Phase V: The Determination of the Active Protein Binding Moiety of EGCG Utilizing Multidimensional NMR Spectroscopy. A Novel Approach Toward A Cure For Cancer.

The goal of this research is to determine the mechanism by which epigallocatechin-3-gallate (EGCG) and small drug molecules (6-fluoro-4H-1,3-benzodioxine-8-carboxylic acid known as RK038) bind to the glutaredoxin protein from Pseudomonas aeruginosa known as PaGRX.

Previous research proved that the EGCG, which is a polyphenol primarily found in green tea, has an anti-cancer effect on breast (MCF7) and cervical (SiHa) cells, but doesn’t negatively affect non-cancerous breast epithelial (HMEC) cells. The reason why this occurs is unknown.

Plasmids containing the rDNA to express PaGRX were transferred to BL21 (DE3) cells (a strain of E. coli). The PaGRX was 15N-labeled (for use with NMR), extracted from the cells via French Press (lysing), and concentrated. NMR spectra were obtained of the protein sample with 0, 0.5, 1, 1.5, and 3 equivalence points of the RK038 ligand. Other NMR spectra were obtained of the PaGRX with EGCG and its associated moieties.

The NMR data shows that the moiety in EGCG that binds to the PaGRX is the gallate portion of the EGCG molecule. The (H,N) chemical shifts for the G65, V53, H70, and A71 amino acids, respectively, were (0.052,0.4), (0.04,0.4), (0.117, 0.4), and (0.067, 0.5). The shifting values are considered significant because the shifts are 0.05ppm or more. The NMR data of gallic acid combined with the PaGRX shows shifting of the hydrogen and nitrogen atoms in histidine-70, alanine-71, glycine-65, and valine-53 which are located in the protein. The NMR data sets of both the catechin and epigallocatechin show no shifting. The NMR data of the RK038 doesn’t show any significant shifting due to bonding, but the amino acid peaks in the spectra show broadening (the peaks change size due to the amino acids being affected, but not moved). This indicates that there is a weak association between the RK038 and the protein, but no true bonding between RK038 and the protein. Because
the RK038 contains a carboxylic acid functional group that didn’t bind, it stands to reason that the acid functional group is not responsible for binding, but the remainder of the gallic acid molecule is the active binding moiety.
The green tea polyphenol, epigallocatechin-3-gallate (EGCG) caused the protein to precipitate out of solution, which means that the protein’s structure was damaged by the EGCG, and therefore it’s functionality.

References:

1. Wanjek, C. Green Tea’s Anti-Cancer Secrets Revealed. Live Science.com, Oct 18, 2012
2. Wilson, A. Cancer Rates in the USA Compared to Japan. Cancer News, Aug 26, 2011
3. Oviedo, N. Regeneration: The Origin of Cancer or a possible Cure? Semin Cell Dev Biol, Jul 20, 2009
4. Rawls, S. Nicotine Behavioral Pharmacology: Clues from Planarians. Drug Alcohol Depend, Nov 1, 2011
5. Baguna, J. Regeneration and Pattern Formation in Planarians.
    University of Barcelona, 107:77-86, 1989
6. Newmark, P. Regeneration in Planaria. Encyclopedia of Life Sciences, 2001: 1-5
7. Stanford University, Ludwig Center. The Stem Cell Theory of Cancer,
    med.stanford.edu/ludwigcenter/overview/theory.html
8. Pagan, O., Toxicity and Behavioral Effect of Dimethylsulfoxide in Planaria, Neurosci Lett., Oct 30;
    407(3):274-8
9. Varghese, G. Neoblast Response to Carcinogen Induced Malignancy in Planarians. New York University,
    Jan 7, 2015
10. Reddien, P. Fundamentals of Planaria Regeneration, Cell Dev. Biol, 2004. 20:725-57
11. Litt, S. An Investigation of the Effect of Polyphenols on Planaria Regeneration, Observations and Results:
     Graph of Polyphenol Concentration vs. Planaria Eye Pixel Count
12. Snitsarev, Vladislav, The Spectral Properties of Epigallocatechin 3-O-Gallate (EGCG) Fluorescence in
      Different Solvents: Dependence of Solvent Polarity, Montclair State University, Nov 22, 2013
13. Du, Guang-Jian, Epigallocatechin Gallate (EGCG) Is the Most Effective Cancer Chemopreventive
      Polyphenol in Green Tea, Nutrients. Nov 2012; 4(11): 1679-1691
14. Lachaier, Emma, Sorafenib Induces Ferroptosis in Human Cancer Cell Lines Originating from Different Solid
      Tumors, Anticancer Research, November 2014 v.34 no.11, 6417-6422
15. Leeper, Thomas, An NMR-Guided Screening Method for Selective Fragment Docking and Synthesis of a
      Warhead Inhibitor, Molecules. July 16, 2016
16. Khattri, Ram, Identifying Ortholog Selective Fragment Molecules for Bacterial Glutaredoxins by NMR and
      Affinity Enhancement by Modification with an Acrylamide Warhead, Molecules, December 30, 2019
17. He, Feng, Glutaredoxin 3 Promotes Nasopharyngeangeal Carcinoma Growth and Metastasis via EGFR/Akt
      Pathway and Independent of ROS, Oncotarget, May 18, 2016

EGCG and How It Affects Cancer Cells

THE EGCG MOLECULE

Research Plan / Project Summary

Phase IV: The Effect of Epigallocatechin-3-Gallate on Breast and Cervical Carcinomas

(Stephen Robert Litt)

The rationale for the experiment is finding a cure for cancer. Breast and Cervical cancers are very aggressive forms of cancer. In Japan, people consume vast amounts of green tea, and has a much lower rate of cancer, over three times lower than the US. It has been proven that polyphenols, which are abundant in green tea, have an anti-cancer effect.

Phase I research proved that a polyphenol in green tea, specifically epigallocatechin-3-gallate (EGCG) inhibits regeneration of planarians by 157.54% after injury. Phase II research proved that the same polyphenol in green tea, epigallocatechin-3-gallate, inhibits planarian (dugesia tigrina) tumorigenesis.

Phase III proved that EGCG (epigallocatechin-3-gallate) eliminates preexisting tumors. Phase III also proved that EGCG can be used as a way to detect active cancer since EGCG has specific fluorescence properties. Brightfield and fluorescence microscopy was be used to prove this. An NMR study, serving as an ultimate control proved that EGCG is not chemically interacting with the carcinogen in solution. This proves that EGCG is a biologically active anti-cancer agent.

For the current phase of research, Phase IV, the goal is to bring the experiment into the realm of human cancer cells. Utilizing the MCF7 (breast) and SiHa (cervical) cancer cell lines, it will be determined if EGCG can induce cell apoptosis. A logarithmic series of EGCG concentrations will be utilized to determine the concentration of EGCG that may be used therapeutically. CytoTox (cell apoptosis) assays of multiple well plates will be used to determine the optical density of positive and negative controls and the cancer cells themselves. Also, HMEC (normal breast epithelial cells) will be used as a control to determine if EGCG has any effect on non-cancerous cells.

Also, all cell colonies will be imaged microscopically before treatment and then after EGCG treatment at 24, 48, and 72 hours.

Bibliography

1. Wanjek, C. Green Tea’s Anti-Cancer Secrets Revealed. Live Science.com, Oct 18, 2012

2. Wilson, A. Cancer Rates in the USA Compared to Japan. Cancer News, Aug 26, 2011

3. Oviedo, N. Regeneration: The Origin of Cancer or a possible Cure? Semin Cell Dev Biol, Jul 20, 2009

4. Rawls, S. Nicotine Behavioral Pharmacology: Clues from Planarians Drug Alcohol Depend, Nov 1, 2011

5. Baguna, J. Regeneration and Pattern Formation in Planarians. University of Barcelona, 107:77-86, 1989

6. Newmark, P. Regeneration in Planaria. Encyclopedia of Life Sciences, 2001: 1-5

7. Stanford University, Ludwig Center. The Stem Cell Theory of Cancer, med.stanford.edu/ludwigcenter/overview/theory.html

8. Pagan, O., Toxicity and Behavioral Effect of Dimethylsulfoxide in Planaria, Neurosci Lett., Oct 30; 407(3):274-8 New York University, Jan 7, 2015

9. Varghese, G. Neoblast Response to Carcinogen Induced Malignancy in Planarians.

10. Reddien, P. Fundamentals of Planaria Regeneration, Cell Dev. Biol, 2004. 20:725-57

11. Litt, S. An Investigation of the Effect of Polyphenols on Planaria Regeneration,Observations and Results: Graph of Polyphenol Concentration vs. Planaria Eye Pixel Count

12. Snitsarev, Vladislav, The Spectral Properties of Epigallocatechin 3-O-Gallate (EGCG) Fluorescence in Different Solvents: Dependence of Solvent Polarity, Montclair State University, Nov 22, 2013

13. Du, Guang-Jian, Epigallocatechin Gallate (EGCG) Is the Most Effective Cancer Chemopreventive Polyphenol in Green Tea, Nutrients. Nov 2012; 4(11): 1679-1691

14. Lachaier, Emma, Sorafenib Induces Ferroptosis in Human Cancer Cell Lines Originating from Different Solid Tumors, Anticancer Research, November 2014 v.34 no.11, 6417-6422