Jennifer Onwukwe's GOx Aptamer Project (2017)

Aptamer Against Glucose Oxidase to Initiate Glucose Starvation and Cell Death in Prostate Cancer Cells

Introduction and Background

Cancer has been the second most leading causes of death in the United States and continues to take the lives of many individuals today (Jin 2016). Cancer is a disease that results from the rapid proliferation of abnormal cells that damage body tissues in various regions of the body. Abnormal cells arise from the damage of DNA inside of the cell. This leads to abnormalities within the cell, which continue invade healthy tissue and cells through rapid growth and division. The mass of cells become a malignant or benign tumor. Benign tumors could be almost harmless; however, the real problem arises from malignant tumors. Metastatic cells on malignant tumors have the ability to detach and spread to various parts of the body (Figure 2).

The fight against cancer has been prevalent in America the mechanisms of cancer are still being researched by doctors and scientists up to today. Various treatments for cancer have been utilized to eliminate metastatic cancer cells from the body, such as injecting a patient with chemotherapeutic drugs to mediate and eventually terminate any signs of cancer within the individual. However, these treatments also damage healthy cells within the body and weaken cancer patients, reducing their chances of survival. The elderly are at a higher risk because of their progressive loss of body protein, decreased ability to repair cell damage, and decreased ability to pull from stem-cell reserves (Repetto, 2003). The elderly, most notably men, are more prone to cancers such as prostate cancer, that would be difficult to treat due to these high risks.

Thus, questions have arose concerning new treatments that would treat cancer with minimal harm done to the patient and lower the mortality rate of cancer. As a result, the use of an enzyme, glucose oxidase (GOx), could eliminate cancer cells with as little harm done to the human body as possible through their enzymatic activity. Since prostate cancer is prevalent in older men, GOx can be used to eliminate these cells. Glucose oxidase is a dimeric protein that has a molecular weight of 160kDa and consists of the cofactor, flavin adenine dinucleotide (FAD) (Fig 3). The enzyme that catalyzes the oxidation of glucose to the products, hydrogen peroxide and D-gluconolactone while FAD serves as a redox complex to promote activity (Holland, Harper, et al, 2012).

The use of aptamers in the medical field have been increasingly popularized for diagnostic and therapeutic purposes. Aptamers are unique oligonucleotide sequences that have a binding affinity for specific targets, such as enzymes, cells, or even small molecules. For instance, aptamers could be used as diagnostic tools for certain diseases to locate and bind to disease-specific biomarkers or pathogens to signal the presence of a disease (Zhu et al, 2015). Thus, unlike antibodies, aptamers have higher binding affinities and specificities for their target and are considered cheaper alternatives that could be mass produced to be extensively used in the medical field. The specific application of an ideal aptamer against glucose oxidase will serve a therapeutic purpose through the ability send glucose oxidase to destroy prostate cancer cells by depriving the cells of the glucose it needs to survive. An aptamer against glucose oxidase will induce the enzyme to break down glucose within prostate cancer cells.

The use of aptamers against glucose oxidase has a variety of applications outside of therapeutics as well. Aptamers have been developed for glucose oxidase by various researchers. A DNA aptamer was developed for glucose oxidase in glucometers as a biosensor that detects the amount of glucose inside of the blood through the detection of elevation levels of ATP consumption (Sitaula et al, 2012). For glucometers that utilize glucose oxidase, an aptamer has been developed, by researchers Sarita Sitaula, Shirmir D. Branch, and Mehnaaz F. Ali, to remove FAD from glucose oxidase to inactive it. Thus, the aptamer-FAD complex had the ability to initiate the reactivation of apo-GOx by releasing the cofactor FAD that is triggered by ATP. The use of this method would determine how much glucose is in the body by measuring the change in the ATP concentration (Sitaula et al, 2012).

Studies have shown that cancer cells display higher intracellular glucose levels than normal body cells. They require higher levels of glucose to perform anaerobic glycolytic metabolism for quick energy to meet their demands for rapid proliferation (Nascimento et al, 2016). Glycolysis is the process in the cytosol of a cell in which glucose molecules are broken down into two reduced pyruvate molecules that enter the mitochondria and are utilized even further to produce more energy. Recent studies show that cancer cells utilize oxidative phosphorylation to produce ATP for growth and function. Although the process of glycolysis is the same for cancer and normal body cells, the respiratory chain complexes in oxidation phosphorylation exert significantly higher flux-control in cancer cells than in normal cells (Moreno-Sáncheza et al, 2014). Thus, an aptamer against glucose oxidase would target these cancer cells and starve them of their critical energy source, inducing them to cell death. To ensure that the GOx-aptamer complex reaches target cells, the aptamer will also be bound to a specialized PSMA aptamer against prostate cancer cells. The PSMA aptamer functions by binding to receptor proteins on the surface of prostate cancer cells and initiating the cell to engulf the aptamer along with whatever the aptamer is attached to (Wu et al, 2011). Thus, the GOx-PSMA aptamer complex will have a high specificity for prostate cancer cells to ensure that it does not affect normal body cells. Once the complex is within the cell, the GOx will be induced to oxidize glucose to produce gluconic acid and hydrogen peroxide. The absence of glucose inhibits glycolysis and leads to a depletion of ATP in cancer cells, which induces p53-dependent apoptosis (Sahra et al, 2010). The p53 protein is a tumor protein that regulates the cell cycle within a cancer cell and assists in triggering apoptosis when the energy sensor AMP kinase fails to detect sufficient production of ATP (Sahra et al, 2010). Therefore, the heavily reliance on glucose by the metastatic cells will cause it to trigger apoptosis if glucose oxidase was introduced to the cell.

Several rounds of the SELEX aptamer selection process will be performed to find an ideal aptamer to serve this therapeutic purpose. Finding an aptamer for glucose oxidase for my application must be done under specific conditions to enhance the affinity of RNA to the protein. For glucose oxidase, however, there are no specific features that prove to be useful in binding nucleic acids. It is not known to bind to nucleic acids, nor does it have an opposite charge to improve the affinity of the RNA to glucose oxidase. Due to the negative charge of RNA, a divalent salt would need to be added to the selection buffer to neutralize the charges between the RNA and the protein so that it would be able to bind. The buffer that will be used is PBS + MgCl2 selection buffer with a pH of 7.3 to mimic the human body. The aptamer selection process starts with bead-based aptamer selection to elute the RNA that bind to the target. The RNA then undergoes a cycle of reverse transcription, amplification, transcription, and purification. The summation of these processes potentially increases the specificity of the RNA pool and increases the likelihood of finding an aptamer with a high binding activity against glucose oxidase.

In the overall aptamer selection process, up cycle course PCR has been performed to determine how many cycles to that are needed to amplify without overamplification the E1. Several PCRs were performed to determine what was contaminating my DNA samples, which resulted in being the Taq DNA polymerase. After troubleshooting this problem, a successful ccPCR revealed that 24 cycles were enough for the amplification of my E1. The E1 was then mass amplified and prepared for the next step. To finish the first round of the aptamer selection process, the amplified DNA will be transcribed and purified. The process will be repeated three to four times before performing a binding assay to observe the binding activity of the RNA on glucose oxidase. Once this an aptamer is found, it could serve a therapeutic approach by utilizing the aptamer to bind to glucose oxidase and enter the prostate cancer cells through the assistance of the PSMA aptamer. This will be a less invasive approach when treating prostate cancer patients and can be further developed to treat other forms of cancers in the elderly.

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References

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