A Thesis entitled. Studying the mechanism of ferroptosis induced by a novel class of small molecules. by Hanan Dhafer Alqahtani

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1 A Thesis entitled Studying the mechanism of ferroptosis induced by a novel class of small molecules by Hanan Dhafer Alqahtani Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biology Dr. William R. Taylor, Committee Chair Dr. LirimShemshedini, Committee Member Dr. Conti, Heather Raquel, Committee Member Dr. L. M. Viranga Tillekeratne, Committee Member Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo July 2016

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3 An Abstract of Studying the mechanism of ferroptosis induced by a novel class of small molecules by Hanan Dhafer Alqahtani Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biology The University of Toledo July 2016 Cancer is a group of diseases characterized by abnormal features including continuous abnormal growth, uncontrolled cell division, and the ability to spread, invade and destroy surrounding tissues and organs in the body. It is the second most common cause of death in the US. Therefore, discovering strategies, signaling pathways, and targeted molecules to eliminate cancer cells has been intensively investigated. Cells in multicellular organisms undergo death for many reasons such as senescence and external chemical stimulus. There are many forms of cell death including necrosis, apoptosis, and autophagy, and each form is morphologically, biochemically, and genetically distinct. In addition, each form is activated differently from each other. We are investigating a recently described form of cell death called Ferrptosis that was originally observed in iii

4 response to the small molecule called Erastin. We hypothesize that Ferroptosis can be exploited to eliminate certain types of tumor cells. In ferroptosis, an iron-dependent form of nonapoptotic cell death, cells die due to the iron-dependent accumulation of Reactive Oxygen Species (ROS). ROS are chemically reactive molecules than contain oxygen, and they are formed naturally as a byproduct of the metabolism of oxygen. We identified a small molecule called 6E that can kill number of cancer cell lines by Ferroptosis. There are many cellular factors that increase the sensitivity of the cells to be killed by 6E such as activated RAS/MAPK pathway, presence of p53 alleles, iron metal, loss of the epithelial gene E-cadherin, and expressing vimentin the mesenchymal gene. In this thesis we study the mechanism of ferroptosis induced by 6E and related compounds. We tested a number of different cancer cell lines toward 6E. Also we measured the level of glutathione inside the cells, and the level of secreted glutamate after 6E treatment. Also, we tested the effect of the iron chelator CPO and the antioxidant trolox in cell death induced by 6E. Together these experiments suggest that 6E induces ferroptosis by inhibiting XC-, a transport that brings cystine into the cell required to replenish the antioxidant glutathione. We also show that mesenchymal cells are particular sensitive to the 6E precursor molecule. Consistent with these, we find that a number of childhood cancer cell lines derived from mesenchymal tissues are sensitive to 6E. iv

5 I dedicate this thesis to my husband, Fahad Almshabab, and my son, Fares Almshabab for their constant love, support and encouragement. v

6 ACKNOWLEDGMENTS First, words would not be enough to express my gratitude to my wonderful husband for encouraging me to study abroad on such an endeavor away from home. Thank you for being a constant source of support, motivation and encouragement at all times. Your unconditional love and inspiration has been my strength in all times, thus I am deeply indebted to you. I am so blessed to have you as my husband and friend. Thank you for your numerous sacrifices and help during this project and life. An enormous thanks to my lovely son, Fares, for all his love, support and being patient, allowing me time to get my project completed. Next, I would like to express my sincere thanks and gratitude to my advisor, Dr. William R. Taylor for his excellent guidance, generous support, endless care and continuous kindness. He has always been there to offer invaluable advice, share his knowledge and ideas, and motivate me through my graduate studies. I am deeply grateful to him for the long discussions that allowed me to develop into a thoughtful and innovative scientist. He is a great scientist and always more than willing to help out in my course study. I would not have been able to complete my thesis without the guidance of him. vi

7 I would like also to thank my committee members, Dr. LirimShemshedini, Dr. Conti, Heather Raquel, and Prof. L. M. Viranga Tillekeratne. Special thanks go to my lab membersmaisha Rashid, and Taylor Monus for the assistance and friendship during my study. Also I would thank Ayad Alhamashi, a PhD candidate in Prof. L.M.Viranga Tillekeratne lab for all his help and advices that he was giving me, and all time he was giving me to explain information that I need to understand especially in his extract project that we are working on together as a collaborator. Special thanks also go to Ejaz Ahmad for all help he provided me when I need any time. He would just help any time I need help. Thanks alot to you and to your beautiful wife and daughter. Last but not least, thanks especially to my dad and mom whose constant prayers, sacrifice and care have sustained me throughout my life, and to my lovely sister Eman and her son Bandar, my brothers and friends for continuously encouraging me with their best wishes throughout this journey. Also, I would thank my father-, mother-, brothers- and sisters-in-law for their support, encouragement, and love during my study. vii

8 Table of Contents Abstract iii Acknowledgements vi Table of Contents viii List of Figures xii 1. Introduction Cancer Statistics Cell lines Cancer Treatment Surgery Chemotherapy Radiation Hormone Therapy Systemic Drugs Drug Discovery 8 2. Ferroptosis is a newly discovered form of cell death 9 3. Other major forms of cell death Apoptosis 13 viii

9 3.2 Necroptosis Autophagy Interaction between the cell death forms and cancer metastasis The explanation of our project Hypotheses Cell lines that are used in this project Breast cancer cell lines Lung cancer cell line Childhood cancer cell lines Materials and Methods Cell culture condition Cell types Drug treatment Measurement of Glutathione level Measurement of secreted glutamate using The Amplex Red 27 Glutamic Acid/Glutamate Oxidase Assay 8.6 Cytotoxicity 28 ix

10 8.7 IC50 calculation Results The chemical structure of our new molecule 6Eand its derivation The cytotoxicity of 6E in different cancer cell lines Cell death induced by 6E is inhibited in the presence of ferroptosis 38 modulators such as Ciclopiroxolamine (CPO) and Trolox 9.4 Mechanism of action of 6E N- acetylcysteine inhibits or stops the cell death induced by 6E Glutathione level is depleted in 6E-treated cells E-treated cells secrete less glutamate Mesenchymal cells tend to be more sensitive to 6E than epithelial cells Some breast cancer cell lines are sensitive to 6E Since mesenchymal cells are more sensitive to 6E, sarcomas from 58 Childhood cancer might also be sensitive Two rhabdomyosarcoma cell lines are sensitive to 6E TC71 (Ewing sarcoma) is sensitive to 6E The protective effect of Z-Vad, 3MA, and ferrostatin-1 in 63 x

11 inhibiting the cell death induced by 6E 9.7 Analysis of 6E analogues to obtain expanded structure activity 66 relationship (SAR) information 10. Natural Product Introduction Anticancer drugs isolated from natural products Techniques used in this project Extraction process Separation process Reverse phase silica chromatography separation Normal phase silica chromatography Size exclusion chromatography Identifying the mechanism of cell death Conclusion of natural products Future direction of natural products Discussion and conclusion References 84 xi

12 List of Figures Figure 1: Natural epothilones and synthetic open-chain epothilones 32 Figure 2: The 4-cyclopentenyl-2-ethynylthiazole compounds. Compound 33 number 58 represents 6E Figure 3: The chemical structure of 6E molecule 35 Figure 4: The IC50 of different cancer cell lines exposed to different concentration 37 of 6E, and the cell viability was analyzed by methylene blue staining. Figure 5: The effect of CPO and Trolox in cell death induced by 6E in MDAMB cell line Figure 6: The antiporter system XC- structure 43 Figure 7: The pathway of erastin in inducing Ferroptosis 44 Figure 8: The pathway of RSL3 molecule in inducing ferroptosis 45 Figure 9: N- acetylcysteine inhibits or stops the cell death induced by 6E 47 Figure 10: 6E depletes glutathione level in NCI-H522 cells 49 Figure 11: 6E-treated cells secrete less glutamate 52 Figure 12: The pathway of 6E in inducing ferroptosis 53 Figure 13: E-Cadherin reduces drug sensitivity 55 Figure 14: MDAMB468 and MDAMB231 breast cancer cell lines were sensitive 57 to 6e, whereas SUM159 cell line was not sensitive to 6E Figure 15: Rh41 and Rh30 childhood cancer cell lines were sensitive to 6E 60 xii

13 Figure 16: TC-71 childhood cancer cell line was sensitive to 6E 62 Figure 17:A- Z-VAD inhibitor did not rescue cell death induced by 6E, whereas 64 ferrostatin-1 did rescue the cell death. Figure 17:B- Autuphagy inhibitor did not rescue cell death induced by 6E 65 Figure 18: A- Analogs of 6E 67 Figure 18: B- The effect of the six analogs of 6E in H522 cells. 68 Figure 19: Testing the sensitivity of four fractions extracted from a 73 natural product Figure 20: Testing the sensitivity of six fractions extracted from a 75 natural product Figure21: Testing the sensitivity of ten fractions extracted from a 77 natural product Figure 22: Testing the effect of cell death inhibitors on the cell 79 death induced by the active fraction D3-8 in H522 cell line xiii

14 1. Introduction 1.1 Cancer Statistics Cancer is a group of diseases characterized by abnormal features including continuous abnormal growth, uncontrolled cell division, and the ability to spread, invade and destroy other tissues and organs in the body. It is the second most common cause of death in the US [1]. The estimated numbers of cancer cases, cancer incidence, mortality, survival statistics, and cancer symptom are provided by American cancer society every year. There are variations in the incidence of cancer cases, mortality, and survival between countries and these differences is due to factors including age, race, ethnicity, lifestyle status. In 2016, there will be an estimated 1,685,210 new cancer cases diagnosed and 595,690 cancer deaths in the USA [2]. Prostate, lung, and colon cancer are common among men, whereas breast, lung, and colon cancer are common among women.breast cancer alone is expected to account for 29% of all new cancers among women [3]. The five years survival rate of all cancer has been decreasing in the past years. These decreases are thought to be the result of treatment advances, earlier detection through screening, and increased awareness among people. Cancers originating in any organ or tissue of your body are called primary cancers. If they spread, they re considered metastatic. There are two primary types of cancer: carcinomas and sarcomas, and they differ in the kind of tissues they originated from. Sarcomas develop in mesodermal 1

15 tissues such as bone, muscle, fat, nerves, cartilage, fibrous tissue, and connective tissue. However, carcinomas originate in epithelial tissue, such as the lining of breast, lung, colon or prostate. Tumors can be benign tumours or malignant tumours. Benign tumors are not cancerous, and the cells in it do not have the ability to spread to other parts of the body. Benign tumours are easily to be removed from the body, and they mostly do not come back. Benign tumours usually surrounded or protected by a sac which makes it stays and not able to move to other parts of the body. This sac is produced by the immune system in human body. Most common types of benign tumors are adenomas, fibromas or fibroids, and Meningiomas. On the other hand, malignant tumours are opposite of the first type benign. Malignant tumours are considered cancerous, and it consists of populations of cells that have the aggressive ability to grow out of control. These cells populations in malignant tumours have the ability to invade surrounding tissues and spread to other parts of the body. Once they spread and move to other parts in the body, they can keep growing and forming another tumour at that site. This is a characteristic of malignant tumours called metastasis. Most common types of malignant tumors are carcinoma and sarcoma. These types of tumors are really hard to detect from the first, and that s why its treatment is harder than benign tumors [3]. Cancer is a very serious and annoying diseases that spread through countries in a very high percentage, so discovering 2

16 updated and effective ways and drugs to kill cancer is really needed. In this way there are many ways and types that have been discovered. 1.2 Cell Lines In this project we tested the sensitivity of different cell lines toward our molecules. There are different factors that determine the level of sensitivity in each cell lines. Cancer cells with high level RAS-RAF-MEK pathway activity or p53 expression may be sensitized to this process [18]. Recent work has identified a number of genes required for ferroptosis, including those involved in lipid and amino acid metabolism [18]. We used breast cancer cell lines such as MDAMB231 cells, MDAMB468 cells, and MCF7 cells. Also we used fibrosarcoma cell line such as HT1080. Colon cancer cell line HCT116 was used. In addition, three childhood cancer cell lines were used, Rh30 cell line, Rh41 cell line, and TC-71 cell line. 1.3 Cancer Treatment Cancer can be treated by many ways such as surgery, radiation therapy, chemotherapy, and hormone therapy [2]. Depending on the type of cancer and how advanced it is, the type of treatment will be determined based on that. 3

17 1.3.1 Surgery The procedure here is to physically remove cancer from the patient s body. Surgery often requires cuts through skin, muscles, and sometimes bone. Sometimes surgery can be performed with no cut involved for example Cryosurgery, Laser therapy, Hyperthermia, and Photodynamic therapy [2]. In cryosurgery liquid nitrogen or argon gas is used to destroy abnormal tissue. In laser therapy, a powerful beam of light is used to cut through tissue and the lasers can focus very accurately on the determined areas. It also can shrink or destroy tumors or growths that might turn into cancer. In Hyperthermia, small determined areas of body tissue are exposed to high temperatures. There are many types of surgery and that depends on many factors such as the purpose of the surgery, the part of the body that requires surgery, the amount of tissue to be removed. Surgery can remove the entire tumor (when the cancer is contained in one area), or it can debulk a tumor which means removing some of the tumor (when removing an entire tumor might damage an organ or the body), or it can remove tumors that cause pain or pressure to the patients [2]. Because surgery can solve just the visible tumor, we need other ways to solve and treat the microscopic tumor deposits. Therefore, new effective chemotherapy drugs are strongly needed. 4

18 1.3.2 Chemotherapy In chemotherapy cancer treatment, drugs are used to kill cancer cells. Chemotherapy kills cancer by stopping or slowing the growth of cancer cells which grow and divide quickly [2]. Some people need just the chemotherapy alone for treatment, but most often people are treated by chemotherapy in combination with other cancer treatments. When chemotherapy is used with other cancer treatments, it can enhance effective uses of other approaches for example, chemotherapy can shrink or reduce the tumor before the surgery is done.chemotherapy can remove cancer cells that might stay after the surgery. Finally chemotherapy also can destroy and kill cancer cells that spread to different parts of the body. Chemotherapy can cause side effects due to damage to the healthy resulting in mouth sores, nausea, and hair loss [2]. Chemotherapy is given in many ways and the common ways are oral, intravenous (IV), injection, intrathecal, intraperitoneal (IP), intraarterial (IA), and topical [2]. Chemotherapy drugs are given based on factors such as the type of cancer patients have and how advanced it is, having chemotherapy before, having health problem such as diabetes or heart disease Radiation Radiation therapy is a cancer treatment that uses high doses of radiation to kill cancer cells and shrink tumors [1]. At low doses, radiation is used in x-rays to see inside the 5

19 body. Radiotherapy is used to either treat cancer or ease its symptoms. Cancer cells are not killed right away after radiotherapy, but it usually takes days and weeks for cells to start dying. There are two main types of radiation therapy, external beam and internal. In external radiation therapy, large machine applies radiation at the cancer, and the machine can send radiation from many direction to the part of the body that has the cancer. The good feature of external beam is that it treats a specific part of your body without affecting the whole body. The second type is the internal radiation therapy where a source of radiation is put inside the body. The radiation source can be liquid or solid. When the radiation source is solid, the radiation is formed as seeds, ribbons, or capsules and placed in your body in or near the cancer [1]. In liquid case, patients receive liquid radiation through an IV line which then travel through the body to reach cancer cells. One of the side effects that radiation can cause is affecting nearby healthy cells, and these healthy cells mostly recover after the treatment is over. Sometimes a patient needs more time to recover and then it is called late effect. Doctors can reduce this side effect by using a small dose of radiation Hormone Therapy Hormone therapy for cancer is the use of medicines to block the effects of hormones.hormones are natural substances made by glands in our bodies, and they have 6

20 lots of effects and one of these is controlling the growth and activity of certain cells and organs. Therefore, some cancers use these hormones to grow. Not all cancers can be treated by hormone therapy, it is used for cancers that are hormone sensitive such as breast cancer, prostate cancer, ovarian cancer, womb cancer, and kidney cancer. Tamoxifen is one of the most common hormone therapies used for breast cancer [1]. Hormone therapy is most often used after surgery (as adjuvant therapy) to help reduce the risk of the cancer coming back, but it can be started before surgery (as neoadjuvant treatment) as well [1]. Hormone therapy might be used in combination with other cancer treatments, such as chemotherapy and radiation therapy Systemic drugs Systemic medications are prescription drugs that work throughout the body. Another definition is a medicine that has effects throughout the body, as opposed to topical drugs that work on the skin. Most medicines that are taken by mouth or by injection are systemic drugs. Systemic drugs are categorized based on the system in the body it works on. Sometimes systemic drugs have side effect on patients or have no effect on cancer treatment; therefore, discovering new drugs is recommended and important for effective treatment [3]. 7

21 1.3.6 Drug discovery Drug discovery is the process, by which new candidate medications are discovered, and these drugs can be synthesized chemically or they can be extracted from natural products [4]. Then chemical libraries of synthetic small molecules, natural products or extracts are screened and experiments conducted in vitro to identify components that have a desirable therapeutic effect in cancer treatment. Drugs usually act on either cellular or genetic chemicals in the body known as targets, and these targets are associated with diseases. This is how the drugs exhibit their biological activities. This tells us that the biological activity of drugs require the presence of specific individual chemicals. Drug can have one target or many targets to interact with to show the activity, and these targets can be proteins, enzymes, hormones, ion channels, and nuclear receptors [4]. There are many techniques that scientists use to identify and isolate targets to learn more about them and how they influence diseases such as high-throughput screening (HTS) where a large libraries of chemicals are tested for their ability to modify the target [4]. So, the process of drug discovery involved identification of candidates, synthesis, characterization, screening, and assays for therapeutic efficacy. Once a compound has shown its value in these tests, the process of drug development prior to clinical trials will begin. In general, the process of discovering a new drug can start from two starting points. The first starting point is having a long-standing history associated with natural product chemistry, and this 8

22 begins when noticing or observing a particular substance exhibits a biological activity that may be of potential therapeutic interest. Although the substance can be any chemical, their source has been small-molecule natural products derived from plants, microbes, marine organisms, and animals [4]. The second starting point begins with an interesting molecular biology or pharmacologic observation. In this case, the observation often involving very large biomolecules or entire physiological systems serves to prompt the pursuit of a substance that can be used to modulate the biological activity in a potentially therapeutic manner. 2. Ferroptosis is a newly discovered form of cell death Ferroptosis is a recently-described form of regulated cell death caused by an irondependent accumulation of lipid reactive oxygen species (ROS) [5]. Ferroptosis is dependent upon intracellular iron, but not other metals. Cells dying through ferropotosis show distinct morphology, and are biochemically distinct from apoptosis, necrosis and autophagy. Ferroptosis is induced through the inhibition of system Xc - antiporter, and this induction is done via two discovered ferroptotic drugs that causing either glutathione (GSH) depletion or glutathione peroxidase (GPX4) inhibition (5). These two ferroptotic drugs are Erastin and RAS- selective lethal molecule 3 (RSL3). System XC - is an amino 9

23 acid antiporter that typically mediates the exchange of extracellular l-cystine and intracellular l-glutamate across the cellular plasma membrane (5). System XC - composed of two subunits and those are SLC7A11 and SLC3A2. Erastin was shown to inhibit this antiporter through direct binding to SLC7A11 subunits thereby blocking the cystine uptake through this antiporter (5). This blocking leads to a low level of cystine inside the cells, and cystine is one of the amino acids used in glutathione synthesis. So glutathione levels will drop in Erastin-treated cells, and it is known that glutathione is very important and crucial for the cells to protect them from oxidative cell death. Glutathione works as an antioxidant that protects and removes the ROS and free radicals that when accumulate in the cells leads to ferroptosis cell death [18]. The other way that ferroptosis can be induced is via RSL3 molecule [19]. RSL3 has different substrate to bind to which is different from erastin s substrate [5]. RSL3 bind to GPX4 [5]. GPX4 is a phospholipid hydroperoxidase that protects cells against membrane lipid peroxidation. Depletion of glutathione causes inactivation of GPX4 [5]. When the activity of the lipid repair enzyme glutathione peroxidase 4 (GPX4) is lost leading to the accumulation of lipid-based reactive oxygen species (ROS), particularly lipid hydroperoxides, the cell dies through ferroptosis [5]. Recent studies have shown that acetaminophen induces cell death by ferropotosis and is characterized by GSH depletion [19]. The RAS pathway is required for ferroptosis induction. RAS is a family of related proteins expressed in all cells, and 10

24 they belong to class of protein called small GTPase. GTPase are involved in transmitting important signals between cells. When signals are received by a cell, these signals switch on the RAS proteins. RAS switchs on other proteins that are involved in cell growth, differentiation and survival. So any mutations in RAS proteins will lead to nonstop activation of these genes and then ultimately to cancer. Cancer cells carrying an active RAS oncogene can be killed by erastin through the process of ferroptosis [5]. When the RAS pathway is inhibited, cells become more resistance to chemotherapy treatment, and interestingly our drug did not show any toxicity in these cells. That Ras can drive oncogenesis through multiple effectors suggests that effective inhibition of Ras will require concurrent inhibition of different effector networks [6]. Resistance to drugs in cancer treatment occurs during chemotherapy treatment. Additionally, drug resistance to chemotherapy can develop over prolonged use as has been seen with doxorubicin and taxol [7]. In a pre-clinical study combining paclitaxel (taxol) and MEK inhibitors in ovarian carcinoma cell lines, results demonstrated enhanced apoptosis and growth inhibition [8]. Deferoxamine preferentially abrogates the intralysosomal accumulation of iron and inhibits oxidative stress-induced lysosomal membrane permeabilization and cell death [9]. p53 inhibits cystine uptake through repression of the SLC7A11 gene to mediate ferroptosis [20]. p53 can induce both apoptosis and ferroptosis upon reactive oxygen species (ROS)-induced stress. Iron is required for cellular physiology, and it works as a 11

25 cofactor for various cellular proteins and enzymes. The absence of its regulation can lead to cell death. Iron homeostasis is maintained via specific cellular pathways. The regulators of iron metabolism include iron-trafficking proteins [21]. It is shown that cargo receptor (NCOA4) is involved in releasing ferritin-bound iron to regulate iron metabolism [21]. The presence of oxygen combined with excess iron contributes to cell death. The hemeoxygenase (HO) is a major intracellular source of iron that accelerates Erastin-induced ferroptotic cell death [22]. Sorafenib induces ferroptosis, and ferrostatin- 1 is capable of blocking the cytotoxic effects of sorafenib [10]. Loss of CARS, the cysteinyl-trnasynthetase, suppresses ferroptosis induced by erastin, which inhibits system XC - [11].Iron-carrier protein transferrin and amino acid glutamine have shown to induce ferroptosis [21]. 3. Other major forms of cell death Cell death can be a result of two reasons. First reason is that cell might get injured by mechanical damage or by exposing to toxic chemicals. Second reason is that a cell goes through suicide process due to internal signals or external signals. Programmed cell-death (or PCD) is when a cell dies in any form, and this PCD is usually mediated by an intracellular program. PCD is an important process during an organism's life-cycle, and it 12

26 serves main functions during tissues development. The number of cells in all organism is tightly regulated by controlling the rate of both cell division and cell death. There are three main forms of cell death which are apoptosis, necroptosis, autophagy, and the new discovered form of cell death called ferroptosis. 3.1 Apoptosis Apoptosis is a process of PCD that takes place in multicellular organisms. Morphological changes happen during apoptosis include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mrna decay [12]. In the average human adult, approximately between 50 and 70 billion cells die every day. Apoptosis is highly regulated process. During apoptosis process first apoptotic bodies are formed then are engulfed by phagocytic cells to remove them. Apoptosis can be initiated via two pathways. First is the intrinsic pathway, which is also called mitochondrial pathway, when the cell senses there is a stress going in the cell, cell kills itself. This pathway depends on proteins that are released from the intermembrane space of mitochondria. Second is extrinsic pathway which is mostly initiated by the binding of an extracellular signals to receptors on the cell membrane to send signals from outside of the cell, and that will ultimately lead to the formation of intracellular apoptosis mechanism such as the formation of the death-inducing signaling complex (DISC) [12]. 13

27 The cell death induced in these two pathways is via activating the caspases. These caspases are also enzymes that are working on degrading proteins, so they are called proteases. Apoptosis is very important biological process, and any defects or errors in this processes will lead to serious problems that are related to many diseases. There are factors that promote or initiate apoptosis such as caspases, Fas cell surface death receptor, tumor necrosis factor alpha (TNFα), whereas there are some proteins that play a role in inhibiting apoptosis by blocking caspases such as Bcl-2 family and, viral inhibitor crma, and inhibitor of apoptosis (IAP s) [12]. Inhibition of apoptosis can lead to cancer and other diseases. Apoptosis plays a major role from embryonic development to senescence. There are examples where apoptosis is needed for proper development such as the formation of the fingers and toes of the fetus, the sloughing off of the inner lining of the uterus, and the formation of the proper connections between neurons in the brain. On the other hand, there are cases where apoptosis is needed to destroy cells such as cells infected with viruses, cancer cells, and cells with DNA damage. If the repair of the damaged DNA is unsuccessful, p53 will induce expression of BCL2 Associated X Protein (BAX). BAX then will cause apoptosis by acting against bcl2 [12]. Flow cytometry is a powerful tool to determine the number of apoptotic cells. Apoptosis requires energy. Z-VAD-FMK is one of a series of well-defined apoptosis modulators, and works by directly inhibiting caspases. 14

28 3.2 Necroptosis Necroptosis was first recognized as a caspase-independent form of cell death that can be triggered by treatment with TNF only in the presence of a pan-caspase inhibitors. Necroptosis is activated through stimulation by tumor necrosis factor α (TNFα), Fas ligand (FasL) which belongs to TNF, and TNF-related apoptosis-inducing ligand(trail). When TNF binds to tumor necrosis factor receptor (TNFR), this binding causes recruitment of RIP1 and RIP3 along with caspase 8 [13]. Receptor interacting protein 1(RIP1) is required in necroptosis regulation with its important kinase activity, and it is known by its role in NF-ҡB activation too. Necrostatin-1 is an inhibitor of the kinase activity of RIP1, so it inhibits necroptosis. There are studies that have focused on finding the substrates of RIP1. RIP3, a member of RIP1 family, is implicated in necroptosis [13]. It has been shown that some cancer cell lines being sensitized to necroptosis when RIP3 is expressed. Although RIP1 and RIP3 proteins share similarity in their kinase domains, Nec-1 inhibits RIP1 but doesn t inhibit RIP3 [13]. To show that kinase activity of RIP3 is involved in necroptosis, reconstitution of RIP3 deficient cells with a kinase dead K50A mutant is unable to restore sensitivity to necroptosis. It is not known that if RIP1is phosphorylated, even though this event is inhibited by Nec-1 [13]. So it is expected that RIP1 mediated RIP3 phosphorylation. Inhibition of caspase 8 allows RIP1 and RIP3 to initiate signals that affect mitochondrial generation of ATP and ROS. RIP1-RIP3 15

29 signaling reduces mitochondrial ATP generation, causes production of ROS, and permeabilizes lysosomal membranes, thereby causing cellular swelling and membrane damage. When cellular contents released because of the damage in the cell membrane, it evokes an inflammatory reaction. Blocking the kinase activity of RIP1, a key druggable target in the necroptosis pathway by Nec-1, inhibits the activation of necroptosis and allows cell survival and proliferation in the presence of death receptor ligands [13]. The function of caspase 8 should be inhibited or disrupted for this process to be activated. Necroptosis is a strong trigger of innate and adaptive immune responses unlike in apoptosis where immune proteins are sequestered. Necroptosis looks like necrosis morphologically and looks like apoptosis mechanistically as a form of programmed cell death. So, upstream signaling of necroptosis activation are known whereas identification of downstream signaling is recommended to better understanding how necroptosis is executed. Mitochondria function has been implicated in downstream signaling of RIP1. Necroptosis is characterized by the increase in cell volume, swelling of organelles, perforation of plasma membrane, cellular collapse, and release of cellular contents [13]. 3.3 Autophagy Autophagy is the process in which a cell sequesters and recycles unnecessary and dysfunctional cellular components and macromolecules. So in a simple definition, the 16

30 cell eats its own components. By this process, autophagy allows degradation and recycling of cellular components. There are three main pathways of autophagy, macroautophagy, microautophagy, and chaperone-mediated autophagy, and every pathway is different from the other two [14]. Autophagy starts in arranged steps, and these are sequestration, transport to lysosomes, degradation, and utilization of degradation products. Each step may exert different function.the autophagy-related (Atg) genes play essential roles at different stages of the autophagic process, including induction, vesicle formation, and autophagosome degradation. Two connected signaling pathways encompassing class-i phosphatidylinositol 3- kinase (PI3k) and (mammalian) target of rapamycin (MTOR) play a central role in controlling macroautophagy in response to starvation [14]. PI3Ks are essential for starvation induced autophagy, and 3- MA, wortmannin, LY target PI3Ks and result in inhibition of autophagy. MTOR is an atypical serine/threonine kinase that is present in two distinct complexes, and it inhibits autophagy when phosphorylated [15]. MTOR also regulates protein and amino acid synthesis. Atgs proteins create autophagic vacuoles, then autophagic vacuoles fuse with lysosomes and content is digested. When cells lose essential nutrients, autophagy is activated to supply the missing components. Studies have clearly shown that autophagy has a very important variety of physiological and pathophysiological roles, such as starvation adaptation, intracellular protein and organelle clearance, development, anti- 17

31 aging, elimination of microorganisms, cell death, and tumor suppression. Autophagy is upscaled during cellular stress, and cellular stress occurs when there is deprivation of nutrients and/or growth factors. Nutrient deprivation and accumulation of long-living proteins trigger autophagy. Autophagy has a dual role in cancer where it can serve in tumor growth or in tumor suppressor. When autophagy serves in tumor suppression mechanism, it helps in protecting cells against DNA damage and genomic instability by removing from the cytoplasm damaged organelles and proteins (major sources of ROS) [14]. Also it may prevent tumor development by regulating the cellular level of p62 [14]. It also can restrict cell proliferation of transformed cells by activating oncogene induced senescence. Finally, it could also act as tumor suppressor as a non-autonomous mechanism by preventing necrosis and subsequent inflammation [14]. However, when autophagy serves in tumor promoting mechanism, it does that by allowing tumor cells to survive in stressful conditions and supporting tumor development by maintaining the survival and self-renewal of cancer stem cells. 3-Methyladenine is a specific autophagy inhibitor. 18

32 4. Interaction between the cell death forms and cancer metastasis In the course of cancer metastasis, malignant cells must overcome a series of unfavorable conditions, including detachment from the ECM, attack by immune cells, hypoxia and a growth factor-lacking environment, which cause increased cellular ROS production and DNA damage and an insufficient energy status [13]. Most often metastatic cells don t acquire the ability to metastasize successfully, therefore; they are killed via apoptosis or necroptosis. On one hand, autophagy greatly improves the fitness of cancer cells under stressful conditions and, thus, attenuates apoptosis and necroptosis, but on the other hand, autophagy antagonizes metastasis by restricting tumor necrosis and subsequent immune cell infiltration [13]. In the same time, increase in autophagy process causes metastasizing cells to die. Therefore, interaction between the cell death forms and cancer metastasis is highly complicated. 5. The explanation of our project Our project is to study the mechanism of our novel molecule in inducing ferroptosis in cancer cells. The novel molecule 6E that was developed in collaboration with Dr. L. M. Viranga Tillekeratne lab at the University of Toledo and related compounds were originally derived from epothilone. Epothilones are cytotoxic agents that induce 19

33 apoptosis and were first isolated from the myxobacteriumsorangiumcellulosum in the 1980s [15]. Epothilones bind tothe α and β tubulin heterodimer subunitsincreasing aggregation of microtubules, while decreasing heterodimer disassociation from microtubules. While designing a library of epothilone derivatives, we have discovered a novel class of small molecules that do not bind to tubulin to induce apoptotic cell death, on the other hand; they kill cells by inducing toxic levels of ROS suggesting that they kill cells by ferroptosis, a newly described form of cell death in which iron is required for ROS formation. After many synthetic changes with these new class of molecules, we finally ended up with four anticancer agents with remarkable selectivity toward cancer cells. These agents have in common a 4-cyclopentenyl-2-ethynylthiazole skeleton (hence called CETZOLES). CETZOLEs kill tumor cells by enhancing the accumulation of (ROS), and this cell death is inhibited when RAS/MAPK pathway is inhibited, features of ferroptosis. Our studies focus on one molecule that shows selective lethal effect on NIH- H522 cells that we named 6E. So, 6E induces cell death through enhancing the ROS, and this cell death is inhibited by Fer-1 inhibitor. Also, the cell death induced by 6E is inhibited when RAS pathway is inhibited. In addition, 6E kills mesenchymal cells, and that s is very important feature of the molecule because it is known that killing cancer cells derived from mesenchymal tissues is more difficult than killing cancer cells derived from epithelial tissues. 20

34 6. Hypotheses First we hypothesis that 6E inhibits system Xc - to induce ferroptosis. To test this hypothesis we will measure glutathione levels in 6E-treated cells, measure glutamate secretion, and the ability of N-acetylcysteine (NAC) to rescue cell death induced by 6E. Second, we hypothesis that cancer cells that derived from mesenchymal tissues are sensitive toward 6E such as some breast cancer cell lines. To test that, we would perform cell variability assay to test the sensitivity of subtypes of breast cancer cells toward 6E. Our third and last hypothesis is that since mesenchymal cells are more sensitive to 6E, childhood sarcomas might also be sensitive. 7. Cell lines that are used in this project 7.1 Breast cancer cell lines Breast cancer is a type of malignant tumors that localize in breast cells. It is found among women mostly, although it can be found among men but this is not common. The risk to get breast cancer depends on many factors such as the age, family history, genetics and hormonal factors, and race. When breast cancer is found in its early stage, the chances for successful treatment will be high. There are symptoms that women can notice and predict to be a sign of having breast cancer, so they can be more careful and talk to their doctors 21

35 as soon as they can before cancer gets worse. Some common breast cancer symptoms are swelling of all or part of the breast, skin irritation or dimpling, breast pain, and nipple pain or the nipple turning inward [16]. Breast imaging tests are an effective way to detect breast cancer in women, and created images allow doctors to see inside the body. About 90% of women newly diagnosed with breast cancer will survive for at least five years [16]. There are five major subtypes of breast cancer that are defined based on the gene expression analyses, and those are LuminalA, Luminal B, HER2-enriched, basal-like, and claudin-low subtypes. It is shown that these subtypes are useful in developing therapies. Most breast cancer are carcinomas from epithelial tissues, even though there is subset of tumors that show mesenchymal gene expression-patterns. These mesenchymal breast cancer are called Claudin-low breast cancer. We used three breast cancer cell lines that are mesenchymal cells.. SUM159 cell line and MDAMB231 cell line are claudin-low, whereas MDAMB468 is basal-type breast cancer cell line. We focus in claudin-low subtype here because they are mesenchymal cells and our novel drug tends to kill mesenchymal cells more effectively than epithelial cells. Claudin-low tumors are characterized by the low to absent expression of luminal differentiation markers, high enrichment for epithelial-to-mesenchymal transition markers, immune response genes and cancer stem cell-like features [17]. 22

36 7.2 Lung Cancer cell line There are two types of lung cancer. First type is non-small cell lung cancer (NSCLC) and it accounts about 80% to 85% of lung cancers [1]. Squamous cell carcinoma, adenocarcinoma, and large cell carcinoma are all subtypes of NSCLC. Second type is small cell lung cancer (SCLC), and it accounts about 10% to 15% of lung cancers. Here we focus on the first type (NSCLC). About 224,390 new cases of lung cancer diagnosed, and about 158,080 deaths cases from lung cancer is estimated by the American Cancer Society [1]. Lung cancer occurs mostly in people who are 65 and older. Lung cancer usually detected in its advanced stages. NCI-H522 cell line is the NSCLC cell line used in our project. 7.3 Childhood cancer cell line 15% of childhood cancers are sarcomas (compared to 1% of adult cancer). About 10,380 children in the United States under the age of 15 will be diagnosed with cancer in 2016 [1]. About 1,250 children younger than 15 years old are expected to die from cancer in The most common cancers in children are (childhood) leukemia (34%), brain tumors (23%), lymphomas (12%), Neuroblastoma (7%, nervous system), Wilms tumor (5%, kidney), Rhabdomyosarcoma (3%, many sites), Retinoblastoma (3%, eye), Osteosarcoma (3%, bone cancer), and Ewing sarcoma (1%, many sites) [1]. The causes 23

37 of it are not known, and about 5% of all cancers in children are caused by an inherited genetic mutation that can be passed from parents to their children. Here, we analyzed two Rhabdomyosarcoma cell lines (Rh-31 and Rh-40) and one Ewing cell line (TC-71) for sensitivity toward 6E. Rhabdomyosarcoma occurs in cells that will develop into skeletal muscles, and it is the most common type of soft tissue sarcoma in children and adolescents less than 20 years old, with an incidence of 4.5 cases per million children/adolescents per year [1]. The Ewing family of tumors is cancers that start in the bones or tissues that are nearby and have same features as bone tissues have. More than 80% of children with cancer now survive five years or more due to the increase in its advanced treatment [1]. 8. Materials and Methods 8.1 Cell culture conditions Cells were grown in Dulbecco s minimal essential medium (Gibco) with 5 ml penicillin/streptomycin and 10% fetal bovine serum and 10% comic calf serum in a humidified atmosphere of 10% CO2 at 37 C. 8.2 Cell types 24

38 We used the non-small cell lung cancer cell line NSCLC (H522) cell line. Also we used three subtypes of breast cancer. MDAMB231 and SUM159 are claudin-low subtype, MDAMB468 is a basal-like subtype. We also use three childhood cancer cell lines. The first two cell lines are Rh41 and Rh30 were derived from Rhabdomyosarcomas. This cancer forms in muscle tissue. The third childhood cancer cell line used was TC-71 belonging to the family of Ewing's Sarcoma. 8.3 Drug treatment Cells were grown in the DMEM that contained either 10% FBS or 10% CCS. Once the cells were confluent in the plate, we aspirated the medium and then washed the cells with sterilized 1X PBS (usually using 5mL/10cm plate). Then we aspirated the 1X PBS and added 1mL Trypsin/EDTA and rotated the plate to allow thetrypsin to cover the whole plate. Then, after cells detached from the plate, we aspirated the trypsin. Then, we resuspended the cells in a small volume of serum-containing medium to inactivate the trypsin. Next, we determined the cell density using ahemacytometer, and transfer the required number of cells to a new tube with the appropriate final volume of new medium. Next, we plated the cells in multi-well plates (in 24 well plates we plated cell per well) and (in 96 multi-well plates we plated cell per well). After plating cells in the multi-well plate, we incubated the plate in a humidified atmosphere of 10% CO2 at 37 C to let the cells attach properly to the bottom of the wells. One day later, we added the 25

39 various treatments and incubated the plate back in a humidified atmosphere of 10% CO2 at 37 C. One to three days later, we measured cell viability. To measure viability, we removed the media from the plate and dried it properly. Then we stained the plate using methylene blue to stain cells left in the wells after treatment. Cells were stained for 30 minutes at room temperature. Then, we removed the dye and allowed the plate to dry. Then, we added 250µL of 0.2M HCL solution into each well, and then incubated the plate at 37 C for 30 minutes. After the incubation time, we transfered 200µL of 0.1M HCL from each well into the wells of 96 well plate to read the absorbance using a Spectramax plate reader. We read the absorbance at 530 nm. 8.4 Measurement of Glutathione levels To detect the amount of glutathione present in the cells we used a thiol- reactive dye monochlorobimane (MCB) which is essentially nonfluorescent until it reacts with a thiol group in glutathione to form a blue fluorescent product. Fluorescence can easily be detected using a 96-well fluorometric plate reader. We prepare a mixture which contains 25mL 1XPBS and 200µL of the prepared dye monochlorobimane. We prepared monochlorobimane by weighting 1.8 gram of monochlorobimane powder and dissolving it in 1.98 ml DMSO. Then we mixed 5 ml PBS in a tube with 50µL of the dissolved solution. Next, we transferred 200µL of the 26

40 mixture into each of 10 Eppendorf tubes that were stored at -20 C. One tube was then used for each experiment to avoid freeze-thaw cycles. To measure glutathione, the media was aspirated from the 96-well plate and the contents of one of the MCB tubes (200µl solution) was mixed with 25mL PBS. Next, 200µL of this dilution was added in all wells which were then covered with foil to protect from light. Plates were incubated for 30 minutes at 37 C. After incubation, we read the fluorescence emission at 400nm using excitation wavelength of 500nm. 8.5 Measurement of secreted glutamate using The Amplex Red Glutamic Acid/Glutamate Oxidase Assay In the assay, glutamate oxidase enzyme oxidizes L-glutamic acid and produces α- ketoglutarate, NH3 and H2O2. Two additional enzymes included in the reaction are L- alanine and L-glutamate pyruvate transaminase. These two enzymes regenerate L- glutamic acid by transamination of α-ketoglutarate which results in multiple repeating of the first reaction and an amplification of the H2O2 produced. Then the H2O2 reacts with 10-acetyl-3,7-dihydroxy phenoxazine (Amplex Red reagent) in a 1:1 stoichiometry, and this reaction is catalyzed by horseradish peroxidase (HRP) to generate resorufin, the highly fluorescent product. First we prepared the samples to carry out the assay. We plated H522 cells at a density cell per well in a 24 well plate. One day after, we 27

41 treated cells with DMSO as negative control, 5µM 6E, 10µM 6E, or 10µM erastin. Three hours later we aspirated the media and washed the wells three times with pre-warmed DMEM (1 minute per wash). After washing, we added 200µL fresh DMEM. Then we started collecting samples (5µL) from the wells starting from 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 10 hours samples. Samples were collected in triplicate and then analyzed for glutamate. The assay was carried out in a 96 well plate by adding 25µL of the sample plus 25µL of the 1X reaction buffer that we prepared and 50µL of the prepared reaction mixture in each well. The final volume in each well is 100µL. We also prepared wells with different concentrations of glutamic acid allowing us to prepare a standard curve to compare the level of glutamic acid in the samples. We covered the plate with foil to protect from light and incubated at 37 C for 30 minutes. Next, fluorescence emission was measured at 590nm using excitation between 530 and 560 nm. 8.6 Cytotoxicity We performed cell viability assay using three childhood cell lines and the NCI-H522 breast cancer cell line. We plated the cells in a 24 well plate at a density cell per well. One day later, cells were exposed to the drugs. One day after drug addition,cell viability was detected using methylene blue staining as describes.similarly, we plated 28

42 TC71 cells in a 24 well plate. One day later, cells exposed to 1µM, 2µM, 10µM, and 20µM concentrations of 6E. After 24 hours of exposure to 6E, cell viability was detected using methylene blue. For the effect of the inhibitors of cell death induced by 6E, we plated H522 cells in a 24 well plate. One day later, cells were exposed to 20µM 6E, 20µM Z-Vad, 2µM ferrostatin-1, or 60µM 3-MA. After 24 hours of exposure, cell viability was determined using methylene blue. 8.7 IC50 calculation We used GraphPad Prism software to calculate IC50s. For this, we collected a series of dose-response data (drug concentrations), and growth inhibition as a percent of vehicle control. IC50 values were the calculated with a non-regression analysis. 9. Results 9.1 The chemical structure of our new molecule 6E and its derivation Our novel molecule 6E that was developed in collaboration with Dr. Tillekeratne lab at the University of Toledo and related compounds were originally derived from epothilones (Figure 1). Epothilones are cytotoxic agents that induce apoptosis and were first isolated from the myxobacteriumsorangiumcellulosum in the 1980s. Epothilones bind totheα and β tubulin heterodimer subunitsincreasing aggregation of microtubules, 29

43 while decreasing heterodimer disassociation from microtubules. While designing a library of epothilone derivatives, we have discovered a novel class of small molecules that do not bind to tubulin to induce apoptotic cell death, on the other hand; they kill cells by inducing toxic levels of ROS. Additional features of these molecules suggested that they kill cells by ferroptosis, a newly described form of cell death in which iron is required for ROS formation. Our molecules resulted from a refinement of the open-chain epothiloneswhich were developed by simplification of the epothilone structure on the basis of available structure-activity relationship (SAR) information(figure 1). After many synthetic changes with these new class of molecules, we finally ended up with four anticancer agents with remarkable selectivity toward cancer cells (Figure 2). These agents have in common a 4-cyclopentenyl-2-ethynylthiazole skeleton (hence called CETZOLES). CETZOLEs kill tumor cellsby enhancing the accumulation of reactive oxygen species (ROS), and this cell death is inhibited when RAS/MAPK pathway is inhibited, features of ferroptosis. Our studies focuson one molecule that shows selective lethal effect on NIH-H522 cells that we named 6E (Figure 3). Stereochemistry of the secondary hydroxyl group of 6E has no effect on activity. The ketone group is slightly less active than the alcohol in 6E, and therefore, H-bond donor appears to be preferred over a H-bond acceptor at this position. These agents contain a terminal alkyne group. 30

44 Preliminary investigations have shown that the terminal alkynegroup acts as a Michael acceptor and can be considered the warhead of the molecule. 31

45 Figure 1: Natural epothilones and synthetic open-chain epothilones. Designing a library of epothilone derivatives, we have discovered a novel class of small molecules (1, 2, 3, and 4) that do not bind to tubulin to induce apoptotic cell death, on the other hand; they kill cells by inducing toxic levels of ROS, suggesting that they kill cells by ferroptosis, a newly described form of cell death in which iron is required for ROS formation. 32

46 Figure.2: The 4-cyclopentenyl-2-ethynylthiazole compounds. Compound number 58 represents 6E. 33

47 Many synthetic changes were performed to the new class of molecules in the previous figure (1), and we finally ended up with four anticancer agents (57, 58, 59, and 60) with remarkable selectivity toward cancer cells. These agents have in common a 4- cyclopentenyl-2-ethynylthiazole skeleton that we named CETZOLES, and CETZOLEs kill tumor cells by enhancing the accumulation of reactive oxygen species (ROS). 34

48 Figure 3: The chemical structure of 6E molecule 35

49 9.2 The cytotoxicity of 6E in different cancer cell lines 6E induces ferroptosis in many cancer cell lines, and it is chemically different from Erastin the first known molecule to induce ferroptosis. Every cell line has a different IC50 toward 6E, and this large difference in sensitivities of these various cell lines suggests that 6Eis not a general toxin, but relies on metabolic properties and/or genetic factors in certain tumor cells to allow sensitivity. We measured the IC50 for different cancer cell lines that were exposed to 6E and analyzed by methylene blue staining 3 days later (Figure 4). Non-small lung cancer cell line H522 was the most sensitive cells to 6E with IC50 of 1.5µM. Also, we observed that sensitivity of the cell line toward 6E is decreasing in cells derived from epithelial tissues whereas sensitivity is increasing in cells derived from mesenchymal tissues. Moreover, 6E induces ferroptosis in RAStransformed cells, so mutations in RAS pathway enhance 6E toxicity. 36

50 Figure 4: The IC50 of different cancer cell lines exposed to different concentration of 6E, and the cell viability was analyzed using methylene blue staining. Data from cell viability assays are not shown here. 37

51 9.3 Cell death induced by 6E is inhibited in the presence of ferroptosis modulators such as Ciclopiroxolamine (CPO) and Trolox Cells die by a number of different mechanisms including programmed cell death. Some of these forms of cell death can be differentiated by the effects of certain inhibitors. For example, apoptosis cell death can be blocked by caspase inhibitors, while necrosis can be blocked by necrostatin. Previous work has uncovered a number of compounds that can modulate ferroptosis.the first important natural chemical involved in ferroptosis inductionis iron which comes from external sources. Although iron is a very important substance for life, in excess it can cause cell death. There are a subset of genes that regulateand maintainiron homeostasis. For example, iron-trafficking proteins [solute carrier family 39, SLC39, also known as ZRT/IRT-like protein, ZIP; and poly-(rc)- binding protein, PCBP] and a cargo receptor (NCOA4) are crucial for iron regulation in cells. Dysregulation of iron metabolism contributes to various human pathologies, including iron overload diseases and cancer.here we tested the effect ofciclopiroxolamine and trolox in the cell death induced by 6E. Ciclopiroxolamine is an antifungal medication and also a strong iron chelator.trolox is an antioxidant like vitamin E and it is used in biological or biochemical applications to reduce oxidative stress or damage. When we treated MDAMB468 breast cancer cells with 10 M 6E in combination with Troloxor Ciclopiroxolamine, we found that these two chemicals inhibit 38

52 the cell death induced by 6E. Since Ciclopiroxolamine is an iron chelator, these observations indicate a requirement for iron in CETZOLE induced killing of human breast cancer cells. Previous work suggests that the role of iron in ferroptosis may be to catalyze a fenton reaction converting hydrogen peroxide to the highly toxic hydroxyl free radical [18]. In thetrolox condition, we also observed inhibition of CETZOLE killing of MDAMB468 cells suggesting that our compounds are killing cells by elevating ROS. (Figure 5). 39

53 Absorbance Units * p < 0.05 versus DMSO Figure 5: MDAMB 468 cells plated in 24 well plate. Day after cells exposed to DMSO as a negative control, 10uM 6E alone, 6E ± CPO or Trolox.After 24 hours of treatment, cell viability analyzed using methylene blue. 40

54 9.4 Mechanism of action of 6E System xc - is an amino acid antiporter that typically mediates the exchange of extracellular l-cystine and intracellular l-glutamate across the cellular plasma membrane. The uptake of l-cystine via this antiporter is very critical and serves in providing the intracellular l-cysteine that is required for glutathione synthesis (GSH). GSH is an important chemical used in protecting cells from oxidative damage (Figure 6). Therefore, any disruption in this system will lead to inadequate level of cysteine inside the cell resulting in depletion of glutathione, increase in ROS, and ultimately cell death. Ferroptosis can occur in two ways depending on the type of compound used.for example, Erastin inhibits system XC - through direct binding to one of its subunits. SLC7A11 is one of the subunits of system XC - that Erastin binds to it to inhibit the antiporter (Figure 7). On the other hand, the compound Ras-Specific-Lethal 3 (RSL3) induces ferroptosis by inhibiting glutathione peroxidase 4 enzyme (GPX4)(Figure 8). GPX4 is a phospholipid hydroperoxidase that protects cells against membrane lipid peroxidation. RSL3 binds directly to GPX4 then results in increased lipid oxidation leading to ferroptosis. Our goal was to determine which way orpathway our drug 6E uses to induce ferroptosis. If 6E blocks XC aserastin does, then we will expect to see less GSH, less glutamate secretion, and rescue with N-acetylcysteine (NAC). NAC works as antioxidant by providing cells cysteine by bypassing the XC - transporter. However, if our drug 6E induces ferroptosis 41

55 via inhibiting GPX4, then we would predict that there would be no effect on GSH, no effect on glutamate secretion, and no rescue with NAC. These predictions are due to the fact that GPX4 essentially works downstream of the cysteine supply. 42

56 Figure 6: Factors that modulate ferroptosis. SLC3A2 and SLC7A11 are the two subunits of system XC -. Uptake of cystine via this antiporter is crucial for glutathione synthesis. GPX4 uses GSH as a substrate to protect cells from oxidative death. Mitochondria NADPH oxidase is responsible for lipid ROS formation and iron is involved in this process. 43

57 Figure 7: The pathway of erastin in inducing ferroptosis. Erastin binds to SLC7A11 subunit of the system XC - leading to decrease in cystine import, glutathione level inside the cells, and less glutamate secretion through the antiporter, and ultimately cells die via ferroptosis. 44

58 Figure 8: The pathway of RSL3 molecule in inducing ferroptosis. RSL3 binds to GPX4 leading to cell death via ferroptosis. Since GPX4 is downstream of GSH, RSL3 and similar compounds do not affect cysteine levels, glutamate secretion of GSH levels. 45

59 9.4.1 N- acetylcysteine inhibits or stops the cell death induced by 6E N- Acetylcysteine is a derivative of the naturally occurring amino acid cysteine, which is deacetylated once it enters cells thereby providing free cysteine. It can work as an antioxidant by providing the building block for glutathione. If N-acetylcysteine rescues cells from CETZOLEs this would suggest that our compounds may alter the availability of cysteine. We plated H522 cells in a 24 well plate at cell per well. After 24 hours, we treated the cells with DMSO as a negative control, 10µM 6E alone, 10µM 6E+10mM NAC, 10µM6E+15mM NAC, 10mM NAC alone, and 15mM NAC alone. 24 hours later, we observed that 6E alone killed 95% of the cells comparing to the control DMSO. When NAC was added in combination with 6E, NAC abolished the toxicity of 6E and inhibited the cell death that induced by 6E. NAC alone did not have any effect on the cells (Figure 9).Therefore, NAC maybe replenishing cysteine that was depleted by 6E. This provides one piece of evidence that 6Eworksby inhibiting system XC - as oppositeof targeting GPX4. 46

60 Figure 9: H522 cells plated in 24 well plate. Day after cells exposed to 10µM 6E ± 10mM and 15mM NAC. After 24 hours, cell viability was analyzed using methylene blue. * presents the P value from student s t-test. 47

61 9.4.2 Glutathione level is depleted in 6E-treated cells Glutathione is an important antioxidant that is able to prevent damage to important cellular components caused by reactive oxygen species such as free radicals, peroxides, lipid peroxides and heavy metals.next we measured the level of glutathione in the cells after treating them with the drug 6E. We plated H522 cells in 96 well plate at density cell/well and leaving them for 24 hours to let the cells attach properly to the bottom of the wells. One day later, we treated the cells with 20 M 6E and 20 M erastin as a positive control and left them for 8 hours. To detect the amount of glutathione present in the cells we use a thiol- reactive dye monochlorobimane (MCB) which is essentially nonfluorescent until it reacts with a thiol group in glutathione to form a blue fluorescent product. Fluorescence can easily be detected using 96-well fluorometric plate reader. After reading the fluorescence, we observed that that cell treated with 6E and erastin had a lower level of glutathione comparedto the cells treated with DMSO (Figure 10). This observation is consistent with CETZOLEs potentially targeting XC - to cause glutathione depletion. 48

62 Figure 10: 6E depletes glutathione. NCI-H522 cells were exposed to either 6E or Erastin in 20µM concentration. After 8 hours, Glutathione was measured using monochlorobimane. 49

63 E-treated cells secrete less glutamate System XC - is an amino acid antiporter that typically mediates the exchange of extracellular cystine and intracellular glutamate across the cellular plasma membrane. When treating cells with 6E, we hypothesize that 6E binds to the antiporter suppressing the secretion of glutamate. To test this idea, we measured the level of secreted glutamate in cells treated with 6E and erastin and compared them with cells treated with DMSO. We used The Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit which provides a method to measure glutamate in culture media using a fluorescence microplate reader or fluorometer. In the assay, glutamate oxidase enzyme oxidizes l-glutamic acid and produces produce three products α-ketoglutarate, NH3 and H2O2. Two enzymes included in the reaction and they are L-alanine and l-glutamate pyruvate transaminase. These two enzymes regenerate l-glutamic acid by transamination of α-ketoglutarate which results in multiple repeating of the first reaction and a considerable amplification of the H2O2 produced. Then the H2O2 reacts with 10-acetyl-3,7-dihydroxy phenoxazine (Amplex Red reagent) in a 1:1 stoichiometry, and this reaction catalyzed by horseradish peroxidase (HRP) to generate resorufin, the highly fluorescent product. So we prepared the samples to carry out the assay. We plated H522 cells at a density cell per well in a 24 wellplate. One day after, we treated cells with DMSO as negative control, 5µM 6E, 10µM 6E, or 10µM erastin. Three hours later we aspirated the media and washed the 50

64 wells three times with pre-warmed DMEM (1 minute per wash). After washing, we add 200µL fresh DMEM. Then we started collecting samples (50µL) from the wells starting from 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 10 hours samples. Sampleswere collected in triplicate and then analyzed for glutamate secretion. The assay was carried out in a 96 well plate by adding 25µL of the sample plus 25µL of the 1X reaction buffer that we prepared and 50µL of the prepared reaction mixture (described in materials and methods section) in each well. So the final volume in each well is 100µL. We also had wells that had different concentrations of glutamic acid, and theseare the standard curve that we used to compare the level of glutamic acid in the samples to the level in these wells.after that we covered the plate with foil to protect from light and incubatedat 37 C for 30 minutes. Next, fluorescence emission at 590 nm was measured using excitation between 530 and 560 nm. What we observed was that cells treated with 6E secreted less glutamate as a result of inhibiting the antiporter, and this result was consistent with what was observed in Erastin-treated cells (Figure 11).Altogether, our results suggest that 6E induces ferroptosis via inhibition of system XC - (Figure 12). 51

65 Figure 11: NCI-H522 cells were plated in a 24 well plate. One daylater, cells were exposed to 5µM + 20µM of 6E, and 10µM erastin for three hours. Then glutamate released from the cells was measured using The Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit. * represents the p values from student s t-test. 52

66 Figure 12: The pathway of 6E in inducing ferroptosis. Our results suggest that 6E inhibits system XC - leading to inhibition of cystine import, decrease in glutathione level, and decreases in glutamate secretion in 6E-treated cells. 53

67 9.5 Mesenchymal cells tend to be more sensitive to 6E than epithelial cells Epithelial cells are bound together in sheets of tissue called epithelia. Epithelia are formed of cells that line the cavities in the body and also cover flat surfaces. These sheets are held together through several types of interactions, including tight junctions, adherens, desmosomes, and gap junctions. Mesenchyme is a type of tissue characterized by loosely associated cells that lack polarity and are surrounded by a large extracellular matrix. The epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells. This process is process co-opted by cancer cells to invade and form metastases. E-cadherin is expressed in epithelial cells whereas vimentin is expressed in many mesenchymal tissues. In one of our first experiments we tested a CETZOLE, 5e, which is a precursor of 6E against the NCI60 panel of cell lines. Next, we correlated sensitivity in this panel of cell lines with expression of E-Cadherin and Vimentin expression obtained from public databases. Using this analysis we observed that cancer cells become more sensitive to 6E when they are in mesenchymal state, whereas cancer cells become more resistance to 6E when they are in epithelial state (Figure 13). 54

68 SURVIVAL SURVIVAL A 150 5e 150 IMATINIB 150 ETOPOSIDE SALINOMYCIN 100 PACLITAXEL 100 GEFITINIB E-CADHERIN EXPRESSION B 150 5e 150 IMATINIB 150 ETOPOSIDE SALINOMYCIN 100 PACLITAXEL 100 GEFITINIB VIMENTIN EXPRESSION Figure 13: E-Cadherin reduces drug sensitivity. (A) Correlation of low E-Cadherin with drug senstivity. Survival data with 5e was obtained from our own 60 cell line NCI screen. Other compounds were queried using CellMiner to obtain NCI-60 drug sensitivity data sets (33, 34). E-Cadherin (cdh1) expression levels were obtained from an NCBI (Geo Dataset GDS4296) (35). (B) Drug sensitivity correlates with high vimentin expression. Expression and survival data was collected as described in A. 55

69 9.5.1 Some breast cancer cell lines are sensitive to 6E Most human cancers are derived from epithelial tissues, however there is a subset of breast cancers that have a mesenchymal gene expression signature. This claudin-low subtype represents ~10% of all breast cancers. Given that CETZOLEs are more toxic to at least some mesenchymal tumor cells we hypothesized that claudin-low breast cancer cells might also be sensitive.therefore, we tested the effect of 6E on claudin-low subtype SUM159 and MDA MB 231 cell lines, along with the basal subtype cell line MDA MB 468. We observed that MDA MB 231 cells and MDA MB 468 cells were sensitive to 6E whereas SUM159 cells were not sensitive (Figure 14). Therefore,not all mesenchymal cell lines are sensitive to 6E. 56

70 6E Erastin Figure 14: SUM159, MDAMB468, and MDAMB231 cells were plated. One day later, cells were exposed to 10µM, 20µM 6E or Erastin. Two days later, cell viability was analyzed using methylene blue. * indicates p values from a student s t-test. 57

71 9.5.2 Since mesenchymal cells are more sensitive to 6E, sarcomas from childhood cancer might also be sensitive. Cancers originating in any organ or tissue of your body are called primary cancers. If they spread, they re considered metastatic.there are two primary types of cancer: carcinomas and sarcomas, and they differ in the kind of tissues they originated from.sarcomas develop in mesodermal tissues such as bone, muscle, fat, nerves, cartilage, fibrous tissue, and connective tissue [1]. Carcinomas originate in epithelial tissue, such as the lining of breast, lung, colon or prostate.childhood cancer is cancer in children. 15% of childhood cancers are sarcomas (compared to 1% of adult cancer).the most common cancers in children are (childhood) leukemia (34%), brain tumors (23%), lymphomas (12%), Neuroblastoma (7%, nervous system), Wilms tumor (5%, kidney), Rhabdomyosarcoma (3%, many sites), Retinoblastoma (3%, eye), Osteosarcoma (3%, bone cancer), and Ewing sarcoma (1%, many sites) [1]. Here, we analyzed two Rhabdomyosarcoma cell lines (Rh-31 and Rh-40) and one Ewing cell line (TC-71) for sensitivity toward 6E Two rhabdomyosarcoma cell lines are sensitive to 6E Rhabdomyosarcoma (RMS) is a malignancy that arises from skeletal muscle precursors. It is the most common type of soft tissue sarcoma in children and adolescents less than 58

72 20 years old, with an incidence of 4.5 cases per million children/adolescents per year. We analyzed two Rhabdomyosarcoma cell lines Rh30 and Rh41 for sensitivity to 6E. Cells were plated, exposed to various concentrations of 6E to determine viability one day post treatment. Both cell lines were killed between 5µM and 10µM concentrations of 6E (Figure15A-B). 59

73 Absorbance Units A- Rh-30 cell line B- Rh-41 cell line * p< 0.05 versus DMSO DMSO 1µM 2µM 5µM 10µM 6E concentrations Figure 15: Rh41 cells (A) andrh30 cells (B) plated in a 24 well plate in density cell per well. One day later, cells were exposed to 1µM, 2µM, 5µM, and 10µM of 6E. One day later, cell viability was determined using methylene blue. 60

74 TC71 (Ewing sarcoma) is sensitive to 6E The Ewing family of tumors is a group of cancers that start in the bones or nearby soft tissues that share some common features. Here we analyzed the sensitivity of TC71 to 6E. So we performed a cell viability assay as described for the sarcoma cell lines.we found that cells were killed by 10µM and 20µM 6E (Figure 16). 61

75 Absorbance Units * p < 0.05 versus DMSO DMSO 1µM 2µM 10µM 20µM 6E concentrations Figure16: TC71 cells were plated in a 24 well plate in density cell per well. Day after cells exposed to 1µM, 2µM, 10µM, and 20µM concentrations of 6E. One day later, cell viability was determined using methylene blue. 62

76 9.6 The protective effect of Z-Vad, 3MA, and ferrostatin-1in inhibiting the cell death induced by 6E To further narrow down the mechanism of death induced by CETZOLEs we tested additional cell death modulators. We use Z-Vad which is apoptosis inhibitor, 3- Methyladenine which is autophagy inhibitor, and ferrostatin-1 which is ferroptosis inhibitor. We plated H522 cells in a 24 well plate. One day later, cells exposed to 20µM 6E, 20µM Z-Vad, 2µM ferrostatin-1, and 60µM 3-MA. After 24 hours exposure time, cell viability was detected using methylene blue. Neither Z-Vad nor 3-MA rescued cell death induced by 6E, whereas ferrostatin-1 did rescue the cell death induced by 6E (Figure 17A-B). This provides additional evidence that 6E induces ferroptosis in cancer cells and may not induce other forms of cell death (apoptosis and/or authophagy). 63

77 Figure 17-A: H522 cells were plated in a 24 well plate at adensity of cell per well. One day after, cells were exposed to 20µM 6E alone, 20µM Z-Vad ± 6E, and 2µM ferrostatin-1 ± 6E. 24 hours later cell viability was detected using methylene blue. * represents the p value from student s t-test. 64

78 Figure 17-B:H522 cells were plated in a 24 well plate at a density of cell per well. One day after, cells were exposed to 20µM 6E alone, and 60µM 3-MA ± 6E. 24 hours later cell viability was detected using methylene blue. 65

79 9.7 Analysis of 6E analogues to obtain expanded structure activity relationship (SAR) information. Our previous studies indicated that the terminal alkyne group attached to the thiazole ring was essential for 6E toxicity. For example replacing the alkyne with a methyl group abolished toxicity (15). To further probe the SAR of 6E, we replaced the alkyne with several additional functional groups including the reactive epoxide moiety (compounds 3 and 6) in (Figure 18-A). Each analog was dissolved in DMSO and tested on H522 cells at 20µM. After 3 days, we observed that only analog 3 retained toxicity (Figure 18-B). Therefore, toxicity is not confined to analogues with the alkyne group, but a reactive function group is required at this position. Surprisingly, the alcohol 6 was not toxic despite the presence of the epoxide group and we currently do not have an explanation for this observation. 66

80 Figure 18-A:Six different analogs of 6E (1, 2, 3, 4, 5 and 6) Changing bonds between the atoms in these molecules and replacing different group between these molecules was done to get these different analogs. We tested the toxicity of these molecules using H522 cells to get an idea of the group that makes the molecule more toxic to the cells. We found that molecule number 3 is the most toxic one between them. 67