The Effect of Ethanol Treatment on the Protein Content of Difi Exosomes

Authors

Kenzie RushingFollow

Publication Date

Spring 3-14-2024

College

Sciences and Mathematics, College of

Department

Chemistry and Physics, Department of

BURS Faculty Advisor

Dr. Amy Ham

Presentation Type

Oral Presentation

Abstract

Colorectal cancer (CRC) is the second most common cause of cancer death in the United States in both men and women combined, second only to lung cancer.1 CRC metastasis is the primary cause of mortality largely due to therapy resistant cancer cells.2 Therefore, detection before metastasis is of great importance and could potentially lead to earlier detection and decreased mortality. Extracellular vesicles (EVs), including exosomes, are lipid bound vesicles secreted by cells3 that are involved in cell-cell communication and have been found to promote CRC progression and metastasis.4 The proteome of exosomes is thought to reflect that of the originating cell, therefore exosomes from cancer cells that are taken up by normal cells, may promote tumor growth, transfer drug resistant traits, and act as a decoy for immunotherapeutic targets.4 Previous research has shown that alcohol consumption over a period of time increases a person’s risk of CRC,5 however the cellular mechanisms that mediate the effects are still unclear. It is hypothesized that exosomes play a role in the proliferation of CRC due to ethanol consumption, which would support exosomes’ role in cancer proliferation and potentially serve as a target for treatment. In this study, DiFi cells (a CRC cell line) were treated with ethanol and exosomes secreted by the cells were isolated using dialysis membrane and purified using electrophoresis. The protein contents were analyzed using a liquid chromatography mass spectrometer (LC-MS/MS). Proteins were identified from the spectra produced from the LC-MS/MS using a database search algorithm. The large set of protein lists were filtered to an acceptable false discovery rate and quantified by spectral counting to look for protein differences in control vs. ethanol treated cells. Results showed proteins upregulated in the ethanol treated cells included: TMP4, VASP, ACTBL2, and PLXNB2. Which are involved in cell structure, cell migration, and cell signaling respectively. Proteins upregulated in the control cells include PTGFRN which inhibits inflammation and RPL7A, a component of the large ribosome subunit. Analysis also showed exosomal markers including EGFR, CD9, CD81, ANXA2, and MVP which indicate that proteins from exosomes were being analyzed. Although these results support the role of exosomes in ethanol-mediated CRC progression, they were not highly reproducible due to an inconsistency in the duration of each step of exosome preparation. Further studies are needed to increase the reproducibility of the results and verify the purity of exosome preparation.

Recommended Citation

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