CD24 low cancer stem cell immunophenotype, trypsonize MDA-MB-231 cells with 0.25%trypsin for five minutes at 37 degrees Celsius. After counting the cells using a hemocytometer, seed the cells at three million cells per dish in 10 milliliter of medium as described in the text protocol. Then add vehicle or GTCpFE to the cells and incubate at 37 degrees Celsius for 72 hours.

Following incubation trypsinize the cells and distribute one million trypsinized cells in five milliliter polystyrene tubes. The tubes should contain two milliliters of 1x HBSS buffer supplemented with 2%FBS and be labeled as test or control. To stain for the surface marker CD44 and CD24, spin the cells down, aspirate the buffer, then add 20 microliters of each conjugated antibody and 80 microliters of the HBSS buffer to test tubes for a final volume of 100 microliters.

Next, add 100 microliters of the same buffer to the control tubes. Also include CD44 APC-conjugated antibody and CD24 PE antibody single stained contols or the ITG immuno isotype controls. Incubate the cells in the dark at four degrees Celsius for 30 minutes.

Spin down the cells for five minutes at 400 times g and reconstitute them in 200 microliters of buffer. Keep the cells on ice and in the dark. FACS analysis can now be performed.

GTCpFE inhibits NFkappaB-RE luciferase activity and expression of NFkappaB target genes, such as Intercellular Adhesion Molecule 1, Chemokine C-C Motif Ligand 2, and Tumor Necrosis Factor in MCF-7 breast cancer cells. The calculated inhibitory concentration at 50%of both endpoints is approximately 20 micromolar GTCpFE. By comparison, 200 micromolar aspirin shows no inhibitory activity in breast cancer cells.

This indicates that the prodrug strategy of adding the Fumarate pharmacore to aspirin significantly improves it's anti-NFkappaB activity. GTCpFE inhibits mammosphere formation of MDA-MB-231 breast cancer cells in a dose dependent manner. Similar to NFkappaB pathway inhibition in adherent cultures, the IC50 value for mammosphere formation is approximately 20 micromolar.

Also measured was the population of cells expressing the CD44 high, CD24 low immunophenotype which is a bonafide cancer stem cell surface marker in breast cancer. GTCpFE pretreament resulted in a significant depletion of the CD44 high, CD24 low population in MDA-MB-231 cells. Together these results establish GTCpFE's ability to effectively target breast cancer stem cells.

After watching this video, you should have a good understanding of how the design and synthesize in aspirin fumarate prodrug and how to characterize its anti-NFkappaB and anti-cancer stem cell activity on breast cancer cells. We first had the idea for this method when aspirin failed to inhibit the NFkappaB pathway in breast cancer cells as it was previously reported in the literature. The implications of this technique extend toward anti-cancer stem cell therapy, because we showed that by targeting multiple proinflammatory pathways we can in fact, effectively eradicate breast cancer stem cells.

Although this method can provide insight into how to design and synthesize and characterize anti-inflammatory agents in breast cancer, this can be applied to other malignancies where multiple proinflammatory pathways are active and contribute to the pathology of the disease. Following this procedure, other methods such as the aldefluor assay and in vivo tumorigenicity can be utilized in order to asses the effect of the drug on breast cancer stem cells. The technique can pave the way for researchers in cancer therapeutics to explore prodrug design of antiinflammatory agents.

GTCpFE may serve as a prototype for developing new fumarate and aspirin based antiinflammatory agents. And this could also be the basis of a new class of anti-cancer stem cell agents.