Rhea Sudhakaran's HRP Aptamer Project (2019)

Therapeutic Aptamer for Horseradish Peroxidase in Conjugation with Indole-3-Acetic Acid to Induce Apoptosis in Epithelial Cancer Cells

Introduction and Background:

Epithelial cancers, or carcinomas, are the most common cancer type in the world, making up approximately 90% of all cancer cases (Cooper, 2000). Cancers of the skin, lungs, breasts, lungs, colon, prostrate, and bladder are all forms of this cancer and are some of the most fatal in the world, with mortality rates reaching up to 98% in pancreatic cancers (Ilic & Ilic, 2016). There are currently treatments for these diseases, such as surgical removal of tumors and chemotherapy to destroy carcinogenic cells (Hori et. al, 2019). However, these treatments can fail in eradicating the cancer fully or can become non-specific and attack healthy cells as well as the cancerous cells (Hori et. al, 2018). An alternative to these treatments is prodrug/enzyme targeted therapy, where an enzyme with its substrate are used in combination to precisely deliver a catalyzed drug to a specific cell (Kim et. al, 2004). Previous studies have shown a possible application of this therapy utilizing the enzyme horseradish peroxidase and the substrate indole-3-acetic acid (Kim et. al, 2004).

Horseradish peroxidase is an enzyme naturally found in Amoracia Rusticana root cells and catalyzes hydrogen peroxide to oxidize various organic and inorganic compounds (“Horseradish Peroxidase” n.d.). In this case, HRP can break down hydrogen peroxide to then oxidize indole-3-acetic acid or IAA. Though IAA acts mainly as a plant growth hormone (auxin), when oxidized by HRP in the human body, it produces free radicals that can induce apoptosis in cytotoxic cells (Kim et. al, 2004). Previous studies have determined the specific pathways are death-receptor mediated and mitochondrial apoptotic (Kim et. al, 2004). The structures of HRP and IAA make it ideal for use in the human body as they are non-toxic and relatively stable (Wardman, 2002). Though HRP and IAA can chemically be used to treat cancer, an issue remains in finding a targeted delivery method to minimize cell death to only cancerous cells. To target cancer cells specifically in epithelial cancers, an aptamer can be found that binds to horseradish peroxidase which can then bind to another aptamer that binds to overexpressed MUC1 receptors found on tumorigenic epithelial cells. Since digestive enzymes could potentially denature horseradish peroxidase structure, HRP-linked aptamers can be delivered via injection directly into bloodstream. Selection conditions would then match the blood environment, 37C and 7.4 pH (Blombäck, 1978).

MUC1, or mucin-1, is a glycoprotein found on epithelial cells that creates a barrier between the cell and its environment (Hori et. al, 2018). Though MUC1 is found on most epithelial cell surfaces, it is overexpressed in 900,000 of the 1.2 million cancers diagnosed (Hori et. al, 2018). With its overexpression, the structure of MUC1 changes, becoming TA-MUC1 or tumor-associated MUC1. By finding an aptamer that binds to TA-MUC1 and another aptamer that binds to horseradish peroxidase, the two aptamers can link to indirectly bind the horseradish peroxidase to the tumorigenic epithelial cancer cells. By then treating the body with indole-3-acetic-acid, the horseradish peroxidase can then break IAA down into cytotoxic molecules only for the cells that are cancerous (as seen in Figure 1.2).

Though antibodies are traditionally used when delivering targeted treatments, aptamers can perform a similar function with several advantages. Aptamers are unique oligonucleotides, such as RNA or DNA, that have a high affinity for a specific target (Gold et. al, n.d.). Since itsinception two decades ago in the Tuerk lab, aptamers have led to immense discovery and progress in the scientific community, lending itself in diagnostic, therapeutic, drug delivery, and systems biology applications (Gold et. al, n.d.). Aptamers act similarly to antibodies as both are molecules that bind to a specific target; however, aptamers have several advantages. Aptamers can be isolated and amplified in vivo and are stable for long-term storage (Jayasena 1999). Aptamers are also more diverse and easier to isolate as a single base pair change in nucleic acid sequence can lead to a vastly different binding capacity (Jayasena, 1999). They can also be amplified easily and inexpensively through PCR or polymerase chain reaction, which generates many copies of DNA that can be transcripted back into RNA (Jayasena, 1999).

These advantageous have made aptamers a prime area of research. In this experiment, the “toggle” SELEX method will be done where the selection method is alternated every round (White et. al, 2016). The target, horseradish peroxidase, will be biotinylated, and streptavidin bead-based selection will be done first followed by filter-based selection to ensure aptamers being selected for only bind to the target of HRP and not the streptavidin beads. The structure of horseradish peroxidase makes it optimal for aptamer selection, as it is a protein with a binding site and several metal ligands, so aptamers have available binding sites (“Horseradish Peroxidase”, n.d.). Aptamers have been found for horseradish peroxidase with various applications, with some examples being in the HRP- linked aptamer assay for detecting levels of aflatoxin, a carcinogen, in various food products (Sun & Zhao, 2018) and using a HRP sandwich assay to measure human thrombin levels which naturally causes blood clots but can lead to deep vein thrombosis if unchecked (Li, Lee, & Corn, 2007). Though aptamers have been found for horseradish peroxidase, most of the applications are for detection. This project aims to find an aptamer for horseradish peroxidase for therapeutic application. By finding an aptamer that binds to horseradish peroxidase and pairing it with an aptamer that binds to MUC1 receptors on epithelial cancer cells, targeted cancer cell apoptosis can be induced with the catalysis of indole-3-acetic acid.

Aptamer selection has begun, with one round of SELEX currently in progress. Binding and selection were done using streptavidin bead-based selection with a 200 pmol horseradish peroxidase to 200 pmol of N71 RNA pool, or a 1:1 ratio so all aptamers with affinity to target have place to bind. ccPCR is being done to determine cycle number for optimal amplification. After many rounds, an aptamer will be selected for horseradish peroxidase which can be paired with an aptamer bound to overexpressed MUC1 receptors in epithelial cancer cells. Indole-3-acetic acid can then be intravenously given to the patient to be oxidized by horseradish peroxidase and produce cytotoxic molecules to induce apoptosis in epithelial cancer cells, paving a more targeted way of cancer treatment using aptamers.

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References:

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