Cell and Molecular Methods

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  Lab Rats

  Samantha Aragon, Cristina Herrera, Felicity Rodriguez, Riley Smith, and Terry Shackleford

  Ewing sarcoma is an aggressive cancer that affects children & adolescents. Common treatments include surgery, radiation, and chemotherapy, yet individuals have a poor survival rate and often develop side effects from treatment, including fertility issues, secondary cancer, heart problems, and growth abnormalities.1SOX18 is a gene identified to aid in cell proliferation and cell differentiation, leading to the development of several cancers, such as gastric, bladder, and lung cancer.2,3 SOX18 resides in the JAK2/STAT3 pathway as a regulator, along with another gene named KIT that instigates the formation of the cell signaling pathway.4KIT is a transmembrane tyrosine kinase, which instigates cell growth, differentiation, and survival.5,6Thus far, no relationship has been stated between SOX18 and KIT. Due to shared presence in the same pathway, this project proposes that when KIT is knocked out, then EWS cells will have an increased expression of SOX18, leading to increased cell growth and proliferation.

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  Test Tubes

  Victoria Castillo, Melany Cervantes, Nicole Robles, and Terry Shackleford

  SOX18, a transcription factor with a DNA-binding HMG domain, plays a critical role in regulatory processes linked to cancer progression. Exhibiting oncogenic properties, SOX18 has been linked to various cancers, where it promotes tumor growth by enhancing cell invasion, uncontrolled proliferation, and resistance to apoptosis through dysregulated signaling pathways. STAT1, a protein crucial for immune system regulation, is prominently expressed in immune-associated tissues like the lymph nodes, and bone marrow, suggesting it could play a role in tumor growth. Ewing Sarcoma (EWS), an aggressive cancer that targets bone and soft tissue, carries a high mortality rate of 90% without treatment and 75-85% even with standard therapies, with a substantial risk of relapse. Given the involvement of SOX18 in oncogenesis and the immune-regulatory role of STAT1, we hypothesized that SOX18 expression would result in increased STAT1 and that knockdown of STAT1 would reduce cell viability and increase apoptosis. This would suggest that SOX18 contributes to the survival and proliferation of EWS cells. Furthermore, if SOX18 is overexpressed into the cell line of STAT1, which is associated with necroptosis, it may decrease cell viability and produce more cancerous cells.

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  Minimal Anti-Cancer Effects of Mushroom-Derived Compounds Hispidin and Chaga Extract on Ewing Sarcoma Cells

  Jordan Cosgrove, Fiona Coulbourne, Iris Reyna, Giselle Serna-Flores, and Terry Jo Shackleford

  Ewing sarcoma is a rare and aggressive cancer of adolescents and young adults driven by the EWSR1–FLI1 gene fusion. Standard treatments include chemotherapy, surgery, and radiation, though survival rates remain low. Natural compounds such as hispidin, derived from fungi, and Chaga mushroom extract have shown potential anti-cancer effects by disrupting cell cycle progression and promoting apoptosis in other cancer models. In this study, we tested the effects of hispidin and Chaga extract on Ewing sarcoma cells to evaluate their ability to suppress cell growth and identify potential therapeutic benefits.

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  A Bitter Brew For Cancer: EGCG’s Impact on Ewing Sarcoma Cells

  Lizzeth Holguin, Mary-Esther Leblanc, Mariana Reyes, and Terry Jo Shackleford

  Epigallocatechin Gallate (EGCG), a powerful antioxidant found in green tea, is an abundant treatment to treat Ewing Sarcoma cells, a pediatric cancer affecting the skeletal system. Given EGCG’s known role in other cancer treatments, these tests aim to investigate its effects as a standalone natural compound on Ewing Sarcoma cells. Focusing on the PI3K/AKT pathway, if the PI3K/AKT pathway is inhibited by EGCG, then it will lead to reduced cell survival and proliferation of EW8 cells. Using cell culture techniques, IncuCyte-based IC50 analysis, caspase 3/7, RNA purification, cDNA synthesis, and qRT-PCR, EGCG’s impact was assessed on cell apoptosis and gene expression. Results showed that EGCG did affect Ewing sarcoma cells by inducing apoptosis in a dose-dependent manner, though its effects were less potent than hypothesized. These findings suggest that while EGCG exhibits anticancer properties, it may be more effective in a synergistic manner rather than as a standalone therapy.

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  The Effects of Hispidin and Eribulin Mesylate on Cell Proliferation of Ewing Sarcoma Cell Lines

  Elena Mares, Angelina Juarrita, Sierra Munoz, and Terry Jo Shackleford

  Ewing sarcoma is a type of cancer that targets the cells within bones and soft tissue and is most prevalent in younger populations. It is important to look at this specific cancer because there is no cure yet, and the risks increase, even after a patient has completed their treatment. We hypothesize that if we treat Ewing sarcoma cell lines with natural compounds, specifically Hispidin and Eribulin Mesylate, then the cell lines will decrease in cell viability and there will be an increase in apoptosis of the cancerous cell lines. We performed a series of experiments, which included IC50 analysis, Caspase-3/7 analysis, and qRTPCR. Our results showed that there was cell inhibition for both Hispidin and Eribulin Mesylate, apoptosis was induced for both Hispidin and Eribulin Mesylate, and there were changes in gene expression for both Hispidin and Eribulin Mesylate. With our gathered data, we were able to prove our hypothesis was supported due to the decrease in viable cells, increase in apoptosis, and changes in gene expression.

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  The Effect of Epigallocatechin Gallate (EGCG) and Alpha L-Mangostin on TP53, BAX, and VIM Gene Expression and Apoptosis in Ewing Sarcoma Cells

  Victoria Valdez and Terry Jo Shackleford

  Ewing Sarcoma (ES) is a malignant pediatric bone tumor driven by chromosomal translocations and fusion oncogenes with limited targeted treatment options. This study investigated the chemopreventive potential of two natural compounds, Epigallocatechin Gallate (EGCG) found in green tea, and Alpha L-Mangostin from mangosteen, on apoptosis and gene expression in ES cells. We hypothesized that EGCG and Alpha L-Mangostin treatment would induce apoptosis and downregulate cancer-promoting genes. To test this, we performed tissue culture, IC50 assays, caspase-based apoptosis detection, RNA purification, cDNA synthesis, and qRT-PCR on ES cell lines treated with a high and low concentration of the two compounds. Our findings showed that both compounds induced caspase-mediated apoptosis and altered the expression of target genes such as TP53, BAX, and VIM. Notably higher concentrations of EGCG reduced TP53 and VIM expression, while Alpha L-Mangostin significantly reduced BAX expression. These results support our hypothesis and suggest that EGCG and Alph L-Mangostin may contribute to cancer treatment strategies via gene regulation and apoptosis induction in Ewing Sarcoma cells.

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  The Effects of Panobinostat on Cellular Signaling Pathways and How it Relates to Antitumor Activities in Ewing Sarcoma Cancer Cells

  Crystal Valenzuela, Andrew Martini, Hannah Navarro, Mario Flores, and Terry Jo Shackleford

  Ewing sarcoma is a rare and aggressive cancer that primarily affects the bones and surrounding soft tissues, most commonly in children and young adults. Although the exact etiology remains unclear, a well-documented cause involves a chromosomal translocation resulting in the fusion of the EWSR1 and FLI1 genes. This fusion produces abnormal proteins that disrupt normal gene expression, cell signaling, and RNA processing, contributing to tumorigenesis. Prognosis varies significantly depending on the extent of metastasis, with 5-year survival rates ranging from 82% in localized cases to 39% in metastatic cases. Standard treatments include chemotherapy typically involving vincristine, doxorubicin, etoposide, and cyclophosphamide—surgical tumor resection, and limited use of radiation therapy. Chemotherapy often results in adverse side effects such as nausea, alopecia, mucositis, and diarrhea, particularly concerning in pediatric patients. Emerging therapeutic approaches include the use of histone deacetylase (HDAC) inhibitors such as Panobinostat. HDAC inhibition can restore the expression of genes involved in tumor suppression, including p53 and histones, promoting apoptosis in cancer cells. This mechanism has shown promise in various cancers, including prostate and breast cancer, and may offer potential benefits for Ewing sarcoma treatment by reactivating antitumor pathways suppressed by HDAC overexpression.

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  Fig Neutrons

  Allie Woods, Samuel De La Cruz, Catherine Garcia, and Terry Shackleford

  Multiple experiments were conducted to measure the effectiveness of certain genes on cell growth. In the exploration of this process, RAC3 acted as the target gene and was applied to both SOX18 overexpressing and normal expressing cells (control). RAC3 is a gene that typically controls the intercellular signaling pathway of a cell. This means it helps with the movement of cells and allows for migration.The purpose of these experiments were to determine how the gene RAC3 would work when introduced to carcinogenic cells, specifically Ewing Sarcoma. Typically, RAC3 acts as a messenger for the cell, but when in conjunction with cancer cells, it acts as pathway that controls cell growth in response to disease. By Targeting RAC3 inside of the ES cells that are overexpressed with SOX18, there should be an efficacy in the knockdown of the overexpressing genes. Thus, Ewing Sarcoma will no longer be able to produce cancer causing cells.