Chantel Blake's GOx Aptamer Selection Project (2018)

RNA Aptamer Against Glucose Oxidase for the Starvation of Cancerous Cells

Introduction and Background

Cancer is plaguing the health of humans and affecting mortality rates of all people, regardless of income or access to healthcare (Siegel, 2017). This disease has ranked itself as the second most common cause of death globally, causing it to become one of the most significant public health challenges of this century. Brain cancer, in particular, has a high mortality with patients having a five-year life expectancy (American Cancer Society, 2018).

The purpose behind my research is to develop a therapeutic application that will reduce the growth of and starve cells in both malignant and benign tumors. The focus will be on brain cancer, mainly astrocytomas and their characteristic mutation of their IDH genes. The mutated cells create a mass in the brain, compressing, invading, and destroying brain parenchyma. This disrupts the normal function of the parenchyma by hijacking the nutrients and metabolic products of normal cells. The therapeutic application being researched will allow for the direct targeting of astrocytomas via the use of the IDH mutation biomarkers on their surfaces, successfully delivering glucose oxidase. This application will prompt this use of an aptamer. An aptamer is an oligonucleotide (RNA or DNA) that specifically bind to their target, the target of this experiment being glucose oxidase. The aptamer application method provides multiple advantages over other methods, such as antibiotics. Aptamer selection has a high specificity, it has maximum suitability for the desired environment, its structure and function can be easily modified, and it is economically efficient in comparison to antibiotics.

Glucose is a necessity for growth in both benign and malignant tumors, while fermentation and respiration are both used by tumor cells for survival (Warburg, 1927). To stunt the growth of, starve, and suffocate tumor cells of the energy and nutrients necessary to its survival, fermentation and respiration will have to be interrupted along with the depletion of the main food source, glucose. This dependence can be exploited by utilizing glucose oxidase. It functions as a catalyst for glucose, stunting the growth of the tumor cells and stopping fermentation. Also, the enzyme can convert oxygen into hydrogen peroxide, which stops respiration from occurring and causes apoptosis of tumor cells due to the high concentration of hydrogen peroxide (Bankar, 2009).

Glucose Oxidase (GOx) is a glycoprotein with a molecular weight of approximately 160 kDa. GOx functions as a dimer, with two subunits, one subunit which binds to the substrate, and the second, which binds to the cofactor flavin adenine dinucleotide (FAD) (Sandip, 2009). Glucose Oxidase is a stable enzyme which oxidizes glucose into D-glucono-1,5-lactone and once hydrolyzed becomes gluconic acid (Goodsell, 2006). This acid converts oxygen into hydrogen peroxide, a process shown in Figure 2.

Figure 2: Reaction Catalyzed by Glucose Oxidase ("Reaction Catalyzed by Glucose Oxidase", 2015)

The application of this aptamer will be challenging. The utilization of a biomarker on the surface of a cancerous cell is one method being highly considered. Biomarkers are preferable because it allows for the anti-GOx aptamer to be delivered to the correct cells for a drug delivery application, a complex which can be seen in Figure 1. Once an anti-GOx aptamer is detected, an aptamer that attaches to a receptor on the cancer cells surface will be selected for. For the brain cancer biomarker in question, the mutations in the IDH1 or IDH2 genes will be utilized as they are common in most all astrocytomas, oligodendrogliomas, and glioblastomas (Brandner, 2018).

Glucose oxidase can catalyze the glucose present in tumor cells, starving the cells of their energy supply and increase the amount of hydrogen peroxide in the tumor cells, killing the cells (Wenguo, 2017). Currently, the second round of selection against Glucose Oxidase for chemotherapeutic application has been started. With selection rounds being performed, methods on delivering this aptamer to cancer cells without harming healthy cells are being researched along with the issue of passing glucose oxidase through the blood brain barrier, as it is a very large protein. For this issue of crossing the blood brain barrier, the utilization of transferins are being considered as they have been researched to allow for drug delivery across the blood brain barrier. The overarching goal of this experiment is to discover an anti-GOx aptamer to deliver GOx to affected astrocytomas in the brain, with the hopes of starving malignant tumors in the brain.

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Works Cited

“Cancer Facts & Figures 2018.” American Cancer Society, www.cancer.org/research/cancer- facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2018.html.

Blind, M., & Blank, M. (2015). Aptamer Selection Technology and Recent Advances. Molecular Therapy. Nucleic Acids, 4(1), e223–. http://doi.org/10.1038/mtna.2014.74

Blind, M. (2015, January 13). The different steps (S1–S5) of the SELEX process. [Digital image]. Retrieved April 1, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4345306/

Brandner, S. and Jaunmuktane, Z. (2018), IDH mutant astrocytoma: biomarkers for prognostic stratification and the next frontiers. Neuropathol Appl Neurobiol. Accepted Author Manuscript. . doi:10.1111/nan.12521

Goodsell, D. (2006, May). Protein Structure of Glucose Oxidase [Digital image]. Retrieved April 1, 2018, from http://pdb101.rcsb.org/motm/77

Goodsell, D. S. (2006, May). Glucose Oxidase. Retrieved April 1, 2018, from http://pdb101.rcsb.org/motm/77

Reaction Catalyzed by Glucose Oxidase [Digital Image]. (2015, April 11). Retrieved April 1, 2018, from https://commons.wikimedia.org/wiki/File:Glucose_oxidase_rxn.svg#/media/File:Glucose_oxidase_rxn.svg

Sandip B. Bankar, Mahesh V. Bule, Rekha S. Singhal, Laxmi Ananthanarayan, Glucose oxidase — An overview, Biotechnology Advances, Volume 27, Issue 4, 2009, Pages 489-501, ISSN 0734-9750, https://doi.org/10.1016/j.biotechadv.2009.04.003.

Sever, R., & Brugge, J. S. (2015). Signal Transduction in Cancer. Cold Spring Harbor

Perspectives in Medicine, 5(4), a006098. http://doi.org/10.1101/cshperspect.a006098 Sever, R. (2015, April). Cancer Progression [Digital image]. Retrieved April 1, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382731/figure/A006098F1/

Siegel, R. L., Miller, K. D. and Jemal, A. (2017), Cancer statistics, 2017. CA: A Cancer Journal for Clinicians, 67: 7-30. doi:10.3322/caac.21387

Warburg, O.; Wind, F.; Negelein, E. The Metabolism of Tumors in the Body J. Gen. Physiol. 1927, 8 (6) 519– 530 DOI: 10.1085/jgp.8.6.519

Wenguo Zhao, Jin Hu, and Weiping Gao, ACS Applied Materials & Interfaces 2017 9 (28), 23528-23535, DOI: 10.1021/acsami.7b06814