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
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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.
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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.
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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
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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.
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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 distinction between the biology and function of cancerous human melanocytes and healthy, normal human melanocytes is vital to developing treatments for such diseases. It is imperative that the drug or treatment being used effectively destroys cancerous human cells while not effecting the normal functioning cells.
In a healthy skin cell, there are mechanisms and enzymes that have essential roles in restricting the growth of melanocytes and promote healthy cellular death.1 In mutated melanocytes where these mechanisms are not functioning properly, cells can become malignant.
Cancerous cells are typically targeted with traditional medical treatments including prescription drugs, radiation therapy, and chemotherapy.16 Cancers can become resistant to conventional treatments, and patients often have issues with these commonly used treatments because they can Menapace 9 decrease the quality of life and cause adverse side effects.16 Parabens have the potential to be used as a new treatment option to overcome resistance and destroy these cancerous cells. This research aims to determine if cyclohexylmethylparaben can cause apoptotic cellular death in
M624 melanoma cells and therefore possibly be used as a future treatment for melanoma cancer.
The final fate of every dead cell undergoing apoptosis or necrosis is engulfment by phagocytes. Cells that undergo apoptosis exhibit several unique characteristics, including plasma membrane blebbing and formation of apoptotic bodies, which can assist in the phagocytosis process and result in efficient clearance of cellular components after death.19 Cells undergoing apoptotic cell death also display caspase-dependent activation and nuclear translocation of caspase-activated DNase that results in internucleosomal DNA cleavage.19 Activation of these apoptotic proteins can be used to identify apoptosis and distinguishes this type of cell death from other forms. Apoptosis is also one of the most important regulatory mechanisms in the growth and development of tissues.6 This form of cellular death can be activated by many factors that include deficiency of nutrients, damage to DNA, exposure to excessive heat, and ligation of cell- surface death receptors.21 The ligation of extracellular surface receptors can trigger caspase cascade pathways which can induce apoptosis within cells. The main characteristics of apoptosis are the condensation of chromatin and subsequent plasma membrane blebbing, which results in a cell separating into apoptotic bodies containing cytoplasm and organelles.11 These apoptotic bodies are then phagocytized by cells such as macrophages, preventing intracellular components of the dead cells from causing an inflammatory response in surrounding tissues, which is a characteristic most commonly seen in necrotic cellular death.11
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Cell death signaling
Caspases are proteases that are crucial in mediating programmed cell death, or apoptosis.23 Caspase-3 is activated in both the extrinsic and intrinsic pathways of apoptotic cell death. Caspase-3 is a protease that is essential in apoptotic cell death, responsible for DNA fragmentation and apoptotic chromatin condensation.23 In mammals, caspase-3 is activated as a result of a protease cascade that promotes the rapid activation or disablement of vital proteins, homeostatic enzymes, repair enzymes and signaling enzymes.23 The involvement of this specific protease in apoptosis is essential in distinguishing if a cell undergoes apoptotic cell death, or if it undergoes a different, less preferred type of cell death such as necrosis. In this study, caspase-3 activation in M624 melanoma cells treated with cyclohexylmethylparaben will be analyzed using caspase-3 colorimetric assays.
Many modern anti-cancer treatments such as radiation therapy and chemotherapy have been shown to trigger apoptotic cellular death either through an extrinsic or intrinsic pathway.
The extrinsic, or death receptor, pathways of apoptosis involve activation of death receptors embedded in the plasma membranes of cells, and these death receptors are members of the tumor necrosis factor, or TNF, receptor superfamily.6 Various types of receptors exist within the TNF superfamily, including Fas, DR4 and DR5.14 These death receptors bind an extracellular death ligand and transmit a death signal to intracellular signaling pathways from the surfaces of cells.6
CD95 and TNR1 are two types of death receptors, and binding of CD95L to CD95 and TNFa to
TNR1 is the first step in activating the signaling cascade which activates caspase-8 and caspase-
3.6 The activation of the signaling cascade will result in cellular death via an extrinsic apoptotic pathway. These apoptotic signaling cascades can be activated without binding of a ligand to a death receptor is some cases.6 Some compounds used in cancer treatment induce apoptosis by Menapace 11 binding to death receptors and activating these signaling cascades. If the aforementioned paraben is able to bind and activate death receptors of the plasma membrane of a cell, then it might be capable of activating an extrinsic apoptotic pathway.
The binding of a death ligand to a receptor allows for Fas-associated death domain
(FADD) protein to bind to the intracellular domain of the receptor and then FADD recruits procaspase-8 through dimerization of the death domain and promotes apoptosis.23 FADD is an adapter protein that consists of an N-terminal death effector domain (DED) and a C-terminal death domain (DD).26 FADD is recruited to an intracellular death domain of trimerized death receptors (DR) which causes a conformational change in FADD, exposing its DED to bind procaspase-8.26 Procaspase-8 will associate with the exposed DED of FADD, forming a death- inducing signaling complex (DISC) which then stimulates the binding of additional procaspase-8 molecules to FADD.26 The initial procaspase-8 associates with the exposed DED through a pocket in DED1 of FADD and additional procaspase-8 molecules bind through a second DED1 pocket to a motif located in DED2 of the initial procaspase-8.26 These additional procaspase-8 molecules form a unidirectional filament that will allow for homodimerization of the proteolytic domains of the interacting procaspase-8 molecules.26 Autoproteolytic cleavage of an aspartate residue between the small subunit and linker in the complex will partially activate procaspase-8 to caspase-8 and a second cleavage of an additional aspartate residue connecting the large subunit of the complex to DED2 will release a fully active caspase-8.26 Caspase-8 can then activate procaspase-3 to caspase-3 by cleaving between the large and small subunits of procaspase-3 allowing for apoptosis to be initiated.26
Apoptosis can also be activated through an intrinsic, or mitochondrial, mechanism that involves the permeabilization of the outer membrane of the mitochondria, which releases Menapace 12 cytochrome c into the cytoplasm of the cell. Cytochrome c is released from the mitochondria upon stimulation from proapoptotic stimuli that promote the permeabilization of the outer mitochondrial membrane. Cytochrome c is responsible for mediating the allosteric activation of apoptosis-protease activating factor 1, or Apaf-1, which is necessary for the proteolytic cleavage and activation of caspase-3 and caspase-9.13 The activation of these caspases creates a signal cascade within a cell, leading to destruction due to apoptosis.13 Permeabilization of the mitochondrial membrane is related to activation of proapoptotic proteins that belong to the Bcl protein family.6 Activated Bcl proteins cause cytochrome c to be released which then binds to the c-terminus of a cytosolic protein, Apaf-1, that activates caspase-9.6 Caspase-9, acting as an initiator caspase, activates caspase-3 which cleaves essential substrates in the cell that produce biochemical and intracellular events that cause apoptotic cellular death.6 If the aforementioned paraben is able to cross the plasma membrane of a cell, then it might cause permeabilization of the mitochondrial membrane, leading to apoptosis through an intrinsic pathway.
Caspase-9 is activated when it binds to the N-terminal caspase-recruitment domain
(CARD) through a CARD-CARD interaction, which results in an autocatalytic intrachain cleavage that activates caspase-9.6 Exposure of this N-terminal domain is facilitated by the binding of cytochrome c to Apaf-1 which causes dATP to associate with Apaf-1, causing the N- terminal CARD to oligomerize and form a platform on which caspase-9 can bind.6 This complex is known as an apoptosome and caspase-3 is recruited to the apoptosome where it is cleaved and activated by caspase-9.6 When caspase-3 is activated, it will cleave and activate ICAD/DFF45 which is typically an inhibitor of caspase-activated DNase.23 When ICAD/DFF45 is activated,
CAD acts as an endonuclease and fragments DNA, and such fragmentation is a key characteristic and signal of apoptotic cellular death.23 Menapace 13
Necrosis is a cell death pathway that can be characterized by rapid cytoplasmic swelling due to extreme physiological stress.19 It is important to note that cells undergoing necrosis don’t express caspase-3 or any other apoptotic signaling proteins, which helps to distinguish necrosis from apoptosis. Cells that undergo necrosis typically display cytoplasmic swelling, which is also different from cells that undergo apoptotic death because those cells display cytoplasmic shrinking before forming apoptotic bodies. Necrotic cell death results in the breakdown of organelles and the rupturing of the plasma membrane of dying cells, which makes phagocytosis of its components rather difficult and can lead to damage to surrounding cells in addition to inflammatory responses in the body.19 This type of cellular death is less preferable than apoptotic cell death due to the pathologies that can occur. Some additional characteristics associated with necrosis are the formation of cytoplasmic blebs, ruptured mitochondria, and disruption of the cellular membrane.11
Necroptosis is a form of regulated necrotic cell death characterized by the swelling of organelles and an increase in cell volume, permeabilization of the plasma membrane, and cellular collapse.2 One of the most common descriptions of necroptosis is cellular death mediated by TNF receptors and chemical suppression of caspases.2 This type of cellular death acts primarily against pathogen-mediated infections and its initiation is mediated by immune ligands such as LPS, Fas, and TNF, which leads to activation of receptor-interacting serine/threonine- protein kinase 3, or RIPK3.8 During the formation of a typical necrosome, RIPK3 will interact with RIPK1 to form the necrosome complex through an RHIM domain, and proteins such as
TRIF and DAI can form the necrosome complex of a non-traditional necrosome.8 RIPK3 will phosphorylate and activate a mixed lineage kinase domain-like pseudokinase, or MLKL, which is then translocated into the plasma membrane and decreases the integrity of cells, leading to Menapace 14 necroptotic death.8 This type of necrotic cell death exhibits morphological characteristics of both necrosis and apoptosis but it only occurs in tissues that express RIPK3 and MLKL.8 Human keratinocytes are one cell type in which RIPK3 and MLKL can be expressed.10 Malignant melanoma cells lines are also capable of expressing RIPK-1, RIPK3 and MLKL, which can induce necroptosis when they are activated (Geserick et al., 1). Cells that express RIPK3 must also express inhibition of caspase-8 to undergo necroptosis because caspase-8 is responsible for inducing exogenous apoptosis while also deactivating necroptosis via inactivation of RIPK3.8
Overview
The paraben used in this study is cyclohexylmethylparaben which contains a cyclohexane alkyl group. A study done by Wood et al. showed that melanoma cells were destroyed when exposed to methylparaben.27 This was the first study to demonstrate that methylparaben can kill human M624 melanoma cells.27 Propylparaben has been shown to cause damaging effects and cell death in HepG2 human liver carcinoma cells.25 Exposure of these liver carcinoma cells to propylparaben was shown to cause damage to mitochondria that led to the healthy apoptotic death.25 It has been demonstrated that both propylparaben and methylparaben can cause cell death in cancerous human cell lines, and this research will determine the capability of cyclohexylmethylparaben to cause cell death in M624 melanoma cells. The purpose of investigating this specific paraben is because of its large hydrocarbon alkyl group which might increase its lipid solubility and enhance its absorption into the skin. This is the first study to investigate cyclohexylmethylparaben and its effects on M624 melanoma cells or any line of cells. This study aims to determine the IC50 value of cyclohexylmethylparaben in M624 cells and the type of induced cellular death. The IC50 value, known as the biochemical half maximal Menapace 15 inhibitory concentration, is the concentration of paraben required to cause a 50% inhibition of cellular growth in M624 cells.18
This study will investigate the cytotoxic effects of cyclohexylmethylparaben on M624 melanoma cells using clonogenic assays. A clonogenic assay in a cell survival assay performed in vitro and can be used to determine the viability and proliferation of cells after treatment with compounds that can cause cell death through apoptosis, chromosomal damage, etc.12 A key component of this assay is crystal violet dye, which is used to stain the nuclei of adherent, viable cells.24 This method is useful in monitoring the influence of a wide range of compounds such as
IAP antagonists, chemotherapeutic drugs, or other compounds, such as cyclohexylmethylparaben.24 The basic principle of this procedure is to allow cells to adhere to a surface, such as the surface of wells on a 12-well cell culture plate, and then treat them with a given compound, stain the adherent cells with crystal violet, and remove dead, non-adherent cells from the wells by washing with phosphate-buffered saline (PBS). By measuring the absorbance of the crystal violet in the adherent cells, treated groups can be standardized to a control to measure cell viability.
This study will also investigate whether or not caspase-3 is activated by cyclohexylmethylparaben in M624 melanoma cells using caspase-3 colorimetric assays. Results from this analysis will indicate whether cyclohexylmethylparaben can induce apoptotic death in
M624 cells. Caspase-3 colorimetric assays measure the activity of caspase-3 based on the hydrolysis of the DEVD-pNA substrate, also known as acetyl-Asp-Glu-Val-Asp p-nitroaniline
(Ac-DEVD-pNA), in the reaction mixture.15 The reaction mixture of the caspase-3 colorimetric assay used in this study also includes dithiothreitol (DTT), reaction buffer, and cell lysis buffer.
The purpose of DTT is to maintain thiol groups in a reduced state while also reducing disulfide Menapace 16 bonds.9 Caspase-3 has a cysteine residue in its active site, so DTT is used to preserve the function of caspase-3 while reducing disulfide bonds in other proteins to ensure that any activity measured by the assay is due to caspase-3 hydrolyzing the DEVD-pNA substrate and not another protein hydrolyzing the substrate.17 Reaction buffer is used in these assays to provide a reaction environment with an ideal pH where caspase-3 can function effectively while cell lysis buffer is used as a detergent to break open cell membranes and release caspase-3 into solution from the cytoplasm of a cell. The amount of pNA in solution after hydrolysis is calculated by measuring the absorbance of the solution at either 400 or 405 nm and the comparison of a treated, or induced, sample is compared to an uninduced control sample to determine the fold increase in activity of caspase-3.15
Preliminary Data
The effects of cyclohexylmethylparaben on the viability of human M624 melanoma cells was first investigated during summer of 2019 at Marietta College. This research included preliminary clonogenic assays with M624 cells aimed at determining an IC50 value for cyclohexylmethylparaben in M624 melanoma cells. Cells were originally treated with 2.5, 5.0, and 7.5 mM cyclohexylmethylparaben, but the paraben was not soluble in solution at these concentrations so the treatment concentrations were decreased to 0.1, 0.25, 0.35, and 0.45 mM.
The exposure time for each treatment group was 10 minutes and the results for each treatment are seen in figure 2.
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120
100
80
60
40
Percentage of Viable CellsM624 20
0 Control Ethanol 0.1 mM 0.25 mM 0.35 mM 0.45 mM Cyclohexylmethylparaben Concentration
Figure 2. Preliminary results for clonogenic assays of human M624 melanoma cells treated with cyclohexylmethylparaben (and ethanol) for 10 minutes. Standard deviations are represented by error bars and each treatment group result is based on three sets of data performed in triplicate.
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Methods
Cell culture
M624 human melanoma cells were cultured and maintained in 100 mm cell culture plates with Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10 % fetal bovine serum
(FBS) and 1% penicillin-streptomycin in an incubator at 37° C with humidity and 5% CO2.
Cyclohexylmethylparaben synthesis
Cyclohexylmethylparaben was synthesized in Dr. Kevin Pate’s organic synthesis laboratory. To synthesize cyclohexylmethylparaben, 0.75mL of concentrated sulfuric acid was added to a 100mL round bottom flask (RBF), then 2.5g of para-Hydroxybenzoic acid (pHBA) was added. 10mL of cyclohexylmethanol was added and reagents were mixed and then refluxed over 90 minutes with the variac set at 50% power of 140v. After the reflux, the mixture was allowed to cool to room temperature and then washed in a separatory funnel with 20mL of roH2O. 15mL of chilled diethyl ether was added and the separatory funnel was inverted and vented carefully several times until gas release was minimal. Once layer separation occurred, the crude ester, or paraben, was in the top layer, which was then washed with 15mL of chilled roH2O. 15mL of 5%M sodium carbonate was slowly added and then the bottom was removed after separation. The crude ester was washed again with 15mL of 5% sodium carbonate and the bottom layer was removed again.
Crude ester remaining in the separatory funnel was pH tested and determined to be close to pH=7. 15mL of supersaturated sodium chloride was added to the separatory funnel, the layers were allowed to separate, the bottom layer was drained and the purified ester was poured out of the top into an Erlenmeyer flask. 4 mm pellets of anhydrous calcium chloride were added to the
Erlenmeyer flask with the ester for drying. Once the ester was dried, it was poured into a beaker Menapace 19 and was left in the hood for 3 days to allow the paraben to crystallize. Once crystals had formed, vacuum filtration was performed, and the crystals were washed twice using 5mL of cold diethyl ether. The final product on the filter paper was pure CHMP. The cyclohexylmethylparaben reaction had an average yield of 50%.
Cyclohexylmethylparaben solution preparation
Four cyclohexylmethylparaben solutions were prepared for cell treatment in this study.
0.0281g of cyclohexylmethylparaben was dissolved in 1.2mL of ethanol to produce a 100 mM solution. This solution was used to prepare a 0.35 mM, 0.45 mM, 0.55 mM, and 0.65 mM solution of cyclohexylmethylparaben by dissolving the 100 mM solution in DMEM containing
10% FBS and 1% penicillin-streptomycin. These solutions were used to treat M624 melanoma cells for clonogenic assays. A control, containing only DMEM containing 10% FBS and 1% penicillin-streptomycin, was also produced, in addition to a “solvent control” made by diluting
45.5µL of ethanol in 7mL of DMEM containing 10% FBS and 1% penicillin-streptomycin, creating a solution which contains the same amount of ethanol as the highest concentrated cell treatment solution, 0.65 mM.
Clonogenic assays
Clonogenic assays were performed with M624 cells after the cells reached 75% – 80% confluence in 100 mm culture plates. M624 cells were removed from the plates using trypsin and were transferred to 12 well culture plates. Cells were incubated in the 12 well culture plates for
24 hours and then the media was replaced with DMEM solution containing cyclohexylmethylparaben to treat samples in addition to control media solutions being added.
Each of the solution concentrations was tested in triplicate. After 10 minutes of exposure and incubation, all media solutions were removed by vacuum. Each well was then washed with 1x Menapace 20 phosphate-buffered saline (PBS) and then stained with 0.5% w/v crystal violet dye. The cells were exposed to the crystal violet dye for 5 minutes and then each well was washed three more times with 1x PBS. Plates were analyzed on a BioTek Epoch plate reader with Gen5 2.04 software using area scanning at 550 nm. All results were averaged and then standardized against the control sample to determine the viability of the results.
Cell treatment for caspase-3 colorimetric assays
M624 melanoma cells were incubated until they reached approximately 80-85% confluence in 100 mm culture plates. Treatment groups included 0.35, 0.45, 0.55, and 0.65 mM cyclohexylmethylparaben along with a control group not treated with any paraben. Each treatment group consisted of two plates containing approximately 5.0•106 cells. The media from each paraben treatment group was discarded and both plates in each treatment group were treated with 3.5mL per plate of the appropriate cycloheyxlmethylparaben solution for 10 minutes. The cyclohexylmethylparaben solution from each paraben treatment group was collected and centrifuged for 10 minutes at maximum speed to collect non-viable M624 cells for analysis.
0.5mL cold PBS was added to each plate and cells were then scraped from the plate with the backside of a 0.5mL pipet tip in order to collect adhered M624 cells which were then centrifuged to pellet and collect viable M624 cells. Cells were lysed according to the instructions provided with the Caspase-3 Colorimetric Assay Kit from BioVision Inc. The media from each centrifuge tube was decanted and the pellets of dead cells were transferred to the tubes containing the scraped cells using 0.5mL cold PBS. The tube of cells was then centrifuged again, the resulting supernatant was decanted and the pellet was resuspended in solution using 100µL of cell lysis buffer (proprietary buffer containing Triton X-100). The resuspended pellet was incubated in the lysis buffer for 10 minutes on ice before being centrifuged for 3 minutes at full speed. The Menapace 21 resulting supernatants were transferred to fresh microcentrifuge tubes and were either stored at -
20°C or placed on ice for an immediate analysis of protein concentration and caspase-3 activity.
Bradford protein concentration assays
A Bradford protein concentration assay was performed to determine the amount of protein in the M624 cell lysate samples. The Bradford assay was performed by combining 5µL of each lysate sample with 100µL of Bradford reagent (0.01% (w/v) Coomassie Brilliant Blue G-
250 in 4.75% (v/v) ethanol and 8.50% (v/v) phosphoric acid). Each sample was analyzed in triplicate, and the absorbance of each sample was measured at 595 nm using the BioTek Epoch plate reader. A standard curve was created using Bovine Serum Albumin by measuring the absorbance of 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL BSA in triplicate at 595 nm and the resulting line equation from the standard curve was used to calculate the protein concentration of each lysate sample. Absorbance of each lysate sample was plugged in for “y” in the line equation and solving for “x” determined the concentration of protein in each sample.
Caspase-3 colorimetric assays
The caspase-3 colorimetric assay (Caspase-3 Colorimetric Assay Kit from BioVision
Inc.) was performed according to the manufacturer’s instructions. The volume of each lysate sample required to provide 50µg of protein for the caspase-3 activity assay was calculated using the data from the Bradford protein concentration assays. Each lysate sample was diluted to an identical volume using cell lysis buffer and then an identical volume of 2x reaction buffer containing 10 mM DTT was added to each lysate sample. A 4 mM DEVD-pNA substrate (200
µM final concentration) was added to each of the lysate samples which were then vortexed and incubated at 37° C for 1 hour and 30 minutes. A buffer control sample containing cell lysis buffer, 2x reaction buffer with 10 mM DTT, and 4 mM DEVD-pNA was prepared and assayed Menapace 22 in order to measure background buffer absorbance to be subtracted from readings from lysate sample absorbance. The volume of this blank was equal to the volume of the lysate samples.
Each lysate sample was centrifuged at maximum speed for 30 seconds and then the entire sample was plated. Lysate sample absorbance was measured at 400 nm in a 96 well plate using the
Epoch plate reader. Absorbance of lysate samples corresponded to the activity of caspase-3, so high absorbance indicated high caspase-3 activity which would indicate M624 cells died via apoptosis. The fold-increase in caspase-3 activity was determined by standardizing the treatment group results to the control sample results.4
Statistical analysis
Three individual clonogenic assays and two individual caspase-3 assays were performed.
Data was averaged and standard deviations were calculated. Individual hypothesis tests were performed to see if there was a difference in each of the treatment groups when compared to the control cells and the p-values were calculated for each sample in the clonogenic assays and caspase-3 assays.
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Results
The cytotoxic effects of cyclohexylmethylparaben on M624 cells were studied using clonogenic assays and after 12 well plates had been stained with crystal violet, the results were quantified using a BioTek Epoch plate reader. Data produced from this analysis are shown in figure 3. This data shows that treatment with 0.35 mM cyclohexylmethylparaben resulted in approximately 77% cell viability while treatment with 0.45 mM cyclohexylmethylparaben resulted in approximately 44% M624 cell viability. Treatment with 0.55 mM and 0.65 mM cyclohexylmethylparaben resulted in approximately 35% and 30% cell viability, respectively.
Following the completion of clonogenic assays, the IC50 value was calculated for cyclohexylmethylparaben in the M624 cells. Individual hypothesis tests were performed to see if there was a difference in each of the treatment groups when compared to the control cells and the p-value was less than 0.0001 for all samples but the ethanol solution control sample.
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120
100 y = -16.058x + 119.71 80 R² = 0.9386
60
40
Percentage of Viable CellsM624 20
0 Control Ethanol 0.35 mM 0.45 mM 0.55 mM 0.65 mM Cyclohexylmethylparaben Concentrations
Figure 3. Quantified data of clonogenic assays for cyclohexylmethylparaben-treated human M624 melanoma cells after 10 minutes. Error bars are representative of standard deviations and results are based on three sets of triplicate data for each treatment group.
Using the data from the clonogenic assays excluding the control sample and the line equation seen in figure 4, it was determined that the IC50 value for cyclohexylmethylparaben was
0.4768 mM in M624 melanoma cells.
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90
80
70
60
50 y = -149.63x + 121.35 R² = 0.8364 40
30
20 Percentage of Viable CellsM624
10
0 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 Cyclohexylmethylparaben Concentrations
Figure 4. Calculation of the IC50 value for cyclohexylmethylparaben-treated human M624 melanoma cells. Data points represent 0.35 mM, 0.45 mM, 0.55 mM, and 0.65 mM treatment groups. Data points are averages from three individual experiments performed in triplicate.
Following an analysis of the cytotoxic effects of cyclohexylmethylparaben on M624 melanoma cells, a caspase-3 colorimetric assay kit was used to measure the activation of caspase-
3 in each treatment group. This data is presented in figure 5 and indicates that caspase-3 was activated in each of the treatment groups, but the highest level of activation was seen in M624 melanoma cells treated with 0.35 mM cyclohexylmethylparaben. The relationship between caspase-3 activity and cyclohexylmethylparaben appears to rely on the concentration of paraben used to treat the M624 cells. An increase in concentration of cyclohexylmethylparaben results in a decrease in the overall activity of caspase-3, which indicates that cyclohexylmethylparaben is more effective at inducing apoptosis in M624 cells at lower concentrations. Individual hypothesis Menapace 26 tests were performed to see if there was a difference in each of the treatment groups when compared to the control cells and the p-value was less than 0.0001 for all samples.
16
14
12 3 Activity - 10
8
6
4
2 Fold Increase of Caspase 0 Control 0.35 mM 0.45 mM 0.55 mM 0.65 mM Cyclohexylmethylparaben Concentrations
Figure 5. Caspase-3 Activation in M624 Melanoma Cells. The absorbance of each treatment group was measured at 400 nm after incubation at 37°C and 5% CO2 for 1 hour and 30 minutes.
Absorbance of each treatment group was standardized to the control to show fold increase of caspase-3 activity and represents averages from two independent experiments. Error bars represent standard deviation in each treatment group.
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Discussion
The data from the clonogenic assays indicate that cyclohexylmethylparaben was cytotoxic to human M624 melanoma cells when compared to the control and solvent control. This data supports the hypothesis that M624 melanoma cell viability would decrease as a result of treatment with cyclohexylmethylparaben. Cyclohexylmethylparaben, which is a novel and newly synthesized paraben compound, killed human M624 melanoma cells and displayed greater cytotoxic effects on M624 cells as cyclohexylmethylparaben concentrations were increased.
Cytotoxicity of cyclohexylmethylparaben to M624 cells and their subsequent death indicates that cell death is occurring through either apoptosis or necrosis.
These cells were also hypothesized to be undergoing apoptotic cell death, which is a pathway that is mediated by the activation of caspase-3, an executioner caspase, activated by proteolytic cleavage by either caspase-8 or caspase-9. Apoptosis is a mechanism of cell death that is tightly regulated within the human body to regulate the number of cells in an organism and to prevent tissue from becoming cancerous.5 It was hypothesized that cyclohexylmethylparaben would cause apoptotic cell death via the activation of caspase-3 and the results from the caspase-3 colorimetric assay kit showed that the 0.35 mM sample of cyclohexylmethylparaben induced the highest activity of caspase-3 compared to the control. Caspase-3 activation was also observed in the other samples, but the activation of caspase-3 in these instances might not be associated with apoptotic cell death whereas the high activation of caspase-3 in the 0.35 mM sample is most likely representative of apoptotic death. The activation of caspase-3 in the control sample can be attributed to the normal cell life cycle which ends in apoptotic cell death.
The remaining treatment groups show a decrease in caspase-3 activity as the concentration of cyclohexylmethylparaben increases beyond 0.35 mM. This trend might be attributed to Menapace 28 cyclohexylmethylparaben inducing necrotic cell death, which would result in a decrease of caspase-3. It is possible that another executioner caspase is being expressed in these samples leading to necrotic cell death, forms of which are induced by caspase-1, caspase-4, and caspase-5 in humans.28 The Caspase-3 Colorimetric Assay Kit provided by BioVision Inc. provides data for the induction of caspase-3 activity by an Anti-Fas antibody in Jurkat-T cells where the fold increase in activity of caspase-3 in the induced sample is 13 times greater than the uninduced control sample.4 As a comparison, the fold increase of the 0.35 mM treated sample in this study is
11.69 when compared to the uninduced control sample, which indicates that M624 melanoma cells treated with 0.35 mM cyclohexylmethylparaben are most likely dying as a result of apoptosis.
As indicated by the caspase-3 activation in the melanoma cells treated with 0.35mM cyclohexylmethylparaben, apoptosis is occurring, but it isn’t clear which mechanism is utilized to activate caspase-3: an intrinsic or extrinsic apoptotic pathway. Cycylohexylmethylparaben is amphipathic and therefore lipophilic, so it could cross the membrane of M624 cells, leading to the activation of caspase-3 through an intrinsic mechanism. However, considering that cyclohexylmethylparaben is relatively large and has a polar region, it is unlikely that the compound would be able to enter a cell via simple diffusion in order to trigger apoptosis. It is possible that cyclohexylmethylparaben could induce intrinsic activation of apoptosis if it is transported into a cell using a transmembrane protein, but this paraben needs to be researched further to understand exactly which mechanism it induces.5
The individual hypothesis tests used in this study for the clonogenic assays and caspase-3 colorimetric assays were performed to determine if the results from each treatment group differed significantly from the control results. Since the p-value of each treatment group in this Menapace 29 study was less than 0.0001, the null-hypothesis that the treatment group results are not significantly different from the control group results can be rejected. Cyclohexylmethylparaben, as a result, can be said to have a statistically significant effect on both cell viability and caspase-3 activation compared to control samples. A p-value less than or equal to 0.05 would’ve been sufficient to reject the null hypothesis in this study.
Future studies of cyclohexylmethylparaben’s effects on M624 melanoma cells might investigate the bioavailability of the molecule to the cell and investigate which apoptotic signaling pathway – intrinsic or extrinsic – is being activated in melanoma cells treated with
0.35mM of the paraben. The effects of lower concentrations of cylcohexylmethylparaben over longer exposure times should also be investigated to determine if cyclohexylmethylparaben can effectively induce apoptosis in M624 cells at lower concentrations through activation of caspase-
3.
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References
1. Bandarchi, B.; Jabbari, C.A.; Vedadi, A.; Navab, R. Molecular biology of normal melanocytes and melanoma cells. J. Clin. Pathol. 2013, 66, 644 – 648.
2. Belizário, J.; Vieira-Cordeiro, L.; Enns, S. Necroptotic Cell Death Signaling and Execution Pathway: Lessons from Knockout Mice. Mediators of Inflammation. 2015, 2015, 1 – 15.
3. Cashman, A.L.; Warshaw, E.M. Parabens: A Review of Epidemiology, Structure, Allergenicity, and Hormonal Properties. Dermatitis. 2005, 16, 57 – 66.
4. “Caspase-3/CPP32 Colorimetric Assay Kit.” BioVision, https://www.biovision.com/doc umentation/datasheets/K106.pdf.
5. Cooper, G.M. The Cell: A Molecular Approach. 2nd edition; Sinauer Associates, Inc.: Sunderland, MA, 2000.
6. Debatin, K.M.; Fulda, S. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene. 2006, 25, 4798 - 4811.
7. Dewalque, L.; Pirard, C.; Charlier, C. Measurement of urinary biomarkers of parabens, benzophenone-3, and phthalates in a Belgian population. BioMed Research International. 2014, 1, 1 – 13.
8. Dhuriya, Y.K.; Sharma, D. Necroptosis: a regulated inflammatory mode of cell death. Journal of Neuroinflammation. 2018, 15, 1 – 9.
9. “Dithiothreitol (DTT).” BioVision, https://www.biovision.com/documentation/data sheets/1201.pdf.
10. Duan, X.; Liu, X.; Liu, N.; Huang, Y.; Jin, Z.; Zhang, S.; Ming, Z.; Chen, H. Inhibition of keratinocyte necroptosis mediated by RIPK1/RIPK3/MLKL provides a protective effect against psoriatic inflammation. Cell Death and Disease. 2020, 11, 1 – 14.
11. Elmore, S. Apoptosis: A Review of Programmed Cell Death. Toxicologic Pathol., 2007, 35, 495 – 516.
12. Franken, N.A.P.; Rodermond, H.M.; Stap, J.; Haveman, J.; van Bree, C. Clonogenic assay of cells in vitro. Nature Protocols. 2006, 1, 2315 – 2319.
13. Garrido, C.; Galluzzi, L.; Brunet, M.; Puig, P.E.; Didelot, C.; Kroemer, G. Mechanisms of cytochrome c release from mitochondria. Cell Death Differ. 2006, 13, 1423 – 1433.
14. Gibson, S.B. Epidermal Growth Factor and Trail Interactions in Epithelial-Derived Cells. In Vitamins & Hormones; Litwack, G., Ed.; Elsevier: 2004; Vol. 67; p 207 – 227.
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15. Gregoraszczuk, E.L.; Rak-Mardyla, A.; Ryś, J.; Jakubowicz, J.; Urbański, K. Effect of Chemotherapeutic Drugs on Caspase-3 activity,ad a Key Biomarker for Apoptosis in Ovarian Tumor Cell Cultured as Monolayer. A Pilot Study. Iranian Journal of Pharmaceutical Research. 2014, 14, 1153 – 1161.
16. Housman, G.; Byler, S.; Heerboth, S.; Lapinska, K.; Longacre, M.; Snyder, N.; Sarkar, S. Drug resistance in cancer: an overview. Cancers. 2014, 6, 1769 – 1792.
17. Jahnke, W. Fragment-Based Approaches. In Comprehensive Medicinal Chemistry II; Taylor, J.B.; Triggle, D.J., Eds.; Elsevier: 2007; Vol. 3; pp 939 – 957.
18. Kalliokoski, T.; Kramer, C.; Vulpetti, A.; Gedeck, P. Comparability of Mixed IC50 Data – A Statistical Analysis. PLOS One. 2013, 8, 1 – 12.
19. Krysko, D.V.; Vanden-Berghe, T.; D’Herde, K.; Vandenabeele, P. Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods. 2008, 44, 205 – 221.
20. Ma, X.; Wan, Y.; Wu, M.; Xu, Y.; Xu, Q.; He, Z.; Xia, W. Occurrence of benzophenones, parabens and triclosan in the Yangtze River of China, and the implications for human exposure. Chemosphere. 2018, 213, 517 – 525.
21. Negroni, A.; Cucchiara, S.; Stronati, L. Apoptosis, Necrosis, and Necroptosis in the Gut and Intestinal Homeostasis. Mediators of Inflammation. 2015, 1 – 10.
22. Nelson, D.L.; Cox, M.M. Lehninger Principles of Biochemistry, 7th ed.; W.H. Freeman. New York, New York, 2017, 1 – 1172.
23. Porter, A.G.; Jänicke, R.U. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 1999, 6, 99 – 104.
24. Schilling, R.; Geserick, P.; Leverkus, M. Characterization of the Ripoptosome and Its Components: Implications for Anti-inflammatory and Cancer Therapy. In Methods in Enzymology; Ashkenazi, A.; Wells, J.; Yuan, J., Eds.; Elsevier: 2014; Vol. 545; pp 83 – 102.
25. Szelag, S.; Zablocka, A.; Trzeciak, K.; Drozd, A.; Baranowska-Bosiacka, I.; Kolasa, A.; Goschorska, M.; Chlubek, D.; Gutowska, I. Propylparaben-induced disruption of energy metabolism in human HepG2 cell line leads to increased synthesis of superoxide anions and apoptosis. Toxicol. In Vitro. 2016, 31, 30 – 34.
26. Tummers, B.; Green, D.R. Caspase-8; regulating life and death. Immunol. Rev. 2018, 277, 76 – 89.
27. Wood, R.S.; Greenstein, R.S.; Hildebrandt, I.M.; Parsons, K.S.G. The Apoptotic Effects of Methylparaben and Ultraviolet B Light on M624 Melanoma Cells. J. Med. Chem. 2018, 1, 1 – 13. Menapace 32
28. Yuan, J.; Najafov, A.; Py, B.F. Roles of Caspases in Necrotic Cell Death. Cell. 2016, 167(7), 1693 – 1704.
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Glossary
Parabens – Organic compounds used to prevent microbial growth in commercial products.
Alkyl chain – A chemical chain containing only hydrogens and carbons arranged in a chain.
Apoptosis – Type of normal, healthy cellular death characterized by expression of caspase-3 or other proteins.
Necrosis – Type of cellular death characterized by destruction of cells leading to inflammation and damage to surrounding tissues.
Caspases – Proteins that are activated when apoptotic cellular death occurs
Clonogenic assays – Technique used to measure the absorbance of living cells stained with a crystal violet dye. Allows the percentage of living cells to be calculated relative to a control sample.
Colorimetric assays – Technique used to measure the activity of caspase-3 in treated cell groups. Allows the fold increase of caspase-3 activity to be calculated by measuring the absorbance of the reaction mixture and standardizing the absorbance measurments to the control.