Louisiana Research Day Program Book 2025

Biomedical Research: Section 1

Biomedical Research: Section 1

Nicholas Wong, BS, OMS-II 1 ; Lydia Ta, BS, OMS-II 1 ; Christine Palma, BS, OMS-II 1 Lin Kang, PhD 1 ; K. Adam Morrow, PhD 1 ; Melissa EH Lipsmeyer, MS, PhD 1 1 VCOM-Louisiana 16 INDOLE-3-PROPIONIC ACID MEDIATES EXTRACELLULAR MATRIX DYNAMICS OF TRIPLE NEGATIVE BREAST CANCER CELLS IN AN IN VITRO MODEL OF OBESITY

Erin Knight, OMS-II 1 ; Md Ashiq Mahmud, MSc 2 ; Sarah Voth, PhD 1 ; Rebekah L. Morrow, PhD 1 ; Khalid A El Sayed, PhD 2 ; K. Adam Morrow, PhD 1,2 VCOM-Louisiana; 2 University of Louisiana Monroe College of Pharmacy, Monroe, LA 17 THE PROTECTIVE ROLE OF OLEOCANTHAL AGAINST PSEUDOMONAS AERUGINOSA INFECTION

Context: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks expression of the receptor for the sex steroid hormones estrogen and progesterone and does not overexpress human epidermal growth factor 2 receptor (HER2) protein. TNBC is associated with special characteristics, including aggressiveness, invasiveness, metastatic potential and overall poor prognosis. TNBC’s are 10–20% more common in overweight compared with normal-weight women suggesting overweight/obesity is a contributing factor to the progression of the disease. Emerging data suggest that dysbiosis of the gut microbiome may be causal to exacerbation of TNBC by altering inflammation and the tumor microenvironment as well as shifting energy substrates that promote cell survival. Gut dysbiosis is a well-known phenomenon in obesity and offers an additional avenue by which obesity promotes growth of TNBC. Indole-3-propionic acid (IPA), a metabolite of tryptophan metabolism, is produced by the gut microbiome and has been shown to have anti inflammatory, anti-oxidative stress, and glucose regulatory properties in numerous cell types and cytostatic functions in breast cancer cells.

Furthermore, IPA is reduced in the serum levels of lean individuals compared to those with overweight/obesity. This suggests reduction of serum IPA levels in obesity may be a contributing factor to the aggressive properties of TNBC. Objective: The objective of this study is to analyze the effect of indole-3-propionic acid on a triple-negative breast cancer cell line to discover novel functions of this metabolite in breast cancer cells and to determine if IPA can mitigate obesity-induced alterations in an in vitro model of obesity. Methods: These experiments were performed utilizing MDA-MB-231 TNBC cells in an in vitro model of obesity. For this model, cells are cultured in adipocyte conditioned media (ACM) that has been conditioned for 48 hours by mature human adipocytes. Cells are also cultured in the presence or absence of physiological levels of IPA (1uM) for 72 hours. After treatment, we deployed next generation RNA sequencing to determine global transcriptional changes induced by ACM and to determine if IPA can reverse any pathogenic

pathways. Genes with a significant p-value (<0.05) and a log-fold greater than 2 or less than 0.5 were selected and analyzed in a public bioinformatic tool. Results: Pathway enrichment analysis revealed treatment of MDA-MB-231 cells with ACM significantly altered gene networks related to cellular processes such as apoptosis, cellular senescence and inflammation. However, co-treatment of cells with ACM+IPA induced significant alterations in extracellular-matrix, adherens junctions, and regulation of the actin cytoskeleton. Conclusions: The addition of IPA to adipocyte-conditioned MDA-MB-231 cells appears to alter the expression of extra cellular matrix related genes that will potentially discourage metastatic behavior. Future studies focusing on these specific gene expression changes are in progress.

Context: Pseudomonas aeruginosa is a Gram-negative, aerobic bacterium responsible for approximately 51,000 nosocomial infections annually in the United States. Primary complications include nosocomial pneumonia and urinary tract infections, as well as septicemia and surgical wound infections. P. aeruginosa has a high mortality rate of 38% in patients who acquire ventilator-associated pneumonia. Additionally, previous studies have shown that patients who recover from pneumonia present later with effects of end-organ damage and a higher risk of cardiovascular-related deaths from myocardial infarction and stroke. One potential cause of end-organ damage in the context of Pseudomonas infection is the release and propagation of the type-three secretion system effectors ExoU- or ExoY induced amyloidogenic proteins from the initial infection site to secondary organs. S-(-)-Oleocanthal, a monophenolic compound found exclusively in extra-virgin olive oil, protects the brain from damage in models of neurodegenerative disease, thought to be caused by amyloidogenic proteins.

Hypothesis: This study investigates the hypothesis that pre-treatment of lung endothelial cells with oleocanthal will impair ExoY activity, decreasing the level of the cyclic nucleotides (cGMP and cAMP) generated, number of amyloidogenic proteins produced, and cytotoxicity of resulting supernatant. Methods: Rat pulmonary microvascular endothelial cells were grown to 12-24 hours post-confluence and serum starved for one hour in Hanks Balanced Salt Solution. Cells were then treated for two hours with either an oleocanthal concentration of 0.5 or 1.0 µM, or dimethyl sulfoxide vehicle control. Next, cells were inoculated with ExoY with or without oleocanthal for 3.5 hours, at which point cell lysates and supernatants were collected. Supernatants were centrifuged, filter-sterilized, and heat and cold-shocked prior to transfer to another set of confluent endothelial cells to observe cytotoxic propagation. ELISA and Western blotting were employed to identify the presence of intracellular ExoY and the amyloidogenic proteins amyloid-beta and Tau in the cell lysates and supernatants. Densitometry was used to quantify the results from the Western blots.

Results: Densitometry of Western blot results revealed that oleocanthal treatment did not impact the level of ExoY present following inoculation. However, a decrease in intracellular amyloid precursor protein (APP) was seen at both 0.5 and 1.0 µM of oleocanthal, and a decrease in various Tau species was seen with oleocanthal. Oleocanthal treatments appeared to protect against the cytotoxicity of the cell supernatant when applied to the naïve cell monolayers. Conclusions: Results suggest that the olive oil phenolic oleocanthal hinders ExoY’s ability to induce protein aggregation and its downstream cytotoxic effect without impacting the amount of ExoY injected into the host cells.

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2025 Research Recognition Day