Author ORCID Identifier

https://orcid.org/0009-0000-0784-1513

Date Available

9-2-2026

Year of Publication

2025

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Medicine

Department/School/Program

Microbiology, Immunology, and Molecular Genetics

Faculty

Dr. M. Ashfaqul Alam

Faculty

Dr. Beth A. Garvy

Abstract

Frequently, alterations in the composition of the gut microbiota and its metabolites are linked with intestinal diseases, including inflammatory bowel disease and colorectal cancer. However, the direct connections between alterations in the microbiome and disease states often remain elusive. Prior research has identified increases in both polyamines, which are a class of microbial metabolite, and in the abundance of bacteria that can produce polyamines, in a murine colorectal cancer model. Understanding the significance of the interaction between these microbial metabolites and intestinal epithelial cells (IECs) will provide new and expanded evidence of the importance of the gut microbiome in colorectal cancer.

My work aims to determine how polyamines can induce physiological changes in IECs as well as a potential mechanism for these phenomena. Gene expression data revealed several initial pathways to pursue, including cell proliferation, migration, changes in signaling pathways, and alterations in metabolic processes. Addition of exogenous spermidine to cultured IECs resulted in increased cell proliferation, measured via EdU assay. I additionally proposed a signaling pathway that spermidine could be acting on to increase cellular proliferation, and using immunofluorescence staining and Western blot, discovered an increase in protein phosphorylation with increasing spermidine treatment. There are multiple sources that polyamines can be derived from, including gut bacteria and human cells. I used an ornithine decarboxylase inhibitor to prevent cultured cells from producing polyamines, thus isolating the polyamine source to the exogenously added spermidine. In this case, the IECs still demonstrated increased proliferation; when a polyamine transport blocker, AMXT-1501, was added, the cells demonstrated decreased proliferation. Similar results were found in cell migration experiments. Exogenous spermidine increased cell migration, but when inhibitors were added, cell migration was suppressed. Mutation is also a key hallmark of cancer. When polyamines are broken down, they produce acrolein and hydrogen peroxide, two compounds that can damage DNA. I identified increased DNA damage in polyamine-treated IECS. Finally, I utilized fluorescently tagged spermidine to analyze the localization of spermidine after inhibition and harnessed siRNA to knock down one of two proposed spermidine transporters.

Spermidine is a vital contributor to many IEC functions, including proliferation and migration. These, along with DNA damage, are several key hallmarks of cancer. Using an established inhibitor of polyamine production and a polyamine transport inhibitor that is novel in colorectal cancer, I was able to reduce cell proliferation and migration, identify potential signaling mechanisms for these changes, and knockdown a polyamine transporter. These studies highlight the potential contribution of polyamines and their transporters to the development and progression of colorectal cancer and identifies a method that could eventually become an option for a microbial-based treatment for colorectal cancer.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2025.456

Funding Information

This study was supported in part by the National Institute of Health National Institute of Diabetes and Digestive and Kidney Diseases grants R56DK136728 (A.A.), 2P20GM130456-06 and P20GM130456 (A.A.; Alam Project ID9790), K01DK114391 (A.A.), ACS IRG (A.A.), and Elsa U. Pardee Foundation Grant (A.A.)

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Available for download on Wednesday, September 02, 2026

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