DETERMINATION OF THE EFFECT OF CYCLOHEXYLMETHYLPARABEN ON ACTIVATION OF APOPTOTIC CASPASE-3 IN M624 MELANOMA CELLS Ryan M. Menapace This thesis is submitted in partial fulfillment of the requirements of the Research Honors Program in the Department of Chemistry and Biochemistry Marietta College Marietta, Ohio May 6, 2020 Menapace 1 This Research Honors thesis has been approved for the Department of Chemistry and Biochemistry and the Honors and Investigative Studies Committee by Suzanne Parsons 5/6/2020 Faculty thesis advisor Date David Brown______________________ 5/6/2020 Thesis committee member Date Menapace 2 Foreword Due to the outbreak of COVID-19, work on this project came to an abrupt end before additional caspase-3 activity assays could be completed in triplicate with statistical significance. Data associated with the caspase-3 activity assays is representative of 2 individual sets of data. The inability to receive additional materials for assays from the manufacturer and the suspension of all academic activities at Marietta College have limited the conclusions to this study, but the data presented is as accurate and thorough as possible under the given circumstances. Menapace 3 Acknowledgements This project would not have been possible without the support of Marietta College’s Department of Chemistry and Biochemistry in addition to the support of the Honors Program and Investigative Studies Program. The resources provided by all of these programs were vital to the completion of my project. The knowledge I obtained throughout my education in biochemistry and chemistry were invaluable tools for this process. Special thanks to Dr. David Brown for serving on my thesis committee and providing insight on my project throughout its completion. I would also like to express the highest amount of gratitude and thanks to Dr. Kimberly Suzanne George Parsons for working with me on this project, providing invaluable knowledge, and for being an exceptional mentor. I could not have come this far without all of the help that you both have provided. Also, I would like to extend a thank you to my family, roommates, and girlfriend for supporting me throughout this project. Finally, I would like to dedicate this project to my late uncle. His battle with cancer not only inspired me to begin this research, but it also motivated me to complete my work in order to contribute to the scientific community and further research on cancer treatments. Menapace 4 Table of Contents Abstract...........................................................................................................................................5 Introduction....................................................................................................................................6 Cyclohexylmethylparaben and membrane permeability………...6 Melanoma and cell death…………………………………………...8 Cell death signaling…………………………………………………10 Overview………………………………………………………….....14 Preliminary Data……………………………………………………16 Methods…….................................................................................................................................18 Cell Culture…………………………………………………………18 Cyclohexylmethylparaben synthesis………………………………18 Cyclohexylmethylparaben solution preparation………………….19 Clonogenic assays…………………………………………………...19 Cell treatment for caspase-3 colorimetric assays…………………20 Bradford protein concentration assays……………………………21 Caspase-3 colorimetric assays……………………………………...21 Statistical analysis…………………………………………………...22 Results...........................................................................................................................................23 Discussion.....................................................................................................................................27 References.....................................................................................................................................30 Glossary........................................................................................................................................33 Menapace 5 Abstract Melanoma is the deadliest form of skin cancer that affects the melanin-producing cells in the human body and it accounts for 60 – 80% of all skin cancer-related deaths. The prevalence of this disease has been increasing during the past several decades and the desired type of cell death in the elimination of these cells is apoptosis. Parabens are a class of organic, antimicrobial compounds widely used in industrial, food, and cosmetic products and have also displayed cytotoxic effects towards cancerous human cells. Cyclohexylmethylparaben is a newly synthesized paraben that has been shown to be cytotoxic to human M624 melanoma cells. Human M624 melanoma cells were treated with 0.35, 0.45, 0.55, and 0.65 mM cyclohexylmethylparaben and then cell viability was analyzed with clonogenic assays using crystal violet dye. An IC50 value of 0.4768 mM was calculated for cyclohexylmethylparaben in human M624 melanoma cells. Caspase-3 activity assays were performed to analyze the activation of the caspase-3 pathway to determine if apoptosis occurred in treated human M624 melanoma cells. Cyclohexylmethylparaben was found to induce its highest levels of caspase-3 activation at 0.35 mM when compared to the other treatment groups and the control group. This data suggests that cyclohexylmethylparaben could be a useful compound in the activation of apoptosis and treatment of human melanoma. Menapace 6 Introduction Cyclohexylmethylparaben and membrane permeability Parabens are organic antimicrobial preservatives that are found in industrial products, pharmaceuticals, and food commodities and can also be found in widely used personal care and cosmetic goods.20 These organic compounds are one of three categories of preservatives found in the aforementioned products in addition to triclosan and benzophenones.20 Parabens are a family of alkyl esters and paraben stands for para-hydroxybenzoic acid.3 There are five variations of parabens that are widely used in consumer products: ethylparaben, butylparaben, benzylparaben, methylparaben, and propylparaben.3 These parabens differ in one part of their chemical structure that alters their solubility, which can affect the ability of the compound to be absorbed through the skin, allowing the paraben to affect skin cells.3 Increasing the length or size of the alkyl chain, or functional group, in parabens can increase the lipid solubility of the compounds which allows for increased penetration of the human epidermis.3 The penetration of human epidermis is one way that these organic compounds can be introduced into the skin and potentially kill cancerous cells.3 Cyclohexylmethylparaben is an amphipathic compound - it has both a polar and nonpolar region, and this nonpolar region is hydrophobic so it can easily dissolve in the lipid bilayer of a cell membrane.22 This lipophilicity could allow the paraben to diffuse through cell membranes and penetrate cancerous cells, possibly triggering apoptosis through an intrinsic mechanism. Lipophilic molecules have the capability to flip between the inner and outer leaflets, or layers, of the lipid bilayer membrane; this leaflet flip can facilitate diffusion of a compound into the cell and explain how such compounds can enter the cell and trigger apoptosis through an intrinsic mechanism.22 These Menapace 7 compounds can interact with the plasma membrane because they don’t have polar regions and are therefore not repelled by the hydrophilic headgroups of the phospholipids.22 Alkyl functional group Figure 1. Structure of cyclohexylmethylparaben Cyclohexylmethylparaben is an amphipathic compound, so it also contains a polar region consisting of ester and hydroxyl functional groups containing electronegative oxygen atoms. A polar, hydrophilic compound is not usually able to pass through the plasma membrane of cells due to the hydrophobic nature of the interior of the lipid bilayer plasma membrane.22 The phospholipids of the lipid bilayer orient themselves with their hydrophilic, polar head on the exterior of the bilayer. The polar nature of the head group on a phospholipid repels polar, hydrophilic molecules and prevents them from crossing the plasma membrane and entering a cell.22 For this reason, polar compounds are less likely to pass across a plasma membrane and might trigger the apoptotic cellular pathway via an extrinsic mechanism by binding to a receptor on the surface of a cell. Menapace 8 Since cyclohexylmethylparaben is amphipathic, it has both hydrophilic and hydrophobic characteristics that might affect its solubility in a cell membrane. Therefore, its size must also be considered as a factor impacting its ability to trigger apoptosis through either an intrinsic or extrinsic mechanism. Cyclohexylmethylparaben has a molecular weight of 234.30 g/mol and a chemical formula of C14H18O3. The cell membrane is typically permeable to small nonpolar 5 molecules such as CO2 and O2 and to small uncharged polar molecules such as H2O. On the other hand, the cell membrane is impermeable to the simple diffusion of large, polar molecules such as amino acids and glucose, which require transmembrane proteins to cross the cell membrane.5 Melanoma and cell death Over the last several decades, the prevalence of malignant cutaneous melanoma has increased around the world.7 It is one of the most aggressive forms of cancer and is responsible for approximately 60 – 80% of all skin cancer-related deaths.1 Making a