Study of Alpha Mangostin as a Chemoprotective Agent for Breast Cancer via Activation of the P53 Pathway

v LITERATURE REVIEW . 1 Breast Carcinoma and Treatments . 1 P53 Gene & Cascade . 3 P53 Mutation & Treatments . 3 P53 Activator Current Research . 4 Chemotherapy & Chemoprotection Treatment . 5 Alpha Mangostin as a Chemoprotectant . 6 Hypothesis. 7 MATERIALS & METHODS . 7 Culturing of MCF10A Cells . 7 Determining the Toxicity of Alpha Mangostin and Paclitaxel . 8 Differentiating between Viable, Apoptotic, and Necrotic Cells. 8 Development and Use of Cell Counting Program . 9 RESULTS. 9 DISCUSSION . 17 BIBLIOGRAPHY . 21 APPENDIX I . 23 ALPHA MANGOSTIN AS A CHEMOPROTECTANT iv LIST OF FIGURES Figure 1. Various Outcomes of the p53 Pathway . 2 Figure 2. The p53 Cascade . 3 Figure 3. Fluorescent Microscopy of MCF10A Wild-Type Cells after treatment with Paclitaxel . 10 Figure 4. Wild-Type MCF10A Paclitaxel Toxicity Curve . 11 Figure 5. Fluorescent Microscopy of MCF10A Wild-Type Cells after treatment with Alpha Mangostin . 12 Figure 6. Wild-Type MCF10A Alpha Mangostin Dose Response Curve . 13 Figure 7. Wild-Type MCF10A Alpha Mangostin & Paclitaxel Dual Treatment . 14 Figure 8. Fluorescent Microscopy of Wild-Type MCF10A Paclitaxel Treatment vs Dual Treatment with Alpha Mangostin . 14 Figure 9. Fluorescent Microscopy of MCF10A p53 (-/-) Cells after treatment with Alpha Mangostin . 15 Figure 10. MCF10A p53 (-/-) Alpha Mangostin Dose Response Curve . 16 Figure 11. Qualitative Observation of Percent of Open Surface Area in Well for Increasing Treatments of Alpha Mangostin . 17 ALPHA MANGOSTIN AS A CHEMOPROTECTANT v ABSTRACT Breast carcinoma is the most frequently diagnosed cancer among women and causes over 400,000 deaths each year worldwide. Current treatments such as chemotherapy are not selective for cancerous tissues but are destructive to normal tissues as well. This causes a range of side effects including pain, nausea, hair loss, weakness, and more. Inactivation of p53 is a very common mutation within human cancer cells. The ability to activate the p53 pathway which protects cells from tumor formation is lost in 50% of cancers. Due to the prevalence of this mutation, p53 is a uniquely valuable target for applied research. Alpha mangostin is an extract from a southeast Asian fruit, Garcinia mangostana. It has potential to be an effective p53 activator in which the small molecule disrupts the binding of p53 to MDM2, a negative regulator, inducing the p53 cascade which results in cell cycle arrest for low level stressors. This protects the cells from paclitaxel, a chemotherapy agent that only kills actively dividing cells. Here, we hypothesized that alpha mangostin protects wild-type, but not p53 (-/-), MCF10A breast cancer cells from the chemotherapeutic agent paclitaxel. When MCF10A wild-type cells were cotreated with alpha mangostin and paclitaxel, alpha mangostin exhibited a protective effect on the cells. However, when MCF10A P53 knockout cells were treated with alpha mangostin, cell viability decreased indicating a loss of protective effect in the p53 distressed cancer cells. These results further support treatments that target chemoprotection via p53 pathway in wild-type cells and the use of alpha mangostin warrants further study. Running Head: ALPHA MANGOSTIN AS A CHEMOPROTECTANT