Investigation of Dinitroparaben-Induced Apoptotic Death in M624 Melanoma Cells

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

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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

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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.

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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

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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.

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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 This electron-withdrawing characteristic is due both to resonance and inductive effects.13 Primarily, addition of electron-withdrawing groups promotes nucleophilic aromatic substitution over aromatic substitution.13

Figure 1. Structure of Dinitroparaben Unlike most other parabens, dinitroparaben is a yellow crystalline powder, suggesting a higher degree of conjugation than existing parabens, which are usually white or colorless. This is a result of the resonance from the two nitro groups.

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Melanoma

While it is a relatively rare form of skin cancer, over 80% of skin cancer deaths are attributed to melanoma.15, 24 Though it varies across different geographic locations and demographic groups, metastatic melanoma can have a 5-year survival rate as low as 10%. 15, 24

Exposure to UV light is the strongest risk factor for melanoma, aside from genetic risk factors.18Another well-known risk factor is the presence of atypical nevi, commonly known as moles.18, 24

UV exposure triggers the synthesis of melanin, which helps protect the skin from UV radiation.18 This is why stronger intermittent exposure is more dangerous than weaker continuous exposure.18, 24 When exposed to low doses over time, it results in gradual protection due to the continued synthesis of melanin. Strong intermittent exposure is more dangerous because it bypasses this protective mechanism and can induce the genetic damage that leads to cancer.18

Often this damage leads to disruption of the pathways that regulate apoptosis, also known as programmed cell death.13 While there are many factors that may facilitate or inhibit apoptosis, metastatic melanoma often lacks the ability to regulate this process such that the cells do not undergo apoptosis and instead continue to proliferate.13

While increasing awareness of the dangers of sun exposure has helped lower the incidence and mortality of melanoma, it still remains one of the most severe forms of skin cancer.24 Current research focuses on identifying genetic risk factors and developing new therapies.24

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Mechanisms of Cell Death

Apoptosis and necrosis are the two main types of cell death.13 Other forms of cell death also occur, but many have overlapping characteristics with apoptosis or necrosis, leading some to

9,13 describe apoptosis and necrosis as more of a continuum than a dichotomy.

While it is not the only form of regulated cell death, apoptosis is known as programmed cell death because it is a mechanism for cells to trigger their own death, which is why it is often referred to as a homeostatic mechanism.9,13 This process is signaled through two major pathways, one intrinsic and one extrinsic. A granzyme-mediated pathway is also utilized by certain immune cells.9

Apoptosis is characterized by chromatin condensation, membrane blebbing, cell shrinkage, and DNA fragmentation.13, 23 In the first steps of apoptosis, the cell shrinks as

9 chromatin condenses. In the next steps, the cell membrane begins to protrude in a process known as blebbing.9 The nucleus and rest of the cell fragments into apoptotic bodies, which are then engulfed and removed by phagosomes.13

Necrosis is the other common form of cell death. This mechanism is also described as oncosis or oncotic necrosis.9,13 The key characteristic of necrosis is cell swelling, as well as

9 organelle swelling and distension, which can eventually lead to rupture of the cell membrane.

The cytoplasmic components of the cell are released into the surrounding area, resulting in the recruitment of immune cells that trigger inflammation.9 Because the cell contents in apoptosis are contained within apoptotic bodies, inflammation does not occur.9 Necrosis and apoptosis can occur separately or simultaneously.9,13 It is possible for cells to utilize both mechanisms in McNeer 11 response to the same stimuli; certain stimuli can trigger apoptosis, but the same stimuli can trigger necrosis at higher concentrations or strengths.9 McNeer 12

Signaling and Initiation of Apoptosis

In the extrinsic pathway, apoptosis is triggered by the activation of death receptors.13

These receptors are part of the tumor necrosis factor gene superfamily, which share a cytoplasmic domain known as the death domain.9

Examples of these receptors include fibroblast-associated (Fas), tumor necrosis factor receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptor (TRAIL-R).9,13,23 Binding of ligands to these receptors triggers association of an adaptor molecule, such as Fas-associated protein with death domain (FADD) or tumor necrosis factor receptor type 1-associated death domain (TRADD).4,13 These adaptor proteins bind procaspase-8 through association of their death effector domains (DEDs).4,13

This results in formation of the death-inducing signaling complex (DISC), which includes the death domain of the receptor, the death effector domain of the adaptor protein, and procaspase 8.13 When DISC binds to procaspase-8, procaspase-8 undergoes autolytic cleavage and becomes active caspase-8. Active caspase-8 can then activate caspase-3 to complete the initiation of apoptosis.

In the intrinsic signaling of apoptosis, certain stimuli, such as interactions with immune cells, trigger the release of cytochrome c from the mitochondria.13,23 These stimuli can be positive signals, like DNA damage, reactive oxygen species, and toxins. 7 These stimuli can also be negative signals, usually due to a lack of certain hormones and growth factors that normally promote cell survival.7

Several proteins are responsible for relaying the signal to the mitochondria, which mediates the intrinsic activation of apoptosis.13 These proteins are all members of the Bcl-2 McNeer 13 family. Within this group of proteins are those that promote apoptosis, such as Bcl-2 interacting domain (BID), Bcl-XL/ Bcl-2-associated death promoter (BAD), PMA-induced protein (NOXA), and p53-upregulated modulator of apoptosis (PUMA).7,9,13 Similarly, there are several proteins in the Bcl-2 family that inhibit apoptosis.9

This series of non-receptor protein interactions promotes pro-apoptotic Bcl-2 associated

X protein (BAX) and Bcl-2 antagonist killer 1 (BAK) to trigger the formation of a pore in the mitochondrial membrane that allows for the release of cytochrome c into the cytosol. 7,9

Additional proteins are released through this pore that inhibit IAPs, inhibitors of apoptosis, thus promoting apoptosis.7

Once cytochrome c is released, it binds to adapter protein apoptotic protease activating factor 1 (APAF-1), which trigger a series of conformational changes that exposes the normally hidden caspase recruitment (CARD) domain of APAF1.7,13 Additionally, these conformational changes also expose a domain that allows for formation of an APAF1 oligomer known as the apoptosome.7, 9 Several procaspase-9 proteins can then bind to the exposed CARD domains in the apoptosome.7 Once activated, caspase 9 can activate caspase 3 to finish the initiation of apoptosis. 7

In some cases, both the intrinsic and extrinsic pathways can be used to signal apoptosis.

In some cells, low recruitment of adaptor protein and caspase-8 in DISC leads to insufficient signaling of apoptosis.14 However, even a low amount of caspase-8 activated in DISC can cleave and activate BID in the cytosol.14 Cleaved BID is transported to the mitochondria and signals apoptosis following the usual remaining steps of the intrinsic signaling pathway.14 Another example of interaction between the two pathways is DNA-damage induced apoptosis, in which p53 activates both the intrinsic and extrinsic pathways to execute apoptosis.14 McNeer 14

Figure 2. Signaling Pathways of Apoptosis. The extrinsic pathway is shown on the left, characterized by the binding of a death ligand to its death receptor. The intrinsic pathway is shown in the middle section, triggered by a self-signal. The perforin/granzyme pathway is shown on the right. Execution of Apoptosis

Figure 3. Execution Pathway of Apoptosis.

During the execution phase of apoptosis, cells undergo changes that eventually result in the formation of apoptotic bodies, which are later marked for phagocytosis and destroyed. The McNeer 15 start of this phase is the activation of caspase-3, signaled through either the extrinsic or intrinsic pathway. Caspase-3 is therefore part of a group of caspases known as effector caspases.14

Once procaspase-3 is cleaved to its active form, caspase-3, it is responsible for the cleavage of several proteins in order to complete apoptosis. One of these proteins is poly (ADP- ribose) polymerase, abbreviated PARP. PARP is a protein normally responsible for DNA repair.

It recognizes and binds to DNA strand breaks. After binding to DNA, it adds ADP-ribose polymers to a series of nuclear proteins to help facilitate DNA repair.2,14 However, this process uses high amounts of NAD+ and ATP, and therefore depletes the cell’s energy storage.14

When PARP is cleaved by caspase-3, it splits into an 89 kDa fragment and a 24 kDa fragment.2 The 89 kDa fragment contains the active site for the enzyme, while the 24 kDa fragment contains the DNA binding domain.2 Thus, cleavage of PARP inactivates its DNA repair activity and conserves ATP and NAD+, which are later needed to finish execution of apoptosis.2 Depletion of these energy reserves can instead trigger necrosis.14 Some studies have shown that necrosis can be regulated and induced partly through activation of PARP and the resulting depletion of ATP.28

Caspase-3 and other executioner caspases also cleave a number of other proteins to complete apoptosis. For example, when the proteins fodrin and gelsolin are cleaved, the actin cytoskeleton is disrupted.14 Other proteins of the cytoskeleton, including intermediate filament proteins, are also cleaved.14 This helps the cell begin to lose its shape in preparation for the formation of apoptotic bodies. Cleavage of nuclear lamins leads to chromatin condensation and shrinkage, which is a necessary step before the formation of apoptotic bodies.14 Finally, cleavage and inactivation of a series of proteins responsible for cell-cell communications help promote apoptosis by interrupting or blocking certain survival signals.14 McNeer 16

Preliminary Data The purpose of the first investigation of the effects of dinitroparaben was to determine if dinitroparaben was lethal to M624 melanoma cells. A clonogenic assay, a type of cell viability assay, was used to quantify the number of living cells after treatment with dinitroparaben. The goal of this assay was to calculate an LC50, the concentration of dinitroparaben needed to induce death in 50% of the cells. Initial treatment concentrations were 2.5 mM, 5.0 mM, and 7.5 mM.

The first set of studies showed that the highest concentration, 7.5 mM, was not sufficient to induce 50% of the cells to die. Thus, an additional treatment group, 10.0 mM was also tested for the data shown below in Figure 1.

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Figure 4. Clonogenic Assay. Cells were plated in 12-well plates, treated with dinitroparaben, and incubated for 24 hours. The cells were then stained with crystal violet, and the absorbance was read at 550 nm. Data were standardized to the control. The results were tested for statistical significance, signified by *, using a single-factor ANOVA with Tukey HSD post hoc tests, with p < 0.01.

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The initial solvent used to dissolve the solid dinitroparaben was methanol. However, the results of these assays showed that methanol alone was inducing death in the cells, making it impossible to tell how many cells had died from dinitroparaben itself, and not methanol.

Additional solvents tested included ethanol, DMSO, dioxane, TMF, and acetonitrile.

Dinitroparaben was only soluble in 50% (v/v) acetonitrile, but this solvent had a similar effect to methanol.

The solution to this issue was to adjust the protocol for the preparation of dinitroparaben solutions to treat the cells. Initially, dinitroparaben was dissolved in pure solvent to obtain a 100 mM dinitroparaben solution. The appropriate volume of this stock solution was then added to cell media to create the four concentrations of dinitroparaben, which could then be applied to the cells. However, this method was the one that led to unreliable data due to the high concentration of solvent.

The adjusted protocol did not require the use of a solvent at all and instead dissolved the necessary amount of dinitroparaben directly into the medium Instead of trying to dissolve a relatively high amount of solid in a small volume of solvent, the dinitroparaben was added directly to a much larger volume of cell media. While it initially appeared that dinitroparaben was not soluble in media, this was when a solubility test was performed following the first method, in which dinitroparaben was dissolved in only 1-2 mL of media to create the 100 mM stock solution. Dinitroparaben could dissolve directly into the volume of media needed to make a

10 mM stock solution because this volume was 50 to 100 times greater than the volume used in the first method. The lower concentrations were subsequently prepared using a dilution series, which also helped to maintain consistency between the concentrations of each treatment group. McNeer 18

The average value for each concentration was fitted linearly and used to calculate the

LC50. This value was 7.20 mM, indicating the concentration of dinitroparaben needed to cause

50% of the treated cells to die. The results were tested for statistical significance compared to the control using a single-factor ANOVA with Tukey HSD post hoc tests, with p < 0.01.

C 2.5 5.0 7.5 10.0 PARP (113 kDa)

Cleaved (89 kDa)

β-actin (42 kDa)

Figure 5. Western Blot. SDS-PAGE was used to separate the proteins based on molecular weight. The gel was then electroblotted onto a nitrocellulose membrane and blocked with 5% (w/v) dry milk in TBST for 30 minutes. Primary antibody was added in the correct dilution and the membrane was incubated overnight at 4° C. After incubation with the primary antibody, a secondary antibody was added, and the membrane was incubated at room temperature for one hour. Chemiluminescence was used to visualize the protein bands. The density of the bands was quantified using ImageJ.

Using the clonogenic assay, it was determined that dinitroparaben did induce cell death in the melanoma cells. The next step of the investigation was to determine the method of cell death.

Proposed as a potential cancer treatment, it was important to learn if the treatment triggered death via apoptosis or necrosis. Many current cancer treatments have a wide variety of side effects, some severe. The ideal compound would trigger apoptosis, as this would result in fewer harmful side effects than necrosis.

As discussed previously, after apoptosis is triggered either through the extrinsic or intrinsic pathway, caspase-3 is eventually activated. Once active, caspase-3 binds to and cleaves

PARP. Thus, cleavage of PARP indicates that apoptosis has occurred through either activation pathway. Western blot was used to detect cleaved PARP in cell lysate samples from M624 cells McNeer 19 treated with dinitroparaben. Cells were treated with the four dinitroparaben concentrations, lysed, and prepared with protein loading buffer before electrophoresis. The preparation of these samples and the western blot procedure are described in detail in the methods section.

The results of the PARP western blot showed that PARP was being cleaved in the samples treated with dinitroparaben. Additionally, the results showed a decrease in PARP cleavage at 10.0 mM compared to the lower concentrations.

Figure 6. PARP Western Blot. Results were tested for statistical significance, signified by *, using a single-factor ANOVA with Tukey HSD post hoc tests, with p <0.01.

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Objectives The preliminary clonogenic assay data showed that dinitroparaben did induce cell death in the M624 cells. Further studies showed that PARP was cleaved in the treated cells, indicating that the mechanism of cell death was apoptosis.

The next step in this investigation was to verify that apoptosis is the mechanism of cell death by confirming that caspase-3 is activated to cleave PARP. This was measured using a colorimetric assay that provided the substrate for caspase-3 and recreated the reaction conditions.

To further investigate the apoptosis signaling pathway, western blot was used to measured total cytochrome c.

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Methods Cell Culture

M624 human melanoma cells were cultured in 100 mm tissue plates using Dulbecco’s

Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin and penicillin at 37°C with 5% CO2.

Paraben Synthesis

Dinitroparaben was synthesized in Dr. Kevin Pate’s lab. 1.088 g of 4-hydroxy-methyl- benzoate and 2.25 ml of concentrated sulfuric acid were added to a 50 ml Erlenmeyer flask on ice. The solution was stirred until the solid dissolved.

0.75 ml of concentrated nitric acid and 0.75 ml concentrated sulfuric acid were combined and cooled on ice. This mixture was added dropwise to the Erlenmeyer flask over 10-15 minutes.

The solution was stirred for an additional five minutes, then left to cool on ice for an additional five minutes without stirring.

The solution was poured directly into ice in a new beaker and the ice was allowed to melt. The resulting orange/yellow crystals were separated using vacuum filtration and washed with 5 mL of cold distilled water twice, then 1.5 mL of cold methanol twice. The product was then transferred to a watch glass and dried.

Western Blot Sample Preparation

In addition to a control, four concentrations of dinitroparaben were prepared. These concentrations were 2.5 mM, 5.0 mM, 7.5 mM, and 10.0 mM. M624 cells were cultured until there were at least four 100 mm tissue plates per dinitroparaben concentration at 90% confluency. The highest concentration solution, 10.0 mM was prepared by dissolving McNeer 22 dinitroparaben directly into the cell medium in a sterile culture flask. The three subsequent concentrations were prepared via dilution series to maintain consistency between the concentration increments. The cell media was removed and 6 ml of the appropriate dinitroparaben solution was added to each plate. The same volume of fresh media was added to the control. The cells were then incubated for 24 hours.

After incubation, the media from each plate was collected in a 15 ml centrifuge tube on ice. Each plate was washed using 0.5 mL of cold phosphate-buffered saline (PBS), which was also collected and added to the appropriate 15 ml centrifuge tube. To collect adherent cells, 1 mL of cold PBS was added to each plate. The plates were scraped using the back of 1 ml plastic pipettor tip and the cells were collected in 1.5 mL microcentrifuge tubes. Both the 15 ml and 1.5 ml tubes were centrifuged at 5000 x g for 10 minutes. The supernatant from each tube was removed and discarded. 0.5 ml of cold PBS was used to combine cell pellets of the same concentration in a 1.5 ml microcentrifuge tube. These were centrifuged at 5000 x g for ten minutes.

After centrifugation, the supernatant was discarded. Lysis buffer with 1% Triton-X-100 and HALT protease inhibitors (Thermo Fisher, #78430) were added to each pellet. The pellet was resuspended and incubated on ice for 15 minutes, mechanically sheared using a 20-gauge needle 20 times, then incubated on ice for an additional 15 minutes. The samples were centrifuged at 5000 x g for 5 minutes. The supernatant, which contained the proteins, was collected in new microcentrifuge tubes.

The remaining volume was prepared for electrophoresis using 5X protein loading buffer.

The correct volume of buffer was added to each sample in a microcentrifuge tube, which was McNeer 23 vortexed, boiled for five minutes, and then vortexed again to complete the preparation of the protein samples for Western Blot.

Western Blot

Proteins were separated using discontinuous SDS-PAGE at 200 V for 45 minutes, with

4% polyacrylamide stacking gel buffered at pH= 6.8 and 10% polyacrylamide separating gel buffered at pH = 8.8 The gel was then electroblotted onto a nitrocellulose membrane at 100 V for

35 minutes. The membrane was blocked with 5% (w/v) dry milk in TBST for 30 minutes.

Primary antibody was added in the correct dilution and the membrane was incubated overnight at

4° C with shaking. (β-actin: Sigma Aldrich a5441, working dilution 1:6667; PARP: Invitrogen,

PIMA515031 working dilution 1:1000; cytochrome c: Santa Cruz Biotechnology, sc-13156, working dilution 1:500).

After incubation with the primary antibody, the membrane was rinsed and washed with

1X Tris-buffered saline with 0.1% Tween 20 detergent (TBST) three times. Secondary antibody was added, and the membrane was incubated at room temperature for one hour (anti-mouse:

Sigma Aldrich, a9044; anti-rabbit: Invitrogen, G-21234). The secondary antibody was removed, and the membrane was rinsed and washed three times with TBST. Chemiluminescence solution was applied for five minutes (ClarityTM Western ECL Substrate, Biorad, 170- 5061). The membrane was exposed to film for 5-30s. The film was immersed in developer and exposed to the air 20 times, rinsed with distilled water, then immersed in fixer. The protein ladder was used to identify the molecular weight and identity of the bands. The density of each band was quantified using ImageJ, an image processing software from the National Institutes Health.

Caspase-3 Assay Sample Preparation McNeer 24

The activation of caspase-3 was measured using a colorimetric assay from BioVision (K-

106). Cells were treated using the same procedure as the samples for Western Blot and lysed in a similar manner. Instead of adding lysis buffer and protease inhibitor, 50 μL of the assay kit’s cell lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol) was added to each pellet. The cell pellet was resuspended and allowed to sit on ice for 10 minutes. The samples were then centrifuged for 5 minutes at speed 7. The supernatant was collected in new microcentrifuge tubes.

Bradford Assay

Bradford reagent, containing 0.01% (w/v) Coomassie Brilliant Blue G-250 in 4.75% (v/v) ethanol and 8.50% (v/v) phosphoric acid was prepared. Standards of 0, 0.2, 0.4, 0.6, 0.8, and 1.0

μg/μL protein were prepared using bovine serum albumin (BSA). Each standard was plated in a

96-well plate. The caspase samples were diluted 1:10, then plated in triplicate. Bradford reagent was added to each standard and sample well. Any large bubbles were popped with a needle. The absorbance was read at 595nm on a Biotek Epoch microplate spectrophotometer. A standard curve was used to quantify the protein in each sample.

Caspase-3 Assay

Based on the results of the Bradford Assay, the volume of each sample needed to obtain

50 μg of protein was determined. This volume was added to a new microcentrifuge tube and diluted to 50 μL using the kit’s cell lysis buffer. 50 μL of 2X reaction buffer with 10 mM DTT and 5 μL of DVED-pNA were added. This procedure was repeated for each sample. If there was sufficient volume, solutions were prepared in duplicate or triplicate for each concentration. The McNeer 25 solutions were incubated at 37 °C for 1.5 hours, then the entire volume of each solution was plated in a 96-well plate. The absorbance was read at 400nm.

Statistical Analysis

Samples were tested for significance from each other using a single-factor ANOVA

(Analysis of Variance) with p < 0.05. Tukey HSD (Honest Significant Difference) tests were used to determine which concentrations were significantly different from the control, with p <

0.01.

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Results The activation of caspase-3 was measured using a colorimetric assay. The absorbance of pNA was used to quantify the amount of caspase activation. The results, shown below in Figure

7, showed that the absorbance increased in the cells treated with dinitroparaben, especially in the

5.0, 7.5, and 10.0 mM concentrations. The average absorbance value for the control was about

0.1, while the highest value reported was about 0.16, or about a 60% increase. Thus, activation of caspase-3 also increased in the treated cells. Statistical analysis showed that the results for the

5.0, 7.5, and 10.0 mM concentrations were significantly different from the control.

* * *

Figure 7. Caspase 3 Assay. The data shown represents eight sets of data. The results were tested for statistical significance, signified by *, using a single-factor ANOVA with Tukey HSD post hoc tests, with p < 0.01.

Results for the western blot for cytochrome c showed that the amount of cytochrome c decreased in the cells treated with dinitroparaben. The untreated control had the highest amount of cytochrome c, while the 7.5 mM and 10.0 mM concentrations had very little or almost no McNeer 27 cytochrome c. The data shown represents one trial. A second set of data is available in Appendix

A; however, the background signal made it impossible to obtain reliable data using ImageJ.

Overall, that data showed the same decreasing trend.

Figure 8. Western Blot for Cytochrome C. B-actin was used as a standard.

Figure 9. Data for the western blot for Cytochrome C. Data shown represents one trial.

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Discussion The first hypothesis in this investigation was that dinitroparaben would induce cell death in M624 human melanoma cells. The results of the preliminary data, specifically the clonogenic assay, showed that treatment with dinitroparaben caused a linear decrease in cell viability as the concentration increased, with the 10.0 mM concentration resulting in the most cell death.

Statistical analysis showed that the data for the 5.0, 7.5, and 10.0 mM concentrations were significantly different than the control, further strengthening support for the hypothesis.

Therefore, the conclusion was that dinitroparaben did induce cell death in the M624 cells.

The second hypothesis of this investigation was that dinitroparaben induces cell death via apoptosis, as opposed to another form of cell death. The first experiment performed to test this hypothesis was a western blot for PARP, specifically cleaved PARP. It was hypothesized that

PARP cleavage would increase as the concentration of dinitroparaben increased.

PARP is cleaved by caspase-3 during the execution phase of apoptosis and thus serves as an indication that apoptosis has occurred. The results of this experiment showed that PARP was cleaved in the treated cells. Statistical analysis showed that the results for the 5.0 mM, 7.5 mM, and 10.0 mM concentrations were significantly different than the control. Therefore, it was concluded that there was strong evidence that the mechanism of cell death was apoptosis.

The results also showed that PARP cleavage decreased in the 10.0 mM treatment group, which was not part of the original hypothesis based on the clonogenic assay results. One possible explanation for this is that at higher concentrations, dinitroparaben may trigger an alternate mechanism of cell death such as necrosis. This would account for the apparent decrease in PARP cleavage and resulting decrease in apoptosis, but the continued increase in overall cell death as the concentration of dinitroparaben increased. McNeer 29

A second experiment, the caspase-3 assay, was performed to verify that apoptosis was occurring. This assay tests for activation of caspase-3 by providing its substrate, DEVD-pNA.

DEVD is an abbreviation for the amino acids aspartate-glutamine-valine-aspartate, referring to the sequence of amino acids on PARP that caspase-3 binds to in order to cleave PARP. When caspase-3 binds to this substrate, it cleaves the pNA, a chromophore whose absorbance can be read at 400-405nm in order to quantify the activity of caspase-3.

The results of this assay showed that activity of caspase-3 increased in the treated cells, especially in the higher concentrations. Any activity of caspase-3 in the control can be attributed to cells undergoing natural death, which occurs through apoptosis. One source of this could be overcrowding in the culture plate, which can result in cell death. However, this activity was minimal compared to the treated cells and statistical analysis showed that the 5.0, 7.5, and 10.0 concentrations were significantly different than the control.

While the data for the caspase-3 assay overall was reliable, a potential source of error was the color of the dinitroparaben. The dinitroparaben is a yellow solid that results in a bright yellow solution when mixed with medium for treatment of the cells. During the preparation of the samples for the assay, the solutions retain this yellow color when the cells are centrifuged.

Thus, the original assay resulted in protein samples with a yellow color, which absorbs light in the same 400-405 nm range as pNA. The results of this specific assay showed an extreme increase in activation compared to the control, which was most likely due to the yellow color of the solution rather than the absorbance of pNA.

This problem was addressed by washing the cell pellets with PBS several times until the pellet and solution were colorless, before adding cell lysis buffer. While this appeared to fix the color problem, small fractions of the cell pellet were lost in each wash, making the final protein McNeer 30 sample less concentrated. This meant that for each cell treatment cycle, only one set of data could be collected, as opposed to collecting data in duplicate or triplicate. To account for this, the assay was performed eight times to collect enough data for analysis. These assays yielded good data with low standard deviations, suggesting that washing the pellets was a reliable solution to the problem of the color of the dinitroparaben.

Results of the western blot for cytochrome c showed that cytochrome c decreased in the cells treated with dinitroparaben, with little to none in the 7.5, and 10.0 mM concentrations. This was the opposite of the expected result, where it was hypothesized that cytochrome c would increase if apoptosis was being signaled through the intrinsic pathway.

Ferraro et al. reported that cytochrome c was degraded by a ubiquitin-proteasome mechanism.10 The experiment showed that in cells modified to lack APAF-1, such that cytochrome c could not bind and form the apoptosome, cytochrome c was degraded.10 Further experiments showed that cytochrome c was also degraded in cells with functional APAF-1 and apoptosome formation. Ubiquitination was confirmed using immunoprecipitation targeted at ubiquitinated cytochrome c.10 Gama et al. found that cytochrome c was degraded via the proteasome in several mouse cell lines and that signaling of apoptosis through the intrinsic pathway prevented this degradation.12

Therefore, one possible explanation for the results of the cytochrome c western blot is that apoptosis is being signaled through the extrinsic pathway and that cytochrome c is then ubiquitinated and marked for degradation. Higher concentrations of cytochrome c are seen in the control because apoptosis is not induced, and cytochrome c continues to carry out its normal functions in the mitochondria. In the higher concentrations of dinitroparaben, more cytochrome c is degraded upon apoptosis. The results presented by Gama et al. suggest that this degradation McNeer 31 would have been prevented if apoptosis was signaled through the intrinsic pathway, suggesting that dinitroparaben-induced apoptosis may be signaled through the extrinsic pathway and cytochrome c is degraded. However, Ferraro et al. found that cytochrome c could also be degraded in the intrinsic pathway.

Jemmerson et al. found that cytochrome c was not degraded in apoptotic cells, but instead was present in the cell medium after release from the mitochondria.14 While further studies would be needed for this investigation to determine which of these hypotheses are supported both explain why cytochrome c might have decreased in the western blot.

One final consideration is the fate of cytochrome c in other mechanisms of cell death. As mentioned previously, decreases in levels of PARP cleavage and caspase-3 activation suggest that apoptosis decreases in the higher concentrations, and another form of cell death may be occurring. In order for the cytochrome c results to support this hypothesis, cytochrome c must also be degraded in the second mechanism of cell death that takes place. Jemmerson et al. reported that cytochrome c was found in the cell media in necrotic cells, in addition to apoptotic cells.14 Additionally, this study showed that cytochrome c is released from the mitochondria during necrosis, which means it could possibly be marked for degradation as suggested by

Ferraro et al.10,14 Therefore, the consistent decrease in cytochrome c could also be a result of necrosis and support the results of the PARP western blot and the caspase- 3 assay.

Future Studies Further investigation of the signaling pathway of apoptosis would be needed to make a conclusion about which pathway is being used in dinitroparaben-induced apoptosis. Testing for cytochrome c on isolated mitochondrial and cytoplasmic fractions would help determine if cytochrome c is truly released from the mitochondria. If these results show that it is not, further McNeer 32 experiments could be conducted to test for signs of the extrinsic pathway, such as testing for the activation of death receptors.

After investigation of dinitroparaben is complete, the next step would be to test dinitroparaben against a non-cancerous human skin cell line. These results would help determine if dinitroparaben could be a viable cancer treatment. If dinitroparaben is harmful to the non- cancerous cells, this could mean that it will not be an ideal treatment. However, it would be useful to investigate the mechanism of cell death that occurs in the non-cancerous cells to determine if the benefits outweigh the side effects on the surrounding healthy tissue.

Additionally, the results of this experiment and testing against non-cancerous skin cells may help inform the synthesis of novel parabens in the future.

McNeer 33

Appendix A. As noted in the results, only one clear image of cytochrome c western blot was acquired.

However, one additional image with a significant amount of background was obtained. Several attempts to remove background were made in hopes of obtaining a clearer image, but none of these efforts resulted in an image that was clear enough to be analyzed by Image J. This image is shown below.

Figure 10. Cytochrome c western blot. Image was enhanced using the Remove Background and Increase Contrast features in Image J.

Figure 11. Western Blot for Cytochrome C with high levels of background. While the same overall trend is observed, a significant amount of signal is present in the higher concentrations that was not present in the data in Figure 9. McNeer 34

While the data is not quantifiable, the image appears to show a decrease in cytochrome c. The signal in the 5.0, 7.5,and 10.0 mM concentrations is relatively higher than the signal in the other set of data (Figure 9), but this may be due to background. However, the same overall trend is shown in which the amount of cytochrome c decreases compared to the control. Thus, while not quantifiable, this result supports the conclusions made about the role of cytochrome c in this experiment.

McNeer 35

Appendix B As noted in the possible future directions for this study, testing dinitroparaben against healthy, non-cancerous, human skin cells would be necessary to determine jf dinitroparaben is a viable cancer treatment. If dinitroparaben effectively kills the melanoma cells, but also kills the healthy skin cells, it will not be an ideal treatment. To this end, preliminary studies of dinitroparaben with HaCaT human keratinocytes were carried out.

* * *

Figure 11. Clonogenic Assay for HaCaT. Data shown represent two trials. The results were tested for statistical significance, signified by *, using a single-factor ANOVA with Tukey HSD post hoc tests, with p < 0.01. Figure 6 shows the results of the clonogenic assay on HaCaT cells. The data was analyzed the same as the M624 clonogenic assay. The calculated LC50 value was 7.42 mM.

While ideally the HaCaT cells would be unaffected, the results show a steep decrease in viability when treated with dinitroparaben, especially in the higher concentrations. Further studies are needed to determine if apoptosis or necrosis is occurring, as dinitroparaben could potentially still McNeer 36 be used as a cancer treatment depending on the severity of the side effects caused to healthy skin cells.

Additionally, while dinitroparaben may not be the ideal treatment, these results are significant for future studies regarding the synthesis of novel parabens and may inform future researchers of structures to alter or avoid when developing new parabens as potential treatments for cancer.

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