Name: synthesis and characterization of an aspirin-fumarate prodrug that inhibits nf&#954;b activity and breast cancer stem cells
Uploaded: 2017-01-18T12:30:00
Description: Watch this Scientific Journal Video about Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFÃŽÂºB Activity and Breast Cancer Stem Cells at JoVE.com

This work was supported by grants provided by the National Institutes of Health (NIH), R01 CA200669 to JF and R01 CA121107 to GRJT, and by a postdoctoral fellowship grant from Susan G. Komen for the Cure to IK (PDF12229484).

1. Synthesis of Aspirin-fumarate Prodrug GTCpFE
<ol>
	<li>Using a plastic plunger syringe, measure 0.81 ml (20 mmol) of methanol and mix it in water (10 ml) in a round bottom flask. Cool the resulting mixture to 0 &#176;C by placing the flask in an ice-water bath. Add 4-hydroxybenzyl alcohol (2.48 mg, 20 mmol) and stir the reaction mixture until the solution is clear.</li>
	<li>Prepare a solution of O-acetylsalicyloyl chloride (3.77 mg, 19 mmol) in anhydrous toluene (10 ml) by weighing the desired amount of <e...

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+frequency+and+duration+of+aspirin+use+and+hormone+receptor+status+with+breast+cancer+risk.">Association of frequency and duration of aspirin use and hormone receptor status with breast cancer risk.</a> JAMA. 291, 2433-2440 (2004).</li><li>Wang, D., Dubois, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclooxygenase-2:+a+potential+target+in+breast+cancer.">Cyclooxygenase-2: a potential target in breast cancer.</a> Semin Oncol. 31, 64-73 (2004).</li><li>Howe, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+breast+cancer.+Cyclooxygenase/prostaglandin+signaling+and+breast+cancer.">Inflammation and breast cancer. Cyclooxygenase/prostaglandin signaling and breast cancer.</a> Breast Cancer Res. 9. 9, 210 (2007).</li><li>Kopp, E., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+NF-kappa+B+by+sodium+salicylate+and+aspirin.">Inhibition of NF-kappa B by sodium salicylate and aspirin.</a> Science. 265, 956-959 (1994).</li><li>Yin, M. J., Yamamoto, Y., Gaynor, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anti-inflammatory+agents+aspirin+and+salicylate+inhibit+the+activity+of+I(kappa)B+kinase-beta.">The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta.</a> Nature. 396, 77-80 (1998).</li><li>Pierce, J. W., Read, M. A., Ding, H., Luscinskas, F. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cancer+stem+cells+by+cytokine+networks:+attacking+cancer's+inflammatory+roots.">Regulation of cancer stem cells by cytokine networks: attacking cancer's inflammatory roots.</a> Clin Cancer Res. 17, 6125-6129 (2011).</li><li>Hinohara, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ErbB+receptor+tyrosine+kinase/NF-kappaB+signaling+controls+mammosphere+formation+in+human+breast+cancer.">ErbB receptor tyrosine kinase/NF-kappaB signaling controls mammosphere formation in human breast cancer.</a> Proc Natl Acad Sci U S A. 109, 6584-6589 (2012).</li><li>Kendellen, M. F., Bradford, J. W., Lawrence, C. L., Clark, K. S., Baldwin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canonical+and+non-canonical+NF-kappaB+signaling+promotes+breast+cancer+tumor-initiating+cells.">Canonical and non-canonical NF-kappaB signaling promotes breast cancer tumor-initiating cells.</a> Oncogene. 33, 1297-1305 (2014).</li><li>Yamamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB+non-cell-autonomously+regulates+cancer+stem+cell+populations+in+the+basal-like+breast+cancer+subtype.">NF-kappaB non-cell-autonomously regulates cancer stem cell populations in the basal-like breast cancer subtype.</a> Nat Commun. 4, 2299 (2013).</li><li>Rio, D. C., Ares, M., Hannon, G. J., Nilsen, T. 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In this protocol, we demonstrated the synthesis of an ASA prodrug, GTCpFE, where the fumarate pharmacophore was incorporated to improve the anti-NF&#312;B activity in breast cancer cells. GTCpFE is an effective NF&#312;B inhibitor, whereas ASA itself is not, even at much higher concentrations. The fumarate moiety has anti-inflammatory properties as shown by its ability to inhibit NF&#312;B signaling in a variety of cell lines and tissues14,25-29. The prodrug strategy described herein, is amendable to other mal...

Inflammation is a hallmark that underlies multiple aspects of tumorigenesis, such as incidence and promotion, and eventually progression to metastasis1. In breast cancer, this is further supported by epidemiological observations showing that regular use of the classical non-steroidal anti-inflammatory drug aspirin (acetyl salicylic acid, ASA) is associated with a reduction in both breast cancer incidence, and risk of metastasis and recurrence2,3. ASA acts primarily by inhibiting cyclooxygenase-2 activity, which is often upregulated in breast cancer4,5. However, the anti-cancer effects of ASA may also be mediated by suppressing aberrant nuclear factor &#954;B (NF&#954;B) signaling6-8. This is important because a deregulated NF&#954;B pathway promotes tumor cell survival, proliferation, migration, invasion, angiogenesis, and resistance to therapy9-11. NF&#954;B pathway activation is also critical for mounting an immune response. Therefore, for anti-cancer therapy where prolonged NF&#954;B inhibition is required, one must consider the detrimental side effects involving long-lasting immune suppression. Hence, ASA may serve as a good starting point for therapeutic optimization.
One limitation for ASA application in cancer therapy is the elevated doses required for cyclooxygenase 2 and NF&#954;B inhibition, which are associated with gastrointestinal toxicity, such as ulcers and stomach bleeding12,13. However, converting ASA into as ester prodrug, may reduce ASA&#39;s gastrointestinal toxicity. To further enhance potency and/or add functionality, additional structural elements or ancillary pharmacophores may also be incorporated into ester prodrug design. One such pharmacophore added to enhance ASA potency against the NF&#954;B pathway is fumarate, which we have previously shown to be important for NF&#954;B pathway inhibition14,15.
We synthesized an aspirin-fumarate prodrug15, GTCpFE, and hypothesized that such hybrid molecule would be safe yet potent against the NF&#954;B pathway. We tested its anti-NF&#954;B activity in breast cancer cells and its ability to block breast cancer stem cells (CSCs)15, which rely on NF&#954;B signaling for survival and growth16-21. We find that the potency of GTCpFE against the NF&#954;B pathway is significantly improved over ASA15. In addition, GTCpFE blocks mammosphere formation and attenuates the CSC surface marker CD44+CD24- immunophenotype, indicating that GTCpFE is capable of eradicating CSCs15. These results establish the aspirin-fumarate prodrug as an effective anti-inflammatory agent that can also target breast CSCs. In terms of breast cancer therapy, GTCpFE may have the potential to treat aggressive and deadly disease.

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			<td height="33" style="height:33px;width:216px;">RPMI 1640 Red Medium</td>
			<td style="width:123px;">Life Technologies&#160;</td>
			<td style="width:119px;">11875-093</td>
			<td style="width:205px;">Warm up to 37&#176;C before use</td>
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			<td height="33" style="height:33px;width:216px;">IMEM</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">10-024-CV</td>
			<td style="width:205px;">Warm up to 37&#176;C before use</td>
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			<td height="33" style="height:33px;width:216px;">DMEM / F12 Medium</td>
			<td style="width:123px;">ThermoFisher</td>
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			<td style="width:205px;">Warm up to 37&#176;C before use</td>
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			<td height="33" style="height:33px;width:216px;">MEM NEAA 10mM 100X</td>
			<td style="width:123px;">Life Technologies&#160;</td>
			<td style="width:119px;">11140</td>
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			<td height="33" style="height:33px;width:216px;">Penicillin Streptomycin</td>
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			<td height="33" style="height:33px;width:216px;">L-Glutamine 100X</td>
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			<td height="33" style="height:33px;width:216px;">Insulin</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">I-1882</td>
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			<td height="33" style="height:33px;width:216px;">Fetal Bovine Serum&#160;</td>
			<td style="width:123px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11150</td>
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			<td height="33" style="height:33px;width:216px;">Trypsin 2.5% 10X</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">15090046</td>
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			<td height="33" style="height:33px;width:216px;">Methyl Cellulose</td>
			<td style="width:123px;">Sigma&#160;</td>
			<td style="width:119px;">M0262</td>
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			<td height="33" style="height:33px;width:216px;">B27 Supplement 50X</td>
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			<td height="33" style="height:33px;width:216px;">EGF</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:119px;">PHG0311L</td>
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			<td height="33" style="height:33px;">NF&#954;B-RE Luciferase Construct&#160;</td>
			<td>Clontech&#160;</td>
			<td style="width:119px;">pGL4.32</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Renilla Luciferase Construct&#160;</td>
			<td style="width:123px;">Promega</td>
			<td style="width:119px;">pGL4.70</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Lipofectamine 2000</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:119px;">11668-019</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Dual-Luciferase Reporter Assay&#160;</td>
			<td style="width:123px;">Promega</td>
			<td style="width:119px;">120000032</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">NanoDrop Spectrophotometer</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:119px;"></td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Eppendorf Mastercycler&#160;</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td style="width:205px;"></td>
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		<tr height="33">
			<td height="33" style="height:33px;">StepOne Real Time PCR System</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Eclipse Microscope</td>
			<td style="width:123px;">Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:205px;"></td>
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		<tr height="33">
			<td height="33" style="height:33px;">CyAn ADP Analyzer&#160;</td>
			<td style="width:123px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td style="width:205px;"></td>
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		<tr height="33">
			<td height="33" style="height:33px;">QCapture Software</td>
			<td style="width:123px;">QImaging</td>
			<td style="width:119px;"></td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Summit Software</td>
			<td style="width:123px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">GraphPad Software</td>
			<td style="width:123px;">Prism</td>
			<td style="width:119px;"></td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">TRIzol</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:119px;">15596-018</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">M-MLV Reverse Transcriptase</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">28025-013</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">100 mM dNTP Set</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">10297-018</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Random Hexamers&#160;</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">48190-011</td>
			<td style="width:205px;"></td>
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		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Fast SYBR Green Master Mix</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:119px;">4385612</td>
			<td></td>
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		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Costar 96W, ultra low attachment&#160;</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">3474</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">HBSS, 1X</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:119px;">14025134</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">CD44-APC conjugated antibody&#160;</td>
			<td style="width:123px;">BD Pharmingen</td>
			<td style="width:119px;">560990</td>
			<td style="width:205px;">Store at 4&#176;C, protect from light</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">CD24-PE antibody</td>
			<td style="width:123px;">BD Pharmingen</td>
			<td style="width:119px;">560991</td>
			<td style="width:205px;">Store at 4&#176;C, protect from light</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Recombinant Human TNF&#945;</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">210TA100&#160;</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">CCL2 Primer Forward Sequence</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:205px;">AGAATCACCAGCAGCAAGTGTCC</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">CCL2 Primer Reverse Sequence</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:205px;">TCCTGAACCCACTTCTGCTTGG</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">ICAM1 Primer Reverse Sequence</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:205px;">TGACGAAGCCAGAGGTCTCAG</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">ICAM1 Primer Forward Sequence</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:205px;">AGCGTCACCTTGGCTCTAGG</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">TNF Primer Forward Sequence</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:205px;">AAGGGTGACCGACTCAGCG</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">TNF Primer Reverse Sequence</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:205px;">ATCCCAAAGTAGACCTGCCCA</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">36B4 Primer Forward Sequence</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:205px;">GTGTTCGACAATGGCAGCAT</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">36B4 Primer Reverse Sequence</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:205px;">GACACCCTCCAGGAAGCGA</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Sodium Hydroxide</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5881-500G</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">4-Hydroxybenzyl Alcohol</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">20806-10G</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">O-Acetylsalicyloyl Chloride</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">165190-5G</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Phosphorous Pentoxide</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">79610-100G</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Ethyl Fumaroyl Chloride</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">669695-1G</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Sodium Sulfate</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">246980-500G</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">4-Dimethylaminopyridine</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">714844-100ML</td>
			<td style="width:205px;">0.5&#160;M in tetrahydrofuran</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Triethylamine</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">T0886-100ML</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Toluene</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">244511-100ML</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Ethyl Acetate</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">270989-100ML</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Tetrahydrofuran</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">401757-2L</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">400 MHz FT NMR spectrometer&#160;</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:205px;">See Bruker&#8217;s Avance User&#8217;s Manual, version 000822 for details</td>
		</tr>
	</tbody>
</table>

synthesis and characterization of an aspirin-fumarate prodrug that inhibits nf&#954;b activity and breast cancer stem cells

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an anti-inflammatory drug to standard cancer regiments may improve patient outcome. One such drug, aspirin (acetylsalicylic acid, ASA), has been explored for cancer chemoprevention and anti-tumor activity. Besides inhibiting the cyclooxygenase 2-prostaglandin axis, ASA&#39;s anti-cancer activities have also been attributed to nuclear factor &#312;B (NF&#312;B) inhibition. Because prolonged ASA use may cause gastrointestinal toxicity, a prodrug strategy has been implemented successfully. In this prodrug design the carboxylic acid of ASA is masked and additional pharmacophores are incorporated.
This protocol describes how we synthesized an aspirin-fumarate prodrug, GTCpFE, and characterized its inhibition of the NF&#312;B pathway in breast cancer cells and attenuation of the cancer stem-like properties, an important NF&#312;B-dependent phenotype. GTCpFE effectively inhibits the NF&#312;B pathway in breast cancer cell lines whereas ASA lacks any inhibitory activity, indicating that adding fumarate to ASA structure significantly contributes to its activity. In addition, GTCpFE shows significant anti-cancer stem cell activity by blocking mammosphere formation and attenuating the cancer stem cell associated CD44+CD24- immunophenotype. These results establish a viable strategy to develop improved anti-inflammatory drugs for chemoprevention and cancer therapy.

In Figure 1, the chemical structure of aspirin-fumarate prodrug, GTCpFE, and its inhibitory activity on cytokine induced NF&#312;B pathway in breast cancer cells are indicated. GTCpFE inhibits both NF&#312;B endpoints, NF&#312;B-RE luciferase activity (Figure 1B) and expression of NF&#312;B target genes, such as Intercellular Adhesion Molecule 1 (ICAM1), Chemokine C-C Motif Ligand 2 (CCL2), and Tumor Necrosis Factor (TNF) (Figure 1C) in M...

Watch this Scientific Journal Video about Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFÃŽÂºB Activity and Breast Cancer Stem Cells at JoVE.com

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFÃƒÅ½Ã‚ÂºB Activity and Breast Cancer Stem Cells

This procedure will demonstrate how we synthesized and characterized the anti-NF&#954;B and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

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