INVESTIGATION OF DINITROPARABEN-INDUCED APOPTOTIC DEATH IN M624 MELANOMA CELLS Sarah K. McNeer 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 3 May 2021 McNeer 2 This Research Honors thesis has been approved for the Department of Chemistry and Biochemistry and the Honors and Investigative Studies Committee by Kimberly Suzanne George Parsons 5/3/2021 Faculty Thesis Advisor Date David Brown 5/3/2021 Thesis Committee Member Date McNeer 3 Acknowledgements I would like to thank the Honors and Investigative Studies Program at Marietta College, which provided opportunities to work on this project during the summer, as well as providing financial support during research fellowships and for supplies. Additionally, I would like to thank the Marietta College Department of Chemistry and Biochemistry for supporting my career as an undergraduate researcher and providing funding for supplies and a summer research fellowship. I would also like to thank Dr. Kevin Pate, Chris Rasnake, Sophia Traussi, and Talitha Hochstetler for designing and synthesizing the paraben in this project. This project would not have been possible without their ideas and continued support. Special thanks to Dr. David Brown, for guiding me through my undergraduate research at various stages through Investigative Studies and Honors Thesis, and for serving on my Thesis committee. Lastly, I would like to thank Dr. Suzanne Parsons. Her mentorship has helped me reach both my personal and professional goals. She has been instrumental in the development of my skills as a scientific researcher, and it is thanks to her support that I will be pursuing a career in scientific research in the future. McNeer 4 Table of Contents Abstract ____________________________________________________________________ 5 Introduction _________________________________________________________________ 6 Parabens _________________________________________________________________________ 6 Dinitroparaben ____________________________________________________________________ 8 Melanoma ________________________________________________________________________ 9 Mechanisms of Cell Death __________________________________________________________ 10 Signaling and Initiation of Apoptosis __________________________________________________ 12 Execution of Apoptosis _____________________________________________________________ 14 Preliminary Data ____________________________________________________________ 16 Objectives _________________________________________________________________ 20 Methods ___________________________________________________________________ 21 Cell Culture ______________________________________________________________________ 21 Paraben Synthesis _________________________________________________________________ 21 Western Blot Sample Preparation _____________________________________________________ 21 Western Blot _____________________________________________________________________ 23 Caspase-3 Assay Sample Preparation __________________________________________________ 23 Bradford Assay ___________________________________________________________________ 24 Caspase-3 Assay __________________________________________________________________ 24 Results ____________________________________________________________________ 26 Discussion _________________________________________________________________ 28 Appendix A. ________________________________________________________________ 33 Appendix B ________________________________________________________________ 35 Literature Cited ____________________________________________________________ 37 McNeer 5 Abstract Parabens are organic compounds commonly used in cosmetic and food products due to their antimicrobial properties. Recent studies suggest that parabens accumulate in the body and have the potential to cause harm to healthy cells. Therefore, parabens could possibly be utilized to target cancer cells. M624 human melanoma cells were treated with dinitroparaben, a novel paraben. Cell viability was determined using clonogenic assay, which showed that dinitroparaben induced cell death. Western blot was used to detect and quantify the cleaved form of poly(ADP-ribose) polymerase, which indicates that apoptosis is occurring. Results showed that PARP was cleaved in the treated cells, suggesting that the melanoma cells underwent apoptosis. In the signaling pathway for apoptosis, PARP is cleaved by caspase-3. The current project employed an enzyme activity assay to measure the activation of caspase-3 in M624 cells treated with dinitroparaben. Results showed that caspase-3 activation increased in the treated cells, confirming apoptosis as the method of cell death. Western Blot was used to measure cytochrome c, which indicates involvement of the mitochondria and also serve as further verification that apoptosis is occurring. Results showed that total cytochrome c decreased as the concentration of dinitroparaben increased. Possible explanations for this decrease were that cytochrome c was degraded or transported out of the cell during the signaling of apoptosis. McNeer 6 Introduction Parabens Parabens are esters of p-hydroxybenzoic acid, commonly known for their use as preservatives in cosmetics and other personal-care products.25 They are also used in food products and some pharmaceuticals.25 Small amounts also occur naturally in the environment, produced by certain plants and bacteria.21 While parabens are generally regarded as safe, recent studies have found that they exhibit weak estrogenic activity and may act as a carcinogen.3,5 Although there is not sufficient evidence to prove parabens are more harmful than beneficial, these studies prompted many industries to alter products to a “paraben-free” formula. However, parabens are still classified as “generally regarded as safe (GRAS)” by the U.S Food and Drug Administration and are approved for use by the European Union.1,8 The EU limits the amount of a single paraben or paraben mixture allowed in each cosmetic product, and while the FDA recommends similar limits, there is no official regulation in the United States.6 Parabens differ in structure by the length of the alkyl chain of the ester. The most common parabens used are methylparaben, ethylparaben, propylparaben, and butylparaben, with methyl and propyl most prevalent in personal-care products and cosmetics.6,21 They can be used alone or in combination with other parabens; a mixture of methylparaben and propylparaben is common.1,8,21 The solubility of different parabens is related to the length of the alkyl chain. Methylparaben, for example, is the most soluble in water, while butylparaben is significantly less soluble.1,8,21. However, parabens with longer alkyl chains have better antimicrobial effects. Thus, combinations of different parabens are used to achieve ideal solubility and anti-bacterial properties in the desired product.3 McNeer 7 Parabens are an ideal preservative for multiple reasons. They have low or minimal toxicity, and are chemically inert.8, 25 They are relatively inexpensive, a driving factor in their widespread use by multiple industries.13 Parabens also have no taste or odor, and are usually white or colorless crystals or powders, making them ideal for food and cosmetic products.8,25 Compared to other available preservatives, like formaldehyde and urea compounds, parabens are relatively safe and present a much lower risk.21 A key point of interest is the mechanism of paraben absorption and metabolism. Multiple studies have shown that parabens are absorbed through the skin and digestive tract when ingested.8,25 Additionally, conjugates of parabens have been detected in human urine, supporting the idea that parabens are metabolized after being absorbed, rather than accumulating in their original forms.26 While the carcinogenicity of parabens is still disputed, some studies have shown that certain parabens can be toxic at high concentrations.1,21 While methylparaben and ethylparaben show very low toxicity, parabens with longer alkyl chains are more toxic.1,21 Butylparaben, and its branched form, isobutylparaben, are the most toxic, while propylparaben and isopropylparaben were noted as moderately toxic.1,21 However, it is important to note that these observations were due to the parent parabens, rather than their metabolized forms.21 Parabens are usually hydrolyzed by a group of esterases in the skin and liver.3 Inhibition of these esterases increased toxicity of propylparaben, suggesting that the parental forms of the parabens are responsible for toxicity.3,21 Methylparaben, while relatively harmless on its own, was shown to induce cell death after exposure to UV light.8 Since many parabens are used in topical products, exposure to UV light is a plausible mechanism for paraben toxicity. This study aims to harness this ability to determine if adjusting the structure of parabens can alter its toxicity such that it can be used to target cancer cells and induce cell death via apoptosis. McNeer 8 Dinitroparaben Dinitroparaben, a novel paraben, differs from currently available variants. Instead of altering the length or structure of the alkyl chain, dinitroparaben has two nitro functional groups at the 3,5 positions. While almost all other forms of parabens do not affect the aromaticity of the ring, the nitro groups are strong electron-withdrawing groups and significantly alter chemical reactivity.13