The Effect of Ethanol Treatment on the Protein Content of Difi Exosomes
Sciences and Mathematics, College of
Chemistry and Physics, Department of
SURS Faculty Advisor
Dr. Amy Ham
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 Colorectal cancer metastasis is the primary cause of mortality largely due to therapy resistant cancer cells.2 Therefore, detection before the cancer metastasizes is of great importance that 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 that 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, as well as 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. TMP4 binds to actin filaments and provides cell structure. VASP is involved in cell migration, while ACTBL2 and PLXNB2 are involved in cell signaling. Proteins upregulated in the control cells include: PTGFRN which inhibits inflammation and RPL7A which is a component of the large ribosome subunit responsible for the synthesis of proteins. 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. Further studies are needed, not only to increase the reproducibility of the results, but also to verify the purity of exosome preparation.
1. CA A Cancer J Clinicians, Volume: 72, Issue: 1, Pages: 7-33, First published: 12 January 2022, DOI: (10.3322/caac.21708)
2. Shin, A. E., Giancotti, F. G., & Rustgi, A. K. (2023). Metastatic colorectal cancer: Mechanisms and emerging therapeutics. Trends in Pharmacological Sciences, 44(4), 222–236. https://doi.org/10.1016/j.tips.2023.01.003
3. 7. Doyle, L., & Wang, M. (2019). Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells, 8(7), 727. https://doi.org/10.3390/cells8070727
4.Milman N, Ginini L, Gil Z. Exosomes and their role in tumorigenesis and anticancer drug resistance. Drug Resist Updat. 2019 Jul;45:1-12.
doi:10.1016/j.drup.2019.07.003. Epub 2019 Jul 23. PMID: 31369918.
5. Na, H.-K., & Lee, J. (2017). Molecular basis of alcohol-related gastric and colon cancer. International Journal of Molecular Sciences, 18(6), 1116. https://doi.org/10.3390/ijms18061116
Rushing, Kenzie, "The Effect of Ethanol Treatment on the Protein Content of Difi Exosomes" (2023). Science University Research Symposium (SURS). 128.